Sickle cell mice exhibit mechanical allodynia and enhanced responsiveness in light touch cutaneous mechanoreceptors

ABSTRACT: BACKGROUND: Sickle cell disease (SCD) is associated with both acute vaso-occlusive painful events as well as chronic pain syndromes, including heightened sensitivity to touch. We have previously shown that mice with severe SCD (HbSS mice; express 100% human sickle hemoglobin in red blood cells; RBCs) have sensitized nociceptors, which contribute to increased mechanical sensitivity. Yet, the hypersensitivity in these neural populations alone may not fully explain the mechanical allodynia phenotype in mouse and humans. FINDINGS: Using the Light Touch Behavioral Assay, we found HbSS mice exhibited increased responses to repeated application of both innocuous punctate and dynamic force compared to control HbAA mice (100% normal human hemoglobin). HbSS mice exhibited a 2-fold increase in percent response to a 0.7mN von Frey monofilament when compared to control HbAA mice. Moreover, HbSS mice exhibited a 1.7-fold increase in percent response to the dynamic light touch "puffed" cotton swab stimulus. We further investigated the mechanisms that drive this behavioral phenotype by focusing on the cutaneous sensory neurons that primarily transduce innocuous, light touch. Low threshold cutaneous afferents from HbSS mice exhibited sensitization to mechanical stimuli that manifested as an increase in the number of evoked action potentials to suprathreshold force. Rapidly adapting (RA) Abeta and Adelta D-hair fibers showed the greatest sensitization, each with a 75% increase in suprathreshold firing compared to controls. Slowly adapting (SA) Abeta afferents had a 25% increase in suprathreshold firing compared to HbAA controls. CONCLUSIONS: These novel findings demonstrate mice with severe SCD exhibit mechanical allodynia to both punctate and dynamic light touch and suggest that this behavioral phenotype may be mediated in part by the sensitization of light touch cutaneous afferent fibers to suprathreshold force. These findings indicate that Abeta fibers can be sensitized to mechanical force and should potentially be examined for sensitization in other tissue injury and disease models.     

Effect of cigarette smoke exposure and structural modifications on the α-1 Antitrypsin interaction with caspases

α-1 Antitrypsin (A1AT) is a serpin with a major protective effect against cigarette smoke-induced emphysema development, and patients with mutations of the A1AT gene display a markedly increased risk for developing emphysema. We reported that A1AT protects lung endothelial cells from apoptosis and inhibits caspase-3 activity. It is not clear if cigarette smoking or A1AT mutations alter the caspase-3 inhibitory activity of A1AT and if this serpin alters the function of other caspases. We tested the hypothesis that the caspase-3 inhibitory activity of A1AT is impaired by cigarette smoking and that the A1AT RCL, the key antiprotease domain of the serpin, is required for its interaction with the caspase. We examined the caspase-3 inhibitory activity of human A1AT purified from plasma of actively smoking and nonsmoking individuals, either affected or unaffected with chronic obstructive pulmonary disease. We also tested the caspase inhibitory activity of two mutant forms of A1AT, the recombinant human piZZ and the RCL-deleted (RCL-null) A1AT forms. A1AT purified from the blood of active smokers exhibited marked attenuation in its caspase-3 inhibitory activity, independent of disease status. In vitro exposure of the normal (MM) form of A1AT to cigarette smoke extract reduced its ability to interact with caspase-3, measured by isothermal titration calorimetry, as did the deletion of the RCL, but not the ZZ point mutation. In cell-free assays A1AT was capable of inhibiting all executioner caspases, -3, -7 and especially -6, but not the initiator or inflammatory caspases. The inhibitory effect of A1AT against caspase-6 was tested in vivo, where overexpression of both human MM and ZZ-A1AT via adeno-associated virus transduction significantly protected against apoptosis and against airspace damage induced by intratracheal instillation of caspase-6 in mice. These data indicate a specific inhibitory effect of A1AT on executioner caspases, which is profoundly attenuated by active exposure to cigarette smoking and is dependent on the protein RCL, but is not affected by the PiZZ mutation.     

