This study investigated the effect of bacteriophages (phages) e11/2 and e4/1c against Escherichia coli O157:H7 in an ex vivo rumen model and in cattle in vivo. In the ex vivo rumen model, samples were inoculated with either 103 or 106 CFU/ml inoculum of E. coli O157:H7 and challenged separately with each bacteriophage. In the presence of phage e11/2, the numbers of E. coli O157:H7 bacteria were significantly (P < 0.05) reduced to below the limit of detection within 1 h. Phage e4/1c significantly (P < 0.05) reduced E. coli O157:H7 numbers within 2 h of incubation, but the number of surviving E. coli O157:H7 bacteria then remained unchanged over a further 22-h incubation period. The ability of a phage cocktail of e11/2 and e4/1c to reduce the fecal shedding of E. coli O157:H7 in experimentally inoculated cattle was then investigated in two cattle trials. Cattle (yearlings, n = 20 for trial one; adult fistulated cattle, n = 2 for trial two) were orally inoculated with 1010 CFU of E. coli O157:H7. Animals (n = 10 for trial one; n = 1 for trial two) were dosed daily with a bacteriophage cocktail of 1011 PFU for 3 days postinoculation. E. coli O157:H7 and phage numbers in fecal and/or rumen samples were determined over 7 days postinoculation. E. coli O157:H7 numbers rapidly declined in all animals within 24 to 48 h; however, there was no significant difference (P > 0.05) between the numbers of E. coli O157:H7 bacteria shed by the phage-treated or control animals. Phages were recovered from the rumen but not from the feces of the adult fistulated animal in trial two but were recovered from the feces of the yearling animals in trial one. While the results from the rumen model suggest that phages are effective in the rumen, further research is required to improve the antimicrobial effectiveness of phages for the elimination of E. coli O157:H7 in vivo.

Images

We demonstrate that potassium 1-13C-phosphoenolpyruvate becomes hyperpolarized potassium 1-13C –phospholactate with 13C T1 = 36 s after molecular hydrogenation by PASADENA (Parahydrogen and Synthesis Allows Dramatically Enhanced Nuclear Alignment). This proof-of-principle study was conducted with a fully protonated molecular precursor. 13C was polarized to a level of 1 %, corresponding to nearly 4,000 fold sensitivity enhancement at 3 T. The relevant homo- and heteronuclear spin-spin couplings are reported.

A total of 220 lactic acid bacteria isolates were screened for antifungal activity using Aspergillus fumigatus and Aspergillus niger as the target strains. Four Lactobacillus strains exhibited strong inhibitory activity on agar surfaces. All four were also identified as having strong inhibitory activity against the human pathogenic fungi Microsporum canis, Microsporum gypseum and Epidermophyton floccosum. One of the four lactobacilli, namely Lb. reuteri ee1p exhibited the most inhibition against dermatophytes. Cell-free culture supernatants of Lb. reuteri ee1p and of the non-antifungal Lb. reuteri M13 were freeze-dried and used to access and compare antifungal activity in agar plate assays and microtiter plate assays. Addition of the Lb. reuteri ee1p freeze-dried cell-free supernatant powder into the agar medium at concentrations greater than 2% inhibited all fungal colony growth. Addition of the powder at 5% to liquid cultures caused complete inhibition of fungal growth on the basis of turbidity. Freeze-dried supernatant of the non-antifungal Lb. reuteri M13 at the same concentrations had a much lesser effect. As Lb. reuteri M13 is very similar to the antifungal strain ee1p in terms of growth rate and final pH in liquid culture, and as it has little antifungal activity, it is clear that other antifungal compounds must be specifically produced (or produced at higher levels) by the anti-dermatophyte strain Lb. reuteri ee1p. Reuterin was undetectable in all four antifungal strains. The cell free supernatant of Lb. reuteri ee1p was analyzed by LC-FTMS using an Accela LC coupled to an LTQ Orbitrap XL mass spectrometer. The high mass accuracy spectrum produced by compounds in the Lb. reuteri ee1p strain was compared with both a multianalyte chromatogram and individual spectra of standard anti-fungal compounds, which are known to be produced by lactic acid bacteria. Ten antifungal metabolites were detected.

