Activation of IP3R in atrial cardiomyocytes leads to generation of cytosolic cAMP.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Excessive stimulation of the IP3 signalling pathway has been linked to AF through abnormal calcium handling. However, little is known about the mechanisms involved in this process. We expressed the fluorescence resonance energy transfer (FRET) based cytosolic cAMP sensor EPAC-SH187 in neonatal rat atrial myocytes (NRAMs) and neonatal rat ventricular myocytes (NRVMs). In NRAMs, addition of the α-1 agonist phenylephrine (PE, 3 µM) resulted in a FRET change 21.20 ± 7.43 % and addition of membrane permeant IP3 derivative, 2,3,6-tri-O-Butyryl-myo-IP3(1,4,5)-hexakis(acetoxymethyl)ester (IP3-AM, 20 µM) resulted in a peak of 20.31 ± 6.74 %. These FRET changes imply an increase in cAMP. Prior application of IP3 receptor (IP3R) inhibitors 2-Aminoethyl diphenylborinate (2-APB, 2.5µM) or Xestospongin-C (0.3 µM) significantly inhibited the change in FRET in NRAMs in response to PE. Xestospongin-C (0.3 µM) significantly inhibited the change in FRET in NRAMs in response to IP3-AM. The FRET change in response to PE in NRVMs were not inhibited by 2-APB or Xestospongin-C. Finally, the localisation of cAMP signals was tested by expressing the FRET-based cAMP sensor, AKAP79-CUTie, which targets the intracellular surface of the plasmalemma. We found in NRAMs that PE led to FRET change corresponding to an increase in cAMP that was inhibited by 2-APB and Xestospongin C. These data support further investigation of the pro-arrhythmic nature and components of IP3 induced cAMP signalling to identify potential pharmacological targets.

Co-observation of germline pathogenic variants in breast cancer predisposition genes: Results from analysis of the BRIDGES sequencing dataset.

Co-observation of a gene variant with a pathogenic variant in another gene that explains the disease presentation has been designated as evidence against pathogenicity for commonly used variant classification guidelines. Multiple variant curation expert panels have specified, from consensus opinion, that this evidence type is not applicable for the classification of breast cancer predisposition gene variants. Statistical analysis of sequence data for 55,815 individuals diagnosed with breast cancer from the BRIDGES sequencing project was undertaken to formally assess the utility of co-observation data for germline variant classification. Our analysis included expected loss-of-function variants in 11 breast cancer predisposition genes and pathogenic missense variants in BRCA1, BRCA2, and TP53. We assessed whether co-observation of pathogenic variants in two different genes occurred more or less often than expected under the assumption of independence. Co-observation of pathogenic variants in each of BRCA1, BRCA2, and PALB2 with the remaining genes was less frequent than expected. This evidence for depletion remained after adjustment for age at diagnosis, study design (familial versus population-based), and country. Co-observation of a variant of uncertain significance in BRCA1, BRCA2, or PALB2 with a pathogenic variant in another breast cancer gene equated to supporting evidence against pathogenicity following criterion strength assignment based on the likelihood ratio and showed utility in reclassification of missense BRCA1 and BRCA2 variants identified in BRIDGES. Our approach has applicability for assessing the value of co-observation as a predictor of variant pathogenicity in other clinical contexts, including for gene-specific guidelines developed by ClinGen Variant Curation Expert Panels.

Adiponectin receptor 1 regulates endometrial receptivity via the adenosine monophosphate­activated protein kinase/E­cadherin pathway.

