Endogenously produced itaconate negatively regulates innate-driven cytokine production and drives global ubiquitination in human macrophages.

A wide variety of electrophilic derivatives of itaconate, the Kreb's cycle-derived metabolite, are immunomodulatory, yet these derivatives have overlapping and sometimes contradictory activities. Therefore, we generated a genetic system to interrogate the immunomodulatory functions of endogenously produced itaconate in human macrophages. Endogenous itaconate is driven by multiple innate signals restraining inflammatory cytokine production. Endogenous itaconate directly targets cysteine 13 in IRAK4 (disrupting IRAK4 autophosphorylation and activation), drives the degradation of nuclear factor κB, and modulates global ubiquitination patterns. As a result, cells unable to make itaconate overproduce inflammatory cytokines such as tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and IL-1ß in response to these innate activators. In contrast, the production of interferon (IFN)ß, downstream of LPS, requires the production of itaconate. These data demonstrate that itaconate is a critical arbiter of inflammatory cytokine production downstream of multiple innate signaling pathways, laying the groundwork for the development of itaconate mimetics for the treatment of autoimmunity.

Earth-Moon dynamics from cyclostratigraphy reveals possible ocean tide resonance in the Mesoproterozoic era.

Cyclostratigraphy is an important observational window into the history of the Earth-Moon system. However, there is limited information from the Mesoproterozoic era (1.0 to 1.6 billion years ago); accordingly, only weak constraints on Earth-Moon separation and tidal dissipation are available for this time. To close this knowledge gap, we analyze cyclostratigraphy from the Yemahe Formation (~1.2 billion years ago), Wumishan Formation (~1.5 billion years ago), and Chuanlinggou Formation (~1.6 billion years ago) in China. We use a Bayesian inversion method to analyze the three cyclostratigraphic sections. We combine previous results with these three estimates to construct an updated Earth-Moon system evolution and tidal dissipation history after 2.5 billion years ago. The results show a tidal dissipation peak that is consistent with the model predictions within the error range but also that there may be an additional resonance fluctuation in the Mesoproterozoic era.

Isomeric Effects of Au28(S-c-C6H11)20 Nanoclusters on Photoluminescence: Roles of Electron-Vibration Coupling and Higher Triplet State.

The exploration of near-infrared photoluminescence (PL) from atomically precise nanoclusters is currently a prominent area of interest owing to its importance in both fundamental research and diverse applications. In this work, we investigate the near-infrared (NIR) photoluminescence mechanisms of two structural isomers of atomically precise gold nanoclusters of 28 atoms protected by cyclohexanethiolate (CHT) ligands, i.e., Au28i(CHT)20 and Au28ii(CHT)20. Based on their structures, analysis of 3O2 (triplet oxygen) quenching of the nanocluster triplet states, temperature-dependent photophysical studies, and theoretical calculations, we have elucidated the intricate processes governing the photoluminescence of these isomeric nanoclusters. For Au28i(CHT)20, its emission characteristics are identified as phosphorescence plus thermally activated delayed fluorescence (TADF) with a PL quantum yield (PLQY) of 0.3% in dichloromethane under ambient conditions. In contrast, the Au28ii(CHT)20 isomer exhibits exclusive phosphorescence with a PLQY of 3.7% in dichloromethane under ambient conditions. Theoretical simulations reveal a larger singlet (S1)-triplet (T1) gap in Au28ii than that in Au28i, and the higher T2 state plays a critical role in both isomers' photophysical processes. The insights derived from this investigation not only contribute to a more profound comprehension of the fundamental principles underlying the photoluminescence of atomically precise gold nanoclusters but also provide avenues for tailoring their optical properties for diverse applications.

Scarless Baculovirus Genome Editing Using Lambda-Red Recombineering in E. coli.

