Comparing two data collection methods to track vital events in maternal and child health via community health workers in rural Nepal.

BACKGROUND: Timely tracking of health outcomes is difficult in low- and middle-income countries without comprehensive vital registration systems. Community health workers (CHWs) are increasingly collecting vital events data while delivering routine care in low-resource settings. It is necessary, however, to assess whether routine programmatic data collected by CHWs are sufficiently reliable for timely monitoring and evaluation of health interventions. To study this, we assessed the consistency of vital events data recorded by CHWs using two methodologies-routine data collected while delivering an integrated maternal and child health intervention, and data from a birth history census approach at the same site in rural Nepal. METHODS: We linked individual records from routine programmatic data from June 2017 to May 2018 with those from census data, both collected by CHWs at the same site using a mobile platform. We categorized each vital event over a one-year period as 'recorded by both methods,' 'census alone,' or 'programmatic alone.' We further assessed whether vital events data recorded by both methods were classified consistently. RESULTS: From June 2017 to May 2018, we identified a total of 713 unique births collectively from the census (birth history) and programmatic maternal 'post-delivery' data. Three-fourths of these births (n = 526) were identified by both. There was high consistency in birth location classification among the 526 births identified by both methods. Upon including additional programmatic 'child registry' data, we identified 746 total births, of which 572 births were identified by both census and programmatic methods. Programmatic data (maternal 'post-delivery' and 'child registry' combined) captured more births than census data (723 vs. 595). Both methods consistently classified most infants as 'living,' while infant deaths and stillbirths were largely classified inconsistently or recorded by only one method. Programmatic data identified five infant deaths and five stillbirths not recorded in census data. CONCLUSIONS: Our findings suggest that data collected by CHWs from routinely tracking pregnancies, births, and deaths are promising for timely program monitoring and evaluation. Despite some limitations, programmatic data may be more sensitive in detecting vital events than cross-sectional census surveys asking women to recall these events.

Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant.

BACKGROUND: The prevention of bleeding with adequately sustained levels of clotting factor, after a single therapeutic intervention and without the need for further medical intervention, represents an important goal in the treatment of hemophilia. METHODS: We infused a single-stranded adeno-associated viral (AAV) vector consisting of a bioengineered capsid, liver-specific promoter and factor IX Padua (factor IX-R338L) transgene at a dose of 5×1011 vector genomes per kilogram of body weight in 10 men with hemophilia B who had factor IX coagulant activity of 2% or less of the normal value. Laboratory values, bleeding frequency, and consumption of factor IX concentrate were prospectively evaluated after vector infusion and were compared with baseline values. RESULTS: No serious adverse events occurred during or after vector infusion. Vector-derived factor IX coagulant activity was sustained in all the participants, with a mean (±SD) steady-state factor IX coagulant activity of 33.7±18.5% (range, 14 to 81). On cumulative follow-up of 492 weeks among all the participants (range of follow-up in individual participants, 28 to 78 weeks), the annualized bleeding rate was significantly reduced (mean rate, 11.1 events per year [range, 0 to 48] before vector administration vs. 0.4 events per year [range, 0 to 4] after administration; P=0.02), as was factor use (mean dose, 2908 IU per kilogram [range, 0 to 8090] before vector administration vs. 49.3 IU per kilogram [range, 0 to 376] after administration; P=0.004). A total of 8 of 10 participants did not use factor, and 9 of 10 did not have bleeds after vector administration. An asymptomatic increase in liver-enzyme levels developed in 2 participants and resolved with short-term prednisone treatment. One participant, who had substantial, advanced arthropathy at baseline, administered factor for bleeding but overall used 91% less factor than before vector infusion. CONCLUSIONS: We found sustained therapeutic expression of factor IX coagulant activity after gene transfer in 10 participants with hemophilia who received the same vector dose. Transgene-derived factor IX coagulant activity enabled the termination of baseline prophylaxis and the near elimination of bleeding and factor use. (Funded by Spark Therapeutics and Pfizer; ClinicalTrials.gov number, NCT02484092 .).

Adjusted restricted mean survival times in observational studies.

