Influence of exposure time to saliva and antioxidant treatment on bond strength to enamel after tooth bleaching: an in situ study

Objectives

This study evaluated the influence of different exposure times to saliva in situ in comparison with an antioxidant treatment on composite resin bond strength to human enamel restored after tooth bleaching.

Material and Methods

Forty human teeth specimens measuring 5x5 mm were prepared and randomly allocated into 5 groups with 8 specimens each: Gct (control group, restored on unbleached enamel); Gbl (restored immediately after bleaching); Gsa (bleached, treated with 10% sodium ascorbate gel for 60 min and restored); G7d (bleached, exposed to saliva in situ for 7 days and restored); and G14d (bleached, exposed to saliva in situ for 14 days and restored). Restored samples were cut into 0.8 mm² sticks that were tested in microtensile. Specimens were microscopically analyzed and failure modes were classified as adhesive, cohesive, or mixed. Pretest and cohesive failures were not considered in the statistical analysis, which was performed with one-way ANOVA and Tukey''s post-hoc test (α=0.05), with the dental specimen considered as the experimental unit.

Results

Mean bond strength results found for Gbl in comparison with Gct indicated that bleaching significantly reduced enamel adhesiveness (P<0.01). However, no statistically significant differences were found between Gct, Gsa and G7d (P>0.05). Bond strength found for G14d was significantly higher than for Gsa (P<0.01). Fractures modes were predominantly of a mixed type.

Conclusions

Bonding strength to bleached enamel was immediately restored with the application of sodium ascorbate and exposure to human saliva in situ for at least 7 days. Best results were obtained with exposure to human saliva in situ for 14 days. Treatment with sodium ascorbate gel for 60 min may be recommended in cases patients cannot wait for at least 7 days for adhesive techniques to be performed.     

Muscle biopsy findings predictive of malignancy in rare infiltrative dermatomyositis

Objective

The characteristic pathological muscular findings of polymyositis (PM) and dermatomyositis (DM) have been shown to reflect their different pathogeneses. Here, we characterized the muscle biopsy findings of PM and DM patients with or without malignancy.

Methods

We evaluated the muscle biopsy findings of 215 consecutive PM and DM patients admitted to our hospital between 1970 and 2009. Pathology of the lesion biopsy sections was classified into 3 types: endomysial infiltration-type, perivascular infiltration-type, and rare-infiltrative-type.

Results

There was no difference between the muscle pathology of PM patients with and without malignancy. However, the incidence of rare-infiltrative type muscle pathology in DM patients with malignancy was significantly higher than in those without such tumors (p = 0.0345).

Conclusion

The incidence of rare-infiltrative type muscle pathology may be a predictive marker of DM with malignancy.     

Risk factors associated with respiratory disorders in late preterm infants

Objective: Late preterm infants are still high risk for respiratory problems. The aim of this study was to identify risk factors associated with respiratory problems in Japanese late preterm infants.

Methods: In this retrospective multicenter study, we included singleton late preterm deliveries at 34+^0/7–36+^6/7 weeks of gestation. We excluded cases with congenital anomalies. We defined neonatal respiratory disorders (NRD) as the combination of the need for mechanical ventilation or the use of nasal continuous positive airway pressure. We examined the perinatal risk factors associated with NRD.

Results: We included 683 late preterm infants. We found that 13.7%, 6.8% and 2.6% of the infants with NRD were born at 34, 35 and 36 weeks of gestation, respectively. In a multivariate logistic regression analysis adjusting for confounders, the gestational age (GA) at birth (adjusted odds ratio 0.40 per week [95% confidence interval, 0.25–0.61]), cesarean birth (4.18 [2.11–8.84]), and a low Apgar score (33.3 [9.93–121.3]) were independent risk factors associated with NRD.

Conclusions: An earlier GA, cesarean delivery, and a low Apgar score are independent risk factors associated with NRD in singleton late preterm infants. Patients with late preterm deliveries exhibiting these risk factors should be managed in the intensive delivery setting.     

