Child immunization status according to number of siblings and birth order in 85 low- and middle-income countries: a cross-sectional study.

Background: Identification of unvaccinated children is important for preventing deaths due to infections. Number of siblings and birth order have been postulated as risk factors for zero-dose prevalence. Methods: We analysed nationally representative cross-sectional surveys from 85 low and middle-income countries (2010-2020) with information on immunisation status of children aged 12-35 months. Zero-dose prevalence was defined as the failure to receive any doses of DPT (diphtheria-pertussis-tetanus) vaccine. We examined associations with birth order and the number of siblings, adjusting for child's sex, maternal age and education, household wealth quintiles and place of residence. Poisson regression was used to calculate zero-dose prevalence ratios. Findings: We studied 375,548 children, of whom 13.7% (n = 51,450) were classified as zero-dose. Prevalence increased monotonically with birth order and with the number of siblings, with prevalence increasing from 11.0% for firstborn children to 17.1% for birth order 5 or higher, and from 10.5% for children with no siblings to 17.2% for those with four or more siblings. Adjustment for confounders attenuated but did not eliminate these associations. The number of siblings remained as a strong risk factor when adjusted for confounders and birth order, but the reverse was not observed. Among children with the same number of siblings, there was no clear pattern in zero-dose prevalence by birth order; for instance, among children with two siblings, the prevalence was 13.0%, 14.7%, and 13.3% for firstborn, second, and third-born, respectively. Similar results were observed for girls and boys. 9513 families had two children aged 12-35 months. When the younger sibling was unvaccinated, 61.9% of the older siblings were also unvaccinated. On the other hand, when the younger sibling was vaccinated, only 5.9% of the older siblings were unvaccinated. Interpretation: The number of siblings is a better predictor than birth order in identifying children to be targeted by immunization campaigns. Zero-dose children tend to be clustered within families. Funding: Gavi, the Vaccine Alliance.

Influence of Pain Sensitivity on Surgical Outcomes of Arthroscopic Rotator Cuff Repair: A Prospective Cohort Study.

BACKGROUND: The Pain Sensitivity Questionnaire (PSQ) has been found to be a valid tool, and PSQ scores have been shown to be predictive of outcomes after surgery for lumbar stenosis. The effect of pain sensitivity on outcomes of rotator cuff repair (RCR) surgery has not been examined. HYPOTHESIS: PSQ scores would be associated with surgical outcomes after arthroscopic RCR surgery. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: Patients 18 to 80 years old scheduled for RCR were consecutively enrolled. Patients with glenohumeral arthritis grade ≥2 or RCR revision surgery were excluded. PSQ was completed preoperatively. The Disabilities of the Arm, Shoulder and Hand score and American Shoulder and Elbow Surgeons score were used as patient-reported outcome measurements (PROMs), and visual analog scale pain score was documented as well. Active shoulder external rotation (ER), internal rotation, and anterior forward elevation range of motion (ROM) were recorded. PROMs and ROM measurements were recorded preoperatively and at 3 months, 6 months, and 1 year after surgery. Rotator cuff tear size, type of repair, and concomitant procedures were documented. Patients were classified as having high or normal pain sensitivity based on PSQ scores. RESULTS: Of 100 enrolled patients, 38 patients were classified as having high pain sensitivity. Patients with high pain sensitivity had worse American Shoulder and Elbow Surgeons and Disabilities of the Arm, Shoulder and Hand scores preoperatively, 6 months postoperatively, and 1 year postoperatively (P < .01). From the preoperative assessment to 3 months postoperatively, PROMs improved more in patients with high versus normal pain sensitivity. However, for patients with high pain sensitivity, PROMs plateaued after 3 months but continued to improve for patients with normal pain sensitivity (P < .01). Visual analog scale pain scores were higher at all time points for patients with high pain sensitivity (P < .05). Preoperatively, patients with high pain sensitivity had restricted active ROM compared with patients who had normal pain sensitivity for anterior forward elevation, ER, and internal rotation (P = .009, P = .012, and P = .006, respectively). By 1 year after surgery, ER ROM was still restricted in patients with high pain sensitivity. CONCLUSION: Pain sensitivity is an important factor influencing RCR outcomes. Patients with high pain sensitivity undergoing RCR showed less improvement in active ROM and worse PROMs after surgery compared with patients who had normal pain sensitivity. Preoperative PSQ may predict postoperative improvements.