In observational studies with censored data, exposure-outcome associations are commonly measured with adjusted hazard ratios from multivariable Cox proportional hazards models. The difference in restricted mean survival times (RMSTs) up to a pre-specified time point is an alternative measure that offers a clinically meaningful interpretation. Several regression-based methods exist to estimate an adjusted difference in RMSTs, but they digress from the model-free method of taking the area under the survival function. We derive the adjusted RMST by integrating an adjusted Kaplan-Meier estimator with inverse probability weighting (IPW). The adjusted difference in RMSTs is the area between the two IPW-adjusted survival functions. In a Monte Carlo-type simulation study, we demonstrate that the proposed estimator performs as well as two regression-based approaches: the ANCOVA-type method of Tian et al and the pseudo-observation method of Andersen et al. We illustrate the methods by reexamining the association between total cholesterol and the 10-year risk of coronary heart disease in the Framingham Heart Study.

Revised Framingham Stroke Risk Profile to Reflect Temporal Trends.

BACKGROUND: Age-adjusted stroke incidence has decreased over the past 50 years, likely as a result of changes in the prevalence and impact of various stroke risk factors. An updated version of the Framingham Stroke Risk Profile (FSRP) might better predict current risks in the FHS (Framingham Heart Study) and other cohorts. We compared the accuracy of the standard (old) and of a revised (new) version of the FSRP in predicting the risk of all-stroke and ischemic stroke and validated this new FSRP in 2 external cohorts, the 3C (3 Cities) and REGARDS (Reasons for Geographic and Racial Differences in Stroke) studies. METHODS: We computed the old FSRP as originally described and a new model that used the most recent epoch-specific risk factor prevalence and hazard ratios for individuals ≥55 years of age and for the subsample ≥65 years of age (to match the age range in REGARDS and 3C studies, respectively) and compared the efficacy of these models in predicting 5- and 10-year stroke risks. RESULTS: The new FSRP was a better predictor of current stroke risks in all 3 samples than the old FSRP (calibration χ2 of new/old FSRP: in men: 64.0/12.1, 59.4/30.6, and 20.7/12.5; in women: 42.5/4.1, 115.4/90.3, and 9.8/6.5 in FHS, REGARDS, and 3C, respectively). In the REGARDS, the new FSRP was a better predictor among whites compared with blacks. CONCLUSIONS: A more contemporaneous, new FSRP better predicts current risks in 3 large community samples and could serve as the basis for examining geographic and racial differences in stroke risk and the incremental diagnostic utility of novel stroke risk factors.

Comparative genomics of Cp8viruses with special reference to Campylobacter phage vB_CjeM_los1, isolated from a slaughterhouse in Ireland.

Campylobacter phage vB_CjeM_Los1 was recently isolated from a slaughterhouse in the Republic of Ireland using the host Campylobacter jejuni subsp. jejuni PT14, and full-genome sequencing and annotation were performed. The genome was found to be 134,073 bp in length and to contain 169 predicted open reading frames. Transmission electron microscopy images of vB_CjeM_Los1 revealed that it belongs to the family Myoviridae, with tail fibres observed in both extended and folded conformations, as seen in T4. The genome size and morphology of vB_CjeM_Los1 suggest that it belongs to the genus Cp8virus, and seven other Campylobacter phages with similar size characteristics have also been fully sequenced. In this work, comparative studies were performed in relation to genomic rearrangements and conservation within each of the eight genomes. None of the eight genomes were found to have undergone internal rearrangements, and their sequences retained more than 98% identity with one another despite the widespread geographical distribution of each phage. Whole-genome phylogenetics were also performed, and clades were shown to be representative of the differing number of tRNAs present in each phage. This may be an indication of lineages within the genus, despite their striking homology.

Management of Refractory Pediatric Kaposiform Hemangioendothelioma With Sirolimus and Aspirin.

Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor characterized by aggressive local invasion and a syndrome of platelet trapping known as Kasabach-Merritt phenomenon that, through deposition of platelet derived growth factors, may perpetuate the growth of the tumor. Although many cases of KHE are successfully treated with local control or low-intensity chemotherapy, some cases are often refractory even to aggressive treatment. Herein, we describe a patient with a refractory, recurrent KHE despite multiple attempts at local control and intensive chemotherapy, that ultimately was successfully treated with rationally designed and low-intensity combination therapy of sirolimus and aspirin.

A Randomized Controlled Feasibility Trial in Behavioral Weight Management for Underserved Postpartum African American Women: The RENEW Study.

