Predicting COVID-19 severity: Challenges in reproducibility and deployment of machine learning methods.

The increasing use of electronic health records (EHR) based computable phenotypes in clinical research is providing new opportunities for development of data-driven medical applications. Adopted widely in the United States and globally, EHRs facilitate systematic collection of patients' longitudinal information, which serves as one of the important foundations for artificial intelligence applications in medicine. Harmonization of input variables and outcome definitions is critically important for wider clinical applicability of artificial intelligence (AI) methodologies. In this review, we focused on Coronavirus Disease 2019 (COVID-19) severity machine learning prediction models and explored the pipeline for standardizing future disease severity model development using EHR information. We identified 2,967 studies published between 01/01/2020 and 02/15/2022 and selected 135 independent studies that had built machine learning prediction models to predict severity related outcomes of COVID-19 patients based on EHR data for the final review. These 135 studies spanning across 27 counties covered a broad range of severity related prediction outcomes. We observed substantial inconsistency in COVID-19 severity phenotype definitions among models in these studies. Moreover, there was a gap between the outcome of these models and clinician-recognized clinical concepts. Accordingly, we recommend that robust clinical input metrics, with outcome definitions which eliminate ambiguity in interpretation, to reduce algorithmic bias, mitigate model brittleness and improve generalizability of a universal model for COVID-19 severity. This framework can potentially be extended to broader clinical application.

Reference intervals for free testosterone in adult men measured using a standardized equilibrium dialysis procedure.

BACKGROUND: Free testosterone (FT) determination may be helpful in evaluating men suspected of testosterone deficiency especially in conditions with altered binding-protein concentrations. However, methods for measuring FT by equilibrium dialysis and reference intervals vary among laboratories. OBJECTIVE: To determine reference intervals for FT in healthy, nonobese men by age groups as well as in healthy young men, 19-39 years, using a standardized equilibrium dialysis procedure METHODS: We measured FT in 145 healthy, nonobese men, 19 years or older, using a standardized equilibrium dialysis method performed for 16-h at 37°C using undiluted serum and dialysis buffer that mimicked the ionic composition of human plasma. FT in dialysate was measured using a CDC-certified liquid chromatography tandem mass spectrometry assay. RESULTS: In healthy nonobese men, the 2.5th, 10th, 50th, 90th, and 97.5th percentile values for FT were 66, 91, 141, 240, and 309 pg/ml, respectively; corresponding values for men, 19-39 years, were 120, 128, 190, 274, and 368 pg/ml, respectively. FT levels by age groups exhibit the expected age-related decline. FT levels were negatively associated with body mass index, age, and sex hormone-binding globulin (SHBG) levels. Percent FT was lower in middle-aged and older men than young men adjusting for SHBG level. DISCUSSION: Further studies are needed to determine how these reference intervals apply to the diagnosis of androgen deficiency in clinical populations and in men of different races and ethnicities in different geographic regions. CONCLUSION: Reference intervals for free FT levels (normative range 66-309 pg/ml [229-1072 pmol/L] in all men and 120-368 pg/ml [415-1274 pmol/L] in men, 19-39 years), measured using a standardized equilibrium dialysis method in healthy nonobese men, provide a rational basis for categorizing FT levels. These intervals require further validation in other populations, in relation to outcomes, and in randomized trials.

Circulating dihydrotestosterone, testosterone, and free testosterone levels and dihydrotestosterone-to-testosterone ratios in healthy women across the menstrual cycle.

