Urinary metabolomic biomarker candidates for skeletal muscle wasting in patients with rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease that affects the joints, leading to chronic synovial inflammation and local tissue destruction. Extra-articular manifestations may also occur, such as changes in body composition. Skeletal muscle wasting is often observed in patients with RA, but methods for assessing loss of muscle mass are expensive and not widely available. Metabolomic analysis has shown great potential for identifying changes in the metabolite profile of patients with autoimmune diseases. In this setting, urine metabolomic profiling in patients with RA may be a useful tool to identify skeletal muscle wasting. METHODS: Patients aged 40-70 years with RA have been recruited according to the 2010 ACR/EULAR classification criteria. Further, the Disease Activity Score in 28 joints using the C-reactive protein level (DAS28-CRP) determined the disease activity. The muscle mass was measured by Dual X-ray absorptiometry (DXA) to generate the appendicular lean mass index (ALMI) by summing the lean mass measurements for both arms and legs and dividing them by height squared (kg/height2 ). Finally, urine metabolomic analysis by 1 H nuclear magnetic resonance (1 H-NMR) spectroscopy was performed and the metabolomics data set analysed using the BAYESIL and MetaboAnalyst software packages. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were applied to the 1 H-NMR data, followed by Spearman's correlation analysis. The combined receiver operating characteristic curve (ROC) was calculated, as well as the logistic regression analyses to establish a diagnostic model. The significance level at P < 0.05 was set for all analyses. RESULTS: The total set of subjects investigated included 90 patients with RA. Most patients were women (86.7%), with a mean age of 56.5 ± 7.3 years old and a median DAS28-CRP of 3.0 (IQR 1.0-3.0). Fifteen metabolites were identified in the urine samples with high variable importance in projection (VIP scores) by MetaboAnalyst. Of these, dimethylglycine (r = 0.205; P = 0.053), oxoisovalerate (r = -0.203; P = 0.055), and isobutyric acid (r = -0.249; P = 0.018) were significantly correlated with ALMI. Based on the low muscle mass (ALMI ≤6.0 kg/m2 for women and ≤8.1 kg/m2 for men) a diagnostic model have been established with dimethylglycine (area under the curve [AUC] = 0.65), oxoisovalerate (AUC = 0.49), and isobutyric acid (AUC = 0.83) with significant sensitivity and specificity. CONCLUSIONS: Isobutyric acid, oxoisovalerate, and dimethylglycine from urine samples were associated with low skeletal muscle mass in patients with RA. These findings suggest that this group of metabolites may be further tested as biomarkers for identification of skeletal muscle wasting.

Evaluating the impact of a cemetery on groundwater by multivariate analysis.

Water analyses in conjunction with hydrological and geotechnical investigations were carried out to assess the potential for groundwater contamination from the decomposition of buried human bodies. Water samples were collected from 2007 to 2018 in three monitoring wells built within the cemetery area. Water quality was evaluated based on the determination of 25 analytical parameters (20 physical-chemical and 5 microbiological). Laboratory data reported by the local sewage water company for water collected in household cisterns located outside the cemetery area were also evaluated. Multivariate analysis showed a similar pattern between iron content, turbidity, and rainfall data collected at the rainfall station closest to the study area. This behavior is a direct consequence of soil leaching (oxisol). The physical characterization of the soil of the unsaturated area above the aquifer indicates that the absorption of body waste by the soil is favored, preventing surface contaminants from reaching the aquifer. This work also found that the water samples collected outside the cemetery area do not comply with the Brazilian limits for drinking water. In conclusion, water samples collected from monitoring wells located within the cemetery area have little to none impact on both subsurface and underground contamination.

Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.

Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.

Community pharmacists' experience relying on select implementation strategies in the delivery of comprehensive medication management.

