Discovery of the First-in-Class Inhibitors of Hypoxia Up-Regulated Protein†1 (HYOU1) Suppressing Pathogenic Fibroblast Activation.

Fibroblasts are key regulators of inflammation, fibrosis, and cancer. Targeting their activation in these complex diseases has emerged as a novel strategy to restore tissue homeostasis. Here, we present a multidisciplinary lead discovery approach to identify and optimize small molecule inhibitors of pathogenic fibroblast activation. The study encompasses medicinal chemistry, molecular phenotyping assays, chemoproteomics, bulk RNA-sequencing analysis, target validation experiments, and chemical absorption, distribution, metabolism, excretion and toxicity (ADMET)/pharmacokinetic (PK)/in vivo evaluation. The parallel synthesis employed for the production of the new benzamide derivatives enabled us to a) pinpoint key structural elements of the scaffold that provide potent fibroblast-deactivating effects in cells, b) discriminate atoms or groups that favor or disfavor a desirable ADMET profile, and c) identify metabolic "hot spots". Furthermore, we report the discovery of the first-in-class inhibitor leads for hypoxia up-regulated protein 1 (HYOU1), a member of the heat shock protein 70 (HSP70) family often associated with cellular stress responses, particularly under hypoxic conditions. Targeting HYOU1 may therefore represent a potentially novel strategy to modulate fibroblast activation and treat chronic inflammatory and fibrotic disorders.

Intrahepatic Cholangiolitis in Cystic Fibrosis (ICCF): An Under-Appreciated Cause of Persistent Cholestasis in Infancy.

Liver histology in infants with cystic fibrosis (CF) and persistent cholestasis is seldom reported in detail. We extend previous observation of a distinctive intrahepatic cholangiopathy (ICCF) to 3 additional infants homozygous for CFTR pathological variants and a fourth infant with a heterozygous CFTR variant, summarizing our experience in 10 infants with CFTR variants and persistent cholestasis. Cholangiograms demonstrate abnormal extrahepatic ducts in 2 infants with CF, 1 with uniform dilatation interpreted as a choledochal cyst and the other with narrow patent ducts. Liver histology in 3 CF homozygotes had prominent ductular reaction with a focally destructive cholangiolitis (inflammation of small bile ducts). The CFTR heterozygote had generalized portal edema with ductular reaction and paucity but no cholangitis. Cholestasis slowly subsided in all infants. ICCF is characterized by severe ductular reaction, prominent cholangiocyte injury, and multifocal necrotizing cholangiolitis. Local aggregates of portal ceroid might suggest previous bile leakage from damaged ducts. ICCF in liver biopsies from infants with cystic fibrosis and persistent cholestasis is unrelated to the specific CFTR genotype. Liver biopsy findings and intraoperative cholangiogram help rule out biliary atresia. ICCF is an early manifestation of CF, a likely prototype for pathogenesis of cystic fibrosis liver disease later in life.

Discovery of Acylsulfonohydrazide-Derived Inhibitors of the Lysine Acetyltransferase, KAT6A, as Potent Senescence-Inducing Anti-Cancer Agents.

A high-throughput screen designed to discover new inhibitors of histone acetyltransferase KAT6A uncovered CTX-0124143 (1), a unique aryl acylsulfonohydrazide with an IC50 of 1.0 µM. Using this acylsulfonohydrazide as a template, we herein disclose the results of our extensive structure-activity relationship investigations, which resulted in the discovery of advanced compounds such as 55 and 80. These two compounds represent significant improvements on our recently reported prototypical lead WM-8014 (3) as they are not only equivalently potent as inhibitors of KAT6A but are less lipophilic and significantly more stable to microsomal degradation. Furthermore, during this process, we discovered a distinct structural subclass that contains key 2-fluorobenzenesulfonyl and phenylpyridine motifs, culminating in the discovery of WM-1119 (4). This compound is a highly potent KAT6A inhibitor (IC50 = 6.3 nM; KD = 0.002 µM), competes with Ac-CoA by binding to the Ac-CoA binding site, and has an oral bioavailability of 56% in rats.

Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells.

Monogenic disorders of the blood system have the potential to be treated by autologous stem cell transplantation of ex vivo genetically modified hematopoietic stem and progenitor cells (HSPCs). The sgRNA/Cas9 system allows for precise modification of the genome at single nucleotide resolution. However, the system is reliant on endogenous cellular DNA repair mechanisms to mend a Cas9-induced double stranded break (DSB), either by the non-homologous end joining (NHEJ) pathway or by the cell-cycle regulated homology-directed repair (HDR) pathway. Here, we describe a panel of ectopically expressed DNA repair factors and Cas9 variants assessed for their ability to promote gene correction by HDR or inhibit gene disruption by NHEJ at the HBB locus. Although transient global overexpression of DNA repair factors did not improve the frequency of gene correction in primary HSPCs, localization of factors to the DSB by fusion to the Cas9 protein did alter repair outcomes toward microhomology-mediated end joining (MMEJ) repair, an HDR event. This strategy may be useful when predictable gene editing outcomes are imperative for therapeutic success.

Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates.

Site-specific correction of a point mutation causing a monogenic disease in autologous hematopoietic stem and progenitor cells (HSPCs) can be used as a treatment of inherited disorders of the blood cells. Sickle cell disease (SCD) is an ideal model to investigate the potential use of gene editing to transvert a single point mutation at the ß-globin locus (HBB). We compared the activity of zinc-finger nucleases (ZFNs) and CRISPR/Cas9 for editing, and homologous donor templates delivered as single-stranded oligodeoxynucleotides (ssODNs), adeno-associated virus serotype 6 (AAV6), integrase-deficient lentiviral vectors (IDLVs), and adenovirus 5/35 serotype (Ad5/35) to transvert the base pair responsible for SCD in HBB in primary human CD34+ HSPCs. We found that the ZFNs and Cas9 directed similar frequencies of nuclease activity. In vitro, AAV6 led to the highest frequencies of homology-directed repair (HDR), but levels of base pair transversions were significantly reduced when analyzing cells in vivo in immunodeficient mouse xenografts, with similar frequencies achieved with either AAV6 or ssODNs. AAV6 also caused significant impairment of colony-forming progenitors and human cell engraftment. Gene correction in engrafting hematopoietic stem cells may be limited by the capacity of the cells to mediate HDR, suggesting additional manipulations may be needed for high-efficiency gene correction in HSPCs.

Guidelines and checklists for short-term missions in global pediatric surgery: Recommendations from the American Academy of Pediatrics Delivery of Surgical Care Global Health Subcommittee, American Pediatric Surgical Association Global Pediatric Surgery Committee, Society for Pediatric Anesthesia Committee on International Education and Service, and American Pediatric Surgical Nurses Association, Inc. Global Health Special Interest Group.

Pediatric surgeons, anesthesia providers, and nurses from North America and other high-income countries are increasingly engaged in resource-limited areas, with short-term missions as the most common form of involvement. However, consensus recommendations currently do not exist for short-term missions in pediatric general surgery and associated perioperative care. The American Academy of Pediatrics (AAP) Delivery of Surgical Care Subcommittee and American Pediatric Surgical Association (APSA) Global Pediatric Surgery Committee, with the American Pediatric Surgical Nurses Association, Inc. (APSNA) Global Health Special Interest Group, and the Society for Pediatric Anesthesia (SPA) Committee on International Education and Service generated consensus recommendations for short-term missions based on extensive experience with short-term missions. Three distinct, but related areas were identified: (i) Broad goals of surgical partnerships between high-income countries and low- and middle-income countries. A previous set of guidelines published by the Global Paediatric Surgery Network Collaborative (GPSN) was endorsed by all groups; (ii) Guidelines for the conduct of short-term missions were developed, including planning, in-country perioperative patient care, post-trip follow-up, and sustainability; and (iii) travel and safety considerations critical to short-term mission success were enumerated. A diverse group of stakeholders developed these guidelines for short-term missions in low- and middle-income countries. These guidelines may be a useful tool to ensure safe, responsible, and ethical short-term missions given increasing engagement of high-income country providers in this work.

Guidelines and checklists for short-term missions in global pediatric surgery: Recommendations from the American Academy of Pediatrics Delivery of Surgical Care Global Health Subcommittee, American Pediatric Surgical Association Global Pediatric Surgery Committee, Society for Pediatric Anesthesia Committee on International Education and Service, and American Pediatric Surgical Nurses Association, Inc. Global Health Special Interest Group.

INTRODUCTION: Pediatric surgeons, anesthesia providers, and nurses from North America and other high-income countries (HICs) are increasingly engaged in resource-limited areas, with short-term missions (STMs) as the most common form of involvement. However, consensus recommendations currently do not exist for STMs in pediatric general surgery and associated perioperative care. METHODS: The American Academy of Pediatrics (AAP) Delivery of Surgical Care Subcommittee and American Pediatric Surgical Association (APSA) Global Pediatric Surgery Committee, with the American Pediatric Surgical Nurses Association, Inc. (APSNA) Global Health Special Interest Group, and the Society for Pediatric Anesthesia (SPA) Committee on International Education and Service generated consensus recommendations for STMs based on extensive experience with STMs. RESULTS: Three distinct, but related areas were identified: 1) Broad goals of surgical partnerships between HICs- and low and middle-income countries (LMICs). A previous set of guidelines published by the Global Paediatric Surgery Network Collaborative (GPSN), was endorsed by all groups; 2) Guidelines for the conduct of STMs were developed, including planning, in-country perioperative patient care, post-trip follow-up, and sustainability; 3) travel and safety considerations critical to STM success were enumerated. CONCLUSION: A diverse group of stakeholders developed these guidelines for STMs in LMICs. These guidelines may be a useful tool to ensure safe, responsible, and ethical STMs given increasing engagement of HIC providers in this work. LEVEL OF EVIDENCE: 5.

