Vγ9Vδ2 T cells recognize butyrophilin 2A1 and 3A1 heteromers.

Butyrophilin (BTN) molecules are emerging as key regulators of T cell immunity; however, how they trigger cell-mediated responses is poorly understood. Here, the crystal structure of a gamma-delta T cell antigen receptor (γδTCR) in complex with BTN2A1 revealed that BTN2A1 engages the side of the γδTCR, leaving the apical TCR surface bioavailable. We reveal that a second γδTCR ligand co-engages γδTCR via binding to this accessible apical surface in a BTN3A1-dependent manner. BTN2A1 and BTN3A1 also directly interact with each other in cis, and structural analysis revealed formation of W-shaped heteromeric multimers. This BTN2A1-BTN3A1 interaction involved the same epitopes that BTN2A1 and BTN3A1 each use to mediate the γδTCR interaction; indeed, locking BTN2A1 and BTN3A1 together abrogated their interaction with γδTCR, supporting a model wherein the two γδTCR ligand-binding sites depend on accessibility to cryptic BTN epitopes. Our findings reveal a new paradigm in immune activation, whereby γδTCRs sense dual epitopes on BTN complexes.

Monoallelic intragenic POU3F2 variants lead to neurodevelopmental delay and hyperphagic obesity, confirming the gene's candidacy in 6q16.1 deletions.

While common obesity accounts for an increasing global health burden, its monogenic forms have taught us underlying mechanisms via more than 20 single-gene disorders. Among these, the most common mechanism is central nervous system dysregulation of food intake and satiety, often accompanied by neurodevelopmental delay (NDD) and autism spectrum disorder. In a family with syndromic obesity, we identified a monoallelic truncating variant in POU3F2 (alias BRN2) encoding a neural transcription factor, which has previously been suggested as a driver of obesity and NDD in individuals with the 6q16.1 deletion. In an international collaboration, we identified ultra-rare truncating and missense variants in another ten individuals sharing autism spectrum disorder, NDD, and adolescent-onset obesity. Affected individuals presented with low-to-normal birth weight and infantile feeding difficulties but developed insulin resistance and hyperphagia during childhood. Except for a variant leading to early truncation of the protein, identified variants showed adequate nuclear translocation but overall disturbed DNA-binding ability and promotor activation. In a cohort with common non-syndromic obesity, we independently observed a negative correlation of POU3F2 gene expression with BMI, suggesting a role beyond monogenic obesity. In summary, we propose deleterious intragenic variants of POU3F2 to cause transcriptional dysregulation associated with hyperphagic obesity of adolescent onset with variable NDD.

Genomic Diagnosis of Rare Pediatric Disease in the United Kingdom and Ireland.

BACKGROUND: Pediatric disorders include a range of highly penetrant, genetically heterogeneous conditions amenable to genomewide diagnostic approaches. Finding a molecular diagnosis is challenging but can have profound lifelong benefits. METHODS: We conducted a large-scale sequencing study involving more than 13,500 families with probands with severe, probably monogenic, difficult-to-diagnose developmental disorders from 24 regional genetics services in the United Kingdom and Ireland. Standardized phenotypic data were collected, and exome sequencing and microarray analyses were performed to investigate novel genetic causes. We developed an iterative variant analysis pipeline and reported candidate variants to clinical teams for validation and diagnostic interpretation to inform communication with families. Multiple regression analyses were performed to evaluate factors affecting the probability of diagnosis. RESULTS: A total of 13,449 probands were included in the analyses. On average, we reported 1.0 candidate variant per parent-offspring trio and 2.5 variants per singleton proband. Using clinical and computational approaches to variant classification, we made a diagnosis in approximately 41% of probands (5502 of 13,449). Of 3599 probands in trios who received a diagnosis by clinical assertion, approximately 76% had a pathogenic de novo variant. Another 22% of probands (2997 of 13,449) had variants of uncertain significance in genes that were strongly linked to monogenic developmental disorders. Recruitment in a parent-offspring trio had the largest effect on the probability of diagnosis (odds ratio, 4.70; 95% confidence interval [CI], 4.16 to 5.31). Probands were less likely to receive a diagnosis if they were born extremely prematurely (i.e., 22 to 27 weeks' gestation; odds ratio, 0.39; 95% CI, 0.22 to 0.68), had in utero exposure to antiepileptic medications (odds ratio, 0.44; 95% CI, 0.29 to 0.67), had mothers with diabetes (odds ratio, 0.52; 95% CI, 0.41 to 0.67), or were of African ancestry (odds ratio, 0.51; 95% CI, 0.31 to 0.78). CONCLUSIONS: Among probands with severe, probably monogenic, difficult-to-diagnose developmental disorders, multimodal analysis of genomewide data had good diagnostic power, even after previous attempts at diagnosis. (Funded by the Health Innovation Challenge Fund and Wellcome Sanger Institute.).

