Genomic testing for copy number and single nucleotide variants in spermatogenic failure.

PURPOSE: To identify clinically significant genomic copy number (CNV) and single nucleotide variants (SNV) in males with unexplained spermatogenic failure (SPGF). MATERIALS AND METHODS: Peripheral blood DNA from 97/102 study participants diagnosed with oligozoospermia, severe oligozoospermia, or non-obstructive azoospermia (NOA) was analyzed for CNVs via array comparative genomic hybridization (aCGH) and SNVs using whole-exome sequencing (WES). RESULTS: Of the 2544 CNVs identified in individuals with SPGF, > 90% were small, ranging from 0.6 to 75 kb. Thirty, clinically relevant genomic aberrations, were detected in 28 patients (~ 29%). These included likely diagnostic CNVs in 3/41 NOA patients (~ 7%): 1 hemizygous, intragenic TEX11 deletion, 1 hemizygous DDX53 full gene deletion, and 1 homozygous, intragenic STK11 deletion. High-level mosaicism for X chromosome disomy (~ 10% 46,XY and ~ 90% 47,XXY) was also identified in 3 of 41 NOA patients who previously tested normal with conventional karyotyping. The remaining 24 CNVs detected were heterozygous, autosomal recessive carrier variants. Follow-up WES analysis confirmed 8 of 27 (30%) CNVs (X chromosome disomy excluded). WES analysis additionally identified 13 significant SNVs and/or indels in 9 patients (~ 9%) including X-linked AR, KAL1, and NR0B1 variants. CONCLUSION: Using a combined genome-wide aCGH/WES approach, we identified pathogenic and likely pathogenic SNVs and CNVs in 15 patients (15%) with unexplained SPGF. This value equals the detection rate of conventional testing for aneuploidies and is considerably higher than the prevalence of Y chromosome microdeletions. Our results underscore the importance of comprehensive genomic analysis in emerging diagnostic testing of complex conditions like male infertility.

[Place of preperitoneal pelvic packing in severe pelvic traumatisms: 20 cases in a French level 1 trauma center]. / Place du packing pré-péritonéal pelvien dans les traumatismes graves du bassin : étude à partir de 20 cas réalisés dans un trauma center de niveau 1 français.

BACKGROUND: The overall mortality of hemodynamically unstable patients with pelvic trauma is high. Their management is controversial concerning places of arterioembolization and pelvic packing associated with pelvic stabilization. The aim of this study was to collect the pre-peritoneal pelvic packing (PPP) performed in our institution over 10years in order to propose a management algorithm. METHOD: From January 2010 to December 2020, all patients with a hemodynamically unstable pelvic fracture who had PPP combined with pelvic stabilization were included. Data were collected prospectively and analyzed retrospectively. The main judgement criteria were early hemorrhage-induced mortality (<24h) and overall mortality (<30d). RESULTS: Twenty patients had PPP out of 287 polytrauma patients with pelvic fracture. The first-line PPP proposed in our algorithm significantly reduced the number of red blood cells (RBCs) (P=0.0231) and improved systolic blood pressure (SBP) (P<0.001) within 24hours of first-line PPP (compared with preoperative). Six patients (30%) were embolized postoperatively for active bleeding not necessarily pelvic. The overall mortality at 30days was 50% (10/20). CONCLUSION: PPP is a fast, easy, effective and safe procedure for venous, bone and sometimes arterial bleeding. PPP is part of damage control surgery and we propose it as a first-line procedure. AE remains complementary in a second step.

Elevated In Vitro Kinase Activity in Peripheral Blood Mononuclear Cells of Leucine-Rich Repeat Kinase 2 G2019S Carriers: A Novel Enzyme-Linked Immunosorbent Assay-Based Method.

