ResearchPad - specimen-preservation https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Imaging dataset of fresh hydrous plants obtained by field-emission scanning electron microscopy conducted using a protective NanoSuit]]> https://www.researchpad.co/article/elastic_article_7644 Although scanning electron microscopy (SEM) can generate high-resolution images of nanosized objects, it requires a high vacuum to do so, which precludes direct observations of living organisms and often produces unwanted structural changes. It has previously been reported that a simple surface modification gives rise to a nanoscale layer, termed the “NanoSuit”, which can keep small animals alive under the high vacuum required for field-emission scanning electron microscopy (FE-SEM). We have previously applied this technique to plants, and successfully observed healthy petals in a fully hydrated state using SEM. The flower petals protected with the NanoSuit appeared intact, although we still lack a fundamental understanding of the images of other plants observed using FE-SEM. This report presents and evaluates a rich set of images, acquired using the NanoSuit, for a taxonomically diverse set of plant species. This dataset of images allows the surface features of various plants to be analyzed and thus provides a further complementary morphological profile. Image data can be accessed and viewed through Figshare (https://doi.org/10.6084/m9.figshare.c.4446026.v1).

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<![CDATA[Quantitative analysis of F-actin alterations in adherent human mesenchymal stem cells: Influence of slow-freezing and vitrification-based cryopreservation]]> https://www.researchpad.co/article/5c64490fd5eed0c484c2f524

Cryopreservation is an essential tool to meet the increasing demand for stem cells in medical applications. To ensure maintenance of cell function upon thawing, the preservation of the actin cytoskeleton is crucial, but so far there is little quantitative data on the influence of cryopreservation on cytoskeletal structures. For this reason, our study aims to quantitatively describe cryopreservation induced alterations to F-actin in adherent human mesenchymal stem cells, as a basic model for biomedical applications. Here we have characterised the actin cytoskeleton on single-cell level by calculating the circular standard deviation of filament orientation, F-actin content, and average filament length. Cryo-induced alterations of these parameters in identical cells pre and post cryopreservation provide the basis of our investigation. Differences between the impact of slow-freezing and vitrification are qualitatively analyzed and highlighted. Our analysis is supported by live cryo imaging of the actin cytoskeleton via two photon microscopy. We found similar actin alterations in slow-frozen and vitrified cells including buckling of actin filaments, reduction of F-actin content and filament shortening. These alterations indicate limited functionality of the respective cells. However, there are substantial differences in the frequency and time dependence of F-actin disruptions among the applied cryopreservation strategies; immediately after thawing, cytoskeletal structures show least disruption after slow freezing at a rate of 1°C/min. As post-thaw recovery progresses, the ratio of cells with actin disruptions increases, particularly in slow frozen cells. After 120 min of recovery the proportion of cells with an intact actin cytoskeleton is higher in vitrified than in slow frozen cells. Freezing at 10°C/min is associated with a high ratio of impaired cells throughout the post-thawing culture.

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<![CDATA[Cryopreservation of the Mediterranean fruit fly (Diptera: Tephritidae) VIENNA 8 genetic sexing strain: No effect on large scale production of high quality sterile males for SIT applications]]> https://www.researchpad.co/article/5c57e685d5eed0c484ef3571

The sterile insect technique (SIT) integrated in area-wide integrated pest management (AW-IPM) programmes is being used for the successful management of the Mediterranean fruit fly Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) which is a horticultural pest of economic importance in tropical and subtropical countries. All programmes with an SIT component are using the VIENNA genetic sexing strains (GSS), mainly the VIENNA 8 GSS, which have been developed by applying classical genetic approaches. The VIENNA 8 GSS carries two selectable markers, the white pupae and the temperature sensitive lethal genes, which allows the production and release of only males thus increasing the biological efficiency and cost effectiveness of SIT applications. However, mass rearing may affect quality traits of the GSS, in which case replenishment of the colony with wild flies is recommended, a process which is tedious and time consuming. We previously reported the development of a cryopreservation protocol for the VIENNA 8D53+ strain. In the present study, we report on the evaluation of the cryopreserved strain VIENNA 8D53+/Cryo-228L, reared under semi mass rearing conditions, for production parameters, quality control indices and mating competitiveness of males, in a comparative way with the non-cryopreserved VIENNA 8D53+ strain, against wild type males. The VIENNA 8D53+ and VIENNA 8D53+/Cryo-228L strains were similar for production parameters viz. egg production, pupal production, pupal recovery, and quality control indices like fly emergence, sex ratio and flight ability. Males from both strains were equally competitive with males of the wild type strain in achieving mating with wild type females under field cage conditions. Results are discussed in the context of cryopreservation as a potential backup strategy for refreshing the mass rearing colony with biological material from a cryopreserved stock.

