Frozen Samples Research Articles

Abstract 5180: Enhanced and controlled chromatin extraction for chromatin-based epigenetic assays in FFPE tissues

Abstract Background: Epigenetic dysregulation is involved in the etiology and progression of various human diseases. Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples and are hampered by low chromatin yield in FFPE samples due to the lack of reliable and efficient chromatin preparation methods. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays. Results: During rehydration of FFPE tissues, applying tissue-level cross-link reversal to the deparaffinized tissue at 65°C dramatically increased chromatin yield in the soluble fraction. We found the resulting chromatin to be compatible with ChIP-qPCR and ChIP-seq analysis of histone marks (H3K4me3, H3K27me3, and H3K27Ac) and RNA polymerase II. The chromatin prepared by Chrom-EX PE showed a gradual fragmentation pattern with varying incubation temperature. At temperatures below 37°C, the majority of soluble chromatin was over 1 kb in size. The soluble chromatin prepared at 45-60°C showed a typical nucleosomal pattern shown in cell lines and frozen tissues. The majority of the chromatin prepared at 65°C is close to mononucleosomal size. These observations indicate that chromatin preparation from FFPE samples can be controlled for downstream chromatin-based epigenetic assays. Conclusions: Our method achieves efficient extraction of high-quality chromatin from FFPE samples suitable for targeted and genome-wide epigenetic assays while minimizing degradation and damages. This approach provides a new platform for developing chromatin-based assays applicable to archived FFPE samples to circumvent the over-fixed nature of FFPE tissues. This work was supported, in part, by National Institutes of Health grant P01DK068055 and the Mayo Clinic Center for Individualized Medicine Epigenomics Program. Citation Format: Jian Zhong, Zhenqing Ye, Chad Clark, Samuel Lenz, Justin Nguyen, Huihuang Yan, Keith Robertson, Gianrico Farrugia, Zhiguo Zhang, Tamas Ordog, Jeong-Heon Lee. Enhanced and controlled chromatin extraction for chromatin-based epigenetic assays in FFPE tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5180.

Abstract 1811: The long noncoding RNA KIAA0125 is aberrantly expressed in ameloblastomas

Abstract Long noncoding RNAs (lncRNAs) are transcribed RNA molecules that can interact with DNA or RNA, transcription factors, histones and other chromatin modifying proteins, affecting the expression level of a broad spectrum of genes. Because of their tissue-specific expression characteristics, lncRNAs hold strong promise as novel biomarkers and therapeutic targets for diseases. High-density whole-genome microarray analysis in ameloblastomas, an aggressive odontogenic tumor, and in adenomatoid odontogenic tumor (AOT), an indolent one, showed a high frequency of copy number alteration at chromosome band 14q32.33, which encompasses the lncRNA gene KIAA0125. To understand molecular mechanisms associated with their biological behavior and considering the copy-number gains observed at 14q32.33, we aimed to investigate the expression levels of the lncRNA KIAA0125 in ameloblastomas and in AOTs. The University Ethics Committee approved this study and patients signed informed consent. Thirteen frozen samples were included: five solid/multicystic ameloblastomas, four AOT, and four dental follicles. qPCR reactions were performed in triplicates and the relative changes in gene expression were obtained using the 2^-ΔΔCt formula. The reference gene HPRT1 was used for normalization and the human keratinocyte HaCat cell line was used as calibrator. Differences in the relative changes in gene expression between two groups of samples were assessed and the significance level was set at 0.05. All samples expressed more KIAA0125 than the calibrator. The ameloblastoma group showed higher expression levels of KIAA0125 when compared to dental follicles (p=0.042), while there was no difference between the expression in ameloblastomas and AOT (p>0.05). The expression levels of KIAA0125 in AOT were not different from that of the dental follicle. Our findings suggest that lncRNA KIAA0125 is likely involved in ameloblastoma pathobiology. LncRNAs hold strong promise as therapeutic targets, and experimental validation of this lncRNA functions may lead to tailored therapies targeting KIAA0125 in extensive and recurrent ameloblastomas. Citation Format: Silvia F. Sousa, Marina G. Diniz, Josiane A. França, Fabricio A. Vilas-Boas, Fabricio T. Souza, George A. Calin, Ricardo S. Gomez, Carolina C. Gomes. The long noncoding RNA KIAA0125 is aberrantly expressed in ameloblastomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1811.

