The role of multiple vitellogenins in early development of fishes.

Functions of vitellogenins have been in the limelight of fish reproductive physiology research for decades. The Vtg system of acanthomorph teleosts consists of two complete forms of Vtgs (VtgAa and VtgAb) and an incomplete form, VtgC. Insufficient uptake and processing of Vtgs and their yolk proteins lead to inadequate oocyte hydration ensuing failure in acquisition of egg buoyancy and early developmental deficiencies. This review presents a summary of our studies on utilization of multiple Vtgs in species with different egg buoyancy characteristics, as examples. Studies of moronids revealed limited degradation of all three forms of lipovitellin heavy chain derived from their three respective forms of Vtg, by which they contribute to the free amino acid pool driving oocyte hydration during oocyte maturation. In later studies, CRISPR/Cas9 was employed to invalidate zebrafish type I, type II and type III Vtgs, which are orthologs of acanthamorph VtgAa, VtgAb and VtgC, respectively. Results revealed type I Vtg to have essential developmental and nutritional functions in both late embryos and larvae. Genomic disturbance of type II Vtg led to high mortalities during the first 24 h of embryonic development. Despite being a minor form of Vtg in zebrafish and most other species, type III Vtg was also found to contribute essentially to the developmental potential of zebrafish zygotes and early embryos. Apart from severe effects on progeny survival, these studies also disclosed previously unreported regulatory effects of Vtgs on fecundity and fertility, and on embryo hatching. We recently utilized parallel reactions monitoring based liquid chromatography tandem mass spectrometry to assess the processing and utilization of lipovitellins derived from different forms of Vtg in Atlantic halibut and European plaice. Results showed the Lv heavy chain of VtgAa (LvHAa) to be consumed during oocyte maturation and the Lv light chain of VtgAb (LvLAb) to be utilized specifically during late larval stages, while all remaining YPs (LvLAa, LvHAb, LvHC, and LvLC) were utilized during or after hatching up until first feeding in halibut. In plaice, all YPs except LvHAa, which similarly to halibut supports oocyte maturation, are utilized from late embryo to late larval development up until first feeding. The collective findings from these studies affirm substantial disparity in modes of utilization of different types of Vtgs among fish species with various egg buoyancy characteristics, and they reveal previously unknown regulatory functions of Vtgs in maintenance of reproductive assets such as maternal fecundity and fertility, and in embryonic hatching. Despite the progress that has been made over the past two decades by examining multiple Vtgs and their functions, a higher complexity of these systems with much greater diversity between species in modes of Vtg utilization is now evident. Further research is needed to reveal novel ways each species has evolved to utilize these complex multiple Vtg systems and to discover unifying principles for this evolution in fishes of diverse lineages, habitats and life history characteristics.

Shared Biological Pathways and Processes in Patients with Intellectual Disability: A Multicenter Study.

BACKGROUND: Although the underlying genetic causes of intellectual disability (ID) continue to be rapidly identified, the biological pathways and processes that could be targets for a potential molecular therapy are not yet known. This study aimed to identify ID-related shared pathways and processes utilizing enrichment analyses. METHODS: In this multicenter study, causative genes of patients with ID were used as input for Disease Ontology (DO), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes enrichment analysis. RESULTS: Genetic test results of 720 patients from 27 centers were obtained. Patients with chromosomal deletion/duplication, non-ID genes, novel genes, and results with changes in more than one gene were excluded. A total of 558 patients with 341 different causative genes were included in the study. Pathway-based enrichment analysis of the ID-related genes via ClusterProfiler revealed 18 shared pathways, with lysine degradation and nicotine addiction being the most common. The most common of the 25 overrepresented DO terms was ID. The most frequently overrepresented GO biological process, cellular component, and molecular function terms were regulation of membrane potential, ion channel complex, and voltage-gated ion channel activity/voltage-gated channel activity, respectively. CONCLUSION: Lysine degradation, nicotine addiction, and thyroid hormone signaling pathways are well-suited to be research areas for the discovery of new targeted therapies in ID patients.

Inhibition of Sphingosine-1-Phosphate Receptor 2 by JTE013 Enhanced Alveolar Bone Regeneration by Promoting Angiogenesis.

