Randomised Controlled Trial of a Customised Text Messaging and Activity Monitor Program for Lifestyle Improvement after Gestational Diabetes.

Gestational diabetes (GDM) is associated with a long-term risk of diabetes. We aimed to determine whether a text-messaging-based lifestyle support program would improve diabetes risk factors following GDM. Women with GDM were randomised following delivery to receive four text messages per week supporting a healthy lifestyle and parenting for 6 months, with feedback from an activity monitor (intervention), or to receive the activity monitor only (control). The primary outcome was a composite of weight, physical activity and dietary goals. There were 177 women randomised, with 88 intervention and 89 control participants. All the participants experienced COVID-19 lockdowns during the study. Six-month primary outcome data were obtained for 57 intervention participants and 56 controls. There were 7/57 (12%) intervention and 6/56 (11%) control participants who met the primary outcome (relative risk, 1.08; 95%CI, 0.63-1.85; p = 0.79). Two intervention participants met the dietary goals compared to none of the control participants (p = NS). The intervention participants were more likely to record >1000 steps/day (on 102 ± 59 vs. 81 ± 59 days, p = 0.03). When analysed monthly, this was not initially different but became significant 3-6 months post-partum. Interviews and surveys indicated that with the Intervention, healthier choices were made, but these were negatively impacted by COVID-19 restrictions. Participants found the messages motivational (74%) and the activity monitor useful (71%). In conclusion, no improvement in the diabetes risk factors occurred among the women receiving the text messaging intervention when affected by COVID-19 restrictions.

Regression From Early GDM to Normal Glucose Tolerance and Adverse Pregnancy Outcomes in the Treatment of Booking Gestational Diabetes Mellitus Study.

OBJECTIVE: To compare pregnancy outcomes among women with a normal oral glucose tolerance test (OGTT) before 20 weeks' gestation (early) and at 24-28 weeks' gestation (late) (no gestational diabetes mellitus, or No-GDM), those with early GDM randomized to observation with a subsequent normal OGTT (GDM-Regression), and those with GDM on both occasions (GDM-Maintained). RESEARCH DESIGN AND METHODS: Women at <20 weeks' gestation with GDM risk factors who were recruited for a randomized controlled early GDM treatment trial were included. Women with treated early GDM and late GDM (according to the World Health Organization's 2013 criteria) were excluded from this analysis. Logistic regression compared pregnancy outcomes. RESULTS: GDM-Regression (n = 121) group risk factor profiles and OGTT results generally fell between the No-GDM (n = 2,218) and GDM-Maintained (n = 254) groups, with adjusted incidences of pregnancy complications similar between the GDM-Regression and No-GDM groups. CONCLUSIONS: Women with early GDM but normal OGTT at 24-28 weeks' gestation had pregnancy outcomes that were similar to those of individuals without GDM. Identifying early GDM likely to regress would allow treatment to be avoided.

Perinatal Outcomes in Early and Late Gestational Diabetes Mellitus After Treatment From 24-28 Weeks' Gestation: A TOBOGM Secondary Analysis.

