SF Samples Research Articles

The influence of different freezing and thawing conditions on the quality of beef rib primals

This research investigated the influence of freezing/thawing rates on the quality of beef rib primals. Forty ribs were subjected to one of five treatments: control (no freezing), fast freezing/fast thawing (FF), fast freezing/slow thawing (FS), slow freezing/fast thawing (SF), and slow freezing/slow thawing (SS). Ribs were cut, and steaks from the cranial, medial, and caudal locations were assessed for meat quality and ultrastructural changes. Additional steaks from each location underwent a second freezing/thawing cycle and were evaluated similarly. Following the first cycle, FF, FS, SF, and SS samples generally exhibited increased a∗, b∗, drip loss, tenderness, and ultrastructural degradation than the control (P < 0.05). While no major meat quality differences were found across the four freezing/thawing treatments, the FF and SF had lower drip loss than FS and SS (P < 0.05), signifying the importance of the thawing rate. The second cycle further influenced meat quality by decreasing a∗ and b∗, increasing tenderness and TBARS, and accentuating ultrastructural deterioration (P < 0.05). Variations among sampling locations were detected for most tested parameters, but inconsistent results hindered definitive conclusions. Overall, minor differences in meat quality were detected among the freezing rates tested herein, while fast thawing may alleviate quality deterioration.

Formulation and Characterization of New Experimental Dental Composites with Zirconium Filling in Different Forms.

Short glass fibers are generally used in posterior dental restorations to enhance the mechanical properties and improve the material microstructure. Two resin-based composites (S0 and SF) were formulated and characterized to investigate the influence of zirconium in their characteristics and properties. The organic part of the investigated materials was the same (BisGMA, TEGDMA, and a photochemical polymerization system), and in the inorganic part, besides quart, glassA, and hydroxylapatite with Zn, sample S0 contained strontium glass with zirconium and sample SF contained fiber powder of chopped zirconium. The samples were characterized by the degree of conversion (DC), mechanical properties, water sorption (WS), scanning electron microscopy (SEM), atomic force microscopy (AFM) before and after the WS test, and antimicrobial properties. The results obtained were subjected to one-way ANOVA and Tukey's statistical tests. Both samples had a high DC. Regarding the mechanical properties, both samples were very similar, except DTS, which was higher for the composite without fibers. After 14 days, the WS value of the SF sample was lower than that of the S0 sample. Water caused significant changes in the topography of the SF sample, but thanks to its antimicrobial properties and the diffusion phenomenon, SF had a more pronounced antimicrobial effect. This study shows that the addition of appropriate amounts of Sr-Zr-glass powder gives the material in which it is added similar properties to material containing chopped zirconium glass fiber powder. According to the antimicrobial test results, resin composites containing experimental zirconia fillings can be considered in future in vitro clinical studies for posterior reconstructions with significantly improved mechanical properties.

Advancing the Diagnosis of Periprosthetic Joint Infections: Integrated Microfluidic Platform for Alpha-Defensins-Specific Aptamer Selection and Its Analytical Applications.

Periprosthetic joint infections (PJIs) pose a significant challenge in orthopedic surgery, particularly total joint arthroplasty (TJA), due to the potential for implant failure and increased patient morbidity. Early and accurate detection of PJIs is crucial for timely intervention and better patient prognosis. Herein, we successfully screened a high-affinity aptamer targeting alpha-defensin complex human neutrophil protein 1-3 (HNP 1-3; potential PJI biomarkers in synovial fluid [SF]) for the first time using systematic evolution of ligands by exponential enrichment (SELEX) on an integrated microfluidic platform. The compact microfluidic device enabled efficient screening, with each round completed within <2 h, comprising five rounds of positive selection, two rounds of negative selection, and one round of competitive selection. A novel one-aptamer-one-antibody assay was further developed from the optimal aptamer screened, and it could accurately quantify HNP 1-3 in SF within 3 h with only ∼50 μL of SF. The assay demonstrated strong binding affinity and specificity for the target protein in SF. Thirteen PJI SF samples were accurately diagnosed and the assay was accurate over a wide dynamic range (0.32-100 mg/L). This study has showcased a rapid and accurate diagnostic tool for PJI detection, which should see widespread use in the clinic, holding promise for potential analytical applications in orthopedic surgery and improving patient care.