Cell migration is a fundamental biological function, critical during development and regeneration, whereas deregulated migration underlies neurological birth defects and cancer metastasis. MARCKS-like protein 1 (MARCKSL1) is widely expressed in nervous tissue, where, like Jun N-terminal protein kinase (JNK), it is required for neural tube formation, though the mechanism is unknown. Here we show that MARCKSL1 is directly phosphorylated by JNK on C-terminal residues (S120, T148, and T183). This phosphorylation enables MARCKSL1 to bundle and stabilize F-actin, increase filopodium numbers and dynamics, and retard migration in neurons. Conversely, when MARCKSL1 phosphorylation is inhibited, actin mobility increases and filopodium formation is compromised whereas lamellipodium formation is enhanced, as is cell migration. We find that MARCKSL1 mRNA is upregulated in a broad range of cancer types and that MARCKSL1 protein is strongly induced in primary prostate carcinomas. Gene knockdown in prostate cancer cells or in neurons reveals a critical role for MARCKSL1 in migration that is dependent on the phosphorylation state; phosphomimetic MARCKSL1 (MARCKSL1S120D,T148D,T183D) inhibits whereas dephospho-MARCKSL1S120A,T148A,T183A induces migration. In summary, these data show that JNK phosphorylation of MARCKSL1 regulates actin homeostasis, filopodium and lamellipodium formation, and neuronal migration under physiological conditions and that, when ectopically expressed in prostate cancer cells, MARCKSL1 again determines cell movement.

Low levels of ultraviolet (UV)-radiation alter the morphology of plants. UV-B exposure can lead to shorter petioles and shorter, narrower and/or thicker leaf blades. The resulting decrease in leaf area has been associated with inhibitory UV-B effects on biomass accumulation. In Arabidopsis, UV-B effects on leaf area have variously been attributed to altered cell division, cell expansion or combinations of these two processes. A dedicated UV-B sensory system, crosstalk between flavonoids and auxins, endoreduplication and generic Stress Induced Morphogenic Responses (SIMR) have all been proposed to contribute to the UV-B phenotype. Here, we propose that UV-mediated morphogenesis, rather than being controlled by a single regulatory pathway, is controlled by a regulatory blur involving multiple compensatory molecular and physiological feedback interactions.

For a variety of infectious diseases, the richness of the community of potential host species has emerged as an important factor in pathogen transmission, whereby a higher richness of host species is associated with a lowered disease risk. The proposed mechanism driving this pattern is an increased likelihood in species-rich communities that infectious individuals will encounter dead-end hosts. Mosquito-borne pathogen systems potentially are exceptions to such “dilution effects” because mosquitoes vary their rates of use of vertebrate host species as bloodmeal sources relative to host availabilities. Such preferences may violate basic assumptions underlying the hypothesis of a dilution effect in pathogen systems. Here, we describe development of a model to predict exposure risk of sentinel chickens to eastern equine encephalitis virus (EEEV) in Walton County, Florida between 2009 and 2010 using avian species richness as well as densities of individual host species potentially important to EEEV transmission as candidate predictor variables. We found the highest support for the model that included the density of northern cardinals, a highly preferred host of mosquito vectors of EEEV, as a predictor variable. The highest-ranking model also included Culiseta melanura abundance as a predictor variable. These results suggest that mosquito preferences for vertebrate hosts influence pathogen transmission.

Objective

SMAD proteins are downstream effectors of the TGF-β signaling pathway. Smad3 null mice develop colorectal cancer by 6 months of age. In this study, we have examined whether the loss of Smad3 promotes gastric neoplasia in mice.

Design

The stomachs of Smad3−/− mice were compared with age-matched Smad3 heterozygous and wild type mice. E-cadherin, Ki-67, phosphoSTAT3, and TFF2/SP expression was analyzed by immunohistochemisty. The shRNA-mediated knockdown of Smad3 in AGS and MKN28 cells was also performed. In addition, we examined alterations in DCLK1-expressing cells.