Two new secoiridoids from Chelonanthus alatus (Aubl) Pulle (Gentianaceae) / Dos nuevos secoiridoides de Chelonanthus alatus (Aubl) Pulle (Gentianaceae)

The species Chelananthus alatus is an herbaceous plant with known ethno botanical and medicinal properties used in control of fever, especially those produced by malaria. From dried leaves (1.11 Kg), the crude alcoholic extract was fractionated by liquid-liquid partition with different polarity solvents. From the sec-butyl alcohol soluble fraction, by successive application of chromatographic methods, four compounds type iridoid were isolated and identified by spectroscopic techniques. Compound 1 is a new secoiridoid which was identified as sweroside 7-isobutyryloxy, and it is reported here for the first time in the Gentianaceae family; the other secoiridoids which were isolated are known as vogeloside (2), dihydro-chelonanthoside (3) and sweroside (4); vogeloside was identified for the first time in this plant (C. alatus). From the isopropyl acetate extract, in conjunction with the sweroside 7- isobutyryloxy (1), chelonanthoside (5) and sweroside (4), were identified, along with the sweroside 7-isovaleryloxy-(6) as a new side chain isomeric ester of dihydrochelonanthoside (3) . This work presents the spectroscopic analysis of the new structures and some bioactivity data.

La especie Chelonanthus alatus (Gentianaceae) es una hierba de aplicaciones ethnobotánicas reconocidas en medicina tradicional, especialmente en el control de la fiebre producida por la malaria. De las hojas secas (1,11 Kg) se realizó el extracto crudo en alcohol etílico, el cual se fraccionó por partición líquido-líquido (L-L) con disolventes de diferente polaridad. De la fracción soluble en alcohol sec-butílico, se aislaron cuatro compuestos tipo seco-iridoide por aplicación sucesiva de diversos métodos cromatográficos los cuales se identificaron por técnicas espectroscópicas. El compuesto 1 es un nuevo secoiridoide identificado como de 7- isobutiriloxi-swerosido, y se reporta por primera vez en la familia Gentianaceae; los otros tres secoiridoides aislados se conocen como vogelósido (2), dihidrochelonanthosido (3) y swerósido (4); el vogelósido se identificó por primera vez en C. alatus. De la fracción soluble en acetato de isopropilo además del 7-isobutiriloxi-swerosido (1) y el swerosido se aislaron e identificaron, el chelonanthosido (5) y el isovaleriloxi-swerosido (6), el cual es un nuevo isómero del dihidrochelonanthosido. En este trabajo se presenta el análisis espectroscópico que llevó a la elucidación estructural de los compuestos novedosos y algunos datos de bioactividad.

Pseudoaneurysm associated with multiple osteochondromatosis.

The authors present a case of a 12-year-old boy with a known family history of multiple hereditary exostoses who presented with knee swelling. Physical examination and subsequent workup revealed a pseudoaneurysm associated with an osteochondroma. We present this interesting case and review the literature.

Actinomycosis imitating an adrenal tumor.

Abdominal actinomycosis is a rare condition that may mimic malignant disease. The authors report a case of adrenal actinomycosis discovered incidentally by computed tomography scan of the abdomen. Frozen sections and culture of the mass were consistent with actinomycosis. The diagnosis and management of actinomycosis is discussed in detail.

The application of vascular technology to esophageal and airway strictures.

BACKGROUND/PURPOSE: Strictures of the esophagus and airway tract can be dilated if the strictures can be traversed and dilators passed. Unfortunately, using standard methods, not all strictures can be traversed. The authors set out to find a safe, expeditious, and reproducible way to traverse otherwise impassable strictures of the esophagus and airway. METHODS: Eight patients (n = 8), over a 2-year period, with strictures were entered prospectively into the study. One patient (n = 1) had a main stem bronchial stricture, and 7 patients (n = 7) had esophageal strictures from the following etiologies: esophageal atresia/tracheoesophageal fistula (EA/TEF) repair, Lye ingestion (n = 2), EA/TEF with gastroesophageal reflux, esophageal atresia without fistula, lye ingestion with colon interposition (n = 2), and iron pill inhalation lodged in left main bronchus. None of the strictures could be passed with conventional maneuvers or instrumentation including endoscopy, guide wires, Fogarty catheters, and filliform and followers. Results of barium studies showed no flow into the stomach. In the bronchial case, no lumen could be identified at bronchoscopy. RESULTS: Utilizing the "Vascular Surgery Glidewire/Berenstein Catheter System" under fluoroscopy and utilizing the "spinning top" dynamic maneuver intrinsic to this system, all of the strictures were traversed easily. The passage of the wire/catheter system thus allowed sequential dilation of the previously impassable strictures. The mean time to cross the strictures with the wire/catheter system was 1 minute, 10 seconds. (t = 70 seconds). All of the procedures were done in the operating room under general endotracheal anesthesia by the same 2 attending pediatric surgeons. CONCLUSIONS: The use of vascular surgical technology in difficult, otherwise impassable strictures of the esophagus and upper airway proved to be an extremely effective, easy-to-perform, and reproducible method of therapy. This procedure may obviate the need for resectional surgery in this setting.