Impact of typical land use type on the stability and content of carbon and nitrogen of soil aggregates in western Hubei

AbstractIn order to investigate the characteristics of soil structural stability and the factors influencing it under typical land use types in the karst region of western Hubei, soil samples were collected from five land use types (natural mixed forest [NF], cypress forest [CF], stone dike terrace [ST], stone dike forest [SF], and abandoned land [AL]) in Xialaoxi to analyze the particle size distribution of aggregates, structural stability, and the distribution characteristics of carbon, nitrogen, and extracellular enzyme contents. The results showed that the aggregates of NF, CF, SF, and AL were dominated by &gt;2‐mm aggregates, while ST showed mostly 2–0.25‐mm aggregates. The mean weight diameter (EMWD) and the geometric mean diameter (EGMD) of mechanical aggregates showed a trend of CF &gt; NF &gt; SF &gt; AL &gt; ST, whereas the erodibility factor K showed an opposite trend. The total organic carbon (TOC), labile organic carbon (LOC), and total nitrogen (TN) contents of aggregates of each particle size were significantly higher in NF and CF than in ST and AL under the five land types. The content of available nitrogen (AN) was highest in NF and ST. The content of carbon and nitrogen and extracellular hydrolases (β‐glucosidase [BG], cellulose disaccharide hydrolase [CBH], β‐N‐acetyl‐aminoglucosidase [NAG], and leucine aminopeptidase [LAP]) in soil aggregates were mainly concentrated in &lt;0.25–0.053‐mm aggregates. The variation in carbon and nitrogen abundance of each particle size agglomerate mainly originated from the LAP content of that particle size agglomerate and showed significant correlation. The partial least squares path models (PLS‐PM) showed that the main influences on the stability of soil aggregates in small watersheds were land use types, soil physical properties, carbon and nitrogen abundance, and extracellular enzyme activity. In conclusion, the structural stability of soil aggregates in Xialaoxi watershed is the result of the interaction between human disturbance and soil nutrient self‐cycling. In general, NF and CF have better soil aggregate stability and are conducive to soil carbon and nitrogen accumulation, whereas for later development and management of SF, ST, and AL sample plots, scientific cultivation means should be adopted, and attention should be paid to long‐term carbon and nitrogen accumulation and conservation.

Changes in the quality of apple tissue subjected to different freezing rates during long-term frozen storage at different temperatures

In this manuscript, the impact of various parameters including freezing rate, storage temperature and duration on the quality of apples is reported. Freezing rate significantly affected the quality attributes (firmness, drip loss, and morphology of ice crystals). Significant difference in the firmness loss was seen between the static freezing (SF at -18 °C) and rapid freezing (RF at -40 °C and 2 m/s air velocity) and ultra rapid freezing (URF at -72 °C and 1 m/s air velocity) samples on day 0. Regarding drip loss, significant difference could only be seen between SF and URF conditions. Nevertheless, microscopy results showed that ice crystal size reduced with the increase in the freezing rates. Storage of frozen products at two different temperatures (i.e. -12 °C and -18 °C) for 90 days caused serious damage to the product. Whatever the freezing condition and storage temperature, the maximum reduction in firmness happened during the first 30 days of storage. A maximum reduction (85%) in the texture was seen for SF samples stored for 30 days at -12 °C compared to fresh samples. On the other hand, drip loss increased gradually with the storage period for all freezing methods and storage temperatures. After 90 days, SF samples stored at -12 °C recorded the highest drip loss (6.12%), while RF samples stored at -18 °C exhibited the lowest value (5.05%). MANOVA analysis revealed that all three independent variables (i.e. freezing rate, storage time, and storage temperature) significantly affected the drip loss and texture. Lastly, microscopy after 90 days showed a high degree of tissue damage, making it difficult to differentiate the freezing method and storage temperature.

