Self-Powered Wireless Carbohydrate/Oxygen Sensitive Biodevice Based on Radio Signal Transmission

Here for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.     

Regulation of plasmid R1 replication: PcnB and RNase E expedite the decay of the antisense RNA, CopA

The replication frequency of plasmid R1 is controlled by an unstable antisense RNA, CopA, which, by binding to its complementary target, blocks translation of the replication rate-limiting protein RepA. Since the degree of inhibition is directly correlated with the intracellular concentration of CopA, factors affecting CopA turnover can also alter plasmid copy number. We show here that PcnB (PAP I — a poly(A)polymerase of Escherichia coli  ) is such a factor. Previous studies have shown that the copy number of ColE1 is decreased in pcnB mutant strains because the stability of the RNase E processed form of RNAI, the antisense RNA regulator of ColE1 replication, is increased. We find that, analogously, the twofold reduction in R1 copy number caused by a pcnB lesion is associated with a corresponding increase in the stability of the RNase E-generated 3' cleavage product of CopA. These results suggest that CopA decay is initiated by RNase E cleavage and that PcnB is involved in the subsequent rapid decay of the 3' CopA stem-loop segment. We also find that, as predicted, under conditions in which CopA synthesis is unaffected, pcnB mutation reduces RepA translation and increases CopA stability to the same extent.     

Effects of temperature and body size on the clearance rate of Oikopleura dioica

The effects of temperature and body size on the clearance rateof the appendicularian Oikopleura dioica were investigated.The clearance rate increased with the trunk length of the animalsaccording to a power function with exponent 3.2 ± 0.17(range 2.9–3.8). The intercepts of the three regressionswere significantly different with the highest rates at 20°C,corresponding to a Q₁₀ value of 1.78 (10–20°C).     

The composition of the gut microbiota shapes the colon mucus barrier

Two C57BL/6 mice colonies maintained in two rooms of the same specific pathogen-free (SPF) facility were found to have different gut microbiota and a mucus phenotype that was specific for each colony. The thickness and growth of the colon mucus were similar in the two colonies. However, one colony had mucus that was impenetrable to bacteria or beads the size of bacteria—which is comparable to what we observed in free-living wild mice—whereas the other colony had an inner mucus layer penetrable to bacteria and beads. The different properties of the mucus depended on the microbiota, as they were transmissible by transfer of caecal microbiota to germ-free mice. Mice with an impenetrable mucus layer had increased amounts of Erysipelotrichi, whereas mice with a penetrable mucus layer had higher levels of Proteobacteria and TM7 bacteria in the distal colon mucus. Thus, our study shows that bacteria and their community structure affect mucus barrier properties in ways that can have implications for health and disease. It also highlights that genetically identical animals housed in the same facility can have rather distinct microbiotas and barrier structures.     

The gut microbiota modulates host amino acid and glutathione metabolism in mice

The gut microbiota has been proposed as an environmental factor that promotes the progression of metabolic diseases. Here, we investigated how the gut microbiota modulates the global metabolic differences in duodenum, jejunum, ileum, colon, liver, and two white adipose tissue depots obtained from conventionally raised (CONV‐R) and germ‐free (GF) mice using gene expression data and tissue‐specific genome‐scale metabolic models (GEMs). We created a generic mouse metabolic reaction (MMR) GEM, reconstructed 28 tissue‐specific GEMs based on proteomics data, and manually curated GEMs for small intestine, colon, liver, and adipose tissues. We used these functional models to determine the global metabolic differences between CONV‐R and GF mice. Based on gene expression data, we found that the gut microbiota affects the host amino acid (AA) metabolism, which leads to modifications in glutathione metabolism. To validate our predictions, we measured the level of AAs and N‐acetylated AAs in the hepatic portal vein of CONV‐R and GF mice. Finally, we simulated the metabolic differences between the small intestine of the CONV‐R and GF mice accounting for the content of the diet and relative gene expression differences. Our analyses revealed that the gut microbiota influences host amino acid and glutathione metabolism in mice.     