Endometrial receptivity is essential for successful embryo implantation and pregnancy initiation and is regulated via various signaling pathways. Adiponectin, an important adipokine, may be a potential regulator of reproductive system functions. The aim of the present study was to elucidate the regulatory role of adiponectin receptor 1 (ADIPOR1) in endometrial receptivity. The endometrial receptivity between RL95­2 and AN3CA cell lines was confirmed using an in vitro JAr spheroid attachment model. 293T cells were transfected with control or short hairpin (sh)ADIPOR1 vectors and RL95­2 cells were transduced with lentiviral particles targeting ADIPOR1. Reverse transcription­quantitative PCR and immunoblot assays were also performed. ADIPOR1 was consistently upregulated in the endometrium during the mid­secretory phase compared with that in the proliferative phase and in receptive RL95­2 cells compared with that in non­receptive AN3CA cells. Stable cell lines with diminished ADIPOR1 expression caused by shRNA showed reduced E­cadherin expression and attenuated in vitro endometrial receptivity. ADIPOR1 regulated AMP­activated protein kinase (AMPK) activity in endometrial epithelial cells. Regulation of AMPK activity via dorsomorphin and 5­aminoimidazole­4­carboxamide ribonucleotide affected E­cadherin expression and in vitro endometrial receptivity. The ADIPOR1/AMPK/E­cadherin axis is vital to endometrial receptivity. These findings can help improve fertility treatments and outcomes.

Exploring the molecular mechanisms underlying neuroprotective effect of ellagic acid in okadaic acid-induced Alzheimer's phenotype.

Pomegranate polyphenol ellagic acid has medicinal potential in neurodegenerative disorders. The advantageous effect of this polyphenol in improving cognition in okadaic acid (OA)-instigated murine model with unraveling some modes of its action was assessed. Rats received ICV okadaic acid (OA) and post-treated with oral ellagic acid for 3 weeks (25 and 100 mg/kg/day). Cognition was analyzed in behavioral tasks besides assessment of oxidative, apoptotic, and inflammatory factors in addition to hippocampal histochemical analysis. Ellagic acid at a dose of 100 mg/kg properly attenuated cognitive abnormalities in novel object recognition (NOR), Y maze, and Barnes maze tests. Additionally, ellagic acid diminished hippocampal changes of malondialdehyde (MDA), protein carbonyl, reactive oxygen species (ROS), glutathione (GSH), glutathione peroxidase, superoxide dismutase (SOD), apoptotic factors caspases 1 and 3, tumor necrosis factor α (TNFα), and acetylcholinesterase (AChE) and beta secretase 1 (BACE 1) besides reversal of AMP-activated protein kinase (AMPK) and hyperphosphorylated tau (p-tau). Moreover, lower glial fibrillary acidic protein (GFAP) and less injury of hippocampal CA1 pyramidal neurons were observed upon ellagic acid. To conclude, neuroprotective potential of ellagic acid was shown which is somewhat attributable to its reversal of oxidative, apoptotic, and neuroinflammatory events in addition to proper regulation of AMPK and p-tau.

Cyclic GMP-AMP synthase recognizes the physical features of DNA.

Cyclic GMP-AMP synthase (cGAS) is a major cytosolic DNA sensor that plays a significant role in innate immunity. Upon binding to double stranded DNA (dsDNA), cGAS utilizes GTP and ATP to synthesize the second messenger cyclic GMP-AMP (cGAMP). The cGAMP then binds to the adapter protein stimulator of interferon genes (STING) in the endoplasmic reticulum, resulting in the activation of the transcription factor interferon regulatory factor 3 (IRF3) and subsequent induction of type I interferon. An important question is how cGAS distinguishes between self and non-self DNA. While cGAS binds to the phosphate backbone of DNA without discrimination, its activation is influenced by physical features such as DNA length, inter-DNA distance, and mechanical flexibility. This suggests that the recognition of DNA by cGAS may depend on these physical features. In this article we summarize the recent progress in research on cGAS-STING pathway involved in antiviral defense, cellular senescence and anti-tumor response, and focus on DNA recognition mechanisms based on the physical features.

Addition of nucleotide adjuvants enhances the immunogenicity of a recombinant subunit vaccine against the Zika virus in BALB/c mice.

