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Objective To report a developmental-related tooth that the mandibular first molar fused to the second molar with seven canals and treated with a conservative option. Methods CBCT images were used for analyzing the complex morphoanatomy in coronal and root canal systems. The tooth was isolated with a rubber dam and seven canal orifices were identified. Root canal treatment under a microscope was carried out. Results The tooth was diagnosed as an anomalous fused mandibular molar with chronic apical periodontitis and the root canal treatment was successful. Conclusions We reported a rare case of fusion involving permanent mandibular molars and CBCT is a critical aid in efficiently diagnosing and understanding such an anatomically abnormal tooth. Clinical Relevance This study suggests CBCT analysis is important for understanding the abnormal structure and guiding for treating such a malformed tooth before operation. This study also provides a rare and interesting case for endodontics.

Background: Double teeth are usually the result of an abnormality in the developing tooth germ. Double teeth can occur in either the primary or permanent dentition, with the majority of cases concerning permanent teeth reported in the anterior teeth and less frequently in the molar teeth. Case presentation: This report illustrates five cases of double teeth in molars with periapical periodontitis, including one case of geminated teeth and four cases of fused teeth. Radiographic findings revealed the presence of extra teeth on the buccal aspect of the molar in five cases, with or without communication between the two root canal systems. Root canal treatment was performed by using CBCT and a dental operating microscope. The treatment outcome was good in all five cases. Conclusion: The diagnosis and treatment of double teeth requires special attention. The root canal system should be carefully explored to obtain a full understanding of the anatomy, allowing it to be fully cleaned and obturated. Proper anatomical structure analysis prior to treatment facilitates the development of an appropriate treatment plan, thereby increasing the likelihood of successful treatment both aesthetically and functionally.

A member of the Janus kinase (JAK) family, Tyrosine Kinase 2 (TYK2), is crucial in mediating various cytokine-signaling pathways such as interleukin-23 (IL23), interleukin-12 (IL12) and type I Interferons (IFN) which contribute to autoimmune disorders (e.g., psoriasis, lupus, and inflammatory bowel disease). Thus, TYK2 represents an attractive target to develop small-molecule therapeutics for the treatment of cytokine-driven inflammatory diseases. Selective inhibition of TYK2 over other JAK isoforms is critical to achieve a favorable therapeutic index in the development of TYK2 inhibitors. However, designing small molecule inhibitors to target the adenosine triphosphate (ATP) binding site of TYK2 kinase has been challenging due to the substantial structural homology of the JAK family catalytic domains. Here, we employed an approach to target the JAK homology 2 (JH2) pseudokinase regulatory domain of the TYK2 protein. We developed a series of small-molecule TYK2 pseudokinase ligands, which suppress the TYK2 catalytic activity through allosteric regulation. The TYK2 pseudokinase-binding small molecules in this study simultaneously achieve high affinity-binding for the TYK2 JH2 domain while also affording significantly reduced affinity for the TYK2 JAK homology 1 (JH1) kinase domain. These TYK2 JH2 selective molecules, although possessing little effect on suppressing the catalytic activity of the isolated TYK2 JH1 catalytic domain in the kinase assays, can still significantly block the TYK2-mediated receptor-stimulated pathways by binding to the TYK2 JH2 domain and allosterically regulating the TYK2 JH1 kinase. These compounds are potent towards human T-cell lines and primary immune cells as well as in human whole-blood specimens. Moreover, TYK2 JH2-binding ligands exhibit remarkable selectivity of TYK2 over JAK isoforms not only biochemically but also in a panel of receptor-stimulated JAK1/JAK2/JAK3-driven cellular functional assays. In addition, the TYK2 JH2-targeting ligands also demonstrate high selectivity in a multi-kinase screening panel. The data in the current study underscores that the TYK2 JH2 pseudokinase is a promising therapeutic target for achieving a high degree of biological selectivity. Meanwhile, targeting the JH2 domain represents an appealing strategy for the development of clinically well-tolerated TYK2 inhibitors that would have superior efficacy and a favorable safety profile compared to the existing Janus kinase inhibitors against autoimmune diseases.