OBJECTIVE: To characterize the circulating androgen levels across the menstrual cycle in healthy women using highly sensitive and accurate methods and report sex differences in the relative levels of dihydrotestosterone (DHT) to testosterone (T) levels. DESIGN: Prospective cohort study. SETTING: Research clinic, academic teaching hospital. PATIENT(S): Twenty-one healthy premenopausal women, aged 19-40 years, with regular menstrual cycles. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Serum total T and DHT levels measured using liquid chromatography-tandem mass spectrometry, free T levels measured using a standardized equilibrium dialysis method coupled with measurement of the T levels in the dialysate using liquid chromatography-tandem mass spectrometry, and comparison of the DHT-to-T ratio between healthy women and age-matched healthy men. RESULT(S): The serum total and free T levels increased across the follicular phase and peaked at midcycle (total T, 43.6 ± 16.2 ng/dL; free T, 15.6 ± 11.9 pg/mL) and gradually declined in the luteal phase. The DHT level did not significantly change across the menstrual cycle. The DHT-to-T ratios were 1:4 and 1:13 in women and men, respectively. CONCLUSION(S): In healthy premenopausal women, the total and free T levels varied significantly across the menstrual cycle, whereas the DHT levels did not change; the peak total and free T levels in the midcycle period were higher than previously reported, underscoring the importance of establishing menstrual phase-specific reference ranges to avoid misdiagnosis of hyperandrogenism. Women have significantly higher DHT levels relative to total T than men; the significance of this sex difference in the DHT-to-T ratio needs further investigation.

Skeletal Muscle Dysfunction in Experimental Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a serious, progressive, and often fatal disease that is in urgent need of improved therapies that treat it. One of the remaining therapeutic challenges is the increasingly recognized skeletal muscle dysfunction that interferes with exercise tolerance. Here we report that in the adult rat Sugen/hypoxia (SU/Hx) model of severe pulmonary hypertension (PH), there is highly significant, almost 50%, decrease in exercise endurance, and this is associated with a 25% increase in the abundance of type II muscle fiber markers, thick sarcomeric aggregates and an increase in the levels of FoxO1 in the soleus (a predominantly type I fiber muscle), with additional alterations in the transcriptomic profiles of the diaphragm (a mixed fiber muscle) and the extensor digitorum longus (a predominantly Type II fiber muscle). In addition, soleus atrophy may contribute to impaired exercise endurance. Studies in L6 rat myoblasts have showed that myotube differentiation is associated with increased FoxO1 levels and type II fiber markers, while the inhibition of FoxO1 leads to increased type I fiber markers. We conclude that the formation of aggregates and a FoxO1-mediated shift in the skeletal muscle fiber-type specification may underlie skeletal muscle dysfunction in an experimental study of PH.

A deep tensor-based approach for automatic depression recognition from speech utterances.

Depression is one of the significant mental health issues affecting all age groups globally. While it has been widely recognized to be one of the major disease burdens in populations, complexities in definitive diagnosis present a major challenge. Usually, trained psychologists utilize conventional methods including individualized interview assessment and manually administered PHQ-8 scoring. However, heterogeneity in symptomatic presentations, which span somatic to affective complaints, impart substantial subjectivity in its diagnosis. Diagnostic accuracy is further compounded by the cross-sectional nature of sporadic assessment methods during physician-office visits, especially since depressive symptoms/severity may evolve over time. With widespread acceptance of smart wearable devices and smartphones, passive monitoring of depression traits using behavioral signals such as speech presents a unique opportunity as companion diagnostics to assist the trained clinicians in objective assessment over time. Therefore, we propose a framework for automated depression classification leveraging alterations in speech patterns in the well documented and extensively studied DAIC-WOZ depression dataset. This novel tensor-based approach requires a substantially simpler implementation architecture and extracts discriminative features for depression recognition with high f1 score and accuracy. We posit that such algorithms, which use significantly less compute load would allow effective onboard deployment in wearables for improve diagnostics accuracy and real-time monitoring of depressive disorders.

Predicting hospitalization of COVID-19 positive patients using clinician-guided machine learning methods.

