Immigration Brings New Pathology with No Standardized Treatment Protocol Madura Foot Case Studies Phialemonium and Phaeoacremonium.

Madura foot is an uncommon invasive soft-tissue infection that foot and ankle specialists encounter. We present two rare cases of Phialemonium and Phaeoacremonium fungi infections of the foot diagnosed in northern California to inform physicians on the presentation and current treatment options for this unique pathology. The two cases presented outline the clinical presentations, diagnostic data, and surgical and antimicrobial interventions. There is a concentration on the antimicrobial options depending on which of the over 20 species is encountered. The pertinent literature and supporting data are reviewed to create an outline for discussion of treatment protocols when faced with these emerging opportunistic infections.

Angiomotin stabilization by tankyrase inhibitors antagonizes constitutive TEAD-dependent transcription and proliferation of human tumor cells with Hippo pathway core component mutations.

The evolutionarily conserved Hippo inhibitory pathway plays critical roles in tissue homeostasis and organ size control, while mutations affecting certain core components contribute to tumorigenesis. Here we demonstrate that proliferation of Hippo pathway mutant human tumor cells exhibiting high constitutive TEAD transcriptional activity was markedly inhibited by dominant negative TEAD4, which did not inhibit the growth of Hippo wild-type cells with low levels of regulatable TEAD-mediated transcription. The tankyrase inhibitor, XAV939, identified in a screen for inhibitors of TEAD transcriptional activity, phenocopied these effects independently of its other known functions by stabilizing angiomotin and sequestering YAP in the cytosol. We also identified one intrinsically XAV939 resistant Hippo mutant tumor line exhibiting lower and less durable angiomotin stabilization. Thus, angiomotin stabilization provides a new mechanism for targeting tumors with mutations in Hippo pathway core components as well as a biomarker for sensitivity to such therapy.

"Life in Data"--Outcome of a Multi-Disciplinary, Interactive Biobanking Conference Session on Sample Data.

INTRODUCTION: Clinical, biodiversity, and environmental biobanks share many data standards, but there is a lack of harmonization on how data are defined and used among biobank fields. This article reports the outcome of an interactive, multidisciplinary session at a meeting of the European, Middle Eastern, and African Society for Biopreservation and Biobanking (ESBB) designed to encourage a 'learning-from-each-other' approach to achieve consensus on data needs and data management across biobank communities. MATERIALS, METHODS, AND RESULTS: The Enviro-Bio and ESBBperanto Working Groups of the ESBB co-organized an interactive session at the 2013 conference (Verona, Italy), presenting data associated with biobanking processes, using examples from across different fields. One-hundred-sixty (160) diverse biobank participants were provided electronic voting devices with real-time screen display of responses to questions posed during the session. The importance of data standards and robust data management was recognized across the conference cohort, along with the need to raise awareness about these issues within and across different biobank sectors. DISCUSSION AND CONCLUSION: While interactive sessions require a commitment of time and resources, and must be carefully coordinated for consistency and continuity, they stimulate the audience to be pro-active and direct the course of the session. This effective method was used to gauge opinions about significant topics across different biobanking communities. The votes revealed the need to: (a) educate biobanks in the use of data management tools and standards, and (b) encourage a more cohesive approach for how data and samples are tracked, exchanged, and standardized across biobanking communities. Recommendations for future interactive sessions are presented based on lessons learned.

Stable heteroplasmy at the single-cell level is facilitated by intercellular exchange of mtDNA.

Eukaryotic cells carry two genomes, nuclear (nDNA) and mitochondrial (mtDNA), which are ostensibly decoupled in their replication, segregation and inheritance. It is increasingly appreciated that heteroplasmy, the occurrence of multiple mtDNA haplotypes in a cell, plays an important biological role, but its features are not well understood. Accurately determining the diversity of mtDNA has been difficult, due to the relatively small amount of mtDNA in each cell (<1% of the total DNA), the intercellular variability of mtDNA content and mtDNA pseudogenes (Numts) in nDNA. To understand the nature of heteroplasmy, we developed Mseek, a novel technique to purify and sequence mtDNA. Mseek yields high purity (>90%) mtDNA and its ability to detect rare variants is limited only by sequencing depth, providing unprecedented sensitivity and specificity. Using Mseek, we confirmed the ubiquity of heteroplasmy by analyzing mtDNA from a diverse set of cell lines and human samples. Applying Mseek to colonies derived from single cells, we find heteroplasmy is stably maintained in individual daughter cells over multiple cell divisions. We hypothesized that the stability of heteroplasmy could be facilitated by intercellular exchange of mtDNA. We explicitly demonstrate this exchange by co-culturing cell lines with distinct mtDNA haplotypes. Our results shed new light on the maintenance of heteroplasmy and provide a novel platform to investigate features of heteroplasmy in normal and diseased states.