Sphingosine-1-phosphate receptor 2 (S1PR2) is a G protein-coupled receptor that regulates various immune responses. Herein, we report the effects of a S1PR2 antagonist (JTE013) on bone regeneration. Murine bone marrow stromal cells (BMSCs) were treated with dimethylsulfoxide (DMSO) or JTE013 with or without infection by an oral bacterial pathogen Aggregatibacter actinomycetemcomitans. Treatment with JTE013 enhanced vascular endothelial growth factor A (VEGFA), platelet derived growth factor subunit A (PDGFA), and growth differentiation factor 15 (GDF15) gene expression and increased transforming growth factor beta (TGFß)/Smad and Akt signaling. Eight-week-old male C57BL/6J mice were challenged with ligatures around the left maxillary 2nd molar for 15 days to induce inflammatory bone loss. After ligature removal, mice were treated with diluted DMSO or JTE013 in the periodontal tissues 3 times per week for 3 weeks. Calcein was also injected twice to measure bone regeneration. Micro-CT scanning of maxillary bone tissues and calcein imaging revealed that treatment with JTE013 enhanced alveolar bone regeneration. JTE013 also increased VEGFA, PDGFA, osteocalcin, and osterix gene expressions in the periodontal tissues compared to control. Histological examination of periodontal tissues revealed that JTE013 promoted angiogenesis in the periodontal tissues compared to control. Our findings support that inhibition of S1PR2 by JTE013 increased TGFß/Smad and Akt signaling; enhanced VEGFA, PDGFA, and GDF15 gene expression; and subsequently promoted angiogenesis and alveolar bone regeneration.

The Presence of Periodontitis Exacerbates Non-Alcoholic Fatty Liver Disease via Sphingolipid Metabolism-Associated Insulin Resistance and Hepatic Inflammation in Mice with Metabolic Syndrome.

Clinical studies have shown that periodontitis is associated with non-alcoholic fatty liver disease (NAFLD). However, it remains unclear if periodontitis contributes to the progression of NAFLD. In this study, we generated a mouse model with high-fat diet (HFD)-induced metabolic syndrome (MetS) and NAFLD and oral P. gingivalis inoculation-induced periodontitis. Results showed that the presence of periodontitis increased insulin resistance and hepatic inflammation and exacerbated the progression of NAFLD. To determine the role of sphingolipid metabolism in the association between NAFLD and periodontitis, we also treated mice with imipramine, an inhibitor of acid sphingomyelinase (ASMase), and demonstrated that imipramine treatment significantly alleviated insulin resistance and hepatic inflammation, and improved NAFLD. Studies performed in vitro showed that lipopolysaccharide (LPS) and palmitic acid (PA), a major saturated fatty acid associated with MetS and NAFLD, synergistically increased the production of ceramide, a bioactive sphingolipid involved in NAFLD progression in macrophages but imipramine effectively reversed the ceramide production stimulated by LPS and PA. Taken together, this study showed for the first time that the presence of periodontitis contributed to the progression of NAFLD, likely due to alterations in sphingolipid metabolism that led to exacerbated insulin resistance and hepatic inflammation. This study also showed that targeting ASMase with imipramine improves NAFLD by reducing insulin resistance and hepatic inflammation.

Epigallocatechin-3-gallate and resveratrol attenuate hydrogen peroxide induced damage in neuronal cells.

PURPOSE: The purpose of this study is to investigate how the antioxidants resveratrol and epigallocatechin-3-gallate (EGCG) protect SH-SY5Y cells against damage caused by hydrogen peroxide (H2O2). METHODS: SH-SY5Y cells were pretreated with EGCG and resveratrol at concentrations of 0.1 µM, 1 µM, and 10 µM, individually and in various combinations. Cells were exposed to 250 µM H2O2 for 1-hour following a 24-hour pretreatment. The effects of EGCG and resveratrol on cellular survival against hydrogen peroxide toxicity were evaluated with the MTS. Caspase 3 levels were measured with caspase 3 ELISA test for evaluating survival. The clonogenic test was utilized to assess the colony forming capacity. RESULTS: The MTS test revealed that pretreatment of SH-SY5Y cells for 24 hours with EGCG and Resveratrol enhanced cellular survival against hydrogen peroxide damage at all dosages (p < 0.005). The caspase 3 ELISA test revealed that EGCG and resveratrol statistically substantially decreased caspase 3 levels and improved cellular survival via the caspase 3 pathway (p < 0.005). The clonogenic test findings show that resveratrol and EGCG statistically boost SH-SY5Y cells' potential to form colonies (p<0.005). CONCLUSIONS: By reducing caspase 3 levels in exposure to hydrogen peroxide damage, EGCG and resveratrol promote cellular viability (Tab. 1, Fig. 3, Ref. 37). Text in PDF www.elis.sk Keywords: epigallocatechin-3-gallate, resveratrol, hydrogen peroxide, neurodegeneration.