Baculoviruses are widely used for their potential as biological pesticide and as platform for the production of recombinant proteins and gene therapy vectors. The Baculovirus Expression Vector System (BEVS) is used for high level of expression of (multiple) proteins in insect cells. Baculovirus recombinants can be quickly constructed by transposition of the gene(s) of interest into a so-called bacmid, which is a baculovirus infectious clone maintained as single-copy, bacterial artificial chromosome in Escherichia coli. A two-step homologous recombineering technique using the lambda-red system in E. coli allows for scarless editing of the bacmid with PCR products based on sequence homology. In the first step, a selection cassette with 50 bp homology arms, typically generated by PCR, is inserted into the designated locus. In the second step, the selection cassette is removed based on a negative selection marker, such as SacB or rpsL. This lambda-red recombineering technique can be used for multiple gene editing purposes, including (large) deletions, insertions, and even single point mutations. Moreover, since there are no remnants of the editing process, successive modifications of the same bacmid are possible. This chapter provides detailed instructions to design and perform two-step homologous recombineering of baculovirus bacmid DNA in E. coli. We present two case studies demonstrating the utility of this technique for creating a deletion mutant of the chitinase and cathepsin genes and for introducing a single point mutation in the baculovirus gene gp41. This scarless genome editing approach can facilitate functional studies of baculovirus genes and improve the production of recombinant proteins using the BEVS.

Distinct Plasma Metabolomic and Gut Microbiome Profiles after Gestational Diabetes Mellitus Diet Treatment: Implications for Personalized Dietary Interventions.

Gestational diabetes mellitus (GDM) triggers alterations in the maternal microbiome. Alongside metabolic shifts, microbial products may impact clinical factors and influence pregnancy outcomes. We investigated maternal microbiome-metabolomic changes, including over 600 metabolites from a subset of the "Choosing Healthy Options in Carbohydrate Energy" (CHOICE) study. Women diagnosed with GDM were randomized to a diet higher in complex carbohydrates (CHOICE, n = 18, 60% complex carbohydrate/25% fat/15% protein) or a conventional GDM diet (CONV, n = 16, 40% carbohydrate/45% fat/15% protein). All meals were provided. Diets were eucaloric, and fiber content was similar. CHOICE was associated with increases in trimethylamine N-oxide, indoxyl sulfate, and several triglycerides, while CONV was associated with hippuric acid, betaine, and indole propionic acid, suggestive of a healthier metabolome. Conversely, the microbiome of CHOICE participants was enriched with carbohydrate metabolizing genes and beneficial taxa such as Bifidobacterium adolescentis, while CONV was associated with inflammatory pathways including antimicrobial resistance and lipopolysaccharide biosynthesis. We also identified latent metabolic groups not associated with diet: a metabolome associated with less of a decrease in fasting glucose, and another associated with relatively higher fasting triglycerides. Our results suggest that GDM diets produce specific microbial and metabolic responses during pregnancy, while host factors also play a role in triglycerides and glucose metabolism.

Technique for guttural pouch bead removal using a novel three-dimensional (3D)-printed instrument.

OBJECTIVE: The aim of the present study was to determine if a three-dimensional (3D)-printed instrument technique would improve lavage removal of plastic beads (guttural pouch [GP] chondroid mimics) through a dorsal pharyngeal recess (DPR) fenestration. We hypothesized that using a 3D-printed instrument placed through the DPR fenestration would remove more beads, reduce lavage time and incur less soft tissue damage than using a lavage tube control or instrument placement through the salpingopharyngeal ostium (SPO). STUDY DESIGN: Experimental cadaveric study. SAMPLE POPULATION: A total of 30 cadaveric equine heads. METHODS: DPR fenestration was performed using transendoscopic laser and 50 plastic 12 mm beads were placed into one GP of horse heads. Four removal procedures using a 3D-printed instrument or lavage tube control placed through the DPR fenestration or the SPO were compared. Number of beads removed and number of 2-min lavage cycles to recover ≥96% of beads or three consecutive no-yield cycles were recorded. Endoscopic soft tissue damage was graded. Data were compared by generalized estimating equations (GEE) model and Fisher's exact test (p < .05). RESULTS: More beads (median 48 beads; range 0-49) were removed faster (median 24 beads/cycle; range 12-50) using the 3D-printed instrument compared to control (median 6 beads; range 0-29, 0.66 beads/cycle, range 0-49). There was no difference between total beads removed or removal speed between placement sites. There was no difference in soft tissue damage between procedures. CONCLUSION: Our 3D-printed instrument enabled efficient plastic bead removal. CLINICAL SIGNIFICANCE: DPR fenestration and use of our 3D-printed instrument represents an alternative to current chondroid removal techniques, warranting investigation in clinical cases.