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<![CDATA[Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes]]> https://www.researchpad.co/article/5c26977bd5eed0c48470fa8f

Intact red blood cells (RBCs) are required for phenotypic analyses. In order to allow separation (time and location) between subject encounter and sample analysis, we developed a research-specific RBC cryopreservation protocol and assessed its impact on data fidelity for key biochemical and physiological assays. RBCs drawn from healthy volunteers were aliquotted for immediate analysis or following glycerol-based cryopreservation, thawing, and deglycerolization. RBC phenotype was assessed by (1) scanning electron microscopy (SEM) imaging and standard morphometric RBC indices, (2) osmotic fragility, (3) deformability, (4) endothelial adhesion, (5) oxygen (O2) affinity, (6) ability to regulate hypoxic vasodilation, (7) nitric oxide (NO) content, (8) metabolomic phenotyping (at steady state, tracing with [1,2,3-13C3]glucose ± oxidative challenge with superoxide thermal source; SOTS-1), as well as in vivo quantification (following human to mouse RBC xenotransfusion) of (9) blood oxygenation content mapping and flow dynamics (velocity and adhesion). Our revised glycerolization protocol (40% v/v final) resulted in >98.5% RBC recovery following freezing (-80°C) and thawing (37°C), with no difference compared to the standard reported method (40% w/v final). Full deglycerolization (>99.9% glycerol removal) of 40% v/v final samples resulted in total cumulative lysis of ~8%, compared to ~12–15% with the standard method. The post cryopreservation/deglycerolization RBC phenotype was indistinguishable from that for fresh RBCs with regard to physical RBC parameters (morphology, volume, and density), osmotic fragility, deformability, endothelial adhesivity, O2 affinity, vasoregulation, metabolomics, and flow dynamics. These results indicate that RBC cryopreservation/deglycerolization in 40% v/v glycerol final does not significantly impact RBC phenotype (compared to fresh cells).

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<![CDATA[Comparison of GnRH agonist and hCG for priming in vitro maturation cycles in cancer patients undergoing urgent fertility preservation]]> https://www.researchpad.co/article/5c12cf00d5eed0c484913cde

In vitro maturation (IVM) of oocytes retrieved at germinal vesicle or Metaphase I stage, followed by vitrification of Metaphase II (MII) oocytes, has recently emerged as an option for urgent fertility preservation (FP). Priming is usually achieved with an injection of hCG, 10,000 IU, 36 hours before retrieval. This study aimed to assess a new method of priming, using GnRH agonists, and compare it to hCG, in cancer patients undergoing urgent FP. From 2009 to 2015, 373 cancer patients underwent MII oocyte cryopreservation after IVM cycles primed either with GnRHa (triptorelin 0.2 mg) (n = 138) or hCG (10,000 IU) (n = 235). Patients’ characteristics were comparable between the two groups. The number of COC retrieved was significantly higher in the GnRHa group (9.1 ± 6.8 versus 7.7 ± 5.5 oocytes, p = 0.04). However, the maturation rates (59 ±25% versus 64 ±26%, p = 0.07, respectively), and the total number of MII oocytes frozen (5.2 ±4.2 versus 4.9 ±4.0, p = 0.6, respectively) were similar between the GnRha and hCG groups. We did not find any difference between GnRHa and hCG priming for IVM. GnRHa priming is more physiological since it stimulates endogenous FSH and LH activity, and is well suited for FP in hormone-sensitive cancers and urgent cases.