Zika virus (ZIKV) infection remains a global public health problem. After the "Public Health Emergencies of International Concern" declared in February 2016, the incidence of new infections by this pathogen has been decreasing in many areas. However, there is still a likely risk that ZIKV will spread to more countries. To date, there is no vaccine or antiviral drug available to prevent or treat Zika virus infection. In the Zika vaccine development, those based on protein subunits are attractive as a non-replicable platform due to their potentially enhanced safety profile to be used in all populations. However, these vaccines frequently require multiple doses and adjuvants to achieve protective immunity. In this study we show the immunological evaluation of new formulations of the recombinant protein ZEC, which combines regions of domain III of the envelope and the capsid from ZIKV. Two nucleotide-based adjuvants were used to enhance the immunity elicited by the vaccine candidate ZEC. ODN 39M or c-di-AMP was incorporated as immunomodulator into the formulations combined with aluminum hydroxide. Following immunizations in immunocompetent BALB/c mice, the formulations stimulated high IgG antibodies. Although the IgG subtypes suggested a predominantly Th1-biased immune response by the formulation including the ODN 39M, cellular immune responses measured by IFNγ secretion from spleen cells after in vitro stimulations were induced by both immunomodulators. These results demonstrate the capacity of both immunomodulators to enhance the immunogenicity of the recombinant subunit ZEC as a vaccine candidate against ZIKV.

Extract of Phyllanthus emblica L. fruit stimulates basal glucose uptake and ameliorates palmitate-induced insulin resistance through AMPK activation in C2C12 myotubes.

BACKGROUND: The fruit of Phyllanthus emblica L., a traditional medicine in China and India, is used to treat diabetes mellitus. Its water extract (WEPE) has demonstrated hypoglycemic effects in diabetic rats, but its mechanisms on glucose utilization and insulin resistance in skeletal muscle remain unclear. Therefore, this study aims to investigate the effects and underlying mechanisms of WEPE on glucose utilization and insulin resistance using C2C12 myotubes. METHODS: Effects of WEPE on glucose uptake, GLUT4 translocation, and AMPK and AKT phosphorylation were investigated in C2C12 myotubes and palmitate-treated myotubes. An AMPK inhibitor and siRNA were used to explore the mechanisms of WEPE. Glucose uptake was determined using a 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) uptake assay, and protein expression and GLUT4 translocation were assessed via western blotting. RESULTS: In normal myotubes, WEPE significantly stimulated glucose uptake and GLUT4 translocation to the plasma membrane at concentrations of 125 and 250 µg/mL. This was accompanied by an increase in the phosphorylation of AMPK and its downstream targets. However, both compound C and AMPK siRNA blocked the WEPE-induced GLUT4 translocation and glucose uptake. Moreover, pretreatment with STO-609, a calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) inhibitor, inhibited WEPE-induced AMPK phosphorylation and attenuated the WEPE-stimulated glucose uptake and GLUT4 translocation. In myotubes treated with palmitate, WEPE prevented palmitate-induced insulin resistance by enhancing insulin-mediated glucose uptake and AKT phosphorylation. It also restored the insulin-mediated translocation of GLUT4 from cytoplasm to membrane. However, these effects of WEPE on glucose uptake and GLUT4 translocation were blocked by pretreatment with compound C. CONCLUSIONS: WEPE significantly stimulated basal glucose uptake though CaMKKß/AMPK pathway and markedly ameliorated palmitate-induced insulin resistance by activating the AMPK pathway in C2C12 myotubes.

Placental growth factor deficiency initiates obesity- and aging-associated metabolic syndrome.

Obesity often leads to inadequate angiogenesis in expanding adipose tissue, resulting in inflammation and insulin resistance. We explored the role of placental growth factor (PlGF) in metabolic syndrome (MS) using mice models of type 2 diabetes, high-fat diet, or aging. Reduced serum PlGF levels were associated with decreased insulin sensitivity and development of MS features. PlGF was localized within endothelial cells and pericytes of adipose tissue. In vitro, low PlGF levels in hypoxic conditions worsened oxidative stress, apoptosis, and reduced autophagy. This was associated with a reduction in expression of vascular endothelial growth factor (VEGF)-A/VEGF-R1/-R2, which was influenced by a decrease and increase in PlGF/pAMPK/PI3K-pAkt/PLCγ1-iCa++/eNOS and PTEN/GSK3ß axes, respectively. PlGF-knockout mice exhibited MS traits through alterations in the same signaling pathways, and these changes were mitigated by recombinant PlGF and metformin. These enhanced angiogenesis and lipid metabolism, underscoring PlGF's role in age-related MS and its potential as a therapeutic target.