BACKGROUND: Leucine-rich repeat kinase 2 kinase inhibitors are being vigorously pursued as potential therapeutic options; however, there is a critical need for sensitive and quantitative assays of leucine-rich repeat kinase 2 function and target engagement. OBJECTIVES: Our objective was to compare collection and storage protocols for peripheral blood mononuclear cells, and to determine the optimal conditions for downstream analyses of leucine-rich repeat kinase 2 in PD cohorts. METHODS: Here, we describe enzyme-linked immunosorbent assay-based assays capable of detecting multiple aspects of leucine-rich repeat kinase 2 function at endogenous levels in human tissues. RESULTS: In peripheral blood mononuclear cells from both healthy and affected carriers of the G2019S mutation in leucine-rich repeat kinase 2, we report, for the first time, significantly elevated in vitro kinase activity, while detecting a significant increase in pS935/leucine-rich repeat kinase 2 in idiopathic PD patients. CONCLUSIONS: Quantitative assays such as these described here could potentially uncover specific markers of leucine-rich repeat kinase 2 function that are predictive of disease progression, aid in patient stratification, and be a critical component of upcoming clinical trials. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Kinase activity of mutant LRRK2 manifests differently in hetero-dimeric vs. homo-dimeric complexes.

The Parkinson's disease (PD) protein leucine-rich repeat kinase 2 (LRRK2) exists as a mixture of monomeric and dimeric species, with its kinase activity highly concentrated in the dimeric conformation of the enzyme. We have adapted the proximity biotinylation approach to study the formation and activity of LRRK2 dimers isolated from cultured cells. We find that the R1441C and I2020T mutations both enhance the rate of dimer formation, whereas, the G2019S kinase domain mutant is similar to WT, and the G2385R risk factor variant de-stabilizes dimers. Interestingly, we find a marked departure in the kinase activity between G2019S-LRRK2 homo-dimers and wild-type-G2019S hetero-dimers. While the homo-dimeric G2019S-LRRK2 exhibits the typical robust enhancement of kinase activity, hetero-dimers comprised of wild-type (WT) and G2019S-LRRK2 exhibit kinase activity similar to WT. Dimeric complexes of specific mutant forms of LRRK2 show reduced stability following an in vitro kinase reaction, in LRRK2 mutants for which the kinase activity is similar to WT. Phosphorylation of the small GTPase Rab10 follows a similar pattern in which hetero-dimers of WT and mutant LRRK2 show similar levels of phosphorylation of Rab10 to WT homo-dimers; while the levels of pRab10 are significantly increased in cells expressing mutant homo-dimers. Interestingly, while the risk variant G2385R leads to a de-stabilization of LRRK2 dimers, those dimers possess significantly elevated kinase activity. The vast majority of familial LRRK2-dependent PD cases are heterozygous; thus, these findings raise the possibility that a crucial factor in disease pathogenesis may be the accumulation of homo-dimeric mutant LRRK2.

Nurr1:RXRα heterodimer activation as monotherapy for Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic (DAergic) neurons in the substantia nigra and the gradual depletion of dopamine (DA). Current treatments replenish the DA deficit and improve symptoms but induce dyskinesias over time, and neuroprotective therapies are nonexistent. Here we report that Nuclear receptor-related 1 (Nurr1):Retinoid X receptor α (RXRα) activation has a double therapeutic potential for PD, offering both neuroprotective and symptomatic improvement. We designed BRF110, a unique in vivo active Nurr1:RXRα-selective lead molecule, which prevents DAergic neuron demise and striatal DAergic denervation in vivo against PD-causing toxins in a Nurr1-dependent manner. BRF110 also protects against PD-related genetic mutations in patient induced pluripotent stem cell (iPSC)-derived DAergic neurons and a genetic mouse PD model. Remarkably, besides neuroprotection, BRF110 up-regulates tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC), and GTP cyclohydrolase I (GCH1) transcription; increases striatal DA in vivo; and has symptomatic efficacy in two postneurodegeneration PD models, without inducing dyskinesias on chronic daily treatment. The combined neuroprotective and symptomatic effects of BRF110 identify Nurr1:RXRα activation as a potential monotherapeutic approach for PD.

P62/SQSTM1 is a novel leucine-rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity.