Quantitative proteome profiling reveals molecular hallmarks of egg quality in Atlantic halibut: impairments of transcription and protein folding impede protein and energy homeostasis during early development.

BACKGROUND: Tandem mass tag spectrometry (TMT labeling-LC-MS/MS) was utilized to examine the global proteomes of Atlantic halibut eggs at the 1-cell-stage post fertilization. Comparisons were made between eggs judged to be of good quality (GQ) versus poor quality (BQ) as evidenced by their subsequent rates of survival for 12 days. Altered abundance of selected proteins in BQ eggs was confirmed by parallel reaction monitoring spectrometry (PRM-LC-MS/MS). Correspondence of protein levels to expression of related gene transcripts was examined via qPCR. Potential mitochondrial differences between GQ and BQ eggs were assessed by transmission electron microscopy (TEM) and measurements of mitochondrial DNA (mtDNA) levels. RESULTS: A total of 115 proteins were found to be differentially abundant between GQ and BQ eggs. Frequency distributions of these proteins indicated higher protein folding activity in GQ eggs compared to higher transcription and protein degradation activities in BQ eggs. BQ eggs were also significantly enriched with proteins related to mitochondrial structure and biogenesis. Quantitative differences in abundance of several proteins with parallel differences in their transcript levels were confirmed in egg samples obtained over three consecutive reproductive seasons. The observed disparities in global proteome profiles suggest impairment of protein and energy homeostasis related to unfolded protein response and mitochondrial stress in BQ eggs. TEM revealed BQ eggs to contain significantly higher numbers of mitochondria, but differences in corresponding genomic mtDNA (mt-nd5 and mt-atp6) levels were not significant. Mitochondria from BQ eggs were significantly smaller with a more irregular shape and a higher number of cristae than those from GQ eggs. CONCLUSION: The results of this study indicate that BQ Atlantic halibut eggs are impaired at both transcription and translation levels leading to endoplasmic reticulum and mitochondrial disorders. Observation of these irregularities over three consecutive reproductive seasons in BQ eggs from females of diverse background, age and reproductive experience indicates that they are a hallmark of poor egg quality. Additional research is needed to discover when in oogenesis and under what circumstances these defects may arise. The prevalence of this suite of markers in BQ eggs of diverse vertebrate species also begs investigation.

The relationship between dietary elimination and maternal psychopathology in breastfeeding mothers of infants with food allergy.

BACKGROUND: There has been no study evaluating the psychopathology in breastfeeding mothers of infants with food allergy (FA). OBJECTIVE: The aim of the study was to investigate the relationship between dietary elimination and maternal psychopathology, specifically anxiety, depression, and mother-to-infant bonding, in breastfeeding mothers of infants with food allergy. METHODS: Breastfeeding mothers following an elimination diet due to FA in their children aged 1-to-12 months were compared with the healthy controls. The physician-diagnosed FA group was divided into IgE-, non-IgE-mediated, and infants with some minor symptoms which were not enough to make the diagnosis of FA were classified as Indecisive symptoms for FA group. Mothers completed standardized questionnaires including Symptom Checklist 90R, Beck Depression/Anxiety Inventories (BDI/BAI), Postpartum Bonding Questionnaire (Bonding). RESULTS: Of 179 mother and infants, 64 were healthy, 89 were FA, 16 were indecisive symptoms for FA. The mean age of the mothers and infants were 31.1 ± 4.7 years and 6.3 ± 3.6 months, respectively. The physician-diagnosed FA groups had higher scores for anxiety (p = .008), anger (p = .042), depression (p < .001), obsession (p = .002), phobia (p = .008), somatization (p = .002), general symptom index (GSI) (p = .001), BDI (p < .001), BAI (p = .008), and Bonding (attachment [p = .001], anger [p = .019], and total [p = .036]) than the healthy ones. The indecisive symptoms for FA group had a similar score pattern to physician-diagnosed FA, except interpersonal sensitivity, BDI, and attachment. CONCLUSION: Breastfeeding mothers of infants with FA were more anxious, with higher depression scores than controls, and had many psychopathologies which affected bonding. Interventions targeting negativity in caregivers' social relationships are urgently needed.

Porphyromonas gingivalis infection upregulates the endothelin (ET) system in brain microvascular endothelial cells.

