A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions

Fluorescent protein (FP) has enabled the analysis of biomolecular interactions in living cells, and bimolecular fluorescence complementation (BiFC) represents one of the newly developed imaging technologies to directly visualize protein–protein interactions in living cells. Although 10 different FPs that cover a broad range of spectra have been demonstrated to support BiFC, only Cerulean (cyan FP variant), Citrine and Venus (yellow FP variants)-based BiFC systems can be used under 37 °C physiological temperature. The sensitivity of two mRFP-based red BiFC systems to higher temperatures (i.e., 37 °C) limits their applications in most mammalian cell-based studies. Here we report that mLumin, a newly isolated far-red fluorescent protein variant of mKate with an emission maximum of 621 nm, enables BiFC analysis of protein–protein interactions at 37 °C in living mammalian cells. Furthermore, the combination of mLumin with Cerulean- and Venus-based BiFC systems allows for simultaneous visualization of three pairs of protein–protein interactions in the same cell. The mLumin-based BiFC system will facilitate simultaneous visualization of multiple protein–protein interactions in living cells and offer the potential to visualize protein–protein interactions in living animals.     

Paradoxical Effects of All-Trans-Retinoic Acid on Lupus-Like Disease in the MRL/lpr Mouse Model

Roles of all-trans-retinoic acid (tRA), a metabolite of vitamin A (VA), in both tolerogenic and immunogenic responses are documented. However, how tRA affects the development of systemic autoimmunity is poorly understood. Here we demonstrate that tRA have paradoxical effects on the development of autoimmune lupus in the MRL/lpr mouse model. We administered, orally, tRA or VA mixed with 10% of tRA (referred to as VARA) to female mice starting from 6 weeks of age. At this age, the mice do not exhibit overt clinical signs of lupus. However, the immunogenic environment preceding disease onset has been established as evidenced by an increase of total IgM/IgG in the plasma and expansion of lymphocytes and dendritic cells in secondary lymphoid organs. After 8 weeks of tRA, but not VARA treatment, significantly higher pathological scores in the skin, brain and lung were observed. These were accompanied by a marked increase in B-cell responses that included autoantibody production and enhanced expression of plasma cell-promoting cytokines. Paradoxically, the number of lymphocytes in the mesenteric lymph node decreased with tRA that led to significantly reduced lymphadenopathy. In addition, tRA differentially affected renal pathology, increasing leukocyte infiltration of renal tubulointerstitium while restoring the size of glomeruli in the kidney cortex. In contrast, minimal induction of inflammation with tRA in the absence of an immunogenic environment in the control mice was observed. Altogether, our results suggest that under a predisposed immunogenic environment in autoimmune lupus, tRA may decrease inflammation in some organs while generating more severe disease in others.     

NMDA Receptor Antagonist Attenuates Bleomycin-Induced Acute Lung Injury

Background

Glutamate is a major neurotransmitter in the central nervous system (CNS). Large amount of glutamate can overstimulate N-methyl-D-aspartate receptor (NMDAR), causing neuronal injury and death. Recently, NMDAR has been reported to be found in the lungs. The aim of this study is to examine the effects of memantine, a NMDAR channel blocker, on bleomycin-induced lung injury mice.

Methods

C57BL/6 mice were intratracheally injected with bleomycin (BLM) to induce lung injury. Mice were randomized to receive saline, memantine (Me), BLM, BLM plus Me. Lungs and BALF were harvested on day 3 or 7 for further evaluation.

Results

BLM caused leukocyte infiltration, pulmonary edema and increase in cytokines, and imposed significant oxidative stress (MDA as a marker) in lungs. Memantine significantly mitigated the oxidative stress, lung inflammatory response and acute lung injury caused by BLM. Moreover, activation of NMDAR enhances CD11b expression on neutrophils.

Conclusions

Memantine mitigates oxidative stress, lung inflammatory response and acute lung injury in BLM challenged mice.     

