Apparent absence of the amphibian chytrid fungus (Batrachochytrium dendrobatidis) in frogs in Malaita Province, Solomon Islands

A major driver of global biodiversity loss is disease. One of the most devastating wildlife diseases known is chytridiomycosis, which is caused by the amphibian chytrid fungus Batrachochytrium dendrobatidis, and is implicated in population declines in over 500 frog species. Thought to originate in Asia, B. dendrobatidis now has a global distribution, likely due to human movement and trade. The pathogen has yet to be detected in Melanesia, but there have been few surveys for B. dendrobatidis in the region, and none in the Solomon Islands archipelago, a biogeographic region with a unique and culturally important frog fauna. We swabbed 200 frogs of eight species in three genera in lowland and highland sites in East Kwaio on the island of Malaita in the Solomon Islands. All frogs tested negative for the pathogen but it is possible that the pathogen is present despite non-detection, so further surveys for the pathogen are needed throughout the country. Despite this, it is safest to take a precautionary approach and assume that B. dendrobatidis has not yet been introduced to the Solomon Islands, and that naïve native amphibian populations may be at risk of decline if the pathogen is introduced. Protocols are needed to prevent the accidental import of infected frogs via tourism or in logging or mining equipment. Monitoring of frog populations near areas of high risk such as ports is also recommended. The frogs of the Solomon Islands archipelago are biologically unique and culturally significant, and protecting them from the potentially devastating impacts of B. dendrobatidis is vital

Insulin, Ascorbate, and Glucose Have a Much Greater Influence Than Transferrin and Selenous Acid on the In Vitro Growth of Engineered Cartilage in Chondrogenic Media

The primary goal of this study was to characterize the response of chondrocyte-seeded agarose constructs to varying concentrations of several key nutrients in a chondrogenic medium, within the overall context of optimizing the key nutrients and the placement of nutrient channels for successful growth of cartilage tissue constructs large enough to be clinically relevant in the treatment of osteoarthritis (OA). To this end, chondrocyte-agarose constructs (phi4x2.34 mm, 30x106 cells/mL) were subjected to varying supplementation levels of insulin (0× to 30× relative to standard supplementation), transferrin (0x to 30x), selenous acid (0x to 10x), ascorbate (0x to 30x), and glucose (0x to 3x). The quality of resulting engineered tissue constructs was evaluated by their compressive modulus (E-Y), tensile modulus (E+Y), hydraulic permeability (k), and content of sulfated glycosaminoglycans (sGAG) and collagen (COL); DNA content was also quantified. Three control groups from two separate castings of constructs (1x concentrations of all medium constituents) were used. After 42 days of culture, values in each of these controls were, respectively, E-Y=518 plus or minus 78, 401 plus or minus 113, 236 plus or minus 67 kPa; E+Y=1420 plus or minus 430, 1140 plus or minus 490, 1240 plus or minus 280 kPa; k=2.3 plus or minus 0.8x10-3, 5.4 plus or minus 7.0x10-3, 3.3 plus or minus 1.3x10-3 mm4/N times s; sGAG=7.8 plus or minus 0.3, 6.3 plus or minus 0.4, 4.1 plus or minus 0.5%/ww; COL=1.3 plus or minus 0.2, 1.1 plus or minus 0.3, 1.4 plus or minus 0.4%/ww; and DNA=11.5 plus or minus 2.2, 12.1 plus or minus 0.6, 5.2 plus or minus 2.8 μg/disk. The presence of insulin and ascorbate was essential, but their concentrations may drop as low as 0.3x without detrimental effects on any of the measured properties; excessive supplementation of ascorbate (up to 30x) was detrimental to E-Y, and 30x insulin was detrimental to both E+Y and E-Y. The presence of glucose was similarly essential, and matrix elaboration was significantly dependent on its concentration (p less than 10-6), with loss of functional properties, composition, and cellularity observed at less than or equal to 0.3x; excessive glucose supplementation (up to 3x) showed no detrimental effects. In contrast, transferrin and selenous acid had no influence on matrix elaboration. These findings suggest that adequate distributions of insulin, ascorbate, and glucose, but not necessarily of transferrin and selenous acid, must be ensured within large engineered cartilage constructs to produce a viable substitute for joint tissue lost due to OA

Overexpression of the Lung Cancer-Prognostic miR-146b MicroRNAs Has a Minimal and Negative Effect on the Malignant Phenotype of A549 Lung Cancer Cells