Autosomal-dominant, missense mutations in the leucine-rich repeat protein kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson's disease (PD). LRRK2 kinase activity is increased in several pathogenic mutations (N1437H, R1441C/G/H, Y1699C, G2019S), implicating hyperphosphorylation of a substrate in the pathogenesis of the disease. Identification of the downstream targets of LRRK2 is a crucial endeavor in the field to understand LRRK2 pathway dysfunction in the disease. We have identified the signaling adapter protein p62/SQSTM1 as a novel endogenous interacting partner and a substrate of LRRK2. Using mass spectrometry and phospho-specific antibodies, we found that LRRK2 phosphorylates p62 on Thr138 in vitro and in cells. We found that the pathogenic LRRK2 PD-associated mutations (N1437H, R1441C/G/H, Y1699C, G2019S) increase phosphorylation of p62 similar to previously reported substrate Rab proteins. Notably, we found that the pathogenic I2020T mutation and the risk factor mutation G2385R displayed decreased phosphorylation of p62. p62 phosphorylation by LRRK2 is blocked by treatment with selective LRRK2 inhibitors in cells. We also found that the amino-terminus of LRRK2 is crucial for optimal phosphorylation of Rab7L1 and p62 in cells. LRRK2 phosphorylation of Thr138 is dependent on a p62 functional ubiquitin-binding domain at its carboxy-terminus. Co-expression of p62 with LRRK2 G2019S increases the neurotoxicity of this mutation in a manner dependent on Thr138. p62 is an additional novel substrate of LRRK2 that regulates its toxic biology, reveals novel signaling nodes and can be used as a pharmacodynamic marker for LRRK2 kinase activity.

Whole-exome sequencing of the BDR cohort: evidence to support the role of the PILRA gene in Alzheimer's disease.

AIM: Late-onset Alzheimer's disease (LOAD) accounts for 95% of all Alzheimer's cases and is genetically complex in nature. Overlapping clinical and neuropathological features between AD, FTD and Parkinson's disease highlight the potential role of genetic pleiotropy across diseases. Recent genome-wide association studies (GWASs) have uncovered 20 new loci for AD risk; however, these exhibit small effect sizes. Using NGS, here we perform association analyses using exome-wide and candidate-gene-driven approaches. METHODS: Whole-exome sequencing was performed on 132 AD cases and 53 control samples. Exome-wide single-variant association and gene burden tests were performed for 76 640 nonsingleton variants. Samples were also screened for known causative mutations in familial genes in AD and other dementias. Single-variant association and burden analysis was also carried out on variants in known AD and other neurological dementia genes. RESULTS: Tentative single-variant and burden associations were seen in several genes with kinase and protease activity. Exome-wide burden analysis also revealed significant burden of variants in PILRA (P = 3.4 × 10-5 ), which has previously been linked to AD via GWAS, hit ZCWPW1. Screening for causative mutations in familial AD and other dementia genes revealed no pathogenic variants. Variants identified in ABCA7, SLC24A4, CD33 and LRRK2 were nominally associated with disease (P < 0.05) but did not withstand correction for multiple testing. APOE (P = 0.02) and CLU (P = 0.04) variants showed significant burden on AD. CONCLUSIONS: In addition, polygenic risk scores (PRS) were able to distinguish between cases and controls with 83.8% accuracy using 3268 variants, sex, age at death and APOE ε4 and ε2 status as predictors.

Alzheimer's disease.

Alzheimer's disease, the commonest cause of dementia, is a growing global health concern with huge implications for individuals and society. In this review, current understanding of the epidemiology, genetics, pathology and pathogenesis of Alzheimer's disease is outlined, before its clinical presentation and current treatment strategies are discussed. Finally, the review discusses how our enhanced understanding of Alzheimer pathogenesis, including the recognition of a protracted preclinical phase, is informing new therapeutic strategies with the aim of moving from treatment to prevention.

Neuronal death signaling pathways triggered by mutant LRRK2.

Autosomal dominantly inherited mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease. While considerable progress has been made in understanding its function and the many different cellular activities in which it participates, a clear understanding of the mechanism(s) of the induction of neuronal death by mutant forms of LRRK2 remains elusive. Although several in vivo models have documented the progressive loss of dopaminergic neurons of the substantia nigra, more complete interrogations of the modality of neuronal death have been gained from cellular models. Overexpression of mutant LRRK2 in neuronal-like cell lines or in primary neurons induces an apoptotic type of cell death involving components of the extrinsic as well as intrinsic death pathways. While informative, these studies are limited by their reliance upon isolated neuronal cells; and the pathways triggered by mutant LRRK2 in neurons may be further refined or modulated by extracellular signals. Nevertheless, the identification of specific cell death-associated signaling events set in motion by the dominant action of mutant LRRK2, the loss of an inhibitory function of wild-type LRRK2, or a combination of the two, expands the landscape of potential therapeutic targets for future intervention in the clinic.