Gut microbial changes associated with obesity in youth with type 1 diabetes: a Pilot Study.

CONTEXT: Obesity is prevalent in type 1 diabetes (T1D) and is problematic with higher risk for diabetes complications. It is unknown to what extent gut microbiome changes are associated with obesity and T1D. OBJECTIVE: To describe the gut microbiome and microbial metabolite changes associated with obesity in T1D. We hypothesized significant gut microbial and metabolite differences in lean T1D youth (BMI: 5-<85%) vs. those with obesity (BMI: ≥95%). METHODS: We analyzed stool samples for gut microbial (using metagenomic shotgun sequencing) and short-chain fatty acid (SCFA) differences in lean (n=27) and obese (n=21) T1D youth in a pilot study. The mean±SD age was 15.3±2.2yrs, A1c 7.8±1.3%, diabetes duration 5.1±4.4yrs, 42.0% females, and 94.0% were White. RESULTS: Bacterial community composition showed between sample diversity differences (ß-diversity) by BMI group (p=0.013). There was a higher ratio of Prevotella to Bacteroides in the obese group (p=0.0058). There was a differential distribution of significantly abundant taxa in either the lean or obese groups, including increased relative abundance of Prevotella copri, among other taxa in the obese group. Functional profiling showed an upregulation of branched chain amino acid (BCAA) biosynthesis in the obese group and upregulation of BCAA degradation, tyrosine metabolism and secondary bile acid biosynthesis in the lean group. Stool SCFAs were higher in the obese versus the lean group (p<0.05 for all). CONCLUSIONS: Our findings identify a gut microbiome and microbial metabolite signature associated with obesity in T1D. These findings could help identify gut microbiome targeted therapies to manage obesity in T1D.

Effect of a peer-led emergency department behavioral intervention on non-fatal opioid overdose: 18-month outcome in the Navigator randomized controlled trial.

BACKGROUND AND AIMS: Emergency departments (EDs) provide an opportunity to identify people at risk of overdose and reduce the risk. We evaluated the effect of an ED behavioral intervention delivered by peer recovery support specialists (PRSSs) on non-fatal opioid overdose. DESIGN: Two-arm, randomized trial. SETTING: Two EDs in Rhode Island, USA. PARTICIPANTS: ED patients presenting with an opioid overdose, complications of opioid use disorder or a recent history of opioid overdose (November 2018-May 2021). Among 648 participants, the mean age was 36.9 years, 68.2% were male and 68.5% were White. INTERVENTION AND COMPARATOR: Participants were randomized to receive a behavioral intervention from a PRSS (n = 323) or a licensed clinical social worker (LICSW) (n = 325). PRSS and LICSW used evidence-based interviewing and intervention techniques, informed by their lived experience (PRSS) or clinical theory and practice (LICSW). MEASUREMENTS: We identified non-fatal opioid overdoses in the 18 months following the ED visit through linkage to statewide emergency medical services data using a validated case definition. The primary outcome was any non-fatal opioid overdose during the 18-month follow-up period. FINDINGS: Among 323 participants randomized to the PRSS arm, 81 (25.1%) had a non-fatal opioid overdose during follow-up, compared with 95 (29.2%) of 325 participants randomized to the LICSW arm (P = 0.24). There was no statistically significant difference in the effectiveness of randomization to the PRSS arm versus the LICSW arm on the risk of non-fatal opioid overdose, adjusting for the history of previous overdose (relative risk = 0.86, 95% confidence interval = 0.67-1.11). CONCLUSIONS: In Rhode Island, USA, over one-in-four emergency department patients at high risk of overdose experience a non-fatal opioid overdose in the 18 months post-discharge. We found no evidence that the risk of non-fatal opioid overdose differs for emergency department patients receiving a behavioral intervention from a peer recovery support specialist versus a licensed clinical social worker.