We aimed to test the feasibility of an in-person behavioral weight-loss intervention for underserved postpartum African American women with overweight or obesity in an urban hospital setting. Participants were randomized to an intervention of a culturally tailored adaptation of the Diabetes Prevention Program or usual care. The primary outcome was program satisfaction. Women who completed the intervention reported higher levels of satisfaction with the program, despite low attendance rates at group meetings. The intervention was not feasible because of these low rates of attendance and high rates of attrition after randomization. Offering the program electronically and off-site for convenience and more psychosocial support for postpartum women with obesity may improve feasibility.

A Technology-Enabled Solution to Manage Referrals to Hospice and Palliative Care Beds: The Ottawa SMART System as a Case Study.

Ottawa has a 31-bed palliative care unit (PCU) and two residential adult hospices (total 19 beds). In 2013, we initiated a project to improve the referral and triage processes to these beds. Previously, there were two separate paper-based systems with duplication, inefficiencies, delays and inappropriate patient placements. The multipronged approach included clarifying the respective roles of the PCU and hospices, creating a single referral and triage office and developing an e-platform. We leveraged technology that was available in the public-funded system. This paper describes the development processes, lessons learned, and the final system, referred to as System to Manage Access, Referrals and Triage (SMART).

High-level transgene expression in induced pluripotent stem cell-derived megakaryocytes: correction of Glanzmann thrombasthenia.

Megakaryocyte-specific transgene expression in patient-derived induced pluripotent stem cells (iPSCs) offers a new approach to study and potentially treat disorders affecting megakaryocytes and platelets. By using a Gp1ba promoter, we developed a strategy for achieving a high level of protein expression in human megakaryocytes. The feasibility of this approach was demonstrated in iPSCs derived from two patients with Glanzmann thrombasthenia (GT), an inherited platelet disorder caused by mutations in integrin αIIbß3. Hemizygous insertion of Gp1ba promoter-driven human αIIb complementary DNA into the AAVS1 locus of iPSCs led to high αIIb messenger RNA and protein expression and correction of surface αIIbß3 in megakaryocytes. Agonist stimulation of these cells displayed recovery of integrin αIIbß3 activation. Our findings demonstrate a novel approach to studying human megakaryocyte biology as well as functional correction of the GT defect, offering a potential therapeutic strategy for patients with diseases that affect platelet function.

Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond Blackfan anemia patients.

Diamond Blackfan anemia (DBA) is a congenital disorder with erythroid (Ery) hypoplasia and tissue morphogenic abnormalities. Most DBA cases are caused by heterozygous null mutations in genes encoding ribosomal proteins. Understanding how haploinsufficiency of these ubiquitous proteins causes DBA is hampered by limited availability of tissues from affected patients. We generated induced pluripotent stem cells (iPSCs) from fibroblasts of DBA patients carrying mutations in RPS19 and RPL5. Compared with controls, DBA fibroblasts formed iPSCs inefficiently, although we obtained 1 stable clone from each fibroblast line. RPS19-mutated iPSCs exhibited defects in 40S (small) ribosomal subunit assembly and production of 18S ribosomal RNA (rRNA). Upon induced differentiation, the mutant clone exhibited globally impaired hematopoiesis, with the Ery lineage affected most profoundly. RPL5-mutated iPSCs exhibited defective 60S (large) ribosomal subunit assembly, accumulation of 12S pre-rRNA, and impaired erythropoiesis. In both mutant iPSC lines, genetic correction of ribosomal protein deficiency via complementary DNA transfer into the "safe harbor" AAVS1 locus alleviated abnormalities in ribosome biogenesis and hematopoiesis. Our studies show that pathological features of DBA are recapitulated by iPSCs, provide a renewable source of cells to model various tissue defects, and demonstrate proof of principle for genetic correction strategies in patient stem cells.

Clonal genetic and hematopoietic heterogeneity among human-induced pluripotent stem cell lines.