OBJECTIVE: Implementation strategies are methods or techniques that facilitate adoption, implementation, and sustainability of a clinical program or practice. There has been a lack of widespread adoption of comprehensive medication management (CMM) in community pharmacies. The objective of this evaluation was to expand understanding of how select implementation strategies impacted pharmacists' experience implementing and delivering CMM in the community pharmacy setting. METHODS: A community pharmacy CMM initiative, Slice of Partners in Excellence (PIE), employed a number of implementation strategies and key elements from the Institute for Healthcare Improvement's Breakthrough Series to support increased engagement and delivery of CMM within a local payer's CMM program. The program provides incentives to pharmacies for providing CMM to select patients and achieving predetermined quality metrics. To evaluate pharmacists' experience with the implementation strategies and the impact it had on implementing and delivering CMM, a focus group and survey were conducted. RESULTS: Some of the implementation strategies were more frequently highlighted as generating value to participants (coaching, community-wide problem-solving) than others (monthly webinars). Pharmacists identified a need for more formal education regarding billing, documentation and patient engagement supports. Pharmacists expressed a desire for ongoing implementation supports extending beyond this initaitive. CONCLUSION: Understanding the types of implementation strategies that are deemed as influential by pharmacists delivering clinical services in the community pharmacy setting is critical to maximizing patient access to these services in the future.

Metabolomic Biomarker Candidates for Skeletal Muscle Loss in the Collagen-Induced Arthritis (CIA) Model.

There is no consensus for diagnosis or treatment of RA muscle loss. We aimed to investigate metabolites in arthritic mice urine as biomarkers of muscle loss. DBA1/J mice comprised collagen-induced arthritis (CIA) and control (CO) groups. Urine samples were collected at 0, 18, 35, 45, 55, and 65 days of disease and subjected to nuclear magnetic resonance spectroscopy. Metabolites were identified using Chenomx and Birmingham Metabolite libraries. The statistical model used principal component analysis, partial least-squares discriminant analysis, and partial least-squares regression analysis. Linear regression and Fisher's exact test via the MetaboAnalyst website were performed (VIP-score). Nearly 100 identified metabolites had CIA vs. CO and disease time-dependent differences (p < 0.05). Twenty-eight metabolites were muscle-associated: carnosine (VIPs 2.8 × 102) and succinyl acetone (VIPs 1.0 × 10) showed high importance in CIA vs. CO models at day 65; CIA pair analysis showed histidine (VIPs 1.2 × 102) days 55 vs. 65, histamine (VIPs 1.1 × 102) days 55 vs. 65, and L-methionine (VIPs 1.1 × 102) days 0 vs. 18. Carnosine was fatigue- (0.039) related, creatine was food intake- (-0.177) and body weight- (-0.039) related, and both metabolites were clinical score- (0.093; 0.050) and paw edema- (0.125; 0.026) related. Therefore, muscle metabolic alterations were detected in arthritic mice urine, enabling further validation in RA patient's urine, targeting prognosis, diagnosis, and monitoring of RA-mediated muscle loss.

Base editing of haematopoietic stem cells rescues sickle cell disease in mice.

Sickle cell disease (SCD) is caused by a mutation in the ß-globin gene HBB1. We used a custom adenine base editor (ABE8e-NRCH)2,3 to convert the SCD allele (HBBS) into Makassar ß-globin (HBBG), a non-pathogenic variant4,5. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBBS to HBBG. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBBG was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBBS base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse6 and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar ß-globin represented 79% of ß-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBBS-to-HBBG editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes the undesired consequences of double-strand DNA breaks.

Ultrasound Findings and Laboratory Predictors of Early Mortality in Patients With Severe Yellow Fever.

OBJECTIVE. Yellow fever is a hemorrhagic disease caused by an arbovirus endemic in South America; outbreaks have occurred in recent years. The purpose of this study was to describe abdominal ultrasound findings in patients with severe yellow fever and correlate them with clinical and laboratory data. MATERIALS AND METHODS. A retrospective cohort study was performed between January and April 2018. The subjects were patients admitted to an ICU with polymerase chain reaction-confirmed yellow fever. Bedside sonography was performed within 48 hours of admission. Images were independently analyzed by two board-certified radiologists. Laboratory test samples were collected within 12 hours of image acquisition. Multivariable logistic regression analysis was performed to identify 30-day mortality predictors; p < .05 was considered statistically significant. RESULTS. Forty-six patients (40 [87%] men, six [13%] women; mean age, 47.5 ± 15.2 years) were evaluated with bedside sonography. Laboratory tests showed high serum levels of aspartate aminotransferase (5319 U/L), total bilirubin (6.2 mg/dL), and creati-nine (4.3 mg/dL). Twenty-six (56.5%) patients died within 30 days of admission (median time to death, 5 days [interquartile range, 2-9 days]). The most frequent ultrasound findings were gallbladder wall thickening (80.4%), increased renal cortex echogenicity (71.7%), increased liver parenchyma echogenicity (65.2%), perirenal fluid (52.2%), and ascites (30.4%). Increased renal echogenicity was associated with 30-day mortality (84.6% versus 55.0%; p = .046) and was an independent predictor of this outcome after multivariate analysis (odds ratio, 10.89; p = .048). CONCLUSION. Reproducible abdominal ultrasound findings in patients with severe yellow fever may be associated with severity of disease and prognosis among patients treated in the ICU.