Writing standard operating procedures (SOPs) for cryostorage protocols: using shoot meristem cryopreservation as an example.

Standard operating procedures are a systematic way of making sure that biopreservation processes, tasks, protocols, and operations are correctly and consistently performed. They are the basic documents of biorepository quality management systems and are used in quality assurance, control, and improvement. Methodologies for constructing workflows and writing standard operating procedures and work instructions are described using a plant cryopreservation protocol as an example. This chapter is pertinent to other biopreservation sectors because how methods are written, interpreted, and implemented can affect the quality of storage outcomes.

Cryopreservation of shoot tips and meristems: an overview of contemporary methodologies.

Cryopreservation is the storage of viable bioresources at ultra-low temperatures in liquid nitrogen (LN). This chapter provides an overview of those protocols most commonly used to cryopreserve in vitro derived shoot tips and meristems; they are described generically, as sequential technical steps, including preparative and cryogenic treatments and the morphogenetic assessment of recovery. The importance of translating research-generated methods into formal Standard Operating Procedures (SOPs) is considered.

p53-mediated heterochromatin reorganization regulates its cell fate decisions.

p53 is a major sensor of cellular stresses, and its activation influences cell fate decisions. We identified SUV39H1, a histone code 'writer' responsible for the histone H3 Lys9 trimethylation (H3K9me3) mark for 'closed' chromatin conformation, as a target of p53 repression. SUV39H1 downregulation was mediated transcriptionally by p21 and post-translationally by MDM2. The H3K9me3 repression mark was found to be associated with promoters of representative p53 target genes and was decreased upon p53 activation. Overexpression of SUV39H1 maintained higher levels of the H3K9me3 mark on these promoters and was associated with decreased p53 promoter occupancy and decreased transcriptional induction in response to p53. Conversely, SUV39H1 pre-silencing decreased H3K9me3 levels on these promoters and enhanced the p53 apoptotic response. These findings uncover a new layer of p53-mediated chromatin regulation through modulation of histone methylation at p53 target promoters.

Standard preanalytical coding for biospecimens: review and implementation of the Sample PREanalytical Code (SPREC).

The first version of the Standard PREanalytical Code (SPREC) was developed in 2009 by the International Society for Biological and Environmental Repositories (ISBER) Biospecimen Science Working Group to facilitate documentation and communication of the most important preanalytical quality parameters of different types of biospecimens used for research. This same Working Group has now updated the SPREC to version 2.0, presented here, so that it contains more options to allow for recent technological developments. Existing elements have been fine tuned. An interface to the Biospecimen Reporting for Improved Study Quality (BRISQ) has been defined, and informatics solutions for SPREC implementation have been developed. A glossary with SPREC-related definitions has also been added.

Effects of osmotic pretreatments on oxidative stress, antioxidant profiles and cryopreservation of olive somatic embryos.

A three-day pretreatment of olive somatic embryos (SE) with 0.75 M sucrose, combined with cryoprotection (0.5M DMSO, 1M sucrose, 0.5M glycerol and 0.009 M proline) and controlled rate cooling, supported regrowth (as 34.6% fresh weight gain) and resumption of embryo development after cryopreservation. Pretreatment with mannitol or sorbitol did not support regrowth. Profiles of sugars, proline, antioxidant enzymes, Reactive oxygen species (ROS), secondary oxidation products and ethylene were constructed for the most successful (0.75 M) pretreatment series. Sucrose was the optimal pretreatment for supporting recovery, it also elevated glutathione reductase (GR) activity compared to controls, whereas superoxide dismutase (SOD), catalase and guaiacol peroxidase activities remained relatively unchanged. Superoxide dismutase activity was higher in SE pretreated with sucrose, compared with those pretreated with polyols; H(2)O(2) was enhanced in SE pretreated with sorbitol and sucrose compared to mannitol. The overall trend for ethylene and OH production revealed their levels were highest in SE pretreated with polyols albeit, for individual treatments this was not always the case. Generally, pretreatments did not significantly change embryo secondary oxidation profiles of ThioBarbituric Acid Reactive Substances (TBARS) and Schiff's bases. In combination these studies suggest oxidative processes may influence regrowth of cryopreserved olive SE and that optimal pretreatments could, in part, increase tolerance by an overall enhancement of endogenous antioxidants (particularly GR), proline and sugars.

Standard PREanalytical Codes: A New Paradigm for Environmental Biobanking Sectors Explored in Algal Culture Collections.

