Combined approaches, including long-read sequencing, address the diagnostic challenge of HYDIN in primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD), a disorder of the motile cilia, is now recognised as an underdiagnosed cause of bronchiectasis. Accurate PCD diagnosis comprises clinical assessment, analysis of cilia and the identification of biallelic variants in one of 50 known PCD-related genes, including HYDIN. HYDIN-related PCD is underdiagnosed due to the presence of a pseudogene, HYDIN2, with 98% sequence homology to HYDIN. This presents a significant challenge for Short-Read Next Generation Sequencing (SR-NGS) and analysis, and many diagnostic PCD gene panels do not include HYDIN. We have used a combined approach of SR-NGS with bioinformatic masking of HYDIN2, and state-of-the-art long-read Nanopore sequencing (LR_NGS), together with analysis of respiratory cilia including transmission electron microscopy and immunofluorescence to address the underdiagnosis of HYDIN as a cause of PCD. Bioinformatic masking of HYDIN2 after SR-NGS facilitated the detection of biallelic HYDIN variants in 15 of 437 families, but compromised the detection of copy number variants. Supplementing testing with LR-NGS detected HYDIN deletions in 2 families, where SR-NGS had detected a single heterozygous HYDIN variant. LR-NGS was also able to confirm true homozygosity in 2 families when parental testing was not possible. Utilising a combined genomic diagnostic approach, biallelic HYDIN variants were detected in 17 families from 242 genetically confirmed PCD cases, comprising 7% of our PCD cohort. This represents the largest reported HYDIN cohort to date and highlights previous underdiagnosis of HYDIN-associated PCD. Moreover this provides further evidence for the utility of LR-NGS in diagnostic testing, particularly for regions of high genomic complexity.

O blood type is not associated with worse coagulopathy or outcome in exsanguinating trauma.

BACKGROUND: Despite improving understanding of trauma-induced coagulopathy (TIC), mortality and morbidity due to exsanguinating trauma remain high. Increased complications due to hemorrhage have been reported in blood group O, possibly due to reduced levels of von Willebrand factor (vWF). METHODS: An urban level 1 adult trauma center registry was retrospectively queried. Patients receiving ≥6 units of pRBC within 4 â€‹h of presentation were included. Patient demographics, admission labs and outcomes were obtained. Univariate and multiple logistic regression analyses were performed. RESULTS: 562 patients were identified. There were no significant differences in demographics, admission labs, or outcome between different ABO groups. After adjustment, Type A patients were more likely to be hypocoagulable compared to Type O patients (p â€‹= â€‹0.014). No mortality differences were seen between ABO types in multiple regression analysis. CONCLUSIONS: No outcome or mortality differences were seen between ABO types, therefore factors other than vWF expression should be considered to explain coagulopathy in trauma patients.

Synthesis and import of GDP-l-fucose into the Golgi affect plant-water relations.

Land plants evolved multiple adaptations to restrict transpiration. However, the underlying molecular mechanisms are not sufficiently understood. We used an ozone-sensitivity forward genetics approach to identify Arabidopsis thaliana mutants impaired in gas exchange regulation. High water loss from detached leaves and impaired decrease of leaf conductance in response to multiple stomata-closing stimuli were identified in a mutant of MURUS1 (MUR1), an enzyme required for GDP-l-fucose biosynthesis. High water loss observed in mur1 was independent from stomatal movements and instead could be linked to metabolic defects. Plants defective in import of GDP-l-Fuc into the Golgi apparatus phenocopied the high water loss of mur1 mutants, linking this phenotype to Golgi-localized fucosylation events. However, impaired fucosylation of xyloglucan, N-linked glycans, and arabinogalactan proteins did not explain the aberrant water loss of mur1 mutants. Partial reversion of mur1 water loss phenotype by borate supplementation and high water loss observed in boron uptake mutants link mur1 gas exchange phenotypes to pleiotropic consequences of l-fucose and boron deficiency, which in turn affect mechanical and morphological properties of stomatal complexes and whole-plant physiology. Our work emphasizes the impact of fucose metabolism and boron uptake on plant-water relations.

Hepatic Metastasectomy in Pediatric Patients: An Observational Study.

