A Versatile Isocyanate-Mediated Strategy for Appending Chemical Tags onto Drug-Like Small Molecules.

Target identification studies are a major hurdle in probe and drug discovery pipelines due to the need to chemically modify small molecules of interest, which can be time intensive and have low throughput. Here, we describe a versatile and scalable method for attaching chemical moieties to a small molecule, isocyanate-mediated chemical tagging (IMCT). By preparation of a template resin with an isocyanate capture group and a cleavable linker, nucleophilic groups on small molecules can be modified with an enforced one-to-one stoichiometry. We demonstrate a small molecule substrate scope that includes primary and secondary amines, thiols, phenols, benzyl alcohols, and primary alcohols. Cheminformatic analyses predict that IMCT is reactive with more than 25% of lead-like compounds in publicly available databases. To demonstrate that the method can produce biologically active molecules, we generated FKBP12 photoaffinity labeling (PAL) compounds with a wide range of affinities and showed that purified and crude cleavage products can bind to and label FKBP12. This method could be used to rapidly modify small molecules for many applications, including the synthesis of PAL probes, fluorescence polarization probes, pull-down probes, and degraders.

Fifteen years of shared care for paediatric oncology, haematology and palliative patients across Queensland: The role of Regional Case Managers.

OBJECTIVE: A shared care model was implemented in 2006 in Queensland to facilitate paediatric oncology, haematology and palliative care patients receiving care as close to home as possible. Following initial diagnosis, care planning and treatment at the tertiary children's hospital, appropriate local care was coordinated by Regional Case Managers (RCMs) established at each of 10 Shared Care Units (SCUs). This enabled safe and quality regional care supported by a statewide network providing clinical governance and education. This paper examines learnings from 15 years of this shared care. SETTING: Ten hospitals throughout Queensland facilitated a statewide model of shared care for paediatric oncology, haematology and palliative care patients, supported by a tertiary hub in Brisbane. PARTICIPANTS: Regional Case Managers in Shared Care Units and their supporting staff. DESIGN: Staff from SCUs were surveyed and focus group interviews conducted. RESULTS: The paper reviews the attributes, knowledge and experience required for RCMs. Standards of care were supported through education workshops, clinical placements, chemotherapy credentialing, guidelines and standards. RCMs facilitated communication and information sharing with the tertiary centre, advocated for their cohort of patients locally and streamlined and supported the family's experience of care. CONCLUSION: The RCM role provided invaluable clinical leadership for the care of paediatric oncology, haematology and palliative patients across Queensland. As new treatments evolve, the expertise and coordination provided by the RCMs will be even more critical. Achieving high-quality shared care outcomes is underpinned by the RCMs drive to achieve statewide safety and support for this cohort of children.

Pseudodicentric Chromosome Originating from an X-Autosome Translocation in a Male Patient with Cryptozoospermia.

Genetic factors are responsible for 15% of male infertility conditions. Numerical and structural chromosomal anomalies are validated genetic factors leading to spermatogenic quantitative defects, with a frequency depending on the severity of the phenotype. Among the structural chromosomal rearrangements, dicentric chromosomes are generally observed in robertsonian translocations or in cases of Y chromosome isodicentrics. In X-autosome translocations, male carriers are generally infertile, regardless of the position of the breakpoint, due to interrupted spermatogenesis. We report an infertile man bearing an unusual balanced (X;22) translocation, with a centromeric X breakpoint generating a derivative pseudodicentric chromosome psu dic(22;X). Extensive cytogenetic analyses were necessary to determine the precise nature of the derivative chromosome. The likely cause of the reproductive phenotype of the patient is discussed based on meiotic chromosomal conformation.

Embedding of HIV Egress within Cortical F-Actin.