Molecular analysis of the genome segments S1, S4, S6, S7 and S12 of a Rice gall dwarf virus isolate from Thailand; completion of the genomic sequence

Summary The complete nucleotide sequences of the double-stranded RNA segments S1, S4, S6, S7 and S12 of the genome of a Rice gall dwarf virus (RGDV) isolate from Thailand were determined. The segments consisted of 4505, 2622, 1648, 1652 and 853 nucleotides, encoding putative proteins of 1458, 725, 489, 511 and 206 amino acids with molecular masses of approximately 166, 80, 53, 59 and 24 kDa, respectively. Homology searches indicated that each of the putative proteins has a counterpart in isolates of Rice dwarf virus (RDV) and Wound tumor virus, two other species in the genus Phytoreovirus. However, no similarities were found to other registered sequences, including those of other viruses that belong to the family Reoviridae. The identities between homologous structural proteins of RGDV and RDV ranged from 34 to 51% and were thus higher than those between homologous non-structural proteins of RGDV and RDV (16–37%). Among the nonstructural proteins, the highest amino acid sequence identity (37%) was observed for RGDV Pns11 and RDV Pns10, a constituent of tubular inclusions. This observation suggests that a specific amino acid backbone might be required for maintaining not only the three-dimensional structure of virions but also that of inclusions. The entire sequence of the RGDV genome is now available.     

Brain natriuretic peptide as a novel cardiac hormone in humans. Evidence for an exquisite dual natriuretic peptide system, atrial natriuretic peptide and brain natriuretic peptide.

Using a specific radioimmunoassay for human brain natriuretic peptide (hBNP) with a monoclonal antibody, we have investigated its synthesis, secretion, and clearance in comparison with those of atrial natriuretic peptide (ANP) in normal subjects and patients with congestive heart failure (CHF). Mean BNP-like immunoreactivity (-LI) levels in normal atrium and ventricle were 250 and 18 pmol/g, respectively. The plasma BNP-LI level in normal subjects was 0.90 +/- 0.07 fmol/ml, which was 16% of the ANP-LI level. In contrast, the plasma BNP-LI level markedly increased in patients with CHF in proportion to its severity, and surpassed the ANP-LI level in severe cases. There was a significant step-up of the plasma BNP-LI level in the coronary sinus (CS) compared with that in the aortic root (Ao) and the difference between these BNP-LI levels, delta(CS-Ao)BNP, also increased with the severity of CHF. In addition, the step-up of the BNP-LI level in the anterior interventricular vein [delta(AIV-Ao)BNP] was comparable to delta(CS-Ao)BNP, indicating that BNP is secreted mainly from the ventricle. Predominant BNP synthesis in the ventricle was also confirmed by Northern blot analysis. Catheterization and pharmacokinetic studies revealed that hBNP is cleared from the circulation more slowly than alpha-hANP; this was in part attributed to lower (about 7%) binding affinity of hBNP to clearance receptors than that of alpha-hANP. A predominant molecular form of BNP-LI in the heart and plasma was a 3-kD form corresponding to hBNP. These results indicate that BNP is a novel cardiac hormone secreted predominantly from the ventricle, and that the synthesis, secretion and clearance of BNP differ from those of ANP, suggesting discrete physiological and pathophysiological roles of BNP in a dual natriuretic peptide system.     

Association of crossing capillaries in the finger nailfold with diabetic retinopathy in type 2 diabetes mellitus

Aims/IntroductionCrossing capillaries in the finger nailfold might potentially be a novel diabetic retinopathy (DR) biomarker that could be assessed non‐invasively in the clinical setting. However, the association between crossing capillaries and DR is controversial. This study aimed to investigate the association between the percentage of crossing capillaries in the finger nailfold and DR in patients with type 2 diabetes mellitus.Materials and MethodsThis cross‐sectional study enrolled 108 type 2 diabetes mellitus patients (aged 40–75 years) who visited the outpatient diabetic clinic at Osaka University Hospital, Osaka, Japan, between May and October 2019. Capillary morphology was assessed using nailfold capillaroscopy based on the simple capillaroscopic definitions of the European League Against Rheumatism Study Group. Details of DR and other laboratory data were obtained from medical records. The association between the tertile of the percentage of the crossing capillary and DR was analyzed using multivariable logistic regression.ResultsAfter adjusting for age, sex, diabetes duration, glycated hemoglobin, systolic blood pressure, body mass index, and use of renin–angiotensin system inhibitor and antihyperlipidemic medication, the percentage of crossing capillaries was significantly associated with DR (multivariable‐adjusted odds ratios for increasing tertiles of the percentage of crossing capillary: 1 [reference], 2.05 [95% confidence interval 0.53–7.94], and 4.33 [95% confidence interval 1.16–16.21]; P‐trend = 0.028).ConclusionsA higher percentage of crossing capillaries in the nailfold was associated with a higher risk of DR, independent of traditional risk and inhibiting factors, in patients with type 2 diabetes mellitus.     

