Cyto-architecture of Byblis glands and leaf cells based on freeze-substitution and conventional TEM.

BACKGROUND AND AIMS: Byblis liniflora (Byblidaceae) is a carnivorous plant that has developed sticky fly paper traps with two types of glandular trichomes producing digestive enzymes and sticky mucilage. This study aimed to analyze the ultrastructure of these glandular leaf trichomes based on rapid freeze fixation and conventional chemical fixation in the attempt to understand their functional contribution to the carnivorous performance of the plants. METHODS: The Byblis cells were studied in TEM, SEM and STEM using cryo techniques for fixation and substitution in addition to conventional chemical fixation. KEY RESULTS: We show in detail the architecture of both the digestive glands and the mucilage glands with their relevant sets of organelles. Both mitochondria and plastids have a conspicuous plasticity, with branches and constrictions, and they associate to form clusters. The glandular cells appear to be transfer cells with cell wall ingrowths. Digestive glands occur in different states of development. Their cuticle forms discontinuities which are unique among glands of carnivorous plants. They look like cuticular holes -- the cuticle separates from the cell wall in only one spot and then ruptures. Cuticular discontinuities thus differ from cuticular gaps and cuticular pores so far described in carnivorous plants. We therefore propose for them the term cuticular holes. CONCLUSIONS: Application of cryo-techniques made it possible to show the true structure of the cell wall and the relationship between cell wall ingrowths and organelles, as well as the morphology and structure of organelles and their associations.

Delivery of a Healthy Child Through International Gestational Surrogacy 10 Years Following Female Fertility Preservation and In Vitro Fertilization (IVF) for Recurrent Breast Cancer: A Case Report.

Assisted reproduction technology (ART) has made considerable progress in recent years; in particular with regard to cryopreservation, long-term storage, successful thawing, and embryo transfer of cryopreserved embryos. Regarding gestational surrogacy, progress has been made in the areas of awareness, social acceptance, regulation, legislation, availability, streamlining, and optimization of cross-border care. The above is being highlighted in the current presentation of a particularly challenging and novel case. A 43-year-old woman visited our clinic in Greece, seeking international gestational surrogacy due to recurrent breast cancer which rendered her medically unfit for pregnancy. Ten years before her initial visit to our clinic the patient had undergone fertility preservation due to breast cancer, her oocytes had been fertilized with her husband's sperm, and the embryos were cryopreserved and stored in a fertility clinic based in the United Kingdom. The stored embryos were transported to Greece, thawed, and successfully implanted to the selected gestational surrogate. Following an uneventful pregnancy, the surrogate delivered a healthy girl. This successful outcome exemplified innovation, motivation, and hope and may represent a paradigm of team scientific excellence associated with positive patient outcomes. Furthermore, this case constitutes the successful culmination of major advances made in various different sectors of cross-border reproductive care; laboratory, clinical, legal, ethical, and logistical.

Turner Syndrome and Fertility.

Turner syndrome (TS) is tightly associated with hypergonadotropic hypogonadism and ovarian dysgenesis, typically resulting in infertility in the great majority of patients. Therefore females with TS are usually treated with female sex steroids from 11-12years of age until the normal age of natural menopause of around 53-54years of age. Infertility is rated among females with TS as a distressing concern and a detractor from a good quality of life. Options for motherhood for females with TS has expanded during recent years. Originally, only adoption was an option, unless of course for the small minority of TS females that still has ovarian function and are capable of achieving pregnancy through normal means. Oocyte donation has become the mainstream option in many countries and seems to work well, especially if patients have been treated with optimal estrogen and gestagen for a prolonged time before the intervention. It comes with an increased risk of cardiovascular complications and TS oocyte donation pregnancies are viewed as high risk pregnancies necessitating increased vigilance. Oocyte cryopreservation of own oocytes is also becoming an option in a select group of TS and has special challenges. Ovarian tissue cryopreservation is a promising new techniques that has been applied successfully in children with cancer. Currently, several trials are running around the world evaluating this techniques in TS. The genetics and genomics behind the ovarian dysgenesis seen in TS is not understood, but new studies have elucidated global changes in DNA methylation and RNA expression in blood from persons with TS and it is likely that similar changes are present in the ovaries. We still, however, need more thorough research to fully uncover the genetic background of ovarian failure in TS. Gene expression studies and methylation analysis from ovarian TS tissues still needs to be performed.