BACKGROUND: The role of hepatectomy for metastatic disease in children is controversial. Rationales include potential cure, obtaining a diagnosis, and guiding chemotherapy decisions. This study examines the safety and utility of hepatic metastasectomy for children at a single institution. METHODS: After IRB approval (#22-1258), medical records were reviewed from 1995 to 2022 for children undergoing hepatic metastasectomy. En-bloc hepatectomies during primary tumor resection were excluded. RESULTS: Hepatic metastasectomy was performed in 16 patients for a variety of histologies. Median patient age was 12.2 years [IQR 6.9-22.6], and 13/16 patients were female (81 %). Number of hepatic metastases ranged from 1 to 23 and involved between 1 and 8 Couinaud segments. Anatomic resections included 4 hemihepatectomies and 1 sectionectomy. All other resections were nonanatomic. 3/6 resections for germ cell tumor (GCT) revealed only mature teratoma, driving adjuvant therapy decisions. When indicated, median time to adjuvant chemotherapy was 19 days [IQR 11-22]. No patients had Clavien-Dindo Class III or higher perioperative morbidity. Three patients (1 GCT, 1 adrenocortical carcinoma (ACC), and 1 gastric neuroendocrine tumor (GNET) experienced hepatic relapse. The patients with relapsed GCT and GNET are alive with disease at 17 and 135 months, respectively. The patient with ACC died of disease progression and liver failure. One patient with Wilms tumor experienced extrahepatic, retroperitoneal recurrence and died. With a median follow-up of 38 months, 10-year disease-specific and disease-free survival were 77 % and 61 %, respectively. CONCLUSIONS: Hepatic metastasectomy can be accomplished safely in children, may guide adjuvant therapy decisions, and is associated with long-term survival in selected patients. LEVEL OF EVIDENCE: Level IV. TYPE OF STUDY: Treatment Study, Case series with no comparison group.

Oncogenic Cells of Renal Embryonic Lineage Sensitive to the Small-Molecule Inhibitor QC6352 Display Depletion of KDM4 Levels and Disruption of Ribosome Biogenesis.

The histone lysine demethylases KDM4A-C are involved in physiologic processes including stem cell identity and self-renewal during development, DNA damage repair, and cell-cycle progression. KDM4A-C are overexpressed and associated with malignant cell behavior in multiple human cancers and are therefore potential therapeutic targets. Given the role of KDM4A-C in development and cancer, we aimed to test the potent, selective KDM4A-C inhibitor QC6352 on oncogenic cells of renal embryonic lineage. The anaplastic Wilms tumor cell line WiT49 and the tumor-forming human embryonic kidney cell line HEK293 demonstrated low nanomolar QC6352 sensitivity. The cytostatic response to QC6352 in WiT49 and HEK293 cells was marked by induction of DNA damage, a DNA repair-associated protein checkpoint response, S-phase cell-cycle arrest, profound reduction of ribosomal protein gene and rRNA transcription, and blockade of newly synthesized proteins. QC6352 caused reduction of KDM4A-C levels by a proteasome-associated mechanism. The cellular phenotype caused by QC6352 treatment of reduced migration, proliferation, tumor spheroid growth, DNA damage, and S-phase cell-cycle arrest was most closely mirrored by knockdown of KDM4A as determined by siRNA knockdown of KDM4A-C. QC6352 sensitivity correlated with high basal levels of ribosomal gene transcription in more than 900 human cancer cell lines. Targeting KDM4A may be of future therapeutic interest in oncogenic cells of embryonic renal lineage or cells with high basal expression of ribosomal protein genes.

Malignant Solid Pseudopapillary Neoplasm of the Pancreas: An Orthogonal Analysis.

BACKGROUND: Pancreatic solid pseudopapillary neoplasms (SPN) are generally indolent; however, some patients present with "malignant" SPN. An orthogonal analysis of multiple datasets was performed to investigate the utility of complete surgical resection (CSR) for malignant SPN. METHODS: A systematic review was performed for cases of malignant SPN, defined as T4, N1, and/or M1. Malignant SPN was analyzed within the National Cancer Database (NCDB) and compared with T1-3N0M0 SPN. Predictors of malignant SPN were assessed, and treatments were analyzed by using survival analysis. RESULTS: The systematic review yielded 164 cases of malignant SPN. Of 31 children, only one died due to malignant SPN. Among adults, CSR was associated with improved disease-specific survival (DSS) (P = 0.0002). Chemotherapy did not improve malignant SPN DSS, whether resected (P = 0.8485) or not (P = 0.2219). Of 692 adults with SPN within the NCDB, 93 (13.4%) had malignant SPN. Pancreatic head location (odds ratio [OR] 2.174; 95% confidence interval [CI] 1.136-4.166; P = 0.0186) and tumor size (OR 1.154; 95% CI 1.079-1.235; P < 0.0001) associated with the malignant phenotype. Malignant SPN predicted decreased overall survival (OS) compared with T1-3N0M0 disease (P < 0.0001). Resected malignant SPN demonstrated improved OS (P < 0.0001), including resected stage IV malignant SPN (P = 0.0003). Chemotherapy did not improve OS for malignant SPN, whether resected (P = 0.8633) or not (P = 0.5734). Within a multivariable model, resection was associated with decreased hazard of death (hazard ratio 0.090; 95% CI 0.030-0.261; P < 0.0001). CONCLUSIONS: Approximately 13% of patients with SPN present with a malignant phenotype. Pediatric cases may be less aggressive. Resection may improve survival for malignant SPN, which does not appear chemosensitive.

