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This article describes the methods, successes and challenges of engaging parents while studying the impacts of COVID-19 on healthy children and families. Parent partners in a Parent and Clinician Team (PACT) informed study aims, supported feasibility and recommended changes to enhance participation. PACT members stated that they felt a sense of connectedness and purpose by contributing to COVID-19 research. Engagement increased by parents acquiring new roles, attending more frequent meetings and co-creating alternative methods of engagement. Recruiting new PACT members was challenging, likely due to limited time and resources available to parents of young children during a pandemic.

Background The COVID-19 pandemic has uniquely affected children and families by disrupting routines, changing relationships and roles, and altering usual child care, school and recreational activities. Understanding the way families experience these changes from parents’ perspectives may help to guide research on the effects of COVID-19 among children. Main body As a multidisciplinary team of child health researchers, we assembled a group of nine parents to identify concerns, raise questions, and voice perspectives to inform COVID-19 research for children and families. Parents provided a range of insightful perspectives, ideas for research questions, and reflections on their experiences during the pandemic. Conclusion Including parents as partners in early stages of COVID-19 research helped determine priorities, led to more feasible data collection methods, and hopefully has improved the relevance, applicability and value of research findings to parents and children.

Objective To evaluate the relationship between the timing of infant cereal introduction between 4 and 6 months of age and growth and dietary intake in later childhood. Study design A longitudinal cohort study was conducted among healthy children 0-10 years of age participating in The Applied Research Group for Kids cohort study between June 2008 and August 2019 in Toronto, Canada. Results Of 8943 children included, the mean (SD) age of infant cereal introduction was 5.7 (2.1) months. In the primary analysis, children who were introduced to infant cereal at 4 vs 6 months had 0.17 greater body mass index z score (95% CI 0.06-0.28; P = .002) and greater odds of obesity (OR 1.82; 95% CI 1.18-2.80; P = .006) at 10 years of age. In the secondary analysis, children who were introduced to infant cereal at 4 vs 6 months had 0.09 greater height-for-age z score (95% CI 0.04-0.15; P = .002) at 1 year of age, an association that was not observed at 5 or 10 years of age. Children who were introduced to infant cereal at 4 vs 6 months had greater nutrition risk which was primarily determined by a less-favorable eating behavior score at 18 months to 5 years of age (0.18 units higher; 95% CI 0.07-0.29; P = .001). Conclusions Introduction of infant cereal at 4 vs 6 months was associated with greater body mass index z score, greater odds of obesity, similar height-for-age z score, and less favorable eating behavior. These findings support recommendations for introducing solid food around 6 months of age.

Factor V Leiden is a genetic condition that can decrease natural anticoagulant effect in response to activated protein C, increasing thromboembolic risk. We report a 17-year-old man, homozygous for the Factor V Leiden mutation, with sudden onset arm stiffness, pain and swelling caused by a deep vein thrombosis in the subclavian vein. Pediatricians should consider thromboembolic diseases in cases of spontaneous acute limb discomfort and discoloration with no obvious cause. Int J Clin Pediatr. 2020;9(1):1-3 doi: https://doi.org/10.14740/ijcp343

Background Little is known about nutrition outcomes in preschoolers associated with breastfeeding duration beyond 12 mo of age. Objective The aim was to examine the association between total breastfeeding duration and nutrition outcomes at 3 to 5 y of age. Design A cross-sectional study in healthy children, ages 3-5 y, recruited from 9 primary care practices in Toronto was conducted through the TARGet Kids! (The Applied Research Group for Kids) research network. Parents completed standardized surveys, including the Nutrition Screening for Every Preschooler (NutriSTEP) used to assess nutrition risk. Results A total of 2987 children were included. Ninety-two percent of children were breastfed, and the mean ± SD breastfeeding duration was 11.4 ± 8.4 mo. The prevalence of nutrition risk (score >20) was 17.0%. We examined breastfeeding duration as a continuous variable. With the use of restricted cubic spline modeling, we confirmed a nonlinear relation between breastfeeding duration and NutriSTEP score, dietary intake and eating behavior subscores, and sugar-sweetened beverage and sweet-savory snack consumption. Segmented linear regression was used to examine this nonlinear relation in a piecewise approach. We found a decreasing trend in NutriSTEP score for children who were breastfed for 0-6 mo (β = -0.14; 95% CI: -0.29, 0.004), a significant decrease in NutriSTEP score for children breastfed for 6-12 mo (β = -0.20; 95% CI: -0.33, -0.07), and no significant change after 12 mo (β = 0.09; 95% CI: -0.07, 0.24) and beyond. The mean ± SD NutriSTEP scores were 17.1 ± 7.4 for no breastfeeding, 15.9 ± 6.5 for breastfeeding >0-6 mo, 13.9 ± 6.2 for >6-12 mo, 13.7 ± 6.3 for >12-18 mo, 14.6 ± 6.7 for >18-24 mo, and 14.3 ± 6.8 for >24-36 mo. Conclusions Breastfeeding for ≤12 mo was associated with decreased nutrition risk and healthier eating behaviors and dietary intake at 3-5 y of age. We found insufficient evidence of additional benefit for breastfeeding beyond 12 mo of age. The TARGet Kids! practice-based research network is registered at www.clinicaltrials.gov as NCT01869530.