The development of RAF inhibitors targeting cancers with wild type RAF kinase and/or RAS mutation has been challenging due to the paradoxical activation of the RAS-RAF-MEK-ERK cascade following RAF inhibitor treatment. Herein is the discovery and optimization of a series of RAF inhibitors with a novel spiro structure. The most potent spiro molecule 9 showed excellent in vitro potency against b/c RAF enzymes and RAS mutant H358 cancer cells with minimal paradoxical RAF signaling activation. Compound 9 also exhibited good drug-like properties as demonstrated by in vitro cytochrome P450 (CYP), liver microsome stability (LMS) data and moderate oral pharmacokinetics (PK) profiles in rat and mouse.

Background and Overview The incidence rate of severely curved root canals in mandibular molars is low, and the root canal treatment of mandibular molars with this aberrant canal anatomy may be visibly and technically challenging. Case Description A 26-year-old Chinese female patient presented with intermittent and occlusal pain in the left mandibular second molar. The patient had undergone caries for filling restoration before endodontic consultation. With the aid of cone beam computed tomography (CBCT), a large periapical radiolucency was observed, and curved root canals in a mandibular second molar were confirmed, depicting a severe and curved distolingual root. Nonsurgical treatments, including novel individual preparation skills and techniques and the use of bioceramic materials as an apical barrier, were performed, and complete healing of the periapical lesion and a satisfactory effect were achieved. Conclusions and Practical implications A case of a severely curved root canal in a mandibular second molar was successfully treated and reported herein. The complex anatomy of the tooth and the postoperative effect were also evaluated via the three-dimensional (3D) reconstruction of CBCT images, which accurately identified the aberrant canal morphology. Furthermore, new devices and biomaterial applications combined with novel synthesis techniques can increase the success rate of intractable endodontic treatment.

As a noninvasive technique, transcranial sonography (TCS) of substantia nigra (SN) has gradually showed its effectiveness not only in diagnosis but also in understanding clinical features of Parkinson’s Disease (PD). This study aimed to further evaluate TCS for clinical diagnosis of PD, and to explore the association between sonographic manifestations and visual hallucinations (VH). A total of 226 subjects including 141 PD patients and 85 controls were recruited. All participants received TCS. A series of rating scales to evaluate motor and non-motor symptoms were performed in PD patients. Results showed that 172 subjects were successfully assessed by TCS. The area of SN was greater in PD patients than that in controls (P < 0.001). As receiver-operating characteristic (ROC) curve analysis showed, the best cutoff value for the larger SN echogenicity size was 23.5 mm2 (sensitivity 70.3%, specificity 77.0%). Patients with VH had larger SN area (P = 0.019), as well as higher Non-Motor Symptoms Scale (NMSS) scores (P = 0.018). Moreover, binary logistic regression analysis indicated that SN hyperechogenicity (odds ratio = 4.227, P = 0.012) and NMSS scores (odds ratio = 0.027, P = 0.042) could be the independent predictors for VH. In conclusion, TCS can be used as an auxiliary diagnostic tool for Parkinson’s disease. Increased SN echogenicity is correlated with VH in Parkinson’s disease, possibly because the brain stem is involved in the mechanism in the onset of VH. Further studies are needed to confirm these findings.

The RAS-RAF-MEK-ERK signaling pathway plays a key role to regulate multiple cellular functions. Acquired resistance to the first-generation RAF inhibitors that only targeted the bRAF