Uncovering Protein Ensembles: Automated Multiconformer Model Building for X-ray Crystallography and Cryo-EM.

With the advent of AlphaFold, protein structure prediction has attained remarkable accuracy. These achievements resulted from a focus on single static structures. The next frontier in this field involves enhancing our ability to model conformational ensembles, not just the ground states of proteins. Notably, deposited structures result from interpretation of density maps, which are derived from either X-ray crystallography or cryogenic electron microscopy (cryo-EM). These maps represent ensemble averages, reflecting molecules in multiple conformations. Here, we present the latest developments in qFit, an automated computational approach to model protein conformational heterogeneity into density maps. We present algorithmic advancements to qFit, validated by improved Rfree and geometry metrics across a broad and diverse set of proteins. Automated multiconformer modeling holds significant promise for interpreting experimental structural biology data and for generating novel hypotheses linking macromolecular conformational dynamics to function.

Incidence of concussions and helmet use in equestrians.

OBJECTIVES: Equestrians have a high risk of concussions per hospital records. However, most concussions occur in private settings where concussions are not tracked. We determined concussion incidence by self-report, expressed per 1000 h of exposure, and determined helmet usage and concussion knowledge. DESIGN: Descriptive epidemiological study. METHODS: Equestrians were recruited using a snowball method of sampling in which enrolled participants recruited more equestrians. Participants completed a survey of equestrian experience and history of concussion, symptoms and provided estimates of hours spent in various equestrian activities. From these data, incidences of concussions were calculated. In addition, they answered questions regarding helmet usage and willingness to take risks when concussed. RESULTS: 210 participants (203 women) reported 27 ±â€¯14 years of equine experience and 728 concussions, 3.47 ±â€¯5.34 per person (0-55). Incidence while riding was 0.19/1000 h which was greater than the incidence while driving (0.02/1000 h) or handling horses (0.03/1000 h). Riders were helmeted at the time of injury 85% of the time. While concussion knowledge was high, most reported willingness to risk permanent injury by continuing to work with horses while injured. CONCLUSIONS: To our knowledge this is the first study to document incidence of concussions in equestrians: incidence is higher while riding than during football or rugby training. Helmets were far more commonly worn at the time of concussion than reported in hospital data, suggesting that helmets effectively reduce concussions severe enough to warrant urgent medical care.

Lumbar spine intervertebral disc desiccation is associated with medical comorbidities linked to systemic inflammation.

INTRODUCTION: Symptomatic disc degeneration is a common cause of low back pain. Recently, the prevalence of low back pain has swiftly risen leading to increased patient disability and loss of work. The increase in back pain also coincides with a rapid rise in patient medical comorbidities. However, a comprehensive study evaluating a link between patient's medical comorbidities and their influence on lumbar intervertebral disc morphology is lacking in the literature. METHODS: Electronic medical records (EMR) were retrospectively reviewed to determine patient-specific medical characteristics. Magnetic resonance imaging (MRI) was evaluated for lumbar spine intervertebral disc desiccation and height loss according to the Griffith-modified Pfirrmann grading system. Bivariate and multivariable linear regression analyses assessed strength of associations between patient characteristics and lumbar spine Pfirrmann grade severity (Pfirrmann grade of the most affected lumbar spine intervertebral disc) and cumulative grades (summed Pfirrmann grades for all lumbar spine intervertebral discs). RESULTS: In total, 605 patients (304 diabetics and 301 non-diabetics) met inclusion criteria. Bivariate analysis identified older age, diabetes, American Society of Anesthesiologists (ASA) class, hypertension, chronic obstructive pulmonary disease (COPD), peripheral vascular disease, and hypothyroidism as being strongly associated with an increasing cumulative Pfirrmann grades. Multivariable models similarly found an association linking increased cumulative Pfirrmann grades with diabetes, hypothyroidism, and hypertension, while additionally identifying non-white race, heart disease, and previous lumbar surgery. Chronic pain, depression, and obstructive sleep apnea (OSA) were associated with increased Pfirrmann grades at the most affected level without an increase in cumulative Pfirrmann scores. Glucose control was not associated with increasing severity or cumulative Pfirrmann scores. CONCLUSION: These findings provide specific targets for future studies to elucidate key mechanisms by which patient-specific medical characteristics contribute to the development and progression of lumbar spine disc desiccation and height loss. LEVEL OF EVIDENCE: III (retrospective cohort).