Shear-Sensing by C-Reactive Protein: Linking Aortic Stenosis and Inflammation.

BACKGROUND: CRP (C-reactive protein) is a prototypical acute phase reactant. Upon dissociation of the pentameric isoform (pCRP [pentameric CRP]) into its monomeric subunits (mCRP [monomeric CRP]), it exhibits prothrombotic and proinflammatory activity. Pathophysiological shear rates as observed in aortic valve stenosis (AS) can influence protein conformation and function as observed with vWF (von Willebrand factor). Given the proinflammatory function of dissociated CRP and the important role of inflammation in the pathogenesis of AS, we investigated whether shear stress can modify CRP conformation and induce inflammatory effects relevant to AS. METHODS: To determine the effects of pathological shear rates on the function of human CRP, pCRP was subjected to pathophysiologically relevant shear rates and analyzed using biophysical and biochemical methods. To investigate the effect of shear on CRP conformation in vivo, we used a mouse model of arterial stenosis. Levels of mCRP and pCRP were measured in patients with severe AS pre- and post-transcatheter aortic valve implantation, and the presence of CRP was investigated on excised valves from patients undergoing aortic valve replacement surgery for severe AS. Microfluidic models of AS were then used to recapitulate the shear rates of patients with AS and to investigate this shear-dependent dissociation of pCRP and its inflammatory function. RESULTS: Exposed to high shear rates, pCRP dissociates into its proinflammatory monomers (mCRP) and aggregates into large particles. Our in vitro findings were further confirmed in a mouse carotid artery stenosis model, where the administration of human pCRP led to the deposition of mCRP poststenosis. Patients undergoing transcatheter aortic valve implantation demonstrated significantly higher mCRP bound to circulating microvesicles pre-transcatheter aortic valve implantation compared with post-transcatheter aortic valve implantation. Excised human stenotic aortic valves display mCRP deposition. pCRP dissociated in a microfluidic model of AS and induces endothelial cell activation as measured by increased ICAM-1 and P-selectin expression. mCRP also induces platelet activation and TGF-ß (transforming growth factor beta) expression on platelets. CONCLUSIONS: We identify a novel mechanism of shear-induced pCRP dissociation, which results in the activation of cells central to the development of AS. This novel mechanosensing mechanism of pCRP dissociation to mCRP is likely also relevant to other pathologies involving increased shear rates, such as in atherosclerotic and injured arteries.

BTB domain mutations perturbing KCTD15 oligomerisation cause a distinctive frontonasal dysplasia syndrome.