Gut microbiome analysis by post: Evaluation of the optimal method to collect stool samples from infants within a national cohort study.

BackgroundUnderstanding the role of the gut microbiome is pivotal for the future development of therapies for the prevention and management of autoimmune conditions such as type 1 diabetes when sampling during early life may be particularly important. The current standard methods for collecting gut microbiome samples for research is to extract fresh samples or freeze samples immediately after collection. This is often impractical however for population-based studies. The aim of this study was to determine the optimal method for the stabilization of stool bacterial DNA obtained from nappies and transported by post in ambient conditions to the research centre for a national birth cohort study.MethodsFour methods to collect samples were compared to immediate freezing of samples: 1) collecting faeces onto a swab which was immediately frozen, 2) using a commercially available kit with stabilisation solution (OMNIgene•GUT kit) at ambient temperature, 3) collecting onto a swab and 4) collecting into a sterile plain tube. Samples 3) and 4) were returned to the laboratory by post at ambient temperatures. A Bland Altman analysis was used to assess the agreement between the different methods and the frozen standard.ResultsStool samples were collected by parents. For samples transported in ambient conditions, the limits of agreement showed that the OMNIgene•GUT kit had the narrowest 95% limits of agreement with the frozen standard as measured by the number of operational taxonomic units and the Shannon diversity index.ConclusionsAll methods assessed for preserving samples collected from nappies at a distance and delivered by post for gut microbiome analysis showed variation / disagreement from the frozen standard. Overall, the OMNIgene•GUT kit preserved the samples with minimal changes compared to other methods and was practical for parents to use.

Effect of ultrasound-assisted freezing on the textural characteristics of dough and the structural characterization of wheat gluten.

To better understand the effects of ultrasonic treatment in the whole freezing process (UWF) and the maximum ice crystal formation zone (UMF) on the quality of frozen dough, the textural properties of dough and the structure of gluten were investigated. The results showed that the UWF and UMF treatments improved the textural properties of frozen dough and obtain the best effect at the 60W/L power densities. Ultrasound-assisted freezing reduced the destructive effect of disulfide bonds on dough, and led to a state of dynamic equilibrium of hydrophobic groups. UWF treatment at 80W/L and UMF treatment at 40W/L had positive effects prevented the secondary structure from destruction by freezing. The network of gluten treated by ultrasound-assisted freezing was more uniform and smaller than that of traditional freezing samples, which was similar to the network structure of fresh protein. According to Pearson's correlation analysis, there was a high correlation between SH, α-helix content and springiness. There was a significant positive correlation between β-turn and G', G″, and there was a significant negative correlation between β-turn and hardness. These results suggest that ultrasound-assisted freezing improved the process quality of dough though reducing the damage to gluten structure caused by freezing.

The role of tissue type, sampling and nucleic acid purification methodology on the inferred composition of Pacific oyster (Crassostrea gigas) microbiome.

This study evaluated methods to sample and extract nucleic acids from Pacific oysters to accurately determine the microbiome associated with different tissues. Samples were collected from haemolymph, gill, gut and adductor muscle, using swabs and homogenates of solid tissues. Nucleic acids were extracted from fresh and frozen samples using three different commercial kits. The bacterial DNA yield varied between methods (P<0·05) and each tissue harboured a unique microbiota, except for gill and muscle. Higher bacterial DNA yields were obtained by swabbing compared to tissue homogenates and from fresh tissues compared to frozen tissues, without impacting the bacterial community composition estimated by 16S rRNA gene (V1-V3 region) sequencing. Despite the higher bacterial DNA yields with QIAamp® DNA Microbiome Kit, the E.Z.N.A.® Mollusc DNA Kit identified twice as many operational taxonomic units (OTUs) and eliminated PCR inhibition from gut tissues. Sampling and nucleic acid purification substantially affected the quantity and diversity of bacteria identified in Pacific oyster microbiome studies and a fit-for-purpose strategy is recommended. Accurate identification of Pacific oyster microbial diversity is instrumental for understanding the polymicrobial aetiology of Pacific oyster mortality diseases which greatly impact oyster production.

