Whole-Body Imaging with Single-Cell Resolution by Tissue Decolorization

The development of whole-body imaging at single-cell resolution enables system-level approaches to studying cellular circuits in organisms. Previous clearing methods focused on homogenizing mismatched refractive indices of individual tissues, enabling reductions in opacity but falling short of achieving transparency. Here, we show that an aminoalcohol decolorizes blood by efficiently eluting the heme chromophore from hemoglobin. Direct transcardial perfusion of an aminoalcohol-containing cocktail that we previously termed CUBIC coupled with a 10 day to 2 week clearing protocol decolorized and rendered nearly transparent almost all organs of adult mice as well as the entire body of infant and adult mice. This CUBIC-perfusion protocol enables rapid whole-body and whole-organ imaging at single-cell resolution by using light-sheet fluorescent microscopy. The CUBIC protocol is also applicable to 3D pathology, anatomy, and immunohistochemistry of various organs. These results suggest that whole-body imaging of colorless tissues at high resolution will contribute to organism-level systems biology.

Wound healing from the outback: novel wound healing therapeutics from native Australian plants

Introduction Chronic wounds are an area of major concern. The on-going and in-direct costs are substantial, reaching far beyond the costs of the hospitalization and associated care. As a result, pharmacological therapies have been developed to address treatment insufficiencies, however, the availability of drugs capable of promoting the wound repair process still remain limited. The wound healing properties of various herbal plants is well recognised amongst indigenous Australians. Hence, based on traditional accounts, we evaluated the wound healing potential of two Australian native plants. Methods Bioactive compounds were methanol extracted from dried plant leaves that were commercially sourced. Primary keratinocyte (Kc) and fibroblast (Fib) cells (denoted as Kc269, Kc274, Kc275, Kc276 and Fib274) obtained from surgical discarded tissue were cultured in 48-well plates and incubated (37⁰C, 5% CO2) overnight. The growth media was discarded and replaced with fresh growth media plus various concentrations (15.12 µg/mL, 31.25 µg/mL, 62.5 µg/mL, 125 µg/mL, 250 µg/mL and 500 µg/mL) of the plant extracts. Cellular responses were measured using the alamarBlue® assay and the CyQUANT® assay. Plant extracts in the aqueous phase were prepared by boiling whole leaves in water and taking aqueous phase samples at various (1, 2 , 5 minutes boiling) time points. Plant leaves were either added before the water was boiled (cold boiled) or after the water was boiled (hot boiled). The final concentrations of the aqueous plant extracts were 3.3 ng/mL (± 0.3 ng/mL) per sample. The antimicrobial properties of the plant extracts were tested using the well diffusion assay method against Staphylococcus aureus, Klebsiella pnuemoniae and methicillin resistant S. aureus and Bacillus cereus. Results Assay results from the almarBlue® and CYQUANT® assays indicated that extracts from both native plants at various time points (0, 24 and 48 hours) and concentrations (31.25 mg/mL, 62.5 mg/mL, and 125 mg/mL) were significantly higher (n=3, p=0.03 for Kc269, p=0.04 for Kc274, p=0.02 for Fib274, p=0.04 for Kc275 and p=0.001 for Kc276) compared with the untreated controls. Neither plant extract demonstrated cytotoxic effects. Significant antimicrobial activity against methicillin resistant Staphylococcus aureus (p=0.0009 for hot boiled plant A, n=2, p=0.034 for cold boiled plant A, n=2) K. pnuemoniae (p=0.0009 for hot boiled plant A, n=2, p=0.002 for cold boiled plant A, n=2) and B. cereus (p=0.0009 for hot boiled plant A, n=2, p=0.003 for cold boiled plant A, n=2) was observed at concentrations of 3.2 ng/mL for plant A and 3.4 ng/mL for plant B. Conclusion Both native plants contain bioactive compounds that increase cellular metabolic rates and total nucleic acid content. Neither plant was shown to be cytotoxic. Furthermore, both exhibited significant antimicrobial activity.