OBJECTIVE: In most gestational diabetes mellitus (GDM) studies, cohorts have included women combined into study populations without regard to whether hyperglycemia was present earlier in pregnancy. In this study we sought to compare perinatal outcomes between groups: women with early GDM (EGDM group: diagnosis before 20 weeks but no treatment until 24-28 weeks if GDM still present), with late GDM (LGDM group: present only at 24-28 weeks), and with normoglycemia at 24-28 weeks (control subjects). RESEARCH DESIGN AND METHODS: This is a secondary analysis of a randomized controlled treatment trial where we studied, among women with risk factors, early (<20 weeks' gestation) GDM defined according to World Health Organization 2013 criteria. Those receiving early treatment for GDM treatment were excluded. GDM was treated if present at 24-28 weeks. The primary outcome was a composite of birth before 37 weeks' gestation, birth weight ≥4,500 g, birth trauma, neonatal respiratory distress, phototherapy, stillbirth/neonatal death, and shoulder dystocia. Comparisons included adjustment for age, ethnicity, BMI, site, smoking, primigravity, and education. RESULTS: Women with EGDM (n = 254) and LGDM (n = 467) had shorter pregnancy duration than control subjects (n = 2,339). BMI was lowest with LGDM. The composite was increased with EGDM (odds ratio [OR] 1.59, 95% CI 1.18-2.12)) but not LGDM (OR 1.19, 95% CI 0.94-1.50). Induction of labor was higher in both GDM groups. In comparisons with control subjects there were higher birth centile, higher preterm birth rate, and higher rate of neonatal jaundice for the EGDM group (but not the LGDM group). The greatest need for insulin and/or metformin was with EGDM. CONCLUSIONS: Adverse perinatal outcomes were increased with EGDM despite treatment from 24-28 weeks' gestation, suggesting the need to initiate treatment early, and more aggressively, to reduce the effects of exposure to the more severe maternal hyperglycemia from early pregnancy.

The relationship between body mass index and sleep in women with risk factors for gestational diabetes mellitus.

Background: Both obesity and sleep disorders are common among women during pregnancy. Although prior research has identified a relationship between obesity and sleep disorders, those findings are from women later in pregnancy. Objective: To explore the relationships between self-reported sleep duration, insufficient sleep and snoring with body mass index (BMI) among multiethnic women at risk of gestational diabetes mellitus (GDM)in early pregnancy. Methods: Cross-sectional study of baseline data from women at risk of GDM enrolled in the Treatment of BOoking Gestational diabetes Mellitus (TOBOGM) multicentre trial across 12 Australian/Austrian sites. Participants completed a questionnaire before 20 weeks' gestation to evaluate sleep. BMI <25 kg/m2 served as the reference group in multivariable logistic regression. Results: Among the 2865 women included, the prevalence of overweight and obesity classes I-III was 28%, 19%, 11% and 12%, respectively. There was no relationship between sleep duration and BMI. The risk of insufficient sleep >5 days/month was higher in class II and class III obesity (1.38 (1.03-1.85) and 1.34 (1.01-1.80), respectively), and the risk of snoring increased as BMI increased (1.59 (1.25-2.02), 2.68 (2.07-3.48), 4.35 (3.21-5.88) to 4.96 (3.65-6.74), respectively)). Conclusions: Obesity is associated with insufficient sleep among pregnant women at risk of GDM. Snoring is more prevalent with increasing BMI.

Safety and Efficacy of Rapid Methadone Titration for Opioid Use Disorder in an Inpatient Setting: A Retrospective Cohort Study.

OBJECTIVES: Inpatient guidelines for methadone titration do not exist, whereas outpatient guidelines lack flexibility and do not consider individual opioid tolerance. The evaluation of rapid, adaptable titration protocols may allow more patient-centered and effective treatment for opioid use disorder in the fentanyl era. METHODS: This study performed a retrospective chart review of patients 18 years or older with opioid use disorder who were initiated on methadone at a single academic urban hospital using a rapid divided dose protocol between November 2019 and November 2020. The primary outcome was adverse events associated with methadone, specifically opioid toxicity or sedation requiring increased medical observation or intervention. The secondary outcome was total daily dose of methadone received on day 7 of titration. RESULTS: Ninety-eight patients were included for a total of 168 visits. Sixty-five (66%) were male, with a median age of 38 years (interquartile range, 31-42 years). Sedation occurred in 2 patients (1%), who required either naloxone administration or transfer to an intensive care unit for monitoring. Of the 135 visits where patients received at least 7 days of methadone, the mean dose on day 1 was 41 mg (SD, 9.6 mg) and on day 7 was 65 mg (SD, 20.9 mg). CONCLUSIONS: In this inpatient cohort, rapid methadone titration was well tolerated and resulted in patients reaching higher doses of methadone than would be possible with a standard schedule, with few adverse events. Given the known effective dose range, this approach may result in shorter time to clinical stabilization and suggests that alternative methadone titration schedules may be safe and effective in appropriately selected patients.

