Accurate long-read sequencing allows assembly of the duplicated RHD and RHCE genes harboring variants relevant to blood transfusion.

Next-generation sequencing (NGS) technologies have transformed medical genetics. However, short-read lengths pose a limitation on identification of structural variants, sequencing repetitive regions, phasing of distant nucleotide changes, and distinguishing highly homologous genomic regions. Long-read sequencing technologies may offer improvements in the characterization of genes that are currently difficult to assess. We used a combination of targeted DNA capture, long-read sequencing, and a customized bioinformatics pipeline to fully assemble the RH region, which harbors variation relevant to red cell donor-recipient mismatch, particularly among patients with sickle cell disease. RHD and RHCE are a pair of duplicated genes located within an ∼175 kb region on human chromosome 1 that have high sequence similarity and frequent structural variations. To achieve the assembly, we utilized palindrome repeats in PacBio SMRT reads to obtain consensus sequences of 2.1 to 2.9 kb average length with over 99% accuracy. We used these long consensus sequences to identify 771 assembly markers and to phase the RHD-RHCE region with high confidence. The dataset enabled direct linkage between coding and intronic variants, phasing of distant SNPs to determine RHD-RHCE haplotypes, and identification of known and novel structural variations along with the breakpoints. A limiting factor in phasing is the frequency of heterozygous assembly markers and therefore was most successful in samples from African Black individuals with increased heterogeneity at the RH locus. Overall, this approach allows RH genotyping and de novo assembly in an unbiased and comprehensive manner that is necessary to expand application of NGS technology to high-resolution RH typing.

The transcription factor c-Myb primes CD4+CD8+ immature thymocytes for selection into the iNKT lineage.

Type I invariant NKT cells (iNKT cells) are a subset of alphabeta T cells characterized by the expression of an invariant alpha-chain variable region 14-alpha-chain joining region 18 (V(alpha)14J(alpha)18) T cell antigen receptor (TCR) alpha-chain. The iNKT cells derive from CD4(+)CD8(+) double-positive (DP) thymocytes, and their generation requires a long half-life of DP thymocytes to allow V(alpha)14-J(alpha)18 rearrangements, expression of glycolipid-loaded CD1d on DP thymocytes, and signaling through the signaling-activation molecule SLAM-adaptor SAP pathway. Here we show that the transcription factor c-Myb has a central role in priming DP thymocytes to enter the iNKT lineage by simultaneously regulating CD1d expression, the half-life of DP cells and expression of SLAMF1, SLAMF6 and SAP.

AnthOligo: automating the design of oligonucleotides for capture/enrichment technologies.

SUMMARY: A number of methods have been devised to address the need for targeted genomic resequencing. One of these methods, region-specific extraction (RSE) is characterized by the capture of long DNA fragments (15-20 kb) by magnetic beads, after enzymatic extension of oligonucleotides hybridized to selected genomic regions. Facilitating the selection of the most appropriate capture oligos for targeting a region of interest, satisfying the properties of temperature (Tm) and entropy (ΔG), while minimizing the formation of primer-dimers in a pooled experiment, is therefore necessary. Manual design and selection of oligos becomes very challenging, complicated by factors such as length of the target region and number of targeted regions. Here we describe, AnthOligo, a web-based application developed to optimally automate the process of generation of oligo sequences used to target and capture the continuum of large and complex genomic regions. Apart from generating oligos for RSE, this program may have wider applications in the design of customizable internal oligos to be used as baits for gene panel analysis or even probes for large-scale comparative genomic hybridization array processes. AnthOligo was tested by capturing the Major Histocompatibility Complex (MHC) of a random sample.The application provides users with a simple interface to upload an input file in BED format and customize parameters for each task. The task of probe design in AnthOligo commences when a user uploads an input file and concludes with the generation of a result-set containing an optimal set of region-specific oligos. AnthOligo is currently available as a public web application with URL: http://antholigo.chop.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Discovery and optimization of a potent and selective indazolamine series of IRAK4 inhibitors.