Composition and assembly of the bacterial community in the overlying waters of the coral reef of China's Xisha Islands.

Coral reef ecosystems are one of the most diverse and productive habitats on Earth. Microbes in the reef-overlying waters are key players in maintaining this ecosystem through regulating biogeochemical and ecological processes. However, the composition structure and assembly mechanism of microbial community in the reef-overlying waters remain largely unknown. In the present study, the bacterial communities from the overlying waters of atolls and fringing reefs as well as the surface waters of the adjacent open ocean of the Xisha Islands in the South China Sea were investigated using 16S rRNA high-throughput sequencing combined with a size-fractionation strategy. The results showed that environments of all sampling stations were similar, characterized by an almost complete lack of inorganic nutrients such as nitrogen and phosphorus. Proteobacteria, Cyanobacteria and Bacteroidetes were the dominant phyla, and Synechococcus was most abundant at the genus level in both large fraction (LF; 1.6-200 μm) and small fraction (SF; 0.2-1.6 μm) communities. Only a slight difference in community composition between LF and SF samples was observed. The bacterial communities among the three habitat types showed noticeable differences, and the bacterial composition among the atoll reefs was more varied than that among the fringing reefs. The similarity of bacterial communities significantly declined with the increasing geographic distance, and stochastic processes were more important than deterministic processes in bacterial community assembly. This study sheds lights on the bacterial biodiversity of coral reefs and the importance of stochastic process in structuring bacterial communities.

CCL24, CXCL9 and CXCL10 are increased in synovial fluid in patients with juvenile idiopathic arthritis requiring advanced treatment.

Oligoarticular JIA disease progression and outcomes are variable. Our objective is to detect protein markers that would allow for earlier intervention to potentially halt disease progression. In this retrospective study of serial SF samples, elevated expression of CCL24, CXCL9 and CXCL10 was linked to the eventual need for advanced medications. Serial SF samples were selected from patients with persistent and extended oligoarticular JIA. The samples were separated into two groups: those who did and did not receive advanced medications throughout their disease course. Protein antibody arrays and Luminex assays were performed to determine changes in protein expression. CCL24, CXCL9 and CXCL10 expression levels were significantly higher in patients who eventually required advanced treatment than in those who did not. The expression levels of CCL24 and CXCL9 were consistently elevated in paired samples of those who later received advanced medications. In the persistent oligoarticular JIA group, CXCL10 levels remained elevated over time in those who required advanced treatment. Conversely, CCL24 levels decreased in patients who did not require advanced treatment. In the extended samples, the levels of CCL24 and CXCL10 expression increased significantly over time in the patients who ultimately required advanced treatment. In patients with oligoarticular JIA, regardless of disease onset and progression, the consistent elevation of any or all three markers, the CCL24, CXCL9 and CXCL10 in SFs was associated with the future use of advanced therapy, which could be reflective of disease severity.

Hyaluronidase treatment of synovial fluid is required for accurate detection of inflammatory cells and soluble mediators

BackgroundSynovial fluid (SF) is commonly used for diagnostic and research purposes, as it is believed to reflect the local inflammatory environment. Owing to its complex composition and especially the presence of hyaluronic acid, SF is usually viscous and non-homogeneous. In this study, we investigated the importance of homogenization of the total SF sample before subsequent analysis.MethodsSF was obtained from the knee of 29 arthritis patients (26 rheumatoid arthritis, 2 osteoarthritis, and 1 juvenile idiopathic arthritis patient) as part of standard clinical care. Synovial fluid was either treated with hyaluronidase as a whole or after aliquoting to determine whether the concentration of soluble mediators is evenly distributed in the viscous synovial fluid. Cytokine and IgG levels were measured by ELISA or Luminex and a total of seven fatty acid and oxylipin levels were determined using LC-MS/MS in all aliquots. For cell analysis, synovial fluid was first centrifuged and the pellet was separated from the fluid. The fluid was subsequently treated with hyaluronidase and centrifuged to isolate remaining cells. Cell numbers and phenotype were determined using flow cytometry.ResultsIn all patients, there was less variation in IgG, 17-HDHA, leukotriene B4 (LTB4), and prostaglandin E2 (PGE2) levels when homogenization was performed before aliquoting the SF sample. There was no difference in variation for cytokines, 15-HETE, and fatty acids arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Between 0.8 and 70% of immune cells (median 5%) remained in suspension and were missing in subsequent analyses when the cells were isolated from untreated SF. This percentage was higher for T and B cells: 7–85% (median 22%) and 7–88% (median 23 %), respectively.ConclusionsHomogenization of the entire SF sample leads to less variability in IgG and oxylipin levels and prevents erroneous conclusions based on incomplete isolation of synovial fluid cells.