Endothelin-1 (ET-1), the most potent vasoconstrictor identified to date, contributes to cerebrovascular dysfunction and brain ET-1 levels were shown to be related to Alzheimer's disease and related dementias (ADRD) progression. ET-1 also contributes to neuroinflammation, especially in infections of the central nervous system. Recent studies causally linked chronic periodontal infection with an opportunistic anaerobic bacterium Porphyromonas gingivalis (Coykendall et al.) Shah & Collins to AD development. Thus, the goal of the study was to determine the impact of P. gingivalis infection on the ET system and cell senescence in brain microvascular endothelial cells. Cells were infected with a multiplicity of infection 50 P. gingivalis with and without extracellular ATP-induced oxidative stress for 24 h. Cell lysates were collected for analysis of endothelin A receptor (ETA)/endothelin B receptor (ETB) receptor as well as senescence markers. ET-1 levels in cell culture media were measured with enzyme-linked immunosorbent assay. P. gingivalis infection increased ET-1 (pg/mL) secretion, as well as the ETA receptor expression, whereas decreased lamin A/C expression compared to control. Tight junction protein claudin-5 was also decreased under these conditions. ETA or ETB receptor blockade during infection did not affect ET-1 secretion or the expression of cell senescence markers. Current findings suggest that P. gingivalis infection may compromise endothelial integrity and activate the ET system.

Knock out of specific maternal vitellogenins in zebrafish (Danio rerio) evokes vital changes in egg proteomic profiles that resemble the phenotype of poor quality eggs.

BACKGROUND: We previously reported the results of CRISPR/Cas9 knock-out (KO) of type-I and type-III vitellogenins (Vtgs) in zebrafish, which provided the first experimental evidence on essentiality and disparate functioning of Vtgs at different stages during early development. However, the specific contributions of different types of Vtg to major cellular processes remained to be investigated. The present study employed liquid chromatography and tandem mass spectrometry (LC-MS/MS) to meet this deficit. Proteomic profiles of zebrafish eggs lacking three type-I Vtgs simultaneously (vtg1-KO), or lacking only type III Vtg (vtg3-KO) were compared to those of wild type (Wt) eggs. Obtained spectra were searched against a zebrafish proteome database and identified proteins were quantified based on normalized spectral counts. RESULTS: The vtg-KO caused severe changes in the proteome of 1-cell stage zebrafish eggs. These changes were disclosed by molecular signatures that highly resembled the proteomic phenotype of poor quality zebrafish eggs reported in our prior studies. Proteomic profiles of vtg-KO eggs and perturbations in abundances of hundreds of proteins revealed unique, noncompensable contributions of multiple Vtgs to protein and in energy homeostasis. The lack of this contribution appears to have a significant impact on endoplasmic reticulum and mitochondrial functions, and thus embryonic development, even after zygotic genome activation. Increased endoplasmic reticulum stress, Redox/Detox activities, glycolysis/gluconeogenesis, enrichment in cellular proliferation and in human neurodegenerative disease related activities in both vtg1- and vtg3-KO eggs were found to be indicators of the aforementioned conditions. Distinctive increase in apoptosis and Parkinson disease pathways, as well as the decrease in lipid metabolism related activities in vtg3-KO eggs implies compelling roles of Vtg3, the least abundant form of Vtgs in vertebrate eggs, in mitochondrial activities. Several differentially abundant proteins representing the altered molecular mechanisms have been identified as strong candidate markers for studying the details of these mechanisms during early embryonic development in zebrafish and possibly other vertebrates. CONCLUSIONS: These findings indicate that the global egg proteome is subject to extensive modification depending on the presence or absence of specific Vtgs and that these modifications can have a major impact on developmental competence.

Knock out of a major vitellogenin receptor gene with eight ligand binding repeats in medaka (Oryzias latipes) using the CRISPR/Cas9 system.

Recent studies of vitellogenesis engendered a novel model of teleost yolk formation in which multiple yolk precursors, vitellogenins (Vtgs), and their receptors (Vtgrs) interact to ensure proper yolk composition for embryonic development and larval growth. As a step toward verification of this concept, we examined the role of one candidate Vtgr, termed low-density lipoprotein receptor relative with eight ligand-binding repeat (Lr8), in the medaka, a representative teleost and established laboratory model. A homozygous lr8 knock out (lr8-KO) medaka was produced to perform reverse-genetic functional analyses. In ovaries of wild type (WT) medaka, Western blotting detected a putative Lr8 protein band at ~130 kDa, while immunohistochemistry detected the putative Lr8 signal at the periphery of the oocyte underneath the zona radiata. These signals disappeared in ovaries of the lr8-KO group. Offspring of lr8-KO medaka exhibited decreased survival rate compared to WT fish, but KO of lr8 was not 100% lethal. There was no significant difference in total yolk protein content or size of eggs between WT and lr8-KO fish. However, LC-MS/MS analyses revealed a remarkable decrease in the relative abundance of yolk proteins derived from VtgAb in lr8-KO eggs, in conjunction with a compensatory increase in proteins derived from VtgAa1. These findings strongly support the conclusion that Lr8 is an important receptor for VtgAb in medaka. The disruption of proper yolk composition by lr8-KO is possibly one cause of the low offspring survival.