Widespread human exposure to ledanteviruses in Uganda: A population study.

Le Dantec virus (LDV), assigned to the species Ledantevirus ledantec, genus Ledantevirus, family Rhabdoviridae has been associated with human disease but has gone undetected since the 1970s. We describe the detection of LDV in a human case of undifferentiated fever in Uganda by metagenomic sequencing and demonstrate a serological response using ELISA and pseudotype neutralisation. By screening 997 individuals sampled in 2016, we show frequent exposure to ledanteviruses with 76% of individuals seropositive in Western Uganda, but lower seroprevalence in other areas. Serological cross-reactivity as measured by pseudotype-based neutralisation was confined to ledanteviruses, indicating population seropositivity may represent either exposure to LDV or related ledanteviruses. We also describe the discovery of a closely related ledantevirus in blood from the synanthropic rodent Mastomys erythroleucus. Ledantevirus infection is common in Uganda but is geographically heterogenous. Further surveys of patients presenting with acute fever are required to determine the contribution of these emerging viruses to febrile illness in Uganda.

Characterisation of extracellular vesicles in baculovirus infection of Spodoptera frugiperda cells.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an enveloped DNA virus of the Baculoviridae family. This baculovirus is widely exploited for the biological control of insect pest species and as an expression platform to produce recombinant proteins in insect cells. Extracellular vesicles (EVs) are secreted by all cells and are involved in key roles in many biological processes through their cargo consisting of proteins, RNA or DNA. In viral infections, EVs have been found to transfer both viral and cellular cargo that can elicit either a pro- or antiviral response in recipient cells. Here, small EVs (sEVs) released by Spodoptera frugiperda (Sf) insect cells were characterised for the first time. Using S. frugiperda (SfC1B5) cells stably expressing the baculovirus gp64, the viral envelope protein GP64 was shown to be incorporated into sEVs. Sf9 cells were also transfected with a bacmid AcMNPV genome lacking p6.9 (AcΔP6.9) to prevent budded virus production. The protein content of sEVs from both mock- and AcΔP6.9-transfected cells were analysed by mass spectrometry. In addition to GP64, viral proteins Ac-F, ME-53 and viral ubiquitin were identified, as well as many host proteins including TSG101-which may be useful as a protein marker for sEVs.

Insulin Management for Gestational and Type 2 Diabetes in Pregnancy.

Insulin is preferred as the first-line agent for glucose management of gestational diabetes mellitus and type 2 diabetes in pregnancy when nutritional and lifestyle modifications are unable to achieve pregnancy-specific glucose targets. Individual heterogeneity in defects of insulin secretion or sensitivity in liver and muscle, unique genetic influences on pregnancy glycemic regulation, and variable cultural and lifestyle behaviors that affect meal, activity, sleep, and occupational schedules necessitate a personalized approach to insulin regimens. Newer insulin preparations have been developed to mimic the physiologic release of endogenous insulin, maintaining appropriate basal levels to cover hepatic gluconeogenesis and simulate the rapid, meal-related, bolus rise of insulin. Such physiologic basal-bolus dosing of insulin can be administered safely, achieving tighter glycemic control while reducing episodes of hypoglycemia. Insulin initiation and titration require understanding the pharmacodynamics of different insulin preparations in addition to a patient's glycemic profiles, effect of variable nutritional intake and mealtimes, physical activity, stress, timing of sleep cycles, and cultural habits. Educating and empowering patients to learn how their glucose responds to insulin, portion and content of meals, and physical activity can increase personal engagement in therapy, flexibility in eating patterns, and improved glycemic control. This Clinical Expert Series article is focused on optimizing insulin management (initiation, dosing, and titration) of gestational and type 2 diabetes in pregnancy.

Bioavailability of Australian pre-schooler iron intakes at specific eating occasions is low.