OBJECTIVES: The coronavirus disease 2019 (COVID-19) is a resource-intensive global pandemic. It is important for healthcare systems to identify high-risk COVID-19-positive patients who need timely health care. This study was conducted to predict the hospitalization of older adults who have tested positive for COVID-19. METHODS: We screened all patients with COVID test records from 11 Mass General Brigham hospitals to identify the study population. A total of 1495 patients with age 65 and above from the outpatient setting were included in the final cohort, among which 459 patients were hospitalized. We conducted a clinician-guided, 3-stage feature selection, and phenotyping process using iterative combinations of literature review, clinician expert opinion, and electronic healthcare record data exploration. A list of 44 features, including temporal features, was generated from this process and used for model training. Four machine learning prediction models were developed, including regularized logistic regression, support vector machine, random forest, and neural network. RESULTS: All 4 models achieved area under the receiver operating characteristic curve (AUC) greater than 0.80. Random forest achieved the best predictive performance (AUC = 0.83). Albumin, an index for nutritional status, was found to have the strongest association with hospitalization among COVID positive older adults. CONCLUSIONS: In this study, we developed 4 machine learning models for predicting general hospitalization among COVID positive older adults. We identified important clinical factors associated with hospitalization and observed temporal patterns in our study cohort. Our modeling pipeline and algorithm could potentially be used to facilitate more accurate and efficient decision support for triaging COVID positive patients.

Accurate Measurement and Harmonized Reference Ranges for Total and Free Testosterone Levels.

Diagnosing testosterone deficiency requires accurate and precise measurement of total testosterone levels by an accurate method, such as liquid chromatography-tandem mass spectrometry in a laboratory certified by an accuracy-based program (eg, Centers for Disease Control and Prevention's Hormone Standardization (HoST) Program), and, if needed, free testosterone level. Free testosterone level should ideally be measured by equilibrium dialysis method. Testosterone levels should be measured in 2 or more fasting samples obtained in the morning. Harmonized reference ranges for total testosterone can be applied to laboratories that certified by the HoST Program.

Age Trends in Growth and Differentiation Factor-11 and Myostatin Levels in Healthy Men, and Differential Response to Testosterone, Measured Using Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Growth and differentiation factor (GDF)-11 controls embryonic development and has been proposed as an antiaging factor. GDF-8 (myostatin) inhibits skeletal muscle growth. Difficulties in accurately measuring circulating GDF-11 and GDF-8 have generated controversy. METHODS: We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous measurement of circulating GDF-8 and GDF-11 that employs denaturation, reduction, and alkylation; cation-exchange solid-phase extraction; tryptic digestion; followed by separation and quantification using 2 signature peptides for multiple reaction monitoring and C-terminal [13C615N4]-Arg peptides as internal standards. We evaluated age trends in serum GDF-11 and GDF-8 concentrations in community-dwelling healthy men, 19 years or older, and determined the effects of graded testosterone doses on GDF-8 and GDF-11 concentrations in healthy men in a randomized trial. RESULTS: The assay demonstrated linearity over a wide range, lower limit of quantitation 0.5 ng/mL for both proteins, and excellent precision, accuracy, and specificity (no detectable cross-reactivity of GDF-8 in GDF-11 assay or of GDF-11 in GDF-8 assay). Mean ± SD (median ± 1QR) GDF-8 and GDF-11 levels in healthy community-dwelling men, 19 years and older, were 7.2 ±â€…1.9 (6.8 ±â€…1.4) ng/mL. Neither GDF-8 nor GDF-11 levels were related to age or body composition. Testosterone treatment significantly increased serum GDF-8 but not GDF-11 levels. CONCLUSIONS: The LC-MS/MS method for the simultaneous measurement of circulating total GDF-8 and GDF-11 demonstrates the characteristics of a valid assay. Testosterone treatment increased GDF-8 levels, but not GDF-11. Increase in GDF-8 levels by testosterone treatment, which increased muscle mass, suggests that GDF-8 acts as a chalone to restrain muscle growth.

Estradiol induces allosteric coupling and partitioning of sex-hormone-binding globulin monomers among conformational states.