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<![CDATA[From the Past to the Future: Natural Sound Recordings and the Preservation of the Bioacoustics Legacy in Portugal]]> https://www.researchpad.co/article/5989da7bab0ee8fa60b98a0a

The preservation of historical and contemporary data safeguards our scientific legacy. Bioacoustic recordings can have historical as well as scientific value and should be assessed for their conservation requirements. Unpreserved bioacoustics recordings are generally not referenced and are frequently at high risk of loss by material degradation and/or by misplacement. In this study we investigated the preservation status of sets of natural sound recordings made in Portugal from 1983 until 2010 inclusive. We evaluated the recordings on the basis of their rate of loss, the degree to which unpreserved recordings could be preserved, and their risk of loss. Recordists of animal sounds were surveyed (by questionnaire or interview) to identify sets of recordings and to collect information on their quality and state of preservation. Of the 78 recordists identified, we found that 32% of the recordings have an unclear status and that only 9% of the recordings are lost. Of the c. 6 terabytes of unpreserved sound recordings discovered, an estimated 49% were recoverable. Moreover, 95% of the recoverable sets of recordings were at high risk of loss by their being misplaced. These risks can be minimized if recordists are persuaded to deposit their material in an institution committed to long-term curation of such data (e.g. sound archives). Overall, the study identified a considerable body of unpreserved animal sound recordings that could contribute to our scientific heritage and knowledge of the biodiversity found in Portugal. It highlights the need to implement effective policies to promote the deposit of recordings for preservation and to reverse the present scenario so that scientific material can be preserved for future generations.

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<![CDATA[MALDI Imaging Mass Spectrometry Profiling of N-Glycans in Formalin-Fixed Paraffin Embedded Clinical Tissue Blocks and Tissue Microarrays]]> https://www.researchpad.co/article/5989da27ab0ee8fa60b80eb3

A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.

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<![CDATA[Isolation of human explant derived cardiac stem cells from cryopreserved heart tissue]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc79f

The value of preserving high quality bio specimens for fundamental research is significant as linking cellular and molecular changes to clinical and epidemiological data has fueled many recent advances in medicine. Unfortunately, storage of traditional biospecimens is limited to fixed samples or isolated genetic material. Here, we report the effect of cryopreservation of routine myocardial biopsies on explant derived cardiac stem cell (EDC) culture outcomes. We demonstrate that immediate cryopreservation or delayed cryopreservation after suspension within cardioplegia for 12 hours did not alter EDC yields, proliferative capacity, antigenic phenotype or paracrine signature. Cryopreservation had negligible effects on the ability of EDCs to adopt a cardiac lineage, stimulate new vessel growth, attract circulating angiogenic cells and repair injured myocardium. Finally, cryopreservation did not influence the ability of EDCs to undergo genetic reprogramming into inducible pluripotent stem cells. This study establishes a means of storing cardiac samples as a retrievable live cell source for cardiac repair or disease modeling.

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<![CDATA[Rescuing Perishable Neuroanatomical Information from a Threatened Biodiversity Hotspot: Remote Field Methods for Brain Tissue Preservation Validated by Cytoarchitectonic Analysis, Immunohistochemistry, and X-Ray Microcomputed Tomography]]> https://www.researchpad.co/article/5989da0eab0ee8fa60b78b04