IRAK4 is a key mediator of innate immunity. There is a high interest in identifying novel IRAK4 inhibitors for the treatment of inflammatory autoimmune diseases. We describe here a highly potent and selective IRAK4 inhibitor (HS271) that exhibited superior enzymatic and cellular activities, as well as excellent pharmacokinetic properties. HS271 displayed robust in vivo anti-inflammatory efficacy as evaluated in rat models of LPS induced TNFα production and collagen-induced arthritis.

Palladium-Catalyzed Difluoromethylation of Aryl Chlorides and Triflates and Its Applications in the Preparation of Difluoromethylated Derivatives of Drug/Agrochemical Molecules.

A palladium-catalyzed difluoromethylation of a series of aryl chlorides and triflates under mild conditions was described. A variety of common functional groups were tolerated. In addition, by using this protocol, several drug molecules containing an aryl chloride unit were successfully difluoromethylated, thus enabling medicinal chemists to rapidly access novel drug derivatives with potentially improved properties via late-stage functionalization.

Synthesis of 2-morpholinetetraphenylporphyrins and their photodynamic activities.

A series of 2-morpholinetetraphenylporphyrins functionalized with various substituents (Cl, Me, MeO group) at 4-phenyl position were prepared via nucleophilic substitution of 2-nitroporphyrin copper derivatives with morpholine by refluxing under a nitrogen atmosphere and then demetalization. Their basic photophysical properties, intracellular localization, cytotoxicities in vitro and in vivo were also investigated. All synthesized photosensitizers exhibited longer maxima absorption wavelengths than Hematoporphyrin monomethyl ether (HMME). They showed low dark cytotoxicity compared with that of HMME and were more phototoxic than HMME against Eca-109 cells in vitro. M3 also exhibited better photodynamic antitumor efficacy on BALB/c nude mice at a lower concentration. Therefore, M3 is a promising antitumor photosensitizer in photodynamic therapy application.

Inhibition of Laser-Induced Choroidal Neovascularization by Hematoporphyrin Dimethylether-Mediated Photodynamic Therapy in Rats.

This study aimed to investigate the effect of hematoporphyrin dimethylether (HDME)-mediated photodynamic therapy for laser-induced choroidal neovascularization (CNV) in adult Brown Norway rats. HDME was administered via tail vein at 14 d after the laser photocoagulation, and the rats received irradiance with a laser light at 570 nm at 15 min after injection. CNV was evaluated by fundus photography, fundus fluorescein angiography, optical coherence tomography, and hematoxylin and eosin staining. We found that CNV was occurred at 7 d after photocoagulation and reaching peak activity at 14 d after photocoagulation. There is a significant reduction in the total area of the fluorescein leakage and the number of strong fluorescein leakage spots on 7 d after HDME-mediated photodynamic therapy (PDT). The results suggest that HDME-mediated PDT inhibits laser-induced CNV in rats, representing a promising therapy for wet age-related macular degeneration.

Targeting Hippo pathway by specific interruption of YAP-TEAD interaction using cyclic YAP-like peptides.

Hippo signaling pathway is emerging as a novel target for anticancer therapy because it plays key roles in organ size control and tumorigenesis. As the downstream effectors, Yes-associated protein (YAP)-transcriptional enhancer activation domain family member (TEAD) association is essential for YAP-driven oncogenic activity, while TEAD is largely dispensable for normal tissue growth. We present the design of YAP-like peptides (17mer) to occupy the interface 3 on TEAD. Introducing cysteines at YAP sites 87 and 96 can induce disulfide formation, as confirmed by crystallography. The engineered peptide significantly improves the potency in disrupting YAP-TEAD interaction in vitro. To confirm that blocking YAP-TEAD complex formation by directly targeting on TEAD is a valid approach, we report a significant reduction in tumor growth rate in a hepatocellular carcinoma xenograft model after introducing the dominant-negative mutation (Y406H) of TEAD1 to abolish YAP-TEAD interaction. Our results suggest that targeting TEAD is a promising strategy against YAP-induced oncogenesis.

