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The various models used in gross anatomical studies to improve the visualization of blood vessels differ in the amount of manual labour, cost, equipment and time involved. This study aimed to compare chemical and enzymatic maceration processes for soft‐tissue removal from arterial silicone casts on skull scaffolds using ringed seal (Pusa hispida) skull specimens. Both processes produced specimens that covered all anatomical aspects required to visualize the intracranial arterial arrangement on a bone scaffold. Overall, the enzyme maceration process was better for production of such specimens, as this process is easy and safe to perform, is less harmful to the bony parts of the specimen, and the resulting specimens are visually more appealing for display and teaching. Compared with previously published models, the end result varied in the amount of dissolved bone tissue and the visual presentation of the model. [ABSTRACT FROM AUTHOR]

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Medical diagnostic laboratories have come under further scrutiny to ensure quality standards of their service and external quality assurance (EQA) programs involving multiple laboratories have been used to gauge this quality based on a consensus. However, because of the geographical distances within a country or internationally, cell surface marker expressions may change due to time delays and transport temperatures. Attention was given to this issue some decades ago and hence requires a re-evaluation in consideration of updated methods, reagents and instruments for flow cytometry and phenotyping. We have undertaken an extensive study to examine the effects of various conditions on blood storage akin to that experienced by patient samples as well as EQA programs, examining expression of lymphocyte surface markers, CD3, CD4, CD8, CD2, CD19, CD20, CD16/56 and HLA-DR. Assessment of lithium-heparin anticoagulated whole blood showed an increase in percentage of CD3 + and CD8 + T cells and a decrease in CD16/56 + NK cells after storage at room temperature (RT) for 24 and/or 48 h. In comparison, storage at 4°C led to a decrease in percentage of CD4 + and increase in percentage of CD8 + cells. The low temperature also caused an increase in percentage of B cells (CD19 + , CD20 + ). While storage at RT did not alter levels of HLA-DR + CD3 + T cells, there was a significant increase in percentage of these cells after 48 h. Changes were also seen at both temperatures when EDTA was used as an anti-coagulant. Assessment of blood treated with a stabiliser, normally used in the EQA samples (Streck Cell Preservative), reduced the range of lymphocyte subsets affected, with only CD2 + and CD20 + cells being significantly different at both temperatures, We conclude that 24-48 h storage/transport can affect the percentage of CD3 + , CD4 + T cells, CD8 + T cells, B cells, NK cells and HLADR + T cells which can be minimised by using the blood stabiliser as per EQA programs and we emphasise the need to adopt this in the processing of patients' blood samples., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Rotavirus is considered worldwide as one of the most important viral gastrointestinal infections, resulting in potentially life-threatening diarrhoea and death in children under the age of 5 years. Rotavirus can survive and remain infectious for long periods outside of the human body and can be easily transmitted via environmental surfaces. Method: Stool specimens that had been collected and stored since 2010/2011 at 2°C – 8°C instead of −20°C or −80°C were analysed to determine the viability of rotavirus in these specimens after 6 years of improper storage. The specimens were analysed using simple enzyme immunoassay (EIA) methods from two different suppliers at different times throughout the period (2012–2017). Results: The analysis showed similar detection results for the two EIA kits. Conclusion: The rotavirus can be detected after several years of incorrect storage with EIA kits.

Climate warming and organic matter decomposition are connected in a recursive manner; this recursion can be described by temperature sensitivity. We conducted a multifactorial laboratory experiment to quantify the temperature sensitivity of organic carbon (C) and nitrogen (N) decomposition processes of common boreal organic soils. We incubated 36 mor and 36 slightly decomposed Carex-Sphagnum peat samples in a constant moisture and ambient temperature for 6 months. The experiment included three temperature and two moisture levels and two food web manipulations (samples with and without fungivore enchytraeid worms). We determined the release of carbon dioxide (CO2) and dissolved organic carbon (DOC) in seven molecular size classes together with ammonium N and dissolved organic N in low molecular weight and high molecular weight fractions. The temperature sensitivity function Q10 was fit to the data. The C and N release rate was almost an order of magnitude higher in mor than in peat. Soil fauna increased the temperature sensitivity of C release. Soil fauna played a key role in N release; when fauna was absent in peat, the N release was ceased. The wide range of the studied C and N compounds and treatments (68 Q10 datasets) allowed us to recognize five different temperature sensitivity patterns. The most common pattern (37 out of 68) was a positive upwards temperature response, which was observed for CO2 and DOC release. A negative downward pattern was observed for extractable organic nitrogen and microbial C. Sixteen temperature sensitivity patterns represented a mixed type, where the Q10function was not applicable, as this does not allow changing the sign storage change rate with increasing or decreasing temperature. The mixed pattern was typically connected to intermediate decomposition products, where input and output fluxes with different temperature sensitivities may simultaneously change the storage. Mixed type was typical for N processes. Our results provide useful parameterization for ecosystem models that describe the feedback loop between climate warming, organic matter decomposition, and productivity of N-limited vegetation. [ABSTRACT FROM AUTHOR]

Objective: Epidemiological studies suggest vitamin D deficiency as a potential risk factor for rheumatoid arthritis (RA) development, a chronic autoimmune disorder highly prevalent in indigenous North American (INA) population. We therefore profiled the circulating levels of 25-hydroxyvitaminD [25(OH)D], an active metabolite of vitamin D, in a cohort of at-risk first-degree relatives (FDR) of INA RA patients, a subset of whom subsequently developed RA (progressors). Methods: 2007 onward, serum samples from INA RA patients and FDR were collected at the time of a structured baseline visit and stored at -20°C. Anti-citrullinated protein antibodies (ACPA), 25(OH)D, hs-CRP, vitamin-D binding protein (VDBP) and parathyroid hormone (PTH) levels were determined using ELISA and rheumatoid factor (RF) seropositivity was determined by nephelometry. Results: We demonstrate that 25 (OH) D concentrations were lower in winter than summer (P = 0.0538), and that serum 25(OH)D levels were higher in samples collected and stored after 2013 (P<0.0001). Analysis of samples obtained after 2013 demonstrated that 37.6% of study participants were 25(OH)D insufficient (<75nmol/L). Also, seropositive RA patients and FDR had lower 25(OH)D levels compared to ACPA-/FDR (P<0.05, P<0.01 respectively). Linear regression analysis showed 25(OH)D insufficiency was inversely associated with presence of RA autoantibodies. Longitudinal samples from 14 progressors demonstrated a consistent increase in 25(OH)D levels at the time they exhibited clinically detectable joint inflammation, without any significant change in VDBP or PTH levels. Spearman rank correlation analysis showed significant association between 25(OH)D and PTH levels, both in RA patients and progressors at RA onset time. Conclusion: We demonstrate that 25(OH)D levels in serum increased at RA onset in progressors. The potential role that vitamin D metabolites and their downstream effects play in RA transition requires further investigation. [ABSTRACT FROM AUTHOR]

Interception of potential invasive species at ports-of-entry is essential for effective biosecurity and biosurveillance programs. However, taxonomic assessment of the immature stages of most arthropods is challenging; characters for identification are often dependent on adult morphology and reproductive structures. This study aims to strengthen the identification of such specimens through DNA barcoding, with a focus on microlepidoptera. A sample of 241 primarily immature microlepidoptera specimens intercepted at U.S. ports-of-entry from 2007 to 2011 were selected for analysis. From this sample, 201 COI-5P sequences were generated and analyzed for concordance between morphology-based and DNA-based identifications. The retrospective analysis of the data over 10 years (2009 to 2019) using the Barcode of Life Data (BOLD) system demonstrates the importance of establishing and growing DNA barcode reference libraries for use in specimen identification. Additionally, analysis of specimen identification using public data (43.3% specimens identified) vs. non-public data (78.6% specimens identified) highlights the need to encourage researchers to make data publicly accessible. DNA barcoding surpassed morphological identification with 42.3% (public) and 66.7% (non-public) of the sampled specimens achieving a species-level identification, compared to 38.3% species-level identification by morphology. Whilst DNA barcoding was not able to identify all specimens in our dataset, its incorporation into border security programs as an adjunct to morphological identification can provide secondary lines of evidence and lower taxonomic resolution in many cases. Furthermore, with increased globalization, database records need to be clearly annotated for suspected specimen origin versus interception location. [ABSTRACT FROM AUTHOR]

Dextranol, a reduced dextran, prevents damage to stored dry protein samples that unmodified dextran would otherwise cause. Desiccation protectants (xeroprotectants) like the polysaccharide dextran are critical for preserving dried protein samples by forming a rigid glass that protects entrapped protein molecules. Stably dried proteins are important for maintaining critical information in clinical samples like blood serum as well as maintaining activity of biologic drug compounds. However, we found that dextran reacts with both dried serum proteins and lyophilized purified proteins during storage, producing high-molecular weight Amadori-product conjugates. These conjugates appeared in a matter of days or weeks when stored at elevated temperatures (37° or 45°C), but also appeared on a timescale of months when stored at room temperature. We synthesized a less reactive dextranol by reducing dextran’s anomeric carbon from an aldehyde to an alcohol. Serum samples dried in a dextranol-based matrix protected the serum proteins from forming high-molecular weight conjugates. The levels of four cancer-related serum biomarkers (prostate specific antigen, neuropilin-1, osteopontin, and matrix-metalloproteinase 7) decreased, as measured by immunoassay, when serum samples were stored for one to two weeks in dextran-based matrix. Switching to a dextranol-based xeroprotection matrix slightly reduced the damage to osteopontin and completely stopped any detectable damage during storage in the other three biomarkers when stored for a period of two weeks at 45°C. We also found that switching from dextran to dextranol in a lyophilization formulation eliminates this unwanted reaction, even at elevated temperatures. Dextranol offers a small and easy modification to dextran that significantly improves the molecule’s function as a xeroprotectant by eliminating the potential for damaging protein-polysaccharide conjugation. [ABSTRACT FROM AUTHOR]