Biodiversity hotspots, which harbor more endemic species than elsewhere on Earth, are increasingly threatened. There is a need to accelerate collection efforts in these regions before threatened or endangered species become extinct. The diverse geographical, ecological, genetic, morphological, and behavioral data generated from the on-site collection of an individual specimen are useful for many scientific purposes. However, traditional methods for specimen preparation in the field do not permit researchers to retrieve neuroanatomical data, disregarding potentially useful data for increasing our understanding of brain diversity. These data have helped clarify brain evolution, deciphered relationships between structure and function, and revealed constraints and selective pressures that provide context about the evolution of complex behavior. Here, we report our field-testing of two commonly used laboratory-based techniques for brain preservation while on a collecting expedition in the Congo Basin and Albertine Rift, two poorly known regions associated with the Eastern Afromontane biodiversity hotspot. First, we found that transcardial perfusion fixation and long-term brain storage, conducted in remote field conditions with no access to cold storage laboratory equipment, had no observable impact on cytoarchitectural features of lizard brain tissue when compared to lizard brain tissue processed under laboratory conditions. Second, field-perfused brain tissue subjected to prolonged post-fixation remained readily compatible with subsequent immunohistochemical detection of neural antigens, with immunostaining that was comparable to that of laboratory-perfused brain tissue. Third, immersion-fixation of lizard brains, prepared under identical environmental conditions, was readily compatible with subsequent iodine-enhanced X-ray microcomputed tomography, which facilitated the non-destructive imaging of the intact brain within its skull. In summary, we have validated multiple approaches to preserving intact lizard brains in remote field conditions with limited access to supplies and a high degree of environmental exposure. This protocol should serve as a malleable framework for researchers attempting to rescue perishable and irreplaceable morphological and molecular data from regions of disappearing biodiversity. Our approach can be harnessed to extend the numbers of species being actively studied by the neuroscience community, by reducing some of the difficulty associated with acquiring brains of animal species that are not readily available in captivity.

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<![CDATA[Effect of Embryo Banking on U.S. National Assisted Reproductive Technology Live Birth Rates]]> https://www.researchpad.co/article/5989db44ab0ee8fa60bd7f4e

Background

Assisted Reproductive Technology (ART) reports generated by the Centers for Disease Control and Prevention (CDC) exclude embryo banking cycles from outcome calculations.

Methods

We examined data reported to the CDC in 2013 for the impact of embryo banking exclusion on national ART outcomes by recalculating autologous oocyte ART live birth rates. Inflation of reported fresh ART cycle live birth rates was assessed for all age groups of infertile women as the difference between fresh cycle live births with reference to number of initiated fresh cycles (excluding embryo banking cycles), as typically reported by the CDC, and fresh cycle live births with reference to total initiated fresh ART cycles (including embryo banking cycles).

Results

During 2013, out of 121,351 fresh non-donor ART cycles 27,564 (22.7%) involved embryo banking. The proportion of banking cycles increased with female age from 15.5% in women <35 years to 56.5% in women >44 years. Concomitantly, the proportion of thawed cycles decreased with advancing female age (P <0.0001). Exclusion of embryo banking cycles led to inflation of live birth rates in fresh ART cycles, increasing in size in parallel to advancing female age and utilization of embryo banking, reaching 56.3% in women age >44. The inflation of live birth rates in thawed cycles could not be calculated from the publically available CDC data but appears to be even greater.

Conclusions

Utilization of embryo banking increased during 2013 with advancing female age, suggesting a potential age selection bias. Exclusion of embryo banking cycles from national ART outcome reports significantly inflated national ART success rates, especially among older women.

Précis

Exclusion of embryo banking cycles from US National Assisted Reproductive Technology outcome reports significantly inflates reported success rates especially in older women.

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<![CDATA[Conditioned Medium from Placental Mesenchymal Stem Cells Reduces Oxidative Stress during the Cryopreservation of Ex Vivo Expanded Umbilical Cord Blood Cells]]> https://www.researchpad.co/article/5989db06ab0ee8fa60bc84de

Background

The limited cell dose in umbilical cord blood (UCB) necessitates ex vivo expansion of UCB. Further, the effective cryopreservation of these expanded cells is important in widening their use in the clinics. During cryopreservation, cells experience oxidative stress due to the generation of reactive oxygen species (ROS). Conditioned medium from mesenchymal stem cells (MSCs-CM) has been shown to alleviate the oxidative stress during wound healing, Alzheimer’s disease and ischemic disease. This premise prompted us to investigate the influence of MSCs-CM during cryopreservation of expanded UCB cells.