Comparison of cyclic fatigue resistance of Rotate instrument with reciprocating and continuous rotary nickel-titanium instruments at body temperature in relation to their transformation temperatures.

OBJECTIVE: The aim of this study was to compare the cyclic fatigue resistance at body temperature and phase transformation behaviors of novel Rotate instrument (25.06) with rotating Mtwo (25.06) and reciprocating Reciproc Blue (25.08) and Reciproc (25.08) instruments. MATERIALS AND METHODS: The Rotate, Reciproc Blue, Reciproc, and Mtwo instruments free of visible deformations were collected and tested in a static cyclic fatigue test method, which has a ceramic block containing an artificial canal with 60° angle of curvature and a 5-mm radius of curvature at 37 °C (n = 16). All instruments were operated until fracture occurred, and both time to fracture (TF) and the lengths of the fractured fragments were recorded. TF data was analyzed with one-way ANOVA followed by post hoc Tukey tests and Weibull analysis, and fractured fragment length data were subjected to one-way ANOVA and post hoc Tukey tests (P < 0.05). Two unused instruments from each brand were also subjected to differential scanning calorimetry (DSC) analysis to determine their phase transformation temperatures. RESULTS: The Reciproc Blue instruments showed significantly higher TF values and reliability than the other groups (P < 0.05). Rotate instruments exhibited greater cyclic fatigue resistance than the Reciproc and Mtwo instruments (P < 0.05). No significant difference was detected among the fractured fragment lengths (P > 0.05). The lowest austenite finish temperature was exhibited by the Mtwo, which presented a single transformation peak, followed by the Rotate, Reciproc Blue, and Reciproc instruments which all presented two peaks during transformation. CONCLUSIONS: Cyclic fatigue resistance of instruments manufactured from thermally treated Blue wire instruments was superior to those of the Mtwo and Reciproc, whereas reciprocating the Blue wire showed the highest resistance. CLINICAL RELEVANCE: The present study compared the cyclic fatigue resistance of the novel Rotate instrument with similar instruments manufactured from conventional nickel-titanium, m-wire, and Blue wire at body temperature and reported that Blue-treated instruments exhibited superior cyclic fatigue resistance.

Research training program in a Turkish medical school: challenges, barriers and opportunities from the perspectives of the students and faculty members.

BACKGROUND: Providing medical students with opportunities for research experience is challenging for medical schools in developing countries. The Research Training Program (RTP), which is carried out in Ege University Faculty of Medicine (EUFM) parallel to the core curriculum, aims to improve the scientific competencies of the highly motivated students and to provide them with the opportunity to conduct a research. The purpose of this project is to evaluate RTP through the perspectives of students and faculty members. METHODS: This phenomenological study included two groups; students of RTP and faculty members who contributed to the program. Interviews were conducted with the research group whose selection was determined by maximum variation technique. Interviews with new individuals continued until data saturation was reached. Interpretative data analysis started with close reading of the transcripts and generating a list of codes. Coding by two independently, developing categories and themes were the following steps. RESULTS: Twenty-one RTP students and 14 faculty members were interviewed. The main motivation for students to participate was the desire to learn how to do research. The introduction course providing the students with the basic competencies needs to be improved in terms of practical activities. It was reported that during the project process students needed intensive guidance especially in finding a research topic and a mentor. The students' lack of time, deficit of enough mentoring and the fact that conducting a research does not provide a competitive advantage for residency are important obstacles to the completion of the program. The most frequently mentioned achievement of the students is to learn all the stages of the research as well as getting acquainted with critical thinking. CONCLUSIONS: This research showed that it was realistic to implement research programs for highly motivated students in medical schools with conditions like those in EUFM. The solution of mentor shortage emerged in this study is dependent on the adoption of student research as a national policy. Getting acquainted with the interrogative thinking style, conducting research, and making lifelong learning a core value are more important outcomes of research programs than the number of completed projects.

IgE and IgG4 binding to lentil epitopes in children with red and green lentil allergy.