Cap 1 Messenger RNA Synthesis with Co-transcriptional CleanCap® Analog by In Vitro Transcription.

Synthetic messenger RNA (mRNA)-based therapeutics are an increasingly popular approach to gene and cell therapies, genome engineering, enzyme replacement therapy, and now, during the global SARS-CoV-2 pandemic, vaccine development. mRNA for such purposes can be synthesized through an enzymatic in vitro transcription (IVT) reaction and formulated for in vivo delivery. Mature mRNA requires a 5'-cap for gene expression and mRNA stability. There are two methods to add a cap in vitro: via a two-step multi-enzymatic reaction or co-transcriptionally. Co-transcriptional methods minimize reaction steps and enzymes needed to make mRNA when compared to enzymatic capping. CleanCap® AG co-transcriptional capping results in 5 mg/ml of IVT with 94% 5'-cap 1 structure. This is highly efficient compared to first-generation cap analogs, such as mCap and ARCA, that incorporate cap 0 structures at lower efficiency and reaction yield. This article describes co-transcriptional capping using TriLink Biotechnology's CleanCap® AG in IVT. © 2021 Wiley Periodicals LLC. Basic Protocol 1: IVT with CleanCap Basic Protocol 2: mRNA purification and analysis.

The pharmacist as a public health resource: Expanding telepharmacy services to address social determinants of health during the COVID-19 pandemic.

Background: The advent of COVID-19 exacerbated the impact of social determinants of health (SDOH) on patients' ability to manage their health, especially those with chronic conditions. Clinical pharmacists are well positioned to expand the patient care services they already provide to address patients' basic social needs, which may otherwise impede medication access and adherence. Objectives: The purpose of this exploratory study was to evaluate the feasibility of expanding a comprehensive medication management (CMM) telepharmacy service to include SDOH support. This service was offered as part of four primary care clinics in rural and underserved North Carolina communities. More specifically, the study aimed to describe the expanded service, evaluate stakeholders' experience with the service, and assess short-term impact on patients with diabetes. Methods: Data collected over the first 4 months of implementation included administrative data used to describe the expanded service; a clinic survey and interviews to assess clinic team members' experience with the service; and patient surveys to evaluate patient satisfaction, as well as impact on SDOH self-efficacy and diabetes quality of life. Results: Through SDOH screening, the pharmacist identified 26 unresolved COVID-prompted SDOH concerns across 66 patients. These concerns were addressed by the pharmacist through three types of brief interventions, including information provision/education (71%), access to resources (21%), and additional care coordination (7%). Clinic team members perceived the expanded service as highly satisfactory and beneficial. Patients also reported high levels of satisfaction and significantly increased their SDOH self-efficacy and diabetes quality of life as a result of the service. Conclusion: These data provide preliminary insights into the expanded role that pharmacists can play to address current population health gaps that can directly impact patients' engagement with their medication regimen and overall health status.

Risk factors for methicillin-resistant Staphylococcus aureus and extended-spectrum ß-lactamase producing Enterobacterales in patients with diabetic foot infections requiring hospital admission.