Methodology/Principle findings

CM-was collected from cord/placental MSCs(C-MSCs-CM, P-MSC-CM). UCB CD34+cells were expanded as suspension cultures in serum free medium containing cytokines for 10 days. Cells were frozen with/without C-MSCs-CM and or P-MSCs-CM in the conventional freezing medium containing 20%FCS +10%DMSO using a programmable freezer and stored in liquid nitrogen. Upon revival, cells frozen with MSCs-CM were found to be superior to cells frozen in conventional medium in terms of viability, CD34+content and clonogenecity. Priming of revived cells for 48 hrs with MSCs-CM further improved their transplantation ability, as compared to those cultured without MSCs-CM. P-MSCs-CM radically reduced the oxidative stress in cryopreserved cells, resulting in better post thaw functionality of CD34+ cells than with C-MSCs-CM. The observed cryoprotective effect of MSCs-CM was primarily due to anti-oxidative and anti-apoptotic properties of the MSCs-CM and not because of the exosomes secreted by them.

Conclusions/Significance

Our data suggest that MSCs-CM can serve as a valuable additive to the freezing or the priming medium for expanded UCB cells, which would increase their clinical applicability.

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<![CDATA[Pathogen Inactivating Properties and Increased Sensitivity in Molecular Diagnostics by PAXgene, a Novel Non-Crosslinking Tissue Fixative]]> https://www.researchpad.co/article/5989da86ab0ee8fa60b9c4be

Background

Requirements on tissue fixatives are getting more demanding as molecular analysis becomes increasingly relevant for routine diagnostics. Buffered formaldehyde in pathology laboratories for tissue fixation is known to cause chemical modifications of biomolecules which affect molecular testing. A novel non-crosslinking tissue preservation technology, PAXgene Tissue (PAXgene), was developed to preserve the integrity of nucleic acids in a comparable way to cryopreservation and also to preserve morphological features comparable to those of formalin fixed samples.

Methods

Because of the excellent preservation of biomolecules by PAXgene we investigated its pathogen inactivation ability and biosafety in comparison to formalin by in-vitro testing of bacteria, human relevant fungi and human cytomegalovirus (CMV). Guidelines for testing disinfectants served as reference for inactivation assays. Furthermore, we tested the properties of PAXgene for detection of pathogens by PCR based assays.

Results

All microorganisms tested were similarly inactivated by PAXgene and formalin except Clostridium sporogenes, which remained viable in seven out of ten assays after PAXgene treatment and in three out of ten assays after formalin fixation. The findings suggest that similar biosafety measures can be applied for PAXgene and formalin fixed samples. Detection of pathogens in PCR-based diagnostics using two CMV assays resulted in a reduction of four to ten quantification cycles of PAXgene treated samples which is a remarkable increase of sensitivity.

Conclusion

PAXgene fixation might be superior to formalin fixation when molecular diagnostics and highly sensitive detection of pathogens is required in parallel to morphology assessment.

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<![CDATA[Unexpected Decrease in Milk Production after Fenbendazole Treatment of Dairy Cows during Early Grazing Season]]> https://www.researchpad.co/article/5989db20ab0ee8fa60bcf39c

Gastrointestinal nematodes (GIN) infection can impair milk production (MP) in dairy cows. To investigate whether MP would be optimized by spring targeted-selective anthelmintic treatment in grazing cows, we assessed (1) the effect on MP of an anthelmintic treatment applied 1.5 to 2 months after turn-out, and (2) herd and individual indicators associated with the post-treatment MP response. A randomized controlled clinical trial was conducted in 13 dairy farms (578 cows) in western France in spring 2012. In each herd, lactating cows of the treatment group received fenbendazole orally, control cows remained untreated. Daily cow MP was recorded from 2 weeks before until 15 weeks after treatment. Individual serum pepsinogen and anti-Ostertagia antibody levels (expressed as ODR), faecal egg count and bulk tank milk (BTM) Ostertagia ODR were measured at treatment time. Anthelmintic treatment applied during the previous housing period was recorded for each cow. In each herd, information regarding heifers’ grazing and anthelmintic treatment history was collected to assess the Time of Effective Contact (TEC, in months) with GIN infective larvae before the first calving. The effect of treatment on weekly MP averages and its relationships with herd and individual indicators were studied using linear mixed models with two nested random effects (cow within herd). Unexpectedly, spring treatment had a significant detrimental effect on MP (-0.92 kg/cow/day on average). This negative MP response was particularly marked in high producing cows, in cows not treated during the previous housing period or with high pepsinogen levels, and in cows from herds with a high TEC or a high BTM ODR. This post-treatment decrease in MP may be associated with immuno-inflammatory mechanisms. Until further studies can assess whether this unexpected result can be generalized, non-persistent treatment of immunized adult dairy cows against GIN should not be recommended in early grazing season.