BACKGROUND: The consumption of lentil is common in the Mediterranean area and is one of the causes of IgE-mediated food allergy in many countries. Len c 1 is a well-defined allergen of lentil and approximately 80% of the patients with lentil allergy recognize the purified Len c 1 protein. We sought to identify IgE and IgG4 sequential epitopes of Len c 1 in patients with red and/or green lentil allergy. We also aimed to determine IgE and IgG4 binding differences between those patients who had outgrown or remained reactive to lentil. METHODS: Children with IgE-mediated lentil allergy were included in the study. We applied a microarray immunoassay to determine the characterization of positive IgE and IgG4 binding to Len c 1 epitopes in the patients' sera. RESULTS: The peptides specifically recognized by IgE and IgG4 antibodies were mainly detected between peptides 107 and 135 of Len c 1. The signal intensities of positive epitopes were significantly greater in reactive patients than tolerant ones (P = .008 for IgE and P = .002 for IgG4). Moreover, IgE and IgG4 antibodies bound largely the same sequential epitopes in patients who remained reactive or outgrew their allergy. CONCLUSION: IgG4-binding epitopes in lentil allergy were identified and IgE and IgG4 binding to epitopes in both red and green lentils was compared. Our data regarding signal intensity differences between reactive and outgrown patients and overlap binding of IgE and IgG4 antibodies may be important for the development of more accurate diagnostic tests and understanding of natural tolerance development.

Cross-cultural adaptation and validation of the Greek Version of the SARC-F for evaluating sarcopenia in Greek older adults.

OBJECTIVE: To translate and validate into Greek, the SARC-F questionnaire, a screening tool for sarcopenia. METHODS: Questionnaire was back-translated and culturally adapted into Greek according to guidelines proposed by the World Health Organization. A convenience sample of 197 Greek elderly people (71.6±7.8 years, 68.5% women) was recruited, 64 of which were classified as persons at risk of sarcopenia according to the SARC-F. Internal consistency, test-retest and inter-rater reliability were evaluated. Validity (sensitivity, specificity, predictive positive value and predictive negative value) was assessed against the definition from the European Working Group of Sarcopenia in Older People (EWGSOP2), which is considered gold standard. Receiver-operating characteristic analysis was also performed to calculate the area under the curve. RESULTS: SARC-F demonstrated high internal consistency (Cronbach's alpha of 0.93) and excellent inter-rater and test-retest reliability, with intraclass correlation coefficient (ICC) of 0.91 (95% CI 0.79-0.96), and 0.93 (95% CI 0.91-0.95), respectively. According to the definition of sarcopenia from the EWGSOP2, 53 (26.85) participants were identified as probable sarcopenic and 23 (11.6%) as sarcopenic. Sensitivity of the tool for sarcopenia was 34.4 and specificity was 93.2. Positive predictive values were 26.4 and negative predictive values were 66.6%. CONCLUSION: Τhe SARC-F was successfully adapted into Greek language. The Greek SARC-F revealed low sensitivity but high specificity with EWGSOP2 sarcopenia definitions, indicating that it can detect with precision the absence of sarcopenia.

Genome editing reveals reproductive and developmental dependencies on specific types of vitellogenin in zebrafish (Danio rerio).

Oviparous vertebrates produce multiple forms of vitellogenin (Vtg), the major source of yolk nutrients, but little is known about their individual contributions to reproduction and development. This study utilized clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing to assess essentiality and functionality of zebrafish (Danio rerio) type-I and type-III Vtgs. A multiple CRISPR approach was employed to knockout (KO) all genes encoding type-I vtgs (vtg1, 4, 5, 6, and 7) simultaneously (vtg1-KO), and the type-III vtg (vtg3) individually (vtg3-KO). Results of polymerase chain reaction (PCR) genotyping and sequencing, quantitative PCR, liquid chromatography-tandem mass spectrometry, and Western blot analysis showed that only vtg6 and vtg7 escaped Cas9 editing. In fish whose remaining type-I vtgs were incapacitated (vtg1-KO), and in vtg3-KO fish, significant increases in Vtg7 transcript and protein levels occurred in liver and eggs, revealing a heretofore-unknown mechanism of genetic compensation regulating Vtg homeostasis. Egg numbers per spawn were elevated more than 2-fold in vtg1-KO females, and egg fertility was approximately halved in vtg3-KO females. Substantial mortality was evident in vtg3-KO eggs/embryos after only 8 hr of incubation and in vtg1-KO embryos after 5 days. Hatching rate and timing were markedly impaired in embryos from vtg mutant mothers and pericardial and yolk sac/abdominal edema and spinal lordosis were evident in the larvae, with feeding and motor activities also being absent in vtg1-KO larvae. By late larval stages, vtg mutations were either completely lethal (vtg1-KO) or nearly so (vtg3-KO). These novel findings offer the first experimental evidence that different types of vertebrate Vtg are essential and have disparate requisite functions at different times during both reproduction and development.

