The dystonia gene THAP1 controls DNA double-strand break repair choice.

The Shieldin complex shields double-strand DNA breaks (DSBs) from nucleolytic resection. Curiously, the penultimate Shieldin component, SHLD1, is one of the least abundant mammalian proteins. Here, we report that the transcription factors THAP1, YY1, and HCF1 bind directly to the SHLD1 promoter, where they cooperatively maintain the low basal expression of SHLD1, thereby ensuring a proper balance between end protection and resection during DSB repair. The loss of THAP1-dependent SHLD1 expression confers cross-resistance to poly (ADP-ribose) polymerase (PARP) inhibitor and cisplatin in BRCA1-deficient cells and shorter progression-free survival in ovarian cancer patients. Moreover, the embryonic lethality and PARPi sensitivity of BRCA1-deficient mice is rescued by ablation of SHLD1. Our study uncovers a transcriptional network that directly controls DSB repair choice and suggests a potential link between DNA damage and pathogenic THAP1 mutations, found in patients with the neurodevelopmental movement disorder adult-onset torsion dystonia type 6.

53BP1 Enforces Distinct Pre- and Post-resection Blocks on Homologous Recombination.

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.

A tetravalent dengue virus-like particle vaccine induces high levels of neutralizing antibodies and reduces dengue replication in non-human primates.

Dengue virus (DENV) represents a significant global health burden, with 50% of the world's population at risk of infection, and there is an urgent need for next-generation vaccines. Virus-like particle (VLP)-based vaccines, which mimic the antigenic structure of the virus but lack the viral genome, are an attractive approach. Here, we describe a dengue VLP (DENVLP) vaccine which generates a neutralizing antibody response against all four DENV serotypes in 100% of immunized non-human primates for up to 1 year. Additionally, DENVLP vaccination produced no ADE response against any of four DENV serotypes in vitro. DENVLP vaccination reduces viral replication in a non-human primate challenge model. We also show that transfer of purified IgG from immunized monkeys into immunodeficient mice protects against subsequent lethal DENV challenge, indicating a humoral mechanism of protection. These results indicate that this DENVLP vaccine is immunogenic and can be considered for clinical evaluation. Immunization of non-human primates with a tetravalent DENVLP vaccine induces high levels of neutralizing antibodies and reduces the severity of infection for all four dengue serotypes.IMPORTANCEDengue is a viral disease that infects nearly 400 million people worldwide and causes dengue hemorrhagic fever, which is responsible for 10,000 deaths each year. Currently, there is no therapeutic drug licensed to treat dengue infection, which makes the development of an effective vaccine essential. Virus-like particles (VLPs) are a safe and highly immunogenic platform that can be used in young children, immunocompromised individuals, as well as healthy adults. In this study, we describe the development of a dengue VLP vaccine and demonstrate that it induces a robust immune response against the dengue virus for over 1 year in monkeys. The immunity induced by this vaccine reduced live dengue infection in both murine and non-human primate models. These results indicate that our dengue VLP vaccine is a promising vaccine candidate.

An Envelope-Modified Tetravalent Dengue Virus-Like-Particle Vaccine Has Implications for Flavivirus Vaccine Design.

Dengue viruses (DENV) infect 50 to 100 million people each year. The spread of DENV-associated infections is one of the most serious public health problems worldwide, as there is no widely available vaccine or specific therapeutic for DENV infections. To address this, we developed a novel tetravalent dengue vaccine by utilizing virus-like particles (VLPs). We created recombinant DENV1 to -4 (DENV1-4) VLPs by coexpressing precursor membrane (prM) and envelope (E) proteins, with an F108A mutation in the fusion loop structure of E to increase the production of VLPs in mammalian cells. Immunization with DENV1-4 VLPs as individual, monovalent vaccines elicited strong neutralization activity against each DENV serotype in mice. For use as a tetravalent vaccine, DENV1-4 VLPs elicited high levels of neutralization activity against all four serotypes simultaneously. The neutralization antibody responses induced by the VLPs were significantly higher than those with DNA or recombinant E protein immunization. Moreover, antibody-dependent enhancement (ADE) was not observed against any serotype at a 1:10 serum dilution. We also demonstrated that the Zika virus (ZIKV) VLP production level was enhanced by introducing the same F108A mutation into the ZIKV envelope protein. Taken together, these results suggest that our strategy for DENV VLP production is applicable to other flavivirus VLP vaccine development, due to the similarity in viral structures, and they describe the promising development of an effective tetravalent vaccine against the prevalent flavivirus.IMPORTANCE Dengue virus poses one of the most serious public health problems worldwide, and the incidence of diseases caused by the virus has increased dramatically. Despite decades of effort, there is no effective treatment against dengue. A safe and potent vaccine against dengue is still needed. We developed a novel tetravalent dengue vaccine by using virus-like particles (VLPs), which are noninfectious because they lack the viral genome. Previous attempts of other groups to use dengue VLPs resulted in generally poor yields. We found that a critical amino acid mutation in the envelope protein enhances the production of VLPs. Our tetravalent vaccine elicited potent neutralizing antibody responses against all four DENV serotypes. Our findings can also be applied to vaccine development against other flaviviruses, such as Zika virus or West Nile virus.

Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation.

Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43(-/-) salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43(-/-) samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43(-/-) phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.

The effect of sex hormones on peroxisome proliferator-activated receptor gamma expression and activity in mature adipocytes.

Peroxisome proliferator-activated receptor (PPAR) γ plays a major role in the regulation of lipid and carbohydrate metabolism. Pioglitazone is a PPARγ agonist that is widely used for the treatment of type 2 diabetes mellitus. However, female patients have been reported to experience stronger efficacy and adverse effects than male patients. This study evaluated the effects of sex hormones on PPARγ expression and activity in adipocytes. Mouse 3T3-L1 preadipocytes were used after being grown into matured adipocytes. The sex hormones 17ß-estradiol (E2), testosterone (T), or 5α-androstan-17ß-ol-3-one (dihydrotestosterone; DHT) were added to the matured adipocytes and the cells were then maintained for short (24-72 h) or long (1- or 2-weeks) periods. E2 significantly upregulated PPARγ protein expression in a concentration-dependent manner after extended exposure, whereas T and DHT did not have such an effect. When cells were co-treated with pioglitazone and E2, PPARγ protein expression significantly increased in an E2-dependent manner, whereas this expression seemed to be reduced by pioglitazone mono-treatment and co-treatment with DHT at higher concentrations. The secretion levels of adiponectin protein, a major indicator of PPARγ activity, were significantly decreased by DHT, but were not affected by E2. Finally a luciferase assay was performed using a PPAR response element-Luk reporter gene. Transcriptional activity was not changed by any of single sex hormone treatment, but was significantly downregulated by co-treatment with pioglitazone and DHT. Taken together, our results suggest that sex hormones may influence PPARγ expression and function, which may explain the observed sex-specific different effect of pioglitazone.

Mechanical Regulation of Transcription: Recent Advances.

Mechanotransduction is the ability of a cell to sense mechanical cues from its microenvironment and convert them into biochemical signals to elicit adaptive transcriptional and other cellular responses. Here, we describe recent advances in the field of mechanical regulation of transcription, highlight mechanical regulation of the epigenome as a key novel aspect of mechanotransduction, and describe recent technological advances that could further elucidate the link between mechanical stimuli and gene expression. In this review, we emphasize the importance of mechanotransduction as one of the governing principles of cancer progression, underscoring the need to conduct further studies of the molecular mechanisms involved in sensing mechanical cues and coordinating transcriptional responses.

Association of oral HPV16 infection with periodontal inflammation and the oral microbiome in older women.