Religious affiliation as a driver of immunization coverage: Analyses of zero-dose vaccine prevalence in 66 low- and middle-income countries.

Background: The literature on the association between religion and immunization coverage is scant, mostly consisting of single-country studies. Analyses in low and middle-income countries (LMICs) to assess whether the proportions of zero-dose children vary according to religion remains necessary to better understand non-socioeconomic immunization barriers and to inform interventions that target zero-dose children. Methods: We included 66 LMICs with standardized national surveys carried out since 2010, with information on religion and vaccination. The proportion of children who failed to receive any doses of a diphtheria-pertussis-tetanus (DPT) containing vaccine - a proxy for no access to routine vaccination or "zero-dose" status - was the outcome. Differences among religious groups were assessed using a test for heterogeneity. Additional analyses were performed controlling for the fixed effect of country, household wealth, maternal education, and urban-rural residence to assess associations between religion and immunization. Findings: In 27 countries there was significant heterogeneity in no-DPT prevalence according to religion. Pooled analyses adjusted for wealth, maternal education, and area of residence showed that Muslim children had 76% higher no-DPT prevalence than Christian children. Children from the majority religion in each country tended to have lower no-DPT prevalence than the rest of the population except in Muslim-majority countries. Interpretation: Analyses of gaps in coverage according to religion are relevant to renewing efforts to reach groups that are being left behind, with an important role in the reduction of zero-dose children.

A Review of Cutaneous Diseases Observed in Solid Organ Transplant Recipients.

Solid organ transplant recipients are at increased risk for numerous cutaneous conditions that fall within four categories: pre-neoplastic, neoplastic, infectious, or idiopathic. Many of these diseases can be attributed to immunosuppressive medications, including mycophenolate mofetil, cyclosporine, azathioprine, tacrolimus, or glucocorticoids. Iatrogenic lessening of the immune system places the patient at risk of malignancies, opportunistic infections, immune-mediated dermatoses, and adverse effects of medications. As the life expectancy of patients with solid organ transplants continues to increase, dermatologists and transplant physicians must stay abreast of this spectrum of dermatologic conditions, their respective prognoses, prevention, mitigation, and treatment.

Exposure of Zero-Dose Children to Multiple Deprivation: Analyses of Data from 80 Low- and Middle-Income Countries.

The concept of multiple deprivation recognizes that the same individuals, households, and communities are often exposed to several forms of scarcity. We assessed whether lack of immunization is also associated with nutritional, environmental, and educational outcomes. We analyzed data from nationally representative surveys from 80 low- and middle-income countries with information on no-DPT (children aged 12-23 months without any doses of a diphtheria, pertussis and tetanus containing vaccine), stunting, wasting, maternal education and use of contraception, improved water and sanitation, and long-lasting insecticidal nets. Analyses of how these characteristics overlap were performed at individual and ecological levels. Principal component analyses (PCA) provided additional information on indicator clustering. In virtually all analyses, no-DPT children were significantly more likely to be exposed to the other markers for deprivation. The strongest, most consistent associations were found with maternal education, water, and sanitation, while the weakest associations were found for wasting and bed nets. No-DPT prevalence reached 46.1% in the most deprived quintile from first PCA component derived from deprivation indicators. All children were immunized in the two least deprived quintiles of the component. Our analyses provide strong support for the hypothesis that unimmunized children are also affected by other forms of deprivation.