Lineage-dependent role of miR-410-3p as oncomiR in gonadotroph and corticotroph pituitary adenomas or tumor suppressor miR in somatotroph adenomas via MAPK, PTEN/AKT, and STAT3 signaling pathways

PurposemiR-410-3p plays opposite roles in different cancers and may act as an oncomiR or tumor suppressor miR. The purpose of this study was to assess the role of miR-410-3p in somatotroph, gonadotroph, and corticotroph pituitary adenomas.MethodsTissue samples were obtained from 75 patients with pituitary adenoma. miR-410-3p expression was assessed using qRT-PCR performed on RNA isolated from fresh frozen samples. In vitro experiments were performed on cell lines derived from somatotroph (GH3), gonadotroph (RC-4B/C), and corticotroph (AtT-20) pituitary tumors. Cells were transfected with synthetic mimic of miR-410-3p or non-targeting scrambled-miR control. Subsequently, proliferation assays and transwell invasion assays were performed. The expression of cyclin D1, E1, and B1 in cells after transfection was determined using qRT-PCR. The activation of MAPK, PTEN/AKT and STAT3 signaling pathways were assessed using western blot.ResultsWe have found that the level of expression of miR-410-3p differs in particular types of pituitary adenomas. miR-410-3p significantly upregulates proliferation and invasiveness of RC-4B/C and AtT-20 cells, while inhibiting GH3 cells. We observed that the levels of cyclin B1 upon transfection with miR-410-3p mimic were increased in RC-4B/C and AtT-20, yet decreased in GH3 cells. We have shown that miR-410-3p promoted the activation of MAPK, PTEN/AKT, and STAT3 signaling pathways in RC-4B/C and AtT-20 cells, but suppressed their activity in GH3 cells.ConclusionsmiR-410-3p acts as an oncomiR in gonadotroph and corticotroph adenoma cells, while as a tumor suppressor miR in somatotroph adenoma cells.

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue.

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample's refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Enhanced and controlled chromatin extraction from FFPE tissues and the application to ChIP-seq

BackgroundEpigenetic dysregulation is involved in the etiology and progression of various human diseases. Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples, and are hampered by low chromatin yield in FFPE samples due to the lack of a reliable and efficient chromatin preparation method. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays.ResultsDuring rehydration of FFPE tissues, applying a tissue-level cross-link reversal into the deparaffinized tissue at 65 °C dramatically increased chromatin yield in the soluble fraction. The resulting chromatin is compatible with targeted ChIP-qPCR and genome-wide ChIP-seq approaches. The chromatin prepared by Chrom-EX PE showed a gradual fragmentation pattern with varying incubation temperature. At temperatures below 37 °C, the majority of soluble chromatin is over 1 kb. The soluble chromatin prepared in the range of 45–60 °C showed a typical nucleosomal pattern. And the majority of chromatin prepared at 65 °C is close to mononucleosomal size. These observations indicate that chromatin preparation from FFPE samples can be controlled for downstream chromatin-based epigenetic assays.ConclusionsThis study provided a new method that achieves efficient extraction of high-quality chromatin suitable for chromatin-based epigenetic assays with less damage on chromatin. This approach may provide a way to circumvent the over-fixed nature of FFPE tissues for future technology development.

Indicators of Oxic and Anoxic Conditions in the System of the Current Sedimentation of Saline Lake Shira (Khakassia), According to High-Resolution SR XRF Data on Bottom Sediments Frozen In Situ

Frozen upper layers of the bottom sediment of Lake Shira are selected using special sampling equipment. Frozen samples and solid samples prepared from the upper layers of a sediment core are examined by means of X-ray fluorescence at the Local and Scanning X-Ray Fluorescence Elemental Analysis Station of the Siberian Synchrotron and Terahertz Radiation Center’s Shared Resource Center. Analytical data on the initial frozen and processed solid samples demonstrate the good repeatability of the results. Quantitative estimates of the sedimentation regimes are obtained, and geochemical indicators of the change in redox conditions are determined from the analysis data. Lithological and geochemical records are synchronized with regional seasonal and annual variations in weather and climate, and with the hydrological regime of the lake for the last 50 years.