State of the Art in Fertility Preservation for Female Patients Prior to Oncologic Therapies.

Quality of life improvement stands as one of the main goals of the medical sciences. Increasing cancer survival rates associated with better early detection and extended therapeutic options led to the specific modeling of patients' choices, comprising aspects of reproductive life that correlated with the evolution of modern society, and requires better assessment. Of these, fertility preservation and ovarian function conservation for pre-menopause female oncologic patients pose a contemporary challenge due to procreation age advance in evolved societies and to the growing expectations regarding cancer treatment. Progress made in cell and tissue-freezing technologies brought hope and shed new light on the onco-fertility field. Additionally, crossing roads with general fertility and senescence studies proved highly beneficial due to the enlarged scope and better synergies and funding. We here strive to bring attention to this domain of care and to sensitize all medical specialties towards a more cohesive approach and to better communication among caregivers and patients.

Preparation of a standardised faecal slurry for ex-vivo microbiota studies which reduces inter-individual donor bias.

BACKGROUND: In-vitro gut fermentation systems provide suitable models for studying gut microbiota composition and functionality. However, such methods depend on the availability of donors and the assumption of reproducibility between microbial communities before experimental treatments commence. The aim of this study was to develop a frozen standardised inoculum (FSI) which minimizes inter-individual variation and to determine its stability over time using culture-dependent and culture-independent techniques. RESULTS: A method for the preparation difference of a FSI is described which involves pooling the faecal samples, centrifugation and pelleting of the cell biomass and finally homogenising the cell pellets with phosphate buffer and glycerol. Using this approach, no significant difference in total anaerobe cell viability was observed between the fresh standardised inoculum (before freezing) and the 12days post freezing FSI. Moreover, Quantitative PCR revealed no significant alterations in the estimated bacterial numbers in the FSI preparations for any of the phyla. MiSeq sequencing revealed minute differences in the relative abundance at phylum, family and genus levels between the FSI preparations. Differences in the microbiota denoted as significant were limited between preparations in the majority of cases to changes in percentage relative abundance of ±0.5%. The independently prepared FSIs revealed a high degree of reproducibility in terms of microbial composition between the three preparations. CONCLUSIONS: This study provides a method to produce a standardised human faecal inoculum suitable for freezing. Based on culture-dependent and independent analysis, the method ensures a degree of reproducibility between preparations by lessening the effect of inter-individual variation among the donors, thereby making the system more suitable for the accurate interpretation of the effects of experimental treatments.

Criopreservación de espermatozoides de canino con extracto de hojas de arándano (Vaccinium corymbosum L. ) / Cryopreservation of canine spermatozoa with blueberry leaf extract (Vaccinium corymbosum L. )