Antidepressants, but not antipsychotics, modulate GR function in human whole blood: An insight into molecular mechanisms

Clinical studies have demonstrated an impairment of glucocorticoid receptor (GR)-mediated negative feedback on the hypothalamic-pituitary-adrenal (HPA) axis in patients with major depression (GR resistance), and its resolution by antidepressant treatment. Recently, we showed that this impairment is indeed due to a dysfunction of GR in depressed patients (Carvalho et al., 2009), and that the ability of the antidepressant clomipramine to decrease GR function in peripheral blood cells is impaired in patients with major depression who are clinically resistant to treatment (Carvalho et al. 2008). To further investigate the effect of antidepressants on GR function in humans, we have compared the effect of the antidepressants clomipramine, amytriptiline, sertraline, paroxetine and venlafaxine, and of the antipsychotics, haloperidol and risperidone, on GR function in peripheral blood cells from healthy volunteers (n=33). GR function was measured by glucocorticoid inhibition of lypopolysaccharide (LPS)-stimulated interleukin-6 (IL-6) levels. Compared to vehicle-treated cells, all antidepressants inhibited dexamethasone (DEX, 10-100nM) inhibition of LPS-stimulated IL-6 levels (p values ranging from 0.007 to 0.1). This effect was specific to antidepressants, as antipsychotics had no effect on DEX-inhibition of LPS-stimulated IL-6 levels. The phosphodiesterase (PDE) type 4 inhibitor, rolipram, potentiated the effect of antidepressants on GR function, while the GR antagonist, RU-486, inhibited the effect of antidepressants on GR function. These findings indicate that the effect of antidepressants on GR function are specific for this class of psychotropic drugs, and involve second messenger pathways relevant to GR function and inflammation. Furthermore, it also points towards a possible mechanism by which one maybe able to overcome treatment-resistant depression. Research in this field will lead to new insights into the pathophysiology and treatment of affective disorders.

Sodium and lithium storage properties of spray-dried molybdenum disulfide-graphene hierarchical microspheres

Developing nano/micro-structures which can effectively upgrade the intriguing properties of electrode materials for energy storage devices is always a key research topic. Ultrathin nanosheets were proved to be one of the potential nanostructures due to their high specific surface area, good active contact areas and porous channels. Herein, we report a unique hierarchical micro-spherical morphology of well-stacked and completely miscible molybdenum disulfide (MoS2) nanosheets and graphene sheets, were successfully synthesized via a simple and industrial scale spray-drying technique to take the advantages of both MoS2 and graphene in terms of their high practical capacity values and high electronic conductivity, respectively. Computational studies were performed to understand the interfacial behaviour of MoS2 and graphene, which proves high stability of the composite with high interfacial binding energy (−2.02 eV) among them. Further, the lithium and sodium storage properties have been tested and reveal excellent cyclic stability over 250 and 500 cycles, respectively, with the highest initial capacity values of 1300 mAh g−1 and 640 mAh g−1 at 0.1 A g−1.

Calcium phosphate flocs and the clarification of sugar cane juice from whole of crop harvesting

Sugar cane biomass is one of the most viable feedstocks for the production of renewable fuels and chemicals. Therefore, processing the whole of crop (WC) (i.e., stalk and trash, instead of stalk only) will increase the amount of available biomass for this purpose. However, effective clarification of juice expressed from WC for raw sugar manufacture is a major challenge because of the amounts and types of non-sucrose impurities (e.g., polysaccharides, inorganics, proteins, etc.) present. Calcium phosphate flocs are important during sugar cane juice clarification because they are responsible for the removal of impurities. Therefore, to gain a better understanding of the role of calcium phosphate flocs during the juice clarification process,the effects of impurities on the physicochemical properties of calcium phosphate flocs were examined using small-angle laser light scattering technique, attenuated total reflectance Fourier transformed infrared spectroscopy, and X-ray powder diffraction. Results on synthetic sugar juice solutions showed that the presence of SiO2 and Na+ ions affected floc size and floc structure. Starch and phosphate ions did not affect the floc structure; however, the former reduced the floc size, whereas the latter increased the floc size. The study revealed that high levels of Na+ ions would negatively affect the clarification process the most, as they would reduce the amount of suspended particles trapped by the flocs. A complementary study on prepared WC juice using cold and cold/intermediate liming techniques was conducted. The study demonstrated that, in comparison to the one-stage (i.e., conventional) clarification process, a two-stage clarification process using cold liming removed more polysaccharides (≤19%),proteins (≤82%), phosphorus (≤53%), and SiO2 (≤23%) in WC juice but increased Ca2+ (≤136%) and sulfur (≤200%)