Changes in microstructure, quality and water distribution of porcine longissimus muscles subjected to ultrasound-assisted immersion freezing during frozen storage

The effect of ultrasound-assisted immersion freezing at 180 W (UIF-180) on the microstructure, quality and water distribution of porcine longissimus muscles during frozen storage was evaluated. The size of the ice crystals increased with extended storage time, and UIF-180 samples had a smaller size and uniform distribution of ice crystals. The thawing and cooking losses in the UIF-180 sample were significantly lower than that in air freezing (AF) and immersion freezing (IF) samples (P < 0.05). AF samples had a higher cutting force at 0 days. During the 60–180 days of the storage period, the cutting force of UIF-180 samples was significantly higher than that of AF and IF samples (P < 0.05). Low field-nuclear magnetic resonance results showed that UIF-180 decreased water migration during frozen storage. UIF-180 samples had significantly lower lipid oxidation and higher redness than that of the AF and IF samples (P > 0.05). Overall, UIF at particular powers is an efficient method in reducing quality deterioration of muscles during long-term frozen storage.

Cryopreservation of human spermatozoa by vitrification versus conventional rapid freezing: Effects on motility, viability, morphology and cellular defects

ObjectivesSperm cryopreservation has great potential for male infertility treatment as used in assisted reproduction technology (ART). There are a variety of cryopreservation methods in order to preserve sperm in a long term. Although conventional freezing and vitrification now are used widely, they have damage on sperm parameters as well as sperm DNA integrity. It is necessary to answer which method is better and appropriate for sperm cryopreservation. The aim of this study was to compare the effect of conventional freezing and vitrification regarding to motility, vitality and morphology of sperm found in washed and unwashed samples. Study designOne hundred and five human fresh semen samples were divided into washed and unwashed halves using density-gradient centrifugation. Each group then was split into two aliquots: one cryopreserved by conventional freezing and the other by vitrification, using SpermFreeze Solution™ (Vitrolife, Västra Frölunda, Sweden) containing glycerol as a cryoprotectant. The sperm parameters were analyzed and compared between six groups: washed fresh (FW), unwashed fresh (FU), washed conventional freezing (CfW), unwashed conventional freezing (CfU), washed vitrification (VitW) and unwashed vitrification (VitU) samples. ResultsSperm progressive motility, vitality and normal morphology significantly decreased, together with an appreciable increase in sperm head, midpiece and tail defects when comparing to the fresh sperm parameters after thawing in all groups. In conventional freezing method groups, progressive motility and vitality were substantially higher than that in vitrification method groups. However, vitrification gave better results in normal morphology rates. Additionally, sperm head, midpiece and tail defects were significant lower in two vitrification groups in comparison with conventional freezing groups. Interestingly, washed groups had better sperm parameters than unwashed groups so that washing process before frozen seemed to improve sperm parameters. ConclusionConventional freezing method resulted in better motility, viability in both washed/unwashed groups. On the contrary, spermatozoa undergoing vitrification were healthier regarding morphology with less defects than conventional freezing. Sperm washing before frozen was a beneficial preparation to sperm cryopreservation.

Cryopreservation as a Tool for Reef Restoration: 2019.

Throughout the world coral reefs are being degraded at unprecedented rates. Locally, reefs are damaged by pollution, nutrient overload and sedimentation from out-dated land-use, fishing and mining practices. Globally, increased greenhouse gases are warming and acidifying oceans, making corals more susceptible to stress, bleaching and newly emerging diseases. The coupling of climate change impacts and local anthropogenic stressors has caused a widespread and well-recognized reef crisis. While the establishment and enforcement of marine protected areas and preventing the acceleration of climate change are essential to management of these stressors, the inexorable impacts of climate change will continue to cause declines in genetic diversity and population viability. Gamete cryopreservation has already acted as an effective insurance policy to maintain the genetic diversity of many wildlife species, and has now begun to be explored and applied to coral conservation. Cryopreservation can act to preserve reef biodiversity and genetic diversity. To date, we have had a great deal of success with cryopreserving sperm from ~30 coral species of coral species. Moreover, we are creating the basic science to freeze and thaw coral larvae that can soon be used to help secure and restore reefs. Building on these successes, we have established genetic banks using frozen samples and use those samples to help mitigate threats to the Great Barrier Reef and other areas.