Neuroimaging genetic associations between SEMA6D, brain structure, and reading skills

ABSTRACT Specific reading disability (SRD) is defined by genetic and neural risk factors that are not fully understood. The current study used imaging genetics methodology to investigate relationships between SEMA6D, brain structure, and reading. SEMA6D, located on SRD risk locus DYX1, is involved in axon guidance, synapse formation, and dendrite development. SEMA6D’s associations with brain structure in reading-related regions of interest (ROIs) were investigated in a sample of children with a range of reading performance, from sites in Connecticut, CT (n = 67, 6–13 years, mean age = 9.07) and San Francisco, SF (n = 28, 5–8 years, mean age = 6.5). Multiple regression analyses revealed significant associations between SEMA6D’s rs16959669 and cortical thickness in the fusiform gyrus and rs4270119 and gyrification in the supramarginal gyrus in the CT sample, but this was not replicated in the SF sample. Significant clusters were not associated with reading. For white matter volume, combined analyses across both samples revealed associations between reading and the left transverse temporal gyrus, left pars triangularis, left cerebellum, and right cerebellum. White matter volume in the left transverse temporal gyrus was nominally related to rs1817178, rs12050859, and rs1898110 in SEMA6D, and rs1817178 was significantly related to reading. Haplotype analyses revealed significant associations between the whole gene and brain phenotypes. Results suggest SEMA6D likely has an impact on multiple reading-related neural structures, but only white matter volume in the transverse temporal gyrus was significantly related to reading in the current sample. As the sample was young, the transverse temporal gyrus, involved in auditory perception, may be more strongly involved in reading because phonological processing is still being learned. The relationship between SEMA6D and reading may change as different brain regions are involved during reading development. Future research should examine mediating effects, use additional brain measures, and use an older sample to better understand effects.

Neutrophil extracellular traps release in gout and pseudogout depends on the number of crystals regardless of leukocyte count.

Microcrystal-induced arthritis is still an unresolved paradigm for medicine. Overt inflammation may be absent even when crystals occur in SF. Recently, the production of neutrophil extracellular traps (NETs) embedding MSU crystals has been proposed as a possible mechanism of the auto-resolution of the inflammatory phase during gout. We aimed to verify and quantify the release of NETs in SFs during gout and pseudogout attacks and to compare any differences with respect to crystals and neutrophils number, and to analyse activation of necroptosis pathway in SF from crystal-induced arthritis. SF samples were obtained by arthrocentesis from 22 patients presenting acute crystal-induced arthritis, gout or pseudogout (n = 11 each group), and from 10 patients with acute non-crystal arthritis as controls. NETosis was quantified in SF by nucleic acid stain and by quantification of human neutrophil elastase. Activation of phosphorylated MLKL was assessed by western blot. We observed that SF neutrophils encountering MSU and CPPD crystals during episodes of gout and pseudogout release NETs in relation to the number of crystals in SF and irrespective of neutrophil density and type of crystal. This release was accompanied by necroptosis through the activation of the MLKL pathway. Our findings suggest that a role of NETs in crystal-induced arthritis is to 'trap extracellular particles', including microcrystals. Embedding crystals in aggregates of NETs may be the basis of tophi and CPPD deposition, and may have implications for disease evolution rather than for spontaneous resolution of the acute attack.