Development of cyclic peptides with potent in vivo osteogenic activity through RaPID-based affinity maturation

Osteoporosis is caused by a disequilibrium between bone resorption and bone formation. Therapeutics for osteoporosis can be divided into antiresorptives that suppress bone resorption and anabolics which increase bone formation. Currently, the only anabolic treatment options are parathyroid hormone mimetics or an anti-sclerostin monoclonal antibody. With the current global increases in demographics at risk for osteoporosis, development of therapeutics that elicit anabolic activity through alternative mechanisms is imperative. Blockade of the PlexinB1 and Semaphorin4D interaction on osteoblasts has been shown to be a promising mechanism to increase bone formation. Here we report the discovery of cyclic peptides by a novel RaPID (Random nonstandard Peptides Integrated Discovery) system-based affinity maturation methodology that generated the peptide PB1m6A9 which binds with high affinity to both human and mouse PlexinB1. The chemically dimerized peptide, PB1d6A9, showed potent inhibition of PlexinB1 signaling in mouse primary osteoblast cultures, resulting in significant enhancement of bone formation even compared to non-Semaphorin4D–treated controls. This high anabolic activity was also observed in vivo when the lipidated PB1d6A9 (PB1d6A9-Pal) was intravenously administered once weekly to ovariectomized mice, leading to complete rescue of bone loss. The potent osteogenic properties of this peptide shows great promise as an addition to the current anabolic treatment options for bone diseases such as osteoporosis.

Osteoporosis is a common cause of bone fracture in the elderly, costing billions globally due to fractures leading to long-term disability and subsequent exit from the working population (1, 2). Several treatment options are available for osteoporosis which can be divided into antiresorptives and anabolics ranging from orally dosed small molecules to injectable peptides and biologics (2, 3). Antiresorptive and anabolic agents differ in that antiresorptives inhibit or reduce bone resorption, thereby suppressing bone remodeling, whereas anabolics enhance the rate of bone formation while allowing continued resorption and remodeling of bone tissue. Although both treatments result in increased bone mass, resorption and remodeling are key to the microstructural integrity of bone, and emerging evidence points toward anabolics being more effective in reducing fracture (4–6). Currently, the only anabolics in the clinic are the parathyroid hormone and parathyroid hormone-related peptide mimetics (teriparatide and abaloparatide, respectively) and the sclerostin inhibitor monoclonal antibody (romosozumab). Teriparatide and abaloparatide both cannot be administered over 24 mo in a patient’s lifetime due to the risk for developing osteosarcomas, and romosozumab treatment is recommended for 12 mo due to waning efficacy beyond this duration (7, 8). Therefore, with the ever increasing global median age and associated osteoporosis cases, the development of additional anabolic treatment options are of high importance.Bone resorption and bone formation are regulated through communications between osteoclasts and osteoblasts, respectively (9). Among the paracrine factors involved in this process, axon guidance molecules, such as Semaphorin4D (Sema4D) and Semaphorin3A, mediate the regulation of bone cell differentiation. Sema4D, which is expressed and secreted by mature osteoclasts, inhibits osteoblast differentiation through its receptor PlexinB1 (PlxnB1) expressed on osteoblast surfaces. Binding of Sema4D to PlxnB1 leads to the inhibition of the activation of insulin receptor substrate-1 which is downstream of insulin-like growth factor-1 signaling. In addition, Sema4D controls the spatial distribution of bone-forming osteoblasts through PlxnB1-RhoA signaling (10). Mice with genetic deletion of Sema4D or PlxnB1 as well as mice expressing a dominant-negative form of RhoA in osteoblasts exhibit a high bone mass phenotype due to increased bone formation (11). These findings suggest that inhibiting PlxnB1-Sema4D signaling would lead to a bone anabolic effect through enhancement of osteoblastic differentiation while keeping osteoblasts away from osteoclasts to enable efficient osteoclastic bone resorption.We have previously reported a macrocyclic peptide discovery campaign to identify binding sites on PlxnB1 that inhibit its interaction with Sema4D by means of messenger RNA (mRNA) display in combination with genetic code reprogramming, referred to as the RaPID (Random nonstandard Peptides Integrated Discovery) system (12). We successfully identified a 16-mer thioether-macrocyclic peptide, PB1m6, capable of binding human PlxnB1 (hPlxnB1) with single-digit nanomolar-binding affinity and inhibiting its interaction with Sema4D (13). X-ray structural analysis of cocrystals of PB1m6 and hPlxnB1 revealed that PB1m6 is a negative allosteric modulator of the hPlxnB1-Sema4D interaction by binding a cleft distal to the Sema4D-binding interface of hPlxnB1 while still able to inhibit the hPlxnB1-Sema4D interaction. However, PB1m6 was shown to be selective toward hPlxnB1 over mouse PlxnB1 (mPlxnB1) displaying no detectable affinity (dissociation constant (K_D) over 1 µM) regardless of having 88% sequence identity in the N-terminal sema domains of hPlxnB1 and mPlxnB1. Modeling efforts based on the three-dimensional (3D) structure to rationally increase the species cross-reactivity were unsuccessful in our hands. This high selectivity is often observed with RaPID-derived macrocyclic peptides and is generally considered beneficial (13–18). However, in this instance, the high selectivity of PB1m6 hinders its ability to validate the inhibitory mechanism in mouse models. In this study, we used a fragmented saturation mutagenesis approach to create an mRNA library of PB1m6 analogs to be utilized in a RaPID selection campaign against mouse PlxnB1. After five iterative rounds of selection, we discovered a PB1m6 analog, referred to as PB1m6A9, exhibiting enhanced cross-reactivity with 44 nM K_D against mouse PlxnB1 and which, remarkably, also showed 10-fold stronger binding affinity against human PlxnB1 (0.28 nM K_D). To further improve apparent affinity and inhibitory activity, a homodimer of PB1m6A9 was chemically synthesized (PB1d6A9), and it was shown to exhibit potent mPlxnB1-Sema4D inhibitory activity in mouse primary osteoblasts as well as enhanced osteogenesis even when compared to cells not treated with Sema4D. Moreover, once-weekly intravenous (i.v.) administration of palmitoylated PB1d6A9 (PB1d6A9-Pal) in a mouse model of postmenopausal osteoporosis showed significant enhancement of bone formation compared to both vehicle and sham-operated (Sham) control mice. This work presents the facile development of a novel bone anabolic modality which shows promise as an addition to the current repertoire of anabolic agents used to address osteoporosis.     