A novel kinase function of a nucleoside-diphosphate-kinase homologue in Porphyromonas gingivalis is critical in subversion of host cell apoptosis by targeting heat-shock protein 27.

We have previously shown that a homologue of a conserved nucleoside-diphosphate-kinase (Ndk) family of multifunctional enzymes and secreted molecule in Porphyromonas gingivalis can modulate select host molecular pathways including downregulation of reactive-oxygen-species generation to promote bacterial survival in human gingival epithelial cells (GECs). In this study, we describe a novel kinase function for bacterial effector, P. gingivalis-Ndk, in abrogating epithelial cell death by phosphorylating heat-shock protein 27 (HSP27) in GECs. Infection by P. gingivalis was recently suggested to increase phosphorylation of HSP27 in cancer-epithelial cells; however, the mechanism and biological significance of antiapoptotic phospho-HSP27 during infection has never been characterised. Interestingly, using glutathione S-transferase-rNdk pull-down analysed by mass spectrometry, we identified HSP27 in GECs as a strong binder of P. gingivalis-Ndk and further verified using confocal microscopy and ELISA. Therefore, we hypothesised P. gingivalis-Ndk can phosphorylate HSP27 for inhibition of apoptosis in GECs. We further employed P. gingivalis-Ndk protein constructs and an isogenic P. gingivalis-ndk-deficient-mutant strain for functional examination. P. gingivalis-infected GECs displayed significantly increased phospho-HSP27 compared with ndk-deficient-strain during 24 hr infection. Phospho-HSP27 was significantly increased by transfection of GFP-tagged-Ndk into uninfected-GECs, and in vitro phosphorylation assays revealed direct phosphorylation of HSP27 at serines 78 and 82 by P. gingivalis-Ndk. Depletion of HSP27 via siRNA significantly reversed resistance against staurosporine-mediated-apoptosis during infection. Transfection of recombinant P. gingivalis-Ndk protein into GECs substantially decreased staurosporine-induced-apoptosis. Finally, ndk-deficient-mutant strain was unable to inhibit staurosporine-induced Cytochrome C release/Caspase-9 activation. Thus, we show for the first time the phosphorylation of HSP27 by a bacterial effector-P. gingivalis-Ndk-and a novel function of Ndks that is directly involved in inhibition of host cell apoptosis and the subsequent bacterial survival.

Activation of vitamin D in the gingival epithelium and its role in gingival inflammation and alveolar bone loss.

BACKGROUND AND OBJECTIVE: Both chronic and aggressive periodontal disease are associated with vitamin D deficiency. The active form of vitamin D, 1,25(OH)2 D3 , induces the expression of the antimicrobial peptide LL-37 and innate immune mediators in cultured human gingival epithelial cells (GECs). The aim of this study was to further delineate the mechanism by which vitamin D enhances the innate defense against the development of periodontal disease (PD). MATERIALS AND METHODS: Wild-type C57Bl/6 mice were made deficient in vitamin D by dietary restriction. Cultured primary and immortalized GEC were stimulated with 1,25(OH)2 D3 , followed by infection with Porphyromonas gingivalis, and viable intracellular bacteria were quantified. Conversion of vitamin D3 to 25(OH)D3 and 1,25(OH)2 D3 was quantified by ELISA. Effect of vitamin D on basal IL-1α expression in mice was determined by topical administration to the gingiva of wild-type mice, followed by qRT-PCR. RESULTS: Dietary restriction of vitamin D led to alveolar bone loss and increased inflammation in the gingiva in the mouse model. In primary human GEC and established human cell lines, treatment of GEC with 1,25(OH)2 D3 inhibited the intracellular growth of P. gingivalis. Cultured GEC expressed two 25-hydroxylases (CYP27A1 and CYP2R1), as well as 1-α hydroxylase, enabling conversion of vitamin D to both 25(OH)D3 and 1,25(OH)2 D3 . Topical application of both vitamin D3 and 1,25(OH)2 D3 to the gingiva of mice led to rapid inhibition of IL-1α expression, a prominent pro-inflammatory cytokine associated with inflammation, which also exhibited more than a 2-fold decrease from basal levels in OKF6/TERT1 cells upon 1,25(OH)2 D3 treatment, as determined by RNA-seq. CONCLUSION: Vitamin D deficiency in mice contributes to PD, recapitulating the association seen in humans, and provides a unique model to study the development of PD. Vitamin D increases the activity of GEC against the invasion of periodontal pathogens and inhibits the inflammatory response, both in vitro and in vivo. GEC can convert inactive vitamin D to the active form in situ, supporting the hypothesis that vitamin D can be applied directly to the gingiva to prevent or treat periodontal disease.