Effect of hydrothermal carbonization temperature on reactivity and synergy of co-gasification of biomass hydrochar and coal

In this study, co-gasification reactivity, synergy and kinetics of blended char of bituminous coal (SF) and raw/hydrothermal-carbonization-treated (HTC) rice straw (RS/RSH) at different HTC temperatures (180–240 °C) and gasification temperatures (800–950 °C) were investigated. Moreover, mineral matter transformation and chemical structure evolution during co-gasification were quantitatively investigated to reveal the influence mechanism of binary particle interaction on co-gasification synergy behavior. Reactivity results indicate that co-gasification reactivity of SF-RSH blended char was lower than SF-RS blended char and was decreased with increasing HTC temperature. Synergy factor results show that the existence of synergistic effect during co-gasification contributed to the higher actual blended char reactivity than the corresponding calculated value, which was mainly due to the inhibition effect on active K transformation and the positive effect on carbon structure evolution (i.e., the decrease of ordering degree of carbon structure and the increase of amorphous carbon structure amount) resulted from binary particle interaction during co-gasification. Moreover, synergistic effect was weaker for SF-RSH blended char than SF-RS blended char and was reduced as HTC temperature and gasification temperature increased, which could be well correlated by the weakened inhibition effect on active K transformation, the enhanced promotion effect on active Ca transformation and the weakened inhibition effect on carbon structure graphitization process at higher HTC temperature and gasification temperature. Kinetics analysis demonstrates that activation energies of SF, RS, RSH, SF-RS and SF-RSH char samples were 208.21, 144.74, 160.62–174.01, 161.09, 169.20–178.97 kJ·mol−1, respectively. Moreover, binary particle interaction improved blended char co-gasification kinetics with activation energy reduction of 19.96–31.41 kJ·mol−1 than the calculated values.

Biosynthesis of Gold Clusters and Nanoparticles by Using Extracts of Mexican Plants and Evaluation of Their Catalytic Activity in Oxidation Reactions

This work shows the biosynthesis of gold clusters and nanoparticles (AuNPs) from a solution of gold in a mixture of ammonium chloride and nitric acid treated with two different Mexican plants extracts, Toad Leaf (Eryngium heterophyllum, SF) and Cuachalalate (Amphipterygium adstringens, CF) as bioreductants. Subsequently, the gold entities were supported on the ordered mesoporous material SBA-15 functionalized with mercaptopropyl groups (1.6 wt% of sulphur). The gold contents were 0.7 wt% and 1.1 wt% for the CF and SF samples, respectively. UV–vis spectroscopy and TEM show the presence of gold nanoclusters in both samples, while very minor amount of AuNPs is detected only in the sample with the highest Au content (SF). The Au/SBA-15 materials are active in the aerobic oxidation of cyclohexene at 65 °C and atmospheric pressure. Two of the catalytic reaction products come from the addition of oxygen to the double bond of cyclohexene: cyclohexene epoxide and cyclohexanediol, while the other three come from allylic oxidation of the ring, which is the major reaction pathway: 2-cyclohexen-1-ol, 2-cyclohexen-1-one and 2-cyclohexenyl hydroperoxide, being the two first the major stable reaction products, while the enone/enol ratio is nearly 2. It has been found that the gold nanoclusters initially present in the catalysts evolve toward AuNPs during catalytic reaction.

Ruling out septic arthritis risk in a few minutes using mid-infrared spectroscopy in synovial fluids.