Insulin-induced activation of phosphoinositide 3-kinase in Fao cells

Summary Phosphoinositide 3-kinase (PI3-kinase) plays a crucial role in insulin signal transduction. We studied the molecular mechanism of the insulin-induced activation of PI3-kinase in rat hepatoma Fao cells using an antibody against the 110-kDa catalytic subunit (p110) and two against the 85-kDa regulatory subunit (p85). PI3-kinase activity increased 1.6-fold in anti-p85 immunoprecipitates after insulin stimulation, whereas it did not increase when cell lysates were first immunoprecipitated with anti-phosphotyrosine or anti-insulin receptor substrate-1 (IRS-1), then with anti-p85, suggesting that the PI3-kinase which associates with tyrosyl phosphoproteins including IRS-1 is responsible for the increase in kinase activity. The activated PI3-kinase molecules constituted 4–6% of the total PI3-kinase, and their specific activity was 11–14 times higher than that of the basal state. Anti-p110 recognized the catalytically active form of p110, and immunoprecipitated p110 only after exposure to insulin. Hence, the epitope of anti-p110, P200-C215, seems to be included in the portion of p110, the conformation of which is changed by insulin stimulation. We conclude that, in response to insulin stimulation, only a small fraction of p85 in the PI3-kinase pool associates with tyrosyl phosphoproteins including IRS-1, and that the specific activity of p110 is increased presumably through a conformational change including the P200-C215 region.Abbreviations PI3-kinase Phosphoinositide 3-kinase - p85 85-kDa subunit of PI3-kinase - p110 110-kDa subunit of PI3-kinase - IRS-1 insulin receptor substrate-1 - SH2 src homology 2 - SH3 src homology 3 - BCR breakpoint cluster region - PMSF phenylmethylsulphonyl fluoride - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid - PIP phosphatidylinositol phosphate - TLC thin layer chromatography - IP (in figures) immunoprecipitation with the indicated antibody - TBS Tris-buffered saline