Literacy is the ability to read, write and understand print. Proficiency in literacy is fundamental to social inclusion and strongly linked to health outcomes. Thus, improving literacy is important for lifelong health promotion. Poverty, inadequate hearing, speech and vision and learning disabilities may challenge literacy development. In our review, we explore these topics and suggest recommendations to: Mitigate the Effects of Poverty, Access Comprehensive Medical Assessments, Promote Early Childhood Education and Advocate for Early Intervention and Remediation Programs.

Mitigating the harmful effects of adverse social conditions is critical to promoting optimal health and development throughout the life course. Many Canadians worry over food access or struggle with household food insecurity. Public policy positions breastfeeding as a step toward eradicating poverty. Breastfeeding fulfills food security criteria by providing the infant access to sufficient, safe and nutritious food that meets dietary needs and food preferences. Unfortunately, a breastfeeding paradox exists where infants of low-income families who would most gain from the health benefits, are least likely to breastfeed. Solving household food insecurity and breastfeeding rates may be best realized at the public policy level. Notably, the health care provider’s competencies as medical expert, professional, communicator and advocate are paramount. Our commentary aims to highlight the critical link between breastfeeding and household food insecurity that may provide opportunities to affect clinical practice, public policy and child health outcomes.

The 'forgotten years' of middle childhood, from age 6 to 12, represent a critical period in child development. Emotional, social and physical development during this time have a lifelong impact on health and adult contributions to society. Mental health conditions have displaced physical illness as the leading childhood disability. Positive parenting can improve child behaviour, prevent early-onset conduct problems and provide a buffer from adverse childhood events resulting in decreased toxic stress and improved health. Medical homes can play a key role in supporting parents with positive parenting skills that are practical, evidence-based and useful in everyday life. Paediatricians need to explore the domains that promote healthy development, including caring environments, fundamental needs and nurturing relationships. Our objective is to promote high-quality positive parenting through middle childhood by identifying opportunities for paediatricians to frame parenting discussions in the context of development, behaviour and safety and to provide access to valuable parenting resources.Les « années oubliées » de la phase intermédiaire de l’enfance, entre l’âge de six et 12 ans, sont une période critique du développement de l’enfant. Pendant cette période, le développement émotionnel, social et physique a des conséquences permanentes sur la santé et l’apport de l’adulte à la société. Les troubles de santé mentale ont remplacé les maladies physiques comme principale affection de l’enfance. Des pratiques parentales positives peuvent améliorer le comportement de l’enfant, prévenir les troubles des conduites d’apparition précoce et limiter les événements indésirables de l’enfance, réduisant ainsi le stress toxique et améliorant la santé. Les milieux médicaux peuvent beaucoup contribuer à soutenir les parents afin qu’ils acquièrent des compétences parentales positives pratiques, fondées sur des données probantes et utiles dans la vie quoti- dienne. Les pédiatres doivent explorer les sphères qui favorisent un développement sain, y compris les environnements attentionnés, les besoins fondamentaux et les relations aimantes. Afin de promouvoir des pratiques parentales positives de qualité tout au long de la phase intermédiaire de l’enfance, les auteurs visent à cerner les occasions que peuvent saisir les pédiatres pour structurer les discussions avec les parents sur le développement, le comportement et la sécurité et les orienter vers des ressources intéressantes sur ce type de pratiques.