INTRODUCTION:Expression levels of miR-146b-5p and -3p microRNAs in human non-small cell lung cancer (NSCLC) are associated with recurrence of the disease after surgery. To understand this, the effect of miR-146b overexpression was studied in A549 human lung cancer cells. METHODS:A549 cells, engineered with lentiviruses to overexpress the human pre-miR-146b precursor microRNA, were examined for proliferation, colony formation on plastic surface and in soft agar, migration and invasiveness in cell culture and in vivo in mice, chemosensitivity to cisplatin and doxorubicin, and global gene expression. miR-146b expressions were assessed in microdissected stroma and epithelia of human NSCLC tumors. Association of miR-146b-5p and -3p expression in early stage NSCLC with recurrence was analyzed. PRINCIPAL FINDINGS:A549 pre-miR-146b-overexpressors had 3-8-fold higher levels of both miR-146b microRNAs than control cells. Overexpression did not alter cellular proliferation, chemosensitivity, migration, or invasiveness; affected only 0.3% of the mRNA transcriptome; and, reduced the ability to form colonies in vitro by 25%. In human NSCLC tumors, expression of both miR-146b microRNAs was 7-10-fold higher in stroma than in cancerous epithelia, and higher miR-146b-5p but lower -3p levels were predictive of recurrence. CONCLUSIONS:Only a minimal effect of pre-miR-146b overexpression on the malignant phenotype was seen in A549 cells. This could be because of opposing effects of miR-146b-5p and -3p overexpression as suggested by the conflicting recurrence-predictive values of the two microRNAs, or because miR-146b expression changes in non-cancerous stroma and not cancerous epithelia of tumors are responsible for the prognostic value of miR-146b

Integrative miRNA-mRNA Profiling of Adipose Tissue Unravels Transcriptional Circuits Induced by Sleep Fragmentation

Obstructive sleep apnea (OSA) is a prevalent condition and strongly associated with metabolic disorders. Sleep fragmentation (SF) is a major consequence of OSA, but its contribution to OSA-related morbidities is not known. We hypothesized that SF causes specific perturbations in transcriptional networks of visceral fat cells, leading to systemic metabolic disturbances. We simultaneously profiled visceral adipose tissue mRNA and miRNA expression in mice exposed to 6 hours of SF during sleep, and developed a new computational framework based on gene set enrichment and network analyses to merge these data. This approach leverages known gene product interactions and biologic pathways to interrogate large-scale gene expression profiling data. We found that SF induced the activation of several distinct pathways, including those involved in insulin regulation and diabetes. Our integrative methodology identified putative controllers and regulators of the metabolic response during SF. We functionally validated our findings by demonstrating altered glucose and lipid homeostasis in sleep-fragmented mice. This is the first study to link sleep fragmentation with widespread disruptions in visceral adipose tissue transcriptome, and presents a generalizable approach to integrate mRNA-miRNA information for systematic mapping of regulatory networks

MicroRNA-sequence profiling reveals novel osmoregulatory microRNA expression patterns in catadromous eel anguilla marmorata

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that regulate gene expression by post-transcriptional repression of mRNAs. Recently, several miRNAs have been confirmed to execute directly or indirectly osmoregulatory functions in fish via translational control. In order to clarify whether miRNAs play relevant roles in the osmoregulation of Anguilla marmorata, three sRNA libraries of A. marmorata during adjusting to three various salinities were sequenced by Illumina sRNA deep sequencing methods. Totally 11,339,168, 11,958,406 and 12,568,964 clear reads were obtained from 3 different libraries, respectively. Meanwhile, 34 conserved miRNAs and 613 novel miRNAs were identified using the sequence data. MiR-10b-5p, miR-181a, miR-26a-5p, miR-30d and miR-99a-5p were dominantly expressed in eels at three salinities. Totally 29 mature miRNAs were significantly up-regulated, while 72 mature miRNAs were significantly down-regulated in brackish water (10‰ salinity) compared with fresh water (0‰ salinity); 24 mature miRNAs were significantly up-regulated, while 54 mature miRNAs were significantly down-regulated in sea water (25‰ salinity) compared with fresh water. Similarly, 24 mature miRNAs were significantly up-regulated, while 45 mature miRNAs were significantly down-regulated in sea water compared with brackish water. The expression patterns of 12 dominantly expressed miRNAs were analyzed at different time points when the eels transferred from fresh water to brackish water or to sea water. These miRNAs showed differential expression patterns in eels at distinct salinities. Interestingly, miR-122, miR-140-3p and miR-10b-5p demonstrated osmoregulatory effects in certain salinities. In addition, the identification and characterization of differentially expressed miRNAs at different salinities can clarify the osmoregulatory roles of miRNAs, which will shed lights for future studies on osmoregulation in fish

