Genotype-by-environment interactions govern fitness changes associated with adaptive mutations in two-component response systems.

Introduction: Two-component response systems (TCRS) are the main mechanism by which prokaryotes acclimate to changing environments. These systems are composed of a membrane bound histidine kinase (HK) that senses external signals and a response regulator (RR) that activates transcription of response genes. Despite their known role in acclimation, little is known about the role TCRS play in environmental adaptation. Several experimental evolution studies have shown the acquisition of mutations in TCRS during adaptation, therefore here we set out to characterize the adaptive mechanism resulting from these mutations and evaluate whether single nucleotide changes in one gene could induce variable genotype-by-environment (GxE) interactions. Methods: To do this, we assessed fitness changes and differential gene expression for four adaptive mutations in cusS, the gene that encodes the HK CusS, acquired by Escherichia coli during silver adaptation. Results: Fitness assays showed that as the environment changed, each mutant displayed a unique fitness profile with greatest fitness in the original selection environment. RNAseq then indicated that, in ± silver nitrate, each mutant induces a primary response that upregulates cusS, its RR cusR, and constitutively expresses the target response genes cusCFBA. This then induces a secondary response via differential expression of genes regulated by the CusR through TCRS crosstalk. Finally, each mutant undergoes fitness tuning through unique tertiary responses that result in gene expression patterns specific for the genotype, the environment and optimized for the original selection conditions. Discussion: This three-step response shows that different mutations in a single gene leads to individualized phenotypes governed by unique GxE interactions that not only contribute to transcriptional divergence but also to phenotypic plasticity.

Influence of antiseptic lavage during tibial plateau leveling osteotomies on surgical site infection in 1422 dogs.

OBJECTIVE: To determine the influence of preclosure antiseptic versus saline lavage on surgical site infections (SSI) in dogs following tibial plateau leveling osteotomy (TPLO). STUDY DESIGN: A multicenter retrospective study. SAMPLE POPULATION: Dogs treated with TPLO (n = 1422) between December 2019 and October 2021. METHODS: The medical records of dogs treated with TPLO were reviewed for preclosure antiseptic lavage or saline irrigation. Additional collected data included signalment, unilateral or bilateral TPLO, postoperative administration of antimicrobial medications, antibiotic agent, surgeon, and development of SSI within 90 postoperative days. Eleven factors were analyzed for association with SSI using univariate and multivariate analysis. RESULTS: Data were collected from the records of 519 dogs treated with antiseptic lavage and 903 dogs treated with saline lavage during TPLO. Surgical site infections were diagnosed more frequently in dogs that received preclosure antiseptic lavage (77/519, 14.84%) than those with saline irrigation (80/903, 8.86%) (p = .001). Single session bilateral TPLO increased the odds of SSI by 2.5x (p = .004). The odds of SSI increased by 11% (p = .001) for every 5 kg increase in bodyweight. Postoperative administration of antimicrobials decreased the risk of SSI (p = .008). CONCLUSION: The preclosure antiseptic lavage tested here did not decrease the incidence of SSI after TPLO. CLINICAL SIGNIFICANCE: The results of this study do not provide evidence to support preclosure antiseptic lavage during TPLOs.

Reporting off-target effects of recombinant engineering using the pORTMAGE system.

pORTMAGE recombineering is a simple technique for incorporation of novel point mutations into bacterial genomes that eliminates off-target effects. Here we inserted point mutations into the cusS gene from Escherichia coli, then, using Illumina sequencing, report genetic variants in all mutant strains. Several off-site mutations were found at high frequency. Low frequency mutations also show high heterogeneity. This means that it is essential for studies to report all off-target effects and acknowledge the effect that this may have on resultant phenotypes.

Long-Term Immunological Consequences of Radiation Exposure in a Diverse Cohort of Rhesus Macaques.

