Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum

BACKGROUND: The circulating level of the peptide hormone insulin-like factor 3 (INSL3) is a promising diagnostic marker reflecting Leydig cell function in the male. Few commercial immunoassays of varying quality exist. Therefore, we decided to develop and validate a precise method for quantification of INSL3 by mass spectrometry. METHODS: We developed an assay in which the INSL3 A-chain is released from the INSL3 A-B heterodimer by chemical reduction and alkylation. The alkylated INSL3 A-chain is quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as substitute for serum INSL3. The method was compared to a validated and sensitive in-house serum INSL3 immunoassay using 97 serum samples from 12 healthy boys during pubertal transition. Adult levels were determined based on sera from 72 adult healthy males aged 18-40 years. RESULTS: An LC-MS/MS assay with limit of detection and limit of quantification (LOQ) of 0.06 and 0.15 ng/mL, respectively, and intra-assay CVs <9% in the relevant ranges was obtained. The LC-MS/MS compared well with the in-house immunoassay (Deming regression slope: 1.28; Pearson correlation: R=0.86). INSL3 concentrations increased with pubertal maturation in healthy boys. INSL3 concentrations were above the LOQ in all samples from the adult men. The mean (±2 SD range)for serum INSL3 concentrations in the adult men was 2.2 (0.5-3.9) ng/mL. CONCLUSIONS: We have developed a robust and sensitive method suitable for quantitation of serum INSL3 in a clinical setting using LC-MS/MS instrumentation available in modern clinical laboratories. The method paves the way for future studies into the clinical role of serum INSL3 measurements

Assessing a digital technology-supported community child health programme in India using the Social Return on Investment framework.

An estimated 5.0 million children aged under 5 years died in 2020, with 82% of these deaths occurring in sub-Saharan Africa and southern Asia. Over one-third of Mumbai's population has limited access to healthcare, and child health outcomes are particularly grave among the urban poor. We describe the implementation of a digital technology-based child health programme in Mumbai and evaluate its holistic impact. Using an artificial intelligence (AI)-powered mobile health platform, we developed a programme for community-based management of child health. Leveraging an existing workforce, community health workers (CHW), the programme was designed to strengthen triage and referral, improve access to healthcare in the community, and reduce dependence on hospitals. A Social Return on Investment (SROI) framework is used to evaluate holistic impact. The programme increased the proportion of illness episodes treated in the community from 4% to 76%, subsequently reducing hospitalisations and out-of-pocket expenditure on private healthcare providers. For the total investment of Indian Rupee (INR) 2,632,271, the social return was INR 34,435,827, delivering an SROI ratio of 13. The annual cost of the programme per child was INR 625. Upskilling an existing workforce such as CHWs, with the help of AI-driven decision- support tools, has the potential to extend capacity for critical health services into community settings. This study provides a blueprint for evaluating the holistic impact of health technologies using evidence-based tools like SROI. These findings have applicability across income settings, offering clear rationale for the promotion of technology-supported interventions that strengthen healthcare delivery

Revising the WHO verbal autopsy instrument to facilitate routine cause-of-death monitoring.

OBJECTIVE: Verbal autopsy (VA) is a systematic approach for determining causes of death (CoD) in populations without routine medical certification. It has mainly been used in research contexts and involved relatively lengthy interviews. Our objective here is to describe the process used to shorten, simplify, and standardise the VA process to make it feasible for application on a larger scale such as in routine civil registration and vital statistics (CRVS) systems. METHODS: A literature review of existing VA instruments was undertaken. The World Health Organization (WHO) then facilitated an international consultation process to review experiences with existing VA instruments, including those from WHO, the Demographic Evaluation of Populations and their Health in Developing Countries (INDEPTH) Network, InterVA, and the Population Health Metrics Research Consortium (PHMRC). In an expert meeting, consideration was given to formulating a workable VA CoD list [with mapping to the International Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10) CoD] and to the viability and utility of existing VA interview questions, with a view to undertaking systematic simplification. FINDINGS: A revised VA CoD list was compiled enabling mapping of all ICD-10 CoD onto 62 VA cause categories, chosen on the grounds of public health significance as well as potential for ascertainment from VA. A set of 221 indicators for inclusion in the revised VA instrument was developed on the basis of accumulated experience, with appropriate skip patterns for various population sub-groups. The duration of a VA interview was reduced by about 40% with this new approach. CONCLUSIONS: The revised VA instrument resulting from this consultation process is presented here as a means of making it available for widespread use and evaluation. It is envisaged that this will be used in conjunction with automated models for assigning CoD from VA data, rather than involving physicians

Ischaemic strokes in patients with pulmonary arteriovenous malformations and hereditary hemorrhagic telangiectasia: associations with iron deficiency and platelets.

