Insights into Nitrosoalkane Binding to Myoglobin Provided by Crystallography of Wild-Type and Distal Pocket Mutant Derivatives.

Nitrosoalkanes (R-N═O; R = alkyl) are biological intermediates that form from the oxidative metabolism of various amine (RNH2) drugs or from the reduction of nitroorganics (RNO2). RNO compounds bind to and inhibit various heme proteins. However, structural information on the resulting Fe-RNO moieties remains limited. We report the preparation of ferrous wild-type and H64A sw MbII-RNO derivatives (λmax 424 nm; R = Me, Et, Pr, iPr) from the reactions of MbIII-H2O with dithionite and nitroalkanes. The apparent extent of formation of the wt Mb derivatives followed the order MeNO > EtNO > PrNO > iPrNO, whereas the order was the opposite for the H64A derivatives. Ferricyanide oxidation of the MbII-RNO derivatives resulted in the formation of the ferric MbIII-H2O precursors with loss of the RNO ligands. X-ray crystal structures of the wt MbII-RNO derivatives at 1.76-2.0 Å resoln. revealed N-binding of RNO to Fe and the presence of H-bonding interactions between the nitroso O-atoms and distal pocket His64. The nitroso O-atoms pointed in the general direction of the protein exterior, and the hydrophobic R groups pointed toward the protein interior. X-ray crystal structures for the H64A mutant derivatives were determined at 1.74-1.80 Å resoln. An analysis of the distal pocket amino acid surface landscape provided an explanation for the differences in ligand orientations adopted by the EtNO and PrNO ligands in their wt and H64A structures. Our results provide a good baseline for the structural analysis of RNO binding to heme proteins possessing small distal pockets.

Targeting RAS-ERK pathway alterations with MEK inhibitors to improve chemosensitivity in high grade serous ovarian cancers.

OBJECTIVE: Assess if MEK inhibitor blockade of RAS-ERK pathway adaptive response in high grade serous ovarian cancers (HGSOC) improves platinum sensitivity. METHODS: Three HGSOC cell lines and three patient derived organoid (PDOs) samples from ascites of platinum resistant HGSOC patients were collected. Cell lines and PDOs were exposed to carboplatin and MEK inhibitors cobimetinib or trametinib. Cytotoxic effects of MEK inhibitors alone or combined with carboplatin were established. Western blots demonstrated RAS-ERK pathway blockage after MEK inhibitor treatment. RNA sequencing assessed gene expression after MEK inhibitor treatment. Cell line NF1 gene knockdown was performed with corresponding chemosensitivity levels. RESULTS: High carboplatin IC50 levels indicated platinum resistance in cell lines and PDOs. Cobimetinib induced cytotoxicity in cell lines and PDOs, while trametinib was less effective. Western blot confirmed MEK-ERK pathway blockage at minimal concentrations of MEK inhibitors in cell lines and PDOs. Phosphorylated-ERK levels of untreated cells indicated higher levels of RAS-ERK pathway activation in OVSAHO and OVCAR7 compared to OVCAR3. OVSAHO harbors a NF1 mutation and had highest levels of RAS-ERK activation. Cotreatment with carboplatin and MEK inhibitors showed varying synergistic cytotoxic effects at different combinations. Synergistic effect was most prominent in the OVSAHO carboplatin and cobimetinib combination. RNA sequencing identified downregulation of c-MYC and FOXM1 gene expression after MEK inhibitor treatment. NF1 gene knockdown showed an acquired increased IC50 compared to parental cells. CONCLUSION: MEK inhibitors block RAS-ERK pathways in platinum resistant HGSOC cells and PDOs. MEK inhibitors with carboplatin have select synergistic effects which may indicate a strategy to improve platinum sensitivity.

Time to completion of radiation treatment in locally advanced squamous cell carcinoma of the vulva and the impact on survival.

OBJECTIVE: To assess whether radiation completion within a planned timeframe in locally advanced squamous cell vulvar cancer impacts overall survival (OS). METHODS: The National Cancer Database from 2004 to 2017 was used to identify women ≥18 years old with stage II-IVA squamous cell vulvar cancer. We included women who received radiation alone (RT) or concurrent chemoradiation (CRT) for initial vulvar cancer treatment. Primary outcome was overall survival associated with time of delay in radiation completion. RESULTS: There were 2378 women identified (n = 856 RT and n = 1522 CRT). Median age was 67 (IQR 56-78), majority (88.35%) were white with advanced stage III or IVA (72.29%) disease. Median radiation dose was 5720 c-Gray (IQR 5040-6300). Radiation completion with delay ≥7 days resulted in reduction in survival compared to delay of <7 days (unadjusted HR 1.183 [95%CI: 1.066-1.313], p = 0.0016). When delays extended to ≥14 days compared to <14 days there was increased hazard of death (unadjusted HR: 1.263 [95%CI:1.126-1.416], p < 0.0001). Survival improved for patients with <7 versus ≥7 days delay whether treatment was with RT (median OS: 34.9 months versus 21.6 months, p < 0.01) or CRT (Median OS:58 months versus 41.3 months, p < 0.01). Stage IVA disease was associated with the greatest increase in hazard of death (HR 1.759 [95%CI 1.517-2.039], p < 0.0001) compared to stage II. CONCLUSION: Radiation completion with <7 days delay is associated with improved overall survival, independent of concurrent chemotherapy. This suggest that strategies to minimize delays in radiation are crucial in locally advanced vulvar cancer.