INTRODUCTION: KCTD15 encodes an oligomeric BTB domain protein reported to inhibit neural crest formation through repression of Wnt/beta-catenin signalling, as well as transactivation by TFAP2. Heterozygous missense variants in the closely related paralogue KCTD1 cause scalp-ear-nipple syndrome. METHODS: Exome sequencing was performed on a two-generation family affected by a distinctive phenotype comprising a lipomatous frontonasal malformation, anosmia, cutis aplasia of the scalp and/or sparse hair, and congenital heart disease. Identification of a de novo missense substitution within KCTD15 led to targeted sequencing of DNA from a similarly affected sporadic patient, revealing a different missense mutation. Structural and biophysical analyses were performed to assess the effects of both amino acid substitutions on the KCTD15 protein. RESULTS: A heterozygous c.310G>C variant encoding p.(Asp104His) within the BTB domain of KCTD15 was identified in an affected father and daughter and segregated with the phenotype. In the sporadically affected patient, a de novo heterozygous c.263G>A variant encoding p.(Gly88Asp) was present in KCTD15. Both substitutions were found to perturb the pentameric assembly of the BTB domain. A crystal structure of the BTB domain variant p.(Gly88Asp) revealed a closed hexameric assembly, whereas biophysical analyses showed that the p.(Asp104His) substitution resulted in a monomeric BTB domain likely to be partially unfolded at physiological temperatures. CONCLUSION: BTB domain substitutions in KCTD1 and KCTD15 cause clinically overlapping phenotypes involving craniofacial abnormalities and cutis aplasia. The structural analyses demonstrate that missense substitutions act through a dominant negative mechanism by disrupting the higher order structure of the KCTD15 protein complex.

Messing with ßc: A unique receptor with many goals.

Our understanding of the biological role of the ßc family of cytokines has evolved enormously since their initial identification as bone marrow colony stimulating factors in the 1960's. It has become abundantly clear over the intervening decades that this family of cytokines has truly astonishing pleiotropic capacity, capable of regulating not only hematopoiesis but also many other normal and pathological processes such as development, inflammation, allergy and cancer. As noted in the current pandemic, ßc cytokines contribute to the cytokine storm seen in acutely ill COVID-19 patients. Ongoing studies to discover how these cytokines activate their receptor are revealing insights into the fundamental mechanisms that give rise to cytokine pleiotropy and are providing tantalizing glimpses of how discrete signaling pathways may be dissected for activation with novel ligands for therapeutic benefit.

Mechanism of Bloom syndrome complex assembly required for double Holliday junction dissolution and genome stability.

The RecQ-like helicase BLM cooperates with topoisomerase IIIα, RMI1, and RMI2 in a heterotetrameric complex (the "Bloom syndrome complex") for dissolution of double Holliday junctions, key intermediates in homologous recombination. Mutations in any component of the Bloom syndrome complex can cause genome instability and a highly cancer-prone disorder called Bloom syndrome. Some heterozygous carriers are also predisposed to breast cancer. To understand how the activities of BLM helicase and topoisomerase IIIα are coupled, we purified the active four-subunit complex. Chemical cross-linking and mass spectrometry revealed a unique architecture that links the helicase and topoisomerase domains. Using biochemical experiments, we demonstrated dimerization mediated by the N terminus of BLM with a 2:2:2:2 stoichiometry within the Bloom syndrome complex. We identified mutations that independently abrogate dimerization or association of BLM with RMI1, and we show that both are dysfunctional for dissolution using in vitro assays and cause genome instability and synthetic lethal interactions with GEN1/MUS81 in cells. Truncated BLM can also inhibit the activity of full-length BLM in mixed dimers, suggesting a putative mechanism of dominant-negative action in carriers of BLM truncation alleles. Our results identify critical molecular determinants of Bloom syndrome complex assembly required for double Holliday junction dissolution and maintenance of genome stability.

Proximalization of arterial inflow for treatment of hemodialysis access-induced distal ischemia.