Bioinformatic and Genetic Association Analysis of MicroRNA Target Sites in One-Carbon Metabolism Genes

One-carbon metabolism (OCM) is linked to DNA synthesis and methylation, amino acid metabolism and cell proliferation. OCM dysfunction has been associated with increased risk for various diseases, including cancer and neural tube defects. MicroRNAs (miRNAs) are ∼22 nt RNA regulators that have been implicated in a wide array of basic cellular processes, such as differentiation and metabolism. Accordingly, mis-regulation of miRNA expression and/or activity can underlie complex disease etiology. We examined the possibility of OCM regulation by miRNAs. Using computational miRNA target prediction methods and Monte-Carlo based statistical analyses, we identified two candidate miRNA “master regulators” (miR-22 and miR-125) and one candidate pair of “master co-regulators” (miR-344-5p/484 and miR-488) that may influence the expression of a significant number of genes involved in OCM. Interestingly, miR-22 and miR-125 are significantly up-regulated in cells grown under low-folate conditions. In a complementary analysis, we identified 15 single nucleotide polymorphisms (SNPs) that are located within predicted miRNA target sites in OCM genes. We genotyped these 15 SNPs in a population of healthy individuals (age 18–28, n = 2,506) that was previously phenotyped for various serum metabolites related to OCM. Prior to correction for multiple testing, we detected significant associations between TCblR rs9426 and methylmalonic acid (p  =  0.045), total homocysteine levels (tHcy) (p  =  0.033), serum B12 (p < 0.0001), holo transcobalamin (p < 0.0001) and total transcobalamin (p < 0.0001); and between MTHFR rs1537514 and red blood cell folate (p < 0.0001). However, upon further genetic analysis, we determined that in each case, a linked missense SNP is the more likely causative variant. Nonetheless, our Monte-Carlo based in silico simulations suggest that miRNAs could play an important role in the regulation of OCM

Sequence Relationships among C. elegans, D. melanogaster and Human microRNAs Highlight the Extensive Conservation of microRNAs in Biology

microRNAs act in a prevalent and conserved post-transcriptional gene regulatory mechanism that impacts development, homeostasis and disease, yet biological functions for the vast majority of miRNAs remain unknown. Given the power of invertebrate genetics to promote rapid evaluation of miRNA function, recently expanded miRNA identifications (miRBase 10.1), and the importance of assessing potential functional redundancies within and between species, we evaluated miRNA sequence relationships by 5′ end match and overall homology criteria to compile a snapshot overview of miRNA families within the C. elegans and D. melanogaster genomes that includes their identified human counterparts. This compilation expands literature documentation of both the number of families and the number of family members, within and between nematode and fly models, and highlights sequences conserved between species pairs or among nematodes, flies and humans. Themes that emerge include the substantial potential for functional redundancy of miRNA sequences within species (84/139 C. elegans miRNAs and 70/152 D. melanogaster miRNAs share significant homology with other miRNAs encoded by their respective genomes), and the striking extent to which miRNAs are conserved across species—over half (73/139) C. elegans miRNAs share sequence homology with miRNAs encoded also in both fly and human genomes. This summary analysis of mature miRNA sequence relationships provides a quickly accessible resource that should facilitate functional and evolutionary analyses of miRNAs and miRNA families

Steps on a journey to TB control in Solomon Islands: a cross-sectional, mixed methods pre-post evaluation of a local language DVD

Background: In Solomon Islands many people with Tuberculosis (TB) have challenges in accessing services because of socio-cultural, geographic and health service reasons, resulting in delays in TB treatment and low detection rates. The purpose of this project was to (i) develop a local language audio-visual resource (DVD) about TB (ii) share this resource with people in remote villages and (iii) evaluate the process and outcomes.\ud \ud Methods: The project involved the development and evaluation of a DVD in local Kwaio language. The DVD included five short videos based on the Australian Respiratory Council TB Education Flipchart. The DVD also included short videos of: traditional music/chanting (ai'imae); drama that presented an allegory of TB; and a short documentary on the redevelopment of the local TB Ward. A mixed-methods approach evaluated changes in TB knowledge and investigated the impact of the DVD.\ud \ud Results: The DVD was recorded and produced in March – June 2013 and screened in 41 villages and hamlets. The pre-post DVD survey was completed by 64% (255/400) of people who viewed the DVD in the villages. Pre-DVD survey responses showed a moderate to high knowledge about TB signs, symptoms and treatment but 76/255 (30%) stated TB was caused by sorcery and 85/255 (33%) incorrectly stated that TB medication should be stopped when a patient feels better. The post-DVD survey showed a significant increase in people in coastal villages reporting (i) a 3-week cough would trigger a medical assessment and (ii) TB is mainly spread through the air. Statements that TB is not caused by sorcery increased post-DVD in both coastal and mountain villages, however belief in sorcery in mountain villages remained high at 20/70 (29%).\ud \ud Conclusions: The local DVD resource was developed within local cultural understandings and oral traditions of Kwaio people. Using modern but accessible DVD technology enerated a lot of interest about the disease and the stories. The project evaluation indicates that current delays in seeking treatment may be more due to socio-cultural and health service factors than awareness of the disease. Therefore the development of TB services, including TB education, which are culturally sensitive, remains important