Regulatory NADH dehydrogenase-like complex optimizes C4 photosynthetic carbon flow and cellular redox in maize.

C4 plants typically operate a CO2 concentration mechanism from mesophyll (M) cells into bundle sheath (BS) cells. NADH dehydrogenase-like (NDH) complex is enriched in the BS cells of many NADP-malic enzyme (ME) type C4 plants and is more abundant in C4 than in C3 plants, but to what extent it is involved in the CO2 concentration mechanism remains to be experimentally investigated. We created maize and rice mutants deficient in NDH function and then used a combination of transcriptomic, proteomic, and metabolomic approaches for comparative analysis. Considerable decreases in growth, photosynthetic activities, and levels of key photosynthetic proteins were observed in maize but not rice mutants. However, transcript abundance for many cyclic electron transport (CET) and Calvin-Benson cycle components, as well as BS-specific C4 enzymes, was increased in maize mutants. Metabolite analysis of the maize ndh mutants revealed an increased NADPH : NADP ratio, as well as malate, ribulose 1,5-bisphosphate (RuBP), fructose 1,6-bisphosphate (FBP), and photorespiration intermediates. We suggest that by optimizing NADPH and malate levels and adjusting NADP-ME activity, NDH functions to balance metabolic and redox states in the BS cells of maize (in addition to ATP supply), coordinating photosynthetic transcript abundance and protein content, thus directly regulating the carbon flow in the two-celled C4 system of maize.

Lithium-associated primary hyperparathyroidism:: An evaluation of screening and referral patterns in a southeastern veteran population.

BACKGROUND: Long-term lithium therapy has a well-established but under-recognized association with primary hyperparathyroidism. Rates of hypercalcemia, screening for primary hyperparathyroidism, and referral for parathyroidectomy were evaluated among United States veterans on long-term lithium therapy. METHODS: Patients undergoing chronic long-term lithium therapy (>12 months) were identified from 1999 to 2022. Demographics, long-term lithium therapy duration, post-treatment calcium, parathyroid hormone, creatinine, and vitamin D levels were abstracted. Rates of screening for hypercalcemia (calcium ≥10.2 mg/dL), primary hyperparathyroidism (parathyroid hormone ≥30 pg/mL in the setting of hypercalcemia), referral for parathyroidectomy, and outcomes were evaluated. RESULTS: A total of 1,356 patients underwent long-term lithium therapy, 514 of whom received chronic long-term lithium therapy. Baseline characteristics of patients with and without post-treatment hypercalcemia were compared. Of 148 patients with post-treatment hypercalcemia, 112 (74.7%) underwent no further evaluation for primary hyperparathyroidism, while 36 (25.3%) patients had a parathyroid hormone level recorded. Although 33 (91.7%) hypercalcemic patients screened positive for primary hyperparathyroidism, only 5 (13%) were referred for parathyroidectomy. Of the 4 patients who underwent parathyroidectomy, mean calcium was 11.2 mg/dL (range 11.1-11.4), and mean parathyroid hormone was 272 pg/mL (range 108-622). Three patients were localized on preoperative imaging, 2 of whom underwent unilateral exploration with cure, with 1 experiencing recurrence at 31 months. The remaining patient who localized preoperatively underwent bilateral exploration and had 2 ipsilateral glands resected and persistence. The patient who did not localize preoperatively underwent bilateral exploration with 3 gland resection and cure. CONCLUSIONS: Screening for primary hyperparathyroidism and referral for parathyroidectomy are underutilized in United States veterans undergoing chronic long-term lithium therapy. Institutional protocols to standardize screening, surveillance, and referrals to endocrinology/endocrine surgery could benefit this population at increased risk for primary hyperparathyroidism.

Conserved cellular patterning in the mesophyll of rice leaves.

The mesophyll cells of grass leaves, such as rice, are traditionally viewed as displaying a relatively uniform pattern, in contrast to the clear distinctions of palisade and spongy layers in typical eudicot leaves. This quantitative analysis of mesophyll cell size and shape in rice leaves reveals that there is an inherent pattern in which cells in the middle layer of the mesophyll are larger and less circular and have a distinct orientation of their long axis compared to mesophyll cells in other layers. Moreover, this pattern was observed in a range of rice cultivars and species. The significance of this pattern with relation to potential photosynthetic function and the implication of the widespread use of middle layer mesophyll cells as typical of the rice leaf have been investigated and discussed.