The aim of this study was to show the usefulness of a mid-infrared fibre evanescent wave spectroscopy point of care device in the identification of septic arthritis patients in a multicentre cohort, and to apply this technology to clinical practice among physicians. SF samples from 402 patients enrolled in a multicentre cohort were frozen for analysis by mid-infrared fibre evanescent wave spectroscopy. The calibration cohort was divided into two groups of patients (septic arthritis and non-septic arthritis) and relevant spectral variables were used for logistic regression model. Model performances were tested on an independent set of 86 freshly obtained SF samples from patients enrolled in a single-centre acute arthritis cohort and spectroscopic analyses performed at the patient's bedside. The model set-up, using frozen-thawed SFs, provided good performances, with area under the curve 0.95, sensitivity 0.90, specificity 0.90, positive predictive value 0.41 and negative predictive value 0.99. Performances obtained in the validation cohort were area under the curve 0.90, sensitivity 0.92, specificity 0.81, positive predictive value 0.46 and negative predictive value 0.98. The septic arthritis probability has been translated into a risk score from 0 to 4 according to septic risk. For a risk score of 0, the probability of identifying a septic patient is very low (negative predictive value of 1), whereas a risk score of 4 indicates very high risk of septic arthritis (positive predictive value of 1). Mid-infrared fibre evanescent wave spectroscopy could distinguish septic from non-septic synovial arthritis fluids with good performances, and showed particular usefulness in ruling out septic arthritis. Our data supports the possibility of technology transfer. ClinicalTrials.gov, http://clinicaltrials.gov, NCT02860871.

Characterization of carbonaceous compounds emitted from Indonesian surface and sub surface peat burning

Indonesia has been experiencing an annually reoccurring forest fire events over the peatlands in eastern Sumatera and western Kalimantan and the impacts on the air pollution episodes in the surrounding countries gained international concerns. The most notable events were in 1997 and 2015 which caused significant losses both on human health and economy in the region. Fires in peat layers could spread radially due to peat structure and emitted substantial amount of aerosols containing carbonaceous compounds, including BC/EC and OC. In this study, peat samples from different levels of depth were collected from western part of Kalimantan and were burned in a combustion chamber received from Kyoto University. Particulate (PM2.5) samples from burning experiments were collected and the carbonaceous components (i.e. BC/EC and OC) were also analyzed, respectively. The results showed that average concentration of PM2.5 emitted from the combustion of the surface peat (SF) samples was 7,467 ± 3,976 μg/m³. while the corresponding value for the subsurface (SSF) was 5,693 ± 2,137 μg/m³. In average, EC and OC compositions were 1.9% and 70% to the total of PM2.5 concentrations, respectively. Emission factors (EFs, in g/kg) were estimated for PM2.5, EC, and OC for SF samples of 4.51 ± 0.42, 0.21 ± 0.17, and 3.77 ± 0.82, respectively. The associated EFs for SSF samples were 4.53 ± 1.67, 0.1 ± 0.09, and 1.82 ± 0.43 g/kg, respectively. The results may substantially contribute to fill in the gaps of the availability of EFs for both SF and SFF peat fires in the country to improve the existing PM emission database.

The Effect of Aging/Freezing Sequence and Freezing Rate on Quality Attributes of Beef Loins (M. longissimus lumborum)

The objective of this study was to determine the effect of different aging/freezing sequences combined with different freezing rates on quality attributes of beef loins (M. longissimus lumborum). Loins from 1 side of 8 beef carcasses (USDA Low Choice grade) were obtained at 2 d postmortem, cut into 10 steaks (2.54 cm), and randomly assigned into 5 treatments, where factorial combinations of 2 aging/freezing sequences (aging first then freezing/thawing [AFT] and freezing first then thawing/aging [FTA]) and 2 freezing rates (fast freezing [FF] and slow freezing [SF]) were used and compared to an aged only (AO) treatment as a control. Samples frozen using the FF condition were found to have a critical freezing time (Tc) of 21.5 min while the SF counterpart had a Tc of 175 min. Histological observation showed more visually enlarged gaps between muscle fibers in the SF samples, regardless of aging/freezing sequence. The SF-FTA samples had a significantly higher purge/ thaw loss followed by FF-FTA, while the loss from FF-AFT, SF-AFT, and AO were not different from each other (P &gt; 0.05), suggesting that freezing rate effect might be more profound when applied in FTA. Cook loss was higher in AO compared to both FTA treatments (P &lt; 0.05); however, it was not different when compared to AFT (P &gt; 0.05). A lower shear force value and higher troponin-T protein degradation were found in both AFT and FTA compared to AO (P &lt; 0.05), which would be attributed to the ice-crystal damage due to freezing. These results suggest that aging/freezing sequence could be an overriding factor in determining major meat quality attributes of frozen/thawed meat over freezing rate. However, fast freezing could negate some quality defects associated with the FTA process.