F-Actin remodeling is important for the spread of HIV via cell-cell contacts; however, the mechanisms by which HIV corrupts the actin cytoskeleton are poorly understood. Through live cell imaging and focused ion beam scanning electron microscopy (FIB-SEM), we observed F-Actin structures that exhibit strong positive curvature to be enriched for HIV buds. Virion proteomics, gene silencing, and viral mutagenesis supported a Cdc42-IQGAP1-Arp2/3 pathway as the primary intersection of HIV budding, membrane curvature and F-Actin regulation. Whilst HIV egress activated the Cdc42-Arp2/3 filopodial pathway, this came at the expense of cell-free viral release. Importantly, release could be rescued by cell-cell contact, provided Cdc42 and IQGAP1 were present. From these observations, we conclude that a proportion out-going HIV has corrupted a central F-Actin node that enables initial coupling of HIV buds to cortical F-Actin to place HIV at the leading cell edge. Whilst this initially prevents particle release, the maturation of cell-cell contacts signals back to this F-Actin node to enable viral release & subsequent infection of the contacting cell.

Therapeutic approach and outcome of children with Philadelphia chromosome-positive acute lymphoblastic leukemia at first relapse in the era of tyrosine kinase inhibitors: An SFCE retrospective study.

BACKGROUND: Since the introduction of tyrosine kinase inhibitors (TKIs), the profile of pediatric relapse of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) has changed. However, the management of pediatric Ph+ ALL relapses is not currently standardized. PROCEDURE: We retrospectively analyzed the therapeutic strategies and outcomes of pediatric Ph+ ALL patients in first relapse who were initially treated with a TKI-containing regimen in one of the French pediatric hematology centers from 2004 to 2019. RESULTS: Twenty-seven children experienced a Ph+ ALL relapse: 24 (89%) had an overt relapse and three a molecular relapse. Eight involved the central nervous system. A second complete remission (CR2) was obtained for 26 patients (96%). Induction consisted of nonintensive chemotherapy for 13 patients (48%) and intensive chemotherapy for 14 (52%). Thirteen patients (48%) received consolidation. Allogenic hematopoietic stem cell transplantation (alloHSCT) was performed for 21 patients (78%). The TKI was changed for 23 patients (88%), mainly with dasatinib (n = 15). T315I was the most common mutation at relapse (4/7). The 4-year event-free survival and survival rates were 60.9% and 76.1%, respectively. Survival was positively associated with alloHSCT in CR2. CONCLUSION: We show that pediatric first-relapse Ph+ ALL reinduces well with a second course of TKI exposure, despite the use of different therapeutic approaches. The main prognostic factor for survival was alloHSCT in CR2. Because of the small size of the cohort, we could not draw any conclusions about the respective impact of TKIs, but the predominance of the T315I mutation should encourage careful consideration of the TKI choice.

Spontaneous renal artery dissection: an elusive diagnosis.

Spontaneous renal artery dissection is a rare condition with an often non-specific presentation, resulting in a challenging diagnosis for clinicians. This is the case of a 39-year-old man who presented with an acute-onset right flank pain, mild neutrophilia and sterile urine. CT of abdomen and pelvis showed a patchy hypodense area in the right kidney originally thought to represent infection. He was treated as an atypical pyelonephritis with antibiotics and fluids. When his symptoms failed to improve, a diagnosis of renal infarction was considered and CT angiogram of the aorta revealed a spontaneous renal artery dissection. He was managed conservatively with systemic anticoagulation, antihypertensive treatment and analgesia and discharged home with resolution of his symptoms and normal renal function.

Multicolor-FISH Characterization of a Prenatal Mosaicism for a Chromosomal Rearrangement Undetected by Molecular Cytogenetics.