Generation of synthetic antibody fragments with optimal complementarity determining region lengths for Notch-1 recognition.

Synthetic antibodies have been engineered against a wide variety of antigens with desirable biophysical, biochemical, and pharmacological properties. Here, we describe the generation and characterization of synthetic antigen-binding fragments (Fabs) against Notch-1. Three single-framework synthetic Fab libraries, named S, F, and modified-F, were screened against the recombinant human Notch-1 extracellular domain using phage display. These libraries were built on a modified trastuzumab framework, containing two or four diversified complementarity-determining regions (CDRs) and different CDR diversity designs. In total, 12 Notch-1 Fabs were generated with 10 different CDRH3 lengths. These Fabs possessed a high affinity for Notch-1 (sub-nM to mid-nM KDapp values) and exhibited different binding profiles (mono-, bi-or tri-specific) toward Notch/Jagged receptors. Importantly, we showed that screening focused diversity libraries, implementing next-generation sequencing approaches, and fine-tuning the CDR length diversity provided improved binding solutions for Notch-1 recognition. These findings have implications for antibody library design and antibody phage display.

Ethnic disparities in immunisation: analyses of zero-dose prevalence in 64 countries.

BACKGROUND: The Sustainable Development Goals (SDGs) recommend stratification of health indicators by ethnic group, yet there are few studies that have assessed if there are ethnic disparities in childhood immunisation in low-income and middle-income countries (LMICs). METHODS: We identified 64 LMICs with standardised national surveys carried out since 2010, which provided information on ethnicity or a proxy variable and on vaccine coverage; 339 ethnic groups were identified after excluding those with fewer than 50 children in the sample and countries with a single ethnic group. Lack of vaccination with diphtheria-pertussis-tetanus vaccine-a proxy for no access to routine vaccination or 'zero-dose' status-was the outcome of interest. Differences among ethnic groups were assessed using a χ2 test for heterogeneity. Additional analyses controlled for household wealth, maternal education and urban-rural residence. FINDINGS: The median gap between the highest and lowest zero-dose prevalence ethnic groups in all countries was equal to 10 percentage points (pp) (IQR 4-22), and the median ratio was 3.3 (IQR 1.8-6.7). In 35 of the 64 countries, there was significant heterogeneity in zero-dose prevalence among the ethnic groups. In most countries, adjustment for wealth, education and residence made little difference to the ethnic gaps, but in four countries (Angola, Benin, Nigeria and Philippines), the high-low ethnic gap decreased by over 15 pp after adjustment. Children belonging to a majority group had 29% lower prevalence of zero-dose compared with the rest of the sample. INTERPRETATION: Statistically significant ethnic disparities in child immunisation were present in over half of the countries studied. Such inequalities have been seldom described in the published literature. Regular analyses of ethnic disparities are essential for monitoring trends, targeting resources and assessing the impact of health interventions to ensure zero-dose children are not left behind in the SDG era.

No Difference in Complication Rates or Patient-Reported Outcomes Between Bone-Patella Tendon-Bone and Quadriceps Tendon Autograft for Anterior Cruciate Ligament Reconstruction.