The Standard PREanalytical Code (SPREC) was developed by the medical/clinical biobanking sector motivated by the need to harmonize biospecimen traceability in preanalytical processes and enable interconnectivity and interoperability between different biobanks, research consortia, and infrastructures. The clinical SPREC (01) consists of standard preanalytical variable options (7-code elements), which comprise published and (ideally) validated methodologies. Although the SPREC has been designed to facilitate clinical research, the concept could have utility in biorepositories and culture collections that service environmental and biodiversity communities. The SPREC paradigm can be applied to different storage regimes across all types of biorepository. The objective of this article is to investigate adapting the code in nonclinical biobanks using algal culture collections and their cryostorage as a case study. The SPREC (01) is recalibrated as a putative code that might be adopted for biobanks holding different types of biodiversity; it is extended to include optional coding from the point of sample collection to postcryostorage manipulations, with the caveat that the processes are undertaken by biorepository personnel.

Role of progerin-induced telomere dysfunction in HGPS premature cellular senescence.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature-aging syndrome caused by a dominant mutation in the gene encoding lamin A, which leads to an aberrantly spliced and processed protein termed progerin. Previous studies have shown that progerin induces early senescence associated with increased DNA-damage signaling and that telomerase extends HGPS cellular lifespan. We demonstrate that telomerase extends HGPS cellular lifespan by decreasing progerin-induced DNA-damage signaling and activation of p53 and Rb pathways that otherwise mediate the onset of premature senescence. We show further that progerin-induced DNA-damage signaling is localized to telomeres and is associated with telomere aggregates and chromosomal aberrations. Telomerase amelioration of DNA-damage signaling is relatively rapid, requires both its catalytic and DNA-binding functions, and correlates in time with the acquisition by HGPS cells of the ability to proliferate. All of these findings establish that HGPS premature cellular senescence results from progerin-induced telomere dysfunction.

Standard preanalytical coding for biospecimens: defining the sample PREanalytical code.

BACKGROUND: Management and traceability of biospecimen preanalytical variations are necessary to provide effective and efficient interconnectivity and interoperability between Biobanks. METHODS: Therefore, the International Society for Biological and Environmental Repositories Biospecimen Science Working Group developed a "Standard PREanalytical Code" (SPREC) that identifies the main preanalytical factors of clinical fluid and solid biospecimens and their simple derivatives. RESULTS: The SPREC is easy to implement and can be integrated into Biobank quality management systems and databases. It can also be extended to nonhuman biorepository areas. Its flexibility allows integration of new novel technological developments in future versions. SPREC version 01 is presented in this article. CONCLUSIONS AND IMPACT: Implementation of the SPREC is expected to facilitate and consolidate international multicenter biomarker identification research and biospecimen research in the clinical Biobank environment.

Effects of p21 deletion in mouse models of premature aging.

An approach to investigate the role of cellular senescence in organismal aging has been to abrogate signaling pathways known to induce cellular senescence and to assess the effects in mouse models of premature aging. Recently, we reported the effect of loss of function of p21, a gene implicated in p53-induced cellular senescence, in the background of the Ku80(-/-) premature aging mouse (Zhao et al., EMBO Rep 2009). Here, we provide an overview of the effects of p21 deletion in different models of premature aging.

Cryopreservation induces temporal DNA methylation epigenetic changes and differential transcriptional activity in Ribes germplasm.

The physiological and molecular mechanisms associated with acclimation and survival have been examined in four Ribes genotypes displaying differential cryotolerance. Changes in DNA methylation, nucleic acid and nucleoside composition were determined during acclimation and recovery of in vitro shoot-meristems from cryopreservation. DNA methylation was induced in the tolerant genotype, while demethylation was evident in sensitive genotypes. This response initially occurred during sucrose simulated acclimation, with progressive changes as shoots recovered from successive stages of the encapsulation-dehydration protocol. These methylation patterns existed in the initial vegetative cycle but regressed to control values following subculture, indicating the changes in DNA methylation to be a reversible epigenetic mechanism. RNA levels indicating transcriptional activity during the acclimation of nodal tissue are inversely linked to methylation changes, where activity appears to be up-regulated in the cryosensitive genotypes. Conversely, cryopreserved shoots show increased levels of both RNA and DNA methylation in the cryotolerant genotypes. Other nucleosides show post-transcriptional activity corresponds with tolerance during acclimation and cryopreservation. These observations connect physiological attributes to differential molecular changes in Ribes, the implications of which are discussed in relation to cryopreservation-induced apoptosis and genetic stability.

Cellular senescence and organismal ageing in the absence of p21(CIP1/WAF1) in ku80(-/-) mice.