Results

Smad3−/− mouse stomachs at 6 months of age revealed the presence of exophytic growths along the lesser curvature in the proximal fundus. Six-month-old Smad3−− mouse stomachs showed metaplastic columnar glands initiating from the transition zone junction between the forestomach and the glandular epithelium along the lesser curvature. Ten month-old Smad3−/− mice all exhibited invasive gastric neoplastic changes with increased Ki-67, phosphoSTAT3 expression, and aberrant cytosolic E-cadherin staining in papillary glands within the invading submucosal gland. The shRNA-mediated knockdown of Smad3 in AGS and MKN28 cells promoted the expression of phosphoSTAT3. DCLK1-expressing cells, which also stained for the tuft cell marker acetylated-α-tubulin, were observed in 10-month-old Smad3−/− mice within tumors and in fundic invasive lesions.

Conclusion

Smad3 null mice develop gastric tumors in the fundus, which arise from the junction between the forestomach and the glandular epithelium and progress to prominent invasive tumors over time. Smad3 null mice represent a novel model of fundic gastric tumor initiated from forestomach/glandular transition zone along the lesser curvature.

Human disease incidence attributed to arbovirus infection is increasing throughout the world, with effective control interventions limited by issues of sustainability, insecticide resistance and the lack of effective vaccines. Several promising control strategies are currently under development, such as the release of mosquitoes trans-infected with virus-blocking Wolbachia bacteria. Implementation of any control program is dependent on effective virus surveillance and a thorough understanding of virus-vector interactions. Massively parallel sequencing has enormous potential for providing comprehensive genomic information that can be used to assess many aspects of arbovirus ecology, as well as to evaluate novel control strategies. To demonstrate proof-of-principle, we analyzed Aedes aegypti or Aedes albopictus experimentally infected with dengue, yellow fever or chikungunya viruses. Random amplification was used to prepare sufficient template for sequencing on the Personal Genome Machine. Viral sequences were present in all infected mosquitoes. In addition, in most cases, we were also able to identify the mosquito species and mosquito micro-organisms, including the bacterial endosymbiont Wolbachia. Importantly, naturally occurring Wolbachia strains could be differentiated from strains that had been trans-infected into the mosquito. The method allowed us to assemble near full-length viral genomes and detect other micro-organisms without prior sequence knowledge, in a single reaction. This is a step toward the application of massively parallel sequencing as an arbovirus surveillance tool. It has the potential to provide insight into virus transmission dynamics, and has applicability to the post-release monitoring of Wolbachia in mosquito populations.

Invasive species often display different patterns of parasite burden and virulence compared to their native counterparts. These differences may be the result of variability in host-parasite co-evolutionary relationships, the occurrence of novel host-parasite encounters, or possibly innate differences in physiological responses to infection between invasive and native hosts. Here we examine the adaptive, humoral immune responses of a resistant, native bird and a susceptible, invasive bird to an arbovirus (Buggy Creek virus; Togaviridae: Alphavirus) and its ectoparasitic arthropod vector (the swallow bug; Oeciacus vicarius). Swallow bugs parasitize the native, colonially nesting cliff swallow (Petrochelidon pyrrhonota) and the introduced house sparrow (Passer domesticus) that occupies nests in cliff swallow colonies. We measured levels of BCRV-specific and swallow bug-specific IgY levels before nesting (prior to swallow bug exposure) and after nesting (after swallow bug exposure) in house sparrows and cliff swallows in western Nebraska. Levels of BCRV-specific IgY increased significantly following nesting in the house sparrow but not in the cliff swallow. Additionally, house sparrows displayed consistently higher levels of swallow bug-specific antibodies both before and after nesting compared to cliff swallows. The higher levels of BCRV and swallow bug specific antibodies detected in house sparrows may be reflective of significant differences in both antiviral and anti-ectoparasite immune responses that exist between these two avian species. To our knowledge, this is the first study to compare the macro- and microparasite-specific immune responses of an invasive and a native avian host exposed to the same parasites.

Background

Variation in the implementation of complex multilevel interventions can impact on their delivery and outcomes. Few suicide prevention interventions, especially multilevel interventions, have included evaluation of both the process of implementation as well as outcomes. Such evaluation is essential for the replication of interventions, for interpreting and understanding outcomes, and for improving implementation science. This paper reports on a process evaluation of the early implementation stage of an optimised suicide prevention programme (OSPI-Europe) implemented in four European countries.

Methods

The process analysis was conducted within the framework of a realist evaluation methodology, and involved case studies of the process of implementation in four European countries. Datasets include: repeated questionnaires to track progress of implementation including delivery of individual activities and their intensity; serial interviews and focus groups with stakeholder groups; and detailed observations at OSPI implementation team meetings.