Purpose: To compare subjective outcomes and complications of anterior cruciate ligament reconstruction (ACLR) using either bone-patellar tendon-bone (BPTB) or quadriceps tendon (QT) autograft. Methods: A retrospective analysis of prospectively collected data identified consecutive cohorts of patients undergoing ACLR with either BPTB or QT autograft. Patients with less than 12-month follow-up and those undergoing concomitant osteotomies, cartilage restoration, and/or other ligament reconstruction procedures were excluded. Pre- and postsurgical patient-reported outcomes including International Knee Documentation Committee, Knee Injury and Osteoarthritis Outcome Score, Patient-Reported Outcomes Measurement Information System (PROMIS), Single Assessment Numeric Evaluation, Tegner, and Marx were compared between groups. Complications requiring reoperation were recorded. Results: One hundred nineteen patients met inclusion criteria, including 39 QT autografts and 80 BPTB autografts. Demographic information was comparable between groups. Mean follow-up was comparable between groups (QT 22.4 ± 10.6 months vs BPTB 28.5 ± 18.5 months, P = .06). At minimum 12-month follow-up (range 12.0-100.8 months), patients in both groups demonstrated statistically significant improvements in International Knee Documentation Committee (QT 60.0%, P < .0001; BPTB 57.7%, P < .0001), all Knee Injury and Osteoarthritis Outcome Score domains, PROMIS Mobility T-Score (QT 27.2%, P = .0001; BPTB 23.2%, P < .0001), PROMIS Global Physical Health (QT 14.4%, P = .002; BPTB 13.4%, P = .001), PROMIS Physical Function (QT 29.6%, P < .0001; BPTB 37.1%, P < .0001), PROMIS Pain Interference (QT -16.5%, P < .0001; BPTB -20.8%, P < .0001), Single Assessment Numeric Evaluation, (QT 76.9%, P < .0001; BPTB 73.3%, P < .0001), Tegner (QT 92.9%, P = .0002; BPTB 101.4%, P < .0001), and Marx (QT -26.6%, P = .02; BPTB -32.0%, P = .0002) with no statistically significant differences between the 2 groups. Overall postoperative reoperation rate did not differ between groups (QT 12.8% vs BPTB 23.8%, P = .2). Revision ACL reconstruction rate did not differ between groups (QT 5.1% vs BPTB 7.5%, P = .6). Conclusions: Patients undergoing autograft ACLR with either BPTB or QT demonstrated significant subjective improvements in patient-reported outcomes from preoperative values and no statistically significant differences in outcomes between the groups. Complication and revision ACLR rates were similar between the 2 groups. Level of Evidence: III, retrospective cohort study.

Economic-Related Inequalities in Zero-Dose Children: A Study of Non-Receipt of Diphtheria-Tetanus-Pertussis Immunization Using Household Health Survey Data from 89 Low- and Middle-Income Countries.

Despite advances in scaling up new vaccines in low- and middle-income countries, the global number of unvaccinated children has remained high over the past decade. We used 2000-2019 household survey data from 154 surveys representing 89 low- and middle-income countries to assess within-country, economic-related inequality in the prevalence of one-year-old children with zero doses of diphtheria-tetanus-pertussis (DTP) vaccine. Zero-dose DTP prevalence data were disaggregated by household wealth quintile. Difference, ratio, slope index of inequality, concentration index, and excess change measures were calculated to assess the latest situation and change over time, by country income grouping for 17 countries with high zero-dose DTP numbers and prevalence. Across 89 countries, the median prevalence of zero-dose DTP was 7.6%. Within-country inequalities mostly favored the richest quintile, with 19 of 89 countries reporting a rich-poor gap of ≥20.0 percentage points. Low-income countries had higher inequality than lower-middle-income countries and upper-middle-income countries (difference between the median prevalence in the poorest and richest quintiles: 14.4, 8.9, and 2.7 percentage points, respectively). Zero-dose DTP prevalence among the poorest households of low-income countries declined between 2000 and 2009 and between 2010 and 2019, yet economic-related inequality remained high in many countries. Widespread economic-related inequalities in zero-dose DTP prevalence are particularly pronounced in low-income countries and have remained high over the previous decade.

Ligand binding remodels protein side-chain conformational heterogeneity.

