A subset of FG-nucleoporins is necessary for efficient Msn5-mediated nuclear protein export.

The transport of proteins between the cytoplasm and nucleus requires interactions between soluble transport receptors (karyopherins) and phenylalanine-glycine (FG) repeat domains on nuclear pore complex proteins (nucleoporins). However, the role of specific FG repeat-containing nucleoporins in nuclear protein export has not been carefully investigated. We have developed a novel kinetic assay to investigate the relative export kinetics mediated by the karyopherin Msn5/Kap142 in yeast containing specific FG-Nup mutations. Using the Msn5 substrate Crz1 as a marker for Msn5-mediated protein export, we observe that deletions of NUP100 or NUP2 result in decreased rates of Crz1 export, while nup60Δ and nup42Δ mutants do not vary significantly from wild type. The decreased Msn5 export rate in nup100Δ was confirmed using Mig1-GFP as a transport substrate. A nup100ΔGLFG mutant shows defects in nuclear export kinetics similar to a nup100Δ deletion. Removal of FG-repeats from Nsp1 also decreases export kinetics, while a loss of Nup1 FXFGs does not. To confirm that our export data reflected functional differences in protein localization, we performed Crz1 transcription activation assays using a CDRE::LacZ reporter gene that is upregulated upon increased transcription activation by Crz1 in vivo. We observe that expression from this reporter increases in nup100ΔGLFG and nsp1ΔFGΔFXFG strains that exhibit decreased Crz1 export kinetics but resembles wild-type levels in nup1ΔFXFG strains that do not exhibit export defects. These data provide evidence that the export of Msn5 is likely mediated by a specific subset of FG-Nups and that the GLFG repeat domain of Nup100 is important for Msn5-mediated nuclear protein export.

A mosaic activating mutation in AKT1 associated with the Proteus syndrome.

BACKGROUND: The Proteus syndrome is characterized by the overgrowth of skin, connective tissue, brain, and other tissues. It has been hypothesized that the syndrome is caused by somatic mosaicism for a mutation that is lethal in the nonmosaic state. METHODS: We performed exome sequencing of DNA from biopsy samples obtained from patients with the Proteus syndrome and compared the resultant DNA sequences with those of unaffected tissues obtained from the same patients. We confirmed and extended an observed association, using a custom restriction-enzyme assay to analyze the DNA in 158 samples from 29 patients with the Proteus syndrome. We then assayed activation of the AKT protein in affected tissues, using phosphorylation-specific antibodies on Western blots. RESULTS: Of 29 patients with the Proteus syndrome, 26 had a somatic activating mutation (c.49G→A, p.Glu17Lys) in the oncogene AKT1, encoding the AKT1 kinase, an enzyme known to mediate processes such as cell proliferation and apoptosis. Tissues and cell lines from patients with the Proteus syndrome harbored admixtures of mutant alleles that ranged from 1% to approximately 50%. Mutant cell lines showed greater AKT phosphorylation than did control cell lines. A pair of single-cell clones that were established from the same starting culture and differed with respect to their mutation status had different levels of AKT phosphorylation. CONCLUSIONS: The Proteus syndrome is caused by a somatic activating mutation in AKT1, proving the hypothesis of somatic mosaicism and implicating activation of the PI3K-AKT pathway in the characteristic clinical findings of overgrowth and tumor susceptibility in this disorder. (Funded by the Intramural Research Program of the National Human Genome Research Institute.).

Pilot study of peripheral internal jugular venous catheters on a hospitalist-run medicine procedure service.

Providers caring for hospitalized patients with difficult intravenous access (DIVA) frequently use central venous catheters (CVCs). One potential alternative is a peripheral internal jugular (PIJ) catheter, which is less traumatic to place and has fewer lumens than a CVC. We describe the results of 2 years' experience from a pilot project of a medicine procedure service placing PIJ catheters in hospitalized patients with DIVA. We successfully placed 34/35 (97%) PIJ catheters in 32 patients with zero complications. Median duration of use was 2.5 days (range 0-53 days, IQR 1-5). Catheter failure rate within 7 days was 32.4%, though it varied across catheter types: 9.5% in 8-10 cm midline catheters versus 69.2% (p < .001) in 6 cm angiocatheter wire introducers or shorter peripheral intravenous catheters. Our results suggest that PIJ catheters may be an option to reduce the mechanical and infectious risks associated with CVCs in some hospitalized patients with DIVA.