Results

Analysis of local contexts in each of the four countries revealed that the advisory group was a key mechanism that had a substantial impact on the ease of implementation of OSPI interventions, particularly on their ability to recruit to training interventions. However, simply recruiting representatives of key organisations into an advisory group is not sufficient to achieve impact on the delivery of interventions. In order to maximise the potential of high level ‘gatekeepers’, it is necessary to first transform them into OSPI stakeholders. Motivations for OSPI participation as a stakeholder included: personal affinity with the shared goals and target groups within OSPI; the complementary and participatory nature of OSPI that adds value to pre-existing suicide prevention initiatives; and reciprocal reward for participants through access to the extended network capacity that organisations could accrue for themselves and their organisations from participation in OSPI.

Conclusions

Exploring the role of advisory groups and the meaning of participation for these participants revealed some key areas for best practice in implementation: careful planning of the composition of the advisory group to access target groups; the importance of establishing common goals; the importance of acknowledging and complementing existing experience and activity; and facilitating an equivalence of benefit from network participation.

Background

Bocaviruses are classified as a genus within the Parvoviridae family of single-stranded DNA viruses and are pathogenic in some mammalian species. Two species have been previously reported in dogs, minute virus of canines (MVC), associated with neonatal diseases and fertility disorders; and Canine bocavirus (CBoV), associated with respiratory disease.

Findings

In this study using deep sequencing of enriched viral particles from the liver of a dog with severe hemorrhagic gastroenteritis, necrotizing vasculitis, granulomatous lymphadenitis and anuric renal failure, we identified and characterized a novel bocavirus we named Canine bocavirus 3 (CnBoV3). The three major ORFs of CnBoV3 (NS1, NP1 and VP1) shared less than 60% aa identity with those of other bocaviruses qualifying it as a novel species based on ICTV criteria. Inverse PCR showed the presence of concatemerized or circular forms of the genome in liver.

Conclusions

We genetically characterized a bocavirus in a dog liver that is highly distinct from prior canine bocaviruses found in respiratory and fecal samples. Its role in this animal’s complex disease remains to be determined.

The eye has protective barriers (ie, the conjunctival and corneal membranes) and defense mechanisms (ie, reflex tearing, blinking, lacrimal drainage) which present challenges to topical drug delivery. Topical ocular corticosteroids are commonly used in the treatment of anterior segment diseases and inflammation associated with ocular surgery, and manufacturers continually strive to improve their characteristics. We describe the development of a novel ophthalmic gel formulation of loteprednol etabonate (LE), a C-20 ester-based corticosteroid with an established safety profile, in the treatment of ocular inflammatory conditions. The new LE gel formulation is non-settling, eliminating the need to shake the product to resuspend the drug, has a pH close to that of tears, and a low preservative concentration. The rheological characteristics of LE gel are such that the formulation is instilled as a drop and transitions to a fluid upon instillation in the eye, yet retains sufficient viscosity to prolong ocular surface retention. The new formulation provides consistent, uniform dosing as evidenced by dose extrusion studies, while pharmacokinetic studies in rabbits demonstrated rapid and sustained exposure to LE in ocular tissues following instillation of LE gel. Finally, results from two clinical studies of LE gel in the treatment of postoperative inflammation and pain following cataract surgery indicate that it was safe and effective. Most patients reported no unpleasant drop sensation upon instillation, and reports of blurred vision were rare.

New antibacterial agents are urgently needed for the elimination of biofilm-forming bacteria that are highly resistant to traditional antimicrobial agents. Proliferation of such bacteria can lead to significant economic losses in the agri-food sector. This study demonstrates the potential of the bacteriophage-derived peptidase, CHAPK, as a biocidal agent for the rapid disruption of biofilm-forming staphylococci, commonly associated with bovine mastitis. Purified CHAPK applied to biofilms of Staphylococcus aureus DPC5246 completely eliminated the staphylococcal biofilms within 4 h. In addition, CHAPK was able to prevent biofilm formation by this strain. The CHAPK lysin also reduced S. aureus in a skin decolonization model. Our data demonstrates the potential of CHAPK as a biocidal agent for prevention and treatment of biofilm-associated staphylococcal infections or as a decontaminating agent in the food and healthcare sectors.