Progress towards TB control in East Kwaio, Solomon Islands

[Extract] The recently published project report TB questions, East Kwaio answers: community-based participatory research in a remote area of Solomon Islands1, reported that tuberculosis (TB) remains an important issue to communities and health providers. The island of Malaita has the highest rate of TB in Solomon Islands. Many complex issues are contributors to this concerning level of disease. Factors such as remoteness, socioeconomic and sociocultural issues have been described. Massey et al particularly highlighted how cultural differences based on different worldviews had resulted in some TB patients being unable to access routine hospital based services.\ud \ud Since the development of a research-capacity strengthening approach with the Atoifi Adventist Hospital (AAH) in 20094, and the TB project of 20111, important initiatives and outcomes have been seen, including:\ud \ud * Building a new TB Ward that is culturally safer: a site for the new ward, that meets both socio-cultural and health requirements, has been agreed with mountain area Chiefs and AAH. The area has been cleared, plans drawn up and builders engaged.\ud \ud * Food for TB inpatients: AAH now purchases food each week for TB inpatients to enhance treatment. Currently other hospital inpatients provide their own food.\ud \ud * Active case finding and treatment: the AAH TB Team have conducted active case finding in distant TB ‘hotspots’ including the Kwaibaita Valley and remote mountain hamlets. Ten new cases were found during community visits and treated, including one person treated only in the community setting.\ud \ud * Funded Project to develop TB resources: AAH was successful in applying for a grant from the Australian Respiratory Council to develop local TB resources. This project will work with the traditional oral story-telling model of education and communication, but with modern technology. Locally made videos in local language and Pijin are being developed for community based TB education, including taking DVD players and video clips into remote hamlets and villages to stimulate discussion.\ud \ud * Stronger links with the National TB Program: the Provincial TB Coordinator is involved in the TB DVD project and has invited AAH to run research-training workshops in the provincial capital.\ud \ud * Reported TB case numbers increased: Since the commencement of the research capacity-strengthening approach and concentration on TB, annual reported cases of TB at AAH have increased from four cases in 2009 to 34 cases in 2012, increasing the case detection rate

Spatio-temporal relief from hypoxia and production of reactive oxygen species during bud burst in grapevine (Vitis vinifera L.)

Background and Aims: Plants regulate cellular oxygen partial pressures (pO2), together with reduction/ oxidation (redox) state to manage rapid developmental transitions such as bud burst after a period of quiescence. However, our understanding of pO2 regulation in complex meristematic organs such as buds is incomplete, and particularly lacks spatial resolution. Methods: The gradients in pO2 from the outer scales to the primary meristem complex were measured in grapevine (Vitis vinifera L.) buds, together with respiratory CO2 production rates and the accumulation of superoxide and hydrogen peroxide, from ecodormancy through the first 72 h preceding bud burst, triggered by the transition from low to ambient temperatures. Key Results: Steep internal pO2 gradients were measured in dormant buds with values as low as 2.5 kPa found in the core of the bud prior to bud burst. Respiratory CO2 production rates increased soon after the transition from low to ambient temperatures and the bud tissues gradually became oxygenated in a patterned process. Within 3 h of the transition to ambient temperatures, superoxide accumulation was observed in the cambial meristem, co-localising with lignified cellulose associated with pro-vascular tissues. Thereafter, superoxide accumulated in other areas subtending the apical meristem complex, in the absence of significant hydrogen peroxide accumulation, except in the cambial meristem. By 72 h, the internal pO2 gradient showed a biphasic profile, where the minimum pO2 was external to the core of the bud complex. Conclusions: Spatial and temporal control of the tissue oxygen environment occurs within quiescent buds, and the transition from quiescence to bud burst is accompanied by a regulated relaxation of the hypoxic state and accumulation of reactive oxygen species (ROS) within the developing cambium and vascular tissues of the heterotrophic grapevine buds