PURPOSE: Poor bioavailability may contribute to iron deficiency among children in high-resource countries, but iron bioavailability of Australian pre-schooler diets is unknown. This study aimed to estimate the bioavailability of Australian pre-schooler iron intakes across the day and by eating occasions to identify optimal timing for intervention, by using five previously developed algorithms, and to estimate the proportion of children with intakes of absorbable iron below the requirements. METHODS: Dietary data of children aged 2 to < 6 y (n = 812) from the 2011-12 National Nutrition and Physical Activity Survey were collected via two 24-h recalls. Usual food and nutrient intakes were estimated via Multiple Source Method. Phytate, polyphenol, and heme iron values were sourced from international databases or the literature. Five previously published algorithms were applied to observed dietary data to estimate iron bioavailability and calculate the prevalence of children with intakes of absorbable iron below requirements. RESULTS: Pre-schooler daily iron bioavailability was low (2.7-10.5%) and corresponded to intakes of 0.18-0.75 mg/d of absorbable iron. The proportion of children with inadequate intakes of absorbable iron ranged between 32 and 98%. For all eating occasions, dinner offered iron of the greatest bioavailability (4.2-16.4%), while iron consumed at breakfast was of the lowest bioavailability (1.2-5.6%). CONCLUSION: Future strategies are required to improve intakes of bioavailable iron for pre-schoolers to prevent the risk of deficiency. These strategies could include the encouragement of concomitant consumption of enhancers of iron absorption with iron-rich sources, particularly at breakfast.

Tobacco quitline staffs' knowledge and attitudes about connecting quitline callers to lung cancer screening educational materials.

OBJECTIVE: To evaluate the impact of training quitline staff in lung cancer screening (LCS) on knowledge and attitudes towards connecting quitline callers to LCS educational materials. METHODS: We conducted a pre-post evaluation within a larger implementation project in the U.S. to support LCS among quitline callers. From July 2020 to June 2021, staff from four quitline service providers completed surveys before and after training on LCS knowledge. After training, staff completed the acceptability of intervention measure, intervention appropriateness measure, and feasibility of the intervention measure. RESULTS: A total of 245 staff completed the initial demographic survey (analytic sample), 130 completed the pre-training survey, and 225 completed the post-training survey. Staff were on average 47.4 years old and 76.7% were female. LCS knowledge improved after the training (n = 120, mean difference = +26.5%, 95% CI 21.6, 31.4, p < 0.001). Overall, staff felt that connecting quitline callers to LCS education materials was acceptable (M = 4.0, SD = 0.8), appropriate (M = 4.1, SD = 0.7), and feasible (M = 4.0, SD = 0.7). CONCLUSIONS: Receiving training about LCS eligibility and the benefits and harms of screening improved LCS knowledge among quitline staff. Quitline staff found that connecting callers with LCS educational materials is acceptable, appropriate, and feasible, and aligned with their primary mission.

University of Pennsylvania Telehealth Research Center of Excellence.

Drawing from insights from communication science and behavioral economics, the University of Pennsylvania Telehealth Research Center of Excellence (Penn TRACE) is designing and testing telehealth strategies with the potential to transform access to care, care quality, outcomes, health equity, and health-care efficiency across the cancer care continuum, with an emphasis on understanding mechanisms of action. Penn TRACE uses lung cancer care as an exemplar model for telehealth across the care continuum, from screening to treatment to survivorship. We bring together a diverse and interdisciplinary team of international experts and incorporate rapid-cycle approaches and mixed methods evaluation in all center projects. Our initiatives include a pragmatic sequential multiple assignment randomized trial to compare the effectiveness of telehealth strategies to increase shared decision-making for lung cancer screening and 2 pilot projects to test the effectiveness of telehealth to improve cancer care, identify multilevel mechanisms of action, and lay the foundation for future pragmatic trials. Penn TRACE aims to produce new fundamental knowledge and advance telehealth science in cancer care at Penn and nationally.

Characterization of medium and small-scale gold processing operations, wastewaters, and tailings in the Arequipa region of Peru.