Whole-body cryotherapy (extreme cold air exposure) for preventing and treating muscle soreness after exercise in adults

Background Recovery strategies are often usedwith the intention of preventing orminimisingmuscle soreness after exercise. Whole-body cryotherapy, which involves a single or repeated exposure(s) to extremely cold dry air (below -100 °C) in a specialised chamber or cabin for two to four minutes per exposure, is currently being advocated as an effective intervention to reduce muscle soreness after exercise. Objectives To assess the effects (benefits and harms) of whole-body cryotherapy (extreme cold air exposure) for preventing and treating muscle soreness after exercise in adults. Search methods We searched the Cochrane Bone, Joint and Muscle Trauma Group Specialised Register, the Cochrane Central Register of Controlled Trials, MEDLINE, EMBASE, CINAHL, the British Nursing Index and the Physiotherapy Evidence Database. We also searched the reference lists of articles, trial registers and conference proceedings, handsearched journals and contacted experts. The searches were run in August 2015. Selection criteria We aimed to include randomised and quasi-randomised trials that compared the use of whole-body cryotherapy (WBC) versus a passive or control intervention (rest, no treatment or placebo treatment) or active interventions including cold or contrast water immersion, active recovery and infrared therapy for preventing or treating muscle soreness after exercise in adults. We also aimed to include randomised trials that compared different durations or dosages of WBC. Our prespecified primary outcomes were muscle soreness, subjective recovery (e.g. tiredness, well-being) and adverse effects. Data collection and analysis Two review authors independently screened search results, selected studies, assessed risk of bias and extracted and cross-checked data. Where appropriate, we pooled results of comparable trials. The random-effects model was used for pooling where there was substantial heterogeneity.We assessed the quality of the evidence using GRADE. Main results Four laboratory-based randomised controlled trials were included. These reported results for 64 physically active predominantly young adults (mean age 23 years). All but four participants were male. Two trials were parallel group trials (44 participants) and two were cross-over trials (20 participants). The trials were heterogeneous, including the type, temperature, duration and frequency of WBC, and the type of preceding exercise. None of the trials reported active surveillance of predefined adverse events. All four trials had design features that carried a high risk of bias, potentially limiting the reliability of their findings. The evidence for all outcomes was classified as ’very low’ quality based on the GRADE criteria. Two comparisons were tested: WBC versus control (rest or no WBC), tested in four studies; and WBC versus far-infrared therapy, also tested in one study. No studies compared WBC with other active interventions, such as cold water immersion, or different types and applications of WBC. All four trials compared WBC with rest or no WBC. There was very low quality evidence for lower self-reported muscle soreness (pain at rest) scores after WBC at 1 hour (standardised mean difference (SMD) -0.77, 95% confidence interval (CI) -1.42 to -0.12; 20 participants, 2 cross-over trials); 24 hours (SMD -0.57, 95%CI -1.48 to 0.33) and 48 hours (SMD -0.58, 95% CI -1.37 to 0.21), both with 38 participants, 2 cross-over studies, 1 parallel group study; and 72 hours (SMD -0.65, 95% CI -2.54 to 1.24; 29 participants, 1 cross-over study, 1 parallel group study). Of note is that the 95% CIs also included either no between-group differences or a benefit in favour of the control group. One small cross-over trial (9 participants) found no difference in tiredness but better well-being after WBC at 24 hours post exercise. There was no report of adverse events. One small cross-over trial involving nine well-trained runners provided very low quality evidence of lower levels of muscle soreness after WBC, when compared with infrared therapy, at 1 hour follow-up, but not at 24 or 48 hours. The same trial found no difference in well-being but less tiredness after WBC at 24 hours post exercise. There was no report of adverse events. Authors’ conclusions There is insufficient evidence to determine whether whole-body cryotherapy (WBC) reduces self-reportedmuscle soreness, or improves subjective recovery, after exercise compared with passive rest or no WBC in physically active young adult males. There is no evidence on the use of this intervention in females or elite athletes. The lack of evidence on adverse events is important given that the exposure to extreme temperature presents a potential hazard. Further high-quality, well-reported research in this area is required and must provide detailed reporting of adverse events.