Genetic and epigenetic features of bilateral Wilms tumor predisposition in patients from the Children's Oncology Group AREN18B5-Q.

Developing synchronous bilateral Wilms tumor suggests an underlying (epi)genetic predisposition. Here, we evaluate this predisposition in 68 patients using whole exome or genome sequencing (n = 85 tumors from 61 patients with matched germline blood DNA), RNA-seq (n = 99 tumors), and DNA methylation analysis (n = 61 peripheral blood, n = 29 non-diseased kidney, n = 99 tumors). We determine the predominant events for bilateral Wilms tumor predisposition: 1)pre-zygotic germline genetic variants readily detectable in blood DNA [WT1 (14.8%), NYNRIN (6.6%), TRIM28 (5%), and BRCA-related genes (5%)] or 2)post-zygotic epigenetic hypermethylation at 11p15.5 H19/ICR1 that may require analysis of multiple tissue types for diagnosis. Of 99 total tumor specimens, 16 (16.1%) have 11p15.5 normal retention of imprinting, 25 (25.2%) have 11p15.5 copy neutral loss of heterozygosity, and 58 (58.6%) have 11p15.5 H19/ICR1 epigenetic hypermethylation (loss of imprinting). Here, we ascertain the epigenetic and genetic modes of bilateral Wilms tumor predisposition.

CLIP-Seq analysis enables the design of protective ribosomal RNA bait oligonucleotides against C9ORF72 ALS/FTD poly-GR pathophysiology.

Amyotrophic lateral sclerosis and frontotemporal dementia patients with a hexanucleotide repeat expansion in C9ORF72 (C9-HRE) accumulate poly-GR and poly-PR aggregates. The pathogenicity of these arginine-rich dipeptide repeats (R-DPRs) is thought to be driven by their propensity to bind low-complexity domains of multivalent proteins. However, the ability of R-DPRs to bind native RNA and the significance of this interaction remain unclear. Here, we used computational and experimental approaches to characterize the physicochemical properties of R-DPRs and their interaction with RNA. We find that poly-GR predominantly binds ribosomal RNA (rRNA) in cells and exhibits an interaction that is predicted to be energetically stronger than that for associated ribosomal proteins. Critically, modified rRNA "bait" oligonucleotides restore poly-GR-associated ribosomal deficits and ameliorate poly-GR toxicity in patient neurons and Drosophila models. Our work strengthens the hypothesis that ribosomal function is impaired by R-DPRs, highlights a role for direct rRNA binding in mediating ribosomal dysfunction, and presents a strategy for protecting against C9-HRE pathophysiological mechanisms.

Beyond gene-disease validity: capturing structured data on inheritance, allelic requirement, disease-relevant variant classes, and disease mechanism for inherited cardiac conditions.

BACKGROUND: As the availability of genomic testing grows, variant interpretation will increasingly be performed by genomic generalists, rather than domain-specific experts. Demand is rising for laboratories to accurately classify variants in inherited cardiac condition (ICC) genes, including secondary findings. METHODS: We analyse evidence for inheritance patterns, allelic requirement, disease mechanism and disease-relevant variant classes for 65 ClinGen-curated ICC gene-disease pairs. We present this information for the first time in a structured dataset, CardiacG2P, and assess application in genomic variant filtering. RESULTS: For 36/65 gene-disease pairs, loss of function is not an established disease mechanism, and protein truncating variants are not known to be pathogenic. Using the CardiacG2P dataset as an initial variant filter allows for efficient variant prioritisation whilst maintaining a high sensitivity for retaining pathogenic variants compared with two other variant filtering approaches. CONCLUSIONS: Access to evidence-based structured data representing disease mechanism and allelic requirement aids variant filtering and analysis and is a pre-requisite for scalable genomic testing.

Same day discharge after minimally invasive adrenalectomy: a national study.