Real-world Accuracy of CGM in Inpatient Critical and Noncritical Care Settings at a Safety-Net Hospital.

OBJECTIVE: We sought to determine real-world accuracy of inpatient continuous glucose monitoring (CGM) at multiple levels of acuity in a large safety-net hospital. RESEARCH DESIGN AND METHODS: We analyzed records from hospitalized patients on Dexcom G6 CGM, including clinical, point of care (POC), and laboratory (Lab) glucose, and CGM data. POC/Lab values were matched to the closest timed CGM value. Encounters were divided into not critically ill (NCI) versus critically ill (CI). CGM accuracy was evaluated. RESULTS: Paired readings (2,744 POC-CGM; 3,705 Lab-CGM) were analyzed for 233 patients with 239 encounters (83 NCI, 156 CI). POC-CGM aggregated and average mean absolute relative differences (MARD) were 15.1% and 17.1%. Lab-CGM aggregated and average MARDs were 11.4% and 12.2%. Accuracy for POC-CGM and Lab-CGM was 96.5% and 99.1% in Clarke Error Grid zones A/B. CONCLUSIONS: Real-world accuracy of inpatient CGM is acceptable for NCI and CI patients. Further exploration of conditions associated with lower CGM accuracy in real-world settings is warranted.

Glycerol as a precursor for hepatic de novo glutathione synthesis in human liver.

BACKGROUND: Glycerol is a substrate for gluconeogenesis and fatty acid esterification in the liver, processes which are upregulated in obesity and may contribute to excess fat accumulation. Glycine and glutamate, in addition to cysteine, are components of glutathione, the major antioxidant in the liver. In principle, glycerol could be incorporated into glutathione via the TCA cycle or 3-phosphoglycerate, but it is unknown whether glycerol contributes to hepatic de novo glutathione biosynthesis. METHODS: Glycerol metabolism to hepatic metabolic products including glutathione was examined in the liver from adolescents undergoing bariatric surgery. Participants received oral [U-13C3]glycerol (50 mg/kg) prior to surgery and liver tissue (0.2-0.7g) was obtained during surgery. Glutathione, amino acids, and other water-soluble metabolites were extracted from the liver tissue and isotopomers were quantified with nuclear magnetic resonance spectroscopy. RESULTS: Data were collected from 8 participants (2 male, 6 female; age 17.1 years [range 14-19]; BMI 47.4 kg/m2 [range 41.3-63.3]). The concentrations of free glutamate, cysteine, and glycine were similar among participants, and so were the fractions of 13C-labeled glutamate and glycine derived from [U-13C3]glycerol. The signals from all component amino acids of glutathione - glutamate, cysteine and glycine - were strong and analyzed to obtain the relative concentrations of the antioxidant in the liver. The signals from glutathione containing [13C2]glycine or [13C2]glutamate derived from the [U-13C3]glycerol drink were readily detected, and 13C-labelling patterns in the moieties were consistent with the patterns in corresponding free amino acids from the de novo glutathione synthesis pathway. The newly synthesized glutathione with [U-13C3]glycerol trended to be lower in obese adolescents with liver pathology. CONCLUSIONS: This is the first report of glycerol incorporation into glutathione through glycine or glutamate metabolism in human liver. This could represent a compensatory mechanism to increase glutathione in the setting of excess glycerol delivery to the liver.

Safety and predictors of the success of lumbar punctures performed by a medicine procedure service.

BACKGROUND: Medicine procedure services (MPS) increasingly perform bedside procedures, including lumbar punctures (LPs). Success rates and factors associated with LP success performed by MPS have not been well described. OBJECTIVE: We identified patients undergoing LP by an MPS September 2015 to December 2020. We identified demographic and clinical factors, including patient position, body mass index (BMI), use of ultrasound, and trainee participation. We performed multivariable analysis to identify factors associated with LP success and complications. MAIN OUTCOME AND MEASURES: We identified 1065 LPs among 844 patients. Trainees participated in 82.2%; ultrasound guidance was used in 76.7% of LPs. The overall success rate was 81.3% with 7.8% minor and 0.1% major complications. A minority of LPs were referred to radiology (15.2%) or were traumatic (11.1%). In multivariable analysis, BMI > 30 kg/m2 (odds ratio [OR] 0.32, 95% confidence interval [CI] 0.21-0.48), prior spinal surgery (OR 0.50, 95% CI 0.26-0.87), and Black race (OR 0.62, 95% CI 0.41-0.95) were associated with decreased odds of successful LP; trainee participation (OR 2.49, 95% CI 1.51-4.12) was associated with increased odds. Ultrasound guidance (OR 0.53, 95% CI 0.31-0.89) was associated with lower odds of traumatic LP. RESULTS: In a large cohort of patients undergoing LP by an MPS, we identified high success and low complication rates. Trainee participation was associated with increased odds of success, while obesity, prior spinal surgery, and Black race were associated with decreased odds of success. Ultrasound guidance was associated with lower odds of a traumatic LP. Our data may help proceduralists in planning and assist in shared decision-making.