PURPOSE: The aim of this study was to develop an improved understanding of the delayed immunologic effects of acute total body irradiation (TBI) using a diverse cohort of nonhuman primates as a model for an irradiated human population. METHODS AND MATERIALS: Immune recovery was evaluated in 221 rhesus macaques either left unirradiated (n = 36) or previously irradiated (n = 185) at 1.1 to 8.5 Gy TBI (median, 6.5 Gy) when aged 2.1 to 15.5 years (median, 4.2 years). Blood was drawn annually for up to 5 years total between 0.5 and 14.3 years after exposure. Blood was analyzed by complete blood count, immunophenotyping of monocytes, dendritic cells (DC) and lymphocytes by flow cytometry, and signal joint T-cell receptor exclusion circle quantification in isolated peripheral blood CD4 and CD8 T cells. Animals were categorized by age, irradiation status, and time since irradiation. Sex-adjusted means of immune metrics were evaluated by generalized estimating equation models to identify cell populations altered by TBI. RESULTS: Overall, the differences between irradiated and nonirradiated animals were subtle and largely restricted to younger animals and select cell populations. Subsets of monocytes, DC, T cells, and B cells showed significant interaction effects between radiation dose and age after adjustment for sex. Irradiation at a young age caused transient increases in the percentage of peripheral blood myeloid DC and dose-dependent changes in monocyte balance for at least 5 years after TBI. TBI also led to a sustained decrease in the percentage of circulating memory B cells. Young irradiated animals exhibited statistically significant and prolonged disruption of the naïve/effector memory/central memory CD4 and CD8 T-cell equilibrium and exhibited a dose-dependent increase in thymopoiesis for 2 to 3 years after exposure. CONCLUSIONS: This study indicates TBI subtly but significantly alters the circulating proportions of cellular mediators of adaptive immune memory for several years after irradiation, especially in macaques under 5 years of age and those receiving a high dose of radiation.

Long-Term Recovery of the Adaptive Immune System in Rhesus Macaques After Total Body Irradiation.

PURPOSE: Ionizing radiation causes acute damage to hematopoietic and immune cells, but the long-term immunologic consequences of irradiation are poorly understood. We therefore performed a prospective study of the delayed immune effects of radiation using a rhesus macaque model. METHODS AND MATERIALS: Ten macaques received 4 Gy high-energy x-ray total body irradiation (TBI) and 6 control animals received sham irradiation. TBI caused transient lymphopenia that resolved over several weeks. Once white blood cell counts recovered, flow cytometry was used to immunophenotype the circulating adaptive immune cell populations 4, 9, and 21 months after TBI. Data were fit using a mixed-effects model to determine age-dependent, radiation-dependent, and interacting effects. T cell receptor (TCR) sequencing and quantification of TCR Excision Circles were used to determine relative contributions of thymopoiesis and peripheral expansion to T cell repopulation. Two years after TBI, the cohort was vaccinated with a 23-valent pneumococcal polysaccharide vaccine and a tetravalent influenza hemagglutinin vaccine. RESULTS: Aging, but not TBI, led to significant changes in the frequencies of dendritic cells, CD4 and CD8 T cells, and B cells. However, irradiated animals exhibited increased frequencies of central memory T cells and decreased frequencies of naïve T cells. These consequences of irradiation were time-dependent and more prolonged in the CD8 T cell population. Irradiation led to transient increases in CD8+ T cell TCR Excision Circles and had no significant effect on TCR sequence entropy, indicating T cell recovery was partially mediated by thymopoiesis. Animals that were irradiated and then vaccinated showed normal immunoglobulin G binding and influenza neutralization titers in response to the 4 protein antigens but weaker immunoglobulin G binding titers to 10 of the 23 polysaccharide antigens. CONCLUSIONS: These findings indicate that TBI causes subtle but long-lasting immune defects that are evident years after recovery from lymphopenia.

T cell-depleted cultured pediatric thymus tissue as a model for some aspects of human age-related thymus involution.

Human age-related thymus involution is characterized by loss of developing thymocytes and the thymic epithelial network that supports them, with replacement by adipose tissue. The mechanisms that drive these changes are difficult to study in vivo due to constant trafficking to and from the thymus. We hypothesized that the loss of thymocytes that occurs during human thymic organ cultures could model some aspects of thymus involution and begin to identify mechanisms that drive age-related changes in the thymic microenvironment. Potential mechanistically important candidate molecules were initially identified by screening conditioned media from human thymus organ cultures using antibody microarrays. These candidates were further validated using cultured tissue extracts and conditioned media. Results were compared with gene expression studies from a panel of well-characterized (non-cultured) human thymus tissues from human donors aged 5 days to 78 years. L-selectin released into conditioned media was identified as a biomarker for the content of viable thymocytes within the cultured thymus. Levels of the chemokines CCL21 and CXCL12, likely produced by surviving thymic epithelial cells, increased markedly in conditioned media as thymocytes were lost during culture. Native non-cultured thymus from adults older than 18 years also showed a strong trend toward increased CCL21 expression, in conjunction with significant decreases in thymocyte-related mRNAs compared with thymus from subjects younger than 18 years. Together, these findings demonstrate that use of postnatal human thymus organ cultures can model some aspects of human age-related thymic involution.

