Effects of long term feeding of Quillaja saponins on sex ratio,muscle and serum cholesterol and LH levels in Nile tilapia (Oreochromis niloticus (L))

Seventeen-day-old Nile tilapia fry were fed a standard diet (C) or diets containing 50-700 mg kg(-1) Quillaja saponin (QS) extract (groups S50, S150, S300, S500 and S700). After the first 8 weeks, 30 randomly selected tilapia from each of the treatments were placed in separate aquaria and fed the standard diet without saponins from then on (these were designated S50/C, S150/C, S300/C, S500/C and S700/C). The fish grew from an initial average weight of approximately 30 mg to a final average weight of 79 g during the 6-month feeding period. The difference between the average weight of C-fed tilapia and the treatment with the highest average weight after 6 months was 53.5%. The sex ratio of tilapia in the saponin-fed groups deviated from the normal 50:50 male:female ratio, with the S700 group showing a significantly higher number of males. Quillaja saponin stimulated LH release from dispersed tilapia pituitary cells in vitro. This effect was abolished in the presence of dilute calf serum. Serum LH values did not show any diet-dependent trend in either male or female tilapia in vivo. In both continuously saponin-fed and only-initially saponin-fed groups, the average serum (but not muscle) cholesterol levels in males showed an increasing trend (R(2) values of 0.62 and 0.69) with increasing dietary saponin level. It was concluded that dietary QS has the potential to change the sex-ratio in favour of males. More investigations are required to determine the mechanism of action and the optimum dietary level of QS for maximum effects.     

Ric-8B is a GTP-dependent G protein alphas guanine nucleotide exchange factor

ric-8 (resistance to inhibitors of cholinesterase 8) genes have positive roles in variegated G protein signaling pathways, including Gα(q) and Gα(s) regulation of neurotransmission, Gα(i)-dependent mitotic spindle positioning during (asymmetric) cell division, and Gα(olf)-dependent odorant receptor signaling. Mammalian Ric-8 activities are partitioned between two genes, ric-8A and ric-8B. Ric-8A is a guanine nucleotide exchange factor (GEF) for Gα(i)/α(q)/α(12/13) subunits. Ric-8B potentiated G(s) signaling presumably as a Gα(s)-class GEF activator, but no demonstration has shown Ric-8B GEF activity. Here, two Ric-8B isoforms were purified and found to be Gα subunit GDP release factor/GEFs. In HeLa cells, full-length Ric-8B (Ric-8BFL) bound endogenously expressed Gα(s) and lesser amounts of Gα(q) and Gα(13). Ric-8BFL stimulated guanosine 5'-3-O-(thio)triphosphate (GTPγS) binding to these subunits and Gα(olf), whereas the Ric-8BΔ9 isoform stimulated Gα(s short) GTPγS binding only. Michaelis-Menten experiments showed that Ric-8BFL elevated the V(max) of Gα(s) steady state GTP hydrolysis and the apparent K(m) values of GTP binding to Gα(s) from ～385 nm to an estimated value of ～42 μM. Directionality of the Ric-8BFL-catalyzed Gα(s) exchange reaction was GTP-dependent. At sub-K(m) GTP, Ric-BFL was inhibitory to exchange despite being a rapid GDP release accelerator. Ric-8BFL binds nucleotide-free Gα(s) tightly, and near-K(m) GTP levels were required to dissociate the Ric-8B·Gα nucleotide-free intermediate to release free Ric-8B and Gα-GTP. Ric-8BFL-catalyzed nucleotide exchange probably proceeds in the forward direction to produce Gα-GTP in cells.     

The Drosophila blood brain barrier is maintained by GPCR-dependent dynamic actin structures

The blood brain barrier (BBB) is essential for insulation of the nervous system from the surrounding environment. In Drosophila melanogaster, the BBB is maintained by septate junctions formed between subperineurial glia (SPG) and requires the Moody/G protein-coupled receptor (GPCR) signaling pathway. In this study, we describe novel specialized actin-rich structures (ARSs) that dynamically form along the lateral borders of the SPG cells. ARS formation and association with nonmuscle myosin is regulated by Moody/GPCR signaling and requires myosin activation. Consistently, an overlap between ARS localization, elevated Ca(2+) levels, and myosin light chain phosphorylation is detected. Disruption of the ARS by inhibition of the actin regulator Arp2/3 complex leads to abrogation of the BBB. Our results suggest a mechanism by which the Drosophila BBB is maintained by Moody/GPCR-dependent formation of ARSs, which is supported by myosin activation. The localization of the ARSs close to the septate junctions enables efficient sealing of membrane gaps formed during nerve cord growth.     