Somatostatin receptor expression and clinical outcome of multilineage pituitary tumours expressing PIT1 and SF1.

The application of transcription factor immunohistochemistry to pituitary neuroendocrine tumour (PitNET) assessment has allowed identification of tumours that do not conform to a single lineage. Multilineage pituitary transcription factor 1 (PIT1) and steroidogenic factor 1 (SF1) PitNETs are a rare and relatively newly described tumour subtype. These tumours express both transcription factors and may also express combinations of hormones corresponding to both lineages. Histological and clinical characteristics can vary, and overall clinical behaviour and prognosis is not known. We describe the clinical outcomes and somatostatin receptor status (SSTR) of a series of nine cases identified from our cohort of pituitary tumours at Westmead Hospital. Eight PitNETs (88.9%) expressed growth hormone and caused acromegaly at presentation. Of the seven macrotumours that caused acromegaly, one had cavernous sinus invasion. The Ki-67 labeling index score ranged from 0.6% to 3.6%. About 88% of tumours that secreted excess growth hormone exhibited strong immunostaining for SSTR 2 and all tumours displayed weak immunoreactivity for SSTR5. In 62.5% of patients with acromegaly, cure was achieved after surgical resection. Somatostatin receptor ligands resulted in clinical remission in cases where medical treatment was initiated. There was no new tumour recurrence or regrowth over an overall mean follow-up period of 62.5 months.

Revealing KRas4b topology on the membrane surface.

KRas4b is a membrane-bound regulatory protein belonging to the family of small GTPases that function as a molecular switch, facilitating signal transduction from activated membrane receptors to intracellular pathways controlling cell growth and proliferation. Oncogenic mutations locking KRas4b in the active GTP state are responsible for nearly 85% of all Ras-driven cancers. Understanding the membrane-bound state of KRas4b is crucial for designing new therapeutic approaches targeting oncogenic KRas-driven signaling pathways. Extensive research demonstrates the significant involvement of the membrane bilayer in Ras-effector interactions, with anionic lipids playing a critical role in determining protein conformations The preferred topology of KRas4b for interacting with signaling partners has been a long-time question. Computational studies suggest a membrane-proximal conformation, while other biophysical methods like neutron reflectivity propose a membrane-distal conformation. To address these gaps, we employed FRET measurements to investigate the conformation of KRas4b. Using fully post-translationally modified KRas4b, we designed a Nanodisc based FRET assay to study KRas4b-membrane interactions. We suggest an extended conformation of KRas4b relative to the membrane surface. Measurement of FRET donor - acceptor distances reveal that a negatively charged membrane surface weakly favors closer association with the membrane surface. Our findings provide insights into the role of anionic lipids in determining the dynamic conformations of KRas4b and shed light on the predominant conformation of its topology on lipid headgroups.

Treatment of Gestational Diabetes Mellitus Diagnosed Early in Pregnancy.