Induced pluripotent stem cells (iPSCs) hold great promise for modeling human hematopoietic diseases. However, intrinsic variability in the capacities of different iPSC lines for hematopoietic development complicates comparative studies and is currently unexplained. We created and analyzed 3 separate iPSC clones from fibroblasts of 3 different normal individuals using a standardized approach that included excision of integrated reprogramming genes by Cre-Lox mediated recombination. Gene expression profiling and hematopoietic differentiation assays showed that independent lines from the same individual were generally more similar to one another than those from different individuals. However, one iPSC line (WT2.1) exhibited a distinctly different gene expression, proliferation rate, and hematopoietic developmental potential relative to all other iPSC lines. This "outlier" clone also acquired extensive copy number variations (CNVs) during reprogramming, which may be responsible for its divergent properties. Our data indicate how inherent and acquired genetic differences can influence iPSC properties, including hematopoietic potential.

Peptide spectra in Wilms tumor that associate with adverse outcomes.

BACKGROUND: The 2013 Children's Oncology Group (COG) blueprint for renal tumor research challenges investigators to develop new, risk-specific biological therapies for unfavorable histology and higher-risk Wilms tumor (WT) in an effort to close a persistent survival gap and to reduce treatment toxicities. As an initial response to this call from the COG, we used imaging mass spectrometry to determine peptide profiles of WT associated with adverse outcomes. MATERIALS AND METHODS: We created a WT tissue microarray containing 2-mm punches of formalin-fixed, paraffin-embedded specimens archived from 48 sequentially treated WT patients at our institutions. Imaging mass spectrometry was performed to compare peptide spectra between three patient groups as follows: unfavorable versus favorable histology, treatment success versus failure, and COG higher- versus lower-risk disease. Statistically significant peptide peaks differentiating groups were identified and incorporated into a predictive model using a genetic algorithm. RESULTS: One hundred thirty-one peptide peaks were differentially expressed in unfavorable versus favorable histology WT (P < 0.05). Two hundred three peaks differentiated treatment failure from success (P < 0.05). Seventy-one peaks differentiated COG higher-risk disease from the very-low, low, and standard-risk groups (P < 0.05). These peaks were used to develop predictive models that could differentiate among patient groups 98.49%, 94.46%, and 98.55% of the time, respectively. Spectral patterns were internally cross-validated using a leave-20% out model. CONCLUSIONS: Peptide spectra can discriminate adverse behavior of WT. After future external validation and refinement, these models could be used to predict WT behavior and to stratify intensity of chemotherapy regimens. Furthermore, peptides discovered in the model could be sequenced to identify potential risk-specific drug targets.

Association of exhaled carbon monoxide with subclinical cardiovascular disease and their conjoint impact on the incidence of cardiovascular outcomes.

AIMS: Whereas endogenous carbon monoxide (CO) is cytoprotective at physiologic levels, excess CO concentrations are associated with cardiometabolic risk and may represent an important marker of progression from subclinical to clinical cardiovascular disease (CVD). METHODS AND RESULTS: In 1926 participants of the Framingham Offspring Study (aged 57 ± 10 years, 46% women), we investigated the relationship of exhaled CO, a surrogate of blood CO concentration, with both prevalent subclinical CVD and incident clinical CVD events. Presence of subclinical CVD was determined using a comprehensive panel of diagnostic tests used to assess cardiac and vascular structure and function. Individuals with the highest (>5 p.p.m.) compared with lowest (≤4 p.p.m.) CO exposure were more likely to have subclinical CVD [odds ratios (OR): 1.67, 95% CI: 1.32-2.12; P < 0.001]. During the follow-up period (mean 5 ± 3 years), 193 individuals developed overt CVD. Individuals with both high CO levels and any baseline subclinical CVD developed overt CVD at an almost four-fold higher rate compared with those with low CO levels and no subclinical disease (22.1 vs. 6.3%). Notably, elevated CO was associated with incident CVD in the presence [hazards ration (HR): 1.83, 95% CI: 1.08-3.11; P = 0.026] but not in the absence (HR: 0.80, 95% CI: 0.42-1.53; P = 0.51) of subclinical CVD (Pinteraction = 0.019). Similarly, subclinical CVD was associated with incident CVD in the presence of high but not low CO exposure. CONCLUSION: Our findings in a community-based sample suggest that elevated CO is a marker of greater subclinical CVD burden and, furthermore, a potential key component in the progression from subclinical to clinical CVD.

Low serum magnesium and the development of atrial fibrillation in the community: the Framingham Heart Study.

