Computational analyses reveal spatial relationships between nuclear pore complexes and specific lamins

Nuclear lamin isoforms form fibrous meshworks associated with nuclear pore complexes (NPCs). Using datasets prepared from subpixel and segmentation analyses of 3D–structured illumination microscopy images of WT and lamin isoform knockout mouse embryo fibroblasts, we determined with high precision the spatial association of NPCs with specific lamin isoform fibers. These relationships are retained in the enlarged lamin meshworks of Lmna^−/− and Lmnb1^−/− fibroblast nuclei. Cryo-ET observations reveal that the lamin filaments composing the fibers contact the nucleoplasmic ring of NPCs. Knockdown of the ring-associated nucleoporin ELYS induces NPC clusters that exclude lamin A/C fibers but include LB1 and LB2 fibers. Knockdown of the nucleoporin TPR or NUP153 alters the arrangement of lamin fibers and NPCs. Evidence that the number of NPCs is regulated by specific lamin isoforms is presented. Overall the results demonstrate that lamin isoforms and nucleoporins act together to maintain the normal organization of lamin meshworks and NPCs within the nuclear envelope.     

Shiga toxin production and translocation during microaerobic human colonic infection with Shiga toxin‐producing E. coli O157:H7 and O104:H4

Haemolytic uraemic syndrome caused by Shiga toxin‐producing E. coli (STEC) is dependent on release of Shiga toxins (Stxs) during intestinal infection and subsequent absorption into the bloodstream. An understanding of Stx‐related events in the human gut is limited due to lack of suitable experimental models. In this study, we have used a vertical diffusion chamber system with polarized human colon carcinoma cells to simulate the microaerobic (MA) environment in the human intestine and investigate its influence on Stx release and translocation during STEC O157:H7 and O104:H4 infection. Stx2 was the major toxin type released during infection. Whereas microaerobiosis significantly reduced bacterial growth as well as Stx production and release into the medium, Stx translocation across the epithelial monolayer was enhanced under MA versus aerobic conditions. Increased Stx transport was dependent on STEC infection and occurred via a transcellular pathway other than macropinocytosis. While MA conditions had a similar general effect on Stx release and absorption during infection with STEC O157:H7 and O104:H4, both serotypes showed considerable differences in colonization, Stx production, and Stx translocation which suggest alternative virulence strategies. Taken together, our study suggests that the MA environment in the human colon may modulate Stx‐related events and enhance Stx absorption during STEC infection.     

Effect of ionic liquids on enzymatic synthesis of caffeic acid phenethyl ester

Although caffeic acid phenethyl ester (CAPE), an active flavonoid, plays an important role in the antioxidant activity of honeybee propolis, the isolation of CAPE from honeybee propolis is time-consuming due to wide variety of impurities present. Therefore, biochemical method to synthesize CAPE was investigated in this study. Since ionic liquids (ILs) possess some unique characteristics as appreciated alternatives to conventional solvents for certain biotransformation, the effect of ILs as reaction media for enzymatic synthesis of CAPE was assessed. Several factors including substrate molar ratio, and reaction temperature affecting the conversion yield of lipase-catalyzed CAPE synthesis were also investigated. Reaction yields were significantly higher in hydrophobic ILs than in hydrophilic ILs (almost zero). Among nine hydrophobic ILs tested, the highest conversion of synthetic reaction was obtained in 1-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([Emim][Tf(2)N]). A reaction temperature of 70 °C was found to give high conversion. In addition, optimal substrate molar ratio between phenethyl alcohol and caffeic acid (CA) was decreased significantly from 92:1 to 30:1 when ILs were used instead of isooctane.     

Apolipoprotein A-II/A-I ratio is a key determinant in vivo of HDL concentration and formation of pre-beta HDL containing apolipoprotein A-II

Overexpression of human apolipoprotein A-II (apo A-II) in mice induced postprandial hypertriglyceridemia and marked reduction in plasma HDL concentration and particle size [Boisfer et al. (1999) J. Biol. Chem. 274, 11564-11572]. We presently compared lipoprotein metabolism in three transgenic lines displaying plasma concentrations of human apo A-II ranging from normal to 4 times higher, under ad libitum feeding and after an overnight fast. Fasting dramatically decreased VLDL and lowered circulating human apo A-II in transgenic mice; conversely, plasma HDL levels increased in all genotypes. The apo A-I content of HDL was inversely related to the expression of human apo A-II, probably reflecting displacement of apo A-I by an excess of apo A-II. Thus, the molar ratios of apo A-II/A-I in HDL were significantly higher in fed as compared with fasted animals of the same transgenic line, while endogenous LCAT activity concomitantly decreased. The number and size of HDL particles decreased in direct proportion to the level of human apo A-II expression. Apo A-II was abundantly present in all HDL particles, in contrast to apo A-I mainly present in large ones. Two novel findings were the presence of pre-beta migrating HDL transporting only human apo A-II in the higher-expressing mice and the increase of plasma HDL concentrations by fasting in control and transgenic mice. These findings highlight the reciprocal modifications of VLDL and HDL induced by the feeding-fasting transition and the key role of the molar ratio of apo A-II/A-I as a determinant of HDL particle metabolism and pre-beta HDL formation.     

Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

NK cells utilize a large array of receptors to screen their surroundings for aberrant or virus‐infected cells. Given the vast diversity of receptors expressed on NK cells we seek to identify receptors involved in the recognition of HIV‐1‐infected cells. By combining an unbiased large‐scale screening approach with a functional assay, we identify TRAIL to be associated with NK cell degranulation against HIV‐1‐infected target cells. Further investigating the underlying mechanisms, we demonstrate that TRAIL is able to elicit multiple effector functions in human NK cells independent of receptor‐mediated induction of apoptosis. Direct engagement of TRAIL not only results in degranulation but also IFNγ production. Moreover, TRAIL‐mediated NK cell activation is not limited to its cognate death receptors but also decoy receptor I, adding a new perspective to the perceived regulatory role of decoy receptors in TRAIL‐mediated cytotoxicity. Based on these findings, we propose that TRAIL not only contributes to the anti‐HIV‐1 activity of NK cells but also possesses a multifunctional role beyond receptor‐mediated induction of apoptosis, acting as a regulator for the induction of different effector functions.     

Down-regulation of miR-17 family expression in response to retinoic acid induced neuronal differentiation

Whole-genome microRNA and gene expression analyses were used to monitor changes during retinoic acid induced differentiation of neuroblasts in vitro. Interestingly, the entire miR-17 family was over-represented among the down-regulated miRNA. The implications of these changes are considerable, as target gene prediction suggests that the miR-17 family is involved in the regulation of the mitogen-activated protein kinase (MAPK) signaling pathway, synaptic plasticity and other markers of neuronal differentiation. Significantly, many of the target responses predicted by changes in miRNA expression were supported by the observed changes in gene expression. As expected, markers of neuronal differentiation such as anti-apoptotic protein B-cell lymphoma 2 (BCL2), myocyte enhancer factor-2D (MEF2D) and zipper protein kinase (MAP3K12; aka ZPK/MUK/DLK) were each up-regulated in response to differentiation. The expression of these genes was also reduced in response to miR-17 and miR-20a transfection, and more specifically they were also shown to contain functional miRNA recognition elements for members of the miR-17 family by reporter gene assay. This suggests that the miR-17 family have an integral role in fine-tuning the pathways involved in the regulation of neuronal differentiation.     

Identification of ATP-Binding Regions in the RyR1 Ca2+ Release Channel

ATP is an important modulator of gating in type 1 ryanodine receptor (RyR1), also known as a Ca²⁺ release channel in skeletal muscle cells. The activating effect of ATP on this channel is achieved by directly binding to one or more sites on the RyR1 protein. However, the number and location of these sites have yet to be determined. To identify the ATP-binding regions within RyR1 we used 2N₃ATP-2′,3′-Biotin-LC-Hydrazone (BioATP-HDZ), a photo-reactive ATP analog to covalently label the channel. We found that BioATP-HDZ binds RyR1 specifically with an IC₅₀ = 0.6±0.2 mM, comparable with the reported EC50 for activation of RyR1 with ATP. Controlled proteolysis of labeled RyR1 followed by sequence analysis revealed three fragments with apparent molecular masses of 95, 45 and 70 kDa that were crosslinked by BioATP-HDZ and identified as RyR1 sequences. Our analysis identified four glycine-rich consensus motifs that can potentially constitute ATP-binding sites and are located within the N-terminal 95-kDa fragment. These putative nucleotide-binding sequences include amino acids 699–704, 701–706, 1081–1084 and 1195–1200, which are conserved among the three RyR isoforms. Located next to the N-terminal disease hotspot region in RyR1, these sequences may communicate the effects of ATP-binding to channel function by tuning conformational motions within the neighboring cytoplasmic regulatory domains. Two other labeled fragments lack ATP-binding consensus motifs and may form non-canonical ATP-binding sites. Based on domain topology in the 3D structure of RyR1 it is also conceivable that the identified ATP-binding regions, despite their wide separation in the primary sequence, may actually constitute the same non-contiguous ATP-binding pocket within the channel tetramer.     

Monitoring occurrence,extinction, and colonization probabilities for gibbon populations

All gibbon species (Family: Hylobatidae) are considered threatened with extinction and recognized on the International Union for Conservation of Nature Red List of Threatened Species. Because gibbons are one of the most threatened families of primates, monitoring their status is now critically important. Long-term monitoring programs applying occupancy approaches, in addition to assessing occurrence probability, improves understanding of other population parameters such as site extinction or colonization probabilities, which elucidate temporal and spatial changes and are therefore important for guiding conservation efforts. In this study, we used multiple season occupancy models to monitor occurrence, extinction, and colonization probabilities for northern yellow-cheeked crested gibbon Nomascus annamensis in three adjacent protected areas in the Central Annamites mountain range, Vietnam. We collected data at 30 listening posts in 2012, 2014, and 2016 using the auditory point count method. Occurrence probabilities were highest in 2012 (0.74, confidence interval [CI]: 0.56–0.87) but slightly lower in 2014 (0.66, CI: 0.51–0.79) and 2016 (0.67, CI: 0.49–0.81). Extinction probabilities during the 2012–2014 and 2014–2016 intervals were 0.26 (0.14–0.44) and 0.25 (0.12–0.44), respectively. Colonization probabilities during 2012–2014 were 0.44 (0.19–0.73) and between 2014 and 2016 was 0.51 (0.26–0.75). Although local site extinctions have occurred, high recolonization probability helped to replenish the unoccupied sites and kept the occurrence probability stable. Long-term monitoring programs which use occurrence probability alone might not fully reveal the true dynamics of gibbon populations. We strongly recommend including multiple season occupancy models to monitor occurrence, extinction, and colonization probabilities in long-term gibbon monitoring programs.     

Boosted Growth Performance,Mucosal and Serum Immunity,and Disease Resistance Nile Tilapia ( Oreochromis niloticus ) Fingerlings Using Corncob-Derived Xylooligosaccharide and Lactobacillus plantarum CR1T5

The present work, herein, studied the effects of corncob-derived xylooligosaccharides (CDXOS) and Lactobacillus plantarum CR1T5 (LP) integrated into fish d