BACKGROUND: Whether treatment of gestational diabetes before 20 weeks' gestation improves maternal and infant health is unclear. METHODS: We randomly assigned, in a 1:1 ratio, women between 4 weeks' and 19 weeks 6 days' gestation who had a risk factor for hyperglycemia and a diagnosis of gestational diabetes (World Health Organization 2013 criteria) to receive immediate treatment for gestational diabetes or deferred or no treatment, depending on the results of a repeat oral glucose-tolerance test [OGTT] at 24 to 28 weeks' gestation (control). The trial included three primary outcomes: a composite of adverse neonatal outcomes (birth at <37 weeks' gestation, birth trauma, birth weight of ≥4500 g, respiratory distress, phototherapy, stillbirth or neonatal death, or shoulder dystocia), pregnancy-related hypertension (preeclampsia, eclampsia, or gestational hypertension), and neonatal lean body mass. RESULTS: A total of 802 women underwent randomization; 406 were assigned to the immediate-treatment group and 396 to the control group; follow-up data were available for 793 women (98.9%). An initial OGTT was performed at a mean (±SD) gestation of 15.6±2.5 weeks. An adverse neonatal outcome event occurred in 94 of 378 women (24.9%) in the immediate-treatment group and in 113 of 370 women (30.5%) in the control group (adjusted risk difference, -5.6 percentage points; 95% confidence interval [CI], -10.1 to -1.2). Pregnancy-related hypertension occurred in 40 of 378 women (10.6%) in the immediate-treatment group and in 37 of 372 women (9.9%) in the control group (adjusted risk difference, 0.7 percentage points; 95% CI, -1.6 to 2.9). The mean neonatal lean body mass was 2.86 kg in the immediate-treatment group and 2.91 kg in the control group (adjusted mean difference, -0.04 kg; 95% CI, -0.09 to 0.02). No between-group differences were observed with respect to serious adverse events associated with screening and treatment. CONCLUSIONS: Immediate treatment of gestational diabetes before 20 weeks' gestation led to a modestly lower incidence of a composite of adverse neonatal outcomes than no immediate treatment; no material differences were observed for pregnancy-related hypertension or neonatal lean body mass. (Funded by the National Health and Medical Research Council and others; TOBOGM Australian New Zealand Clinical Trials Registry number, ACTRN12616000924459.).

Successful live birth following a short course of glucocorticoid suppression in a patient with autoimmune polyglandular syndrome type 2 and premature ovarian insufficiency: A case report.

A 32-year-old nulliparous woman with premature ovarian insufficiency POI and autoimmune polyglandular syndrome type 2 (APS-2), presented to our fertility center with a 2.5-year history of amenorrhoea. Controlled ovarian hyperstimulation (COH), with high dose gonadotropins, failed to promote antral follicle growth. The patient was given a short, 4-week course of 2 mg dexamethasone prior to a repeat COH cycle, which resulted in the retrieval of good oocyte numbers and eventual live birth from a thawed embryo transfer.

Widespread genetic heterogeneity and genotypic grouping associated with fungicide resistance among barley spot form net blotch isolates in Australia.

Spot form net blotch, caused by Pyrenophora teres f. maculata, is a major foliar disease of barley worldwide. Knowledge of the pathogen's genetic diversity and population structure is critical for a better understanding of inherent evolutionary capacity and for the development of sustainable disease management strategies. Genome-wide, single nucleotide polymorphism data of 254 Australian isolates revealed genotypic diversity and an absence of population structure, either between states, or between fields and cultivars in different agro-ecological zones. This indicates there is little geographical isolation or cultivar directional selection and that the pathogen is highly mobile across the continent. However, two cryptic genotypic groups were found only in Western Australia, predominantly associated with genes involved in fungicide resistance. The findings in this study are discussed in the context of current cultivar resistance and the pathogen's adaptive potential.

Engineering C-C Bond Cleavage Activity into a P450 Monooxygenase Enzyme.

The cytochrome P450 (CYP) superfamily of heme monooxygenases has demonstrated ability to facilitate hydroxylation, desaturation, sulfoxidation, epoxidation, heteroatom dealkylation, and carbon-carbon bond formation and cleavage (lyase) reactions. Seeking to study the carbon-carbon cleavage reaction of α-hydroxy ketones in mechanistic detail using a microbial P450, we synthesized α-hydroxy ketone probes based on the physiological substrate for a well-characterized benzoic acid metabolizing P450, CYP199A4. After observing low activity with wild-type CYP199A4, subsequent assays with an F182L mutant demonstrated enzyme-dependent C-C bond cleavage toward one of the α-hydroxy ketones. This C-C cleavage reaction was subject to an inverse kinetic solvent isotope effect analogous to that observed in the lyase activity of the human P450 CYP17A1, suggesting the involvement of a species earlier than Compound I in the catalytic cycle. Co-crystallization of F182L-CYP199A4 with this α-hydroxy ketone showed that the substrate bound in the active site with a preference for the (S)-enantiomer in a position which could mimic the topology of the lyase reaction in CYP17A1. Molecular dynamics simulations with an oxy-ferrous model of CYP199A4 revealed a displacement of the substrate to allow for oxygen binding and the formation of the lyase transition state proposed for CYP17A1. This demonstration that a correctly positioned α-hydroxy ketone substrate can realize lyase activity with an unusual inverse solvent isotope effect in an engineered microbial system opens the door for further detailed biophysical and structural characterization of CYP catalytic intermediates.