Gold cyanidation facilities in the Arequipa Region of Peru are challenged by the availability and quality of water for processing in an arid environment. The facilities reuse decant water which recycles residual cyanide but also undesirable constituents. To understand the impact of intensive water recycling on cyanide and metals concentrations, we collected barren water, decant water, and tailings samples from six gold cyanidation facilities with ore capacities of 10-430 tons per day. Processing facilities in Arequipa recycle all effluents, with decant waters making up 58 ± 11 % of process waters. Decant water contained non-target metals: copper (394 ± 161 mg/L), iron (59 ± 34 mg/L), and zinc (74 ± 42 mg/L). In addition, decant water mean free and complexed cyanide concentrations were 534 ± 129 mg/L and 805 ± 297 mg/L, respectively. Complexed cyanide concentrations remained more constant than free cyanide concentrations with 786 ± 299 mg/L for barren water and 805 ± 297 mg/L for decant water. Cyanide mass balances showed between 21 % and 42 % of unaccounted free cyanide from the start of gold cyanidation and discharge to the tailings storage facility (TSF). Free cyanide estimated losses due to volatilization were 0.8 kg and 2.5 kg of hydrogen cyanide per ton of ore processed at barren water pH of 10.1 and 9.7. Together these results indicate two acute hazards: 1) volatilization of free cyanide during processing and 2) loading and retention of cyanides and metals into TSFs. This study elucidates the extent of uncontrolled vapor phase cyanide release during gold processing operation and contaminant concentrations in the tailings storage facilities. The data highlights the need for improvement oversight, accountability, and regulation of gold processing facilities practicing intensive recycling and zero discharge.

Alzheimer's-linked axonal changes accompany elevated antidromic action potential failure rate in aged mice.

Alzheimer's disease (AD) affects both grey and white matter (WM), but considerably more is known about the former. Interestingly, WM disruption has been consistently observed and thoroughly described using imaging modalities, particularly MRI which has shown WM functional disconnections between the hippocampus and other brain regions during AD pathogenesis when early neurodegeneration and synapse loss are also evident. Nonetheless, high-resolution structural and functional analyses of WM during AD pathogenesis remain scarce. Given the importance of the myelinated axons in the WM for conveying information across brain regions, such studies will provide valuable information on the cellular drivers and consequences of WM disruption that contribute to the characteristic cognitive decline of AD. Here, we employed a multi-scale approach to investigate hippocampal WM disruption during AD pathogenesis and determine whether hippocampal WM changes accompany the well-documented grey matter losses. Our data indicate that ultrastructural myelin disruption is elevated in the alveus in human AD cases and increases with age in 5xFAD mice. Unreliable action potential propagation and changes to sodium channel expression at the node of Ranvier co-emerged with this deterioration. These findings provide important insight to the neurobiological substrates and functional consequences of decreased WM integrity and are consistent with the notion that hippocampal disconnection contributes to cognitive changes in AD.

Do Substances Used in Adolescence Predict the Persistence of Substance Use Disorders in Adulthood? A 15-Year Study of Youth After Detention.

PURPOSE: Investigate if the type of substance use disorder (SUD) in adolescence predicts SUDs in adulthood and examine sex and racial/ethnic differences in the persistence of SUDs. METHODS: Data are from the Northwestern Juvenile Project, a 15-year longitudinal study of 1829 youth randomly sampled from detention in Chicago, IL (1995-1998). Interviewers assessed SUDs using structured diagnostic interviews. RESULTS: Compared with females without an SUD at detention, females with cannabis alone, comorbid alcohol and cannabis, or SUDs other than alcohol and cannabis at detention had higher odds of having an SUD 5 years later (25%, 32%, and 36% vs. 15%, adjusted odds ratio [AOR] = 1.94, 95% confidence interval [CI] 1.11-3.40; AOR = 2.76, 95% CI 1.58-4.83; AOR = 3.46, 95% CI 1.56-7.66, respectively). Males and females with SUDs other than alcohol and cannabis at detention had greater odds of having an SUD 15 years later, compared with those without an SUD at detention (males: 36% vs. 14%, AOR = 2.98, 95% CI 1.14-7.83; females: 29% vs. 8%, AOR = 4.77, 95% CI 1.85-12.30). Among youth with an SUD at detention, males were more likely than females to have an SUD 15 years later (AOR = 1.84, 95% CI 1.03-3.29); non-Hispanic White and Hispanic males were more likely to persist than Black males (AOR = 3.32, 95% CI 1.50-7.35; AOR = 2.32, 95% CI 1.04-5.18, respectively). DISCUSSION: The type of SUD during adolescence matters. Youth with SUDs such as cocaine and opioids fared the worst. Healthcare providers must collaborate with correctional officials to increase service provision.