The present preliminary study aimed to investigate the association between oral human papillomavirus type 16 (HPV16) DNA prevalence and periodontal inflammation in older women. The association between oral HPV16 infection and oral health status has not been fully elucidated in older Japanese women. The present study investigated older women aged ≥60 years who visited Hiroshima University Hospital. The present study excluded subjects with clinical factors affecting HPV infection, such as current smoking, oral cancer and pre-malignant lesions, and immunodeficiency. Finally, 46 female patients (mean age, 74.6 years) were analyzed. Quantitative PCR analysis was performed to detect HPV16 DNA in oral rinse samples. A total of 4 participants (8.7%) were HPV16 DNA positive. There was a significant association between the HPV16 DNA positivity rate and bleeding on probing (P=0.03). Additionally, Prevotella intermedia positive cases exhibited a significantly higher HPV16 DNA positivity rate than negative cases (33.3 vs 3.8%). Furthermore, analysis of 16S ribosomal RNA in bacterial flora was performed to examine microbiome diversity in participants with ≥6 mm periodontal pockets and bleeding on probing. Importantly, the average percentage of Porphyromonas was significantly higher in HPV16 DNA positive cases compared with in HPV16 DNA negative cases (5.57 vs. 1.44%). By contrast, the average percentage of Veillonella was significantly lower in HPV16 DNA positive cases than in HPV16 DNA negative cases (2.43 vs. 8.51%). Prevotella was also lower in HPV16 DNA positive cases than in HPV16 DNA negative cases (4.0 vs. 8.23%). These results indicated that people with both deep periodontal pocket inflammation and oral HPV16 infection may not have Prevotella- or Veillonella-dominant oral microbiomes, and their microbiomes may exhibit their own distinctive characteristics. In conclusion, the results suggested that oral HPV16 infection may be associated with periodontal inflammation in older Japanese women. Further research is required to clarify the detailed association between oral HPV infection and the oral microbiome.

The Associations of Periodontopathic Bacteria and Oral Candida with Periodontal Inflamed Surface Area in Older Adults Receiving Supportive Periodontal Therapy.

The periodontal inflamed surface area (PISA) has been proposed for assessment of the total periodontal inflammatory status in people with periodontitis. This study was performed to investigate the associations of periodontopathic bacteria and candida with PISA in older people. We enrolled 100 patients aged ≥ 60 years who visited Hiroshima University Hospital. PISA and periodontal epithelial surface area (PESA) were calculated in each patient. Oral rinse samples were collected for DNA extraction. Periodontopathic bacteria and candida were detected by polymerase chain reaction. The mean values of PISA and PESA were significantly greater in T.forsythia-positive patients than in T.forsythia-negative patients. T.forsythia/C. albicans double-positive patients exhibited significantly greater PISA values than did non-double-positive patients. Additionally, PISA values were significantly greater in T. forsythia//T. denticola/C. albicans triple-positive patients than in T. forsythia//T. denticola/C. albicans non-triple-positive patients (p = 0.02). Propensity score-matching was performed between periodontopathic bacteria-positive and -negative patients using propensity scores generated from clinical factors. Importantly, T.forsythia/T. denticola double-positive patients exhibited significantly greater PISA values than non-double-positive patients among 72 propensity score-matched patients. Our preliminary results highlight the importance of the presence of T.forsythia and T. denticola for periodontal inflammation severity in older Japanese people.

Pannexin 3 ER Ca2+ channel gating is regulated by phosphorylation at the Serine 68 residue in osteoblast differentiation.

Pannexin 3 (Panx3) is a regulator of bone formation. Panx3 forms three distinct functional channels: hemichannels, gap junctions, and endoplasmic reticulum (ER) Ca2+ channels. However, the gating mechanisms of the Panx3 channels remain unclear. Here, we show that the Panx3 ER Ca2+ channel is modulated by phosphorylation of the serine 68 residue (Ser68) to promote osteoblast differentiation. Among the 17 candidate phosphorylation sites identified, the mutation of Ser68 to Ala (Ser68Ala) was sufficient to inhibit Panx3-mediated osteoblast differentiation via reduction of Osterix and ALP expression. Using a Ser68 phospho-specific antibody (P-Panx3) revealed Panx3 was phosphorylated in prehypertrophic, hypertrophic chondrocytes, and bone areas of the newborn growth plate. In osteogenic C2C12 cells, P-Panx3 was located on the ER membranes. Importantly, the Ser68Ala mutation only affected Panx3 ER Ca2+ channel function. Ser68 on Panx3 was phosphorylated by ATP stimulation and PI3K/Akt signaling. Finally, real-time FRET imaging and ratio analysis revealed that the Panx3 channel conformation was sensitive to ATP. Together, the phosphorylation of Panx3 at Ser68 is an essential step controlling the gating of the Panx3 ER Ca2+ channel to promote osteogenesis.

Reducing effect of feeding powdered nacre of Pinctada maxima on the visceral fat of rats.

An abdominal fat accumulation complicated by high blood triglycerides is regarded as a risk factor of metabolic syndrome. Feeding powdered nacre, mother of pearl, from Pinctada maxima, resulted in reduced body weight, visceral fat amount, and blood triglyceride level without influencing the food intake, body length, or amount of muscular tissue, suggesting that nacre powder specifically could decrease visceral fat.