The preservation of biological samples for an extended time period of days to weeks after initial collection is important for the identification, screening, and characterization of bacterial pathogens. Traditionally, preservation relies on cold-chain infrastructure; however, in many situations this is impractical or not possible. Thus, our goal was to develop alternative bacterial sample preservation and transport media that are effective without refrigeration or external instrumentation. The viability, nucleic acid stability, and protein stability of Bacillus anthracis Sterne 34F2, Francisella novicida U112, Staphylococcus aureus ATCC 43300, and Yersinia pestis KIM D27 (pgm-) was assessed for up to 28 days. Xanthan gum (XG) prepared in PBS with L-cysteine maintained more viable F. novicida U112 cells at elevated temperature (40°C) compared to commercial reagents and buffers. Viability was maintained for all four bacteria in XG with 0.9 mM L-cysteine across a temperature range of 22–40°C. Interestingly, increasing the concentration to 9 mM L-cysteine resulted in the rapid death of S. aureus. This could be advantageous when collecting samples in the built environment where there is the potential for Staphylococcus collection and stabilization rather than other organisms of interest. F. novicida and S. aureus DNA were stable for up to 45 days upon storage at 22°C or 40°C, and direct analysis by real-time qPCR, without DNA extraction, was possible in the XG formulations. XG was not compatible with proteomic analysis via LC-MS/MS due to the high amount of residual Xanthomonas campestris proteins present in XG. Our results demonstrate that polysaccharide-based formulations, specifically XG with L-cysteine, maintain bacterial viability and nucleic acid integrity for an array of both Gram-negative and Gram-positive bacteria across ambient and elevated temperatures. [ABSTRACT FROM AUTHOR]

Post-harvest change in the colour of green field pea (Pisum sativum L.) is undesirable as this impacts the visual quality and market value of the seed. To date, there is no standard, objective method to determine bleaching. Therefore, the aim of this study was to develop an objective method for scoring bleaching based on colour reflectance spectra, measured both by spectrophotometer and multispectral Image Analysis (IA). Green field pea seeds were sorted into samples of uniform colour and these were used to train the model. Spectra calculated from multispectral images (with colour bands at 405,470,530,590,660 and 850nm) were matched to the spectrophotometer output through multiple linear regression. All spectra were transformed to emphasize the wavelength regions most impacted during bleaching, following which two critical reflectance values were scaled to a single bleaching score. The bleaching assessment method was tested in a time-course experiment comprising seeds from five green-pea genotypes stored for six months. Each sample was divided into two so that half of the seeds were stored in the dark and the remainder were exposed to controlled light to exaggerate bleaching. Throughout this period, the samples were imaged at six-weekly intervals. Assessment of bleaching by the IA method agreed well with spectrophotometer measurements, achieving a Lin’s concordance statistic of 0.99 and 0.96 for the calibration and time-course samples respectively. The IA method proved more versatile because assessments could be made on individual seeds enabling the computation of bleaching uniformity within each sample. This method captured differences between genotypes in the extent, rate and uniformity of bleaching. All genotypes exhibited susceptibility to bleaching when stored under the controlled light conditions. Excell was observed to be the most susceptible genotype with the greatest bleaching-rate and OZB1308 displayed the most colour-stability. [ABSTRACT FROM AUTHOR]

In recent years, the sweet potato cultivar Jishu 25 has exhibited good characteristics for starch processing in northern China. The storage root dry matter yields of this cultivar can exceed one ton per mu (1/15 of a hectare) at nitrogen (N) rates of 60–90 kg ha-1 based on soil nutrient content. However, the effect of N fertilizer on the physicochemical properties of starches isolated from this cultivar has not been reported. In order to evaluate these effects, three different N rates, 0 (control, N0), 75 (N1), and 150 kg ha-1 (N2), were selected for a field experiment in 2017. The results showed that N1 exhibited the highest storage root yield and starch yield. Compared to the control group, N fertilizer significantly increased the total starch content while no significant difference was found in these between the N1 and N2 groups. The amylose (AM) content was highest in the N2 group and lowest in the N0 group. In addition, N fertilizer exhibited no significant effects on the values of [D(v, 0.9)], D [4, 3] and D [3, 2]. Compared to the control group, N1 demonstrated significantly higher setback viscosity (SV), while N2 showed significantly higher peak viscosity (PV), cold paste viscosity (CPV) and SV. However, there were no significant differences in the hot paste viscosity (HPV), peak time and pasting temperature between the N1 and N2 groups. For the thermal properties of starch, there were no significant differences in peak temperature (Tp), conclusion temperature (Tc) or gelatinization enthalpy (ΔH) between the N1 and N2 groups. Overall, for the starch samples of cultivar Jishu 25, N fertilizer exerts significant effects on the starch content, AM content and viscosity properties but little effect on the particle size distribution and ΔH. 75 kg N ha-1 can easily lead to substantial planting benefits from the high storage root yield, dry matter yield and total starch content of this cultivar. [ABSTRACT FROM AUTHOR]

The measurement of hair cortisol is increasingly used to understand the effect of natural and anthropogenic stressors on wild animals, but it is potentially confounded by individual, seasonal and sex-dependant variations in baseline cortisol secretion. This study validated an enzyme-linked immunoassay for hair cortisol measurement and characterized its baseline variation in a wild population of Egyptian mongoose. The analysis encompassed individuals of both sexes and all ages, across a range of geographic, environmental and seasonal conditions that the species experiences in Portugal allowing us to account for spatial, temporal and biological factors that contribute to hair cortisol variation. Our results showed that age, sex and storage time had an effect on hair cortisol, but season did not. Hair cortisol was higher in early stage juveniles compared to other age cohorts, in males when compared to females, and decreased with longer storage time. By identifying the factors that influence baseline hair cortisol in this wild population, we establish the basis for its application as an indicator of the effect of natural and anthropogenic stressors. [ABSTRACT FROM AUTHOR]

Early infant diagnosis (EID) of HIV provides an opportunity for early HIV detection and access to appropriate Antiretroviral treatment (ART). Dried Blood Spot (DBS) samples are used for EID of exposed infants, born to HIV-positive mothers. However, DBS rejection rates in Zimbabwe have been exceeding the target of less than 2% per month set by the National Microbiology Reference Laboratory (NMRL), in Harare. The aim of this study was to determine the DBS sample rejection rate, the reasons for rejection and the possible associations between rejection and level of health facility where the samples were collected. This is an analytical cross-sectional study using routine DBS sample data from the NMRL in Harare, Zimbabwe, between January and December 2017.A total of 34 950 DBS samples were received at the NMRL. Of these, 1291(4%) were rejected. Reasons for rejection were insufficient specimen volume (72%), missing request form (11%), missing sample (6%), cross-contamination (6%), mismatch of information (4%) and clotted sample (1%). Samples collected from clinics/rural health facilities were five times more likely to be rejected compared to those from a central hospital. Rejection rates were above the set target of <2%. The reasons for rejection were ‘pre-analytical’ errors including labelling errors, missing or inconsistent data, and insufficient blood collected. Samples collected at primary healthcare facilities had higher rejection rates. [ABSTRACT FROM AUTHOR]

The purpose of this study was to examine the effect of storage temperature, duration, and storage vessel seal on 24 h urinary hydration markers. Twenty-one males (n = 8) and females (n = 13) (mean±SD; age, 24±5 y; body mass, 68.9±24.2 kg; height, 160.2±32.1 cm) without a history of renal disease or currently taking any medications or supplements known to affect the accuracy of urinary hydration markers were enrolled in this study. Participants provided a 24 h urine sample in a clean container with each urine sample being separate into four separate containers, two in each of the following temperatures: 7°C and 22°C. One specimen container at each temperature was either sealed using the manufacturers cap (single sealed) or the manufacturers cap plus laboratory wrapping film (double sealed). Each sample was analyzed after 1, 2, 3, 7 and 10 days. Urine samples were assessed for urine osmolality (UOSMO), urine specific gravity (USG) and urine color (UCOL). UOSMO was stable at 7°C for two days (mean difference [95% CI]; +1 mmol·kg-1 [0+3], p>0.05) and three days (+1 mmol·kg-1 [0, +3], p>0.05) for single sealed and double sealed containers, respectively. USG measures were stable for singled sealed and double sealed for up to ten days when stored at 22°C. UCOL measures were maintained for up to three days in all storage methods (p>0.05). In conclusion, if immediate analysis is unavailable, such as in the case of field based or longitudinal research, it is recommended that 24 h urine samples are stored in a refrigerated environment and hydration markers (UOSMO and UCOL) be assessed within 48 h. [ABSTRACT FROM AUTHOR]