Sex-hormone-binding globulin (SHBG) regulates the transport and bioavailability of estradiol. The dynamics of estradiol's binding to SHBG are incompletely understood, although it is believed that estradiol binds to each monomer of SHBG dimer with identical affinity (Kd ∼2 nM). Contrary to the prevalent view, we show that estradiol's binding to SHBG is nonlinear, and the "apparent" Kd changes with varying estradiol and SHBG concentrations. Estradiol's binding to each SHBG monomer influences residues in the ligand-binding pocket of both monomers and differentially alters the conformational and energy landscapes of both monomers. Monomers are not energetically or conformationally equivalent even in fully bound state. Estradiol's binding to SHBG involves bidirectional, inter-monomeric allostery that changes the distribution of both monomers among various energy and conformational states. Inter-monomeric allostery offers a mechanism to extend the binding range of SHBG and regulate hormone bioavailability as estradiol concentrations vary widely during life.

Free testosterone and cardiometabolic parameters in men: comparison of algorithms.

OBJECTIVE: Calculating the free testosterone level has gained increasing interest and different indirect algorithms have been suggested. The objective was to compare free androgen index (FAI), free testosterone estimated using the linear binding model (Vermeulen: cFTV) and the binding framework accounting for allosterically coupled SHBG monomers (Zakharov: cFTZ) in relation to cardiometabolic conditions. DESIGN: A prospective cohort study including 5350 men, aged 30-70 years, participating in population-based surveys (MONICA I-III and Inter99) from 1982 to 2001 and followed until December 2012 with baseline and follow-up information on cardiometabolic parameters and vital status. RESULTS: Using age-standardized hormone levels, FAI was higher among men with baseline cardiometabolic conditions, whereas cFTV and cFTZ levels were lower compared to men without these conditions as also seen for total testosterone. Men in highest quartiles of cFTV or cFTZ had lower risk of developing type 2 diabetes (cFTV: HR = 0.74 (0.49-1.10), cFTZ: HR = 0.59 (0.39-0.91)) than men in lowest quartile. In contrast, men with highest levels of FAI had a 74% (1.17-2.59) increased risk of developing type 2 diabetes compared to men in lowest quartile. CONCLUSION: The association of estimated free testosterone and the studied outcomes differ depending on algorithm used. cFTV and cFTZ showed similar associations to baseline and long-term cardiometabolic parameters. In contrast, an empiric ratio, FAI, showed opposite associations to several of the examined parameters and may reflect limited clinical utility.

Allosterically Coupled Multisite Binding of Testosterone to Human Serum Albumin.

Human serum albumin (HSA) acts as a carrier for testosterone, other sex hormones, fatty acids, and drugs. However, the dynamics of testosterone's binding to HSA and the structure of its binding sites remain incompletely understood. Here, we characterize the dynamics of testosterone's binding to HSA and the stoichiometry and structural location of the binding sites using 2-dimensional nuclear magnetic resonance (2D NMR), fluorescence spectroscopy, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt partitioning, and equilibrium dialysis, complemented by molecular modeling. 2D NMR studies showed that testosterone competitively displaced 18-[13C]-oleic acid from at least 3 known fatty acid binding sites on HSA that also bind many drugs. Binding isotherms of testosterone's binding to HSA generated using fluorescence spectroscopy and equilibrium dialysis were nonlinear and the apparent dissociation constant varied with different concentrations of testosterone and HSA. The binding isotherms neither conformed to a linear binding model with 1:1 stoichiometry nor to 2 independent binding sites; the binding isotherms were most consistent with 2 or more allosterically coupled binding sites. Molecular dynamics studies revealed that testosterone's binding to fatty acid binding site 3 on HSA was associated with conformational changes at site 6, indicating that residues in in these 2 distinct binding sites are allosterically coupled. There are multiple, allosterically coupled binding sites for testosterone on HSA. Testosterone shares these binding sites on HSA with free fatty acids, which could displace testosterone from HSA under various physiological states or disease conditions, affecting its bioavailability.