Gastric mucosal levels of prostaglandins and leukotrienes in patients with gastric ulcer after treatment with rabeprazole in comparison to treatment with ranitidine.

AIM: Prostaglandins (PGs) and leukotrienes (LTs) are major factors involved in the defense of the gastric mucosa against ulcer formation. However, little is still known about the gastromucosa-protecting action of proton pump inhibitors (PPIs) and histamine H(2) receptor antagonists (H(2) blockers) in patients with gastric ulcer. We therefore examined the effectiveness of a PPI in protecting the gastric mucosa. METHODS: We compared the PGE(2) and LTB(4) levels and the expression levels of cyclooxygenase (COX)-1 and COX-2 mRNA in the gastric mucosa in gastric ulcer patients between the group treated for 8 weeks with a PPI, rabeprazole (PPI group; n=5), and the group treated for 8 weeks with an H(2) blocker, ranitidine (H(2) blocker group; n=6), as well as in nonulcer subjects (control group; n=5). RESULTS: The mucosal levels of PGE(2) and COX-2 mRNA expression were significantly lower in the ulcer patients than those in the nonulcer patients, whereas the LTB(4) level was significantly higher in the ulcer patients than that in the nonulcer patients, and it was also significantly lower in the ulcerated mucosa than that in the nonulcerated mucosa. The PPI group had a significantly increased PGE(2) and decreased LTB(4) levels in comparison to the H(2) blocker group during the ulcer-healing stage. The COX-1 mRNA expression showed no difference among the PPI and H(2) blocker groups or between before and after the treatment. However, the COX-2 mRNA expression increased in the PPI group more than that in the H(2) blocker group during the ulcer-healing stage. CONCLUSION: These findings demonstrated the significant gastric-mucosa-protecting effect of PPI by increasing the PGE(2) production and reducing the LTB(4) production.

Development of a Novel Virus-Like Particle Vaccine Platform That Mimics the Immature Form of Alphavirus.

Virus-like particles (VLPs) are noninfectious multiprotein structures that are engineered to self-assemble from viral structural proteins. Here, we developed a novel VLP-based vaccine platform utilizing VLPs from the chikungunya virus. We identified two regions within the envelope protein, a structural component of chikungunya, where foreign antigens can be inserted without compromising VLP structure. Our VLP displays 480 copious copies of an inserted antigen on the VLP surface in a highly symmetric manner and is thus capable of inducing strong immune responses against any inserted antigen. Furthermore, by mimicking the structure of the immature form of the virus, we altered our VLP's in vivo dynamics and enhanced its immunogenicity. We used the circumsporozoite protein (CSP) of the Plasmodium falciparum malaria parasite as an antigen and demonstrated that our VLP-based vaccine elicits strong immune responses against CSP in animals. The sera from immunized monkeys protected mice from malaria infection. Likewise, mice vaccinated with P. yoelii CSP-containing VLPs were protected from an infectious sporozoite challenge. Hence, our uniquely engineered VLP platform can serve as a blueprint for the development of vaccines against other pathogens and diseases.

Intestinal Behcet's disease with esophageal ulcers and colonic longitudinal ulcers.

Intestinal Behcet's disease in a 38-year-old woman was diagnosed because of the history of recurrent oral aphthous ulcers, erythema nodosum-like eruptions, genital ulcer, and endoscopic findings of esophageal and ileocolonic punched-out ulcers with colonic longitudinal ulcers. Esophageal lesions and colonic longitudinal ulcers are rarely seen in intestinal Behcet's disease. The ulcers of esophagus and ileocolon healed with 3 wk of treatment with prednisolone and mesalazine without any adverse effect. Mesalazine may decrease the total dose of prednisolone required to treat the disease.

Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation.