Phenotyping Gamma Delta T cells in Bladder Cancer

Event Abstract Back to Event Phenotyping Gamma Delta T cells in Bladder Cancer Magdalene Joseph1*, Fidelma Cahill2, Harriet Wylie2, Deborah Enting2, Mieke Van Hemelrijck1 and Adrian Hayday1 1 King's College London, United Kingdom 2 Guy's and St Thomas' NHS Foundation Trust, United Kingdom Background Bladder cancer is known to be immunologically modulated since the 1970s when Bacille-Calmette Guerin (BCG) was shown effective in treating superficial bladder tumours. Moreover, immune checkpoint inhibitors have now demonstrated survival benefit in advanced bladder cancer and are considered standard treatment for chemotherapy-resistant disease. Of special interest in immuno-oncology are unconventional, innate-like T cells bearing the gamma delta T cell receptor. They are found enriched at the epithelial surfaces and are capable of rapid, non-MHC restricted responses to various stressors. These cells are found to be the strongest correlate of positive prognosis in a recent transcriptomic analysis of over 18,000 tumours 1. Studies on mouse models of bladder cancer have shown that survival can be prolonged by injecting gamma delta T cells intravesically2 and that responses to BCG are abolished in gamma delta T cell knockout mice suggesting an anti-tumorigenic role3. This project aims to characterise the presence and phenotype of gamma delta T cells in the blood and tissue in human bladder cancer to understand their contribution to the immune regulation of the disease. Methods Mononuclear cells from peripheral blood of healthy, age-matched controls and patients with bladder cancer will be phenotyped by flow cytometry with a focus on gamma delta T cells to elucidate the differences between health and disease. Tissue infiltrating lymphocytes will also be isolated from bladder tumour tissue and peri-tumoural normal tissue using a cell-foam matrix culture system described previously4 and phenotyped in a similar fashion. Phenotypes from flow cytometry will be confirmed by polymerase chain reaction targeting the markers of interest. The localisation of gamma delta T cells within the tissue will be examined by immunohistochemistry. Results Preliminary results from healthy volunteers show differences in the expression of various markers between different gamma delta T cell populations and differences between fresh and frozen samples. Conclusion The aim is to understand gamma delta T cell mediated immune regulation of bladder cancer. Acknowledgements The King's Health Partners Urological Cancer Biobank and the King's College London Infectious Diseases Biobank for their help in obtaining patient and control samples. References

Mitochondrial Function in Oxidative and Glycolytic Bovine Skeletal Muscle Postmortem

ObjectivesMitochondrial function in postmortem muscle is affected by decreasing oxygenation. Functional properties relating to energy production and integrity of mitochondria may influence development of meat quality characteristics. Therefore, the objective was to evaluate changes in mitochondrial function in oxidative and glycolytic muscles during the first 24h postmortem.Materials and MethodsSteers (n = 6) of primarily Angus (80 to 100%) genetics were harvested at approximately 18.5 mo and 630 kg live weight. Samples from the longissimus lumborum (LL) and diaphragm (Dia) were collected at 1, 3, and 24h postmortem. Fresh-preserved muscle samples were permeabilized using saponin, and muscle bundles (2–4 mg) were transferred to a high-resolution oxygraph for respiration measurements (oxygen consumption rate, OCR, pmol/sec/mg of tissue). Samples were assessed in duplicate under hyperoxia. First, pyruvate and malate were added to support the TCA cycle and assess leak respiration. Then, ADP was added to support electron flow through complex I. The influence of glutamate on NADH production (complex I) was tested, followed by complex II activation by succinate. Integrity of the mitochondria outer membrane was tested with cytochrome c. Next, an uncoupler (FCCP) was added to force the electron transport system (ETS) to maximum capacity. Citrate synthase (CS) activity (nmol/min/mg tissue) was determined in frozen samples and used as a marker of mitochondria content. Subsequently, respiration data were normalized to CS activity (pmol/sec/U CS) to account for differences in mitochondria content. Coupling efficiency of oxidative phosphorylation was calculated as 1– (Leak/ADP-stimulated oxidative phosphorylation capacity). Raw and normalized OCR were analyzed in a randomized block design, with slaughter date as block and fixed effects of muscle, time, and the interaction. Time was considered a repeated measure.ResultsMuscle type affected (P = 0.0002) leak OCR, with Dia showing a higher rate than LL. After ADP was added, mitochondria from Dia exhibited higher OCR at all times tested and at all steps, with OCR being 4 times higher after FCCP addition. Mitochondrial content, evidenced by greater (P < 0.0001) CS activity in Dia, largely explained differences in OCR between muscles. After OCR was normalized to CS activity, the 1 and 3h postmortem OCR from Dia and LL were similar (P > 0.05). However, at 24h postmortem, OCR after ADP, glutamate, and FCCP additions were greater (P < 0.05) in Dia mitochondria. Time, but not muscle, affected cytochrome c response. At 1h postmortem, cytochrome c increased OCR by 6.6%, supporting that mitochondria outer membrane integrity is not compromised. However, cytochrome c response at 3h postmortem increased 52.4%, indicating outer membrane damage. Coupling efficiency is different between muscles (P = 0.005) with Dia exhibiting greater efficiency.ConclusionDespite inherent metabolic differences between the LL and Dia, mitochondria from both muscles are intact and coupled at 1h postmortem. However, by 24h postmortem, functional properties of LL mitochondria are reduced compared to Dia. Declining mitochondrial function may be associated with calcium overload, mitochondrial fragmentation, and protease activation.