While protein conformational heterogeneity plays an important role in many aspects of biological function, including ligand binding, its impact has been difficult to quantify. Macromolecular X-ray diffraction is commonly interpreted with a static structure, but it can provide information on both the anharmonic and harmonic contributions to conformational heterogeneity. Here, through multiconformer modeling of time- and space-averaged electron density, we measure conformational heterogeneity of 743 stringently matched pairs of crystallographic datasets that reflect unbound/apo and ligand-bound/holo states. When comparing the conformational heterogeneity of side chains, we observe that when binding site residues become more rigid upon ligand binding, distant residues tend to become more flexible, especially in non-solvent-exposed regions. Among ligand properties, we observe increased protein flexibility as the number of hydrogen bonds decreases and relative hydrophobicity increases. Across a series of 13 inhibitor-bound structures of CDK2, we find that conformational heterogeneity is correlated with inhibitor features and identify how conformational changes propagate differences in conformational heterogeneity away from the binding site. Collectively, our findings agree with models emerging from nuclear magnetic resonance studies suggesting that residual side-chain entropy can modulate affinity and point to the need to integrate both static conformational changes and conformational heterogeneity in models of ligand binding.

Proteins are the workhorses of our cells. They are large molecules that 'fold' into specific, often highly complex, three-dimensional configurations. These structures are not static, but rather dynamic and flexible. In other words, proteins can shift between different three-dimensional shapes to perform their tasks within the cell. To perform their roles, many proteins have to bind to small molecule ligands. Many ligands are drugs, which means that their effectiveness depends on their ability to bind to and impact the proteins involved in the disease they are treating. When a ligand binds to a protein, it can reshape the protein. For example, certain conformations of the protein, which were difficult for the protein to be in on its own, may become more stable when the ligand binds. Additionally, upon ligand binding, some parts of the protein may move relative to each other. Previous studies have shown that these movements can affect the interaction between ligand and protein. However, these studies only examined a small number of proteins. Therefore, Wankowicz et al. set out to determine, in greater detail, what happens to protein flexibility upon ligand binding. First, a pipeline was created to model alternative configurations of the protein both with and without ligands attached. These models measured flexibility within protein structures. The models revealed that when ligands bind to proteins, the flexibility of different regions of the protein changes ­ and does so in a consistent way. Proteins that become more rigid in the region interacting with their ligands become less rigid in other, distant regions, and vice versa. In other words, the rest of the protein is able to compensate for any changes in flexibility caused by ligand binding, which may contribute to how well a ligand binds to a protein. This study demonstrates the ability of ligands to affect the entire structure of the proteins they bind to, and therefore sheds new light on the role of proteins' innate conformational flexibility during this process. These results will contribute to our understanding of how the ligands and proteins involved in different cellular processes interact with each other ­ and, potentially, how these interactions can be manipulated.

Children of more empowered women are less likely to be left without vaccination in low- and middle-income countries: A global analysis of 50 DHS surveys.

Background: To help provide a global understanding of the role of gender-related barriers to vaccination, we have used a broad measure of women's empowerment and explored its association with the prevalence of zero-dose children aged 12-23 months across many low- and middle-income countries, using data from standardized national household surveys. Methods: We used data from Demographic and Health Surveys (DHS) of 50 countries with information on both women's empowerment and child immunisation. Zero-dose was operationally defined as the proportion of children who failed to receive any doses of the diphtheria, pertussis, and tetanus containing vaccines (DPT). We measured women's empowerment using the SWPER Global, an individual-level indicator estimated for women aged 15-49 years who are married or in union and with three domains: social independence, decision-making and attitude towards violence. We estimated two summary measures of inequality, the slope index of inequality (SII) and the concentration index (CIX). Results were presented for individual and pooled countries. Results: In the country-level (ecological) analyses we found that the higher the proportion of women with high empowerment, the lower the zero-dose prevalence. In the individual level analyses, overall, children with highly-empowered mothers presented lower prevalence of zero-dose than those with less-empowered mothers. The social independence domain presented more consistent associations with zero-dose. In 42 countries, the lowest zero-dose prevalence was found in the high empowerment groups, with the slope index of inequality showing significant results in 28 countries. When we pooled all countries using a multilevel Poisson model, children from mothers in the low and medium levels of the social independence domain had respectively 3.3 (95% confidence interval (CI) = 2.3, 4.7) and 1.8 (95% CI = 1.5, 2.1) times higher prevalence of zero-dose compared to those in the high level. Conclusions: Our country-level and individual-level analyses support the importance of women's empowerment for child vaccination, especially in countries with weaker routine immunisation programs.