Although the literature on the Maillard reaction in infant formulas is extensive, most studies have focused on model systems, and in only a few cases on real food systems. Therefore, the objective of the present study was to determine the status of the Maillard reaction, both the early and advanced phases, in a variety of commercial infant formulas available on the Swedish market. Ten powder and liquid milk-based infant formulas from three manufacturers were selected to determine available lysine and CML contents, the two established indicators of the reaction. The products were also characterized with respect to protein content, carbohydrates composition, water content and water activity. In order to be able to compare the impact of different processing steps applied on powder and liquid formulas, the solid formulas contained similar ingredients as their corresponding liquid ones. Our findings showed that powder and liquid formulas contained similar available lysine concentrations regardless of the manufacturer, showing 27.14–36.57% decrease in the available lysine, compared to the reference skim milk powder in this study. The CML concentrations were in a broad range of 68.77–507.99 mg / kg protein. In the case of one manufacturer, liquid infant formulas had significantly higher CML content, compared to the powder products (p < 0.05). The results from this study are a step taken towards better understanding of the extent of the Maillard reaction in real complex systems of infant formulas. [ABSTRACT FROM AUTHOR]

Background: Viral load testing is key to monitoring response to anti-retroviral therapy (ART). However, in lower and middle income countries with large epidemics, pre-analytical challenges threaten the quality of testing. It is unknown how much delayed processing and adverse storage affects the validity of results. The aim of this study was to determine the impact of delayed testing and warmer storage conditions on HIV RNA stability in diagnostic samples. Methods: 1194 samples, collected in EDTA or plasma preparation (PPT) tubes, were studied. Immediately after initial testing, primary tubes were stored for 72, 96 or 168 hours at 4°C, 20°C or 30°C. The viral load was then repeated and the 2 results were compared. Results: Viral loads were very stable, with <0.5 log copies/ml median difference noted between paired tests for all storage times and temperatures. The viral load in samples stored for up to a week reliably differentiated between ART-suppressed and failing patients in 98.83% of instances. However, re-centrifugation immediately prior to repeat testing was essential to avoid falsely elevated readings, probably due to contamination of plasma with cell-associated viral nucleic acids. Approximately 20% of samples with initially undetectable viral loads were weakly positive (<100 copies/mL) on repeat. This was not exacerbated by duration or temperature of storage. Conclusion: Viral RNA in diagnostic samples is stable well beyond currently recommended limits. However, when testing stored primary samples, contamination of plasma with cellular material easily occurs. Low viral loads (<100copies/mL) in samples stored in this way should be interpreted with caution. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small RNA molecules that modulate gene and protein expression in hematopoiesis. Platelets are known to contain a fully functional miRNA machinery. While platelets used for transfusion are normally stored at room temperature, recent evidence suggests more favorable effects under a cold-storage condition, including higher adhesion and aggregation properties. Thus, we sought to determine whether functional differences in platelets are associated with the differential profiling of platelet miRNA expressions. To obtain the miRNA expression profile, next-generation sequencing was performed on human platelets obtained from 10 healthy subjects. The miRNAs were quantified after being stored in three different conditions: 1) baseline (before storage), 2) stored at 22°C with agitation for 72 h, and 3) stored at 4°C for 72 h. Following the identification of miRNAs by sequencing, the results were validated at the level of mature miRNAs from 18 healthy subjects, by using quantitative polymerase chain reaction (qPCR). Differential expression was observed for 125 miRNAs that were stored at 4°C and 9 miRNAs stored at 22°C as compared to the baseline. The validation study by qPCR confirmed that storage at 4°C increased the expression levels (fold change 95% CI) of mir-20a-5p (1.87, p<0.0001), mir-10a-3p (1.88, p<0.0001), mir-16-2-3p (1.54, p<0.01), and mir-223-5p (1.38, p<0.05), compared with those of the samples stored at 22°C. These results show that miRNAs correlate with platelet quality under specific storage conditions. The data indicate that miRNAs could be potentially used as biomarkers of platelet quality. [ABSTRACT FROM AUTHOR]

In addition to conventional cytology, liquid-based cytology (LBC) is also used for immunocytochemistry and gene analysis. However, an appropriate method to obtain high quality DNA for next-generation sequencing (NGS) using LBC specimens remains controversial. We determined the optimal conditions for fixation with an alcohol-based fixative for LBC and DNA extraction using cultured cancer cell lines and clinical specimens. The extracted DNA was processed for NGS after the DNA quality was confirmed based on the DNA concentration and degree of degradation. The optimal conditions for cultured cells to obtain high quality DNA were to fix the cells at a density of 6 × 103 or 2 × 104 cells/mL and to use the magnetic bead-based DNA extraction method. Even after storing the fixed cells for 90 days, DNA extracted using the above and other extraction kits, including membrane-based methods, did not undergo degradation. Furthermore, 5-year-old residual LBC samples demonstrated high DNA quality that was suitable for NGS. Furthermore, a cancer genome panel analysis was successfully performed with DNA extracted from cultured cells fixed at 6 × 103 cells/mL for 90 days, and with DNA from residual LBC samples even after 1 year of storage. Residual LBC samples may be a useful source of DNA for clinical NGS to promote genome-based cancer medicine. [ABSTRACT FROM AUTHOR]

Ground beef makes up more than half of the beef consumed in the U.S. market. Although numerous studies have been conducted on microbial safety and shelf life of ground beef limited work has been done using a culture-independent approach. While past studies have allowed for the evaluation of a few organisms of interest, there is limited work on the microbial community associated with fresh ground beef. In order to have a more complete picture of the microbial ecology of the product, a culture-independent approach utilizing 16S rRNA gene amplicon sequencing was used. The objectives of this study were to characterize the fresh ground beef microbiome and the effect that antimicrobial interventions and antioxidants, applied to beef trim before grinding, and product storage have on community composition using 16S rRNA gene amplicon sequencing. Beef trimmings were treated with antimicrobials and an antioxidant. Samples were ground, loafed, and overwrapped before being packaged in modified-atmosphere packaging. Samples were in dark storage for 21 days followed by five days in retail display. Periodically during storage, samples were collected for microbiological analysis and DNA isolation. Due to low microbial biomass, only 52 of 210 samples were included in the final analysis. These samples represented two antimicrobial treatments (peroxyacetic acid, and a sulfuric acid and sodium sulfate blend) and a control, from day-15 of dark storage and day-5 of retail display. As sample age increased, so did the number of raw reads (P < 0.001) and aerobic plate counts (P < 0.001), which were correlated (r = 0.94, P = 0.017). Across all samples, lactic acid bacteria were most abundant followed by Enterobacteriaceae; several rare taxa were also identified (namely Geobacillus, Thermus, and Sporosarcina). Antimicrobial treatment altered the bacterial alpha (P < 0.001) and beta (P = 0.001) diversity, while storage day altered alpha (P = 0.001) diversity. Enterobacteriaceae relative abundance differed (P < 0.05) among treatments and was highest in control samples. In addition to confirming previously described dominant microbial differences in culture-dependent results, these data identified genera not typically associated with ground beef and allowed for study of shifts in the entire microbiome and not just a subset of indicator organisms. [ABSTRACT FROM AUTHOR]

Background: Whole-genome sequencing (WGS) of heart transplant recipient- and donor-derived cardiac biopsies may facilitate organ matching, graft failure prediction, and immunotolerance research. The objective of this study was to determine the feasibility of WGS based on formalin-fixed paraffin-embedded endomyocardial biopsies. Methods and results: The study included serial donor- and recipient samples from patients who had undergone heart transplantation at Skane University Hospital, Lund, Sweden, between 1988 and 2009. DNA extraction and WGS were conducted. Additional WGS sequencing quality metrics and coverage were obtained with the Genome Analysis Toolkit (GATK). 455 endomyocardial samples from 37 heart transplant recipients were acquired from routine rejection monitoring and stored as formalin-fixed paraffin-embedded samples. They were analyzed after 3–26 years of storage. DNA was extracted from 114 samples and WGS was run on 85 samples. DNA extraction yielded 313 ng (IQR 96–601) for all samples. A coverage of 11.3x (IQR 9.0–15.9) was recorded for all WGS samples. Three samples stored for > 25 years yielded a coverage of > 25x. Data were generated for 1.7 billion reads per sample (IQR 1.4–2.7). A Transition/Transversion (TiTv) ratio of 2.09 ± 0.05 was calculated for all WGS samples. No associations were found among storage time, DNA yield, or sequencing quality metrics. Conclusions: The present study demonstrated the feasibility of whole-genome sequencing based on endomyocardial biopsies. This process could enable large-scale retrospective genomic studies using stored histopathological samples. [ABSTRACT FROM AUTHOR]

Fresh produce outbreaks due to Shiga toxin-producing Escherichia coli (STEC) continue to occur in the United States (US). Manure-amended soils can pose a public health risk when used for growing raw agricultural commodities. Knowing the prevalence and concentration of STEC in untreated biological soil amendments of animal origin (BSAAO) is important to help guide the most appropriate pre-harvest interval(s) following application to limit risks from these soil amendments. Bovine manure samples were collected from 12 farms in Florida, including samples from piles, lagoons, barns, and screened solids. Two methods were used to detect stx1/2 and rfbE genes in samples. A prevalence rate of 9% for stx1 and/or stx2 and 19% for rfbE was observed from the 518 bovine manure samples evaluated. A most probable number (MPN) assay was performed on stx+ samples when applicable. The geometric mean for stx+ samples (n = 20) was 3.37 MPN g-1 (0.53 log MPN g-1) with a maximum value of 6,800 MPN g-1 (3.83 log MPN g-1). This research was part of a larger nationwide geographical study on the prevalence and concentration of STEC in bovine manure to help guide regulations on feasible pre-harvest intervals for the application of untreated BSAAO. [ABSTRACT FROM AUTHOR]