Knockout of SlTOM1 and SlTOM3 results in differential resistance to tobamovirus in tomato

During tobamovirus–host coevolution, tobamoviruses developed numerous interactions with host susceptibility factors and exploited these interactions for replication and movement. The plant‐encoded TOBAMOVIRUS MULTIPLICATION (TOM) susceptibility proteins interact with the tobamovirus replicase proteins and allow the formation of the viral replication complex. Here CRISPR/Cas9‐mediated mutagenesis allowed the exploration of the roles of SlTOM1a, SlTOM1b, and SlTOM3 in systemic tobamovirus infection of tomato. Knockouts of both SlTOM1a and SlTOM3 in sltom1a/sltom3 plants resulted in an asymptomatic response to the infection with recently emerged tomato brown rugose fruit virus (ToBRFV). In addition, an accumulation of ToBRFV RNA and coat protein (CP) in sltom1a/sltom3 mutant plants was 516‐ and 25‐fold lower, respectively, than in wild‐type (WT) plants at 12 days postinoculation. In marked contrast, sltom1a/sltom3 plants were susceptible to previously known tomato viruses, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV), indicating that SlTOM1a and SlTOM3 are not essential for systemic infection of TMV and ToMV in tomato plants. Knockout of SlTOM1b alone did not contribute to ToBRFV and ToMV resistance. However, in triple mutants sltom1a/sltom3/sltom1b, ToMV accumulation was three‐fold lower than in WT plants, with no reduction in symptoms. These results indicate that SlTOM1a and SlTOM3 are essential for the replication of ToBRFV, but not for ToMV and TMV, which are associated with additional susceptibility proteins. Additionally, we showed that SlTOM1a and SlTOM3 positively regulate the tobamovirus susceptibility gene SlARL8a3. Moreover, we found that the SlTOM family is involved in the regulation of plant development.     

Characterization of Two New CTX-M-25-Group Extended-Spectrum β-Lactamase Variants Identified in Escherichia coli Isolates from Israel

Objectives

We characterized two new CTX-M-type extended-spectrum β-lactamase (ESBL) variants in Escherichia coli isolates from stool samples of two elderly patients admitted at the Tel Aviv Sourasky Medical Center, Israel. Both patients underwent treatment with cephalosporins prior to isolation of the E. coli strains.

Methods

ESBLs were detected by the double-disk synergy test and PCR-sequencing of β-lactamase genes. The bla _CTX-M genes were cloned into the pCR-BluntII-TOPO vector in E. coli TOP10. The role of amino-acid substitutions V77A and D240G was analyzed by site-directed mutagenesis of the bla _CTX-M-94 and bla _CTX-M-100 genes and comparative characterization of the resulting E. coli recombinants. MICs of β-lactams were determined by Etest. Plasmid profiling, mating experiments, replicon typing and sequencing of bla _CTX-M flanking regions were performed to identify the genetic background of the new CTX-M variants.

Results

The novel CTX-M β-lactamases, CTX-M-94 and -100, belonged to the CTX-M-25-group. Both variants differed from CTX-M-25 by the substitution V77A, and from CTX-M-39 by D240G. CTX-M-94 differed from all CTX-M-25-group enzymes by the substitution F119L. Glycine-240 was associated with reduced susceptibility to ceftazidime and leucine-119 with increased resistance to ceftriaxone. bla _CTX-M-94 and bla _CTX-M-100 were located within ISEcp1 transposition units inserted into ∼93 kb non-conjugative IncFI and ∼130 kb conjugative IncA/C plasmids, respectively. The plasmids carried also different class 1 integrons.

Conclusions

This is the first report on CTX-M-94 and -100 ESBLs, novel members of the CTX-M-25-group.     