Assessing the overlap between immunisation and other essential health interventions in 92 low- and middle-income countries using household surveys: opportunities for expanding immunisation and primary health care.

BACKGROUND: Unvaccinated children may live in households with limited access to other primary health care (PHC) services, and routine vaccination services may provide the opportunity to bring caregivers into contact with the health system. We aimed to investigate the overlap between not being vaccinated and failing to receive other PHC services in low- and middle-income countries (LMICs). METHODS: Using Demographic and Health Surveys (DHS) and Multiple Indicator Cluster Surveys (MICS) data between 2010-2019 from 92 LMICs, we analysed six vaccination indicators based on the bacille Calmette-Guérin (BCG), polio, diphtheria-pertussis-tetanus (DPT) and measles vaccines and their overlap with four other PHC indicators - at least four antenatal care (ANC) visits, institutional delivery, careseeking for common childhood illnesses or symptoms and place for handwashing in the home - in 211,141 children aged 12-23 months. Analyses were stratified according to wealth quintiles and World Bank income levels. FINDINGS: Unvaccinated children and their mothers were systematically less likely to receive the other PHC interventions. These associations were particularly marked for 4+ ANC visits and institutional delivery and modest for careseeking behaviour. Our stratified analyses confirm a systematic disadvantage of unvaccinated children and their families with respect to obtaining other health services in all levels of household wealth and country income. INTERPRETATION: We suggested that lack of vaccination goes hand in hand with missing out on other health interventions. This represents an opportunity for integrated delivery strategies that may more efficiently reduce inequalities in health service coverage. FUNDING: Bill & Melinda Gates Foundation, Gavi, the Vaccine Alliance, The Wellcome Trust, Associação Brasileira de Saúde Coletiva and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

PIIKA 2.5: Enhanced quality control of peptide microarrays for kinome analysis.

Peptide microarrays consisting of defined phosphorylation target sites are an effective approach for high throughput analysis of cellular kinase (kinome) activity. Kinome peptide arrays are highly customizable and do not require species-specific reagents to measure kinase activity, making them amenable for kinome analysis in any species. Our group developed software, Platform for Integrated, Intelligent Kinome Analysis (PIIKA), to enable more effective extraction of meaningful biological information from kinome peptide array data. A subsequent version, PIIKA2, unveiled new statistical tools and data visualization options. Here we introduce PIIKA 2.5 to provide two essential quality control metrics and a new background correction technique to increase the accuracy and consistency of kinome results. The first metric alerts users to improper spot size and informs them of the need to perform manual resizing to enhance the quality of the raw intensity data. The second metric uses inter-array comparisons to identify outlier arrays that sometimes emerge as a consequence of technical issues. In addition, a new background correction method, background scaling, can sharply reduce spatial biases within a single array in comparison to background subtraction alone. Collectively, the modifications of PIIKA 2.5 enable identification and correction of technical issues inherent to the technology and better facilitate the extraction of meaningful biological information. We show that these metrics demonstrably enhance kinome analysis by identifying low quality data and reducing batch effects, and ultimately improve clustering of treatment groups and enhance reproducibility. The web-based and stand-alone versions of PIIKA 2.5 are freely accessible at via http://saphire.usask.ca.