BackgroundGlutamate racemase (MurI) is a cofactor-independent enzyme that is essential to the bacterial peptidoglycan biosynthesis pathway and has therefore been considered an attractive target for the development of antimicrobial drugs. While in our previous study the essentiality of themurIgene was shown inStreptococcus mutans, the primary aetiologic agent of human dental caries, studies onS. mutansMurI have not yet provided definitive results. This study aimed to produce and characterize the biochemical properties of the MurI from theS. mutansUA159 genome.MethodsStructure characterization prediction and multiple sequence alignment were performed by bioinformatic analysis. Recombinant His6-taggedS. mutansMurI was overexpressed in the expression vector pColdII and further purified using a Ni2+affinity chromatography method. Protein solubility, purity and aggregation state were analyzed by SDS–PAGE, Western blotting, native PAGE and SEC-HPLC. Kinetic parameters were assessed by a circular dichroism (CD) assay. Kinetic constants were calculated based on the curve fit for the Michaelis–Menten equation. The effects of temperature and pH on enzymatic activity were determined by a series of coupled enzyme reaction mixtures.ResultsThe glutamate racemase gene fromS. mutansUA159 was amplified by PCR, cloned and expressed inEscherichia coliBL21 (DE3). The 264-amino-acid protein, as a mixture of dimeric and monomeric enzymes, was purified to electrophoretic homogeneity. In the CD assay,S. mutansMurI displayed unique kinetic parameters (Km,d-Glu→l-Glu= 0.3631 ± 0.3205 mM,Vmax,d-Glu→l-Glu= 0.1963 ± 0.0361 mM min−1,kcat,d-Glu→l-Glu= 0.0306 ± 0.0065 s−1,kcat/Km,d-Glu→l-Glu= 0.0844 ± 0.0128 s−1mM−1, withd-glutamate as substrate;Km,l-Glu→d-Glu= 0.8077 ± 0.5081 mM,Vmax,l-Glu→d-Glu= 0.2421 ± 0.0418 mM min−1,kcat,l-Glu→d-Glu= 0.0378 ± 0.0056 s−1,kcat/Km,l-Glu→d-Glu= 0.0468 ± 0.0176 s−1mM−1, withl-glutamate as substrate).S. mutansMurI possessed an assay temperature optimum of 37.5 °C and its optimum pH was 8.0.ConclusionThe findings of this study provide insight into the structure and biochemical traits of the glutamate racemase inS. mutansand supply a conceivable guideline for employing glutamate racemase in anti-caries drug design.

Ti-10Ta-2Nb-2Zr (TTNZ) alloy has been previously suggested to be a promising alternate biomaterial for implant applications. However, its mismatch in elastic modulus with human bone and its lack of sufficient bioactivity are two concerns that need to be addressed further. In this study, for the first time, we attempted to use macroporous structures on TTNZ alloy scaffolds manufactured by selective laser melting (SLM) to reduce their modulus of elasticity and strengthen bone interlocking. Then, we modified scaffold surfaces with hydroxyapatite (HA) coating by microarc oxidation (MAO) to promote osseointegration. The microstructure, surface morphology, chemical composition, mechanical properties, biological activities and osteogenesis performance of this scaffold were systematically investigated in vitro and in vivo. The results revealed that scaffolds with this novel porous alloy possess a lower elastic modulus than solid structures. A micro/nanostructured layer with HA was successfully formed, which was verified by scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and energy dispersive spectroscopy (EDS). At the same time, this surface has increased hydrophilicity and roughness compared to unmodified surfaces and commercial pure titanium (CP-Ti). Furthermore, in vitro studies found that compared with MAOTTNZ samples with lower porosity, bare TTNZ samples, and CP-Ti samples, MAOTTNZ with adequate porosity could improve protein adsorption, cell proliferation, adhesion, alkaline phosphatase (ALP) activity and osteogenesis-related gene expression. In vivo studies, including those in which microcomputerized tomography, histological examination and biomechanical testing of macroporous MAOTTNZ scaffolds were performed, demonstrated that the formation of new bones was significantly boosted in rabbits after implantation. Consequently, this research presents an option for conventional titanium alloys with improved osteogenesis and osseointegration that could be used in further dental implant applications.

Treatment of hepatitis C patients with direct-acting antiviral drugs involves the combination of multiple small-molecule inhibitors of distinctive mechanisms of action. ACH-806 (or GS-9132) is a novel, small-molecule inhibitor specific for hepatitis C virus (HCV). It inhibits viral RNA replication in HCV replicon cells and was active in genotype 1 HCV-infected patients in a proof-of-concept clinical trial (1). Here, we describe a potential mechanism of action (MoA) wherein ACH-806 alters viral replication complex (RC) composition and function. We found that ACH-806 did not affect HCV polyprotein translation and processing, the early events of the formation of HCV RC. Instead, ACH-806 triggered the formation of a homodimeric form of NS4A with a size of 14 kDa (p14) both in replicon cells and in Huh-7 cells where NS4A was expressed alone. p14 production was negatively regulated by NS3, and its appearance in turn was associated with reductions in NS3 and, especially, NS4A content in RCs due to their accelerated degradation. A previously described resistance substitution near the N terminus of NS3, where NS3 interacts with NS4A, attenuated the reduction of NS3 and NS4A conferred by ACH-806 treatment. Taken together, we show that the compositional changes in viral RCs are associated with the antiviral activity of ACH-806. Small molecules, including ACH-806, with this novel MoA hold promise for further development and provide unique tools for clarifying the functions of NS4A in HCV replication.