Fetal mosaicism for chromosomal rearrangements remains a challenge to diagnose, even in the era of whole-genome sequencing. We present here a case of fetal mosaicism for a chromosomal rearrangement explored in amniocytes and fetal muscle, consisting of a major cell population (95%) with partial monosomy 4q and a minor population (5%) with additional material replacing the 4qter deleted segment. Molecular techniques (MLPA, array-CGH) failed to assess the origin of this material. Only multicolor-FISH identified the additional segment on chromosome 4 as derived from chromosome 17. Due to the poor prognosis, the couple chose to terminate the pregnancy. Because of low-level mosaicism, chromosomal microarray analysis (CMA), now considered as first-tier prenatal genetic analysis, did not allow the identification of the minor cell line. In case of large CNVs (>5 Mb) detected by CMA, karyotyping may be considered to elucidate the mechanism of the underlying rearrangement and eliminate mosaicism.

New insights into the genetic basis of premature ovarian insufficiency: Novel causative variants and candidate genes revealed by genomic sequencing.

Ovarian deficiency, including premature ovarian insufficiency (POI) and diminished ovarian reserve (DOR), represents one of the main causes of female infertility. POI is a genetically heterogeneous condition but current understanding of its genetic basis is far from complete, with the cause remaining unknown in the majority of patients. The genes that regulate DOR have been reported but the genetic basis of DOR has not been explored in depth. Both conditions are likely to lie along a continuum of degrees of decrease in ovarian reserve. We performed genomic analysis via whole exome sequencing (WES) followed by in silico analyses and functional experiments to investigate the genetic cause of ovarian deficiency in ten affected women. We achieved diagnoses for three of them, including the identification of novel variants in STAG3, GDF9, and FANCM. We identified potentially causative FSHR variants in another patient. This is the second report of biallelic GDF9 and FANCM variants, and, combined with functional support, validates these genes as bone fide autosomal recessive "POI genes". We also identified new candidate genes, NRIP1, XPO1, and MACF1. These genes have been linked to ovarian function in mouse, pig, and zebrafish respectively, but never in humans. In the case of NRIP1, we provide functional support for the deleterious nature of the variant via SUMOylation and luciferase/ß-galactosidase reporter assays. Our study provides multiple insights into the genetic basis of POI/DOR. We have further elucidated the involvement of GDF9, FANCM, STAG3 and FSHR in POI pathogenesis, and propose new candidate genes, NRIP1, XPO1, and MACF1, which should be the focus of future studies.

Pan-HDAC Inhibitors Restore PRDM1 Response to IL21 in CREBBP-Mutated Follicular Lymphoma.

PURPOSE: Follicular lymphoma arises from a germinal center B-cell proliferation supported by a bidirectional crosstalk with tumor microenvironment, in particular with follicular helper T cells (Tfh). We explored the relation that exists between the differentiation arrest of follicular lymphoma cells and loss-of-function of CREBBP acetyltransferase.Experimental Design: The study used human primary cells obtained from either follicular lymphoma tumors characterized for somatic mutations, or inflamed tonsils for normal germinal center B cells. Transcriptome and functional analyses were done to decipher the B- and T-cell crosstalk. Responses were assessed by flow cytometry and molecular biology including ChIP-qPCR approaches. RESULTS: Conversely to normal B cells, follicular lymphoma cells are unable to upregulate the transcription repressor, PRDM1, required for plasma cell differentiation. This defect occurs although the follicular lymphoma microenvironment is enriched in the potent inducer of PRDM1 and IL21, highly produced by Tfhs. In follicular lymphoma carrying CREBBP loss-of-function mutations, we found a lack of IL21-mediated PRDM1 response associated with an abnormal increased enrichment of the BCL6 protein repressor in PRDM1 gene. Moreover, in these follicular lymphoma cells, pan-HDAC inhibitor, vorinostat, restored their PRDM1 response to IL21 by lowering BCL6 bound to PRDM1. This finding was reinforced by our exploration of patients with follicular lymphoma treated with another pan-HDAC inhibitor. Patients showed an increase of plasma cell identity genes, mainly PRDM1 and XBP1, which underline the progression of follicular lymphoma B cells in the differentiation process. CONCLUSIONS: Our data uncover a new mechanism by which pan-HDAC inhibitors may act positively to treat patients with follicular lymphoma through the induction of the expression of plasma cell genes.