Whole-genome screening in ankylosing spondylitis: Evidence of non-MHC genetic-susceptibility loci

Ankylosing spondylitis (AS) is a common inflammatory arthritis predominantly affecting the axial skeleton. Susceptibility to the disease is thought to be oligogenic. To identify the genes involved, we have performed a genomewide scan in 185 families containing 255 affected sibling pairs. Two-point and multipoint nonparametric linkage analysis was performed. Regions were identified showing "suggestive" or stronger linkage with the disease on chromosomes 1p, 2q, 6p, 9q, 10q, 16q, and 19q. The MHC locus was identified as encoding the greatest component of susceptibility, with an overall LOD score of 15.6. The strongest non-MHC linkage lies on chromosome 16q (overall LOD score 4.7). These results strongly support the presence of non-MHC genetic-susceptibility factors in AS and point to their likely locations.

Whole-genome linkage analysis of rheumatoid arthritis susceptibility loci in 252 affected sibling pairs in the United Kingdom

Objective. To undertake a systematic wholegenome screen to identify regions exhibiting genetic linkage to rheumatoid arthritis (RA). Methods. Two hundred fifty-two RA-affected sibling pairs from 182 UK families were genotyped using 365 highly informative microsatellite markers. Microsatellite genotyping was performed using fluorescent polymerase chain reaction primers and semiautomated DNA sequencing technology. Linkage analysis was undertaken using MAPMAKER/SIBS for single-point and multipoint analysis. Results. Significant linkage (maximum logarithm of odds score 4.7 [P = 0.000003] at marker D6S276, 1 cM from HLA-DRB1) was identified around the major histocompatibility complex (MHC) region on chromosome 6. Suggestive linkage (P < 7.4 × 10-4) was identified on chromosome 6q by single- and multipoint analysis. Ten other sites of nominal linkage (P < 0.05) were identified on chromosomes 3p, 4q, 7p, 2 regions of 10q, 2 regions of 14q, 16p, 21q, and Xq by single-point analysis and on 3 sites (1q, 14q, and 14q) by multipoint analysis. Conclusion. Linkage to the MHC region was confirmed. Eleven non-HLA regions demonstrated evidence of suggestive or nominal linkage, but none reached the genome-wide threshold for significant linkage (P = 2.2 × 10-5). Results of previous genome screens have suggested that 6 of these regions may be involved in RA susceptibility.

Whole blood transcriptional profiling in ankylosing spondylitis identifies novel candidate genes that might contribute to the inflammatory and tissue-destructive disease aspects

Introduction: A number of genetic-association studies have identified genes contributing to ankylosing spondylitis (AS) susceptibility but such approaches provide little information as to the gene activity changes occurring during the disease process. Transcriptional profiling generates a 'snapshot' of the sampled cells' activity and thus can provide insights into the molecular processes driving the disease process. We undertook a whole-genome microarray approach to identify candidate genes associated with AS and validated these gene-expression changes in a larger sample cohort. Methods: A total of 18 active AS patients, classified according to the New York criteria, and 18 gender- and age-matched controls were profiled using Illumina HT-12 whole-genome expression BeadChips which carry cDNAs for 48,000 genes and transcripts. Class comparison analysis identified a number of differentially expressed candidate genes. These candidate genes were then validated in a larger cohort using qPCR-based TaqMan low density arrays (TLDAs). Results: A total of 239 probes corresponding to 221 genes were identified as being significantly different between patients and controls with a P-value <0.0005 (80% confidence level of false discovery rate). Forty-seven genes were then selected for validation studies, using the TLDAs. Thirteen of these genes were validated in the second patient cohort with 12 downregulated 1.3- to 2-fold and only 1 upregulated (1.6-fold). Among a number of identified genes with well-documented inflammatory roles we also validated genes that might be of great interest to the understanding of AS progression such as SPOCK2 (osteonectin) and EP300, which modulate cartilage and bone metabolism. Conclusions: We have validated a gene expression signature for AS from whole blood and identified strong candidate genes that may play roles in both the inflammatory and joint destruction aspects of the disease.