Diagnostic dilemmas are ubiquitous in pediatric emergency medicine because of the varied and often insidious presentations of many pediatric conditions. Point-of-care ultrasound (POCUS) in emergency departments is being used for some of these diagnostic challenges and can often provide rapid and valuable information to supplement a physician's clinical assessment. Intussusception is a pediatric condition that may be challenging to recognize because of its subtle and varied presenting symptoms. An unrecognized or delayed diagnosis of intussusception can be catastrophic, with complications including bowel obstruction, bowel perforation, and death. Here we present two challenging cases of intussusception, one due to its atypical age of presentation and the other due to its unique symptoms. These cases demonstrate the benefits of point-of-care ultrasound for diagnostically challenging cases in the emergency department.

As an infant grows, human milk may become the substrate for cariogenic bacteria. Here's what to watch for and how to counsel parents.

Although coarctation of the aorta (CoAo) is a congenital anomaly, it can pose a diagnostic challenge in those presenting beyond neonatal period, as some patients can remain asymptomatic until complications of hypertension develop later in life. Careful physical examination can play an important role in timely diagnosis and prevention of complications.We present 2 cases of adolescents with undiagnosed CoAo who both presented with nonspecific headaches and hypertension. Both were initially misdiagnosed as essential hypertension until careful detailed physical examination later detected weak/absent femoral pulses and discrepancy in upper and lower limb blood pressures. These findings raised the clinical suspicion of CoAo, which was confirmed on further investigations.Our cases highlight the importance of considering CoAo in the differential diagnosis of hypertension in adolescents. These cases also stress the significance of detailed cardiac examination including 4-limb blood pressure and femoral pulses in a patient presenting to the emergency department with high blood pressure irrespective of the primary complaint.

In 1998, the Centers for Disease Control and Prevention Adverse Childhood Experiences study established the profound effects of early childhood adversity on life course health. The burden of cumulative adversities can affect gene expression, immune system development and condition stress response. A scientific framework provides explanation for numerous childhood and adult health problems and high-risk behaviours that originate in early life. In our review, we discuss adverse childhood experiences, toxic stress, the neurobiological basis and multigenerational and epigenetic transmission of trauma and recognized health implications. Further, we outline building resilience, screening in the clinical setting, primary care interventions, applying trauma-informed care and future directions. We foresee that enhancing knowledge of the far-reaching effects of adverse childhood events will facilitate mitigation of toxic stress, promote child and family resilience and optimize life course health trajectories.

Qualitative studies have suggested that food insecurity adversely affects infant feeding practices. We aimed to determine how household food insecurity relates to breastfeeding initiation, duration of exclusive breastfeeding and vitamin D supplementation of breastfed infants in Canada.We studied 10 450 women who had completed the Maternal Experiences - Breastfeeding Module and the Household Food Security Survey Module of the Canadian Community Health Survey (2005-2014) and who had given birth in the year of or year before their interview. We used multivariable Cox proportional hazards models and logistic regression to examine the relation between food insecurity and infant feeding practices, adjusting for sociodemographic characteristics, maternal mood disorders and diabetes mellitus.Overall, 17% of the women reported household food insecurity, of whom 8.6% had moderate food insecurity and 2.9% had severe food insecurity (weighted percentages). After adjustment for sociodemographic factors, women with food insecurity were no less likely than others to initiate breastfeeding or provide vitamin D supplementation to their infants. Half of the women with food insecurity ceased exclusive breastfeeding by 2 months, whereas most of those with food security persisted with breastfeeding for 4 months or more. Relative to women with food security, those with marginal, moderate and severe food insecurity had significantly lower odds of exclusive breastfeeding to 4 months, but only women with moderate food insecurity had lower odds of exclusive breastfeeding to 6 months, independent of sociodemographic characteristics (odds ratio 0.60, 95% confidence interval 0.39-0.92). Adjustment for maternal mood disorder or diabetes slightly attenuated these relationships.Mothers caring for infants in food-insecure households attempted to follow infant feeding recommendations, but were less able than women with food security to sustain exclusive breastfeeding. Our findings highlight the need for more effective interventions to support food-insecure families with newborns.

Congenital diaphragmatic hernia (CDH) is associated with high early mortality. However, advances in newborn care have led to improved survival into adolescence and early adulthood. We report a 17-year-old adolescent boy with recurrent small bowel obstruction after CDH repair at 2 days of age. Emergency physicians should be aware of late complications after neonatal CDH repair.