Helicobacter pylori is the strongest known risk factor for the development of gastric adenocarcinoma. H. pylori expresses a repertoire of virulence factors that increase gastric cancer risk, including the cag pathogenicity island and the vacuolating cytotoxin (VacA). One host element that promotes carcinogenesis within the gastrointestinal tract is Krüppel-like factor 5 (KLF5), a transcription factor that mediates key cellular functions. To define the role of KLF5 within the context of H. pylori-induced inflammation and injury, human gastric epithelial cells were co-cultured with the wild-type cag+ H. pylori strain 60190. KLF5 expression was significantly upregulated following co-culture with H. pylori, but increased expression was independent of the cag island or VacA. To translate these findings into an in vivo model, C57BL/6 mice were challenged with the wild-type rodent-adapted cag+ H. pylori strain PMSS1 or a PMSS1 cagE− isogenic mutant. Similar to findings in vitro, KLF5 staining was significantly enhanced in gastric epithelium of H. pylori-infected compared to uninfected mice and this was independent of the cag island. Flow cytometry revealed that the majority of KLF5+ cells also stained positively for the stem cell marker, Lrig1, and KLF5+/Lrig1+ cells were significantly increased in H. pylori-infected versus uninfected tissue. To extend these results into the natural niche of this pathogen, levels of KLF5 expression were assessed in human gastric biopsies isolated from patients with or without premalignant lesions. Levels of KLF5 expression increased in parallel with advancing stages of neoplastic progression, being significantly elevated in gastritis, intestinal metaplasia, and dysplasia compared to normal gastric tissue. These results indicate that H. pylori induces expression of KLF5 in gastric epithelial cells in vitro and in vivo, and that the degree of KLF5 expression parallels the severity of premalignant lesions in human gastric carcinogenesis.

Background

Fetal Alcohol Spectrum Disorders (FASD) are a collection of disorders resulting from fetal ethanol exposure, which causes a wide range of physical, neurological and behavioral deficits including heightened susceptibility for alcoholism and addictive disorders. While a number of mechanisms have been proposed for how ethanol exposure disrupts brain development, with selective groups of neurons undergoing reduced proliferation, dysfunction and death, the induction of a new neurotransmitter phenotype by ethanol exposure has not yet been reported.

Principal Findings

The effects of embryonic and larval ethanol exposure on brain development were visually monitored using transgenic zebrafish expressing cell-specific green fluorescent protein (GFP) marker genes. Specific subsets of GFP-expressing neurons were highly sensitive to ethanol exposure, but only during defined developmental windows. In the med12 mutant, which affects the Mediator co-activator complex component Med12, exposure to lower concentrations of ethanol was sufficient to reduce GFP expression in transgenic embryos. In transgenic embryos and larva containing GFP driven by an oxytocin-like (oxtl) promoter, ethanol exposure dramatically up-regulated GFP expression in a small group of hindbrain neurons, while having no effect on expression in the neuroendocrine preoptic area.

Conclusions

Alcohol exposure during limited embryonic periods impedes the development of specific, identifiable groups of neurons, and the med12 mutation sensitizes these neurons to the deleterious effects of ethanol. In contrast, ethanol exposure induces oxtl expression in the hindbrain, a finding with profound implications for understanding alcoholism and other addictive disorders.

Molecular imaging biomarkers of proliferation hold great promise for quantifying response to personalized medicine. One such approach utilizes the positron emission tomography (PET) tracer 3′-deoxy-3′ [18F]-fluorothymidine ([18F]FLT), an investigational agent whose uptake reflects thymidine salvage-dependent DNA synthesis. The goal of this study was to evaluate [18F]FLT-PET in the setting of Ménétrier’s disease (MD), a rare, premalignant hyperproliferative disorder of the stomach treatable with cetuximab therapy. Over 15 months, a patient with confirmed MD underwent cetuximab therapy and was followed with sequential [18F]FLT-PET. For comparison to MD, an [18F]FLT-PET study was conducted in another patient to quantify uptake in a normal stomach.Prior to cetuximab therapy, stomach tissue in MD was easily visualized with [18F]FLT-PET, with pre-treatment uptake levels exceeding normal stomach uptake by approximately 4-fold. Diminished [18F]FLT-PET in MD was observed following the initial and subsequent doses of cetuximab and correlated with clinical resolution of the disease. To our knowledge, this study reports the first clinical use of [18F]FLT-PET to assess proliferation in a premalignant disorder. We illustrate that the extent of MD involvement throughout the stomach could be easily visualized using [18F]FLT-PET, and that response to cetuximab could be followed quantitatively and non-invasively in sequential [18F]FLT-PET studies. Thus, [18F]FLT-PET appears to have potential to monitor response to treatment in this and potentially other hyperproliferative disorders.