Hypoglycemia in children and young adults with cystic fibrosis during oral glucose tolerance testing vs. continuous glucose monitoring.

BACKGROUND: Hypoglycemia is common in people with cystic fibrosis (pwCF) during oral glucose tolerance tests (OGTTs) and in the free-living setting, yet its pathophysiology remains unclear. OBJECTIVE: To evaluate hypoglycemia in children and young adults with CF by OGTT and continuous glucose monitoring (CGM). METHODS: A 3-h OGTT was performed in children and young adults with CF and healthy controls (HC). Individuals were classified as experiencing hypoglycemia on OGTT (glucose <70 mg/dL) or not. Insulin, C-peptide, glucose, glucagon, and incretins were measured. CGM was performed for 7 days in the free-living setting. Measures of insulin sensitivity, beta cell function accounting for insulin sensitivity, and insulin clearance were calculated. RESULTS: A total of 57 participants (40 CF and 17 HC) underwent assessment. Rates of hypoglycemia by OGTT were similar in pwCF (53%, 21/40) compared to HC (35%, 6/17), p = 0.23. PwCF compared to HC had higher A1c; on OGTT higher and later glucose peaks, later insulin peaks; and on CGM more glucose variability. CF Hypo+ versus CF Hypo- had higher lung function, higher insulin sensitivity, higher beta cell function accounting for insulin sensitivity, and decreased CGM variability. When comparing CF Hypo+ to HC Hypo+, although rates of hypoglycemia are similar, pwCF had blunted glucagon responses to hypoglycemia. OGTT hypoglycemia was not associated with CGM hypoglycemia in any group. CONCLUSION: Youth with CF have increased insulin sensitivity and impaired glucagon response to hypoglycemia on OGTT. Hypoglycemia on OGTT did not associate with free-living hypoglycemia.

Brave new E-world: Medical students' preferences for and usage of electronic learning resources during two different phases of their education.

E-learning strategies have become an important part of biomedical education. However, why and how medical students select hardware tools and software formats during their preclinical education has not been sufficiently evaluated. These aspects should be considered when designing or offering new e-learning modalities to learners. Two medical school classes at a major US medical school were surveyed about their use of e-learning resources during their first year of medical school or their preparation for their first licensing examination (USMLE® Step 1), respectively. Their responses were analyzed for patterns and significant changes. Students' answers indicated that computers and tablets were considered the most important hardware devices to support students' learning. During the first year, students often preferred resources that were tailored to the specific courses in their curriculum. In contrast, some preferences changed when students prepared for the USMLE Step 1, with students shifting almost exclusively to a solitary learning strategy using commercial e-learning resources. Across all phases of medical school education queried, peer advice was the major determinant influencing e-learning resource selection with faculty only playing a minor role. Videos were the most popular e-learning modality, and students cited efficient acquisition of knowledge and preparation for examinations as major reasons for e-learning tool utilization. These factors should be considered when offering e-learning resources to medical students during different phases of their preclinical training.

Participation in varsity sports, dance, and Greek life associated with menstrual disturbance in a national sample of female college students.

Objective: The objective of this study was to identify university-sponsored activities associated with increased odds of menstrual disturbance among female college students. Participants: The sample included 3,277 female-identifying students at 12 colleges and universities participating in the Healthy Bodies Study during the 2013-2014 and 2014-2015 academic years. Methods: Crude and adjusted logistic regression models were run to report odds of any menstrual disturbance and amenorrhea (AMN) for participants in sports, dance, and Greek Life. Survey weights were used to account for response bias. Results: In the adjusted models, participation in varsity sport (OR = 1.82, CI 1.37, 2.37), dance (OR = 1.68, CI 1.37, 2.05) and Greek life (OR= 2.12, CI 0.13,0.18) was significantly associated with increased odds of menstrual disturbance, compared to non-participants. Conclusions: Findings suggest additional need for prevention and intervention programming against menstrual disturbance in varsity sports, dance, and Greek life activities.

