H-Ferritin-Regulated MicroRNAs Modulate Gene Expression in K562 Cells

In a previous study, we showed that the silencing of the heavy subunit (FHC) offerritin, the central iron storage molecule in the cell, is accompanied by a modification in global gene expression. In this work, we explored whether different FHC amounts might modulate miRNA expression levels in K562 cells and studied the impact of miRNAs in gene expression profile modifications. To this aim, we performed a miRNA-mRNA integrative analysis in K562 silenced for FHC (K562^shFHC) comparing it with K562 transduced with scrambled RNA (K562^shRNA). Four miRNAs, namely hsa-let-7g, hsa-let-7f, hsa-let-7i and hsa-miR-125b, were significantly up-regulated in silenced cells. The remarkable down-regulation of these miRNAs, following FHC expression rescue, supports a specific relation between FHC silencing and miRNA-modulation. The integration of target predictions with miRNA and gene expression profiles led to the identification of a regulatory network which includes the miRNAs up-regulated by FHC silencing, as well as91 down-regulated putative target genes. These genes were further classified in 9 networks; the highest scoring network, “Cell Death and Survival, Hematological System Development and Function, Hematopoiesis”, is composed by 18 focus molecules including RAF1 and ERK1/2. We confirmed that, following FHC silencing, ERK1/2 phosphorylation is severely impaired and that RAF1 mRNA is significantly down-regulated. Taken all together, our data indicate that, in our experimental model, FHC silencing may affect RAF1/pERK1/2 levels through the modulation of a specific set of miRNAs and add new insights in to the relationship among iron homeostasis and miRNAs.     

Survey of Physicians’ Perspectives and Knowledge about Diagnostic Tests for Bloodstream Infections

Background

Physicians rely on blood culture to diagnose bloodstream infections (BSI) despite its limitations. As new technologies emerge for rapid BSI diagnosis, optimization of their application to patient care requires an understanding of clinicians’ perspectives on BSI diagnosis and how a rapid test would influence medical decisions.

Methods

We administered a 26-question survey to practitioners in infectious diseases/microbiology, critical care, internal medicine, and hematology/oncology services in USA and Germany about current standards in diagnosing and treating BSI and a hypothetical rapid BSI test.

Results

Responses from 242 providers had roughly equal representation across specialties. For suspected BSI patients, 78% of practitioners would administer empiric broad spectrum antibiotics although they estimated, on average, that 31% of patients received incorrect antibiotics while awaiting blood culture results. The ability of blood culture to rule in or rule out infection was very/extremely acceptable in 67% and 36%, respectively. Given rapid test results, 60–87% of practitioners would narrow the spectrum of antimicrobial therapy depending on the microorganism detected, with significantly higher percentages when resistance determinants were also tested. Over half of respondents felt a rapid test would be very/extremely influential on clinical practice.

Conclusions

Limitations of blood culture were perceived as a barrier to patient care. A rapid test to diagnose BSI would impact clinical practice, but the extent of impact may be limited by prevailing attitudes and practices. Opportunities exist for interventions to influence practitioners’ behaviors in BSI management particularly with emergence of newer diagnostic tests.     

Clinical Features of Reported Ethylene Glycol Exposures in the United States

Background

Ethylene glycol is highly toxic and represents an important cause of poisonings worldwide. Toxicity can result in central nervous system dysfunction, cardiovascular compromise, elevated anion gap metabolic acidosis and acute kidney injury. Many states have passed laws requiring addition of the bittering agent, denatonium benzoate, to ethylene glycol solutions to reduce severity of exposures. The objectives of this study were to identify differences between unintentional and intentional exposures and to evaluate the utility of denatonium benzoate as a deterrent.