OBJECTIVE: Methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) may complicate the treatment of diabetic foot infections (DFIs). The aim of this study was to determine the risk factors for these pathogens in DFIs. METHODS: This was a prospective observational study of 167 consecutive adult patients with DFIs. The diagnosis and severity of DFIs were based on the Infectious Disease Society of America (IDSA) classification system. Multivariate analyses were performed in order to identify risk factors for MRSA and ESBL-E infections. RESULTS: S. aureus was the most isolated pathogen (n=82, 37.9 %) followed by Escherichia coli (n= 40, 18.5%). MRSA accounted for 57.3% of all S. aureus and 70% of Klebsiella pneumoniae and 25% of E. coli were ESBL producers, respectively. Deep ulcer [OR 8,563; 95% CI (1,068-4,727)], previous use of fluoroquinolones [OR 2,78; 95% CI (1,156-6,685)] and peripheral vasculopathy [OR 2,47; 95% CI (1.068-4.727)] were the independent predictors for MRSA infections; and osteomyelitis [OR 6,351; 95% CI (1,609-25,068)] and previous use of cephalosporins [OR 5,824; 95% CI (1,517-22,361)] for ESBL-E infections. CONCLUSIONS: MRSA and ESBL-E have adquired a great clinical relevance in DFIs. The availability of their risk factors is very convenient to choose the empirical treatment in severe forms.

Using a Thermal Camera to Measure Heat Loss Through Bird Feather Coats.

Feathers are essential to insulation, and therefore to the cost of thermoregulation, in birds. There is robust literature on the energetic cost of thermoregulation in birds across a variety of ecological circumstances. However, few studies characterize the contribution of the feathers alone to thermoregulation. Several previous studies have established methods for measuring the insulation value of animal pelts, but they require destructive sampling methods that are problematic for birds, whose feathers are not distributed evenly across the skin. More information is needed about 1) how the contribution of feathers to thermoregulation varies both across and within species and 2) how feather coats may change over space and time. Reported here is a method for rapidly and directly measuring the thermal performance of feather coats and the skin using dried whole skin specimens, without the need to destroy the skin specimen. This method isolates and measures the thermal gradient across a feather coat in a way that measurements of heat loss and metabolic cost in live birds, which use behavioral and physiological strategies to thermoregulate, cannot. The method employs a thermal camera, which allows the rapid collection of quantitative thermal data to measure heat loss from a stable source through the skin. This protocol can easily be applied to various research questions, is applicable to any avian taxa, and does not require destruction of the skin specimen. Finally, it will further the understanding of the importance of passive thermoregulation in birds by simplifying and accelerating the collection of quantitative data.

Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope.

CRISPR-Cas9-associated base editing is a promising tool to correct pathogenic single nucleotide mutations in research or therapeutic settings. Efficient base editing requires cellular exposure to levels of base editors that can be difficult to attain in hard-to-transfect cells or in vivo. Here we engineer a chemically modified mRNA-encoded adenine base editor that mediates robust editing at various cellular genomic sites together with moderately modified guide RNA, and show its therapeutic potential in correcting pathogenic single nucleotide mutations in cell and animal models of diseases. The optimized chemical modifications of adenine base editor mRNA and guide RNA expand the applicability of CRISPR-associated gene editing tools in vitro and in vivo.

Systematic assessment of commercially available low-input miRNA library preparation kits.

High-throughput sequencing is increasingly favoured to assay the presence and abundance of microRNAs (miRNAs) in biological samples, even from low RNA amounts, and a number of commercial vendors now offer kits that allow miRNA sequencing from sub-nanogram (ng) inputs. Although biases introduced during library preparation have been documented, the relative performance of current reagent kits has not been investigated in detail. Here, six commercial kits capable of handling <100ng total RNA input were used for library preparation, performed by kit manufactures, on synthetic miRNAs of known quantities and human total RNA samples. We compared the performance of miRNA detection sensitivity, reliability, titration response and the ability to detect differentially expressed miRNAs. In addition, we assessed the use of unique molecular identifiers (UMI) sequence tags in one kit. We observed differences in detection sensitivity and ability to identify differentially expressed miRNAs between the kits, but none were able to detect the full repertoire of synthetic miRNAs. The reliability within the replicates of all kits was good, while larger differences were observed between the kits, although none could accurately quantify the relative levels of the majority of miRNAs. UMI tags, at least within the input ranges tested, offered little advantage to improve data utility. In conclusion, biases in miRNA abundance are heavily influenced by the kit used for library preparation, suggesting that comparisons of datasets prepared by different procedures should be made with caution. This article is intended to assist researchers select the most appropriate kit for their experimental conditions.

IgG1+ B-cell immunity predates IgE responses in epicutaneous sensitization to foods.