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<![CDATA[Stimulus-triggered enhancement of chilling tolerance in zebrafish embryos]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdb9d6

Background

Cryopreservation of zebrafish embryos is still an unsolved problem despite market demand and massive efforts to preserve genetic variation among numerous existing lines. Chilled storage of embryos might be a step towards developing successful cryopreservation, but no methods to date have worked.

Methods

In the present study, we applied a novel strategy to improve the chilling tolerance of zebrafish embryos by introducing a preconditioning hydrostatic pressure treatment to the embryos. In our experiments, 26-somites and Prim-5 stage zebrafish embryos were chilled at 0°C for 24 hours after preconditioning. Embryo survival rate, ability to reach maturation and fertilizing capacity were tested.

Results

Our results indicate that applied preconditioning technology made it possible for the chilled embryos to develop normally until maturity, and to produce healthy offspring as normal, thus passing on their genetic material successfully. Treated embryos had a significantly higher survival and better developmental rate, moreover the treated group had a higher ratio of normal morphology during continued development. While all controls from chilled embryos died by 30 day-post-fertilization, the treated group reached maturity (~90–120 days) and were able to reproduce, resulting in offspring in expected quantity and quality.

Conclusions

Based on our results, we conclude that the preconditioning technology represents a significant improvement in zebrafish embryo chilling tolerance, thus enabling a long-time survival. Furthermore, as embryonic development is arrested during chilled storage this technology also provides a solution to synchronize or delay the development.

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<![CDATA[Operator Dependent Choice of Prostate Cancer Biopsy Has Limited Impact on a Gene Signature Analysis for the Highly Expressed Genes IGFBP3 and F3 in Prostate Cancer Epithelial Cells]]> https://www.researchpad.co/article/5989daf4ab0ee8fa60bc24db

Background

Predicting the prognosis of prostate cancer disease through gene expression analysis is receiving increasing interest. In many cases, such analyses are based on formalin-fixed, paraffin embedded (FFPE) core needle biopsy material on which Gleason grading for diagnosis has been conducted. Since each patient typically has multiple biopsy samples, and since Gleason grading is an operator dependent procedure known to be difficult, the impact of the operator's choice of biopsy was evaluated.

Methods

Multiple biopsy samples from 43 patients were evaluated using a previously reported gene signature of IGFBP3, F3 and VGLL3 with potential prognostic value in estimating overall survival at diagnosis of prostate cancer. A four multiplex one-step qRT-PCR test kit, designed and optimized for measuring the signature in FFPE core needle biopsy samples was used. Concordance of gene expression levels between primary and secondary Gleason tumor patterns, as well as benign tissue specimens, was analyzed.

Results

The gene expression levels of IGFBP3 and F3 in prostate cancer epithelial cell-containing tissue representing the primary and secondary Gleason patterns were high and consistent, while the low expressed VGLL3 showed more variation in its expression levels.

Conclusion

The assessment of IGFBP3 and F3 gene expression levels in prostate cancer tissue is independent of Gleason patterns, meaning that the impact of operator's choice of biopsy is low.

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<![CDATA[Long-term cryopreservation of decellularised oesophagi for tissue engineering clinical application]]> https://www.researchpad.co/article/5989db5dab0ee8fa60be04f1