La criopreservación espermática induce daño por estrés oxidativo en las células, lo que conlleva a un deterioro de la calidad del semen descongelado. Los espermatozoides pueden ser protegidos de este daño, por la adición de antioxidantes al medio de congelación. El objetivo de este estudio fue determinar el efecto de la adición de extracto de hojas de arándano (EHA) al medio de congelación, sobre la calidad de espermatozoides de canino criopreservados. Espermatozoides desprovistos del plasma seminal fueron congelados con diferentes concentraciones de EHA (0 %, control; 1 %, EHA1; 2 %, EHA2; 4 %, EHA4 y 6 %, EHA6) adicionadas al medio de congelación. Post descongelación se evalúo la motilidad progresiva; la viabilidad e integridad de la membrana plasmática (SYBR-14/PI) e integridad de la membrana acrosomal (FITC-PNA/PI) por citometría de flujo. La motilidad progresiva fue similar al control con las concentraciones de EHA1y EHA4 (P >0,05), mientras que con las concentraciones de EHA2 y EHA6 se observó una disminución significativa de este parámetro comparado con el control (P <0,01 y P <0,001 respectivamente). La adición de EHA1, EHA2 y EHA4 al medio de congelación no presentó diferencias significativas respecto al control sobre la viabilidad e integridad de la membrana plasmática (P >0,05); por el contrario, con la adición de EHA6 se observaron valores significativamente menores (P <0,001). Los valores de integridad de la membrana acrosomal, con las diferentes concentraciones de EHA no presentaron diferencias significativas respecto al control. En conclusión, los resultados obtenidos en este estudio revelaron que las concentraciones de EHA utilizadas no fueron eficaces en mejorar la calidad del semen canino descongelado.

During cryopreservation, oxidative stress damage leads to a deterioration of the quality of thawed semen, which could be reduced by the addition of antioxidants to freezing extender. This study was designed to determine the effect of the addition of blueberry leaf extract (EHA) to freezing extender, on quality of cryopreserved canine sperms. Sperm devoid from seminal plasma were frozen with different concentrations of EHA (0 %, control; 1 %, EHA1; 2 %, EHA2; 4 %, EHA4 y 6 %, EHA6) added to freezing extender. Post-thawing progressive motility was evaluated; the viability and plasma membrane integrity (SYBR-14/PI) and acrosomal membrane integrity (FITC-PNA/PI) were assessed by flow cytometry. Progressive motility was similar to the control with concentrations of EHA1 and EHA4 (P >0.05); at concentrations of EHA2 and EHA6 a significant decrease of this parameter compared to control (P <0.01and P <0.001, respectively) was observed. The addition of EHA1, EHA2 and EHA4 to the freezing extender showed no significant differences with respect to the control on viability and plasma membrane integrity (P >0.05); however with the addition of EHA6 values significantly lower (P <0.001) were exhibited. The concentrations of EHA used showed no significant differences with respect to the control on acrosome membrane integrity. In conclusion, the results of this study revealed that none of the concentrations of EHA used were effective in improving canine thawed semen quality.

Quality control of nucleic acids and protein of freeze-preserving gastric cancer samples / 中国肿瘤临床

Objective:To explore the quality of inventory samples of a biobank stored in a deep freezer from 0 to over 10 years in Shanghai Ruijin Hospital. Methods:We extracted 24 pairs of stocked gastric cancer samples between 2003 and 2014. We used 1%aga-rose gel electrophoresis to analyze DNA and RNA purity and integrity while adding the RNA integrity number (RIN) for precise analysis. Bicinchonininc acid (BCA) assay was used for protein concentration evaluation. Coomassie brilliant blue method was used for protein integrity assay. Results: The samples were divided into four groups according to cryopreservation period (9 years). No significant difference in DNA integrity was found between the groups (P>0.05);however, DNA degradation in normal gastric mucosa was faster than that in gastric cancer tissue (P=0.023). The RIN significantly declined when the storage period was 6 years or longer (P=0.018). No significant difference in protein concentration was observed between different groups. Using Coo-massie brilliant blue method, we found significant differences in preserved proteins with different molecular weights. Proteins with varying molecular weights were detected in the groups with the following cryopreservation periods:>9 years, a small number of low-molecular-weight (average 36.5 KD) proteins;6-8 years, medium-molecular-weight (average 65.63KD) proteins;3-5 years, high-molecu-lar-weight (average 127.5 KD) proteins;<2 years, high-molecular-weight (average 160 KD) proteins. Conclusion:Cryopreservation does not exert an obvious effect on DNA. If the cryopreservation period is more than 5 years, serious degradation of RNA should occur;like-wise, degradation of proteins with higher molecular weight should occur.