Loss of ARNT in skeletal muscle limits muscle regeneration in aging.

The ability of skeletal muscle to regenerate declines significantly with aging. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT), a critical component of the hypoxia signaling pathway, was less abundant in skeletal muscle of old (23-25 months old) mice. This loss of ARNT was associated with decreased levels of Notch1 intracellular domain (N1ICD) and impaired regenerative response to injury in comparison to young (2-3 months old) mice. Knockdown of ARNT in a primary muscle cell line impaired differentiation in vitro. Skeletal muscle-specific ARNT deletion in young mice resulted in decreased levels of whole muscle N1ICD and limited muscle regeneration. Administration of a systemic hypoxia pathway activator (ML228), which simulates the actions of ARNT, rescued skeletal muscle regeneration in both old and ARNT-deleted mice. These results suggest that the loss of ARNT in skeletal muscle is partially responsible for diminished myogenic potential in aging and activation of hypoxia signaling holds promise for rescuing regenerative activity in old muscle.

Associations of Endogenous Sex Hormones with Carotid Plaque Burden and Characteristics in Midlife Women.

CONTEXT: Endogenous sex hormones may be involved in the pathogenesis of cardiovascular disease (CVD) in women. Carotid plaque characteristics, such as echogenicity, an ultrasound measure that reflects plaque composition, may identify unstable plaques that are more likely to rupture, precipitating a CVD event. However, few studies have considered sex steroids in relation to carotid plaque and its characteristics. OBJECTIVE: To evaluate estrone (E1), estradiol (E2), testosterone (T), sex hormone binding globulin (SHBG), and free T (FT) in relation to carotid plaque in women. DESIGN, SETTING, AND PARTICIPANTS: In MsHeart, a cross-sectional study of 304 women aged 40 to 60 years, participants underwent a carotid artery ultrasound assessment. The current analysis included MsHeart participants with carotid plaque (n = 141, 46%). E1, E2, and T were assayed using liquid chromatography-tandem mass spectrometry; FT was estimated using ensemble allostery models. Regression models were adjusted for sociodemographic characteristics and CVD risk factors. MAIN OUTCOMES: Carotid plaque burden (number of plaques, total plaque area [TPA]) and characteristics (calcification, echogenicity) were determined using semi-automated software. RESULTS: SHBG was inversely related to TPA (odds ratio [OR] 0.39; 95% confidence interval [CI] 0.21, 0.74; multivariable) and higher FTs were associated with greater TPA (OR 2.89; 95% CI 1.31, 6.37; multivariable). Higher E1 was related to echogenicity (OR 2.31; 95% CI 1.26, 4.33; multivariable), characteristic of more stable plaque. CONCLUSIONS: SHBG and FT are related to TPA while E1 is related to plaque echogenicity, suggesting these hormones have different roles in the development of carotid plaque. Our findings highlight the importance of sex hormones in the development of carotid plaque in midlife women.

Acute primary testicular failure due to radiotherapy increases risk of severe postoperative adverse events in rectal cancer patients.

AIM: The aim of this study is to analyze postoperative adverse events (AE) in relation to acute primary testicular failure after radiotherapy (RT) for rectal cancer. METHOD: This relation was assessed in 104 men, included in a previous prospective cohort study of men treated with surgical resection of the rectum for rectal cancer stage I-III. Postoperative AE were graded according to Clavien-Dindo (2004). Grade 3 or more was set as cut-off for severe postoperative AE. The impact of primary testicular failure on postoperative AE was related to the cumulative mean testicular dose (TD) and the change in Testosterone (T) and Luteinizing hormone (LH) sampled at baseline and after RT. RESULTS: Twenty-six study participants (25%) had severe postoperative AE. Baseline characteristics and endocrine testicular function did not differ significantly between groups with (AE+) and without severe postoperative AE (AE-). After RT, the LH/T-ratio was higher in AE+, 0.603 (0.2-2.5) vs 0.452 (0.127-5.926) (p = 0.035). The longitudinal regression analysis showed that preoperative change in T (OR 0.844, 95% CI 0.720-0.990, p = 0.034), LH/T-ratio (OR 2.020, 95% CI 1.010-4.039, p = 0.047) and low T (<8 nmol/L, OR 2.605, 95 CI 0.951-7.139, p = 0.063) were related to severe postoperative AE. CONCLUSION: Preoperative decline in T due to primary testicular failure induced by preoperative RT could be a risk factor regarding short-term outcome of surgery in men with rectal cancer.