HSP60 is transported through the secretory pathway of 3-MCA-induced fibrosarcoma tumour cells and undergoes N-glycosylation

There is increasing evidence localizes the mitochondrial chaperone heat shock protein (HSP)60, outside the cell, where it mediates interactions between immune cells and other body tissues. However, the mechanisms by which HSP60 is secreted into the extracellular environment are not fully understood. Recent studies have shown that HSP60 is actively released by a nonconventional secretion mechanism, the lipid raft-exosome pathway. In the present study, we show for the first time that HSP60, produced by 3-methylcholantrene-induced fibrosarcoma tumour cells, is secreted through the conventional endoplasmic reticulum-Golgi secretory pathway. Confocal microscopy using anti-TGN38 and anti-HSP60 antibodies together with monensin, a Golgi transport inhibitor, demonstrated the relocation of HSP60 to the Golgi of malignant cells but not primary fibroblast cells subjected to heat shock or fibroblast cell lines. Transmission electron microscopy, flow cytometry and cell fractionation of cell treated with brefeldin A, an inhibitor of endoplasmic reticulum to Golgi protein transport, further indicated that HSP60 is present both in the endoplasmic reticulum and the Golgi complex of malignant cells. We found a single mRNA with a mitochondrial targeting sequence encoding for HSP60 in the malignant cells but two HSP60 translation products, namely the native unmodified protein and a protein post-translationally modified by N-glycosylation. The N-glycans observed were composed of high-mannose structures and bi-, tri- and tetra-antennary complex type structures occupying sites of the three potential glycosylation sites present on HSP60. Accordingly, we propose that HSP60 in malignant cells is transported through the endoplasmic reticulum-Golgi secretion pathway, where it acquires N-glycans, and thus can affect the immunological properties of the proteins in the tumour microenvironment.     

The structural flexibility of the human copper chaperone Atox1: Insights from combined pulsed EPR studies and computations

Metallochaperones are responsible for shuttling metal ions to target proteins. Thus, a metallochaperone's structure must be sufficiently flexible both to hold onto its ion while traversing the cytoplasm and to transfer the ion to or from a partner protein. Here, we sought to shed light on the structure of Atox1, a metallochaperone involved in the human copper regulation system. Atox1 shuttles copper ions from the main copper transporter, Ctr1, to the ATP7b transporter in the Golgi apparatus. Conventional biophysical tools such as X‐ray or NMR cannot always target the various conformational states of metallochaperones, owing to a requirement for crystallography or low sensitivity and resolution. Electron paramagnetic resonance (EPR) spectroscopy has recently emerged as a powerful tool for resolving biological reactions and mechanisms in solution. When coupled with computational methods, EPR with site‐directed spin labeling and nanoscale distance measurements can provide structural information on a protein or protein complex in solution. We use these methods to show that Atox1 can accommodate at least four different conformations in the apo state (unbound to copper), and two different conformations in the holo state (bound to copper). We also demonstrate that the structure of Atox1 in the holo form is more compact than in the apo form. Our data provide insight regarding the structural mechanisms through which Atox1 can fulfill its dual role of copper binding and transfer.     

Detecting neural assemblies in calcium imaging data

Background

Activity in populations of neurons often takes the form of assemblies, where specific groups of neurons tend to activate at the same time. However, in calcium imaging data, reliably identifying these assemblies is a challenging problem, and the relative performance of different assembly-detection algorithms is unknown.

Results

To test the performance of several recently proposed assembly-detection algorithms, we first generated large surrogate datasets of calcium imaging data with predefined assembly structures and characterised the ability of the algorithms to recover known assemblies. The algorithms we tested are based on independent component analysis (ICA), principal component analysis (Promax), similarity analysis (CORE), singular value decomposition (SVD), graph theory (SGC), and frequent item set mining (FIM-X). When applied to the simulated data and tested against parameters such as array size, number of assemblies, assembly size and overlap, and signal strength, the SGC and ICA algorithms and a modified form of the Promax algorithm performed well, while PCA-Promax and FIM-X did less well, for instance, showing a strong dependence on the size of the neural array. Notably, we identified additional analyses that can improve their importance. Next, we applied the same algorithms to a dataset of activity in the zebrafish optic tectum evoked by simple visual stimuli, and found that the SGC algorithm recovered assemblies closest to the averaged responses.

Conclusions

Our findings suggest that the neural assemblies recovered from calcium imaging data can vary considerably with the choice of algorithm, but that some algorithms reliably perform better than others. This suggests that previous results using these algorithms may need to be reevaluated in this light.