Increased level of E protein activity during invariant NKT development promotes differentiation of invariant NKT2 and invariant NKT17 subsets.

E protein transcription factors and their natural inhibitors, Id proteins, play critical and complex roles during lymphoid development. In this article, we report that partial maintenance of E protein activity during positive selection results in a change in the cell fate determination of developing iNKT cells, with a block in the development of iNKT1 cells and a parallel increase in the iNKT2 and iNKT17 subsets. Because the expression levels of the transcription factors that drive these alternative functional fates (GATA-3, RORγT, T-bet, and Runx-3) are not altered, our results suggest that E protein activity controls a novel checkpoint that regulates the number of iNKT precursors that choose each fate.

Regulation of GATA-3 expression during CD4 lineage differentiation.

GATA-3 is necessary for the development of MHC class II-restricted CD4 T cells, and its expression is increased during positive selection of these cells. TCR signals drive this upregulation, but the signaling pathways that control this process are not well understood. Using genetic and pharmacological approaches, we show that GATA-3 upregulation during thymocyte-positive selection is the result of additive inputs from the Ras/MAPK and calcineurin pathways. This upregulation requires the presence of the transcription factor c-Myb. Furthermore, we show that TH-POK can also upregulate GATA-3 in double-positive thymocytes, suggesting the existence of a positive feedback loop that contributes to lock in the initial commitment to the CD4 lineage during differentiation.

Characteristics and influencing factors of economic resilience in industrial parks.

Economic resilience has been a popular issue in recent years. Along with the consideration of severe shocks caused by the financial crisis of 2007-2008 and globalization of industry and the upgradation of knowledge and technology, economic resilience has brought in much attention. About 50 years of development of planned industrial parks in Taiwan has resulted in considerable economic scale; however, due to changes in interior demands and the exterior environment with time, rearrangement and industrial transformation have made the development of industrial parks difficult. Accordingly, the resilience of planned industrial parks in Taiwan, when encountering a variety of shocks, need to be reviewed and examined. This study selects 12 planned industrial parks from Tainan and Kaohsiung, in southern Taiwan, as subjects and had a complete understanding of economic resilience and factors that influence economic resilience from literature reviews. Four quadrant model constituted by the indicators of economic resistance and recovery as well as discriminant analysis are implemented to analyze the resilience of industrial parks with different backgrounds and various shocks, as well as the elements influencing the resilience. Analytical results indicate that planned industrial parks with industrial structures based on specialized variety or with a steady input of knowledge and innovation to research and development benefited the industrial parks in better resilience, while complete infrastructure planning and governance are fundamental conditions for resilience.

Discovery of Linvencorvir (RG7907), a Hepatitis B Virus Core Protein Allosteric Modulator, for the Treatment of Chronic HBV Infection.

Described herein is the first-time disclosure of Linvencorvir (RG7907), a clinical compound and a hepatitis B virus (HBV) core protein allosteric modulator, for the treatment of chronic HBV infection. Built upon the core structure of hetero aryl dihydropyrimidine, RG7907 was rationally designed by combining all the drug-like features of low CYP3A4 induction, potent anti-HBV activity, high metabolic stability, low hERG liability, and favorable animal pharmacokinetic (PK) profiles. In particular, the chemistry strategy to mitigate CYP3A4 induction through introducing a large, rigid, and polar substituent at the position that has less interaction with the therapeutic biological target (HBV core proteins herein) is of general interest to the medicinal chemistry community. RG7907 demonstrated favorable animal PK, pharmacodynamics, and safety profiles with sufficient safety margins supporting its clinical development in healthy volunteers and HBV-infected patients.

Discovery of 4,5,6,7-Tetrahydropyrazolo[1.5-a]pyrizine Derivatives as Core Protein Allosteric Modulators (CpAMs) for the Inhibition of Hepatitis B Virus.