Using hydrodynamic focusing to predictably alter the diameter of synthetic silk fibers.

Spiders and silkworms provide a model of superior processing for multifunctional and highly versatile high-performance fibers. Mimicking the spider's complex control system for chemical and mechanical gradients has remained an ongoing obstacle for synthetic silk production. In this study, the use of hydrodynamic fluid focusing within a 3D printed biomimetic spinning system to recapitulate the biological spinneret is explored and shown to produce predictable, small diameter fibers. Mirroring in silico fluid flow simulations using a hydrodynamic microfluidic spinning technique, we have developed a model correlating spinning rates, solution viscosity and fiber diameter outputs that will significantly advance the field of synthetic silk fiber production. The use of hydrodynamic focusing to produce controlled output fiber diameter simulates the natural silk spinning process and continues to build upon a 3D printed biomimetic spinning platform.

Influence of the d/l configuration of N-acyl-homoserine lactones (AHLs) and analogues on their Lux-R dependent quorum sensing activity.

Whereas l-3-oxo-hexanoyl homoserine lactone (OHHL) is the active enantiomer of the of LuxR-regulated quorum sensing (QS) autoinducer, its d isomer is implicitly considered as inactive. The present work aims to clarify this l-specificity and investigate whether it extends to some analogues in the acyl homoserine lactone (AHL) family. For this purpose, OHHL and a series of AHL analogs were synthesized in racemic and enantiomerically pure d and l forms and their ability to induce or attenuate bioluminescence in the LuxR-dependent QS system was evaluated. In this study, l-isomers are confirmed as either the only, or as the most active, enantiomers. However, in several cases, especially for the natural ligand of LuxR (OHHL) and the very similar AHL agonist analogue 2, the d-isomer cannot be considered as totally inactive on QS. Molecular modelling suggests that when the lactone moiety of the d-isomer is able to twist, enabling the lactone carbonyl group and the amide function to interact with the key residues in the binding site, then the d-isomer can exhibit some activity.

Spatial and age-related changes in the microstructure of dystrophic and healthy diaphragms.

Duchenne muscular dystrophy (DMD) is a progressive degenerative disease that results in fibrosis and atrophy of muscles. The main cause of death associated with DMD is failure of the diaphragm. The diaphragm is a dome-shaped muscle with a fiber microstructure that differs across regions of the muscle. However, no studies to our knowledge have examined spatial variations of muscle fibers in dystrophic diaphragm or how aging affects those variations in DMD. In this study, diaphragms were obtained from mdx and healthy mice at ages three, seven, and ten months in the dorsal, midcostal, and ventral regions. Through immunostaining and confocal imaging, we quantified sarcomere length, interstitial space between fibers, fiber branching, fiber cross sectional area (CSA), and fiber regeneration measured by centrally located nuclei. Because DMD is associated with chronic inflammation, we also investigated the number of macrophages in diaphragm muscle cross-sections. We saw regional differences in the number of regenerating fibers and macrophages during the progression of DMD in the mdx diaphragm. Additionally, the number of regenerating fibers increased with age, while CSA and the number of branching fibers decreased. Dystrophic diaphragms had shorter sarcomere lengths than age-matched controls. Our results suggest that the dystrophic diaphragm in the mdx mouse is structurally heterogeneous and remodels non-uniformly over time. Understanding regional changes in dystrophic diaphragms over time will facilitate the development of targeted therapies to prevent or minimize respiratory failure in DMD patients.

Socioeconomic Inequalities and Severity of Plaque Psoriasis at a First Consultation in Dermatology Centers.