BACKGROUND: The generation of IgE-mediated food allergy in humans is silent and only diagnosed upon manifestation of clinical symptoms. While experimental models have been used to investigate some mechanisms of allergic sensitization, the generation of humoral immunity and memory remains to be elucidated. Here, we defined the evolution of allergen-specific B-cell responses during epicutaneous sensitization to foods. METHODS: Wild-type and genetic knockout animals, and drug or antibody strategies for cell depletion and immunoglobulin signaling blockade were used to investigate epicutaneous sensitization and disease progression; we analyzed allergen-specific germinal centers and IgG1+ memory B cells by flow cytometry, evaluated humoral responses, and determined clinical reactivity (anaphylaxis). RESULTS: Epicutaneous sensitization caused microscopic skin damage, inflammation, and recruitment of activated dendritic cells to the draining lymph nodes. This process generated allergen-specific IgG1+ germinal center B cells, serum IgG1, and anaphylaxis that was mediated by the alternative pathway. Whether we used peanut and/or ovalbumin from the egg white for sensitization, the allergen-specific IgG1+ memory compartment predominantly exhibited an immature, pro-germinal center phenotype (PDL-2- CD80- CD35+ CD73+ ). Subsequent subclinical exposures to the allergen induced IgE+ germinal center B cells, serum IgE, and likely activated the classical pathway of anaphylaxis. CONCLUSIONS: Our data demonstrate that IgG1+ B-cell immunity against food allergens in epicutaneous sensitization precedes the generation of IgE responses. Therefore, the assessment of allergen-specific cellular and humoral IgG1+ immunity may help to identify individuals at risk of developing IgE-mediated food allergy and hence provide a window for therapeutic interventions.

Genomics and epidemiology for gastric adenocarcinomas (GE4GAC): a Brazilian initiative to study gastric cancer

Gastric cancer (GC) is the fifth most common type of cancer worldwide with high incidences in Asia, Central, and South American countries. This patchy distribution means that GC studies are neglected by large research centers from developed countries. The need for further understanding of this complex disease, including the local importance of epidemiological factors and the rich ancestral admixture found in Brazil, stimulated the implementation of the GE4GAC project. GE4GAC aims to embrace epidemiological, clinical, molecular and microbiological data from Brazilian controls and patients with malignant and pre-malignant gastric disease. In this letter, we summarize the main goals of the project, including subject and sample accrual and current findings

Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification.

The Cas9/guide RNA (Cas9/gRNA) system is commonly used for genome editing. mRNA expressing Cas9 can induce innate immune responses, reducing Cas9 expression. First-generation Cas9 mRNAs were modified with pseudouridine and 5-methylcytosine to reduce innate immune responses. We combined four approaches to produce more active, less immunogenic second-generation Cas9 mRNAs. First, we developed a novel co-transcriptional capping method yielding natural Cap 1. Second, we screened modified nucleotides in Cas9 mRNA to identify novel modifications that increase Cas9 activity. Third, we depleted the mRNA of uridines to improve mRNA activity. Lastly, we tested high-performance liquid chromatography (HPLC) purification to remove double-stranded RNAs. The activity of these mRNAs was tested in cell lines and primary human CD34+ cells. Cytokines were measured in whole blood and mice. These approaches yielded more active and less immunogenic mRNA. Uridine depletion (UD) most impacted insertion or deletion (indel) activity. Specifically, 5-methoxyuridine UD induced indel frequencies as high as 88% (average ± SD = 79% ± 11%) and elicited minimal immune responses without needing HPLC purification. Our work suggests that uridine-depleted Cas9 mRNA modified with 5-methoxyuridine (without HPLC purification) or pseudouridine may be optimal for the broad use of Cas9 both in vitro and in vivo.

Efficient Production of On-Target Reads for Small RNA Sequencing of Single Cells Using Modified Adapters.