OBJECTIVE: Hemodialysis access-induced distal ischemia (HAIDI) is a known complication of hemodialysis (HD) access. Distal revascularization and interval ligation (DRIL) is the preferred treatment for HAIDI by many surgeons. Proximalization of arterial inflow (PAI) is a promising alternative technique that, unlike DRIL, preserves the native arterial inflow. The purpose of this study is to report our experience with PAI on a series of 64 patients. METHODS: This is a single-center, retrospective cohort study of patients with both arteriovenous (AV) fistulas and grafts who underwent PAI for HAIDI from 2017 to 2023. A 4 × 7 tapered polytetrafluoroethylene (PTFE) graft was used to connect HD access inflow to the axillary artery in the majority of cases. The primary outcome of the study is resolution of HAIDI (complete, partial, or no resolution). Secondary outcomes include functional patency (primary and secondary) and 30-day complications following PAI. RESULTS: Of the 71 patients identified to have had PAI between May 2017 to August 2023, seven were lost to follow-up. In total, 64 patients were included, with an average age of 65 years (standard deviation, 15 years), 59.4% (38/64) female, and 37.5% (24/64) African American. The study population was notable for numerous comorbid conditions including 95.3% (61/64) hypertension; 50% (32/64) coronary artery disease; 79.7% (51/64) diabetes; and 43.8% (28/64) smoking history. Following PAI intervention for HAIDI, 55 of 64 patients (85.9%) experienced complete resolution of ischemic symptoms; five of 64 patients (7.8%) had partial resolution; two of 64 patients (3.1%) had no resolution, and two of 64 patients (3.1%) had unknown resolution. Primary patency at 1, 12, and 24 months was 94%, 81%, and 71%, respectively. Secondary patency at 1, 12, and 24 months was 97%, 87%, and 84%, respectively. The 30-day complication rate was 10.9% (7/64), with five of 64 (7.8%) thromboses, one of 64 (1.6%) thrombosis and infection, and one of 64 (1.6%) upper extremity swelling secondary to central venous stenosis (resolved with angioplasty of central venous system). Failure rate due to thrombosis at 12 and 24 months was 14% (9/64) and 15.6% (10/64), respectively. CONCLUSIONS: Our study, the largest case series of PAI to date, demonstrates that PAI is a reliable option for HAIDI intervention and has comparable safety and efficacy results to DRIL, despite the use of a synthetic graft. Furthermore, PAI has the added benefit of maintaining the native arterial pathway. Further investigation of PAI is warranted as a promising alternative to DRIL for HAIDI management.

Inhibitors of histone acetyltransferases KAT6A/B induce senescence and arrest tumour growth.

Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function1. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF)2,3. KAT6A has essential roles in normal haematopoietic stem cells4-6 and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia7,8. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers8. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus9,10, a function that requires its KAT activity10. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days11. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.

'Something that helped the whole picture': Experiences of parents offered rapid prenatal exome sequencing in routine clinical care in the English National Health Service.

OBJECTIVES: In October 2020, rapid prenatal exome sequencing (pES) was introduced into routine National Health Service (NHS) care in England. This study aimed to explore parent experiences and their information and support needs from the perspective of parents offered pES and of health professionals involved in its delivery. METHODS: In this qualitative study, semi-structured interviews were conducted with 42 women and 6 male partners and 63 fetal medicine and genetic health professionals. Interviews were transcribed verbatim and analysed using thematic analysis. RESULTS: Overall views about pES were positive and parents were grateful to be offered the test. Highlighted benefits of pES included the value of the additional information for pregnancy management and planning for future pregnancies. An anxious wait for results was common, often associated with the need to make decisions near to 24 weeks in pregnancy when there are legal restrictions for late termination. Descriptions of dealing with uncertainty were also common, even when results had been returned. Many parents described pES results as informing decision-making around whether or not to terminate pregnancy. Some professionals were concerned that a non-informative result could be overly reassuring and highlighted that careful counselling was needed to ensure parents have a good understanding of what the result means for their pregnancy. Emotional support from professionals was valued; however, some parents felt that post-test support was lacking. CONCLUSION: Parents and professionals welcomed the introduction of pES. Results inform parents' decision-making around the termination of pregnancy. When there are no diagnostic findings or uncertain findings from pES, personalised counselling that considers scans and other tests are crucial. Directing parents to reliable online sources of information and providing emotional support throughout could improve their experiences of care.

Bioethics and the value of disagreement.