Implementation of Web-Based Respondent Driven Sampling among Men Who Have Sex with Men in Sweden

BackgroundRespondent driven sampling (RDS) was designed to study ‘hidden’ populations, for which there are no available sampling frame. RDS has been shown to recruit far into social networks of the study population and achieve unbiased estimates when certain assumptions are fulfilled. Web-based respondent driven sampling (WebRDS) has been implemented among MSM in Vietnam and produced a sufficient sample of MSM. In order to see if WebRDS could work in a ‘hidden’ population in a high-income setting, we performed a WebRDS among MSM in Sweden to study a sensitive topic, sexual risk behaviour for HIV/STI and Internet use.MethodsA cross-sectional survey was implemented between July 11, 2012 and January 21, 2013 by using a WebRDS software. Men, fifteen years old or above, who reported having ever had sex with another man were included. The web-survey explored sociodemographics, sexual risk behaviour for HIV/STI and Internet use.ResultsThe WebRDS process created a sample of 123 eligible respondents. The mean age among participants was 32 years old. All respondents reported having had unprotected anal intercourse (UAI) with at least one regular and one casual sex partner during the last 12 months. On average participants reported having had UAI with three casual sexual partners and in total having had seven casual sex partners during the last 12 months.ConclusionThe WebRDS produced a sample of Internet-using MSM in Sweden who all reported sexual risk behaviour for HIV/STI during the last 12 months. It holds promise for future online studies among MSM and a possibility to reach MSM at risk for HIV/STI with interventions or information. Some challenges were found including short recruitment chains, and further research need to address how to optimize WebRDS online recruitment methods in high income settings.     

High BRAF Mutation Frequency and Marked Survival Differences in Subgroups According to KRAS/BRAF Mutation Status and Tumor Tissue Availability in a Prospective Population-Based Metastatic Colorectal Cancer Cohort

RAS and BRAF mutations impact treatment and prognosis of metastatic colorectal cancer patients (mCRC), but the knowledge is based on trial patients usually not representative for the general cancer population. Patient characteristics, treatment and efficacy according to KRAS, BRAF and MSI status were analyzed in a prospectively collected unselected population-based cohort of 798 non-resectable mCRC patients. The cohort contained many patients with poor performance status (39% PS 2-4) and elderly (37% age>75), groups usually not included in clinical trials. Patients without available tissue micro array (TMA) (42%) had worse prognostic factors and inferior survival (all patients; 7m vs 11m, chemotherapy-treated;12m vs 17m). The 92 patients (21%) with BRAF mutation had a poor prognosis regardless of microsatellite instability, but receipt of 1-2^nd chemotherapy was similar to wildtype BRAF patients. Median survival in this cohort varied from 1 month in BRAF mutated patients not given chemotherapy to 26 months in wildtype KRAS/BRAF patients <75 years in good PS. TMA availability, BRAF mutation and KRAS mutation were all independent prognostic factors for survival. The observed 21% BRAF mutation incidence is higher than the previously and repeatedly reported incidence of 5-12% in mCRC. Screening for BRAF mutations before selection of treatment is relevant for many patients, especially outside clinical trials. A BRAF mutation only partly explained the very poor prognosis of many mCRC patients. Survival in unselected metastatic colorectal cancer patients is extremely variable and subgroups have an extremely short survival compared to trial patients. Patients without available TMA had worse prognostic factors and shorter survival, which questions the total generalizability of present TMA studies and implies that we lack information on the biologically worst mCRC cases. Lack of available tissue is an important underexposed issue which introduces sample bias, and this should be recognized more clearly when conclusions are made from translational mCRC studies.     