Whole exome sequencing is an efficient and sensitive method for detection of germline mutations in patients with phaeochromcytomas and paragangliomas

Background Genetic testing is recommended when the probability of a disease-associated germline mutation exceeds 10%. Germline mutations are found in approximately 25% of individuals with phaeochromcytoma (PCC) or paraganglioma (PGL); however, genetic heterogeneity for PCC/PGL means many genes may require sequencing. A phenotype-directed iterative approach may limit costs but may also delay diagnosis, and will not detect mutations in genes not previously associated with PCC/PGL. Objective To assess whether whole exome sequencing (WES) was efficient and sensitive for mutation detection in PCC/PGL. Methods Whole exome sequencing was performed on blinded samples from eleven individuals with PCC/PGL and known mutations. Illumina TruSeq™ (Illumina Inc, San Diego, CA, USA) was used for exome capture of seven samples, and NimbleGen SeqCap EZ v3.0 (Roche NimbleGen Inc, Basel, Switzerland) for five samples (one sample was repeated). Massive parallel sequencing was performed on multiplexed samples. Sequencing data were called using Genome Analysis Toolkit and annotated using annovar. Data were assessed for coding variants in RET, NF1, VHL, SDHD, SDHB, SDHC, SDHA, SDHAF2, KIF1B, TMEM127, EGLN1 and MAX. Target capture of five exome capture platforms was compared. Results Six of seven mutations were detected using Illumina TruSeq™ exome capture. All five mutations were detected using NimbleGen SeqCap EZ v3.0 platform, including the mutation missed using Illumina TruSeq™ capture. Target capture for exons in known PCC/PGL genes differs substantially between platforms. Exome sequencing was inexpensive (<$A800 per sample for reagents) and rapid (results <5 weeks from sample reception). Conclusion Whole exome sequencing is sensitive, rapid and efficient for detection of PCC/PGL germline mutations. However, capture platform selection is critical to maximize sensitivity.

The potential for production of freeze-dried oral vaccines using alginate hydrogel microspheres as protein carriers

Oral administration of dry vaccine formulations is acknowledged to offer major clinical and logistical benefits by eliminating the cold chain required for liquid preparations. A model antigen, bovine serum albumin (BSA) was encapsulated in alginate microspheres using aerosolisation. Hydrated microspheres 25 to 65 μm in size with protein loading of 3.3 % w/w were obtained. Environmental scanning electron microscopy indicated a stabilizing effect of encapsulated protein on alginate hydrogels revealed by an increase in dehydration resistance. Freeze drying of alginate microspheres without use of a cryoprotectant resulted in fragmentation and subsequent rapid loss of the majority of the protein load in simulated intestinal fluid in 2 h, whereas intact microspheres were observed following freeze-drying of BSA-loaded microspheres in the presence of maltodextrin. BSA release from freeze-dried preparations was limited to less than 7 % in simulated gastric fluid over 2 h, while 90 % of the protein load was gradually released in simulated intestinal fluid over 10 h. SDS-PAGE analysis indicated that released BSA largely preserved its molecular weight. These findings demonstrate the potential for manufacturing freeze-dried oral vaccines using alginate microspheres.

Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging

Here we describe a protocol for advanced CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis). The CUBIC protocol enables simple and efficient organ clearing, rapid imaging by light-sheet microscopy and quantitative imaging analysis of multiple samples. The organ or body is cleared by immersion for 1–14 d, with the exact time required dependent on the sample type and the experimental purposes. A single imaging set can be completed in 30–60 min. Image processing and analysis can take <1 d, but it is dependent on the number of samples in the data set. The CUBIC clearing protocol can process multiple samples simultaneously. We previously used CUBIC to image whole-brain neural activities at single-cell resolution using Arc-dVenus transgenic (Tg) mice. CUBIC informatics calculated the Venus signal subtraction, comparing different brains at a whole-organ scale. These protocols provide a platform for organism-level systems biology by comprehensively detecting cells in a whole organ or body.