Psoriasis has major physical, psychological, and social impacts: its management should not be restricted by individual financial considerations in Western countries as these have well-structured health systems and social/insurance coverage. We investigated if the socioeconomic characteristics of patients were associated with severity of psoriasis and access to healthcare. In a cross-sectional study, we included 903 patients with psoriasis that were consulting for the first time. We showed that low educational level was associated with severity of disease in multivariate analyses. Moreover, patients of lower class and lower educational level, with severe psoriasis, had seen fewer physicians and had less frequently received a systemic treatment. Thus, physicians need to be vigilante of patients with a low socioeconomic status. Both low socioeconomic status and less access to dermatologists are associated with clinical severity of psoriasis at a first consultation.

Preclinical Assessment of Safety and Efficacy of Fluorescent Dye for Detecting Dermal Injuries (the injuries were both abrasions and incision) in a Murine Model.

Objective documentation of tears and abrasions to the external genitalia after sexual assault is an important part of the forensic examination. A 1% aqueous solution of toluidine blue (TB) dye is often used to highlight injury, but the dye can be difficult to see on dark skin. We evaluated a fluorescent dye for detecting injuries on all skin colors in a relevant preclinical murine model. We compared the ability of trained, blinded observers to detect unstained wounds and wounds stained with 1% fluorescein sodium (FL) and with TB. We also evaluated the cutaneous wound healing response after application of both dyes. The no-dye group had significantly fewer (p < 0.05) true-positive identifications compared to all the dye groups. No differences in effectiveness were detected between dye treatments. All groups exhibited statistically similar healing rates. These findings support the evaluation of fluorescein sodium in a future clinical study with human subjects.

Clinical and molecular delineation of Tetrasomy 9p syndrome: report of 12 new cases and literature review.

Tetrasomy 9p is a generic term describing the presence of a supernumerary chromosome incorporating two copies of the 9p arm. Two varieties exist: isodicentric chromosome 9p (i(9p)), where the two 9p arms are linked by a single centromeric region, and pseudodicentric 9p (idic(9p)), where one active and one inactive centromere are linked together by a proximal segment of 9q that may incorporate euchromatic material. In living patients, i(9p) and idic(9p) are usually present in a mosaic state. Fifty-four cases, including fetuses, have been reported, of which only two have been molecularly characterized using array-CGH. Tetrasomy 9p leads to a variable phenotype ranging from multiple congenital anomalies with severe intellectual disability and growth delay to subnormal cognitive and physical developments. Hypertelorism, abnormal ears, microretrognathia and bulbous nose are the most common dysmorphic traits. Microcephaly, growth retardation, joint dislocation, scoliosis, cardiac and renal anomalies were reported in several cases. Those physical anomalies are often, but not universally, accompanied by intellectual disability. The most recurrent breakpoints, defined by conventional cytogenetics, are 9p10, 9q12 and 9q13. We report on 12 new patients with tetrasomy 9p (3 i(9p), 8 idic(9p) and one structurally uncharacterized), including the first case of parental germline mosaicism. All rearrangements have been characterized by DNA microarray. Based on our results and a review of the literature, we further delineate the prenatal and postnatal clinical spectrum of this imbalance. Our results show poor genotype-phenotype correlations and underline the need of precise molecular characterization of the supernumerary marker.

Severe androgenetic alopecia as a proxy of metabolic syndrome in male psoriatic patients older than 59 years.

BACKGROUND: Whereas several studies have underlined the association between severe psoriasis and metabolic syndrome (MetS), the association of androgenetic alopecia (AGA) and MetS have yielded inconsistent results. OBJECTIVE: To investigate the relationship between AGA and the components of MetS in a population of psoriatic male patients. METHODS: A non-interventional, cross-sectional, multicenter study was conducted in France. A standardized questionnaire was completed, including information on components of MetS and other possible risk factors. MetS was defined in this study as a combination of three or more of the four components of MetS: waist circumference, hyperlipidemia, diabetes mellitus and hypertension. In addition, a standardized simplified Norwood classification limited into 5 grades (0-4) was used. RESULTS: In a total of 1073 male patients, hypertension, high waist circumference, diabetes mellitus and hyperlipidemia were observed in 28%, 59%, 11%, and 31%, respectively. In age-adjusted multivariate analysis, severe AGA (grade 3-4 versus grade 0) was associated with the presence of at least one component of MetS. By groups of age, a statistically significant association of severe AGA and MetS was demonstrated in patients over 59 years. Severe AGA was also associated with a first degree familial history of major cardiovascular event in patients older than 59 years. CONCLUSIONS: Our study, based on a simplified but stringent definition of MetS, confirmed the link between severe AGA and individual components of MetS in psoriatic patients. This argues for careful follow-up with regular screening in male psoriatic patients with severe AGA in order to early detect determinants of MetS.