Evidence-based recommendations for gene-specific ACMG/AMP variant classification from the ClinGen ENIGMA BRCA1 and BRCA2 Variant Curation Expert Panel.

The ENIGMA research consortium develops and applies methods to determine clinical significance of variants in hereditary breast and ovarian cancer genes. An ENIGMA BRCA1/2 classification sub-group, formed in 2015 as a ClinGen external expert panel, evolved into a ClinGen internal Variant Curation Expert Panel (VCEP) to align with Food and Drug Administration recognized processes for ClinVar contributions. The VCEP reviewed American College of Medical Genetics and Genomics/Association of Molecular Pathology (ACMG/AMP) classification criteria for relevance to interpreting BRCA1 and BRCA2 variants. Statistical methods were used to calibrate evidence strength for different data types. Pilot specifications were tested on 40 variants and documentation revised for clarity and ease of use. The original criterion descriptions for 13 evidence codes were considered non-applicable or overlapping with other criteria. Scenario of use was extended or re-purposed for eight codes. Extensive analysis and/or data review informed specification descriptions and weights for all codes. Specifications were applied to pilot variants with pre-existing ClinVar classification as follows: 13 uncertain significance or conflicting, 14 pathogenic and/or likely pathogenic, and 13 benign and/or likely benign. Review resolved classification for 11/13 uncertain significance or conflicting variants and retained or improved confidence in classification for the remaining variants. Alignment of pre-existing ENIGMA research classification processes with ACMG/AMP classification guidelines highlighted several gaps in the research processes and the baseline ACMG/AMP criteria. Calibration of evidence strength was key to justify utility and strength of different data types for gene-specific application. The gene-specific criteria demonstrated value for improving ACMG/AMP-aligned classification of BRCA1 and BRCA2 variants.

Phosphorylation of Insig-2 mediates inhibition of fatty acid synthesis by polyunsaturated fatty acids.

Insig-1 and Insig-2 are endoplasmic reticulum (ER) proteins that inhibit lipid synthesis by blocking transport of sterol regulatory element-binding proteins (SREBP-1 and SREBP-2) from ER to Golgi. In the Golgi, SREBPs are processed proteolytically to release their transcription-activating domains, which enhance the synthesis of fatty acids, triglycerides, and cholesterol. Heretofore, the two Insigs have redundant functions, and there is no rationale for two isoforms. The current data identify a specific function for Insig-2. We show that eicosapentaenoic acid (EPA), a polyunsaturated fatty acid, inhibits fatty acid synthesis in human fibroblasts and rat hepatocytes by activating adenylate cyclase, which induces protein kinase A (PKA) to phosphorylate serine-106 in Insig-2. Phosphorylated Insig-2 inhibits the proteolytic processing of SREBP-1, thereby blocking fatty acid synthesis. Phosphorylated Insig-2 does not block the processing of SREBP-2, which activates cholesterol synthesis. Insig-1 lacks serine-106 and is not phosphorylated at this site. EPA inhibition of SREBP-1 processing was reduced by the replacement of serine-106 in Insig-2 with alanine or by treatment with KT5720, a PKA inhibitor. Inhibition did not occur in mutant human fibroblasts that possess Insig-1 but lack Insig-2. These data provide an Insig-2-specific mechanism for the long-known inhibition of fatty acid synthesis by polyunsaturated fatty acids.

Crystallographic fragment screen of the c-di-AMP-synthesizing enzyme CdaA from Bacillus subtilis.