Performance enhancement: A whole of person approach to first year dance students’ transition into tertiary training

The transition into university presents very particular challenges for students. The First Year Experience (FYE) is a transitional liminal phase, fraught with uncertainty, ripe with potential. The complexity inherent in this initial phase of tertiary education is well documented and continues to be interrogated. Providing timely and effective support and interventions for potentially at-risk first year students as they transition into tertiary study is a key priority for universities across the globe (Gale et al., 2015). This article outlines the evolution of an established and highly successful Transitional Training Program (TTP) for first year tertiary dance students, with particular reference to the 2015 iteration of the program. TTP design embraces three dimensions: physical training in transition, learning in transition, and teaching for transition, with an emphasis on developing and encouraging a mindset that enables information to be transferred into alternative settings for practice and learning throughout life. The aim of the 2015 TTP was to drive substantial change in first year Dance students’ satisfaction, connectedness, and overall performance within the Bachelor of Fine Arts (BFA) Dance course, through the development and delivery of innovative curriculum and pedagogical practices that promote the successful transition of dance students into their first year of university. The program targeted first year BFA Dance students through the integration of specific career guidance; performance psychology; academic skills support; practical dance skills support; and specialized curricula and pedagogy.

The multiple sclerosis whole blood mRNA transcriptome and genetic associations indicate dysregulation of specific T cell pathways in pathogenesis

Multiple sclerosis (MS) is an autoimmune disease with a genetic component, caused at least in part by aberrant lymphocyte activity. The whole blood mRNA transcriptome was measured for 99 untreated MS patients: 43 primary progressive MS, 20 secondary progressive MS, 36 relapsing remitting MS and 45 age-matched healthy controls. The ANZgene Multiple Sclerosis Genetics Consortium genotyped more than 300 000 SNPs for 115 of these samples. Transcription from genes on translational regulation, oxidative phosphorylation, immune synapse and antigen presentation pathways was markedly increased in all forms of MS. Expression of genes tagging T cells was also upregulated (P < 10-12) in MS. A T cell gene signature predicts disease state with a concordance index of 0.79 with age and gender as co-variables, but the signature is not associated with clinical course or disability. The ANZgene genome wide association screen identified two novel regions with genome wide significance: one encoding the T cell co-stimulatory molecule, CD40; the other a region on chromosome 12q13-14. The CD40 haplotype associated with increased MS susceptibility has decreased gene expression in MS (P < 0.0007). The second MS susceptibility region includes 17 genes on 12q13-14 in tight linkage disequilibrium. Of these, only 13 are expressed in leukocytes, and of these the expression of one, FAM119B, is much lower in the susceptibility haplotype (P tdthomlt; 10-14). Overall, these data indicate dysregulation of T cells can be detected in the whole blood of untreated MS patients, and supports targeting of activated T cells in therapy for all forms of MS.

Combined analysis of three whole genome linkage scans for Ankylosing Spondylitis

Objective. Ankylosing spondylitis (AS) is a debilitating chronic inflammatory condition with a high degree of familiality (λs=82) and heritability (>90%) that primarily affects spinal and sacroiliac joints. Whole genome scans for linkage to AS phenotypes have been conducted, although results have been inconsistent between studies and all have had modest sample sizes. One potential solution to these issues is to combine data from multiple studies in a retrospective meta-analysis. Methods: The International Genetics of Ankylosing Spondylitis Consortium combined data from three whole genome linkage scans for AS (n=3744 subjects) to determine chromosomal markers that show evidence of linkage with disease. Linkage markers typed in different centres were integrated into a consensus map to facilitate effective data pooling. We performed a weighted meta-analysis to combine the linkage results, and compared them with the three individual scans and a combined pooled scan. Results: In addition to the expected region surrounding the HLA-B27 gene on chromosome 6, we determined that several marker regions showed significant evidence of linkage with disease status. Regions on chromosome 10q and 16q achieved 'suggestive' evidence of linkage, and regions on chromosomes 1q, 3q, 5q, 6q, 9q, 17q and 19q showed at least nominal linkage in two or more scans and in the weighted meta-analysis. Regions previously associated with AS on chromosome 2q (the IL-1 gene cluster) and 22q (CYP2D6) exhibited nominal linkage in the meta-analysis, providing further statistical support for their involvement in susceptibility to AS. Conclusion: These findings provide a useful guide for future studies aiming to identify the genes involved in this highly heritable condition. . Published by on behalf of the British Society for Rheumatology.