Neuropathic pain in cancer: systematic review, performance of screening tools and analysis of symptom profiles.

BACKGROUND: The objectives of this study were to evaluate the methodological quality of rigorous neuropathic pain assessment tools in applicable clinical studies, and determine the performance of screening tools for identifying neuropathic pain in patients with cancer. METHODS: Systematic literature search identified studies reporting use of Leeds Assessment of Neuropathic Symptoms and Signs (LANSS), Douleur Neuropathique en 4 (DN4) or painDETECT (PDQ) in cancer patients with a clinical diagnosis of neuropathic or not neuropathic pain. Individual patient data were requested to examine descriptor item profiles. RESULTS: Six studies recruited a total of 2301 cancer patients of which 1564 (68%) reported pain. Overall accuracy of screening tools ranged from 73 to 94%. There was variation in description and rigour of clinical assessment, particularly related to the rigour of clinical judgement of pain as the reference standard. Individual data from 1351 patients showed large variation in the selection of neuropathic pain descriptor items by cancer patients with neuropathic pain. LANSS and DN4 items characterized a significantly different neuropathic pain symptom profile from non-neuropathic pain in both tumour- and treatment-related cancer pain aetiologies. CONCLUSIONS: We identified concordance between the clinician diagnosis and screening tool outcomes for LANSS, DN4 and PDQ in patients with cancer pain. Shortcomings in relation to standardized clinician assessment are likely to account for variation in screening tool sensitivity, which should include the use of the neuropathic pain grading system. Further research is needed to standardize and improve clinical assessment in patients with cancer pain. Until the standardization of clinical diagnosis for neuropathic cancer pain has been validated, screening tools offer a practical approach to identify potential cases of neuropathic cancer pain.

An open-label pilot study of oral vitamin C as an opioid-sparing agent in patients with chronic pain secondary to cancer.

PURPOSE: The purpose of this study is to assess the efficacy of oral Vitamin C as an opioid-sparing agent when used in conjunction with opioids and standard adjuvant therapy in the management of chronic cancer pain. METHODS: An open-label pilot study of patients ≥18 years of age with chronic pain secondary to cancer and/or its treatment and a Brief Pain Inventory average pain score of ≥3/10. In addition to opioid analgesia, patients received 1 g of vitamin C twice daily over 3 days (total daily dose of 2 g). Patients' usual medications, including breakthrough medications, were continued throughout the study period. The primary endpoint was total daily opioid use during vitamin C administration compared with that immediately prior to study. RESULTS: Thirty-four patients were enrolled in the study. Seven failed to complete the trial. Across the 17 evaluable patients, the median daily opioid consumption was 360 mg oral morphine equivalents (OME) on the days prior to vitamin C and 390 mg when administered with vitamin C. CONCLUSION: This study failed to demonstrate any clinically significant benefit from vitamin C in conjunction with opioids in cancer-related pain and does not provide support for embarking on a larger randomised trial to determine efficacy.

Integration of oncology and palliative care: setting a benchmark.

BACKGROUND: Integration of oncology and palliative care (PC) should be the standard model of care for patients with advanced cancer. An expert panel developed criteria that constitute integration. This study determined whether the PC service within this Health Service, which is considered to be fully "integrated", could be benchmarked against these criteria. METHODS: A survey was undertaken to determine the perceived level of integration of oncology and palliative care by all health care professionals (HCPs) within our cancer centre. An objective determination of integration was obtained from chart reviews of deceased patients. Integration was defined as >70% of all respondents answered "agree" or "strongly agree" to each indicator and >70% of patient charts supported each criteria. RESULTS: Thirty-four HCPs participated in the survey (response rate 69%). Over 90% were aware of the outpatient PC clinic, interdisciplinary and consultation team, PC senior leadership, and the acceptance of concurrent anticancer therapy. None of the other criteria met the 70% agreement mark but many respondents lacked the necessary knowledge to respond. The chart review included 67 patients, 92% of whom were seen by the PC team prior to death. The median time from referral to death was 103 days (range 0-1347). The level of agreement across all criteria was below our predefined definition of integration. CONCLUSION: The integration criteria relating to service delivery are medically focused and do not lend themselves to interdisciplinary review. The objective criteria can be audited and serve both as a benchmark and a basis for improvement activities.