Oesophageal tissue engineering is a therapeutic alternative when oesophageal replacement is required. Decellularised scaffolds are ideal as they are derived from tissue-specific extracellular matrix and are non-immunogenic. However, appropriate preservation may significantly affect scaffold behaviour. Here we aim to prove that an effective method for short- and long-term preservation can be applied to tissue engineered products allowing their translation to clinical application. Rabbit oesophagi were decellularised using the detergent-enzymatic treatment (DET), a combination of deionised water, sodium deoxycholate and DNase-I. Samples were stored in phosphate-buffered saline solution at 4°C (4°C) or slow cooled in medium with 10% Me2SO at -1°C/min followed by storage in liquid nitrogen (SCM). Structural and functional analyses were performed prior to and after 2 and 4 weeks and 3 and 6 months of storage under each condition. Efficient decellularisation was achieved after 2 cycles of DET as determined with histology and DNA quantification, with preservation of the ECM. Only the SCM method, commonly used for cell storage, maintained the architecture and biomechanical properties of the scaffold up to 6 months. On the contrary, 4°C method was effective for short-term storage but led to a progressive distortion and degradation of the tissue architecture at the following time points. Efficient storage allows a timely use of decellularised oesophagi, essential for clinical translation. Here we describe that slow cooling with cryoprotectant solution in liquid nitrogen vapour leads to reliable long-term storage of decellularised oesophageal scaffolds for tissue engineering purposes.

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<![CDATA[Does Enhanced External Counterpulsation (EECP) Significantly Affect Myocardial Perfusion?: A Systematic Review & Meta-Analysis]]> https://www.researchpad.co/article/5989daf0ab0ee8fa60bc0e49

Background

Enhanced external counterpulsation (EECP) is currently applied for treating coronary artery disease (CAD) patients. However, the mechanism(s) by which EECP ameliorates angina pectoris and long-term left ventricular function remain largely unknown. The aim of this study will be to assess whether EECP significantly affects myocardial perfusion in CAD patients through a systematic review and meta-analysis of the available literature.

Methods

MEDLINE, EMBASE, and Cochrane CENTRAL databases were searched for prospective studies on CAD patients that underwent EECP and reported myocardial perfusion data pre- and post-EECP. The impact of EECP was assessed based on the weighted mean difference (WMD) in myocardial perfusion from pre-EECP to post-EECP. Statistical heterogeneity was assessed by the I2 index. Publication bias was assessed through visual inspection of the funnel plot as well as Begg’s and Egger’s testing.

Results

Standard EECP therapy (i.e., 35–36 one-hour sessions within a seven-week period) significantly increased myocardial perfusion in CAD patients (pooled WMD: -0.19, 95% CI: -0.38 to 0.00, p = 0.049). A random effects analysis was applied on account of significant heterogeneity (I2 = 89.1%, p = 0.000). There was no evidence of significant publication bias (Begg’s p = 0.091; Egger’s p = 0.282).

Conclusions

Standard EECP therapy significantly increases myocardial perfusion in CAD patients. This study’s findings support the continued use of standard EECP therapy in CAD patients and provides one putative physiological mechanism to help explain the improvements in angina pectoris and long-term left ventricular function observed in CAD patients after EECP therapy.

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<![CDATA[Raman-microscopy investigation of vitrification-induced structural damages in mature bovine oocytes]]> https://www.researchpad.co/article/5989db5cab0ee8fa60bdff6e

Although oocyte cryopreservation has great potentials in the field of reproductive technologies, it still is an open challenge in the majority of domestic animals and little is known on the biochemical transformation induced by this process in the different cellular compartments. Raman micro-spectroscopy allows the non-invasive evaluation of the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. The aim of this work was to assess the biochemical modifications of both the zona pellucida and cytoplasm of vitrified/warmed in vitro matured bovine oocytes at different post-warming times. By taking advantage of Principal Component Analysis, we were able to shed light on the biochemical transformation induced by the cryogenic treatment, also pointing out the specific role of cryoprotective agents (CPs). Our results suggest that vitrification induces a transformation of the protein secondary structure from the α-helices to the β-sheet form, while lipids tend to assume a more packed configuration in the zona pellucida. Both modifications result in a mechanical hardening of this cellular compartment, which could account for the reduced fertility rates of vitrified oocytes. Furthermore, biochemical modifications were observed at the cytoplasmic level in the protein secondary structure, with α-helices loss, suggesting cold protein denaturation. In addition, a decrease of lipid unsaturation was found in vitrified oocytes, suggesting oxidative damages. Interestingly, most modifications were not observed in oocytes exposed to CPs, suggesting that they do not severely affect the biochemical architecture of the oocyte. Nevertheless, in oocytes exposed to CPs decreased developmental competence and increased reactive oxygen species production were observed compared to the control. A more severe reduction of cleavage and blastocyst rates after in vitro fertilization was obtained from vitrified oocytes. Our experimental outcomes also suggest a certain degree of reversibility of the induced transformations, which renders vitrified oocytes more similar to untreated cells after 2 h warming.