Hepatitis B Virus (HBV) core protein allosteric modulators (CpAMs) are an attractive class of potential anti-HBV therapeutic agents. Here we describe the efforts toward the discovery of a series of 4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine (THPP) compounds as HBV CpAMs that effectively inhibit a broad range of nucleos(t)ide-resistant HBV variants. The lead compound 45 demonstrated inhibition of HBV DNA viral load in a HBV AAV mouse model by oral administration.

Control of early stages in invariant natural killer T-cell development.

Natural killer T (NKT) cells develop in the thymus from the same precursors as conventional CD4(+) and CD8(+) αß T cells, CD4(+) CD8(+) double-positive cells. In contrast to conventional αßT cells, which are selected by MHC-peptide complexes presented by thymic epithelial cells, invariant NKT cells are selected by lipid antigens presented by the non-polymorphic, MHC I-like molecule CD1d, present on the surface of other double-positive thymocytes, and require additional signals from the signalling lymphocytic-activation molecule (SLAM) family of receptors. In this review, we provide a discussion of recent findings that have modified our understanding of the NKT cell developmental programme, with an emphasis on events that affect the early stages of this process. This includes factors that control double-positive thymocyte lifespan, and therefore the ability to generate the canonical Vα rearrangements that characterize this lineage, as well as the signal transduction pathways engaged downstream of the T-cell receptor and SLAM molecules.

Next-generation sequencing technologies: An overview.

Since the days of Sanger sequencing, next-generation sequencing technologies have significantly evolved to provide increased data output, efficiencies, and applications. These next generations of technologies can be categorized based on read length. This review provides an overview of these technologies as two paradigms: short-read, or "second-generation," technologies, and long-read, or "third-generation," technologies. Herein, short-read sequencing approaches are represented by the most prevalent technologies, Illumina and Ion Torrent, and long-read sequencing approaches are represented by Pacific Biosciences and Oxford Nanopore technologies. All technologies are reviewed along with reported advantages and disadvantages. Until recently, short-read sequencing was thought to provide high accuracy limited by read-length, while long-read technologies afforded much longer read-lengths at the expense of accuracy. Emerging developments for third-generation technologies hold promise for the next wave of sequencing evolution, with the co-existence of longer read lengths and high accuracy.

Discovery and Optimization of Highly Potent and Selective AT2R Antagonists to Relieve Peripheral Neuropathic Pain.

The angiotensin II type 2 receptor (AT2R) has attracted much attention as a potential target for the relief of neuropathic pain, which represents an area of unmet clinical need. A series of 1,2,3,4-tetrahydroisoquinolines with a benzoxazole side-chain were discovered as potent AT2R antagonists. Rational optimization resulted in compound 15, which demonstrated both excellent antagonistic activity against AT2R in vitro and analgesic efficacy in a rat chronic constriction injury model. Its favorable physicochemical properties and oral bioavailability make it a promising therapeutic candidate for neuropathic pain.

Complex Linkage Disequilibrium Effects in HLA-DPB1 Expression and Molecular Mismatch Analyses of Transplantation Outcomes.