A New Approach to Segment Both Main and Peripheral Retinal Vessels Based on Gray-Voting and Gaussian Mixture Model

Vessel segmentation in retinal fundus images is a preliminary step to clinical diagnosis for some systemic diseases and some eye diseases. The performances of existing methods for segmenting small vessels which are usually of more importance than the main vessels in a clinical diagnosis are not satisfactory in clinical use. In this paper, we present a method for both main and peripheral vessel segmentation. A local gray-level change enhancement algorithm called gray-voting is used to enhance the small vessels, while a two-dimensional Gabor wavelet is used to extract the main vessels. We fuse the gray-voting results with the 2D-Gabor filter results as pre-processing outcome. A Gaussian mixture model is then used to extract vessel clusters from the pre-processing outcome, while small vessels fragments are obtained using another gray-voting process, which complements the vessel cluster extraction already performed. At the last step, we eliminate the fragments that do not belong to the vessels based on the shape of the fragments. We evaluated the approach with two publicly available DRIVE (Staal et al., 2004) and STARE (Hoover et at., 2000) datasets with manually segmented results. For the STARE dataset, when using the second manually segmented results which include much more small vessels than the first manually segmented results as the “gold standard,” this approach achieved an average sensitivity, accuracy and specificity of 65.0%, 92.1% and 97.0%, respectively. The sensitivities of this approach were much higher than those of the other existing methods, with comparable specificities; these results thus demonstrated that this approach was sensitive to detection of small vessels.     

Is Demography Destiny? Application of Machine Learning Techniques to Accurately Predict Population Health Outcomes from a Minimal Demographic Dataset

For years, we have relied on population surveys to keep track of regional public health statistics, including the prevalence of non-communicable diseases. Because of the cost and limitations of such surveys, we often do not have the up-to-date data on health outcomes of a region. In this paper, we examined the feasibility of inferring regional health outcomes from socio-demographic data that are widely available and timely updated through national censuses and community surveys. Using data for 50 American states (excluding Washington DC) from 2007 to 2012, we constructed a machine-learning model to predict the prevalence of six non-communicable disease (NCD) outcomes (four NCDs and two major clinical risk factors), based on population socio-demographic characteristics from the American Community Survey. We found that regional prevalence estimates for non-communicable diseases can be reasonably predicted. The predictions were highly correlated with the observed data, in both the states included in the derivation model (median correlation 0.88) and those excluded from the development for use as a completely separated validation sample (median correlation 0.85), demonstrating that the model had sufficient external validity to make good predictions, based on demographics alone, for areas not included in the model development. This highlights both the utility of this sophisticated approach to model development, and the vital importance of simple socio-demographic characteristics as both indicators and determinants of chronic disease.     

Effects of low dietary protein on the metabolites and microbial communities in the caecal digesta of piglets

Thirty-six healthy piglets (weighing 10 ± 1 kg; three animals per pen) were randomly allocated to two treatments: (i) a low protein diet (14% crude protein [CP]) supplemented with lysine, methionine, threonine and tryptophan (Group LP) and (ii) a normal protein diet (20% CP, Group NP), resulting in six replicate pens per treatment. One piglet from each pen was slaughtered at days 10, 25 and 45 of the experiment. For the whole experimental period of 45 d, Group LP had lower feed intake and daily gain and a higher feed-to-gain ratio compared with Group NP. At day 10, no effects on measured caecum metabolites were observed, but at days 25 and 45 in Group LP the concentration of ammonia-N, cadaverine, branched chain fatty acids and acetate were reduced. This was also true for the concentration of short chain fatty acids at day 45. The results of denaturing gradient gel electrophoresis showed that microbial diversity in Group LP was less abundant at day 25, but there was no difference at days 10 and 45. An unweighted pair group mean average analysis showed that the similarities were lower between Groups LP and NP at day 10 and higher at days 25 and 45. Quantitation results indicated that the numbers of Firmicutes and Clostridium cluster IV were lower in Group LP than in Group NP at day 25, but there were no differences at days 10 and 45. In conclusion, the low protein diet markedly reduced the metabolites of protein and carbohydrate fermentation and altered microbial communities in the caecal digesta of piglets.     

A Lipoxygenase from Red Alga Pyropia haitanensis,a Unique Enzyme Catalyzing the Free Radical Reactions of Polyunsaturated Fatty Acids with Triple Ethylenic Bonds