Although single-cell mRNA sequencing has been a powerful tool to explore cellular heterogeneity, the sequencing of small RNA at the single-cell level (sc-sRNA-seq) remains a challenge, as these have no consensus sequence, are relatively low abundant, and are difficult to amplify in a bias-free fashion. We present two methods of single-cell-lysis that enable sc-sRNA-seq. The first method is a chemical-based technique with overnight freezing while the second method leverages on-chip electrical lysis of plasma membrane and physical extraction and separation of cytoplasmic RNA via isotachophoresis. We coupled these two methods with off-chip small RNA library preparation using CleanTag modified adapters to prevent the formation of adapter dimers. We then demonstrated sc-sRNA-seq with single K562 human leukemic cells. Our approaches offer a relatively short hands-on time of 6 h and efficient generation of on-target reads. The sc-sRNA-seq with our approaches showed detection of miRNA with various abundances ranging from 16 000 copies/cell to about 10 copies/cell. We anticipate this approach will create a new opportunity to explore cellular heterogeneity through small RNA expression.

Collagen-induced arthritis as an animal model of rheumatoid cachexia.

BACKGROUND: Rheumatoid arthritis is characterized by chronic polyarticular synovitis and presents systemic changes that impact quality of life, such as impaired muscle function, seen in up to 66% of the patients. This can progress to severely debilitating state known as rheumatoid cachexia-without loss of fat mass and body weight-for which there is little consensus in terms of diagnosis or treatment. This study aims to evaluate whether the collagen-induced arthritis (CIA) animal model also develops clinical and functional features characteristic of rheumatoid cachexia. METHODS: Male DBA1/J mice were randomly divided into 2 groups: healthy animals (CO, n = 11) and CIA animals (n = 13). The clinical score and edema size, animal weight and food intake, free exploratory locomotion, grip strength, and endurance exercise performance were tested 0, 18, 35, 45, 55, and 65 days after disease induction. After euthanasia, several organs, visceral and brown fat, and muscles were dissected and weighed. Muscles were used to assess myofiber diameter. Ankle joint was used to assess arthritis severity by histological score. Statistical analysis were performed using one-way and two-way analyses of variance followed by Tukey's and Bonferroni's test or t-test of Pearson and statistical difference were assumed for a P value under 0.05. RESULTS: The CIA had significantly higher arthritis scores and larger hind paw edema volumes than CO. The CIA had decreased endurance exercise performance total time (fatigue; 23, 22, 24, and 21% at 35, 45, 55, and 65 days, respectively), grip strength (27, 55, 63, 60, and 66% at 25, 35, 45, 55, and 65 days, respectively), free locomotion (43, 57, 59, and 66% at 35, 45, 55, and 65 days, respectively), and tibialis anterior and gastrocnemius muscle weight (25 and 24%, respectively) compared with CO. Sarcoplasmic ratios were also reduced in CIA (TA: 23 and GA: 22% less sarcoplasmic ratio), confirming the atrophy of skeletal muscle mass in these animals than in CO. Myofiber diameter was also reduced 45% in TA and 41% in GA in CIA when compared with the CO. Visceral and brown fat were lighter in CIA (54 and 39%, respectively) than CO group. CONCLUSIONS: The CIA model is a valid experimental model for rheumatoid cachexia given that the clinical changes observed were similar to those described in patients with rheumatoid arthritis.

CleanTag Adapters Improve Small RNA Next-Generation Sequencing Library Preparation by Reducing Adapter Dimers.

Next-generation small RNA sequencing is a valuable tool which is increasing our knowledge regarding small noncoding RNAs and their function in regulating genetic information. Library preparation protocols for small RNA have thus far been restricted due to higher RNA input requirements (>10 ng), long workflows, and tedious manual gel purifications. Small RNA library preparation methods focus largely on the prevention or depletion of a side product known as adapter dimer that tends to dominate the reaction. Adapter dimer is the ligation of two adapters to one another without an intervening library RNA insert or any useful sequencing information. The amplification of this side reaction is favored over the amplification of tagged library since it is shorter. The small size discrepancy between these two species makes separation and purification of the tagged library very difficult. Adapter dimer hinders the use of low input samples and the ability to automate the workflow so we introduce an improved library preparation protocol which uses chemically modified adapters (CleanTag) to significantly reduce the adapter dimer. CleanTag small RNA library preparation workflow decreases adapter dimer to allow for ultra-low input samples (down to approx. 10 pg total RNA), elimination of the gel purification step, and automation. We demonstrate how to carry out this streamlined protocol to improve NGS data quality and allow for the use of sample types with limited RNA material.