An evaluation of the geochemical characteristics of 102 storage jar sherds by k0-neutron activation analysis (k0-NAA) from archaeological contexts in Cambodia and reference samples from stoneware production centres in Thailand provides a new perspective on regional and global trade in mainland Southeast Asia. Identification of seven geochemical groups enables distinctions between production centres, and articulation of their role in trade between northern and central Thailand, South China and Cambodia. Storage jars from Thailand and South China are known in archaeological contexts worldwide because of their durability and intrinsic functional and cultural values. Evidenced by a novel application of k0-NAA, analogous stoneware sherds at Longvek connect the Cambodian capital to a global trading network. Additional proof of ceramics from an undocumented Cambodian kiln demonstrates the gradual and complex transition between the Angkorian past and the Early Modern period. [ABSTRACT FROM AUTHOR]

Formalin-fixed paraffin-embedded (FFPE) tissues are among the most widely available clinical specimens. Their potential utility as a source of RNA for transcriptome studies would greatly enhance population-based cancer studies. Although preliminary studies suggest FFPE tissue may be used for RNA sequencing, the effect of storage time on these specimens needs to be determined. We conducted this study to determine whether RNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries was present in sufficient quantity and quality for RNA-Seq analysis. FFPE tissues, stored from 7 to 32 years, were obtained from three SEER sites. RNA was extracted, quantified, quality assessed, and subjected to RNA-Seq (a whole transcriptome sequencing technology). FFPE specimens stored for longer periods of time had poorer RNA sample quality as indicated by negative correlations between specimen storage time and fragment distribution values (DV). In addition, sample contamination was a common issue among the RNA, with 41 of 67 samples having 5% to 48% bacterial contamination. However, regardless of specimen storage time and bacterial contamination, 60% of the samples yielded data that enabled gene expression quantification, identifying more than 10,000 genes, with the correlations among most biological replicates above 0.7. This study demonstrates that FFPE high-grade ovarian serous adenocarcinomas specimens stored in repositories for up to 32 years and under varying storage conditions are a promising source of RNA for RNA-Seq. We also describe certain caveats to be considered when designing RNA-Seq studies using archived FFPE tissues. [ABSTRACT FROM AUTHOR]

Background: The presence of ≥3 oligoclonal bands (OCB) in the cerebrospinal fluid (CSF) without corresponding bands in serum represents a definite pathological pattern, whereas the clinical significance of 1–2 CSF bands (borderline pattern) is poorly investigated. Methods: We screened 1986 consecutive CSF and serum samples which were collected over a four-year time period and had results of isoelectric focusing (IEF) available. Of patients with borderline OCB we reviewed individual medical charts for assessment of clinical diagnoses. Where feasible, IEF was replicated and results of follow-up samples were obtained. IEF was performed using polyacrylamide gel followed by immunoblotting and IgG-specific antibody staining. Additionally, we performed a systematic literature review of the diagnostic specificity of OCB using different cut-offs for CSF-restricted bands. Results: Out of 253 patients with borderline OCB, 21.7% had an inflammatory neurological disease (IND) of the central nervous system, comprising 4% multiple sclerosis patients, and 14.2% had a peripheral IND, whereas the remaining 64.1% of patients showed non-inflammatory diseases. Frequency of one or two CSF bands without corresponding serum bands did not differ between the disease groups. In a subgroup of 100 patients IEF was repeated. Of those, 73% were OCB negative, while no sample was positive. In 26 patients IEF results were available of a follow-up sample collected after a median of 27 months. Of those, 4 (15.4%) turned positive. Systematic literature review revealed a diagnostic specificity of OCB of 97% and 92% using a cut-off ≥3 and ≥2 CSF bands in patients with mainly non-inflammatory neurological diseases. Conclusion: The clinical significance of one or two CSF-restricted bands is moderate and, hence, indicates a possible but not reliable proof of intrathecal B-cell activity. Sample re-testing, introduction of an additional diagnostic category, e.g. “possible intrathecal IgG synthesis”, and follow-up lumbar puncture might be possible options to address this scenario. [ABSTRACT FROM AUTHOR]

Background: The standard treatment of acute ischemic stroke patients is thrombolytic therapy within 60 minutes of a patient’s arrival in stroke center hospitals. Based on the policy of the Lampang Referral System Committee, blood samples of suspected stroke patients need to be collected before transfer to the stroke center (Lampang Hospital). It was still questionable as to whether these blood samples are valid for clinical use and the present study aimed to confirm or deny their validity. Methods: A diagnostic study was conducted from June 2015 to May 2016. After exclusion, 340 patients were deemed eligible for analysis. Blood samples were collected just before normal saline infusion at referring hospitals and stored in blood collecting tube boxes set during transportation. At the stroke center, informed consents was requested, blood samples were re-collected to serve as a ‘gold standard’. Prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (aPTT), platelet count, hemoglobin (Hb), hematocrit (Hct), blood urea nitrogen (BUN), and creatinine (Cr) were compared using paired t-tests. Binary regression was used to analyze for accuracy (%) to adjust for extraneous influences and was presented by modified Bland-Altman plots. Results: The laboratory results of referring hospitals vs. the stroke center were: PT, 12.4±3.2 vs. 12.5±3.0 sec; INR: 1.0±0.3 vs. 1.0±0.3; and platelet count: 239.8±77.1 vs. 239.8±74.8 (x103/μL). The adjusted accuracy of the PT, INR, and platelet counts were 96.8%, 96.8%, and 95.3% respectively. Conclusion: Laboratory tests from referring hospital were determined to be valid. Blood samples should thus be collected at referring hospitals in order to avoid unnecessary blood collection at the stroke center. [ABSTRACT FROM AUTHOR]

Human sample, from patients or healthy donors, are a valuable link between basic research and clinic. Especially in translational research, they play an essential role in understanding development and progression of diseases as well as in developing new diagnostic and therapeutic tools. Stored in biobanks, fast access to appropriate material becomes possible. However, biobanking in a clinical context faces several challenges. In practice, collecting samples during clinical routine does not allow to strictly adhere to protocols of sample collection in all aspects. This may influence sample quality to variable degrees. Time from sample draw to asservation is a variable factor, and influences of prolonged storage at ambient temperature of tissues are not well understood. We investigated whether delays between 5 minutes and 3 hours, and the use of RNAlater RNA-preserving reagent would lead to a relevant drop in sample quality, measured by quantitative mRNA expression analysis. Our findings suggest that even under ambient conditions, delays up to 3 hours do not have a major impact on sample quality as long as the tissue remains intact. [ABSTRACT FROM AUTHOR]

Background: Schistosoma mansoni is the main species causing hepatic and intestinal schistosomiasis in Sub-Saharan Africa, and it is the only species in South America. Adult stages of the parasite reside in the mesenteric venous plexus of infected hosts, and eggs are shed in feces. Collecting patient stool samples for S. mansoni diagnostic purposes is difficult in large-scale field trials. Urine samples would be an alternative approach for molecular S. mansoni detection since they have several advantages over stool samples, including better handling, management and storage. Additionally, loop-mediated isothermal amplification (LAMP) technology is a powerful molecular diagnostic tool for infectious diseases, particularly under field conditions in developing countries. The present study aimed to assess the effectiveness of our previously developed LAMP assay (SmMIT-LAMP) for S. mansoni-specific detection in clinical urine samples. Methodology/Principal findings: The sensitivity of SmMIT-LAMP in urine was established in simulated fresh human urine samples artificially spiked with genomic DNA from S. mansoni. LAMP for 120 min instead of 60 min improved the sensitivity, reaching values of 0.01 fg/μL. A set of well-defined frozen stored human urine samples collected from Sub-Saharan immigrant patients was selected from a biobank to evaluate the diagnostic validity of SmMIT-LAMP. The set included urine samples from patients with microscopy-confirmed infections with S. mansoni, S. haematobium and other nonschistosome parasites, as well as urine samples from patients with microscopy-negative eosinophilia without a confirmed diagnosis. The SmMIT-LAMP was incubated for 60 and 120 min. A longer incubation time was shown to increase the LAMP-positive results in patient urine samples. We also tested urine samples from mice experimentally infected with S. mansoni, and LAMP-positive results were obtained from the third week after infection. A real-time LAMP assay was also performed with three individual urine samples. Conclusions/Significance: The SmMIT-LAMP could effectively detect S. mansoni DNA in mouse urine samples and produced promising results for human clinical samples. The detection of S. mansoni DNA in mouse urine samples from the third week after infection indicates that early diagnosis of active S. mansoni infection is possible using urine as a source of DNA. Further studies are still needed, but our method could be used as a promising molecular tool applicable to urine samples to diagnose human intestinal schistosomiasis caused by S. mansoni. [ABSTRACT FROM AUTHOR]