Muscle strength is increased in mice that are colonized with microbiota from high-functioning older adults.

Evidence in support of a gut-muscle axis has been reported in rodents, but studies in older adult humans are limited. Accordingly, the primary goals of the present study were to compare gut microbiome composition in older adults that differed in terms of the percentage of whole body lean mass and physical functioning (high-functioning, HF, n = 18; low-functioning, LF, n = 11), and to evaluate the causative role of the gut microbiome on these variables by transferring fecal samples from older adults into germ-free mice. Family-level Prevotellaceae, genus-level Prevotella and Barnesiella, and the bacterial species Barnesiella intestinihominis were higher in HF older adults at the initial study visit, at a 1-month follow-up visit, in HF human fecal donors, and in HF-colonized mice, when compared with their LF counterparts. Grip strength was significantly increased by 6.4% in HF-, when compared with LF-colonized mice. In contrast, despite significant differences for the percentage of whole body lean mass and physical functioning when comparing the human fecal donors, the percentage of whole body lean mass and treadmill endurance capacity were not different when comparing human microbiome-containing mice. In sum, these data suggest a role for gut bacteria on the maintenance of muscle strength, but argue against a role for gut bacteria on the maintenance of the percentage of whole body lean mass or endurance capacity, findings that collectively add to elucidation of the gut-muscle axis in older adults.

Effects of an ActRIIB.Fc Ligand Trap on Cardiac Function in Simian Immunodeficiency Virus-Infected Male Rhesus Macaques.

An important safety consideration in the use of antagonists of myostatin and activins is whether these drugs induce myocardial hypertrophy and impair cardiac function. The current study evaluated the effects of a soluble ActRIIB receptor Fc fusion protein (ActRIIB.Fc), a ligand trap for TGF-ß/activin family members including myostatin, on myocardial mass and function in simian immunodeficiency virus (SIV)-infected juvenile rhesus macaques (Macaca mulatta). Fourteen pair-housed, juvenile male rhesus macaques were inoculated with SIVmac239; 4 weeks postinoculation, they were treated with weekly injections of 10 mg/kg ActRIIB.Fc or saline for 12 weeks. Myocardial mass and function were evaluated using two-dimensional echocardiography at baseline and after 12 weeks. The administration of ActRIIB.Fc was associated with a significantly greater increase in thickness of left ventricular posterior wall and interventricular septum both in diastole and systole. Cardiac output and cardiac index increased with time, more in animals treated with ActRIIB.Fc than in those treated with saline, but the difference was not statistically significant. The changes in ejection fraction, fractional shortening, and stroke volume did not differ significantly between groups. The changes in end-diastolic and end-systolic volumes did not differ between groups. In addition to a large reduction in IGF1 mRNA expression in the ActRIIB.Fc-treated animals, complex changes were detected in the myocardial expression of proteins related to calcium transport and storage. In conclusion, ActRIIB.Fc administration for 12 weeks was associated with increased myocardial mass but did not adversely affect myocardial function in juvenile SIV-infected rhesus macaques. Further studies are necessary to establish long-term cardiac safety.

Reproductive Hormones and Subclinical Cardiovascular Disease in Midlife Women.