Objective Health care policy positions breastfeeding as an important part of the solution to household food insecurity; however, there are critical gaps in our knowledge of the relationship between breastfeeding duration (exposure variable) and household food insecurity (outcome variable). Our objective was to examine this relationship. Methods A cross-sectional study was conducted from 2008 to 2016 of healthy urban children (N = 3838) who were 0 to 3 years old and recruited from The Applied Research Group for Kids (TARGet Kids!), a practice-based research network in Toronto, Canada. Total breastfeeding duration was collected from parent-reported questionnaires. Household food insecurity was measured using 1-item and 2-item food insecurity screens. Multivariable regression analysis was performed adjusting for prespecified covariates. Results The median total breastfeeding duration was 10.5 months (interquartile range, 6.0–14.0), and 14.7% of households were food insecure. After adjusting for child characteristics (age, sex), maternal characteristics (age, ethnicity, education, employment), and family characteristics (number of children, single parent family, neighborhood equity score), there was no significant association between total breastfeeding duration and household food insecurity (odds ratio, 0.99; 95% confidence interval, 0.98–1.01). Although low-income families had an increased odds of being household food insecure (P ≤ .001), we found no significant association between total breastfeeding duration and household food insecurity at varying income levels. Conclusions We found no association between breastfeeding duration and household food insecurity, regardless of family income. Although breastfeeding is associated with improved child health outcomes and considered to be part of the solution to household food insecurity, interventions focused on social determinants may provide more promising targets for the prevention of household food insecurity.

Congenital heart disease is a significant cause of infant mortality. Epidemiology and social context play a crucial role in conditioning disease burden and modulating outcomes, while diagnosis and treatment remain resource intensive. This review will address the role of social demographics, environmental exposure, epigenetics and nutrition in the aetiology of congenital heart disease. We then discuss the determinant effect of social factors on the provision and outcomes of care for congenital heart disease and implications for practice. It is our hope that enhanced knowledge of the intersection of social determinants of health and congenital heart disease will facilitate effective preventative strategies at the individual and population levels to optimize heart health outcomes across the life course.

Early childhood caries (ECC) is the most common chronic disease affecting young children in Canada. ECC may lead to pain and infection, compromised general health, decreased quality of life and increased risk for dental caries in primary and permanent teeth. A multidisciplinary approach to prevent and identify dental disease is recommended by dental and medical national organizations. Young children visit primary care providers at regular intervals from an early age. These encounters provide an ideal opportunity for primary care providers to educate clients about their children’s oral health and its importance for general health. We designed an office-based oral health screening guide to help primary care providers identify ECC, a dental referral form to facilitate dental care access and an oral health education resource to raise parental awareness. These resources were reviewed and trialled with a small number of primary care providers.

To examine the association between body mass index (BMI) and iron deficiency in early childhood, while considering the influence of low-grade systemic inflammation.Healthy children ages 1-3 years were included in a cross-sectional analysis. Age- and sex-standardized World Health Organization BMI z score (zBMI) was calculated using height/length and weight measurements; iron status was assessed by serum ferritin; inflammation was assessed by C-reactive protein (CRP). Children with CRP ≥10 mg/L were excluded because this may indicate acute systemic inflammation. Adjusted multivariable regression analyses were used to investigate the association between zBMI and both serum ferritin (µg/L), and iron deficiency (serum ferritin12 µg/L). We performed prespecified subgroup analyses according to CRP level (normal [≤1.0 mg/L] and low-grade inflammation [1.0 mg/L to10.0 mg/L]).Of 1607 children included, 20% were categorized as with zBMI1, 13% had iron deficiency, and 18% had low-grade inflammation. Higher zBMI was associated with lower serum ferritin (-1.51 µg/L, 95% CI -2.23, -0.76, P .0001) and increased odds of iron deficiency (OR 1.28, 95% CI 1.10, 1.50, P = .002). Though there was no interaction between zBMI and CRP for the adjusted linear regression model (P = .79) or logistic regression model (P = .43), children with low-grade inflammation had a higher serum ferritin (P .0001).Higher zBMI is associated with increased risk for iron deficiency in children between 1 and 3 years, and should be considered as a risk factor in targeted screening. Further research is needed to better understand the relationship between serum ferritin and CRP for children in all weight categories.ClinicalTrials.gov: NCT01869530.