Comparison of blood serum protein analysis by MALDI-MS from either conventional frozen samples or storage disc-deposited samples: A study with human serum from pregnant donors and from patients with intrauterine growth restriction

Mass spectrometric profiling of intact serum proteins, i.e. determination of relative protein abundance differences, was performed using two different serum sample preparation methods: one with frozen and thawed serum, the other with at room temperature deposited and dried serum. Since in a typical clinical setting freezing of serum is difficult to achieve, sampling at room temperature is preferred and can be met when using the Noviplex™ card system. Once deposited and dried, serum proteins can be stored and shipped at room temperature. After resolubilization of serum proteins from "dried serum spots", mass spectra of high quality have been recorded comparable to those that were obtained using fresh-frozen and subsequently thawed serum samples. Differentiation between patients with intrauterine growth restriction and control individuals was achievable, independent from the sample work-up procedure. Having at hand a reliable and robust method for serum storage and shipment which works at room temperature bridges the gap between the clinics and the protein analysis laboratory. Our novel serum handling protocol reduces costs for both, storage and shipping, and ultimately enables clinical risk assessment based on mass spectrometric determination of intact protein abundance profiles.

14 Sperm membrane integrity in ejaculates from young bulls may be improved by single-layer centrifugation

Sperm quality in the first ejaculates of young bulls is often poor, leading to rejection of these samples for commercial freezing. It may be several weeks before sperm quality reaches the thresholds regarded as being acceptable for commercial production. Single-layer centrifugation (SLC) has been shown to separate robust bull spermatozoa from the rest of the ejaculate (Goodla et al. 2015 J. Dairy Sci. 97, 2204-2212; Nongbua et al. 2017 Reprod Domest. Anim. 52, 596-602). The aim of this study was to test whether SLC can be used to select spermatozoa from the ejaculates of young bulls and thus freeze samples earlier than is currently possible. Ejaculates (at least 3 per bull; n=33) were collected from 9 young Holstein or Swedish Red bulls, 255 to 415 days old. The sperm concentration was adjusted to 69×106 spermatozoa mL−1 with Andromed™ (Minitub, Tiefenbach, Germany) before layering part of the sample over 4mL of Bovicoll colloid. The remaining sample served as the unselected control. After centrifugation at 300×g for 20min, the sperm pellet was aspirated into fresh extender. Both control (uncentrifuged) and SLC samples were frozen and stored in LN pending analysis. Membrane integrity was evaluated by flow cytometry after staining for 10min at 37°C with SYBR14/propidium iodide (Live-Dead Sperm Viability Kit, L-7011; Invitrogen, Carlsbad, CA, USA). Paired t-test was used to compare results for control and SLC samples, and Pearson correlation was used for age and membrane integrity. The median age at which the samples were taken was 319 days (range: 255-415 days). Membrane integrity varied considerably among bulls (range: 11-72%). The mean proportion of membrane-intact spermatozoa in controls and SLC samples was 40±15 and 47±16%, respectively (P&amp;lt;0.01). In 21 of the 33 ejaculates (64%), the SLC sample had a higher membrane integrity than the controls (Table 1). Age in days was positively correlated with membrane integrity for SLC samples (r=0.40; P&amp;lt;0.05) but not for controls. These results suggest that SLC might be a useful technique for selecting membrane-intact spermatozoa from the ejaculates of young bulls. However, there is considerable variation among bulls, possibly due to age, which is currently being investigated. Other parameters of sperm quality, such as chromatin integrity, mitochondrial membrane potential reactive oxygen species production, and motility, are also being evaluated. Table 1.Age and membrane integrity (mean±standard deviation) in ejaculates from young bulls before and after SLC (n=33) This project was funded by the Royal Swedish Academy of Agriculture and Forestry, Viking Genetics, and the Faculty for Veterinary Medicine and Animal Health, SLU. We thank Viking Genetics for supplying the semen samples.