BACKGROUND: Same day discharge (SDD) may be considered in some patients undergoing minimally invasive adrenalectomy (MIA). Recent studies have demonstrated similar outcomes between SDD and admitted patients; however, most excluded pheochromocytoma and adrenal metastases. This study evaluates 30-day complications and hospital readmission in a large cohort of patients undergoing MIA. METHODS: Adult patients undergoing MIA (2010-2020) for benign adrenal disorders, pheochromocytoma, and adrenal metastases were identified within the ACS-NSQIP database. Comparisons between patients having SDD versus admission were performed. Factors associated with 30-day complications and unplanned readmission were evaluated by multivariable regression modeling. RESULTS: Of 7316 patients who underwent MIA, 254 had SDD. Baseline characteristics were similar between groups, although SDD patients had lower ASA class (p < 0.001) and were more likely to undergo MIA for nonfunctioning adenoma or primary aldosteronism (p = 0.001). After adjusting for covariates, higher ASA class and presence of medical comorbidities were associated with increased complications (p < 0.001; p < 0.05) and unplanned readmission (p < 0.001; p < 0.05). Additionally, prolonged operative time was associated with 30-day complications (p < 0.001). Notably, SDD was not associated with increased complications (OR 0.78, 95% CI 0.38-1.61, p = 0.502) or unplanned readmission (OR 0.76, 95% CI 0.35-1.64, p = 0.490). The rate of SDD for MIA increased from 1.48% in 2017 to 10.81% in 2020. CONCLUSIONS: Not all patients undergoing MIA should have SDD; however, the current analysis demonstrates a trend toward SDD and supports its safety in select patients with adrenal metastases and benign adrenal disorders including pheochromocytoma.

Lymph node metastases in young patients with gastrointestinal stromal tumor: A nationwide analysis.

BACKGROUND: Children, adolescents, and young adults (CAYA) (age ≤39 years) with GIST have high rates of LNM, but their clinical relevance is undefined. This study analyzed the impact of LNM on overall survival (OS) for CAYA with GIST. METHODS: The National Cancer Database was queried for patients with resected GIST and pathologic nodal staging data from 2004-2019. Factors associated with LNM were identified. Survival was assessed stratified by presence of LNM. RESULTS: Of 4420 patients with GIST, 238 were CAYA (5.4%). When compared to older adults, CAYA more often had small intestine primaries (51.8% vs. 36.6%, p < 0.0001), T4 tumors (30.7% vs. 24.5%, p = 0.0275) and pN1 disease (11.3% vs. 4.7%, p < 0.0001). Within a multivariable Cox proportional hazards regression model adjusting for age, comorbid disease, mitotic rate, tumor size, and primary site, LNM were associated with increased hazard of death for older adults (hazard ratio [HR]: 1.83; confidence interval [CI]: 1.35-2.42; p < 0.0001), but not CAYA (HR: 3.38; CI: 0.50-14.08; p = 0.13). For CAYA, only high mitotic rate predicted mortality (HR: 4.68; CI: 1.41-18.37: p = 0.02). CONCLUSIONS: LNM are more commonly identified among CAYA with resected GIST who undergo lymph node evaluations, but do not appear to impact OS as observed in older adults. High mitotic rate remains a predictor of poor outcomes for CAYA with GIST.

Segmental bile duct resection versus pancreatoduodenectomy for middle and distal third bile duct cancer. A systematic review and meta-analysis of comparative studies.

INTRODUCTION: Data regarding oncologic outcomes of segmental bile duct resection (SBDR) versus pancreatoduodenectomy (PD) for bile duct cancers (BDC) are conflicting. We compared SBDR and PD for BDC utilizing pooled data analysis. MATERIALS AND METHODS: A comprehensive PRISMA 2020 systematic review was performed. Studies comparing SBDR with PD for BDC were included. Pooled mean differences (MD), odds ratios (OR), and risk ratios (RR) with 95% confidence intervals (CI) were calculated. Subgroup analyses were performed. Study quality, bias, heterogeneity, and certainty were analyzed. RESULTS: Twelve studies from 2004 to 2021 were included, comprising 533 SBDR and 1,313 PD. SBDR was associated with positive proximal duct margins (OR 1.56; CI 1.11-2.18; P = .01), and distal duct margins (OR 43.25; CI 10.38-180.16; P < .01). SBDR yielded fewer lymph nodes (MD -6.93 nodes; CI -9.72-4.15; P < .01) and detected fewer nodal metastases (OR 0.72; CI 0.55-0.94; P = .01). SBDR portended less perioperative morbidity (OR 0.31; CI 0.21-0.46; P < .01), but not mortality (OR 0.52; CI 0.20-1.32; P = .17). SBDR was associated with locoregional recurrences (OR 1.88; CI 1.01-3.53; P = .02), and lymph node recurrences (OR 2.13; CI 1.42-3.2; P = .04). SBDR yielded decreased 5-year OS (OR 0.75; CI 0.65-0.85; P < .01). CONCLUSIONS: Despite decreased perioperative morbidity, SBDR appears to provide inferior oncologic control for BDC.