What does it mean to be a bioethicist? How should the role(s) of bioethics be understood in the context of a world of intense value conflict and polarisation? Bioethics is-in all its various forms and traditions-potentially well-positioned to contribute to addressing many of the most pressing challenges of value polarisation and conflict in diverse societies. However, realising this potential is going to require moving beyond currently foregrounded methods and developing new models for engaging with moral disagreement. This paper proposes an approach, 'adversarial cooperation,' drawing on the concepts of 'adversarial collaboration' from the sciences and 'antagonistic cooperation' from the humanities. Adversarial cooperation aims to combine the rigour and structured methodology of adversarial collaboration with the cultural sensitivity and expansive vision of antagonistic cooperation. The paper also addresses key challenges to adversarial cooperation, including ethical considerations, tensions between substantive and procedural values, the problem of misinformation and the need for decision-making amidst ongoing disagreement. Ultimately, adversarial cooperation suggests a reimagining of bioethical expertise, emphasising skills in mediation, the arts and humanities and participatory decision-making alongside established philosophical competencies. This implies a model of normative bioethical authority grounded in the ability to facilitate inclusive and trustworthy processes of moral deliberation. Realising the potential of adversarial cooperation will require significant changes in bioethics training and practice, as well as a commitment to reflexivity, humility and the amplification of marginalised voices. By embracing this approach, bioethics can play a vital role in navigating the complex moral landscapes of pluralistic societies.

Ethical preparedness in genomic medicine: how NHS clinical scientists navigate ethical issues.

Much has been published about the ethical issues encountered by clinicians in genetics/genomics, but those experienced by clinical laboratory scientists are less well described. Clinical laboratory scientists now frequently face navigating ethical problems in their work, but how they should be best supported to do this is underexplored. This lack of attention is also reflected in the ethics tools available to clinical laboratory scientists such as guidance and deliberative ethics forums, developed primarily to manage issues arising within the clinic.We explore what ethical issues are being experienced by clinical scientists, how they think such issues could be best analysed and managed, and whether their practice might be enhanced by more situated approaches to ethics deliberation and practice such as ethical preparedness. From thematic analysis of cases presented by clinical scientists at a specially convened meeting of the UK Genethics Forum, we derived three main ethical themes: (1) the redistribution of labour and responsibilities resulting from the practice of genomic medicine; (2) the interpretation and certainty of results and (3) the proposal that better standardisation and consistency of ethical approaches (for example, more guidelines and policy) could resolve some of the challenges arising.We argue that although standardisation is important for promoting shared understandings of good (including ethical) practice, supplementary approaches to enhance and sustain ethical preparedness will be important to help clinical scientists and others in the recently expanded genetic/genomic medicine environment foster quality ethical thinking.

Getting rights right: implementing 'Martha's Rule'.

The UK government has recently committed to adopting a new policy-dubbed 'Martha's Rule'-which has been characterised as providing patients the right to rapidly access a second clinical opinion in urgent or contested cases. Support for the rule emerged following the death of Martha Mills in 2021, after doctors failed to admit her to intensive care despite concerns raised by her parents. We argue that framing this issue in terms of patient rights is not productive, and should be avoided. Insofar as the ultimate goal of Martha's Rule is the provision of a clinical service that protects patient safety, an approach that focuses on the obligations of the health system-rather than the individual rights of patients-will better serve this goal. We outline an alternative approach that situates rapid clinical review as part of a suite of services aimed at enhancing and protecting patient care. This approach would make greater progress towards addressing the difficult systemic issues that Martha's Rule does not, while also better engaging with the constraints of clinical practice.

Design of proteasome inhibitors with oral efficacy in vivo against Plasmodium falciparum and selectivity over the human proteasome.

The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) ß5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.

Influence of balloon type and lesion location on outcomes following fistuloplasty.