The high mutation rate of RNA viruses enables a diverse genetic population of viral genotypes to exist within a single infected host. In-host genetic diversity could better position the virus population to respond and adapt to a diverse array of selective pressures such as host-switching events. Multiple new coronaviruses, including SARS, have been identified in human samples just within the last ten years, demonstrating the potential of coronaviruses as emergent human pathogens. Deep sequencing was used to characterize genomic changes in coronavirus quasispecies during simulated host-switching. Three bovine nasal samples infected with bovine coronavirus were used to infect human and bovine macrophage and lung cell lines. The virus reproduced relatively well in macrophages, but the lung cell lines were not infected efficiently enough to allow passage of non lab-adapted samples. Approximately 12 kb of the genome was amplified before and after passage and sequenced at average coverages of nearly 950×(454 sequencing) and 38,000×(Illumina). The consensus sequence of many of the passaged samples had a 12 nucleotide insert in the consensus sequence of the spike gene, and multiple point mutations were associated with the presence of the insert. Deep sequencing revealed that the insert was present but very rare in the unpassaged samples and could quickly shift to dominate the population when placed in a different environment. The insert coded for three arginine residues, occurred in a region associated with fusion entry into host cells, and may allow infection of new cell types via heparin sulfate binding. Analysis of the deep sequencing data indicated that two distinct genotypes circulated at different frequency levels in each sample, and support the hypothesis that the mutations present in passaged strains were “selected” from a pre-existing pool rather than through de novo mutation and subsequent population fixation.

BACKGROUND

Craving is a hallmark of addiction and characterized by obsessive thoughts about, and compulsive urges to use, a substance. While craving is frequently thought of as primarily being a feature of acute withdrawal, there is evidence to suggest that it increases in strength over extended periods of abstinence. While several measures are available to assess acute craving states, there remains a lack of clinical measures appropriate for capturing the enduring cognitive aspects of urges to use drugs. The present study was designed to develop and validate a measure of obsessive-compulsive thoughts in cocaine-dependent individuals.

METHODS

The proposed 14-item Obsessive Compulsive Cocaine Use Scale (OCCUS) was administered to 107 individuals; 55 participants meeting diagnostic criteria for cocaine dependence and 52 recreational users of cocaine. In addition to the OCCUS, participants also completed the Drug Abuse Screening Test, Cocaine Craving Questionnaire-Now, and Social Desirability Scale of the California Personality Inventory.

RESULTS

Results of confirmatory factor analysis indicated that the OCCUS fit the two-factor structure of the Obsessive Compulsive Drinking Scale on which it was based, independently assessing the “obsessive” and “compulsive” aspects of cocaine dependence. The OCCUS demonstrated good internal consistency reliability and convergent, discriminant, and criterion validity.

CONCLUSION

The proposed measure is a promising step towards the successful capture of the long-term cognitive features of craving for cocaine via self-report, and should represent a useful tool for clinical and research use.

The present investigation examined the main and interactive effects of anxiety sensitivity and behavioral distress tolerance, indexed using the breath-holding task, in relation to PTSD symptom severity among trauma-exposed adults. Participants were 88 adults (63.6% women; Mage = 22.9, SD = 9.1, Range = 18-62), recruited from the community, who met DSM-IV-TR PTSD Criterion A for lifetime trauma exposure. Covariates included number of potentially traumatic events, nonclinical panic attack history, and participant sex. Anxiety sensitivity was significantly incrementally associated with PTSD total symptom severity, as well as Avoidance and Hyperarousal symptom severity (p's < .01). Breath-holding duration was not significantly related to PTSD symptom severity (p's > .05). However, breath-holding duration emerged as a significant moderator of the association between anxiety sensitivity and PTSD Avoidance symptom severity, such that lower breath-holding duration exacerbated the effect of heightened anxiety sensitivity with regard to PTSD Avoidance symptom severity.