Comparison of two Lynch screening strategies in endometrial cancer in a California health system.

OBJECTIVE: Compare detection of Lynch syndrome in endometrial cancer between regions of a health care system with different screening strategies. METHODS: A retrospective study of endometrial cancer (EC) cases from 2 regions of an integrated health care system (Kaiser Permanente Northern (KPNC) and Southern (KPSC) California). Within KPNC, immunohistochemistry tumor screening (IHC) was physician ordered and risk-based; within KPSC, IHC was universal and automated. Clinical risk factors associated with abnormal IHC and Lynch Syndrome (LS) were identified. RESULTS: During the study, there were 2045 endometrial cancers: 1399 in the physician-order group and 646 in the universal testing group. In the physician-order group: among women < age 60, 34% underwent IHC; 9.6% were abnormal, and 3% were possible LS after methylation testing; among women ≥60, 11% underwent IHC, 3% were abnormal and <1% were possible LS. In the universal group, 87% of women age <60 had IHC, 19.4% were abnormal, and 6% were possible LS; Among women age ≥60, 82% underwent IHC, 26% were abnormal, and 2% were possible LS. There were no differences in LS cases between the physician-order group and the universal group in either age strata (<60: 3% vs. 3.6%, p=0.62; ≥60: <1% vs. 1%, p=0.63) Factors associated with LS were younger age (odds ratio (OR) 0.11, 95% confidence interval (CI) 0.04-0.29) and lower body mass index (BMI), (OR 0.38 95% CI 0.18-0.80). CONCLUSIONS: Universal IHC screening did not result in increased LS detection in EC.

Alternative Treatment Utilization Before Hysterectomy for Benign Gynecologic Conditions at a Large Integrated Health System.

STUDY OBJECTIVE: To investigate rates of utilization of alternative treatments before hysterectomy for benign gynecologic indications within a large integrated health care system. DESIGN: Retrospective cohort study of patients who underwent hysterectomies for benign gynecologic conditions between 2012 and 2014 (Canadian Task Force classification II-2). SETTING: Kaiser Permanente Northern California, a community-based integrated health system. PATIENTS: Women who underwent hysterectomy for a benign gynecologic condition between 2012 and 2014. INTERVENTIONS: From an eligible cohort of 6892 patients who underwent hysterectomy, a stratified random sample of 1050 patients were selected for chart review. Stratification was based on the proportion of indications for hysterectomy. MEASUREMENTS AND MAIN RESULTS: The primary outcome was the use of alternative treatments before hysterectomy. Alternative treatments included oral hormone treatment, leuprolide, medroxyprogesterone intramuscular injections, a levonorgestrel intrauterine device, hormonal subdermal implants, endometrial ablation, uterine artery embolization, hysteroscopy, and myomectomy. Of the 1050 charts reviewed, 979 (93.2%) met the criteria for inclusion in this study. The predominant indication for hysterectomy was symptomatic myomas (54.4%), followed by abnormal uterine bleeding (29.0%), endometriosis (5.8%), pelvic pain (3.1%), dysmenorrhea (3.4%), and other (4.3%). The major routes of hysterectomy were laparoscopy (68.7%) and vaginal hysterectomy (13.4%). Before hysterectomy, 81.2% of patients tried at least 1 type of alternative treatment (33.8% with 1 treatment and 47.4% with at least 2 treatments), and 99.3% of patients were counseled regarding alternative treatments. Compared with younger women age <40 years, women age 45 to 49 years were less likely to use alternative treatments before hysterectomy (adjusted odds ratio, 0.41; 95% confidence interval, 0.21-0.76). There were no variations in treatment rates by socioeconomic status or between major racial and ethnic groups. The final pathological analysis identified myomas as the most common pathology (n = 637; 65.1%); 96 patients (9.8%) had normal uterine pathology. CONCLUSION: More than 80% of patients received alternative treatments before undergoing hysterectomy for a benign gynecologic condition. Additional investigation is warranted to assess alternative treatment use as it relates to preventing unnecessary hysterectomies.

Health Literacy Teaching in U.S. Family Medicine Residency Programs: A National Survey.