BACKGROUND: Low serum magnesium has been linked to increased risk of atrial fibrillation (AF) after cardiac surgery. It is unknown whether hypomagnesemia predisposes to AF in the community. METHODS AND RESULTS: We studied 3530 participants (mean age, 44 years; 52% women) from the Framingham Offspring Study who attended a routine examination and were free of AF and cardiovascular disease. We used Cox proportional hazard regression analysis to examine the association between serum magnesium at baseline and risk of incident AF. Analyses were adjusted for conventional AF risk factors, use of antihypertensive medications, and serum potassium. During up to 20 years of follow-up, 228 participants developed AF. Mean serum magnesium was 1.88 mg/dL. The age- and sex-adjusted incidence rate of AF was 9.4 per 1000 person-years (95% confidence interval, 6.7-11.9) in the lowest quartile of serum magnesium (≤1.77 mg/dL) compared with 6.3 per 1000 person-years (95% confidence interval, 4.1-8.4) in the highest quartile (≥1.99 mg/dL). In multivariable-adjusted models, individuals in the lowest quartile of serum magnesium were ~50% more likely to develop AF (adjusted hazard ratio, 1.52; 95% confidence interval, 1.00-2.31; P=0.05) compared with those in the upper quartiles. Results were similar after the exclusion of individuals on diuretics. CONCLUSIONS: Low serum magnesium is moderately associated with the development of AF in individuals without cardiovascular disease. Because hypomagnesemia is common in the general population, a link with AF may have potential clinical implications. Further studies are warranted to confirm our findings and to elucidate the underlying mechanisms.

A sensitive and specific histopathologic prognostic marker for H3F3A K27M mutant pediatric glioblastomas.

Pediatric glioblastomas (GBM) are highly aggressive and lethal tumors. Recent sequencing studies have shown that ~30 % of pediatric GBM and ~80 % of diffuse intrinsic pontine gliomas show K27M mutations in the H3F3A gene, a variant encoding histone H3.3. H3F3A K27M mutations lead to global reduction in H3K27me3. Our goal was to develop biomarkers for the histopathologic detection of these tumors. Therefore, we evaluated the utility of measuring H3K27me3 global reduction as a histopathologic and prognostic biomarker and tested an antibody directed specifically against the H3.3 K27M mutation in 290 samples. The study cohort included 203 pediatric (including 38 pediatric high-grade astrocytomas) and 38 adult brain tumors of various subtypes and grades and 49 non-neoplastic reactive brain tissues. Detection of H3.3 K27M by immunohistochemistry showed 100 % sensitivity and specificity and was superior to global reduction in H3K27me3 as a biomarker in diagnosing H3F3A K27M mutations. Moreover, cases that stained positive for H3.3 K27M showed a significantly poor prognosis compared to corresponding negative tumors. These results suggest that immunohistochemical detection of H3.3 K27M is a sensitive and specific surrogate for the H3F3A K27M mutation and defines a prognostically poor subset of pediatric GBM.

Assessing the incremental predictive performance of novel biomarkers over standard predictors.

It is unclear to what extent the incremental predictive performance of a novel biomarker is impacted by the method used to control for standard predictors. We investigated whether adding a biomarker to a model with a published risk score overestimates its incremental performance as compared to adding it to a multivariable model with individual predictors (or a composite risk score estimated from the sample of interest) and to a null model. We used 1000 simulated datasets (with a range of risk factor distributions and event rates) to compare these methods, using the continuous net reclassification index (NRI), the integrated discrimination index (IDI), and change in the C-statistic as discrimination metrics. The new biomarker was added to the following: null model, model including a published risk score, model including a composite risk score estimated from the sample of interest, and multivariable model with individual predictors. We observed a gradient in the incremental performance of the biomarker, with the null model resulting in the highest predictive performance of the biomarker and the model using individual predictors resulting in the lowest (mean increases in C-statistic between models without and with the biomarker: 0.261, 0.085, 0.030, and 0.031; NRI: 0.767, 0.621, 0.513, and 0.530; IDI: 0.153, 0.093, 0.053 and 0.057, respectively). These findings were supported by the Framingham Study data predicting atrial fibrillation using novel biomarkers. We recommend that authors report the effect of a new biomarker after controlling for standard predictors modeled as individual variables.