Crystallographic fragment screening has become a pivotal technique in structure-based drug design, particularly for bacterial targets with a crucial role in infectious disease mechanisms. The enzyme CdaA, which synthesizes an essential second messenger cyclic di-AMP (c-di-AMP) in many pathogenic bacteria, has emerged as a promising candidate for the development of novel antibiotics. To identify crystals suitable for fragment screening, CdaA enzymes from Streptococcus pneumoniae, Bacillus subtilis and Enterococcus faecium were purified and crystallized. Crystals of B. subtilis CdaA, which diffracted to the highest resolution of 1.1 Å, were used to perform the screening of 96 fragments, yielding data sets with resolutions spanning from 1.08 to 1.87 Å. A total of 24 structural hits across eight different sites were identified. Four fragments bind to regions that are highly conserved among pathogenic bacteria, specifically the active site (three fragments) and the dimerization interface (one fragment). The coordinates of the three active-site fragments were used to perform an in silico drug-repurposing screen using the OpenEye suite and the DrugBank database. This screen identified tenofovir, an approved drug, that is predicted to interact with the ATP-binding region of CdaA. Its inhibitory potential against pathogenic E. faecium CdaA has been confirmed by ITC measurements. These findings not only demonstrate the feasibility of this approach for identifying lead compounds for the design of novel antibacterial agents, but also pave the way for further fragment-based lead-optimization efforts targeting CdaA.

Antimicrobial and immunomodulatory activities of porcine cathelicidin Protegrin-1.

Antimicrobial peptides (AMPs) are a promising alternative to antibiotics in the fight against multi-drug resistant and immune system-evading bacterial infections. Protegrins are porcine cathelicidins which have been identified in porcine leukocytes. Protegrin-1 is the best characterized family member and has broad antibacterial activity by interacting and permeabilizing bacterial membranes. Many host defense peptides (HDPs) like LL-37 or chicken cathelicidin 2 (CATH-2) have also been shown to have protective biological functions during infections. In this regard, it is interesting to study if Protegrin-1 has the immune modulating potential to suppress unnecessary immune activation by neutralizing endotoxins or by influencing the macrophage functionality in addition to its direct antimicrobial properties. This study showed that Protegrin-1 neutralized lipopolysaccharide- (LPS) and bacteria-induced activation of RAW macrophages by binding and preventing LPS from cell surface attachment. Furthermore, the peptide treatment not only inhibited bacterial phagocytosis by murine and porcine macrophages but also interfered with cell surface and intracellular bacterial survival. Lastly, Protegrin-1 pre-treatment was shown to inhibit the amastigote survival in Leishmania infected macrophages. These experiments describe an extended potential of Protegrin-1's protective role during microbial infections and add to the research towards clinical application of cationic AMPs.

Assessing the antitumor effects of metformin on ovarian clear cell carcinoma.

Developing novel therapies that outperform the existing chemotherapeutic treatments is required for treatment-resistant ovarian clear cell carcinoma. We investigated the antitumor effect of metformin on ovarian clear cell carcinoma, enhancement of the antitumor effect by its combination with chemotherapy, and its molecular regulatory mechanism. First, we evaluated the viability of ovarian clear cell carcinoma lines using the water-soluble tetrazolium-1 assay and found that metformin suppressed cell viability. Cell viability was significantly suppressed by co-treatment with cisplatin and metformin. In contrast, co-treatment with paclitaxel and metformin showed no significant difference in viability compared with the group without metformin. Western blot analysis showed increased phosphorylation of AMP-activated protein kinase in some cell lines and suppressed phosphorylation of the mammalian target of rapamycin in a particular cell line. Flow cytometry analysis revealed a significant increase in the rate of apoptosis in the metformin-treated group and rate of cell cycle arrest at the G2/M phase in a particular cell line. These results indicated that metformin may be effective against cultured ovarian clear cell carcinoma cells, particularly in combination with cisplatin.

Carbon monoxide-loaded red blood cells ameliorate metabolic dysfunction-associated steatohepatitis progression via enhancing AMP-activated protein kinase activity and inhibiting Kupffer cell activation.