Breast milk is considered the gold standard nutritional resource for very low birth weight (VLBW) infants in terms of nutrients and protective factors. If mother's milk is not available, the second choice is donated and fortified human milk (HM) from the Human Milk Bank (HMB). This study hypothesized that HM could be lyophilized and used as an additive to increase the levels of macronutrients and micronutrients available to VLBW infants. This study aimed to constitute a lyophilized HM concentrate and determine the osmolality and the concentration of macronutrients and micronutrients in HM samples at “baseline” and in “HM concentrates”, analyzed immediately (HMCI), and after 3 (HMC3m) and 6 (HMC6m) months of freezing. Osmolality was verified using the freezing point osmometric method. Macronutrient quantification was performed using the MIRIS Human Milk Analyzer. Micronutrients were determined by Flame Atomic Absorption Spectrophotometry and by the automated colorimetric method. Bayesian linear mixed effect models were adjusted using OpenBUGS to estimate mean differences and 95% credibility intervals (CrI) of osmolality and of macro- and micronutrients between the types of HM samples. A comparison of dosage values showed a significant increase between HM baseline and HMCI, HMC3m, and HMC6m. Comparing HM baseline and HMCI highlighted the increase in energy content and the concentration of carbohydrates and total lipids. The Ca and P contents increased and the levels of energy, total lipids, and Cu were reduced in HMC3m compared to HMCI. Ca, Mg, K, Zn, and P increased and the levels of energy, total lipids, and Cu were reduced in HMC6m, compared to HMCI. The present study confirms the possibility of formulation and utilization of the immediate concentrate. Partial stability of HM concentrates generated from freeze-drying of donated milk do not recommend storage. [ABSTRACT FROM AUTHOR]

The study of host associations of mosquitoes (Diptera, Culicidae) provides valuable information to assist in our understanding of a variety of related issues, from their life-history to the entomological surveillance of pathogens. In this study, we identified and characterized mosquito blood meals from both urban and forested areas in the city of Paranaguá, state of Paraná, Brazil, by analyzing the amplification of host DNA ingested by mosquitoes under different storage conditions and digestion levels. Host DNA preservation was evaluated in fresh blood meals according to storage duration (30 to 180 days) and temperature (-20°C / -80°C) and, in digested blood, according the degree of digestion classified on the Sella scale. Molecular analysis of blood meals was based on DNA extraction and amplification of a fragment of the mitochondrial COI gene. We determined that, up to180 days of storage, the evaluated temperatures did not influence the preservation of fresh blood meals DNA, whereas the amplification success was increasingly reduced over the course of the digestion process. The species Anopheles cruzii, Aedes fluviatilis, Aedes scapularis, Psorophora ferox, Culex quinquefasciatus, Culex mollis, and Culex intrincatus, together with specimens representing four subgenera and one genus of Culicidae [Ae. (Ochlerotatus), Cx. (Culex), Cx. (Melanoconion), Cx. (Microculex), and Limatus, respectively] had their blood meals identified. Their diverse host use was evidenced by the identification of 19 species of vertebrate host, namely two amphibians, three mammals and 14 birds. Birds were the most commonly identified host in blood meals. These results not only show the diversity of mosquito hosts, but also underscore the challenges involved in monitoring arboviruses of public health importance, given potential combinations of host use for each mosquito species. [ABSTRACT FROM AUTHOR]

Despite the critical need for non-invasive tools to improve monitoring of wildlife populations, especially for endangered and elusive species, faecal genetic sampling has not been adopted as regular practice, largely because of the associated technical challenges and cost. Substantial work needs to be undertaken to refine sample collection and preparation methods in order to improve sample set quality and provide cost-efficient tools that can effectively support wildlife management. In this study, we collected an extensive set of forest elephant (Loxodonta cyclotis) faecal samples throughout Gabon, Central Africa, and prepared them for genotyping using 107 single-nucleotide polymorphism assays. We developed a new quantitative polymerase chain reaction (PCR) assay targeting a 130-bp nuclear DNA fragment and demonstrated its suitability for degraded samples in all three elephant species. Using this assay to compare the efficacy of two sampling methods for faecal DNA recovery, we found that sampling the whole surface of a dung pile with a swab stored in a small tube of lysis buffer was a convenient method producing high extraction success and DNA yield. We modelled the influence of faecal quality and storage time on DNA concentration in order to provide recommendations for optimized collection and storage. The maximum storage time to ensure 75% success was two months for samples collected within 24 hours after defecation and extended to four months for samples collected within one hour. Lastly, the real-time quantitative PCR assay allowed us to predict genotyping success and pre-screen DNA samples, thus further increasing the cost-efficiency of our approach. We recommend combining the validation of an efficient sampling method, the build of in-country DNA extraction capacity for reduced storage time and the development of species-specific quantitative PCR assays in order to increase the cost-efficiency of routine non-invasive DNA analyses and expand the use of next-generation markers to non-invasive samples. [ABSTRACT FROM AUTHOR]

Clinical flow cytometry is a reliable methodology for whole blood cell phenotyping for different applications. The BD FACSLyric™ system comprises a flow cytometer available in different optical configurations, BD FACSuite™ Clinical software, and optional BD FACS™ Universal Loader. BD FACSuite Clinical software used with BD™ FC Beads and BD CS&T Beads enable universal setup for performance QC, instrument control, data acquisition/storage, online/offline data analysis, and instrument standardization. BD Biosciences sponsored the clinical evaluation of the BD FACSLyric 10-color configuration at seven clinical sites using delinked and de-identified blood specimens from HIV-infected and uninfected subjects to enumerate T-, B-, and NK-lymphocytes with the BD Multitest™ reagents (BD Multitest IMK kit and BD Multitest 6-color TBNK). Samples were analyzed on the BD FACSLyric system with BD FACSuite Clinical software, and on the BD FACSCanto™ II system with BD FACSCanto clinical software and BD FACS 7-Color Setup beads. For equivalency between methods, data (n = 362) were analyzed with Deming regression for absolute count and percentage of lymphocytes. Results gave R2 ≥0.98, with slope values ≥0.96, and slope ranges between 0.90–1.05. The percent (%) bias values were <10% for T- and NK cells and <15% for B- cells. The between-site (n = 4) total precision was tested for 5 days (2 runs/day), and gave %coefficient of variation below 10% for absolute cell counts. The stability claims were confirmed (n = 186) for the two BD Multitest reagents. The reference intervals were re-established in male and female adults (n = 134). The analysis by gender showed statistically significant differences for CD3+ and CD4+ T-cell counts and %CD4. In summary, the BD FACSLyric and the BD FACSCanto II systems generated comparable measurements of T-, B-, and NK-cells using BD Multitest assays. [ABSTRACT FROM AUTHOR]

For polymer-particle composites, limited thermodynamic compatibility of polymers and particles often leads to poor dispersal and agglomeration of the particles in the matrix, which negatively impacts the mechanics of composites. To study the impact of particle compatibility in polymer matrices on the mechanical properties of composites, we here study composite silica- protein based hydrogels. The polymer used is a previously studied telechelic protein-based polymer with end groups that form triple helices, and the particles are silica nanoparticles that only weakly associate with the polymer matrix. At 1mM protein polymer, up to 7% of silica nanoparticles can be embedded in the hydrogel. At higher concentrations the system phase separates. Oscillatory rheology shows that at high frequencies the particles strengthen the gels by acting as short-lived multivalent cross-links, while at low frequencies, the particles reduce the gel strength, presumably by sequestering part of the protein polymers in such a way that they can no longer contribute to the network strength. As is generally observed for polymer/particle composites, shear-induced polymer desorption from the particles leads to a viscous dissipation that strongly increases with increasing particle concentration. While linear rheological properties as function of particle concentration provide no signals for an approaching phase separation, this is very different for the non-linear rheology, especially fracture. Strain-at-break decreases rapidly with increasing particle concentration and vanishes as the phase boundary is approached, suggesting that the interfaces between regions of high and low particle densities in composites close to phase separation provide easy fracture planes. [ABSTRACT FROM AUTHOR]

Background: Several sensitive methods are available for red blood cell (RBC) antibody screening. Among these, gel and glass card systems have demonstrated comparably good performance in retrospective studies and are widely used in routine patient diagnostics, but their performance in prospective studies has not been sufficiently characterised. Patients and methods: Gel card (Bio-Rad DiaMed) and glass bead-based (Ortho Clinical Diagnostics) column agglutination technologies were used to screen for antibodies prospectively (group A) and for antibody identification in stored and fresh samples known to contain RBC antibodies retrospectively (group B). Untreated reagent RBCs and either papain-treated (Bio-Rad) or ficin-treated panel C cells (Ortho) were used for antibody identification. Results: RBC-reactive antibodies were detected in 22 of 1000 group A samples, three of which tested positive only by gel card agglutination, and four only by glass bead agglutination (including one false positive each). Group B comprised 202 sera with known antibodies: 33 of these samples contained 36 antibodies detected only by gel card agglutination, whereas 9 samples contained antibodies detectable only by glass bead-based agglutination. Discrepancies mostly involved weak antibodies reactive by enzyme only. Two sera contained antibody mixtures that neither system detected completely. Of note, in antibody differentiation batches one and two, anti-Lua was reactive in 7 of 7 and 1 of 8 samples, respectively. Conclusion: Both column agglutination tests for red cell antibodies had equal sensitivity and specificity with unstored samples. In stored samples, weak and enzyme-only antibodies were more frequently detected with the gel card system. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the potential for waste extracts from the pequi (Caryocar brasiliense) and juçara (Euterpe edulis) to reduce oxidatiove processes in antibiotic-free broiler meat. The use of natural antioxidants extracted from fruit-processing wastes has been neglected. Although these residues contain high amounts of these bioactive compounds, they are often discarded by industry. Meat samples were exposed previously submitted to UV-C radiation at 1.161 mW / cm2 for 10 minutes to accelerate the rancidity process. Pequi and juçara waste extracts were obtained by microwave-assisted extraction (MAE). A total of four conditions were tested using antibiotic-free broiler thighs and drumstick meat: BN–with no antioxidant (negative control), BP–with BHT (Butylated hydroxytoluene) (positive control), BE–with juçara extract, BC–with pequi extract. The color, pH, lipid and protein oxidation (days 0, 2, 4, 6, 8 and 10), antioxidant contents and activity (days 0 and 10), and proximal composition and fatty acid profile (day 0) were tested, followed by principal component analysis (PCA). Pequi waste extract presented the highest antioxidant content and activity. BE and BC treatments presented the highest total phenolic (TPC) and flavonoid (TFC) content, and BE presented the highest total monomeric anthocyanin content (TAC). TFC increased during storage in all treatments. The waste extracts of C. brasiliense presented the highest antioxidant activity against lipid oxidation in the antibiotic-free broiler meat. Moreover, both extracts presented high antioxidant activity against protein oxidation. Although the pequi peel extract had a better effect in terms of suppressing both types of oxidation, either this extract or the jussara waste extract could be used as a technological strategy to reduce the oxidative processes in antibiotic-free broiler meat for the poultry industry. Thus, waste extracts can be a potential technology to reduce the oxidative processes in antibiotic-free broiler meat. [ABSTRACT FROM AUTHOR]