Health care providers, including medical residents, often lack adequate knowledge and skills to work effectively with patients who have limited health literacy. Little is known about the degree to which medical residents are trained to communicate effectively with people who have limited health literacy. This study aimed to assess the status of health literacy training for physicians in U.S. family medicine residency programs. We conducted an online survey of residency directors at 444 U.S. family medicine residencies. Among 138 respondents (31% response rate), 58 programs (42%) reported teaching residents about health literacy as part of the required curriculum. Most instruction occurred during the 1st year of training. Hours of instruction ranged from 2 to 5 during Years 1 through 3. Skills-based training (e.g., plain language techniques) was taught by most programs. Not having access to a faculty authority on health literacy was strongly associated with lack of a required health literacy curriculum. Respondents overwhelmingly agreed that increasing health literacy training for medical students and residents would help improve residents' clinical skills. This study provides a baseline snapshot of health literacy curricula in U.S. family medicine residencies and likely overestimates the prevalence of such curricula. Additional studies are needed to determine the quality of health literacy instruction in U.S. family medicine residencies and the most effective methods for teaching residents about health literacy.

Outcomes in pregnancies complicated by methamphetamine use.

OBJECTIVE: Methamphetamine use is widespread. Our goal was to examine the effects of methamphetamine use on various maternal and neonatal outcomes. STUDY DESIGN: We conducted a retrospective cohort study looking at all pregnancies between 2005 and 2008 in the state of California that were associated with a diagnosis of methamphetamine use. Outcomes examined included gestational hypertension, preeclampsia, preterm birth, small for gestational age, birthweight, abruption, intrauterine fetal death, neonatal death, infant death, jaundice, and gestational diabetes mellitus. Statistical analysis included chi-squared tests and multivariable logistic regression analyses. RESULTS: After adjustment for multiple confounding variables on multivariable regression analysis, results indicated that compared with control subjects, methamphetamine users had greater odds of gestational hypertension (odds ratio [OR], 1.8; 95% confidence interval [CI], 1.6-2.0), preeclampsia (OR, 2.7; 95% CI, 2.4-3.0), intrauterine fetal death (OR, 5.1; 95% CI, 3.7-7.2), and abruption (OR, 5.5; 95% CI, 4.9-6.3). Additionally, these patients had higher odds of preterm birth (OR, 2.9; 95% CI, 2.7-3.1), neonatal death (OR, 3.1; 95% CI, 2.3-4.2), and infant death (OR, 2.5; 95% CI, 1.7-3.7). CONCLUSION: Methamphetamine use in pregnancy was found to be associated with specific patterns of increased maternal and fetal morbidity and death. With these results in mind, further work can be done to improve the care of pregnancies that are complicated by methamphetamine use in hopes of reducing these complications.

Perspectives of gynecologic oncology fellowship training and preparedness for practice.

We aimed to examine the preparedness of recent gynecologic oncology fellowship graduates for independent practice.We conducted a web-based survey study using REDCap targeting Society of Gynecologic Oncology (SGO) members who graduated gynecologic oncology fellowship within the last six years. The survey included 52 items assessing fellowship training experiences, level of comfort in performing core gynecologic oncology surgical procedures and administering cancer-directed therapies. Questions also addressed factors driving participants' selection of fellowship programs, educational experience, research and preparedness for independent practice. A total of 296 participants were invited to complete the survey. Response rate was 42% with n = 124 completed surveys included for analysis. The highest ranked factor for fellowship selection was fit with program 36% (n = 45). Upon completing fellowship, most were uncomfortable performing ureteral conduit formation 84% (n = 103), ureteroneocystostomy 77% (n = 94), exenteration 68% (n = 83), splenectomy 67% (n = 83) and lower anterior resection 41% (n = 51). Most were comfortable managing intraoperative complications 85% (n = 104) and standard cancer staging procedures (range: 61%-99%). Majority were comfortable providing cancer directed therapies with chemotherapy 99% (n = 123), immunotherapy 84% (n = 104), and poly ADP-ribose polymerase (PARP) inhibitors 97% (n = 120). Upon completing fellowship, 77% (n = 95) report having mentorship that met their expectations during fellowship and 94% (n = 116) felt they were ready for independent practice. Majority of fellowship graduates were prepared for independent practice and felt comfortable performing routine surgical procedures and cancer directed treatment. However, most are not comfortable with ultra-radical gynecologic oncology procedures. Maximizing surgical opportunities during fellowship training and acquiring early career mentorship may help.

How should the margin of stability during walking be expressed to account for body size?