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive form of nonalcoholic fatty liver disease characterised by fat accumulation, inflammation, oxidative stress, fibrosis, and impaired liver regeneration. In this study, we found that heme oxygenase-1 (HO-1) is induced in both MASH patients and in a MASH mouse model. Further, hepatic carbon monoxide (CO) levels in MASH model mice were >2-fold higher than in healthy mice, suggesting that liver HO-1 is activated as MASH progresses. Based on these findings, we used CO-loaded red blood cells (CO-RBCs) as a CO donor in the liver, and evaluated their therapeutic effect in methionine-choline deficient diet (MCDD)-induced and high-fat-diet (HFD)-induced MASH model mice. Intravenously administered CO-RBCs effectively delivered CO to the MASH liver, where they prevented fat accumulation by promoting fatty acid oxidation via AMP-activated protein kinase (AMPK) activation and peroxisome proliferator-activated receptor induction. They also markedly suppressed Kupffer cell activation and their corresponding anti-inflammatory and antioxidative stress activities in MASH mice. CO-RBCs also helped to restore liver regeneration in mice with HFD-induced MASH by activating AMPK. We confirmed the underlying mechanisms by performing in vitro experiments in RAW264.7 cells and palmitate-stimulated HepG2 cells. Taken together, CO-RBCs show potential as a promising cellular treatment for MASH.

Kenya Psychosis-Risk Outcomes Study (KePROS): Development of an Accelerated Medicine Partnership Schizophrenia-Aligned Project in Africa.

Background and Hypothesis: The Accelerating Medicines Partnership Schizophrenia (AMP SCZ) funds a longitudinal study of 43 research sites across 5 continents to develop tools to stratify developmental trajectories of youth at clinical high risk for psychosis (CHR) and identify homogenous targets for future clinical trials. However, there are no sites in Africa, leaving a critical gap in our knowledge of clinical and biological outcomes among CHR individuals. Study Design: We describe the development of the Kenya Psychosis-Risk Outcomes Study (KePROS), a 5-year NIH-funded project in Kenya designed to harmonize with AMP SCZ. The study will recruit over 100 CHR and 50 healthy participants and conduct multiple clinical and biomarker assessments over 2 years. Capacity building is a key component of the study, including the construction of an electroencephalography (EEG) laboratory and the upgrading of a local 3 T magnetic resonance imaging (MRI) machine. We detail community recruitment, study methodologies and protocols, and unique challenges with this pioneering research in Africa. Study Results: This paper is descriptive only. Planned future analyses will investigate possible predictors of clinical outcomes and will be compared to results from other global populations. Conclusions: KePROS will provide the research community with a rich longitudinal clinical and biomarker dataset from an African country in the developing Global South, which can be used alongside AMP SCZ data to delineate CHR outcome groups for future treatment development. Training in mental health assessment and investment in cutting-edge biomarker assessment and other technologies is needed to facilitate the inclusion of African countries in large-scale research consortia.

Assessment of ATP metabolism to adenosine by ecto-nucleotidases carried by tumor-derived small extracellular vesicles.

Immunosuppression is a hallmark of cancer progression. Tumor-derived small extracellular vesicles (sEV), also known as TEX, produce adenosine (ADO) and can mediate tumor-induced immunosuppression.Here, the ATP pathway of ADO production (ATP →  ADP →  AMP →  ADO) by ecto-nucleotidases carried on the sEV surface was evaluated by a method using N6-etheno-ATP (eATP) and N6-etheno-AMP (eAMP) as substrates for enzymatic activity. The "downstream" N6-etheno-purines (ePurines) were measured by high performance liquid chromatography with fluorescence detection (HPLC-FL).Human melanoma cell-derived TEX (MTEX) metabolized eATP to N6-etheno-ADP (eADP), eAMP and N6-etheno-Adenosine (eADO) more robustly than control keratinocyte cell-derived sEV (CEX); due to accelerated conversion of eATP to eADP and eADP to eAMP. MTEX and CEX similarly metabolized eAMP to eADO. Blocking of the ATP pathway with the selective CD39 inhibitor ARL67156 or pan ecto-nucleotidase inhibitor POM-1 normalized the ATP pathway but neither inhibitor completely abolished it. In contrast, inhibition of CD73 by PSB12379 or AMPCP abolished eADO formation by both MTEX and CEX, suggesting that targeting CD73 is the preferred approach to eliminating ADO produced by ecto-nucleotidases located on the sEV surface.The noninvasive, sensitive, and specific assay assessing ePurine metabolism ± ecto-nucleotidase inhibitors in TEX enables the personalized identification of ecto-nucleotidase activity primarily involved in ADO production in patients with cancer. The assay could guide precision medicine by determining which purine is the preferred target for inhibitory therapeutic interventions.

cAMP signaling of Bordetella adenylate cyclase toxin blocks M-CSF triggered upregulation of iron acquisition receptors on differentiating CD14+ monocytes.