Aneurysmal subarachnoid haemorrhage in the pituitary fossa of a patient with acromegaly - a rare phenomenon.

Summary: The anatomy of the pituitary fossa is complex. The wall of the fossa can vary, resulting in inconsistencies in the nature and integrity of the sella barrier. Cerebrospinal fluid is generally confined to the subarachnoid space and does not circulate freely in the pituitary fossa. Spontaneous haemorrhage in the fossa typically occurs in the context of pre-existing intrasellar pathology such as a pituitary adenoma. Extravasation of blood into the subarachnoid space can rarely be observed following pituitary apoplexy. We describe the unique occurrence of subarachnoid haemorrhage in a largely empty pituitary fossa after the rupture of a cerebral aneurysm. Learning points: Pituitary apoplexy and subarachnoid haemorrhage (SAH) are both high in the differential diagnosis of sudden onset severe headaches. Haemorrhagic pituitary apoplexy may result in extravasation into the subarachnoid space, resulting in typical SAH symptoms and signs. This is the first reported case of primary SAH resulting in blood pooling in an empty sella arising from previous surgical resection of a large macroadenoma.

A Hospital-based Managed Alcohol Program in a Canadian Setting.

OBJECTIVES: A managed alcohol program (MAP) is a harm reduction strategy that provides regularly, witnessed alcohol to individuals with a severe alcohol use disorder. Although community MAPs have positive outcomes, applicability to hospital settings is unknown. This study describes a hospital-based MAP, characterizes its participants, and evaluates outcomes. METHODS: A retrospective chart review of MAP participants was conducted at an academic hospital in Vancouver, Canada, between July 2016 and October 2017. Data included demographics, alcohol/substance use, alcohol withdrawal risk, and MAP indication. Outcomes after MAP initiation included the change in mean daily alcohol consumption and liver enzymes. RESULTS: Seventeen patients participated in 26 hospital admissions: 76% male, mean age of 54 years, daily consumption prehospitalization of a mean 14 alcohol standard drinks, 59% reported previous nonbeverage alcohol consumption, and 41% participated in a community MAP. Most participants were high risk for severe, complicated alcohol withdrawal and presented in moderate withdrawal. Continuation of community MAP was the most common indication for hospital-based MAP initiation (38%), followed by a history of leaving hospital against medical advice (35%) and hospital illicit alcohol use (15%). Hospital-based MAP resulted in a mean of 5 fewer alcohol standard drinks daily compared with preadmission ( P = 0.002; 95% confidence interval, 2-8) and improvement in liver enzymes, with few adverse events. CONCLUSIONS: Participation in a hospital-based MAP may be an effective safe approach to reduce harms for some individuals with severe alcohol use disorder. Further study is needed to understand who benefits most from hospital-MAP and potential benefits/harms following hospital discharge.

The relationship between falling insulin requirements and serial ultrasound measurements in women with preexisting diabetes: a prospective cohort study.