Recent Advances in Microscale Western Blotting.

Western blotting is a ubiquitous tool used extensively in the clinical and research settings to identify proteins and characterize their levels. It has rapidly become a mainstay in research laboratories due to its specificity, low cost, and ease of use. The specificity arises from the orthogonal processes used to identify proteins. Samples are first separated based on size and then probed with antibodies specific for the protein of interest. This confirmatory approach helps avoid pitfalls associated with antibody cross-reactivity and specificity issues. While the technique has evolved since its inception, the last decade has witnessed a paradigm shift in Western blotting technology. The introduction of capillary and microfluidic platforms has significantly decreased time and sample requirements while enabling high-throughput capabilities. These advances have enabled Western analysis down to the single cell level in highly parallel formats, opening vast new opportunities for studying cellular heterogeneity. Recent innovations in microscale Western blotting are surveyed, and the potential for enhancing detection using advances in label-free biosensing is briefly discussed.

Prevalence and treatment of metabolic syndrome in adolescents with type 2 diabetes.

BACKGROUND: The incidence of obesity and type 2 diabetes among adolescents has risen dramatically in recent years, and it is likely that many of these adolescents also have metabolic syndrome. OBJECTIVE: To investigate the prevalence of metabolic syndrome in the patient population enrolled in a children's hospital type 2 diabetes clinic and to describe baseline pharmacologic treatment and adherence patterns. METHODS: In this retrospective cohort study, the medical charts of 52 adolescents were reviewed. A data collection instrument was used to collect demographic data, laboratory values, medication lists, and documented adherence patterns from the patient's initial clinic visit. Data collected were used to identify patients with metabolic syndrome. Data were entered into a database and analyzed using SPSS 13.0. RESULTS: Data were collected for 52 patients; of these, 40 (76.9%) had characteristics of metabolic syndrome meeting 3 or more of the 5 criteria, although only 8 (15.4%) were diagnosed with the metabolic syndrome. Among patients with characteristics of metabolic syndrome, 92.5% were taking a glucose-lowering drug, with 85% of those receiving metformin. Sixteen (40%) patients were receiving a blood pressure-lowering medication at baseline, and 4 (10%) were taking a lipid-lowering agent. Forty-three percent of these patients reported nonadherence to their prescribed drugs. CONCLUSIONS: Given these treatment patterns, pharmacologic management of metabolic syndrome in adolescents with type 2 diabetes may be suboptimal and may impact cardiovascular outcomes. It is important for clinicians to be aware of the incidence of type 2 diabetes and metabolic syndrome in adolescents so that treatment with lifestyle modifications and pharmacologic therapy may be implemented earlier.

Inappropriate use of inhaled short acting beta-agonists and its association with patient health status.

BACKGROUND: Despite the widespread distribution of guidelines on the proper use of inhaled asthma medication, the overuse of short acting bronchodilators (SABs) persists. This study aims to examine how inhaled asthma medications are used in the US and to examine whether inappropriate use of inhaled SABs is associated with poor patient health. RESEARCH DESIGN AND METHODS: The study design was a retrospective analysis of the Medical Expenditure Panel Survey (MEPS) for asthmatic patients 5 years or older who had used SAB medication during the period from 1996 through 2000. Use of SAB medication was defined as inappropriate when a patient inhaled more than 225 defined daily doses (DDDs) of SABs but less than 45.625 DDDs of corticosteroids per year. Health status was evaluated using survey respondents' perceptions on a 5-point Likert scale. Five functional limitations (activities of daily living, instrumental activities of daily living, walking, social function, and cognitive function) were rated on a dichotomous scale. RESULTS: A total of 2386 asthmatic patients were identified as having used a SAB in the period 1996 through 2000. Of these, 272 (11.4%) used excessive doses of SABs, and of this group of excessive users, 151 (55.5%) underused corticosteroids. Compared to appropriate users of SAB medication, inappropriate users had lower perceptions of their overall health (adjusted mean: 3.21 vs. 2.94, p<0.05) and mental health (adjusted mean: 2.39 vs. 2.13, p<0.05). They were also at an increased risk of limitations in walking (relative risk [RR]: 1.76, 95% confidence interval [CI]:1.15-2.71) and in cognitive function (RR: 2.32, 95% CI: 1.37-3.93). CONCLUSION: Despite the national guidelines concerning the proper use of inhaled asthma medication, over-reliance on SAB medication and under-use of corticosteroids persists in the US. Those not using asthma medication according to the guidelines had poor perceptions of their health and were subject to an increased risk of limitations in walking and cognitive function.