Review: Induced pluripotent stem cell models of frontotemporal dementia.

The increasing prevalence of dementia in the ageing population combined with the lack of treatments and the burden on national health care systems globally make dementia a public health priority. Despite the plethora of important research findings published over the past two decades, the mechanisms underlying dementia are still poorly understood and the progress in pharmacological interventions is limited. Recent advances in cellular reprogramming and genome engineering technologies offer an unprecedented new paradigm in disease modeling. Induced pluripotent stem cells (iPSCs) have enabled the study of patient-derived neurons in vitro, a significant progress in the field of dementia research. The first studies using iPSCs to model dementia have recently emerged, holding promise for elucidating disease pathogenic mechanisms and accelerating drug discovery. In this review, we summarize the major findings of iPSC-based studies in frontotemporal dementia (FTD) and FTD overlapping with amyotrophic lateral sclerosis (FTD/ALS). We also discuss some of the main challenges in the use of iPSCs to model complex, late-onset neurodegenerative diseases such as dementias.

Genetic overlap between Alzheimer's disease and Parkinson's disease at the MAPT locus.

We investigated the genetic overlap between Alzheimer's disease (AD) and Parkinson's disease (PD). Using summary statistics (P-values) from large recent genome-wide association studies (GWAS) (total n=89 904 individuals), we sought to identify single nucleotide polymorphisms (SNPs) associating with both AD and PD. We found and replicated association of both AD and PD with the A allele of rs393152 within the extended MAPT region on chromosome 17 (meta analysis P-value across five independent AD cohorts=1.65 × 10(-7)). In independent datasets, we found a dose-dependent effect of the A allele of rs393152 on intra-cerebral MAPT transcript levels and volume loss within the entorhinal cortex and hippocampus. Our findings identify the tau-associated MAPT locus as a site of genetic overlap between AD and PD, and extending prior work, we show that the MAPT region increases risk of Alzheimer's neurodegeneration.

[Pansclerotic morphea with a rapidly fatal outcome in an 11-year-old girl]. / Morphée pansclérotique d'évolution rapidement létale chez une jeune fille de 11ans.

BACKGROUND: Pansclerotic morphea is a poorly described but extremely debilitating variant of localized scleroderma. We report a case with a rapidly fatal outcome in an 11-year-old girl. PATIENTS AND METHODS: An 11-year-old girl with a 2-year history of morphea presented at our institution in April 2012. The sclerosis had started on her trunk and progressed rapidly to involve her entire skin. Initial treatment with corticosteroids was ineffective and she presented extremely painful ulcerations of the lower limbs. The outcome was rapidly fatal, in early 2014, due to cachexia and sepsis after two amputations and several failed treatments including methotrexate. DISCUSSION: Pansclerotic morphea is characterized by rapidly progressing sclerosis involving the entire skin, trophic cutaneous ulcers, painful contraction and limited joint mobility. The prognosis is poor since the disease has an incapacitating and potentially fatal outcome. No reliably effective treatment has yet been established. CONCLUSION: Our case highlights the clinical characteristics of this uncommon form of localized scleroderma, the extremely severe prognosis, and the therapeutic challenge involved.

Mesenteric lymphatic vessels adapt to mesenteric venous hypertension by becoming weaker pumps.