Objective To determine if there is an association between longer breast-feeding duration and dental caries in healthy urban children. Methods We conducted a cross-sectional study of urban children aged 1 to 6 years recruited through The Applied Research Group for Kids (TARGet Kids!) practice-based research network between September 2011 and August 2013. The main outcome measure was parental report of dental caries. Results The adjusted predicted probability of dental caries was 7%, 8%, 11%, and 16% with total duration of breast-feeding duration of 12, 18, 24, and 36 months, respectively. In the adjusted logistic regression analyses, relative to breast-feeding 0 to 5 months, the odds of dental caries with total breast-feeding duration >24 months was 2.75 (95% confidence interval 1.61–4.72). Conclusions Among healthy urban children, longer breast-feeding duration was associated with higher odds of dental caries. These findings support heightened awareness and enhanced anticipatory guidance for preventive dental care, particularly among children who breast-feed beyond 2 years of age.

ifty years ago, health care for low-income children and families was described as “episodic, fragmented, crisis oriented, and anonymous” (1). Currently, children continue to face similar chal-lenges, and conventional medical care only begins to address a small portion of these concerns. The ‘third era’ of public health will need to focus on optimizing health and well-being through primary prevention, health promotion and community-integrated delivery systems (2).As community paediatricians, we remain powerful advocates for children, families and communities because of respect from parents, altruism and knowledge of resources (3). We understand that advocating for an integrated and high-quality community health system with ‘upstream’ health promotion and prevention requires collaboration with community agencies, multisectorial partners and health care providers (3). By directly addressing social determinants with positive health-promoting influences – such as early childhood education, family support, primary health care and behaviour/mental health services – we can achieve opti-mal population health and health equity by breaking pathways of social risk and poor health outcomes (4). The Canadian model of community health centres (CHCs), in alliance with the evolving role of the community paediatrician, provides an ideal place-based community setting to create innovative solutions and address ongoing concerns, while not attempting to fulfill all characteristics of the community paediatrician (Table 1). This CHCs-community paediatrician model can create a broad gamut of services that range from clinical consultation, school-based health care, child-, adolescent- and family-based programming, to board governance and policy, building needed child health infrastructure.

Proteasomes are responsible for most intracellular protein degradation in eukaryotes. The 20S proteasome comprises a dyad-symmetric stack of four heptameric rings made from 14 distinct subunits. How it assembles is not understood. Most subunits in the central pair of beta-subunit rings are synthesized in precursor form. Normally, the beta5 (Doa3) propeptide is essential for yeast proteasome biogenesis, but overproduction of beta7 (Pre4) bypasses this requirement. Bypass depends on a unique beta7 extension, which contacts the opposing beta ring. The resulting proteasomes appear normal but assemble inefficiently, facilitating identification of assembly intermediates. Assembly occurs stepwise into precursor dimers, and intermediates contain the Ump1 assembly factor and a novel complex, Pba1-Pba2. beta7 incorporation occurs late and is closely linked to the association of two half-proteasomes. We propose that dimerization is normally driven by the beta5 propeptide, an intramolecular chaperone, but beta7 addition overcomes an Ump1-dependent assembly checkpoint and stabilizes the precursor dimer.

Clinically relevant biomarkers are urgently needed for improving patient diagnosis, risk stratification, prognosis and therapeutic treatments. There is a particularly compelling motivation for identifying protein-based indicators of early-stage disease for more effective interventions. Despite recent progress, the proteomic discovery process remains a daunting challenge due to the sheer heterogeneity and skewed protein abundances in biofluids. Even the most advanced mass spectrometry systems exhibit limiting overall dynamic ranges and sensitivities relative to the needs of modern biomedical applications. To this end, we report the development of a robust, rapid, and reproducible high performance ion-exchange liquid chromatography pre-fractionation method that allows for improved proteomic detection coverage of complex biological specimens using basic tandem mass spectrometry screening procedures. This form of sample simplification prior to global proteomic profiling, which we refer to collectively as 'fractionomics', increases the number and diversity of proteins that can be confidently identified in tissue and cell lysates as compared to the straight analysis of unfractionated crude extracts.