When expressing the margin of stability as a distance, it does not directly estimate the perturbation magnitude needed to change stability states. Additionally, it is unknown how body size may influence this measure. Therefore, we propose other expressions of stability margins, including that of an impulse, a change in center of mass velocity, and a scaled, unitless impulse. The purpose of this study was to determine the influence of body size on these margin expressions using walking data from children and adults. We anticipated that margins expressed as an impulse would have strong correlations with body mass and height, as well as large between-group differences. We predicted that scaling this impulse value would result in small correlations and between-group effect sizes. We calculated each stability margin at minimum lateral values and in the anteroposterior directions at mid-swing and foot strike. In the lateral direction, margins expressed as an impulse had strong correlations with body size (r≥0.58, p<0.01) and large between-group differences (|d|≥1.07, p<0.01). The other expressions did not have strong positive correlations (|r|≤0.20) or large between-group effects (|d|≤0.44). In the anteroposterior directions, impulse margins had strong correlations with body size (|r|≥0.83, p<0.01) and large between-group differences (|d|≥1.74, p<0.01). The scaled, unitless impulse margin was the only variable that resulted in small, non-significant differences (|r|≤0.22, p≥0.24) as well as small between-group effect sizes (|d|≤0.46, p≥0.22). We propose expressing stability margins as an impulse. If scaling is needed, we encourage using the scaled, unitless impulse.

Crystal structural investigations of heme protein derivatives resulting from reactions of aryl- and alkylhydroxylamines with human hemoglobin.

Phenylhydroxylamine (PhNHOH) and nitrosobenzene (PhNO) interact with human tetrameric hemoglobin (Hb) to form the nitrosobenzene adduct Hb(PhNO). These interactions also frequently lead to methemoglobin formation in red blood cells. We utilize UV-vis spectroscopy and X-ray crystallography to identify the primary and secondary products that form when PhNHOH and related alkylhydroxylamines (RNHOH; R = Me, t-Bu) react with human ferric Hb. We show that with MeNHOH, the primary product is Hb[α-FeIII(H2O)][ß-FeII(MeNO)], in which nitrosomethane is bound to the ß subunit but not the α subunit. Attempts to isolate a nitrosochloramphenicol (CAMNO) adduct resulted in our isolation of a Hb[α-FeII][ß-FeII-cySOx]{CAMNO} product (cySOx = oxidized cysteine) in which CAMNO was located outside of the protein in the solvent region between the ß2 and α2 subunits of the same tetramer. We also observed that the ßcys93 residue had been oxidized. In the case of t-BuNHOH, we demonstrate that the isolated product is the ß-hemichrome Hb[α-FeIII(H2O)][ß-FeIII(His)2]{t-BuNHOH}, in which the ß heme has slipped ∼4.4 Å towards the solvent exterior to accommodate the bis-His heme coordination. When PhNHOH is used, a similar ß-hemichrome Hb[α-FeIII(H2O)][ß-FeIII(His)2-cySOx]{PhNHOH} was obtained. Our results reveal, for the first time, the X-ray structural determination of a ß-hemichrome in a human Hb derivative. Our UV-vis and X-ray crystal structural result reveal that although Hb(PhNO) and Hb(RNO) complexes may form as primary products, attempted isolation of these products by crystallization may result in the structural determination of their secondary products which may contain ß-hemichromes en route to further protein degradation.

Reliability of Smartphone Accelerometers for Measuring Gait During Data Collection Over Zoom.

This study examined whether gait data could be reliably collected by homebound participants using iPhones under online supervision. Eighteen healthy young adults met with investigators through Zoom and installed an app to record acceleration from their iPhones' accelerometers. Half of the subjects walked normally; the other half walked while spelling words backward. During the gait tasks subjects recorded their anterior-posterior (AP), medial-lateral (ML), and vertical (V) accelerations. Data collection was repeated the following week. Seven maximum and minimum peak accelerations in the AP, ML, and vertical directions associated with events in gait were determined. Significant main effects of week and direction were observed for the first and second vertical acceleration measures. Cronbach alpha values were >0.60 for all acceleration measures, but the maximum and minimum AP accelerations that showed fair to good levels of consistency. The findings suggest gait data collected inside the home setting may be of clinical use.

Genetic heterogeneity in infantile spasms.

Infantile spasms (IS) is a developmental and epileptic encephalopathy with heterogeneous etiologies including many genetic causes. Genetic studies have identified pathogenic variants in over 30 genes as causes of IS. Many of these genetic causes are extremely rare, with only one reported incidence in an individual with IS. To better understand the genetic landscape of IS, we used targeted sequencing to screen 42 candidate IS genes and 53 established developmental and epileptic encephalopathy genes in 92 individual with IS. We identified a genetic diagnosis for 7.6% of our cohort, including pathogenic variants in KCNB1 (n = 2), GNAO1 (n = 1), STXBP1 (n = 1), SLC35A2 (n = 1), TBL1XR1 (n = 1), and KIF1A (n = 1). Our data emphasize the genetic heterogeneity of IS and will inform the diagnosis and management of individuals with this devastating disorder.