Context: Reproductive hormones are important to the pathophysiology of cardiovascular disease (CVD) in women. However, standard estradiol (E2) and testosterone (T) assays lack sensitivity at the levels of postmenopausal women. Objective: Investigate relations of mass spectrometry-assessed estrone (E1), E2, and T and SHBG and subclinical CVD in women. Design, Setting, and Participants: Three hundred and four perimenopausal and postmenopausal women aged 40 to 60 years underwent subclinical CVD measurements. E1, E2, and T were assayed using liquid chromatography-tandem mass spectrometry; free T (FT) was estimated using ensemble allostery models. Regression models were adjusted for CVD risk factors. Main Outcome Measures: Carotid artery intima media thickness, interadventitial diameter (IAD), and plaque; brachial flow mediated dilation (FMD). Results: Higher E1 was related to higher FMD [ß(SE) = 0.77 (0.37), P = 0.04], indicating better endothelial function. Higher E2 was related to lower IAD [ß(SE) = -0.07 (0.02), P = 0.004], indicating less carotid remodeling. Higher SHBG was related to higher FMD [ß(SE) = 1.31 (0.40), P = 0.001], yet higher IAD [ß(SE) = 0.15 (0.06), P = 0.02] and plaque [OR (95% CI) = 1.84 (1.16 to 2.91), P = 0.009]; FT showed a similar yet inverse pattern of relations as SHBG. Thus, higher SHBG and lower FT were associated with better endothelial function, yet greater carotid remodeling and plaque. Conclusions: Endogenous E1 levels were related to endothelial function and E2 to vascular remodeling, suggesting distinct roles of these estrogens. SHBG and FT have complex roles depending on the vessel under study.

A Reappraisal of Testosterone's Binding in Circulation: Physiological and Clinical Implications.

In the circulation, testosterone and other sex hormones are bound to binding proteins, which play an important role in regulating their transport, distribution, metabolism, and biological activity. According to the free hormone hypothesis, which has been debated extensively, only the unbound or free fraction is biologically active in target tissues. Consequently, accurate determination of the partitioning of testosterone between bound and free fractions is central to our understanding of how its delivery to the target tissues and biological activity are regulated and consequently to the diagnosis and treatment of androgen disorders in men and women. Here, we present a historical perspective on the evolution of our understanding of the binding of testosterone to circulating binding proteins. On the basis of an appraisal of the literature as well as experimental data, we show that the assumptions of stoichiometry, binding dynamics, and the affinity of the prevailing models of testosterone binding to sex hormone-binding globulin and human serum albumin are not supported by published experimental data and are most likely inaccurate. This review offers some guiding principles for the application of free testosterone measurements in the diagnosis and treatment of patients with androgen disorders. The growing number of testosterone prescriptions and widely recognized problems with the direct measurement as well as the computation of free testosterone concentrations render this critical review timely and clinically relevant.

Trehalose induces functionally active conformation in the intrinsically disordered N-terminal domain of glucocorticoid receptor.

Glucocorticoid receptor (GR) is a classic member of the nuclear receptor superfamily and plays pivotal roles in human physiology at the level of gene regulation. Various constellations of cellular cofactors are required to associate with GR to activate/repress genes. The effects of specific ligands on the AF2 structure and consequent preferential binding of co-activators or co-repressors have helped our understanding of the mechanisms involved. But the data so far fall short of fully explaining GR actions. We believe that this is because work so far has largely avoided detailed examination of the contributions of AF1 to overall GR actions. It has been shown that the GR containing only the N-terminal domain (NTD) and the DNA-binding domain (GR500) is constitutively quite active in stimulating transcription from simple promoters. However, we are only beginning to understand structure and functions of GR500 in spite of the fact that AF1 located within the NTD serves as major transactivation domain for GR. Lack of this information has hampered our complete understanding of how GR regulates its target gene(s). The major obstacle in determining GR500 structure has been due to its intrinsically disordered NTD conformation, frequently found in transcription factors. In this study, we tested whether a naturally occurring osmolyte, trehalose, can promote functionally ordered conformation in GR500. Our data show that in the presence of trehalose, GR500 is capable of formation of a native-like functionally folded conformation.