BACKGROUND: There is limited evidence supporting the optimal use of fistuloplasty to maintain vascular access at various lesion sites, despite its critical role in facilitating renal replacement therapy and the overall high failure rates of arteriovenous fistulas (AVFs). This study aims to identify covariates affecting primary and secondary patency following fistuloplasty of native upper limb vascular access AVFs. METHODS: This retrospective study included all patients who underwent fistuloplasty at a tertiary vascular centre over 4 years. Baseline characteristics were recorded, and factors associated with primary and secondary patency rates were analysed. RESULTS: A total of 206 patients (88 male, 118 female) with a mean age of 68 (±14) years underwent fistuloplasty during the study period. The prevalence of diabetes, ischaemic heart disease and antiplatelet usage were 33%, 21% and 70%, respectively. The median number of fistuloplasties per access during the follow-up period was 2 [1-3]. Fistulas were classified as radiocephalic (65), brachiocephalic (102) and brachiobasilic transposition (39). Recurrent stenosis (RS) was identified in 60 patients who had previous fistuloplasty before the study period, while 146 patients had de novo stenoses (DNS). Stenosis location significantly differed between RS and DNS (p = .03), with DNS primarily being anastomotic and RS predominantly in central and mixed locations. Younger fistulas were more likely to have anastomotic stenoses compared to those older than 1 year (p = .001). While no significant differences in primary patency (PP) were observed, secondary patency (SP) varied by stenosis location: Central 32 [13-42] months, Fistula vein 20 [12.5-35.5] months, Mixed 25 [13.5-37.5] months and Anastomotic 19 [7-29.5] months (p = .012). CONCLUSION: Stenosis location in AVFs is associated with the age and type of the fistula. Younger fistulas often fail due to anastomotic stenoses, which have lower secondary patency compared to stenoses at other sites. Preliminary data suggest that central stenoses, primarily occurring in older fistulas, exhibit better secondary patency following fistuloplasty than stenoses at other locations.

Assessing the Knowledge and Comfort Level of US Family Medicine Residents Regarding Consenting and Confidentiality in Adolescent Health Care.

OBJECTIVES: Organizations recommend providing confidential adolescent health care to reduce the consequences of high-risk health behaviors such as substance use, unhealthy eating patterns, and high-risk sexual behaviors. Family physicians are uniquely positioned to provide confidential counseling and care to this vulnerable population but must be trained to provide such care. This study describes the impact of formal and informal training on the knowledge of and comfort level in providing confidential adolescent healthcare among a sample of US Family Medicine residents. METHODS: Electronic surveys were distributed to all Family Medicine residents throughout the United States. We used descriptive statistics and χ2 analysis where appropriate to determine the association between resident-reported receipt of training, confidence, and frequency in providing confidential adolescent health care. RESULTS: A total of 714 Family Medicine residents completed the survey. The majority reported no formal training in residency (50.3%). The receipt of formal and informal training in both medical school and residency was associated with a greater degree of comfort in providing confidential adolescent care and a higher likelihood of providing confidential time alone. Those reporting formal training were more likely to always provide confidential care (P = 0.001). CONCLUSIONS: Training focused on confidential adolescent health care in medical school or residency was associated with a greater degree of comfort and a higher likelihood of providing confidential adolescent health care.

Translating the biology of ß common receptor-engaging cytokines into clinical medicine.

The family of cytokines that comprises IL-3, IL-5, and GM-CSF was discovered over 30 years ago, and their biological activities and resulting impact in clinical medicine has continued to expand ever since. Originally identified as bone marrow growth factors capable of acting on hemopoietic progenitor cells to induce their proliferation and differentiation into mature blood cells, these cytokines are also recognized as key mediators of inflammation and the pathobiology of diverse immunologic diseases. This increased understanding of the functional repertoire of IL-3, IL-5, and GM-CSF has led to an explosion of interest in modulating their functions for clinical management. Key to the successful clinical translation of this knowledge is the recognition that these cytokines act by engaging distinct dimeric receptors and that they share a common signaling subunit called ß-common or ßc. The structural determination of how IL-3, IL-5, and GM-CSF interact with their receptors and linking this to their differential biological functions on effector cells has unveiled new paradigms of cell signaling. This knowledge has paved the way for novel mAbs and other molecules as selective or pan inhibitors for use in different clinical settings.

Covalent Inhibition by a Natural Product-Inspired Latent Electrophile.