Population level evidence for seasonality of the human microbiome

The objective of this study is to determine whether human body odors undergo seasonal modulation. We utilized google trends search volume from the United States of America from January 1, 2010 to June 24, 2017 for a number of predetermined body odors. Regression modeling of time series data was completed. Our primary outcome was to determine the proportion of the variability in Internet searches for each unpleasant odor (about the mean) that is explained by a seasonal model. We determined that the seasonal (sinusoidal) model provided a significantly better fit than the null model (best straight line fit) for all searches relating to human body odors (P <.0001 for each). This effect was easily visible to the naked eye in the raw time series data. Seasonality explained 88% of the variability in search volume for flatulence (i.e. R2 = 0.88), 65% of the variability in search volume for axillary odor, 60% of the variability in search volume for foot odor, and 58% of the variability in search volume for bad breath. Flatulence and bad breath tended to peak in January, foot odor in February, and Axillary odor in July. We conclude that searching by the general public for information on unpleasant body odors undergoes substantial seasonal variation, with the timing of peaks and troughs varying with the body part involved. The symptom burden of such smells may have a similar seasonal variation, as might the composition of the commensal bacterial microflora that play a role in creating them     

An electrically-controlled programmable microfluidic concentration waveform generator

Background

Biological systems have complicated environmental conditions that vary both spatially and temporally. It becomes necessary to impose time-varying soluble factor concentrations to study such systems, including cellular responses to pharmaceuticals, inflammation with waxing and waning cytokine concentrations, as well as circadian rhythms and their metabolic manifestations. There is therefore a need for platforms that can achieve time-varying concentrations with arbitrary waveforms.

Results

To address this need, we developed a microfluidic system that can deliver concentration waveforms in a fast and accurate manner by adopting concepts and tools from electrical engineering and fluid mechanics. Specifically, we employed pulse width modulation (PWM), a commonly used method for generating analog signals from digital sources. We implement this technique using three microfluidic components via laser ablation prototyping: low-pass filter (lower frequency signals permitted, high frequency signals blocked), resistor, and mixer. Each microfluidic component was individually studied and iteratively tuned to generate desired concentration waveforms with high accuracy. Using fluorescein as a small-molecule soluble factor surrogate, we demonstrated a series of concentration waveforms, including square, sawtooth, sinusoidal, and triangle waves with frequencies ranging from 100 mHz to 400 mHz.

Conclusion

We reported the fabrication and characterization of microfluidic platform that can generate time-varying concentrations of fluorescein with arbitrary waveforms. We envision that this platform will enable a wide range of biological studies, where time-varying soluble factor concentrations play a critical role. In addition, the technology is expected to assist in the development of biomedical devices that allow precise dosing of pharmaceuticals for enhanced therapeutic efficacy and reduced toxicity.

     

Disruption of a mitochondrial MutS DNA repair enzyme homologue confers drug resistance in the parasite Toxoplasma gondii

MutS homologues (MSHs) are critical components of the eukaryotic mismatch repair machinery. In addition to repairing mismatched DNA, mismatch repair enzymes are known in higher eukaryotes to directly signal cell cycle arrest and apoptosis in response to DNA-damaging agents. Accordingly, mammalian cells lacking certain MSHs are resistant to chemotherapeutic drugs. Interestingly, we have discovered that the disruption of TgMSH-1 , an MSH in the pathogenic parasite, Toxoplasma gondii , confers drug resistance. Through a genetic selection for T. gondii mutants resistant to the antiparasitic drug monensin, we have isolated a strain that is resistant not only to monensin but also to salinomycin and the alkylating agent, methylnitrosourea. We have shown that this phenotype is due to the disruption of TgMSH-1 as the multidrug-resistance phenotype is complemented by a wild-type copy of TgMSH-1 and is recapitulated by a directed disruption of this gene in a wild-type strain. We have also shown that, unlike previously described MSHs involved in signalling, TgMSH-1 localizes to the parasite mitochondrion. These results provide the first example of a mitochondrial MSH that is involved in drug sensitivity and implicate the induction of mitochondrial stress as a mode of action of the widely used drug, monensin.     

Polyphasic Characterization of a Thermotolerant Siderophilic Filamentous Cyanobacterium That Produces Intracellular Iron Deposits