The ability to form endospores allows certain Gram-positive bacteria (e.g. Bacillus subtilis) to challenge the limits of microbial resistance and survival. Thus, B. subtilis is able to tolerate many environmental extremes by transitioning into a dormant state as spores, allowing survival under otherwise unfavorable conditions. Despite thorough study of spore resistance to external stresses, precisely how long B. subtilis spores can lie dormant while remaining viable, a period that potentially far exceeds the human lifespan; is not known although convincing examples of long term spore survival have been recorded. In this study, we report the first data from a 500-year microbial experiment, which started in 2014 and will finish in 2514. A set of vials containing a defined concentration of desiccated B. subtilis spores is opened and tested for viability every two years for the first 24 years and then every 25 years until experiment completion. Desiccated baseline spore samples were also exposed to environmental stresses, including X-rays, 254 nm UV-C, 10% H2O2, dry heat (120°C) and wet heat (100°C) to investigate how desiccated spores respond to harsh environmental conditions after long periods of storage. Data from the first 2 years of storage show no significant decrease in spore viability. Additionally, spores of B. subtilis were subjected to various short-term storage experiments, revealing that space-like vacuum and high NaCl concentration negatively affected spore viability. [ABSTRACT FROM AUTHOR]

Mineral-associated organic matter is associated with a suite of soil minerals that can confer stability, resulting in the potential for long-term storage of carbon (C). Not all interactions impart the same level of protection, however; evidence is suggesting that C in certain mineral pools is dynamic and vulnerable to disturbance in the decades following harvesting. The objective of this research was to describe and characterize organic matter-mineral interactions through depth in horizons of soils of contrasting stand age. Sequential selective dissolutions representing increasingly stable mineral-associated organic matter pools from water soluble minerals (deionized water), organo-metal complexes (Na-pyrophosphate), poorly-crystalline minerals (HCl hydroxylamine), and crystalline secondary minerals (Na-dithionite HCl)) were carried out for Ae, Bf and BC horizons sampled from a Young and Mature forest site (35 and 110 years post-harvest) in Mooseland, Nova Scotia, Canada. Sequential selective dissolution extracts were analyzed for C, δ13C, iron (Fe) and aluminum (Al). Organo-metal complexes (OMC) were the largest mineral-associated OM pool in all horizons. This pool dominated the C distribution in B horizons (~60–70% of Bf bulk C), with a minor contribution from poorly-crystalline (PCrys), crystalline (Crys) minerals and water soluble (WS) associations. C in OMC and PCrys pools explained the variation in bulk C in horizons through depth at both sites. Twice as much C in OMC pools was measured at the Mature site compared to the Young site in the Bf horizons, supported by higher C:(Fe+Al) ratios. Isotopic analysis indicated that this extraction procedure isolated distinct mineral-associated OM pools. δ13C signatures of pyrophosphate-extracted OMC pools ranged from -27‰ to -28‰, similar to δ13C of bulk C and to plant-derived humic acids and associated biomass. The water soluble phase (mean δ13C = -29 ‰) was up to 2 ‰ more depleted, whereas the δ13C of Crys pools were more enriched in 13C (-13‰ to -16 ‰) compared to bulk soil. The results from this study suggest that association with minerals does not necessarily confer stability: organo-metal pools dominate in podzol horizons through depth, and contribute most to C storage, but are potentially susceptible to destabilization following the physical changes resulting from forest harvesting disturbance. [ABSTRACT FROM AUTHOR]

Background: Glucose-6-Phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy worldwide, no reliable bedside diagnostic tests to quantify G6PD activity exist. This study evaluated two novel quantitative G6PD diagnostics. Methods: Participants with known G6PD activity were enrolled in Bangladesh. G6PD activity was measured by spectrophotometry, Biosensor (BS; AccessBio/CareStart, USA) and STANDARD G6PD (SG; SDBiosensor, ROK). G6PD activity was measured repeatedly in a subset of samples stored at room temperature and 4°C. Results: 158 participants were enrolled, 152 samples tested by BS, 108 samples by SG and 102 samples were tested by all three methods. In comparison to spectrophotometry BS had sensitivity and specificity of 72% (95%CI: 53–86) and 100% (95%CI: 97–100) at 30% cut off respectively, while SG had a sensitivity of 100% (95%CI: 88–100) and specificity of 97% (95%CI: 91–99) at the same cut off. The sensitivity and specificity at 70% cut off activity were 71% (95%CI: 59–82) and 98% (95%CI, 92–100) respectively for BS and 89% (95%CI: 77–96) and 93% (95%CI: 83–98) respectively for SG. When an optimal cut-off was applied the sensitivity of the BS at 70 cut off rose to 91% [95%CI: 80–96] and specificity to 82% [95%CI: 83–89]; a diagnostic accuracy comparable to that of the SG (p = 0.879). G6PD activity dropped significantly (-0.31U/gHb, 95%CI: -0.61 to -0.01, p = 0.022) within 24 hours in samples stored at room temperature, but did not fall below 90% of baseline activity until day 13 (-0.87U/gHb, 95%CI: (-1.11 to -0.62), p<0.001). Conclusion: BS and SG are the first quantitative diagnostics to measure G6PD activity reliably at the bedside and represent suitable alternatives to spectrophotometry in resource poor settings. If samples are stored at 4°C, G6PD activity can be measured reliably for at least 7 days after sample collection. [ABSTRACT FROM AUTHOR]

Bacteriophages have been proven as effective antimicrobial agents in the treatment of infectious diseases and in other biocontrol applications including food preservation and disinfection. The extensive use of bacteriophages requires improved methodologies for medium- and long-term storage as well as for easy shipping. To this aim, we have determined the stability of four Staphylococcus phages (phiIPLA88, phiIPLA35, phiIPLA-RODI and phiIPLA-C1C) with antimicrobial potential at different temperatures (20°C/25°C, 4°C, -20°C, -80°C, -196°C) and during lyophilization (freeze drying) using several stabilizing additives (disaccharides, glycerol, sorbitol and skim milk). Differences between phages were observed at different temperatures (20°C/25°C, 4°C and -20°C), where phages were less stable. At lower temperatures (-80°C and -196°C), all phages showed good viability after 24 months regardless of the stabilizer. Differences between phages were also observed after lyophilization although the addition of skim milk yielded a dry powder with a stable titer after 24 months. As an alternative to facilitate storage and transportation, phage encapsulation has been also explored. Phage phiIPLA-RODI encapsulated in alginate capsules retained high viability when stored at 4°C for 6 months and at 20°C for 1 month. Moreover, the spray-dryer technique allowed obtaining dry powders containing viable encapsulated phages (phiIPLA-RODI and phiIPLA88) in both skim milk and trehalose for 12 months at 4°C. Storage of phages at 20°C was less effective; in fact, phiIPLA88 was stable for at least 12 months in trehalose but not in skim milk, while phiIPLA-RODI was stable only for 6 months in either stabilizer. These results suggest that encapsulated phages might be a suitable way for shipping phages. [ABSTRACT FROM AUTHOR]

The main aim of this study was to prepare gelatine-based hydrogels containing entrapped substrate and to examine the applicability of these matrices for detection of enzymes with a specified catalytic activity. The general research concept assumed the use of a substrate that, in the presence of a particular enzyme, will quickly undergo conversion to a coloured product. ortho-Nitrophenyl-β-D-galactopyranoside (ONPG) was used as the immobilized substrate and β-galactosidase from Kluyveromyces lactis as the biocatalyst to be determined. Among other factors, the range of detectable concentrations of galactosidase, the operational pH range, the time necessary to achieve a visible response and the preferred storage conditions for the test were determined. As a result, an effective colourimetric test for β-galactosidase detection was obtained. Its main advantages include (i) the effective detection of the enzyme at concentrations greater than or equal to 0.6 mg.L-1, (ii) the ability to perform initial quantification of the enzyme on the basis of the intensity of the obtained colour (iii) applicability in a wide pH range (from 4.0 to 9.0), (iv) a relatively short response time (from 1 to a maximum of 30 minutes) and (v) stability in long-term storage at 4°C (90 days without loss of specific properties). [ABSTRACT FROM AUTHOR]