Cryopreservation of primary B cells minimally influences their signaling responses

Phospho flow is a powerful approach to detect cell signaling aberrations, identify biomarkers and assess pharmacodynamics, and can be performed using cryopreserved samples. The effects of cryopreservation on signaling responses and the reproducibility of phospho flow measurements are however unknown in many cell systems. Here, B lymphocytes were isolated from healthy donors and patients with the B cell malignancy chronic lymphocytic leukemia and analyzed by phospho flow using phospho-specific antibodies targeting 20 different protein epitopes. Cells were analyzed both at basal conditions and after activation of cluster of differentiation 40 (CD40) or the B cell receptor. Pharmacodynamics of the novel pathway inhibitor ibrutinib was also assessed. At all conditions, fresh cells were compared to cryopreserved cells. Minimal variation between fresh and frozen samples was detected. Reproducibility was tested by running samples from the same donors in different experiments. The results demonstrate reproducibility across different phospho flow runs and support the use of cryopreserved samples in future phospho flow studies of B lymphocytes.

Evaluation of the Quality of the Minced Meat in Egyptian Markets

The aim of the present study is to evaluate the quality of minced meat (fresh – frozen) in Egyptian markets. A total of 90 minced meat samples (45 fresh – 45 frozen) were collected from butchers and retailed supermarkets shops in different locations in Egypt.All collected samples were transferred directly to the laboratory in an ice box under complete aseptic conditions without any delay and then subjected to bacteriological examination to evaluate the quality of minced meat based on their contamination with coliform and staphylococcus spp. The obtained results indicated that the mean value of staphylococcal count and coliform count of the examined fresh minced meat samples were 26.0 ± 2.2× 102 cfu/g and 19.0 ± 0.8 × 103 cfu/g, respectively, while of the frozen minced meat samples were 8.8 ± 0.7×102 cfu/g, and 14.8 ± 0.5 ×103 cfu/g respectively. 100 % of the examined fresh minced meat samples (45 samples) were unaccepted samples and 33.3% of the examined samples identified as Staph. Aureus and 28.9 % of the examined frozen minced meat samples (13 samples) were unaccepted samples and 23.1% identified as Staph. Aureus according to EOS 1694 / 2005 (102 cfu/g). Accepted samples based on their coliforms count were 68.8 % and 88.8 % for fresh and frozen samples , respectively according to EOS (1694/ 2005). These results indicated that the microbiological quality of retailed minced meat samples were unsatisfactory, and have health hazards for consumers as it may be lead to food poisoning.

An experimental parametric study on the liquefaction resistance of sands in spreader dumps of lignite opencast mines

The results of a parametric study on the liquefaction resistance of sands dumped with spreaders in lignite opencast mines using undrained cyclic triaxial tests is presented. Undisturbed samples taken by ground freezing and disturbed samples of the same material reconstituted by different methods were compared. Based on this comparison, a suitable sample preparation method was chosen which reproduces the depositional process in the dumps and leads to a similar initial fabric and thus a similar behaviour under undrained cyclic loading. Numerous tests on more than 50 sands from the dumps or the excavation sites of the mines demonstrated a decrease of the liquefaction resistance with increasing content of plastic fines and with decreasing mean grain size, while no clear trend with uniformity coefficient was observed in the data for a constant relative density. The dependence of the liquefaction resistance on the initial values of mean effective stress and stress ratio is discussed based on respective test series. Furthermore, the increase of the undrained cyclic strength with decreasing degree of saturation and an enhancement caused by a drained monotonic or vibrational preloading is demonstrated. Based on tests with different sample sizes, membrane penetration effects were found to be negligible.