Strategies to target specific protein cysteines are critical to covalent probe and drug discovery. 3-Bromo-4,5-dihydroisoxazole (BDHI) is a natural product-inspired, synthetically accessible electrophilic moiety that has previously been shown to react with nucleophilic cysteines in the active site of purified enzymes. Here, we define the global cysteine reactivity and selectivity of a set of BDHI-functionalized chemical fragments using competitive chemoproteomic profiling methods. Our study demonstrates that BDHIs capably engage reactive cysteine residues in the human proteome and the selectivity landscape of cysteines liganded by BDHI is distinct from that of haloacetamide electrophiles. Given its tempered reactivity, BDHIs showed restricted, selective engagement with proteins driven by interactions between a tunable binding element and the complementary protein sites. We validate that BDHI forms covalent conjugates with glutathione S-transferase Pi (GSTP1) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), emerging anticancer targets. BDHI electrophile was further exploited in Bruton's tyrosine kinase (BTK) inhibitor design using a single-step late-stage installation of the warhead onto acrylamide-containing compounds. Together, this study expands the spectrum of optimizable chemical tools for covalent ligand discovery and highlights the utility of 3-bromo-4,5-dihydroisoxazole as a cysteine-reactive electrophile.

Efficacy and toxicity of primary re-irradiation for malignant spinal cord compression based on radiobiological modelling: a phase II clinical trial.

BACKGROUND: The efficacy and safety of primary re-irradiation for MSCC are not known. Our aim was to establish the efficacy and safety of biologically effective dose-based re-irradiation. METHODS: Patients presenting with MSCC at a previously irradiated spine segment, and not proceeding with surgical decompression, were eligible. A 3 Gray per fraction experimental schedule (minimum 18 Gy/6 fractions, maximum 30 Gy/10 fractions) was used, delivering a maximum cumulative spinal dose of 100 Gy2 if the interval since the last radiotherapy was within 6 months, or 130 Gy2 if longer. The primary outcome was a change in mobility from week 1 to week 5 post-treatment, as assessed by the Tomita score. The RTOG SOMA score was used to screen for spinal toxicity, and an MRI performed to assess for radiation-induced myelopathy (RIM). RESULTS: Twenty-two patients were enroled, of whom eleven were evaluable for the primary outcome. Nine of eleven (81.8%) had stable or improved Tomita scores at 5 weeks. One of eight (12.5%) evaluable for late toxicity developed RIM. CONCLUSIONS: Re-irradiation is an efficacious treatment for MSCC. There is a risk of RIM with a cumulative dose of 120 Gy2. CLINICAL TRIAL REGISTRATION: Cancer Trials Ireland (ICORG 07-11); NCT00974168.

De Novo Variants in SPOP Cause Two Clinically Distinct Neurodevelopmental Disorders.

Recurrent somatic variants in SPOP are cancer specific; endometrial and prostate cancers result from gain-of-function and dominant-negative effects toward BET proteins, respectively. By using clinical exome sequencing, we identified six de novo pathogenic missense variants in SPOP in seven individuals with developmental delay and/or intellectual disability, facial dysmorphisms, and congenital anomalies. Two individuals shared craniofacial dysmorphisms, including congenital microcephaly, that were strikingly different from those of the other five individuals, who had (relative) macrocephaly and hypertelorism. We measured the effect of SPOP variants on BET protein amounts in human Ishikawa endometrial cancer cells and patient-derived cell lines because we hypothesized that variants would lead to functional divergent effects on BET proteins. The de novo variants c.362G>A (p.Arg121Gln) and c. 430G>A (p.Asp144Asn), identified in the first two individuals, resulted in a gain of function, and conversely, the c.73A>G (p.Thr25Ala), c.248A>G (p.Tyr83Cys), c.395G>T (p.Gly132Val), and c.412C>T (p.Arg138Cys) variants resulted in a dominant-negative effect. Our findings suggest that these opposite functional effects caused by the variants in SPOP result in two distinct and clinically recognizable syndromic forms of intellectual disability with contrasting craniofacial dysmorphisms.