Motivation: There is a pressing need for improved proteomic screening methods allowing for earlier diagnosis of disease, systematic monitoring of physiological responses and the uncovering of fundamental mechanisms of drug action. The combined platform of LC-MS (Liquid-Chromatography-Mass-Spectrometry) has shown promise in moving toward a solution in these areas. In this paper we present atechnique for discovering differences in protein signal between two classes of samples of LC-MS serum proteomic data without use of tandem mass spectrometry, gels or labeling. This method works on data from a lower-precision MS instrument, the type routinely used by and available to the community at large today. We test our technique on a controlled (spike-in) but realistic (serum biomarker discovery) experiment which is therefore verifiable. We also develop a new method for helping to assess the difficulty of a given spike-in problem. Lastly, we show that the problem of class prediction, sometimes mistaken as a solution to biomarker discovery, is actually a much simpler problem.Results: Using precision–recall curves with experimentally extracted ground truth, we show that (1) our technique has good performance using seven replicates from each class, (2) performance degrades with decreasing number of replicates, (3) the signal that we are teasing out is not trivially available (i.e. the differences are not so large that the task is easy). Lastly, we easily obtain perfect classification results for data in which the problem of extracting differences does not produce absolutely perfect results. This emphasizes the different nature of the two problems and also their relative difficulties.Availability: Our data are publicly available as a benchmark for further studies of this nature atSupplementary Information: Contact: jenn@cs.toronto.edu

Assembly of the active site of the [NiFe]-hydrogenase enzymes involves a multi-step pathway and the coordinated activity of many accessory proteins. To analyze complex formation between these factors in Escherichia coli, they were genomically tagged and native multi-protein complexes were isolated. This method validated multiple interactions reported in separate studies from several organisms and defined a new complex containing the putative chaperone HybG and the large subunit of hydrogenase 1 or 2. The complex also includes HypE and HypD, which interact with each other before joining the larger complex.

Objective To evaluate the association between breastfeeding duration and early childhood cardiometabolic risk. Study design A cross-sectional study of 1539 healthy children, 3-6 years of age, recruited through The Applied Research Group for Kids! practice-based research network between October 2009 and August 2015. Adjusted multivariable linear regression was used to examine the association between breastfeeding duration and cardiometabolic risk z score and individual cardiometabolic risk factors of waist circumference, systolic blood pressure, glucose, high-density lipoprotein cholesterol, and triglycerides. Results The mean breastfeeding duration was 12.5 months (SD = 8.4). Breastfeeding duration was associated with lower cardiometabolic risk z score (beta = −0.03; 95% CI −0.05, −0.01). In analysis of cardiometabolic risk factors, each additional 3 months of breastfeeding was associated with a 0.13 cm (95% CI −0.20, −0.05) lower waist circumference and 0.16 mm Hg (95% CI −0.30, −0.02) lower systolic blood pressure. Compared with children who breastfed for 6-12 months, those who breastfed for 12-24 months had a lower systolic blood pressure of 1.07 mm Hg (95% CI −2.04, −0.10). There was no association between breastfeeding duration and cardiometabolic risk for those who breastfed beyond 24 months. Conclusions Breastfeeding duration is associated with lower cardiometabolic risk, although the magnitude of association is small. Causation cannot be inferred. Breastfeeding beyond 24 months may not have an added benefit for cardiometabolic health.

Proteomics is the global (genome-wide) study of protein expression in a given living cell, tissue or organism. Multidimensional Protein Identification Technology (MudPIT), gel electrophoresis (GE) and high performance liquid chromatography (HPLC) are the widely employed approaches to survey protein levels in complex biological samples. This article describes an easily implemented, effective, and highly reproducible dual-column HPLC prefractionation method that helps in improving routine proteomic analyses. Keywords: proteomics; multidimensional protein identification technology; high performance liquid chromatography; gel electrophoresis; protein expression

One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans' biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a “systems-wide” functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins., Author Summary One goal of modern biology is to chart groups of proteins that act together to perform biological processes via direct and indirect interactions. Such groupings are sometimes called functional modules. The types of protein interactions within modules include physical interactions that generate protein complexes and biochemical associations that make up metabolic pathways. We have combined proteomic and bioinformatic tools, and used them to decipher a large number of protein interactions, complexes, and functional modules with high confidence. In addition, exploring the topology of the resulting interaction networks, we successfully predicted specific biological roles for a number of proteins with previously unknown functions, and identified some potential drug targets. Although our work is focused on E. coli, our phylogenetic projections suggest that a considerable fraction of our observations and predictions can be extrapolated to many other bacterial taxa. As all the data derived from this study are publicly available, others may build on our work for further hypothesis-driven studies of gene function discovery., A novel resource integrating proteomic and genome context-based tools provides a "systems-wide" functional blueprint ofE. coli, with insights into the biological and evolutionary significance of previously uncharacterized proteins.

Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.