Background: Human milk, with essential nutrients and long chain polyunsaturated fatty acids (LC-PUFAs) such as the omega 3 and 6 fatty acids is important for development of the central nervous system and the retina in very low birth weight infants (<1,500 g). However, breast milk may not be sufficient to meet these needs. The possibility of supplementing breast milk with a lyophilisate of human milk was explored in this study. The objectives of this study were to determine the total lipid content and the lipid profile of the Human Milk on Baseline (HMB) and that of the Concentrates with the Human Milk + lyophilisate (with lyophilisate of milk in the immediate period (HMCI), at 3 months (HMC3m), and at 6 months (HMC6m) of storage). Methods: Fifty donors from the Human Milk Bank of Children’s Hospital provided consent, and donated milk samples. Macronutrient (including total lipids) quantification was performed using the MIRIS® Human Milk Analyzer, and the fatty acid profile was determined by gas chromatography (CG-FID, SHIMADZU®). Results: There was a higher lipid concentration in HMCI relative to HMB. The concentrations of the main fatty acids (% of total) were as follows: palmitic acid (C16:0) HMB, 22.30%; HMCI, 21.46%; HMC3m, 21.54%; and HMC6m, 21.95% (p<0.01); oleic acid (C18:1n-9) HMB, 30.41%; HMCI, 30.47%; HMC3m, 30.55%; and HMC6m, 29.79% (p = 0.46); linoleic acid (C18:2n-6) HMB, 19.62%; HMCI, 19.88%; HMC3m, 19.49%; and HMC6m, 19.45% (p = 0.58); arachidonic acid (C20:4n-6) HMB, 0.35%; HMCI, 0.16%; HMC3m, 0.13%; and HMC6m, 0.15% (p<0.01); α-linolenic acid (C18:3n-3) HMB,1.32%; HMCI, 1.37%; HMC3m, 1.34%; and 1.34% HMC6m (p = 0.14); docosahexaenoic acid (C22:6n-3) HMB, 0.10%; HMCI, 0.06%; HMC3m, 0.05%; and HMC6m, 0.06% (p<0.01). There were no significant changes in the lipid profile when stored. There was no evidence of peroxidation during storage. Conclusions: Freeze-dried human milk fortified with a human milk concentrate brings potential benefits to newborns, mainly by preserving the essential nutrients present only in breast milk; however, further clinical studies are required to evaluate the safety and efficacy of the concentrate as a standard nutritional food option for very low birth weight infants. [ABSTRACT FROM AUTHOR]

Fatty acid (FA) signatures (FAS) are important tools to assess the foraging ecology of wild animals. The present study was conducted to assess how well the general FAS and the proportions of individual FA are preserved in fat samples stored at different temperatures (–196, –80, –20, +4 and +20°C). Using three species (laboratory rat, American mink and rainbow trout), FAS were determined immediately upon sampling. Thereafter, eight subsamples per storage temperature from the inner part of the sample unaffected by oxygen and light were re-analyzed after 1, 2, 3, 7, 28, 84 and 168 days. Each time the remaining sample was sealed in its vial after replacing air with nitrogen gas. The results were tested with the mixed model and discriminant analyses. Generally, the FAS were well preserved regardless of storage temperature, and only a few major FA showed significant changes even after the 6-month period at room temperature. After an initial first-day change in proportions, presumably due to post-mortem enzymatic activities, the remaining minor changes could not be clearly attributed to either further autolysis, decomposition or autoxidation. In the discriminant analysis, the species-specific differences dominated and remained distinct even after 6 months. Furthermore, the analysis mostly classified the samples preserved at sub- and above-freezing temperatures separate from each other, and the general deviation from the initial analysis results was present as early as after 1 day. If FAS are to be analyzed in a very precise manner, the analysis should be performed immediately upon sampling. However, FAS remain adequately reliable for long periods of time even without preservation in deep freeze, widening the availability of potential samples for studies on foraging ecology and related disciplines. [ABSTRACT FROM AUTHOR]

RNA and DNA analyses from paraffin-embedded tissues (PET) are an important diagnostic tool for characterization of a disease, exploring biomarkers and treatment options. Since nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissue are of limited use for molecular analyses due to chemical modifications of biomolecules alternate, formalin-free fixation reagents such as the PAXgene Tissue system are of evolving interest. Furthermore, biomedical research and biomarker development critically relies on using long-term stored PET from medical archives or biobanks to correlate molecular features with long-term disease outcomes. We therefore performed a comparative study to evaluate the effect of long term storage of FFPE and PAXgene Tissue-fixed and paraffin-embedded (PFPE) tissue at different temperatures on nucleic acid stability and usability in PCR. Matched FFPE and PFPE human tissues from routine clinical setting or rat tissues from a highly controlled animal model were stored at room temperature and 4°C, as well as in case of animal tissues frozen at -20°C and -80°C. RNA and DNA were extracted in intervals for up to nine years, and examined for integrity, and usability in quantitative RT-PCR (RT-qPCR) or PCR (qPCR) assays. PET storage at room temperature led to a degradation of nucleic acids which was slowed down by storage at 4°C and prevented by storage at -20°C or -80°C. Degradation was associated with an amplicon length depending decrease of RT-qPCR and qPCR efficiency. Storage at 4°C improved amplifiability in RT-qPCR and qPCR profoundly. Chemically unmodified nucleic acids from PFPE tissue performed superior compared to FFPE tissue, regardless of storage time and temperature in both human and rat tissues. In conclusion molecular analyses from PET can be greatly improved by using a non-crosslinking fixative and storage at lower temperatures such as 4°C, which should be considered in prospective clinical studies. [ABSTRACT FROM AUTHOR]

Study of plant metabolome is a growing field of science that catalogs vast biochemical and functional diversity of phytochemicals. However, collecting and storing samples of plant metabolome, sharing these samples across the scientific community and making them compatible with bioactivity assays presents significant challenges to the advancement of metabolome research. We have developed a RApid Metabolome Extraction and Storage (RAMES) technology that allows efficient, highly compact, field-deployable collection and storage of libraries of plant metabolome. RAMES technology combines rapid extraction with immobilization of extracts on glass microfiber filter discs. Two grams of plant tissue extracted in ethanol, using a specially adapted Dremel® rotary tool, produces 25–35 replicas of 10 mm glass fiber discs impregnated with phytochemicals. These discs can be either eluted with solvents (such as 70% ethanol) to study the metabolomic profiles or used directly in a variety of functional assays. We have developed simple, non-sterile, anti-fungal, anti-bacterial, and anti-oxidant assays formatted for 24-multiwell plates directly compatible with RAMES discs placed inside the wells. Using these methods we confirmed activity in 30 out of 32 randomly selected anti-microbial medicinal plants and spices. Seven species scored the highest activity (total kill) in the anti-bacterial (bacteria from human saliva) and two anti-fungal screens (Fusarium spp. and Saccharomyces cerevisiae), providing functional validation of RAMES technology. RAMES libraries showed limited degradation of compounds after 12 months of storage at -20°C, while others remained stable. Fifty-eight percent of structures characterized in the extracts loaded onto RAMES discs could be eluted from the discs without significant losses. Miniaturized RAMES technology, as described and validated in this manuscript offers a labor, cost, and time-effective alternative to conventional collection of phytochemicals. RAMES technology enables creation of comprehensive metabolomic libraries from various ecosystems and geographical regions in a format compatible with further biochemical and functional studies. [ABSTRACT FROM AUTHOR]

The use of Polymerase Chain Reaction (PCR) assays for pathogen detection in travelers’ diarrhea (TD) field studies is limited by the on-site processing and storage requirements for fecal specimens. The objectives of this investigation were to i) characterize the pathogen distribution in deployed military personnel with TD using the TaqMan® Array Card PCR (TAC) on frozen stool and diarrheal smears on Whatman FTA Elute cards (FTA cards), and to ii) compare TAC detection of enteropathogen targets using smeared FTA cards and frozen stool, using TAC on frozen stool as the ‘reference standard’. Stool samples, obtained from active duty personnel with acute TD enrolled in a field trial, were smeared onto FTA cards and stored at room temperature. A corresponding aliquot of stool was frozen in a cryovial. FTA cards and frozen stool samples were tested at a central lab, using a customized TAC for detection of TD pathogens. 187 paired frozen stool samples and smeared FTA cards were stored for a median of 712 days (IQR 396–750) before testing. Overall detection rates were 78.6% for frozen stool and 73.2% for FTA cards. Diarrheagenic Escherichia coli were the most common bacteria identified. Using the TAC results on frozen stool as the reference, the overall sensitivity and specificity of TAC on FTA cards was 72.9% and 98.0% respectively. TAC on FTA cards demonstrated a decrease in sensitivity with increasing frozen stool quantification cycle (Cq) (90.0% in FTA cards with a corresponding frozen stool Cq < 30, and 72.9% in samples with a corresponding frozen stool Cq < 35). Our findings support the use and further development of FTA cards in combination with a quantitative PCR assay for enteropathogen detection in TD field studies. [ABSTRACT FROM AUTHOR]