Reproductive endocrinology of zoo-housed aardwolves

Knowledge regarding the relationship between endocrine parameters and reproductive activity can offer important insights into how social and environmental factors influence the reproductive success of mammals. Although components of both the physical and social environment affect endocrine regulation of reproduction, less is understood about the potential role of interactions between different endocrine axes on reproductive activity. We evaluated temporal patterns of reproductive and adrenocortical steroids in two male and three female aardwolves (Proteles cristata) housed in captivity at Brookfield Zoo, Chicago, IL, USA. We found seasonal variation in faecal androgens, estrogens, and progestagens, which provide support for previous observations of the aardwolf as a seasonal breeder. However, the timing of peak endocrine activity did not correspond to observations from wild populations. Our interpretation is that this discrepancy is caused by photoperiodic regulation of reproductive activity. We found a positive relationship between faecal androgens and faecal glucocorticoid metabolites in males and a positive relationship between faecal estrogens and faecal glucocorticoid metabolites in females when housed with conspecifics but not when housed alone. We also found a positive but asymptotic relationship between faecal progestagens and faecal glucocorticoid metabolites. We argue that these observations indicate a potential effect of reproductive endocrine activity on the hypothalamic–pituitary–adrenal axis, which could result in interesting physiological trade-offs in male reproductive tactics and female pre-partum maternal investment because of the negative effects of long-term glucocorticoid elevation on reproductive performance. Finally, our results suggest that social and environmental factors interact in regulating many aspects of endocrine fluctuations in this mostly solitary species.     

Inflammatory markers CD11b, CD16, CD66b, CD68, myeloperoxidase and neutrophil elastase in eccentric exercised human skeletal muscles

The aim of the present study was to investigate leucocyte markers, CD11b, CD16, CD66b, CD68, myeloperoxidase and neutrophil elastase on skeletal muscle biopsies from biceps brachii after unaccustomed eccentric exercise followed by the second bout of exercise 3 weeks later. The subjects (10 subjects received COX-2 inhibitor (Celecoxib) and 13 subjects received placebo) were divided into three categories: mild, moderate and severe effect of eccentric exercise, according to the reduction and recovery of muscle force-generating capacity after performing 70 maximal eccentric actions with elbow flexors on an isokinetic dynamometer. The results showed that the CD66b antibody was applicable for localization of neutrophils in human skeletal muscle, whereas the other studied neutrophil markers recognized also other leucocytes than neutrophils. The number of CD66b positive cells in skeletal muscle was very low and was not affected by the exercise. The macrophage marker CD68 showed reactivity also against satellite cells and fibroblast-like cells in skeletal muscle and therefore cannot be applied as a quantitative value for inflammatory cells. Skeletal muscle fibre injury, shown as dystrophin negative fibres, was observed approximately in half of the biopsies at 4 and 7 days after the first exercise bout in the categories moderate and severe effect of eccentric exercise. These subjects represent the most prominent loss in muscle force-generating capacity both at the category and the individual levels. Furthermore, deformed skeletal muscle fibres were observed in five subjects in these categories after the second bout of exercise. The present results suggest that neutrophils are not involved in skeletal muscle fibre injury and the reduction in muscle force-generating capacity after a single bout of eccentric exercise is a good indirect indicator of muscle damage in humans. Furthermore, prolonged regeneration process could be one of the reasons for impaired peripheral muscle function after high-force eccentric exercise.     

Insight into factors directing high production of eukaryotic membrane proteins; production of 13 human AQPs in Pichia pastoris

Membrane proteins are key players in all living cells. To achieve a better understanding of membrane protein function, significant amounts of purified protein are required for functional and structural analyses. Overproduction of eukaryotic membrane proteins, in particular, is thus an essential yet non-trivial task. Hence, improved understanding of factors which direct a high production of eukaryotic membrane proteins is desirable. In this study we have compared the overproduction of all human aquaporins in the eukaryotic host Pichia pastoris. We report quantitated production levels of each homologue and the extent of their membrane localization. Our results show that the protein production levels vary substantially, even between highly homologous aquaporins. A correlation between the extents of membrane insertion with protein function also emerged, with a higher extent of membrane insertion for pure water transporters compared to aquaporin family members with other substrate specificity. Nevertheless, the nucleic acid sequence of the second codon appears to play an important role in overproduction. Constructs containing guanine at the first position of this codon (being part of the mammalian Kozak sequence) are generally produced at a higher level, which is confirmed for hAQP8. In addition, mimicking the yeast consensus sequence (ATGTCT) apparently has a negative influence on the production level, as shown for hAQP1. Moreover, by mutational analysis we show that the yield of hAQP4 can be heavily improved by directing the protein folding pathway as well as stabilizing the aquaporin tetramer.