Biological soft X-ray tomography on beamline 2.1 at the Advanced Light Source.

Beamline 2.1 (XM-2) is a transmission soft X-ray microscope in sector 2 of the Advanced Light Source at Lawrence Berkeley National Laboratory. XM-2 was designed, built and is now operated by the National Center for X-ray Tomography as a National Institutes of Health Biomedical Technology Research Resource. XM-2 is equipped with a cryogenic rotation stage to enable tomographic data collection from cryo-preserved cells, including large mammalian cells. During data collection the specimen is illuminated with `water window' X-rays (284-543 eV). Illuminating photons are attenuated an order of magnitude more strongly by biomolecules than by water. Consequently, differences in molecular composition generate quantitative contrast in images of the specimen. Soft X-ray tomography is an information-rich three-dimensional imaging method that can be applied either as a standalone technique or as a component modality in correlative imaging studies.

The effects of cryopreservation on cells isolated from adipose, bone marrow and dental pulp tissues.

The effects of cryopreservation on mesenchymal stem cell (MSC) phenotype are not well documented; however this process is of increasing importance for regenerative therapies. This study examined the effect of cryopreservation (10% dimethyl-sulfoxide) on the morphology, viability, gene-expression and relative proportion of MSC surface-markers on cells derived from rat adipose, bone marrow and dental pulp. Cryopreservation significantly reduced the number of viable cells in bone marrow and dental pulp cell populations but had no observable effect on adipose cells. Flow cytometry analysis demonstrated significant increases in the relative expression of MSC surface-markers, CD90 and CD29/CD90 following cryopreservation. sqRT-PCR analysis of MSC gene-expression demonstrated increases in pluripotent markers for adipose and dental pulp, together with significant tissue-specific increases in CD44, CD73-CD105 following cryopreservation. Cells isolated from different tissue sources did not respond equally to cryopreservation with adipose tissue representing a more robust source of MSCs.

Biological applications of cryo-soft X-ray tomography.

X-rays are used for imaging many different types of biological specimen, ranging from live organisms to the individual cells and proteins from which they are made. The level of detail achieved as a result of the imaging varies depending on both the sample and the technique used. One of the most recent technical developments in X-ray imaging is that of the soft X-ray microscope, designed to allow the internal structure of individual biological cells to be explored. With a field of view of ∼10-20 × âˆ¼10-20 µm, a penetration depth of ∼10 µm and a resolution of ∼40 nm(3), the soft X-ray microscope neatly fits between the imaging capabilities of light and electron microscopes.

The efficacy of programmed cryo-preservation under pressure in rat periodontal ligament cells / 대한치과보존학회지

The purpose of this study was to evaluate the viability of periodontal ligament cells in rat teeth using slow cryo-preservation method under pressure by means of MTT assay and WST-1 assay. Eighteen teeth of Sprague-Dawley white female rats of 4 week-old were used for each group. Both sides of the first and second maxillary molars were extracted as atraumatically as possible under Tiletamine anesthesia. The experimental groups were group 1 (Immediate control), group 2 (Cold preservation at 4degrees C for 1 week), group 3 (Slow freezing), group 4 (Slow freezing under pressure of 3 MPa). F-medium and 10% DMSO were used as preservation medium and cryo-protectant. For cryo-preservation groups, thawing was performed in 37degrees C water bath, then MTT assay and WST-1 assay were processed. One way ANOVA and Tukey method were performed at the 95% level of confidence. The values of optical density obtained by MTT assay and WST-1 were divided by the values of eosin staining for tissue volume standardization. In both MTT and WST-1 assay, group 4 showed significantly higher viability of periodontal ligament cells than group 2 and 3 (p < 0.05), but showed lower viability than immediate control group. By the results of this study, slow cryo-preservation method under pressure suggests the possibility for long term cryo-preservation of the teeth.