The safe passage study: design, methods, recruitment, and follow-up approach.

BACKGROUND: The Safe Passage Study is a large, prospective, multidisciplinary study designed to (1) investigate the association between prenatal alcohol exposure, sudden infant death syndrome (SIDS), and stillbirth, and (2) determine the biological basis of the spectrum of phenotypic outcomes from exposure, as modified by environmental and genetic factors that increase the risk of stillbirth, SIDS, and in surviving children, fetal alcohol spectrum disorders. METHODS: The results provided are based on an interim assessment of 6004 women enrolled, out of the 12,000 projected, from the Northern Plains, US, and Cape Town, South Africa, areas known to be of high risk for maternal drinking during pregnancy. Research objectives, study design, and descriptive statistics, including consent, recruitment, and retention information, are provided. RESULTS: Overall visit compliance is 87%, and includes prenatal, delivery/newborn, and postnatal contacts through 1 year post-delivery. Pregnancy outcome ascertainment is 98% prior to medical chart review; less than 2% of women withdraw. Consent for the use of DNA and placental tissue exceed 94%, and consent to participate in the autopsy portion of the study is 71%. CONCLUSIONS: The Safe Passage Study is the first multi-site study of SIDS and stillbirth to integrate prospectively collected exposure information with multidisciplinary biological information in the same maternal and fetal/infant dyad using a common protocol. Essential components of the study design and its success are close ties to the community and rigorous systems and processes to ensure compliance with the study protocol and procedures.

Ectopic production of ß-hCG by osteosarcoma: a case report and review of the literature.

Ectopic production of ß-human chorionic gonadotropin (ß-hCG) by nontrophoblastic tumors has been reported but mostly in carcinomas. We report a case of an adolescent female patient with a epithelioid osteosarcoma that was discovered to secrete ß-hCG after routine pregnancy testing. Immunohistochemical staining of her primary tumor biopsy demonstrated immunoreactivity for ß-hCG. Levels of serum ß-hCG were monitored throughout her therapy and demonstrated normalization with effective systemic therapy and local control. She remains disease free 6 months off therapy, with undetectable hormone levels. A review of the available literature on ß-hCG production by sarcomas is also presented.

A genome-wide association study identifies novel loci associated with circulating IGF-I and IGFBP-3.

Insulin-like growth factor-I (IGF-I) and insulin-like growth factor-binding protein-3 (IGFBP-3) are involved in cell replication, proliferation, differentiation, protein synthesis, carbohydrate homeostasis and bone metabolism. Circulating IGF-I and IGFBP-3 concentrations predict anthropometric traits and risk of cancer and cardiovascular disease. In a genome-wide association study of 10 280 middle-aged and older men and women from four community-based cohort studies, we confirmed a known association of single nucleotide polymorphisms in the IGFBP3 gene region on chromosome 7p12.3 with IGFBP-3 concentrations using a significance threshold of P < 5 × 10(-8) (P = 3.3 × 10(-101)). Furthermore, the same IGFBP3 gene locus (e.g. rs11977526) that was associated with IGFBP-3 concentrations was also associated with the opposite direction of effect, with IGF-I concentration after adjustment for IGFBP-3 concentration (P = 1.9 × 10(-26)). A novel and independent locus on chromosome 7p12.3 (rs700752) had genome-wide significant associations with higher IGFBP-3 (P = 4.4 × 10(-21)) and higher IGF-I (P = 4.9 × 10(-9)) concentrations; when the two measurements were adjusted for one another, the IGF-I association was attenuated but the IGFBP-3 association was not. Two additional loci demonstrated genome-wide significant associations with IGFBP-3 concentration (rs1065656, chromosome 16p13.3, P = 1.2 × 10(-11), IGFALS, a confirmatory finding; and rs4234798, chromosome 4p16.1, P = 4.5 × 10(-10), SORCS2, a novel finding). Together, the four genome-wide significant loci explained 6.5% of the population variation in IGFBP-3 concentration. Furthermore, we observed a borderline statistically significant association between IGF-I concentration and FOXO3 (rs2153960, chromosome 6q21, P = 5.1 × 10(-7)), a locus associated with longevity. These genetic loci deserve further investigation to elucidate the biological basis for the observed associations and clarify their possible role in IGF-mediated regulation of cell growth and metabolism.