Analysis of T-Cell Receptor Beta-Constant Region Expression for Rapid Assessment of T-Cell Clonality

IntroductionT-cell malignancies are heterogenous disorders, representing approximately 10-15% of cases of non-Hodgkin's lymphoma and 15% of acute lymphoblastic leukaemia. Diagnosis may be complicated both by the fact that non-malignant oligoclonal T-cell proliferations often mimic T-cell cancers, and due to the lack of consistent markers of malignancy which discriminate healthy and malignant T-cells. B-cell lymphoproliferations, by contrast, can be reliably identified as clonal by confirmation of restricted kappa or lambda light chain expression. We have recently described an analogous method for evaluation of T-cell clonality, by evaluating expression of one of two virtually identical and mutually exclusive alleles at the T-cell receptor beta constant region: TRBC1 or TRBC2. We demonstrated that approximately 35% of normal T-cells and T-cell malignancies expressed TRBC1, using a TRBC1-specific antibody suitable for use in fresh/ frozen tissues. We have now developed a novel antibody with TRBC2 specificity, which recognises an intracellular, non-surface exposed epitope and can be used in both fresh and formalin-fixed paraffin-embedded (FFPE) tissues. Here we demonstrate TRBC1/2 assessment can be used to identify T-cell clonality using immunohistochemistry on FFPE tissues, and confirm our results using reference methods including Sanger and massively parallel sequencing.Methods/ ResultsTo generate anti-TRBC2 antibody, we immunised New Zealand rabbits with a peptide comprising the intracellular portion of the TCR beta-2 chain (VKRKDSRG). Protein A-purified immunoglobulin from the sera of immunised rabbits was tested for reactivity to TRBC1 (VKRKDF) and TRBC2 peptides by ELISA. Strong TRBC2-specificity with limited TRBC1 binding was seen. Immunoglobulin was then depleted of residual TRBC1 reactivity by depletion on TRBC1 peptide-coated agarose beads. Following this, no reactivity to TRBC1 peptide could be detected by ELISA.We then stained fresh frozen and FFPE samples of pelleted TRBC1 and TRBC2 cell lines with the novel, polyclonal anti-TRBC2 antibody. We confirmed that anti-TRBC2 antibody stained only TRBC2 and not TRBC1 cell lines, and that the antibody performed equivalently in fresh or FFPE preparations. We then showed that in a range of normal tissues, including tonsil, spleen and others, a staining pattern restricted to only a proportion of T-cells was seen. In bone marrow sections, some staining of megakaryocytes was also seen, as has been previously demonstrated for other T-cell markers such as linker for activation of T-cells (LAT). No other non-T-cell staining was noted.We obtained paired FFPE and frozen samples of several TCR+ T-cell malignancies. We performed staining for TRBC1 and TRBC2 on both frozen and FFPE samples: TRBC1 staining was performed only on frozen samples. In addition, we extracted DNA from these tissues and performed PCR for TCR beta VDJ rearrangement using standard BIOMED protocols. Clonal proliferations were identified by heteroduplex gel analysis and capillary electrophoresis (Sanger) sequencing was performed on dominant bands. In addition, pooled PCR products were subjected to massively parallel sequencing to quantify clone frequency and to confirm VDJ identity. We demonstrated mutual exclusivity between samples staining positive for TRBC1 and TRBC2, and concordance between TRBC1/2 status identified by Sanger sequencing, NGS and antibody-based methods.Further, in a number of TCR+ malignancies, non-T-cell haematological tumours and reactive lymphoproliferations, where only FFPE material was available, we used anti-TRBC2 antibody to evaluate TRBC2 expression. We demonstrated that in non-T cell tumours, a limited pattern of staining was seen, restricted to a proportion of infiltrating normal T-cells only. T-cell tumours, by contrast, were uniformly TRBC2-negative or positive, across a range of entities. 18/29 (62%) of T-cell neoplasms were TRBC2-positive.ConclusionsWe have generated a new TRBC2-specific polyclonal reagent suitable for use in FFPE tissues, which displays minimal cross-reactivity against other cell types. A monoclonal reagent is in preparation. We suggest that TRBC1/2 assessment can thus be used to rapidly identify clonal T-cell lymphoproliferations and may be a helpful addition to the diagnostic haematopathology toolkit. DisclosuresMaciocia:Autolus: Equity Ownership, Patents & Royalties: UCLB. Neves:Autolus Ltd: Employment. Onuoha:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Pule:UCLB: Patents & Royalties; Autolus: Employment, Equity Ownership.