<div><p>Background</p><p>Pulmonary first pass filtration of particles marginally exceeding ∼7 µm (the size of a red blood cell) is used routinely in diagnostics, and allows cellular aggregates forming or entering the circulation in the preceding cardiac cycle to lodge safely in pulmonary capillaries/arterioles. Pulmonary arteriovenous malformations compromise capillary bed filtration, and are commonly associated with ischaemic stroke. Cohorts with CT-scan evident malformations associated with the highest contrast echocardiographic shunt grades are known to be at higher stroke risk. Our goal was to identify within this broad grouping, which patients were at higher risk of stroke.</p><p>Methodology</p><p>497 consecutive patients with CT-proven pulmonary arteriovenous malformations due to hereditary haemorrhagic telangiectasia were studied. Relationships with radiologically-confirmed clinical ischaemic stroke were examined using logistic regression, receiver operating characteristic analyses, and platelet studies.</p><p>Principal Findings</p><p>Sixty-one individuals (12.3%) had acute, non-iatrogenic ischaemic clinical strokes at a median age of 52 (IQR 41–63) years. In crude and age-adjusted logistic regression, stroke risk was associated not with venous thromboemboli or conventional neurovascular risk factors, but with low serum iron (adjusted odds ratio 0.96 [95% confidence intervals 0.92, 1.00]), and more weakly with low oxygen saturations reflecting a larger right-to-left shunt (adjusted OR 0.96 [0.92, 1.01]). For the same pulmonary arteriovenous malformations, the stroke risk would approximately double with serum iron 6 µmol/L compared to mid-normal range (7–27 µmol/L). Platelet studies confirmed overlooked data that iron deficiency is associated with exuberant platelet aggregation to serotonin (5HT), correcting following iron treatment. By MANOVA, adjusting for participant and 5HT, iron or ferritin explained 14% of the variance in log-transformed aggregation-rate (p = 0.039/p = 0.021).</p><p>Significance</p><p>These data suggest that patients with compromised pulmonary capillary filtration due to pulmonary arteriovenous malformations are at increased risk of ischaemic stroke if they are iron deficient, and that mechanisms are likely to include enhanced aggregation of circulating platelets.</p></div

Quality of medical training and emigration of physicians from India

<p>Abstract</p> <p>Background</p> <p>Physician 'brain drain' negatively impacts health care delivery. Interventions to address physician emigration have been constrained by lack of research on systematic factors that influence physician migration. We examined the relationship between the quality of medical training and rate of migration to the United States and the United Kingdom among Indian medical graduates (1955–2002).</p> <p>Methods</p> <p>We calculated the fraction of medical graduates who emigrated to the United States and the United Kingdom, based on rankings of medical colleges and universities according to three indicators of the quality of medical education (a) student choice, (b) academic publications, and (c) the availability of specialty medical training.</p> <p>Results</p> <p>Physicians from the top quintile medical colleges and of universities were 2 to 4 times more likely to emigrate to the United States and the United Kingdom than graduates from the bottom quintile colleges and universities.</p> <p>Conclusion</p> <p>Graduates of institutions with better quality medical training have a greater likelihood of emigrating. Interventions designed to counter loss of physicians should focus on graduates from top quality institutions.</p

Identifying optimal doses of heart failure medications in men compared with women: a prospective, observational cohort study

Background: Guideline-recommended doses of angiotensin-converting-enzyme (ACE) inhibitors or angiotensin-receptor blockers (ARBs), and β blockers are similar for men and women with heart failure with reduced ejection fraction (HFrEF), even though there are known sex differences in pharmacokinetics of these drugs. We hypothesised that there might be sex differences in the optimal dose of ACE inhibitors or ARBs and β blockers in patients with HFrEF. Methods: We did a post-hoc analysis of BIOSTAT-CHF, a prospective study in 11 European countries of patients with heart failure in whom initiation and up-titration of ACE inhibitors or ARBs and β blockers was encouraged by protocol. We included only patients with left ventricular ejection fraction less than 40%, and excluded those who died within the first 3 months. Primary outcome was a composite of time to all-cause mortality or hospitalisation for heart failure. Findings were validated in ASIAN-HF, an independent cohort of 3539 men and 961 women with HFrEF. Findings: Among 1308 men and 402 women with HFrEF from BIOSTAT-CHF, women were older (74 [12] years vs 70 [12] years, p&lt;0·0001) and had lower bodyweights (72 [16] kg vs 85 [18] kg, p&lt;0·0001) and heights (162 [7] cm vs 174 [8] cm, p&lt;0·0001) than did men, although body-mass index did not differ significantly. A similar number of men and women reached guideline-recommended target doses of ACE inhibitors or ARBs (99 [25%] vs 304 [23%], p=0·61) and β blockers (57 [14%] vs 168 [13%], p=0·54). In men, the lowest hazards of death or hospitalisation for heart failure occurred at 100% of the recommended dose of ACE inhibitors or ARBs and β blockers, but women showed approximately 30% lower risk at only 50% of the recommended doses, with no further decrease in risk at higher dose levels. These sex differences were still present after adjusting for clinical covariates, including age and body surface area. In the ASIAN-HF registry, similar patterns were observed for both ACE inhibitors or ARBs and β blockers, with women having approximately 30% lower risk at 50% of the recommended doses, with no further benefit at higher dose levels. Interpretation: This study suggests that women with HFrEF might need lower doses of ACE inhibitors or ARBs and β blockers than men, and brings into question what the true optimal medical therapy is for women versus men