Lymphangions, the segments of lymphatic vessels between two adjacent lymphatic valves, actively pump lymph. Acute changes in transmural pressure and lymph flow have profound effects on lymphatic pump function in vitro. Chronic changes in pressure and flow in vivo have also been reported to lead to significant changes in lymphangion function. Because changes in pressure and flow are both cause and effect of adaptive processes, characterizing adaptation requires a more fundamental analysis of lymphatic muscle properties. Therefore, the purpose of the present work was to use an intact lymphangion isovolumetric preparation to evaluate changes in mesenteric lymphatic muscle mechanical properties and the intracellular Ca(2+) in response to sustained mesenteric venous hypertension. Bovine mesenteric veins were surgically occluded to create mesenteric venous hypertension. Postnodal mesenteric lymphatic vessels from mesenteric venous hypertension (MVH; n = 6) and sham surgery (Sham; n = 6) animals were isolated and evaluated 3 days after the surgery. Spontaneously contracting MVH vessels generated end-systolic active tension and end-diastolic active tension lower than the Sham vessels. Furthermore, steady-state active tension and intracellular Ca(2+) concentration levels in response to KCl stimulation were also significantly lower in MVH vessels compared with those of the Sham vessels. There was no significant difference in passive tension in lymphatic vessels from the two groups. Taken together, these results suggest that following 3 days of mesenteric venous hypertension, postnodal mesenteric lymphatic vessels adapt to become weaker pumps with decreased cytosolic Ca(2+) concentration.

Pharmacological options for the management of refractory cancer pain-what is the evidence?

Refractory cancer pain that does not respond to standard opioid and/or co-analgesic therapy occurs in 10-20 % of patients. Risk factors include young age, neuropathic pain type, incident pain, psychological distress, previous opioid use, high tolerance, a history of addiction and impaired cognition. The management of patients with refractory pain remains a challenge. Treatment options include opioid manipulation (parenteral delivery, rotation, combination, methadone and buprenorphine), non-opioids and co-analgesics (paracetamol, non-steroidal anti-inflammatory agents, antidepressants and anticonvulsants), NMDA receptor antagonists, cannabinoids, lignocaine and corticosteroids. The evidence of benefit for any of these agents is weak, and each additional agent increases the risk of adverse events. Evidence-based guidelines cannot, therefore, be developed at present. New approaches are recommended including targeted opioid therapy, multimodal analgesia, a goal-oriented approach to pain management and increasing use of the multidisciplinary team and support services.

LRRK2 Parkinson disease mutations enhance its microtubule association.

Dominant missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic causes of Parkinson disease (PD) and genome-wide association studies identify LRRK2 sequence variants as risk factors for sporadic PD. Intact kinase function appears critical for the toxicity of LRRK2 PD mutants, yet our understanding of how LRRK2 causes neurodegeneration remains limited. We find that most LRRK2 PD mutants abnormally enhance LRRK2 oligomerization, causing it to form filamentous structures in transfections of cell lines or primary neuronal cultures. Strikingly, ultrastructural analyses, including immuno-electron microscopy and electron microscopic tomography, demonstrate that these filaments consist of LRRK2 recruited onto part of the cellular microtubule network in a well-ordered, periodic fashion. Like LRRK2-related neurodegeneration, microtubule association requires intact kinase function and the WD40 domain, potentially linking microtubule binding and neurodegeneration. Our observations identify a novel effect of LRRK2 PD mutations and highlight a potential role for microtubules in the pathogenesis of LRRK2-related neurodegeneration.

Pathways to Alzheimer's disease.

Recent trials of anti-amyloid agents have not produced convincing improvements in clinical outcome in Alzheimer's disease; however, the reason for these poor or inconclusive results remains unclear. Recent genetic data continue to support the amyloid hypothesis of Alzheimer's disease with protective variants being found in the amyloid gene and both common low-risk and rare high-risk variants for disease being discovered in genes that are part of the amyloid response pathways. These data support the view that genetic variability in how the brain responds to amyloid deposition is a potential therapeutic target for the disease, and are consistent with the notion that anti-amyloid therapies should be initiated early in the disease process.

ß decay of (38)Ca: sensitive test of isospin symmetry-breaking corrections from mirror superallowed 0+ → 0+ transitions.

We report the first branching-ratio measurement of the superallowed 0+→0+ß transition from Ca38. The result, 0.7728(16), leads to an ft value of 3062.3(68) s with a relative precision of ±0.2%. This makes possible a high-precision comparison of the ft values for the mirror superallowed transitions, Ca38→38mK and K38m→Ar38, which sensitively tests the isospin symmetry-breaking corrections required to extract Vud, the up-down quark-mixing element of the Cabibbo-Kobayashi-Maskawa (CKM) matrix, from superallowed ß decay. The result supports the corrections currently used and points the way to even tighter constraints on CKM unitarity.