INTRODUCTION: A large fall in insulin requirements (FIR) in women with diabetes is associated with adverse clinical outcomes but previous studies have not examined its relation with serial ultrasound parameters. OBJECTIVE: To determine whether FIR is associated with alteration in umbilical artery Doppler parameters and fetal growth restriction (FGR) in women with preexisting diabetes. METHODS: Serial obstetric Doppler ultrasounds were conducted 2 weekly from 28 weeks gestation in women with Type 1 and Type 2 diabetes who were being treated with insulin. Estimated fetal weight (EFW), head circumference:abdominal circumference (HC:AC) ratio and umbilical artery doppler parameters (SD ratio) and pulsatility index (PI) were measured. Information on insulin dose was collected prospectively throughout pregnancy and women with FIR ≥ 15% were considered cases. Linear mixed effect models were used to assess the association between FIR and ultrasound parameters. RESULTS: One hundred and forty two women were included in the study (type 1 diabetes n = 41, type 2 diabetes n = 101). Thirty women demonstrated FIR ≥ 15%. There was no significant difference in the change of S/D ratio or PI over the third trimester in cases with FIR ≥ 15%, compared to the rest of the cohort, before or after adjusting for type of diabetes. Likewise there was no difference in EFW and HC:AC ratio with advancing gestation before or after adjusting for variables known to influence fetal growth. FGR rates (3.3 vs 8% p = 0.298) and high S/D ratio > 95% (13.3 vs 8%, p = 0.296) were similar between the two groups. CONCLUSIONS: FIR ≥ 15% was not associated with changes in placental flow or FGR however larger studies are needed to evaluate this further.

Midazolam as a Probe for Heterotropic Drug-Drug Interactions Mediated by CYP3A4.

Human cytochrome P450 CYP3A4 is involved in the processing of more than 35% of current pharmaceuticals and therefore is responsible for multiple drug-drug interactions (DDI). In order to develop a method for the detection and prediction of the possible involvement of new drug candidates in CYP3A4-mediated DDI, we evaluated the application of midazolam (MDZ) as a probe substrate. MDZ is hydroxylated by CYP3A4 in two positions: 1-hydroxy MDZ formed at lower substrate concentrations, and up to 35% of 4-hydroxy MDZ at high concentrations. The ratio of the formation rates of these two products (the site of metabolism ratio, SOM) was used as a measure of allosteric heterotropic interactions caused by effector molecules using CYP3A4 incorporated in lipid nanodiscs. The extent of the changes in the SOM in the presence of effectors is determined by chemical structure and is concentration-dependent. MD simulations of CYP3A4 in the lipid bilayer suggest that experimental results can be explained by the movement of the F-F' loop and concomitant changes in the shape and volume of the substrate-binding pocket. As a result of PGS binding at the allosteric site, several residues directly contacting MDZ move away from the substrate molecule, enabling the repositioning of the latter for minor product formation.

Insulin-like growth factor role in determining the anti-cancer effect of metformin: RCT in prostate cancer patients.

Objective: Androgen deprivation therapy (ADT), a principal therapy in patients with prostate cancer, is associated with the development of obesity, insulin resistance, and hyperinsulinemia. Recent evidence indicates that metformin may slow cancer progression and improves survival in prostate cancer patients, but the mechanism is not well understood. Circulating insulin-like growth factors (IGFs) are bound to high-affinity binding proteins, which not only modulate the bioavailability and signalling of IGFs but also have independent actions on cell growth and survival. The aim of this study was to investigate whether metformin modulates IGFs, IGF-binding proteins (IGFBPs), and the pregnancy-associated plasma protein A (PAPP-A) - stanniocalcin 2 (STC2) axis. Design and methods: In a blinded, randomised, cross-over design, 15 patients with prostate cancer on stable ADT received metformin and placebo treatment for 6 weeks each. Glucose metabolism along with circulating IGFs and IGFBPs was assessed. Results: Metformin significantly reduced the homeostasis model assessment as an index of insulin resistance (HOMA IR) and hepatic insulin resistance. Metformin also reduced circulating IGF-2 (P < 0.05) and IGFBP-3 (P < 0.01) but increased IGF bioactivity (P < 0.05). At baseline, IGF-2 correlated significantly with the hepatic insulin resistance (r2= 0.28, P < 0.05). PAPP-A remained unchanged but STC2 declined significantly (P < 0.05) following metformin administration. During metformin treatment, change in HOMA IR correlated with the change in STC2 (r2= 0.35, P < 0.05). Conclusion: Metformin administration alters many components of the circulating IGF system, either directly or indirectly via improved insulin sensitivity. Reduction in IGF-2 and STC2 may provide a novel mechanism for a potential metformin-induced antineoplastic effect.

