Unique molecular identifier-based high-resolution HLA typing and transcript quantitation using long-read sequencing

HLA typing provides essential results for stem cell and solid organ transplants, as well as providing diagnostic benefits for various rheumatology, gastroenterology, neurology, and infectious diseases. It is becoming increasingly clear that understanding the expression of patient HLA transcripts can provide additional benefits for many of these same patient groups. Our study cohort was evaluated using a long-read RNA sequencing methodology to provide rapid HLA genotyping results and normalized HLA transcript expression. Our assay used NGSEngine to determine the HLA genotyping result and normalized mRNA transcript expression using Athlon2. The assay demonstrated an excellent concordance rate of 99.7%. Similar to previous studies, for the class I loci, patients demonstrated significantly lower expression o

Focusing Capillary Optics for Use in Solution Small-Angle X-Ray Scattering

Measurements of the global conformation of macromolecules can be carried out using small-angle X-ray scattering (SAXS). Glass focusing capillaries, manufactured at the Cornell High Energy Synchrotron Source (CHESS), have been successfully employed for SAXS measurements on the heme protein cytochrome c. These capillaries provide high X-ray flux into a spot size of tens of micrometres, permitting short exposures of small-volume samples. Such a capability is ideal for use in conjunction with microfluidic mixers, where time resolution may be determined by beam size and sample volumes are kept small to facilitate mixing and conserve material

Utilizing principal component analysis in the identification of clinically relevant changes in patient HLA single antigen bead solid phase testing patterns

Background HLA antibody testing is essential for successful solid-organ allocation, patient monitoring post-transplant, and risk assessment for both solid-organ and hematopoietic transplant patients. Luminex solid-phase testing is the most common method for identifying HLA antibody specificities, making it one of the most complex immunoassays as each panel contains over 90 specificities for both HLA class I and HLA class II with most of the analysis being performed manually in the vendor-provided software. Principal component analysis (PCA), used in machine learning, is a feature extraction method often utilized to assess data with many variables. Methods & findings In our study, solid organ transplant patients who exhibited HLA donor-specific antibodies (DSAs) were used to characterize the utility of PCA-derived analysis when compared to a control group of post-transplant and pre-transplant patients. ROC analysis was utilized to determine a potential threshold for the PCA-derived analysis that would indicate a significant change in a patient’s single antigen bead pattern. To evaluate if the algorithm could identify differences in patterns on HLA class I and HLA class II single antigen bead results using the optimized threshold, HLA antibody test results were analyzed using PCA-derived analysis and compared to the clinical results for each patient sample. The PCA-derived algorithm had a sensitivity of 100% (95% CI, 73.54%-100%), a specificity of 75% (95% CI, 56.30%-92.54%), with a PPV of 65% (95% CI, 52.50%-83.90%) and an NPV of 100%, in identifying new reactivity that differed from the patients historic HLA antibody pattern. Additionally, PCA-derived analysis was utilized to assess the potential over-reactivity of single antigen beads for both HLA class I and HLA class II antibody panels. This assessment of antibody results identified several beads in both the HLA class I and HLA class II antibody panel which exhibit over reactivity from 2018 to the present time. Conclusions PCA-derived analysis would be ideal to help automatically identify patient samples that have an HLA antibody pattern of reactivity consistent with their history and those which exhibit changes in their antibody patterns which could include donor-specific antibodies, de novo HLA antibodies, and assay interference. A similar method could also be applied to evaluate the over-reactivity of beads in the HLA solid phase assays which would be beneficial for lot comparisons and instructive for transplant centers to better understand which beads are more prone to exhibiting over-reactivity and impact patient care

High-flux hard X-ray microbeam using a single-bounce capillary with doubly focused undulator beam

A pre-focused X-ray beam has been used to illuminate a single-bounce capillary in order to generate a high-flux X-ray microbeam at the BioCAT undulator X-ray beamline at the Advanced Photon Source. The combined optical arrangement makes it suitable for many microprobe fluorescence applications and micro-XANES experiments for biological samples

An Assessment of Exposure Models for Bioengineered Microorganisms

Environmental risk assessment performed for chemicals involves exposure assessment, effects assessment, and risk integration. The nature of living microorganisms (growth, replication. and survival), as compared to chemicals, requires that their biological attributes be integrated into risk assessment. Biological attributes have generally been dealt with in effects assessment and not exposure assessment models. Thus, exposure assessment models that characterize source, transport, transformation, and fate (effective environmental concentration, area, and duration of exposure) of genetically engineered microorganisms must incorporate biological factors along with physicochemical factors. A study to assess the state of the art of exposure models for organisms and microorganisms in air, soil, and water was undertaken. Mathematical models developed during the past 15 years were organized into three categories: organism population dynamics, source features and transport, and management and control. One hundred forty-eight models were examined, and 56 were judged to have potential as exposure models. These 56 were screened to 31 models that were then evaluated against eight components that the ideal biotechnology model should have: (a) five state/process components (organism population, source application, exposure site, movement, and imposed management) and (b) three software components (user friendliness, availability/implementability, and flexibility). Each model was rated by individual components, combinations of two components, and total state/process components. An ideal exposure assessment model with high scores in all components was not found. Combining two or more models so that the strong components of one compensate for the weak components of another was concluded to be the best approach for obtaining a predictive model for microorganisms. Potential couplings were ascertained from among the 31 models. Most of these couplings would combine an organism population dynamics model with a model from the U. S. Environmental Protection Agency\u27s Graphical Exposure Modeling System (source and transport oriented models)