Despite the high potential for oxidative stress stimulated by reduced iron, contemporary iron-depositing hot springs with circum-neutral pH are intensively populated with cyanobacteria. Therefore, studies of the physiology, diversity, and phylogeny of cyanobacteria inhabiting iron-depositing hot springs may provide insights into the contribution of cyanobacteria to iron redox cycling in these environments and new mechanisms of oxidative stress mitigation. In this study the morphology, ultrastructure, physiology, and phylogeny of a novel cyanobacterial taxon, JSC-1, isolated from an iron-depositing hot spring, were determined. The JSC-1 strain has been deposited in ATCC under the name Marsacia ferruginose, accession number BAA-2121. Strain JSC-1 represents a new operational taxonomical unit (OTU) within Leptolyngbya sensu lato. Strain JSC-1 exhibited an unusually high ratio between photosystem (PS) I and PS II, was capable of complementary chromatic adaptation, and is apparently capable of nitrogen fixation. Furthermore, it synthesized a unique set of carotenoids, but only chlorophyll a. Strain JSC-1 not only required high levels of Fe for growth (≥40 μM), but it also accumulated large amounts of extracellular iron in the form of ferrihydrite and intracellular iron in the form of ferric phosphates. Collectively, these observations provide insights into the physiological strategies that might have allowed cyanobacteria to develop and proliferate in Fe-rich, circum-neutral environments.Cyanobacteria inhabiting ferrous iron-rich hot springs with circum-neutral pH represent unique models for examining the mechanisms by which early organisms evolved to cope with such habitats common on early Earth. Such organisms have previously been shown to be resistant to Fe²⁺ (37) or Fe³⁺ (6, 7) at concentrations in the micromolar to millimolar range. Moreover, high Fe concentrations (apparent optimum of ∼0.5 mM) stimulated the growth of these cyanobacteria, which were described as siderophilic (having an affinity for iron) cyanobacteria (7).The cyanobacteria inhabiting the Chocolate Pots hot springs in Yellowstone National Park, Wyoming, were shown to have played at least a passive role in contributing to iron deposition by serving as nucleation sites for the accumulation of iron minerals and associated silica deposits (36, 38). The precipitation of external iron that encrusts the cyanobacterial cells inhabiting this hot spring appears to be dependent on the species composition and chemistry of the mat (36, 38); however, multiple anoxygenic phototrophs found in the Chocolate Pots hot springs (8) could also contribute to the formation of Fe oxides (21, 49). Therefore, only iron mineralization experiments with model cyanobacterial strains can demonstrate the role of siderophilic cyanobacteria in the formation of specific, iron-bearing minerals.An additional common feature of circum-neutral iron-depositing hot springs is elevated concentrations of hydrogen peroxide (50). Shcolnick and coauthors (41) showed that a wild type of Synechococcus sp. PCC 6803 was resistant to 8 mM H₂O₂ if grown with 0.3 μM Fe³⁺, while the same concentration of hydrogen peroxide completely inhibited the growth of this cyanobacterium if it was grown with 10 μM Fe³⁺. If a similar correlation between iron concentration and the magnitude of an externally applied oxidative stress were the case for siderophilic cyanobacteria, iron-depositing hot springs should be free of cyanobacteria. However, such springs are very rich with cyanobacteria (38, 7, 36), which suggests that siderophilic cyanobacteria may possess unusual mechanisms of iron homeostasis maintenance and oxidative stress mitigation. Additionally, understanding iron tolerance and phenomena associated with siderophily in oxygenic prokaryotes is also important because such siderophilic organisms might help us find applications for bioremediation of waters polluted with iron.The current work describes the morphology, ultrastructure, physiology, and phylogeny of a previously undescribed, siderophilic cyanobacterium. The results of this polyphasic characterization led to the conclusion that strain JSC-1 represents a new operational taxonomic unit (OTU). (The epithet for JSC-1, Marsacia ferruginose, was chosen in honor of Nicole Tandeau de Marsac.) Additionally, biomineralization of intracellular iron by a cyanobacterium is demonstrated for the first time.     

Production of polyhydroxybutyrate in switchgrass, a value-added co-product in an important lignocellulosic biomass crop

Polyhydroxyalkanoate bio-based plastics made from renewable resources can reduce petroleum consumption and decrease plastic waste disposal issues as they are inherently biodegradable in soil, compost and marine environments. In this paper, the successful engineering of the biomass crop switchgrass ( Panicum virgatum L.) for the synthesis of polyhydroxybutyrate (PHB) is reported. Polymer production was monitored in more than 400 primary transformants grown under in vitro and glasshouse conditions. Plants containing up to 3.72% dry weight of PHB in leaf tissues and 1.23% dry weight of PHB in whole tillers were obtained. Results from the analysis of the polymer distribution at the cellular and whole plant levels are presented, and target areas for the improvement of PHB production are highlighted. Polymer accumulation was also analysed in the T₁ generation obtained from controlled crosses of transgenic plants. This study presents the first successful expression of a functional multigene pathway in switchgrass, and demonstrates that this high-yielding biomass crop is amenable to the complex metabolic engineering strategies necessary to produce high-value biomaterials with lignocellulose-derived biofuels.