Safety and prescribing recommendations for verapamil in newly diagnosed pediatric type 1 diabetes (T1D): The CLVer experience.

Objectives: To report the safety and side effects associated with taking verapamil for beta-cell preservation in children with newly-diagnosed T1D. Research Design and Methods: Eighty-eight participants aged 8.5 to 17.9 years weighing ≥ 30 kg were randomly assigned to verapamil (N = 47) or placebo (N = 41) within 31 days of T1D diagnosis and followed for 12 months from diagnosis, main CLVer study. Drug dosing was weight-based with incremental increases to full dosage. Side effect monitoring included serial measurements of pulse, blood pressure, liver enzymes, and electrocardiograms (ECGs). At study end, participants were enrolled in an observational extension study (CLVerEx), which is ongoing. No study drug is provided during the extension, but participants may use verapamil if prescribed by their diabetes care team. Results: Overall rates of adverse events were low and comparable between verapamil and placebo groups. There was no difference in the frequency of liver function abnormalities. Three CLVer participants reduced or discontinued medication due to asymptomatic ECG changes. One CLVerEx participant (18 years old), treated with placebo during CLVer, who had not had a monitoring ECG, experienced complete AV block with a severe hypotensive episode 6 weeks after reaching his maximum verapamil dose following an inadvertent double dose on the day of the event. Conclusions: The use of verapamil in youth newly-diagnosed with T1D appears generally safe and well tolerated with appropriate monitoring. We strongly recommend monitoring for potential side effects including an ECG at screening and an additional ECG once full dosage is reached.ClinicalTrials.gov number: NCT04233034.

Histopathological phenotyping of cancers in PTEN Hamartoma Tumor Syndrome for improved recognition: A single-center study.

PTEN hamartoma tumor syndrome (PHTS) has a broad clinical spectrum including various benign and malignant tumors at varying age of diagnosis. Many patients remain unrecognized, unaware of their increased cancer risk. We aimed to describe the cancer spectrum, age of onset and histopathological cancer characteristics to assess whether specific cancer characteristics could improve PHTS recognition. Genetic testing results and pathology reports were collected for patients tested for germline PTEN variants between 1997 and 2020 from the diagnostic laboratory and the Dutch nationwide pathology databank (Palga). The cancer spectrum and age of onset were assessed in patients with (PTENpos) and without (PTENneg) a germline PTEN variant. Histopathological cancer characteristics were assessed in a nested cohort. 341 PTENpos patients (56% females) and 2882 PTENneg patients (66% females) were included. PTENpos patients presented mostly with female breast (BC, 30%), endometrial (EC, 6%), thyroid (TC, 4%) or colorectal cancer (4%). PTENpos were significantly younger at cancer onset (43 vs. 47 years) and had more often (46% vs. 18%) a second BC than PTENneg. PTEN detection rates were highest for BC <40 years (9%), TC <20 years (15%) and EC <50 years (28%), and dropped to 6%, 4%, and 15% by age 60. Histopathological characteristics were similar between groups. No histopathological cancer characteristics were distinctive for PHTS. However, PTENpos were significantly younger at cancer onset. Therefore early-onset BC, EC, or TC warrants consideration of PHTS diagnostics either through a pre-screen for other PHTS features or direct germline testing.