Methods and Findings

Using the National Poison Data System, we performed a retrospective analysis of reported cases of ethylene glycol exposures from January 2006 to December 2013. Outcome classification was summed for intentionality and used as a basis for comparison of effect groups. There were 45,097 cases of ethylene glycol exposures resulting in 154 deaths. Individuals more likely to experience major effects or death were older, male, and presented with more severe symptoms requiring higher levels of care. Latitude and season did not correlate with increased exposures; however, there were more exposures in rural areas. Denatonium benzoate use appeared to have no effect on exposure severity or number.

Conclusion

Deaths due to ethylene glycol exposure were uncommon; however, there were major clinical effects and more exposures in rural areas. Addition of denatonium benzoate was not associated with a reduction in exposures. Alternative means to deter ingestion are needed. These findings suggest the need to consider replacing ethylene glycol with alternative and less toxic agents.     

Resistance to desiccation in aquatic invasive snails and implications for their overland dispersal

At least 30 species of nonindigenous freshwater snails have invaded North America. The risk of these snails invading new lakes depends upon their ability to survive overland transport. We first reviewed published laboratory experiments using freshwater snails, which show numerous species are able to tolerate days of air exposure. We then tested tolerance to drying of three species of invasive aquatic snails that are widespread in Wisconsin: Bithynia tentaculata, Cipangopaludina chinensis, and Viviparus georgianus. In a series of seven experiments, we simulated boater transport by placing snails individually in mesh bags, hung outdoors, and confined in a screen tent. The screen roof allowed exposure to both sun and rain, and an on-site weather station recorded temperature, precipitation, and humidity. All three species exhibited high survivorship, with some individuals alive at the end of most experiments: 42 days for B. tentaculata and V. georgianus and 63 days for C. chinensis. Viable young were released by C. chinensis after 54 days of exposure. Overall, our results indicate that these invasive snails should readily survive long periods of transport overland, indicating a need for continued vigilance of recreational boaters entering lakes.     

Enhanced prostacyclin formation and Wnt signaling in sclerostin deficient osteocytes and bone

We show that prostacyclin production is increased in bone and osteocytes from sclerostin (Sost) knockout mice which have greatly increased bone mass. The addition of prostacyclin or a prostacyclin analog to bone forming osteoblasts enhances differentiation and matrix mineralization of osteoblasts. The increase in prostacyclin synthesis is linked to increases in β-catenin concentrations and activity as shown by enhanced binding of lymphoid enhancer factor, Lef1, to promoter elements within the prostacyclin synthase promoter. Blockade of Wnt signaling reduces prostacyclin production in osteocytes. Increased prostacyclin production by osteocytes from sclerostin deficient mice could potentially contribute to the increased bone formation seen in this condition.     

Mutation in the Gene Encoding Ubiquitin Ligase LRSAM1 in Patients with Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth disease (CMT) represents a family of related sensorimotor neuropathies. We studied a large family from a rural eastern Canadian community, with multiple individuals suffering from a condition clinically most similar to autosomal recessive axonal CMT, or AR-CMT2. Homozygosity mapping with high-density SNP genotyping of six affected individuals from the family excluded 23 known genes for various subtypes of CMT and instead identified a single homozygous region on chromosome 9, at 122,423,730–129,841,977 Mbp, shared identical by state in all six affected individuals. A homozygous pathogenic variant was identified in the gene encoding leucine rich repeat and sterile alpha motif 1 (LRSAM1) by direct DNA sequencing of genes within the region in affected DNA samples. The single nucleotide change mutates an intronic consensus acceptor splicing site from AG to AA. Direct analysis of RNA from patient blood demonstrated aberrant splicing of the affected exon, causing an obligatory frameshift and premature truncation of the protein. Western blotting of immortalized cells from a homozygous patient showed complete absence of detectable protein, consistent with the splice site defect. LRSAM1 plays a role in membrane vesicle fusion during viral maturation and for proper adhesion of neuronal cells in culture. Other ubiquitin ligases play documented roles in neurodegenerative diseases. LRSAM1 is a strong candidate for the causal gene for the genetic disorder in our kindred.