The aim of this study was to use contrast-enhanced ultrasound (CEUS) to evaluate renal perfusion after kidney transplantation and investigate the clinical significance of CEUS in monitoring postoperative renal perfusion. Thirty-five patients who underwent kidney transplantations were included in this study and divided into two groups-normal and abnormal-based on their serum creatinine (SCr) levels. Conventional ultrasound and CEUS were used to monitor renal perfusion after kidney transplantation. The differences in the results between the two groups were then compared. Color doppler ultrasonography showed that there were significant differences in the resistance index (RI) and the pulsatility index (PI) of the interlobar artery between the groups. Furthermore, CEUS indicated a significant difference between the two groups regarding the slope rate of the cortical ascending curve (A1), the medullary ascending curve (A2), and the derived peak intensity (DPI1). CEUS precisely showed the characteristics of microcirculation in renal parenchyma after kidney transplantation. It also detected changes in the microcirculation, which was a new method of evaluating tissue perfusion in transplanted kidneys.

Previous work has shown that incorporation of either 1-aminocyclohexanecarboxylic acid (Ac6c) or alpha-methyl-p-phosphonophenylalanine ((alpha-Me)Ppp) in the phosphotyrosyl (pTyr) C-proximal position (pY + 1 residue) of Grb2 SH2 domain binding peptides confers high affinity. The tetralin-based (S)-2-amino-6-phosphonotetralin-2-carboxylic acid (Atc(6-PO3H2)) simultaneously presents structural features of both (alpha-Me)Ppp and Ac6c residues. The current study compares the affinity of this tetralin hybrid Atc(6-PO3H2) versus Ac6c and (alpha-Me)Ppp residues when incorporated into the pY + 1 position of a high-affinity Grb2 SH2 domain binding tripeptide platform. The highest binding affinity (KD = 14.8 nM) was exhibited by the (alpha-Me)Ppp-containing parent, with the corresponding Ac6c-containing peptide being nearly 2-fold less potent (KD = 23.8 nM). The lower KD value was attributable primarily to a 50% increase in off-rate. Replacement of the Ac6c residue with the tetralin-based hybrid resulted in a further 4-fold decrease in binding affinity (KD = 97.8 nM), which was the result of a further 6-fold increase in off-rate, offset by an approximate 45% increase in on-rate. Therefore, by incorporation of the key structural components found in (alpha-Me)Ppp into the Ac6c residue, the tetralin hybrid does enhance binding on-rate. However, net binding affinity is decreased due to an associated increase in binding off-rate. Alternatively, global conformational constraint of an (alpha-Me)Ppp-containing peptide by beta-macrocyclization did result in pronounced elevation of binding affinity, which was achieved primarily through a decrease in the binding off-rate. Mathematical fitting using a simple model that assumed a single binding site yielded an effective KD of 2.28 nM. However this did not closely approximate the data obtained. Rather, use of a complex model that assumed two binding sites resulted in a very close fit of data and provided KD values of 97 pM and 72 nM for the separate sites, respectively. Therefore, although local conformational constraint in the pY + 1 residue proved to be deleterious, global conformational constraint through beta-macrocyclization achieved higher affinity. Similar beta-macrocyclization may potentially be extended to SH2 domain systems other than Grb2, where bend geometries are required.

Reported herein is the first enantioselective preparation of (2S,3R)-3-phenylpipecolic acid as a conformationally constrained phenylalanine analogue bearing N(alpha)-protection suitable for solid-phase peptide synthesis. Stereochemistries at both the 2- and 3-positions are derived inductively from a single chiral center provided by the commercially available Evans chiral auxiliary, (4S)-4-benzyl-1,3-oxazolidin-2-one. By constraining phi and chi(1) torsion angles, this novel amino acid analogue can serve as a useful tool for the induction of defined geometry in phenylalanine-containing peptides.