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<![CDATA[Diagnostic Accuracy of the Enhanced Liver Fibrosis (ELF®) Score Using HCV-Infected Serum Samples Cryopreserved for up to 25 Years]]> https://www.researchpad.co/article/5989da6cab0ee8fa60b931b8

Introduction & Aims

Cryopreservation of serum samples is a standard procedure for biomedical research in tertiary centers. However, studies evaluating the long-term biological stability of direct liver fibrosis markers using cryopreserved samples are scarce.

Methods

We compared the stability of hyaluronic acid (HA), tissue inhibitor of metalloproteinases (TIMP-1) and amino-terminal propeptide of type III procollagen (PIIINP) in 225 frozen serum samples of HCV-infected patients with a paired liver biopsy for up to 25 years (1990–2014). Moreover, we assessed the diagnostic accuracy (AUROC) of the Enhanced Liver Fibrosis (ELF®) score to identify significant fibrosis (F2-4) and its predictive capacity to identify clinical events during follow-up.

Results

Seventy-six patients (39,8%) had mild fibrosis (F0-1) and 115 (60,2%) significant fibrosis (F2-4). HA, PIIINP and TIMP-1 values remained stable during the period from 1995 to 2014 while those of 1990–94 were slightly higher. We did not find significant differences in the median ELF® values during the 20-year period from 1995–2014 in patients with mild (from 8,4 to 8,7) and significant fibrosis (from 9,9 to 10,9) (p = ns between periods and fibrosis stages). The AUROCs of ELF® to identify significant fibrosis were high in all the periods (from 0,85 to 0,91). The ELF® score showed a good predictive capability to identify clinical events during follow-up.

Conclusions

The biological stability of direct serum markers (HA, PIIINP and TIMP-1) using HCV-infected samples cryopreserved for 20 years is good. Therefore, the diagnostic accuracy of the ELF® score to identify significant fibrosis and clinical events during follow-up is very high.

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<![CDATA[Functional Tissue Analysis Reveals Successful Cryopreservation of Human Osteoarthritic Synovium]]> https://www.researchpad.co/article/5989da51ab0ee8fa60b8e0ac

Osteoarthritis (OA) is a degenerative joint disease affecting cartilage and is the most common form of arthritis worldwide. One third of OA patients have severe synovitis and less than 10% have no evidence of synovitis. Moreover, synovitis is predictive for more severe disease progression. This offers a target for therapy but more research on the pathophysiological processes in the synovial tissue of these patients is needed. Functional studies performed with synovial tissue will be more approachable when this material, that becomes available by joint replacement surgery, can be stored for later use. We set out to determine the consequences of slow-freezing of human OA synovial tissue. Therefore, we validated a method that can be applied in every routine laboratory and performed a comparative study of five cryoprotective agent (CPA) solutions. To determine possible deleterious cryopreservation-thaw effects on viability, the synovial tissue architecture, metabolic activity, RNA quality, expression of cryopreservation associated stress genes, and expression of OA characteristic disease genes was studied. Furthermore, the biological activity of the cryopreserved tissue was determined by measuring cytokine secretion induced by the TLR ligands lipopolysaccharides and Pam3Cys. Compared to non frozen synovium, no difference in cell and tissue morphology could be identified in the conditions using the CS10, standard and CryoSFM CPA solution for cryopreservation. However, we observed significantly lower preservation of tissue morphology with the Biofreeze and CS2 media. The other viability assays showed trends in the same direction but were not sensitive enough to detect significant differences between conditions. In all assays tested a clearly lower viability was detected in the condition in which synovium was frozen without CPA solution. This detailed analysis showed that OA synovial tissue explants can be cryopreserved while maintaining the morphology, viability and phenotypical response after thawing, offering enhanced opportunities for human in vitro studies.

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