A Novel Intronic Pathogenic Variant in STAR With a Dominant Negative Mechanism Causes Attenuated Lipoid Congenital Adrenal Hyperplasia.

Lipoid congenital adrenal hyperplasia (LCAH) is typically inherited as an autosomal recessive condition. There are 3 reports of individuals with a dominantly acting heterozygous variant leading to a clinically significant phenotype. We report a 46,XY child with a novel heterozygous intronic variant in STAR resulting in LCAH with an attenuated genital phenotype. The patient presented with neonatal hypoglycemia and had descended testes with a fused scrotum and small phallus. Evaluation revealed primary adrenal insufficiency with deficiencies of cortisol, aldosterone, and androgens. He was found to have a de novo heterozygous novel variant in STAR: c.65-2A>C. We report a case of a novel variant and review of other dominant mutations at the same position in the literature. Clinicians should be aware of the possibility of attenuated genital phenotypes of LCAH and the contribution of de novo variants in STAR at c.65-2 to the pathogenesis of that phenotype.

Markers of Low-Iron Status Are Associated with Female Athlete Triad Risk Factors.

PURPOSE: The Female Athlete Triad (Triad) is common in female athletes. The Triad is caused by low-energy availability (EA), which is often difficult to measure and has been postulated to be associated with low-iron status. Here, we explore whether markers of low-iron status may be associated with indicators of low EA including Triad risk factors. METHODS: A total of 239 female National Collegiate Athletic Association Division I athletes completed preparticipation examinations that included Triad risk factors, medication/supplement use, diagnosis of anemia, and elected to complete dual-energy x-ray absorptiometry scan to measure bone mineral density. The association of markers of low iron (defined as self-report of iron supplementation and/or history of anemia) with each component of the Triad risk assessment score was assessed by stratifying low-iron status across different levels of Triad risk category. Differences across iron status groups were assessed using Fisher exact testing. RESULTS: Every component of the Triad risk assessment score excluding delayed menarche was associated with low-iron status. The proportion of women who reported low iron was 11.5% in the low-risk EA group compared with 50% in the moderate-risk and 66.7% in the high-risk EA groups (P = 0.02); respectively. These numbers were 11.6%, 25.0%, and 66.7% (P = 0.02) for body mass index; 9.7%, 16.7%, and 25.0% (P < 0.05) for oligomenorrhea; 10.3%, 45.5%, and 50.0% (P < 0.01) for bone mineral density; and 10.4%, 20.8%, and 30.8% (P = 0.03) for history of stress reaction or fracture. Lean/endurance athletes were more likely to have low-iron status than other athletes (15.5% vs 3.4%, P = 0.02). CONCLUSIONS: Markers for low-iron status were associated with Triad risk factors. Our study suggests that female athletes with a history of anemia or iron supplementation may require further screening for low EA.

EGFR mutations cause a lethal syndrome of epithelial dysfunction with progeroid features.

The epidermal growth factor receptor (EGFR) is part of a large family of receptors required for communicating extracellular signals through internal tyrosine kinases. Epidermal growth factor (EGF) signaling is required for tissue development, whereas constitutive activation of this signaling pathway is associated with oncogenic transformation. We identified homozygous c.1283G>A (p.Gly428Asp) mutations in the extracellular domain of EGFR in two siblings. The children were born prematurely, had abnormalities in skin and hair, suffered multisystem organ failure, and died in the neonatal period from intestinal perforation. EGF failed to induce mutated receptor phosphorylation in patient-derived fibroblasts and activation of downstream targets was suppressed. The heterologously expressed extracellular domain was impaired in stability and the binding of EGF. Cells from the affected patient undergo early senescence with accelerated expression of ß-galactosidase and shortened telomeres at all passages when compared to controls. A comparison of homozygous inherited regions from a separate report of a patient from the same ethnic background and EGFR genotype confirms the pathogenicity of EGFR mutations in congenital disease.