Bone morphogenetic proteins (BMPs) regulate hard tissue formation, including bone and tooth. Growth differentiation factor 5 (GDF5), a known BMP, is expressed in cartilage and regulates chondrogenesis, and mutations have been shown to cause osteoarthritis. Notably, GDF5 is also expressed in periodontal ligament tissue; however, its role during tooth development is unclear. Here, we used cell culture and in vivo analyses to determine the role of GDF5 during tooth development. GDF5 and its associated BMP receptors are expressed at the protein and mRNA levels during postnatal tooth development, particularly at a stage associated with enamel formation. Furthermore, whereas BMP2 was observed to induce evidently the differentiation of enamel-forming ameloblasts, excess GDF5 induce mildly this differentiation. A mouse model harbouring a mutation in GDF5 (W408R) showed enhanced enamel formation in both the incisors and molars, but not in the tooth roots. Overexpression of the W408R GDF5 mutant protein was shown to induce BMP2-mediated mRNA expression of enamel matrix proteins and downstream phosphorylation of Smad1/5/8. These results suggest that mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-signalling.

Sex hormones influence expression and function of peroxisome proliferator-activated receptor γ in adipocytes: pathophysiological aspects.

Abstract Adipose tissue plays important roles not only in storing fat but also in maintaining metabolic homeostasis by regulating hundreds of biological signaling events and the secretion of various cytokines. One of the central regulators of adipocyte differentiation is peroxisome proliferator-activated receptor γ (PPARγ), which promotes downstream transcriptional activities, such as adiponectin. Disruption of homeostasis leads to the onset of metabolic diseases such as type 2 diabetes and other triggers for metabolic syndrome. Males and post-menopausal females are more likely to be affected with metabolic diseases than pre-menopausal females, suggesting that sex hormones might be involved in the pathogenesis and development of metabolic diseases. Indeed, 17ß-estradiol, testosterone, dihydrotestosterone, and their receptors clearly play a role in adipose regulation: they can alter fat distribution and can modify the expression and activities of PPARγ and its downstream adipocytokines. Furthermore, sex hormones affect inflammatory factors such as nitric oxygen, nitric oxygen synthase, and their surrounding components. Sex hormones are also suggested to be involved with sex differences in the efficacy of the PPARγ agonist thiazolidinediones. Therefore, thorough investigation of how sex hormone-dependent regulation of metabolic homeostasis occurs is necessary in order to develop individualized clinical therapies optimized with regard to each patient's biological condition and drug sensitivities.

Cyclolepis genistoides D. Don (palo azul) promotes differentiation of adipocytes and regulates adipokine expression.

Cyclolepis genistoides D. Don is a herbaceous perennial belonging to the family Asteraceae, and its vernacular name is "palo azul" (palo). Palo has been reported to exhibit many physiological effects that contribute to the prevention of metabolic syndromes, although its mechanism is unclear. Among palo's various activities, we investigated the hypothesis that palo promotes adipocytes differentiation and regulates adipokine profiles in 3T3-L1 adipocytes by modulation of peroxisome proliferator-activated receptor (PPAR) γ, a major regulator of adipose differentiation. 3T3-L1 adipocytes were cultured and differentiated in Dulbecco modified Eagle medium with 50 to 200 µg/mL palo for 7 days or were cultured with palo without differentiation protocol for 14 days. Palo down-regulated the expression of 2 types of expressed/secreted adipokines, leptin and resistin, in a concentration-dependent manner. Under a nondifferentiated condition, palo promoted the accumulation of lipid droplets in cells. Real-time polymerase chain reaction and luciferase reporter assay showed that palo up-regulated expression and transcriptional activity of PPARγ. Furthermore, palo increased the expression of insulin-sensitizing adipokine, adiponectin, which is a directly target of PPARγ, both at the messenger RNA level and at the protein level. In summary, palo demonstrated the potential to improve insulin resistance by promoting adipocyte differentiation via PPARγ activation. Results suggest an increase in adiponectin secretion and a decrease in insulin-resistant factors such as leptin and resistin.

A patient with early syphilis complicated by fatty liver who showed an alleviation of hepatopathy accompanied by jaundice after receiving anti-syphilitic therapy.

A 42-year-old male with visceral obesity and a fatty liver presented with hepatopathy accompanied by jaundice and was diagnosed to have early syphilis based on the results of standard serologic tests and clinical findings. Both the subjective and objective findings including hepatopathy were rapidly alleviated by anti-syphilitic therapy. Severe hepatopathy accompanied by jaundice used to be considered a rare complication. However, due to contemporary unhealthy lifestyle patterns characterized by hypernutrition and a lack of exercise the occurrence of fatty liver is now dramatically increasing. Our findings suggest that the occurrence of fatty liver can exacerbate the damage to the hepatic parenchyma due to a Treponema pallidum infection.