Bordetella pertussis infects the upper airways of humans and disarms host defense by the potent immuno-subversive activities of its pertussis (PT) and adenylate cyclase (CyaA) toxins. CyaA action near-instantly ablates the bactericidal activities of sentinel CR3-expressing myeloid phagocytes by hijacking cellular signaling pathways through the unregulated production of cAMP. Moreover, CyaA-elicited cAMP signaling also inhibits the macrophage colony-stimulating factor (M-CSF)-induced differentiation of incoming inflammatory monocytes into bactericidal macrophages. We show that CyaA/cAMP signaling via protein kinase A (PKA) downregulates the M-CSF-elicited expression of monocyte receptors for transferrin (CD71) and hemoglobin-haptoglobin (CD163), as well as the expression of heme oxygenase-1 (HO-1) involved in iron liberation from internalized heme. The impact of CyaA action on CD71 and CD163 levels in differentiating monocytes is largely alleviated by the histone deacetylase inhibitor trichostatin A (TSA), indicating that CyaA/cAMP signaling triggers epigenetic silencing of genes for micronutrient acquisition receptors. These results suggest a new mechanism by which B. pertussis evades host sentinel phagocytes to achieve proliferation on airway mucosa.IMPORTANCETo establish a productive infection of the nasopharyngeal mucosa and proliferate to sufficiently high numbers that trigger rhinitis and aerosol-mediated transmission, the pertussis agent Bordetella pertussis deploys several immunosuppressive protein toxins that compromise the sentinel functions of mucosa patrolling phagocytes. We show that cAMP signaling elicited by very low concentrations (22 pM) of Bordetella adenylate cyclase toxin downregulates the iron acquisition systems of CD14+ monocytes. The resulting iron deprivation of iron, a key micronutrient, then represents an additional aspect of CyaA toxin action involved in the inhibition of differentiation of monocytes into the enlarged bactericidal macrophage cells. This corroborates the newly discovered paradigm of host defense evasion mechanisms employed by bacterial pathogens, where manipulation of cellular cAMP levels blocks monocyte to macrophage transition and replenishment of exhausted phagocytes, thereby contributing to the formation of a safe niche for pathogen proliferation and dissemination.

Specification of variant interpretation guidelines for inherited retinal dystrophy in Japan.

Accurate interpretation of sequence variants in inherited retinal dystrophy (IRD) is vital given the significant genetic heterogeneity observed in this disorder. To achieve consistent and accurate diagnoses, establishment of standardized guidelines for variant interpretation is essential. The American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for variant interpretation serve as the global "cross-disease" standard for classifying variants in Mendelian hereditary disorders. These guidelines propose a systematic approach for categorizing variants into 5 classes based on various types of evidence, such as population data, computational data, functional data, and segregation data. However, for clinical genetic diagnosis and to ensure standardized diagnosis and treatment criteria, additional specifications based on features associated with each disorder are necessary. In this context, we present a comprehensive framework outlining the newly specified ACMG/AMP rules tailored explicitly to IRD in the Japanese population on behalf of the Research Group on Rare and Intractable Diseases (Ministry of Health, Labour and Welfare of Japan). These guidelines consider disease frequencies, allele frequencies, and both the phenotypic and the genotypic characteristics unique to IRD in the Japanese population. Adjustments and modifications have been incorporated to reflect the specific requirements of the population. By incorporating these IRD-specific factors and refining the existing ACMG/AMP guidelines, we aim to enhance the accuracy and consistency of variant interpretation in IRD cases, particularly in the Japanese population. These guidelines serve as a valuable resource for ophthalmologists and clinical geneticists involved in the diagnosis and treatment of IRD, providing them with a standardized framework to assess and classify genetic variants.