Supercooling preservation on quality of beef steak

A combination of a pulsed electric field (PEF) and oscillating magnetic field (OMF) was used to maintain a supercooled state in beef steak (London Broil) with an internal temperature of approximately −4 °C for up to 14 days. Quality factors such as color, lipid oxidation, drip loss and texture of supercooled beef samples were evaluated and compared with those of refrigerated (4 °C), two frozen (freezing at −10 °C (slow freezing, SF) and −20 °C (rapid freezing, RF)), and fresh samples. In 14 days of storage, there were no significant differences in color parameter values between supercooled and fresh beef samples (P > 0.05). The Thiobarbituric acid reactive substances (TBARS) of supercooled samples after 14 days of storage were significantly lower than those of refrigerated and SF samples (P < 0.05). The cell damage was visualized through optical micrographs. While the drip loss and tenderization due to intracellular ice formation occurred with SF beef, these physical changes were not observed in supercooled beef during the storage.

660. Extraction-Free 16S Ribosomal RNA (rRNA) Gene Amplification and Sequencing from Resected Cardiac Implantable Electronic Device (CIED) Sonicate Fluid

BackgroundWe recently demonstrated that 16S rRNA PCR/sequencing performed on biofilms dislodged from extracted CIEDs into a salt solution—referred to as “sonicate fluid’ (SF)—may be used to detect pathogens in culture-negative CIED infection (Clinical Infectious Diseases, ciz266, doi:10.1093/cid/ciz266). The method we described included a DNA extraction/purification step, which can be time consuming and costly. Here, we evaluated an extraction-free approach to 16S rRNA gene PCR/sequencing.Methods28 SF samples derived from explanted clinically-infected CIEDs were tested. Cases were categorized as “culture-positive” (C-P) if ≥20 cfu/10 mL were recovered, and as “culture-negative” (C-N), if <20 cfu/10 mL were detected in SF culture. The extraction-free method consisted of a single step of lysis at 1,000 rpm, 95°C for 5 minutes using an Eppendorf ThermoMixer®. DNA extraction (the comparator method) was performed using the ZymoBIOMICSTM Kit with modifications. Samples were processed using both methods, followed by amplification of the 16S rRNA gene and bidirectional Sanger sequencing. Crossing points (CPs) generated by the two approaches were compared. Organisms detected by the two PCR methods were compared with those detected with culture.ResultsOf the 28 samples tested, 13 were C-N and 15 C-P. The extraction-free method generated an amplicon in 13/15 C-P cases, with CPs ranging from 26 to 36 cycles vs. 100% (15/15) detected with the DNA extraction method and Cps of 19 to 32. Usable sequence length for the extraction-free method was of 359 (interquartile range, 307–390) vs. 390 (interquartile range, 308–396) base pairs with DNA extraction. Genus-level concordance between bacteria detected by culture in C-P samples and those found using the extraction-free and extraction methods was 92% (12/13) and 93% (14/15), respectively. Bacteria were detected by the extraction method in 2/13 C-N specimens, with none detected with an extraction-free method.ConclusionThe described extraction-free method may be suitable for testing SF derived from CIEDs using 16S rRNA gene PCR/sequencing, saving time and cost. More studies are needed to establish clear cutoffs for interpretation of results and to assess for PCR inhibitors in the studied specimen-type.DisclosuresRobin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.

Metabolomics analysis revealed significantly higher synovial Phe/Tyr ratio in reactive arthritis and undifferentiated spondyloarthropathy.