A device is presented for efficiently enriching parahydrogen by pulsed injection of ambient hydrogen gas. Hydrogen input to the generator is pulsed at high pressure to a catalyst chamber making thermal contact with the cold head of a closed cycle cryostat maintained between 15 and 20 K. The system enables fast production (0.9 standard liters per minute) and allows for a wide range of production targets. Production rates can be systematically adjusted by varying the actuation sequence of high-pressure solenoid valves, which are controlled via an open source microcontroller to sample all combinations between fast and thorough enrichment by varying duration of hydrogen contact in the catalyst chamber. The entire enrichment cycle from optimization to quantification and storage kinetics are also described. Conversion of the para spin-isomer to orthohydrogen in borosilicate tubes was measured at 8 minute intervals over a period of 64 hours with a 12 Tesla NMR spectrometer. These relaxation curves were then used to extract initial enrichment by exploiting the known equilibrium (relaxed) distribution of spin isomers with linear least squares fitting to a single exponential decay curve with an estimated error less than or equal to 1 %. This procedure is time-consuming, but requires only one sample pressurized to atmosphere. Given that tedious matching to external references are unnecessary with this procedure, we find it to be useful for periodic inspection of generator performance. The equipment and procedures offer a variation in generator design that eliminate the need to meter flow while enabling access to increased rates of production. These tools for enriching and quantifying parahydrogen have been in steady use for 3 years and should be helpful as a template or as reference material for building and operating a parahydrogen production facility.

α-synuclein is thought to play a key role in Parkinson’s disease (PD) because it is the major protein in Lewy bodies, and because its gene mutations, duplication, and triplication are associated with early-onset PD. There are conflicting reports as to whether serum and plasma concentrations of α-synuclein and anti-α-synuclein antibodies differ between PD and control subjects. The objectives of this study were to compare the levels of α-synuclein and its antibodies between individuals with typical PD (n = 14), atypical Parkinson syndromes (n = 11), idiopathic rapid eye movement sleep behavior disorder (n = 10), and healthy controls (n = 9), to assess the strength of association between these serum proteins, and to determine group sizes needed for a high probability (80% power) of detecting statistical significance for 25% or 50% differences between typical PD and control subjects for these measurements. Analysis of log-transformed data found no statistically significant differences between groups for either α-synuclein or its antibodies. The concentrations of these proteins were weakly correlated (Spearman rho = 0.16). In subjects with typical PD and atypical Parkinson syndromes, anti-α-synuclein antibody levels above 1.5 µg/ml were detected only in subjects with no more than four years of clinical disease. Power analysis indicated that 236 and 73 samples per group would be required for an 80% probability that 25% and 50% differences, respectively, in mean α-synuclein levels between typical PD and control subjects would be statistically significant; for anti-α-synuclein antibodies, 283 and 87 samples per group would be required. Our findings are consistent with those previous studies which suggested that serum concentrations of α-synuclein and its antibodies are not significantly altered in PD.

Somatic cellular evolution is becoming a popular biological explanation for the common rapid development of resistance to almost every form of cancer therapy and against almost every form of advanced human solid tumors. As a result of the historical power of evolution within nature, this common biological interpretation of the failure to cancer therapy, is leading to a growing despair for many investigators and a stronger turn towards prevention through lifestyle changes. The absolute explosion of molecular scientific discoveries since 1983, in the reductionist identification of specific cancer therapeutic targets, has failed to deliver the impact in the clinic that many of us would have hoped would have result by this time. Personalized molecular medicine may help us reclassify appropriate therapeutic subgroups, but will it significantly impact on the overall specific survival times for all of the cancers combined within the organ type for the entire population? How might we approach this therapeutic dilemma by utilizing new therapeutics insights designed on proven principles of evolution? In other words, can we fight the development of therapeutic resistance in cancer cells by turning established aspects of evolution against the survival of cancer cells within the individual patient. Here we review the concepts of changing the heat habitat and microenvironment of the cancer cell to alter the higher order organization and function of DNA. We have proposed that heat may be a major factor in determining the lasting therapeutic effect on many types of far advanced metastatic tumors.