Ku80 is important in the repair of DNA double-strand breaks by its essential function in non-homologous end-joining. The absence of Ku80 causes the accumulation of DNA damage and leads to premature ageing in mice. We showed that mouse embryonic fibroblasts (MEFs) from ku80(-/-) mice senesced rapidly with elevated levels of p53 and p21. Deletion of p21 delayed the early senescence phenotype in ku80(-/-) MEFs, despite an otherwise intact response of p53. In contrast to ku80(-/-)p53(-/-) mice, which die rapidly primarily from lymphomas, there was no significant increase in tumorigenesis in ku80(-/-)p21(-/-) mice. However, ku80(-/-)p21(-/-) mice showed no improvement with respect to rough fur coat or osteopaenia, and even showed a shortened lifespan compared with ku80(-/-) mice. These results show that the increased lifespan of ku80(-/-) MEFs owing to the loss of p21 is not associated with an improvement of the premature ageing phenotypes of ku80(-/-) mice observed at the organismal level.

Applications of differential scanning calorimetry in developing cryopreservation strategies for Parkia speciosa, a tropical tree producing recalcitrant seeds.

Shoot-tips of Parkia speciosa, a recalcitrant seed producing tropical leguminous tree withstood cryopreservation using encapsulation-vitrification in combination with trehalose preculture. Differential scanning calorimetry (DSC) revealed that trehalose moderated the thermal characteristics of the shoot-tips. A 30 min PVS2 treatment had the lowest glass transition temperature (Tg) (-50.2 +/- 1.1 degree C) when applied in combination with 5% (w/v) trehalose. The Tg increased to -40.2 +/- 1.0 degree C as the sugar concentration was decreased to 2.5 percent (w/v). Tg heat capacity for shoot-tips treated with 2.5 percent and 5 percent (w/v) trehalose and exposed to PVS2 for 30 min increased from 0.17 +/ 0.05 to 0.23 +/- 0.01 J per gram, respectively. Enthalpies of the melt-endotherm varied in proportion to trehalose concentration, for the 30 min PVS2 treatment, whereas the melt enthalpy for control shoots was greater than 150 J per gram and decreased to ca. 60 J per gram with 2.5 percent (w/v) trehalose. For 5 percent and 10 percent (w/v) trehalose treatments, enthalpy declined to ca. 24 and 12 J per gram respectively and freezing points were depressed to -75 degree C and -85 degree C with 2.5 percent and 5 percent trehalose (w/v), respectively. DSC elucidated the critical points at which vitrification occurred in germplasm exposed to trehalose and PVS2. A 60 min PVS2 treatment supporting ca. 70 percent survival was found optimal for stable glass formation during cooling and on rewarming.

Developing cryopreservation for Picea sitchensis (sitka spruce) somatic embryos: a comparison of vitrification protocols.

Two vitrification-based cryopreservation protocols, encapsulation/dehydration and PVS2 were applied to Stage 2 (globular) and Stage 4 (torpedo) somatic embryos (SE) from Picea sitchensis. Two recovery responses: partially differentiated embryogenic suspensor masses (ESM) and dedifferentiated non-embryogenic masses (NEM) were observed following exposure to LN. All genotypes tested, proliferated NEM, approximately 10 to 100% of the total SE cryopreserved. A General Linear Model applied to NEM recovery data demonstrated several different factors (developmental state and genotype, treatment, culture age) interacted at a significant level (P less than 0.05) to influence proliferation. One genotype was capable of proliferating ESM after cryopreservation using encapsulation-dehydration, this response was achieved for Stage 4 embryos derived from the youngest ESM tissue.

Deployment of the encapsulation-dehydration protocol to cryopreserve polar microalgae held at the Czech Republic Academy of Sciences Institute of Botany.

Polar isolates of four chlorococcal microalgae originating from the Arctic and Antarctica withstand cryopreservation using encapsulation-dehydration. Viability assessments, which initially used chloroplhyll fluorescence (Kautsky) induction kinetics, revealed that all strains suffered photosynthetic impairment during early post-cryopreservation recovery. This cryoinjury was reversible, as indicated by cell regrowth in three of the four strains. Lack of growth in the fourth isolate was due to contaminating bacteria rather than cryogenic factors.

Deployment of the encapsulation-dehydration protocol to cryopreserve diverse microalgae held at the Institute of Soil Biology, Academy of Sciences of the Czech Republic.

Twenty-seven strains of soil algae isolated from highly diverse provenances and habitats were assessed for their capacity to withstand cryopreservation using encapsulation/dehydration. Survival was assessed following the release of algae from alginate beads treated with sodium hexametaphosphate and regrowth was assessed using NAJA Image Analysis. Regrowth occurred in 19 strains, with > 50% survival being observed in 15. Algal tolerance to osmotic dehydration and evaporative desiccation was critical to the success of the method. Recovery in five out of the remaining eight recalcitrant strains was enhanced by substituting sorbitol for the osmotic pretreatment or by combining encapsulation with two-step controlled rate cooling.