INTRODUCTION: The complement system is a pivotal player in multiple hematological conditions that include paroxysmal nocturnal hemoglobinuria (PNH). The current standard of care for PNH is intravenous infusion of eculizumab, a humanized monoclonal antibody that targets the terminal complement protein C5 and thereby efficiently impairs intravascular hemolysis. However, a significant fraction of PNH patients in clinical practice respond incompletely to eculizumab due to extravascular hemolysis by immune cells as eculizumab does not prevent erythrocyte opsonization. Additionally, a non-responsive sub-population has been identified with a rare genetic polymorphism in C5 that renders the variant incapable of binding eculizumab. Therefore, an unmet medical need remains for regimens that improve efficacy and that can be administered orally. To achieve this goal, we initiated a discovery program for small molecule inhibitors of complement factor D, a serine protease that is the rate limiting enzyme of the alternative complement pathway. We have obtained high resolution (1.5 Å) crystal structure of inhibitor-bound factor D. Through structure-guided optimization, we have discovered novel inhibitors of factor D that possess high potency and specificity as well as pharmacokinetic properties suitable for oral administration. Herein, we present a biochemical characterization of lead compounds and studies of their activities in various complement-mediated processes. METHODS: The binding affinity to factor D was determined by surface plasmon resonance (Biacore) analysis. The inhibitory effect on factor D protease was evaluated biochemically using both a nonspecific synthetic substrate and a natural substrate consisting of C3b and the complement factor B. The alternative pathway (AP) hemolytic assay was conducted with unsensitized rabbit erythrocytes, 8% human or non-human primate serum, and 10 mM MgEGTA. The classical pathway (CP) hemolytic assay was conducted with antibody-sensitized sheep erythrocytes and 0.5% human serum. To assess inhibition of CP activation through the AP-dependent amplification loop, inhibition of terminal complement complex (TCC) formation by 50% human serum was quantitated following activation by the CP-specific activator HAIGG. Inhibition of complement opsonization and lysis of PNH-like erythrocytes exposed to acidified serum (Ham test) was assessed ex vivo. Finally, selected lead compounds were administered to non-human primates to evaluate the efficacy of factor D inhibitors for blockade of alternative pathway-mediated events. RESULTS: Lead compounds obtained through optimization bound to factor D with Kd values below 1 nM. Tight binding to factor D of the compounds resulted in potent inhibition of factor D proteolytic activity assessed using a synthetic nonspecific substrate as well as a natural substrate. When evaluated in an AP hemolytic assay with human serum, the most potent compounds inhibited cell lysis with EC50 values below 10 nM. Similar inhibitory activity was observed when compounds were assessed in AP hemolytic assays with non-human primate serum, but not with rodent serum. The compounds showed no activity in a CP hemolytic assay using highly diluted human serum and antibody-coated erythrocytes, indicating specificity for factor D. When evaluated in the presence of 50% human serum and with TCC formation as an endpoint, however, the compounds blocked approximately 75% of TCC formation upon CP activation, a consequence of blockage of the AP-dependent amplification loop. Furthermore, the compounds prevented both opsonisation and lysis of PNH-like cells in the Ham test. Finally, when selected compounds were administered to non-human primates, potent inhibition of TCC formation following ex vivo AP activation was observed with EC50 values below 50 nM. CONCLUSIONS: Highly potent and specific factor D inhibitors that block both the terminal pathway and opsonization were discovered using structure-guided design. These orally deliverable inhibitors hold promise as a novel therapeutic approach for treatment of complement-mediated hematological conditions including PNH. Disclosures Morgan: Swedish Orphan Biovitrum: Consultancy, Research Funding; Glaxo Smith Kline: Consultancy; Achillion Pharmaceuticals: Consultancy, Research Funding. Thanassi:Achillion Pharmaceuticals: Employment. Podos:Achillion Pharmaceuticals : Employment. Phadke:Achillion Pharmaceuticals: Employment. Gadhachanda:Achillion Pharmaceuticals: Employment. Pais:Achillion Pharmaceuticals: Employment. Hashimoto:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Chen:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Agarwal:Achillion Pharmaceuticals: Employment. Deshpande:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment. Wiles:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment.

Reported herein is the first enantioselective preparation of (2S,3R)-3-phenylpipecolic acid as a conformationally constrained phenylalanine analogue bearing N(alpha)-protection suitable for solid-phase peptide synthesis. Stereochemistries at both the 2- and 3-positions are derived inductively from a single chiral center provided by the commercially available Evans chiral auxiliary, (4S)-4-benzyl-1,3-oxazolidin-2-one. By constraining phi and chi(1) torsion angles, this novel amino acid analogue can serve as a useful tool for the induction of defined geometry in phenylalanine-containing peptides.