Oral Preexposure Prophylaxis Uptake and Discontinuation in the HIV Vaccine Trials Network 704/HIV Prevention Trials Network 085 Study: Implications for Biomedical Human Immunodeficiency Virus Prevention Trials.

Background: HIV Vaccine Trials Network (HVTN) 704/085, a placebo-controlled clinical trial assessing the efficacy of VRC01 broadly neutralizing antibody infusion for HIV prevention, offered oral preexposure prophylaxis (PrEP) as the standard of prevention at no cost to participants. Methods: We characterized features of- identified factors associated with- PrEP initiation and discontinuation, and the effects of PrEP initiation on HIV incidence. Results: Of 2221 participants, 31.8% initiated oral PrEP during study follow-up, with the highest proportion of PrEP initiations in Brazil (83.2%) and the United States (US) (54.2%). Prior PrEP use was associated with PrEP initiation (hazard ratio [HR], 2.22 [95% confidence interval {CI}, 1.25-3.95]). Participants from Switzerland (HR, 0.5 [95% CI, .3-1.0]) and Peru (HR, 0.08 [95% CI, .06-.1]) had lower likelihood of PrEP initiation compared to the US, while participants from Brazil had higher likelihood (HR, 2.6 [95% CI, 2.0-3.3]). In the US, PrEP initiation was lower in areas with higher unmet need for PrEP (HR, 0.9 per 5 units [95% CI, 0.8-1.0]). PrEP initiators had 58% less risk of acquiring HIV than PrEP noninitiators. Among PrEP initiators, 34.4% discontinued PrEP during study follow-up. Brazil had 63% less likelihood of PrEP discontinuation than the US (HR, 0.37 [95% CI, .22-.60]). Conclusions: When included as standard of prevention in HVTN 704/085, oral PrEP utilization patterns mirrored those observed in real-life settings. Variable effects of oral PrEP on HIV outcomes in clinical trials may be expected based on regional differences in oral PrEP use.

Melinjo (Gnetum gnemon L) Extract Attenuates Colonic Inflammation in a Mouse Colitis Model by Regulating the AMPK/NFκB/Sirt1 Pathway.

Ulcerative colitis (UC) is a subtype of inflammatory bowel disease affecting the colon with idiopathic origin. Melinjo endosperm extract (MeE) contains polyphenolic compounds that have antioxidative and anticancer properties. We examined the effect of MeE on inflammation and mucin expression in the colons of UC of mice treated with dextran sulfate sodium (DSS). C57BL/6J male mice were assigned into four categories: control, DSS + 0% MeE, DSS + 0.1% MeE, and DSS + 0.5% MeE. The control group was provided distilled water and a standard chow diet for 4 weeks. In DSS + 0% MeE, DSS + 0.1% MeE, and DSS + 0.5% MeE groups, the mice were treated with MeE for 3 weeks followed by MeE diets and drinking water containing 3% DSS for a week. Macrophage count, the mucus area stained by Alcian blue (AB), the levels of adenosine monophosphate-activated protein kinase (AMPK), nuclear factor-κB (NFκB) p65, and silent information regulator (Sirt) 1 protein expression, as well as proinflammatory mediators and Mucin 2 mRNA expression were assessed. In the DSS + 0% MeE group, the AB-stained areas and Mucin 2 mRNA expression levels were observed to be lower than those of controls. However, the levels in the +0.5% MeE group were significantly increased. Compared with the control group, the macrophage number, the expression of IL-1ß mRNA, and NFκB p65 protein in the DSS + 0% MeE group showed a significant increase. Conversely, these levels were significantly decreased in the +0.5% MeE group. The phosphorylated AMPK and Sirt1 protein levels were upregulated in the +0.5% MeE group. In conclusion, MeE may alleviate UC injury by reducing macrophage infiltration and regulating the AMPK/NFκB/Sirt1 pathway.