Effect of Maternal Metformin Treatment in Pregnancy on Neonatal Metabolism: Evidence From Newborn Metabolic Screening.

OBJECTIVE: To investigate effects of maternal diabetes and metformin treatment on metabolic newborn screening (NBS) results of infants born to mothers with hyperglycemia during pregnancy. RESEARCH DESIGN AND METHODS: Retrospective case-control study. NBS results of infants born to mothers treated with metformin for hyperglycemia during pregnancy were compared with diet-treated subjects with diabetes and matched normal control subjects. EXCLUSIONS: maternal type 1 diabetes, major fetal anomalies, and incomplete infant data. Inclusions: maternal hyperglycemia in pregnancy treated with diet alone or diet plus metformin. Results from the New South Wales Newborn Screening Program (dried infant blood spot sample, 24-72 h after birth) for 25 routinely studied analytes were measured using mass spectrometry. Data from metformin-exposed and control infants were compared using nonparametric methods and multiples of the median for each analyte. RESULTS: A total of 574 case subjects were compared with 952 diet-treated case subjects with diabetes and 979 control subjects. Metformin-exposed infants had shorter gestational age (266 ± 7 vs. 272 ± 10 vs. 274 ± 9 days) (P < 0.001) and lower birth weights (3.28 ± 0.51 vs. 3.29 ± 0.49 vs. 3.33 ± 0.43 kg) (P = 0.008). Short-, medium-, and one long-chain acylcarntine (tetradecanoylcarnitine [C14]) concentrations were higher in the metformin-exposed group compared with normal control subjects. Comparison with diet-treated control subjects with diabetes (to eliminate confounding by hyperglycemia) continued to show raised butyrylcarnitine (C4), isovalerylcarnitine (C5), and glutarylcarnitine (C5D) in the metformin-exposed group. There was no evidence of vitamin B12 deficiency (low methionine and elevated propionylcarnitine [C3]) in metformin-exposed infants. All results were within normal population limits. CONCLUSIONS: We have identified subtle (nonpathological) changes in neonatal metabolism that represent a signature effect of fetal metformin exposure.

Midazolam as a Probe for Drug-Drug Interactions Mediated by CYP3A4: Homotropic Allosteric Mechanism of Site-Specific Hydroxylation.

We developed an efficient and sensitive probe for drug-drug interactions mediated by human CYP3A4 by using midazolam (MDZ) as a probe substrate. Using global analysis of four parameters over several experimental data sets, we demonstrate that the first MDZ molecule (MDZ1) binds with high affinity at the productive site near the heme iron and gives only hydroxylation at the 1 position (1OH). The second midazolam molecule (MDZ2) binds at an allosteric site at the membrane surface and perturbs the position and mobility of MDZ1 such that the minor hydroxylation product at the 4 position (4OH) is formed in a 1:2 ratio (35%). No increase in catalytic rate is observed after the second MDZ binding. Hence, the site of the 1OH:4OH metabolism ratio is a sensitive probe for drugs, such as progesterone, that bind with high affinity to the allosteric site and serve as effectors. We observe similar changes in the MDZ 1OH:4OH ratio in the presence of progesterone (PGS), suggesting a direct communication between the active and allosteric sites. Mutations introduced into the F-F' loop indicate that residues F213 and D214 are directly involved in allosteric interactions leading to MDZ homotropic cooperativity, and these same residues, together with L211, are involved in heterotropic allosteric interactions in which PGS is the effector and MDZ the substrate. Molecular dynamics simulations provide a mechanistic picture of the origin of this cooperativity. These results show that the midazolam can be used as a sensitive probe for drug-drug interactions in human P450 CYP3A4.