Voltage- and temperature-dependent activation of TRPV3 channels is potentiated by receptor-mediated PI(4,5)P2 hydrolysis

TRPV3 is a thermosensitive channel that is robustly expressed in skin keratinocytes and activated by innocuous thermal heating, membrane depolarization, and chemical agonists such as 2-aminoethyoxy diphenylborinate, carvacrol, and camphor. TRPV3 modulates sensory thermotransduction, hair growth, and susceptibility to dermatitis in rodents, but the molecular mechanisms responsible for controlling TRPV3 channel activity in keratinocytes remain elusive. We show here that receptor-mediated breakdown of the membrane lipid phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) regulates the activity of both native TRPV3 channels in primary human skin keratinocytes and expressed TRPV3 in a HEK-293–derived cell line stably expressing muscarinic M1-type acetylcholine receptors. Stimulation of PI(4,5)P2 hydrolysis or pharmacological inhibition of PI 4 kinase to block PI(4,5)P2 synthesis potentiates TRPV3 currents by causing a negative shift in the voltage dependence of channel opening, increasing the proportion of voltage-independent current and causing thermal activation to occur at cooler temperatures. The activity of single TRPV3 channels in excised patches is potentiated by PI(4,5)P2 depletion and selectively decreased by PI(4,5)P2 compared with related phosphatidylinositol phosphates. Neutralizing mutations of basic residues in the TRP domain abrogate the effect of PI(4,5)P2 on channel function, suggesting that PI(4,5)P2 directly interacts with a specific protein motif to reduce TRPV3 channel open probability. PI(4,5)P2-dependent modulation of TRPV3 activity represents an attractive mechanism for acute regulation of keratinocyte signaling cascades that control cell proliferation and the release of autocrine and paracrine factors

Examining Alternative Measures of Social Disadvantage Among Asian Americans: The Relevance of Economic Opportunity, Subjective Social Status, and Financial Strain for Health

Socioeconomic position is often operationalized as education, occupation, and income. However, these measures may not fully capture the process of socioeconomic disadvantage that may be related to morbidity. Economic opportunity, subjective social status, and financial strain may also place individuals at risk for poor health outcomes. Data come from the Asian subsample of the 2003 National Latino and Asian American Study (n = 2095). Regression models were used to examine the associations between economic opportunity, subjective social status, and financial strain and the outcomes of self-rated health, body mass index, and smoking status. Education, occupation, and income were also investigated as correlates of these outcomes. Low correlations were observed between all measures of socioeconomic status. Economic opportunity was robustly negatively associated with poor self-rated health, higher body mass index, and smoking, followed by financial strain, then subjective social status. Findings show that markers of socioeconomic position beyond education, occupation, and income are related to morbidity among Asian Americans. This suggests that potential contributions of social disadvantage to poor health may be understated if only conventional measures are considered among immigrant and minority populations

PARP Inhibitor PJ34 Protects Mitochondria and Induces DNA-Damage Mediated Apoptosis in Combination With Cisplatin or Temozolomide in B16F10 Melanoma Cells

<p>PARP-1 inhibition has recently been employed in both mono- and combination therapies in various malignancies including melanoma with both promising and contradicting results reported. Although deeper understanding of the underlying molecular mechanisms may help improving clinical modalities, the complex cellular effects of PARP inhibitors make disentangling of the mechanisms involved in combination therapies difficult. Here, we used two cytostatic agents used in melanoma therapies in combination with PARP inhibition to have an insight into cellular events using the B16F10 melanoma model. We found that, when used in combination with cisplatin or temozolomide, pharmacologic blockade of PARP-1 by PJ34 augmented the DNA-damaging and cytotoxic effects of both alkylating compounds. Interestingly, however, this synergism unfolds relatively slowly and is preceded by molecular events that are traditionally believed to support cell survival including the stabilization of mitochondrial membrane potential and morphology. Our data indicate that the PARP inhibitor PJ34 has, apparently, opposing effects on the mitochondrial structure and cell survival. While, initially, it stimulates mitochondrial fusion and hyperpolarization, hallmarks of mitochondrial protection, it enhances the cytotoxic effects of alkylating agents at later stages. These findings may contribute to the optimization of PARP inhibitor-based antineoplastic modalities.</p