Depression and anxiety in systemic lupus erythematosus: The crosstalk between immunological, clinical, and psychosocial factors

Depression and anxiety cause severe loss of quality of life for patients with systemic lupus erythematosus. The causes and factors that contribute to these psychological manifestations in lupus are difficult to disentangle. This study compared clinical, psychological, and demographic factors between lupus patients, depressed patients, and rheumatoid arthritis patients to discover lupus-specific contributors to depression. Lupus-specific manifestations of depression were also investigated. Physiological, clinical, and psychosocial data were collected from 77 patients. ELISA was used to measure cytokine levels. Univariate and Multivariate analyses were used to compare the patient populations and identify correlations between key physical and psychological indicators. The prevalence of depression in the SLE cohort was 6 times greater than the healthy control subjects. Pain, IL-6, and Pittsburgh Sleep Quality index values were all significantly higher in SLE patients compared with the healthy control group (P < .001, P = .038, and P = .005, respectively). Anxiety levels were significantly higher in SLE patients compared to healthy and RA control patients (P = .020 and .011, respectively). Serum IL-10 concentrations, relationship assessment scale, and fatigue severity scale values were found to be correlated with depression among the SLE patients (P = .036, P = .007, and P = .001, respectively). Relationship assessment and fatigue severity scale scores were found to be the best indicators of depression for the SLE patients (P = .042 and .028, respectively). Fatigue Severity, relationship satisfaction, and IL-10 concentrations are indicators of depression in lupus patients. Despite also suffering from the pain and disability that accompanies chronic autoimmune disease, the rheumatoid arthritis patients had less anxiety and better relationship scores.The authors thank the Portugal Fulbright Commission for funding Dr. Pool

Unique Molecular Identifier based High-resolution HLA Typing and Transcript Quantitation using Long-Read Sequencing

HLA typing provides essential results for stem cell and solid organ transplants, as well as providing diagnostic benefits for various rheumatology, gastroenterology, neurology, and infectious diseases. It is becoming increasingly clear that understanding the expression of patient HLA transcripts can provide additional benefits for many of these same patient groups. Our study cohort was evaluated using a long-read RNA sequencing methodology to provide rapid HLA genotyping results and normalized HLA transcript expression. Our assay used NGSEngine to determine the HLA genotyping result and normalized mRNA transcript expression using Athlon2. The assay demonstrated an excellent concordance rate of 99.7%. Similar to previous studies, for the class I loci, patients demonstrated significantly lower expression of HLA-C than HLA-A and -B (Mann–Whitney U, P value = 0.0065 & P value = 0.0154, respectively). In general, the expression of class II transcripts was lower than that of class I transcripts. This study demonstrates a rapid high-resolution HLA typing assay using RNA-Seq that can provide accurate HLA genotyping and HLA allele-specific transcript expression in 7 to 8 hours, a timeline short enough to perform the assay for deceased donors

Influence of Biological Therapeutics, Cytokines, and Disease Activity on Depression in Rheumatoid Arthritis

Purpose: Rheumatoid arthritis (RA) is an often debilitating autoinflammatory disease. Patients with rheumatoid arthritis are often troubled by co-occurring depression or other psychological manifestations. RA patients have a variety of treatment options available, including biologicals that inhibit cytokines or immune cells. If these cytokines influence the psychological symptoms, then the use of cytokine inhibitors should modulate these symptoms.Methods: A cohort of 209 individuals was recruited. This group included 82 RA patients, 22 healthy subjects, 32 depressed control subjects, and 73 subjects with systemic lupus erythematosus. Of the RA patients, 51% were on a biological therapeutic. ELISA was used to measure cytokine levels. A variety of psychological assessments were used to evaluate depression, anxiety, sleep, fatigue, and relationship status. Clinical values were obtained from medical records.Results: IL-10 concentration was associated with depressive symptoms in the RA patients, healthy controls, and the lupus patients. In the patients with primary depression, depressive symptoms were associated with IL-6 and TNF-alpha. In RA patients, Tocilizumab use was associated with decreased depressive symptoms. 14 RA patients who were not using biologicals began using them by a one-month follow-up. In these patients, there was no significant change to any value except for fatigue.Conclusions: A variety of both biological and social factors influences depressive symptoms in RA. IL-10 and IL-6 are likely to be involved, since IL-10 concentration was associated with depression and Tocilizumab decreased depressive symptoms in the RA patients. The roles of these cytokines are different in RA and lupus, as high IL-10 in RA is associated with increased depressive symptoms, but high IL-10 in the lupus patients is associated with decreased depression. IL-6 was also associated with depressive symptoms in the patients with primary depression. These results strongly indicate that disease activity, including cytokine levels, has a strong impact on depressive symptoms.This study was supported by Portugal Fulbright Commission (Dr. Brian D. Poole)