Cyclic fatigue of Reciproc Blue and Reciproc instruments exposed to intracanal temperature in simulated severe apical curvature.

OBJECTIVE: The aim of this study was to evaluate the cyclic fatigue resistance of Reciproc and Reciproc Blue by testing in a severe apical curvature at intracanal temperature. MATERIALS AND METHODS: Eighteen Reciproc R25 (25.08) and Reciproc Blue (25.08) instruments were tested in a cyclic fatigue device at body temperature (37 °C) using a stainless steel block with an artificial canal with a curvature angle of 90° and radius of curvature of 2 mm. The number of cycles to fracture (NCF) was calculated. The instruments were examined by differential scanning calorimetry (DSC). The data were analyzed using both Student's t tests and Weibull analysis. RESULTS: The NCF values of Reciproc R25 were significantly lower than Reciproc Blue R25 (P < 0.05). There was no significant difference between the instruments regarding the length of fractured fragments (P > 0.05). CONCLUSIONS: Reciproc Blue R25 instruments displayed significantly higher NCF than Reciproc R25. CLINICAL RELEVANCE: This study reported that novel reciprocating blue wire instruments exhibited higher cyclic fatigue resistance than its precedence M-wire instrument when tested in severely curvatured canals.

Identification of novel mutations of Insulin Receptor Substrate 1 (IRS1) in tumor samples of non-small cell lung cancer (NSCLC): Implications for aberrant insulin signaling in development of cancer.

Lung cancer is the leading cause of cancer-related death, and NSCLC constitutes nearly 85%-90% of all cases. The IRS proteins function as adaptors and transmit signals from multiple receptors. Upon binding of insulin to the insulin receptor (IR), IRS1 is phosphorylated at several YXXM motifs creating docking sites for the binding of PI3Kp85, which activates AKT kinase. Therefore, we thought that gain of function mutantions of IRS1 could be related to development of lung cancer. In line with this, we wanted determine whether the IRS1 gene was mutated in the coding regions surrounding YXXM motifs. We sequenced the coding regions surrounding YXXM motifs of IRS1 using tumor samples of 42 NSCLC patients and 40 matching controls and found heterozygote p.S668T mutation in nine of 42 samples and four of nine also had the p.D674H mutation. We generated IRS1 expression vectors harboring p.S668T, p.D674H and double mutants. Expression of the mutants differentially affected insulin-induced phosphorylation of IRS1, AKT, ERK, and STAT3. Also, our mutants induced proliferation, glucose uptake, inhibited the migration of 293T cells and affected the responsiveness of the cells to cisplatin and radiation. Our results suggest that these novel mutations play a role in the phenotype of lung cancer.

Unfolding Role of a Danger Molecule Adenosine Signaling in Modulation of Microbial Infection and Host Cell Response.

Ectonucleotidases CD39 and CD73, specific nucleotide metabolizing enzymes located on the surface of the host, can convert a pro-inflammatory environment driven by a danger molecule extracellular-ATP to an adenosine-mediated anti-inflammatory milieu. Accordingly, CD39/CD73 signaling have has strongly implicated in modulating the intensity, duration, and composition of purinergic danger signals delivered to host. Recent studies have eluted potential roles for CD39 and CD73 in selective triggering of a variety of host immune cells and molecules in the presence of pathogenic microorganisms or microbial virulence molecules. Growing evidence also suggests that CD39 and CD73 present complimentary, but likely differential, actions against pathogens to shape the course and severity of microbial infection as well as the associated immune response. Similarly, adenosine receptors A2A and A2B have been proposed to be major immunomodulators of adenosine signaling during chronic inflammatory conditions induced by opportunistic pathogens, such as oral colonizer Porphyromonas gingivalis. Therefore, we here review the recent studies that demonstrate how complex network of molecules in the extracellular adenosine signaling machinery and their interactions can reshape immune responses and may also be targeted by opportunistic pathogens to establish successful colonization in human mucosal tissues and modulate the host immune response.