Lipoxygenases (LOXs) are key enzymes to regulate the production of hormones and defensive metabolites in plants, animals and algae. In this research, a full length LOX gene has been cloned and expressed from the red alga Pyropia haitanensis (Bangiales, Rhodophyta) gametophyte (PhLOX2). Subsequent phylogenetic analysis showed that such LOX enzymes are separated at the early stage of evolution, establishing an independent branch. The LOX activity was investigated at the optimal pH of 8.0. It appears that PhLOX2 is a multifunctional enzyme featuring both lipoxygenase and hydroperoxidase activities. Additionally, PhLOX2 exhibits remarkable substrate and position flexibility, and it can catalyze an array of chemical reactions involving various polyunsaturated fatty acids, ranging from C18 to C22. As a matter of fact, mono-hydroperoxy, di-hydroperoxy and hydroxyl products have been obtained from such transformations, and eicosapentaenoic acid seem to be the most preferred substrate. It was found that at least triple ethylenic bonds are required for PhLOX2 to function as a LOX, and the resulting hydroxy products should be originated from the PhLOX2 mediated reduction of mono-hydroperoxides, in which the hydrogen abstraction occurs on the carbon atom between the second and third double bond. Most of the di-hydroperoxides observed seem to be missing their mono-position precursors. The substrate and position flexibility, as well as the function versatility of PhLOXs represent the ancient enzymatic pathway for organisms to control intracellular oxylipins.     

Growth and Physiological Responses to Water Depths in Carex schmidtii Meinsh

A greenhouse experiment was performed to investigate growth and physiological responses to water depth in completely submerged condition of a wetland plant Carex schmidtii Meinsh., one of the dominant species in the Longwan Crater Lake wetlands (China). Growth and physiological responses of C. schmidtii were investigated by growing under control (non-submerged) and three submerged conditions (5 cm, 15 cm and 25 cm water level). Total biomass was highest in control, intermediate in 5 cm treatment and lowest in the other two submerged treatments. Water depth prominently affected the first-order lateral root to main root mass ratio. Alcohol dehydrogenase (ADH) activity decreased but malondialdehyde (MDA) content increased as water depth increased. The starch contents showed no differences among the various treatments at the end of the experiment. However, soluble sugar contents were highest in control, intermediate in 5 cm and 15 cm treatments and lowest in 25 cm treatment. Our data suggest that submergence depth affected some aspects of growth and physiology of C. schmidtii, which can reduce anoxia damage not only through maintaining the non-elongation strategy in shoot part but also by adjusting biomass allocation to different root orders rather than adjusting root-shoot biomass allocation.     

Prevalence and Molecular Characterization of Glucose-6-Phosphate Dehydrogenase Deficiency at the China-Myanmar Border

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked hereditary disease that predisposes red blood cells to oxidative damage. G6PD deficiency is particularly prevalent in historically malaria-endemic areas. Use of primaquine for malaria treatment may result in severe hemolysis in G6PD deficient patients. In this study, we systematically evaluated the prevalence of G6PD deficiency in the Kachin (Jingpo) ethnic group along the China-Myanmar border and determined the underlying G6PD genotypes. We surveyed G6PD deficiency in 1770 adult individuals (671 males and 1099 females) of the Kachin ethnicity using a G6PD fluorescent spot test. The overall prevalence of G6PD deficiency in the study population was 29.6% (523/1770), among which 27.9% and 30.6% were males and females, respectively. From these G6PD deficient samples, 198 unrelated individuals (147 females and 51 males) were selected for genotyping at 11 known G6PD single nucleotide polymorphisms (SNPs) in Southeast Asia (ten in exons and one in intron 11) using a multiplex SNaPshot assay. Mutations with known association to a deficient phenotype were detected in 43.9% (87/198) of cases, intronic and synonymous mutations were detected alone in 34.8% (69/198) cases and no mutation were found in 21.2% (42/198) cases. Five non-synonymous mutations, Mahidol 487G>A, Kaiping 1388G>A, Canton 1376G>T, Chinese 4 392G>T, and Viangchan 871G>A were detected. Of the 87 cases with known deficient mutations, the Mahidol variant was the most common (89.7%; 78/87), followed by the Kaiping (8.0%; 7/87) and the Viangchan (2.2%; 2/87) variants. The Canton and Chinese 4 variants were found in 1.1% of these 87 cases. Among them, two females carried the Mahidol/Viangchan and Mahidol/Kaiping double mutations, respectively. Interestingly, the silent SNPs 1311C>T and IVS11nt93T>C both occurred in the same 95 subjects with frequencies at 56.4% and 23.5% in tested females and males, respectively (P<0.05). It is noteworthy that 24 subjects carrying the Mahidol mutation and two carrying the Kaiping mutation also carried the 1311C>T/IVS11nt93T>C SNPs. Further studies are needed to determine the enzyme levels of the G6PD deficient people and presence of additional G6PD mutations in the study population.