To compare the synovial phenylalanine/tyrosine (Phe/Tyr) ratio between ReA/uSpA and RA and OA by NMR spectroscopy. Paired SF and serum of 30 patients with ReA/uSpA were collected and analysed using a 1D 1H Carr Purcell Meiboom Gill NMR spectra recorded on 800 MHz NMR spectrometer equipped with a TCI Cryoprobe (at 300 K). Phe and Tyr were quantified. SF from 25 patients with RA fulfilling ACR classification criteria and 21 patients with OA were taken as inflammatory and non-inflammatory controls. The synovial Phe/Tyr ratio was significantly higher in ReA/uSpA compared with RA and OA. Synovial Phe/Tyr ratios were comparable in RA and OA patients. Compared with serum, the Phe/Tyr was significantly higher in the SF in ReA/uSpA. The Phe/Tyr ratio was also found to be positively correlated between serum and SF samples, with a regression coefficient (r2) of 0.287. This NMR-based metabolomics study demonstrates that the synovial Phe/Tyr ratio is specifically elevated in ReA/uSpA.

Characteristics of synovial fluid in patients with juvenile idiopathic arthritis

Synovial fluid (SF) analysis is an important tool for the diagnosis of patients with juvenile idiopathic arthritis (JIA). A retrospective analysis was carried out of cytological features of SF samples obtained from patients with JIA during the period 2008-2016. A total of 102 SF samples from 59 patients were analysed. JIA was more common in females (66%). The most frequent form was persistent oligoarticular JIA (52.5%). The median age at onset was 5 years (IQR 2.4-11.8). SF usually showed an inflammatory pattern (median white blood cells count 11,757/mm3; IQR 4,543-18,800), with a predominance of polymorphonuclear (PMN) cells (61%; IQR 30-75). Eight patients (14%) had white blood cells counts of less than 2,000 cells/mm3, with predominance of mononuclear cells (80%), whereas 3 patients (5%) had white blood cells counts higher than 50,000 cells/mm3, with a predominance of PMN cells (90%). Synovial white blood cells count did not show significant differences among the different forms of JIA. The median synovial white blood cells count in ANA-positive patients was 20% lower than in ANA-negative (9,340 vs. 11,600/mm3; P=.23). The proportion of PMN increased with increasing levels of ESR (P<.001) and/or CRP (P=.03). No significant correlation was found between JADAS-10 and synovial white blood cells count (P=.4). SF obtained from different joints in simultaneous arthrocentesis showed a significant correlation P=.001). SF from JIA patients usually had inflammatory characteristics, although 19% of the patients showed white blood cells counts below 2,000cells/mm3 or higher than 50,000cells/mm3. SF cell count was non-significantly lower in ANA-positive patients, and the proportion of PMN increased with increasing levels of ESR/CRP.

Gut microbiome comparability of fresh-frozen versus stabilized-frozen samples from hospitalized patients using 16S rRNA gene and shotgun metagenomic sequencing

Collection of faecal samples for microbiome analysis in acutely sick patients is logistically difficult, particularly if immediate freezing is required (i.e. fresh-frozen, or FF sampling). Previous studies in healthy/non-hospitalized volunteers have shown that chemical stabilization (i.e. stabilized-frozen, or SF sampling) allows room-temperature storage with comparable results to FF samples. To test this in a hospital setting we compared FF and SF approaches across 17 patients undergoing haematopoietic stem cell transplantation (HSCT) using both 16S rRNA gene and shotgun metagenomic sequencing. A paired (same stool specimen) comparison of FF and SF samples was made, with an overall comparable level in relative taxonomic abundances between the two sampling techniques. Though shotgun metagenomic sequencing found significant differences for certain bacterial genera (P < 0.001), these were considered minor methodological effects. Within-sample diversity of either method was not significantly different (Shannon diversity P16SrRNA = 0.68 and Pshotgun = 0.89) and we could not reject the null hypothesis that between-sample variation in FF and SF were equivalent (P16SrRNA = 0.98 and Pshotgun = 1.0). This indicates that SF samples can be used to reliably study the microbiome in acutely sick patient populations, thus creating and enabling further outcomes-based metagenomic studies on similarly valuable cohorts.