BACKGROUND: HLA molecular mismatch (MM) is a risk factor for de novo donor-specific antibody (dnDSA) development in solid organ transplantation. HLA expression differences have also been associated with adverse outcomes in hematopoietic cell transplantation. We sought to study both MM and expression in assessing dnDSA risk. METHODS: One hundred three HLA-DP-mismatched solid organ transplantation pairs were retrospectively analyzed. MM was computed using amino acids (aa), eplets, and, supplementarily, Grantham/Epstein scores. DPB1 alleles were classified as rs9277534-A (low-expression) or rs9277534-G (high-expression) linked. To determine the associations between risk factors and dnDSA, logistic regression, linkage disequilibrium (LD), and population-based analyses were performed. RESULTS: A high-risk AA:GX (recipient:donor) expression combination (X = A or G) demonstrated strong association with HLA-DP dnDSA (P = 0.001). MM was also associated with HLA-DP dnDSA when evaluated by itself (eplet P = 0.007, aa P = 0.003, Grantham P = 0.005, Epstein P = 0.004). When attempting to determine the relative individual effects of the risk factors in multivariable analysis, only AA:GX expression status retained a strong association (relative risk = 18.6, P = 0.007 with eplet; relative risk = 15.8, P = 0.02 with aa), while MM was no longer significant (eplet P = 0.56, aa P = 0.51). Importantly, these risk factors are correlated, due to LD between the expression-tagging single-nucleotide polymorphism and polymorphisms along HLA-DPB1. CONCLUSIONS: The MM and expression risk factors each appear to be strong predictors of HLA-DP dnDSA and to possess clinical utility; however, these two risk factors are closely correlated. These metrics may represent distinct ways of characterizing a common overlapping dnDSA risk profile, but they are not independent. Further, we demonstrate the importance and detailed implications of LD effects in dnDSA risk assessment and possibly transplantation overall.

Synthesis and structure-activity correlation of natural-product inspired cyclodepsipeptides stabilizing F-actin.

The fundamental role played by actin in the regulation of eukaryotic cell maintenance and motility renders it a primary target for small-molecule intervention. In this arena, a class of potent cytotoxic cyclodepsipeptide natural products has emerged over the last quarter-century to stimulate the fields of biology and chemistry with their unique actin-stabilizing properties and complex peptide-polyketide hybrid structures. Despite considerable research effort, a structural basis for the activity of these secondary metabolites remains elusive, not least for the lack of high-resolution structural data and a reliable synthetic route to diverse compound libraries. In response to this, an efficient solid-phase approach has been developed and successfully applied to the total synthesis of jasplakinolide and chondramide C and diverse analogues. The key macrocylization step was realized using ruthenium-catalyzed ring-closing metathesis (RCM) that in the course of a library synthesis produced discernible trends in metathesis reactivity and E/Z-selectivity. After optimization, the RCM step could be operated under mild conditions, a result that promises to facilitate the synthesis of more extensive analogue libraries for structure-function studies. The growth inhibitory effects of the synthesized compounds were quantified and structure-activity correlations established which appear to be in good alignment with relevant biological data from natural products. In this way a number of potent unnatural and simplified analogues have been found. Furthermore, potentially important stereochemical and structural components of a common pharmacophore have been identified and rationalized using molecular modeling. These data will guide in-depth mode-of-action studies, especially into the relationship between the cytotoxicity of these compounds and their actin-perturbing properties, and should inform the future design of simplified and functionalized actin stabilizers as well.

Small Molecule Dysregulation of TEAD Lipidation Induces a Dominant-Negative Inhibition of Hippo Pathway Signaling.

The transcriptional enhanced associate domain (TEAD) family of transcription factors serves as the receptors for the downstream effectors of the Hippo pathway, YAP and TAZ, to upregulate the expression of multiple genes involved in cellular proliferation and survival. Recent work identified TEAD S-palmitoylation as critical for protein stability and activity as the lipid tail extends into a hydrophobic core of the protein. Here, we report the identification and characterization of a potent small molecule that binds the TEAD lipid pocket (LP) and disrupts TEAD S-palmitoylation. Using a variety of biochemical, structural, and cellular methods, we uncover that TEAD S-palmitoylation functions as a TEAD homeostatic protein level checkpoint and that dysregulation of this lipidation affects TEAD transcriptional activity in a dominant-negative manner. Furthermore, we demonstrate that targeting the TEAD LP is a promising therapeutic strategy for modulating the Hippo pathway, showing tumor stasis in a mouse xenograft model.

Solid-phase based total synthesis of Jasplakinolide by ring-closing metathesis.

The study of classical ring-closing metathesis and relay ring-closing metathesis in a total synthesis of Jasplakinolide and its desbromo analog is described.