Background: Urine poses an attractive non-invasive means for obtaining liquid biopsies for oncological diagnostics. Especially molecular analysis on urinary DNA is a rapid growing field. However, optimal and practical storage conditions that result in preservation of urinary DNA, and in particular hypermethylated DNA (hmDNA), are yet to be determined. Aim: To determine the most optimal and practical conditions for urine storage that result in adequate preservation of DNA for hmDNA analysis. Methods: DNA yield for use in methylation analysis was determined by quantitative methylation specific PCR (qMSP) targeting the ACTB and RASSF1A genes on bisulfite modified DNA. First, DNA yield (ACTB qMSP) was determined in a pilot study on urine samples of healthy volunteers using two preservatives (Ethylenediaminetetraacetic acid (EDTA) and Urine Conditioning Buffer, Zymo Research) at four different temperatures (room temperature (RT), 4°C, -20°C, -80°C) for four time periods (1, 2, 7, 28 days). Next, hmDNA levels (RASSF1A qMSP) in stored urine samples of patients suffering from bladder cancer (n = 10) or non-small cell lung cancer (NSCLC; n = 10) were measured at day 0 and 7 upon storage with and without the addition of 40mM EDTA and/or 20 μl/ml Penicillin Streptomycin (PenStrep) at RT and 4°C. Results: In the pilot study, DNA for methylation analysis was only maintained at RT upon addition of preserving agents. In urine stored at 4°C for a period of 7 days or more, the addition of either preserving agent yielded a slightly better preservation of DNA. When urine was stored at -20 °C or -80 °C for up to 28 days, DNA was retained irrespective of the addition of preserving agents. In bladder cancer and NSCLC samples stored at RT loss of DNA was significantly less if EDTA was added compared to no preserving agents (p<0.001). Addition of PenStrep did not affect DNA preservation (p>0.99). Upon storage at 4°C, no difference in DNA preservation was found after the addition of preserving agents (p = 0.18). The preservation of methylated DNA (RASSF1A) was strongly correlated to that of unmethylated DNA (ACTB) in most cases, except when PCR values became inaccurate. Conclusions: Addition of EDTA offers an inexpensive preserving agent for urine storage at RT up to seven days allowing for reliable hmDNA analysis. To avoid bacterial overgrowth PenStrep can be added without negatively affecting DNA preservation. [ABSTRACT FROM AUTHOR]

Background: The new Xpert MTB/RIF Ultra assay (Ultra, Cepheid, Sunnyvale, USA) is a cartridge-based automated diagnostic test that can simultaneously identify Mycobacterium tuberculosis complex (MTB) and resistance to Rifampicin (RIF). With respect to the previous version Xpert MTB/RIF assay (Xpert), IS6110/IS1081 repetitive elements probes have been added allowing the detection of lower MTB load, defined by the new semi-quantitative category “trace” with indeterminate RIF resistance. The aim of this study was to evaluate performance of the new version Ultra on Xpert-negative, but TB culture-positive clinical samples. Methods: The de-identified frozen samples (-20 °C) collected over a 4-year period (February 2014-October 2017), which had previously resulted smear-negative, Xpert-negative but MTB culture-positive, were analyzed with Ultra. The de-frosted samples were loaded into the cartridge using the same process as the previous version, according to manufacturer’s instruction. Results: During the study period 382 MTB culture-positive samples were archived: 314 resulted Xpert-positive and 68 Xpert-negative. Thirty-one of the 68 Xpert-negative samples resulted positive with Ultra, with an overall improvement in MTB detection of 45.6%. Out of 36 Xpert-negative respiratory samples, 18 resulted Ultra-positive with the following semi-quantitative loads: “low”(n = 1), “very low”(n = 11), “trace”(n = 6), with an improvement in MTB detection of 50%. The best performance was achieved on bronchoalveolar lavage specimens (53.8%). Out of 32 Xpert-negative non-respiratory samples, 13 resulted Ultra-positive with the following semi-quantitative loads: “very low”(n = 7), “trace”(n = 6), with an improvement in MTB detection of 40.6%. The best performance was achieved on biopsies (55.6%) and lymph nodes (50%). The new category “trace” detected 12 out of the 31 Ultra-positive MTB samples; in the remaining 19 samples RIF susceptibility was determined with 100% concordance with the phenotypic susceptibility test. The mean time to positivity of samples found negative by Ultra was significantly longer in comparison to positive samples in liquid culture. Conclusions: Our results are consistent with the few studies published so far and confirm the better performance of Ultra compared to the previous version in both respiratory and non-respiratory smear-negative samples, with an overall improvement of 45.6%. [ABSTRACT FROM AUTHOR]

Tardigrades represent an invertebrate phylum with no circulatory or respiratory system. Their body cavity is filled with free storage cells of the coelomocyte-type, which are responsible for important physiological functions. We report a study comparing the ultrastructure of storage cells in anhydrobiotic and hydrated specimens of the eutardigrade Richtersius coronifer. We also analysed the effect of temperature stress on storage cell structure. Firstly, we verified two types of ultrastructurally different storage cells, which differ in cellular organelle complexity, amount and content of reserve material and connection to oogenetic stage. Type I cells were found to differ ultrastructurally depending on the oogenetic stage of the animal. The main function of these cells is energy storage. Storage cells of Type I were also observed in the single male that was found among the analysed specimens. The second cell type, Type II, found only in females, represents young undifferentiated cells, possibly stem cells. The two types of cells also differ with respect to the presence of nucleolar vacuoles, which are related to oogenetic stages and to changes in nucleolic activity during oogenesis. Secondly, this study revealed that storage cells are not ultrastructurally affected by six months of desiccation or by heating following this desiccation period. However, heating of the desiccated animals (tuns) tended to reduce animal survival, indicating that long-term desiccation makes these animals more vulnerable to heat stress. We confirmed the degradative pathways during the rehydration process after desiccation and heat stress. Our study is the first to document two ultrastructurally different types of storage cells in tardigrades and reveals new perspectives for further studies of tardigrade storage cells. [ABSTRACT FROM AUTHOR]

Background: The human intestinal microbiota contains a vast community of microorganisms increasingly studied using high-throughput DNA sequencing. Standardized protocols for storage and DNA extraction from fecal samples have been established mostly for bacterial microbiota analysis. Here, we investigated the impact of storage and DNA extraction on bacterial and fungal community structures detected concomitantly. Methods: Fecal samples from healthy adults were stored at -80°C as such or diluted in RNAlater® and subjected to 2 extraction protocols with mechanical lysis: the Powersoil® MoBio kit or the International Human Microbiota Standard (IHMS) Protocol Q. Libraries of the 12 samples targeting the V3-V4 16S and the ITS1 regions were prepared using Metabiote® (Genoscreen) and sequenced on GS-FLX-454. Sequencing data were analysed using SHAMAN (). The bacterial and fungal microbiota were compared in terms of diversity and relative abundance. Results: We obtained 171869 and 199089 quality-controlled reads for 16S and ITS, respectively. All 16S reads were assigned to 41 bacterial genera; only 52% of ITS reads were assigned to 40 fungal genera/section. Rarefaction curves were satisfactory in 3/3 and 2/3 subjects for 16S and ITS, respectively. PCoA showed important inter-individual variability of intestinal microbiota largely overweighing the effect of storage or extraction. Storage in RNAlater® impacted (downward trend) the relative abundances of 7/41 bacterial and 6/40 fungal taxa, while extraction impacted randomly 18/41 bacterial taxa and 1/40 fungal taxon. Conclusion: Our results showed that RNAlater® moderately impacts bacterial or fungal community structures, while extraction significantly influences the bacterial composition. For combined bacterial and fungal intestinal microbiota analysis, immediate sample freezing should be preferred when feasible, but storage in RNAlater® remains an option under unfavourable conditions or for concomitant metatranscriptomic analysis; and extraction should rely on protocols validated for bacterial analysis, such as IHMS Protocol Q, and including a powerful mechanical lysis, essential for fungal extraction. [ABSTRACT FROM AUTHOR]

Solidified self-nanoemulsifying drug delivery systems (SNEDDS) offer strong option to enhance both drug aqueous solubility and stability. The current study was designed to evaluate the potential stabilization benefits of solidifying cinnarizine (CN) liquid SNEDDS into single and multi-layer self-nanoemulsifying pellets (SL-SNEP and ML-SNEP, respectively). The selected formulations were enrolled into accelerated, intermediate and long-term stability studies. The chemical stability was assessed based on the % of intact CN remaining in formulation. The physical stability was assessed by monitoring the in-vitro dissolution and physical appearance of the formulations. The degradation pathway of CN within lipid-based formulation was proposed to involve a hydroxylation reaction of CN molecule. The chemical stability study revealed significant CN degradation in liquid SNEDDS, SL-SNEP and ML-SNEP (lacking moisture-sealing) within all the storage conditions. In contrast, the moisture sealed ML-SNEP showed significant enhancement of CN chemical stability within the formulation. In particular, ML-SNEP coated with Kollicoat Smartseal 30D showed superior CN stabilization and no significant decrease in dissolution efficiency, at all the storage conditions. The observed stability enhancement is owing to the complete isolation between CN and SNEDDS layer as well as the effective moisture protection provided by Kollicoat Smartseal 30D. Hence, the degradation problem could be eradicated completely. The incorporation of silicon dioxide had an important role in the inhibition of pellet agglomeration upon storage. Accordingly, ML-SNEP coated with Kollicoat Smartseal 30D and/or silicon dioxide could be an excellent dosage form that combine dual enhancement of CN solubilization and stabilization. [ABSTRACT FROM AUTHOR]