Dynamic estrogen receptor interactomes control estrogen-responsive trefoil Factor (TFF) locus cell-specific activities.

Estradiol signaling is ideally suited for analyzing the molecular and functional linkages between the different layers of information directing transcriptional regulations: the DNA sequence, chromatin modifications, and the spatial organization of the genome. Hence, the estrogen receptor (ER) can bind at a distance from its target genes and engages timely and spatially coordinated processes to regulate their expression. In the context of the coordinated regulation of colinear genes, identifying which ER binding sites (ERBSs) regulate a given gene still remains a challenge. Here, we investigated the coordination of such regulatory events at a 2-Mb genomic locus containing the estrogen-sensitive trefoil factor (TFF) cluster of genes in breast cancer cells. We demonstrate that this locus exhibits a hormone- and cohesin-dependent reduction in the plasticity of its three-dimensional organization that allows multiple ERBSs to be dynamically brought to the vicinity of estrogen-sensitive genes. Additionally, by using triplex-forming oligonucleotides, we could precisely document the functional links between ER engagement at given ERBSs and the regulation of particular genes. Hence, our data provide evidence of a formerly suggested cooperation of enhancers toward gene regulation and also show that redundancy between ERBSs can occur.

Postnatal diagnosis of 9q interstitial imbalances involving PTCH1, resulting from a familial intrachromosomal insertion.

Insertions are rare chromosomal rearrangements resulting from a three breaks mechanism. The risk of chromosomal imbalance in the offspring is estimated to be 15-50%. We have identified a familial history of direct, paracentric intrachromosomal 9q insertion, balanced in healthy members. For intrachromosomal insertions, unbalanced products in the offspring are always recombinants and in our case, reciprocal deletion and duplication of the inserted segment (9q22.31-9q31.1) were observed. These imbalances involved several genes, including PTCH1. PTCH1 haploinsufficiency causes Gorlin syndrome, an autosomal dominant disorder usually linked to the gene mutation but sometimes due to a 9q deletion. Clinical findings are different in 9q deletions and duplications including PTCH1, notably concerning the predisposition to benign and malignant tumors reported in the Gorlin syndrome. Furthermore, some features may be reciprocal. This history of intrachromosomal insertion highlights the importance of morphological cytogenetic analyses to provide an accurate genetic counseling.

MLL-SEPT5 fusion transcript in infant acute myeloid leukemia with t(11;22)(q23;q11).

Chromosomal rearrangements involving the MLL gene at band 11q23 are the most common genetic alteration encountered in infant acute myeloid leukemia. Reciprocal translocation represents the most frequent form of MLL rearrangement. Currently, more than 60 partner genes have been identified. We report here a case of de novo acute myeloid leukemia with a t(11;22)(q23;q11) in a 23-month-old child. Fluorescence in situ hybridization study revealed that the 3'MLL segment was translocated onto the derivative chromosome 22 and the breakpoint on chromosome 22 was located in or near the SEPT5 gene at 22q11.21. Long distance inverse-polymerase chain reaction was used to identify precisely the MLL partner gene and confirmed the MLL-SEPT5 fusion transcript. Involvement of the SEPT5 gene in MLL rearrangement occurs very rarely. Clinical, cytogenetic and molecular features of acute myeloid leukemia with a MLL-SEPT5 fusion gene are reviewed.