Activity profiling of barley vacuolar processing enzymes provides new insights into the plant and cyst nematode interaction

Vacuolar processing enzymes (VPEs) play an important role during regular growth and development and defence responses. Despite substantial attempts to understand the molecular basis of plant–cyst nematode interaction, the mechanism of VPEs functioning during this interaction remains unknown. The second-stage Heterodera filipjevi juvenile penetrates host roots and induces the formation of a permanent feeding site called a syncytium. To investigate whether infection with H. filipjevi alters plant host VPEs, the studies were performed in Hordeum vulgare roots and leaves on the day of inoculation and at 7, 14 and 21 days post-inoculation (dpi). Implementing molecular, biochemical and microscopic methods we identified reasons for modulation of barley VPE activity during interaction with H. filipjevi. Heterodera filipjevi parasitism caused a general decrease of VPE activity in infected roots, but live imaging of VPEs showed that their activity is up-regulated in syncytia at 7 and 14 dpi and down-regulated at 21 dpi. These findings were accompanied by tissue-specific VPE gene expression patterns. Expression of the barley cystatin HvCPI-4 gene was stimulated in leaves but diminished in roots upon infestation. External application of cyclotides that can be produced naturally by VPEs elicits in pre-parasitic juveniles vesiculation of their body, enhanced formation of granules, induction of exploratory behaviour (stylet thrusts and head movements), production of reactive oxygen species (ROS) and final death by methuosis. Taken together, down-regulation of VPE activity through nematode effectors promotes the nematode invasion rates and leads to avoidance of the induction of the plant proteolytic response and death of the invading juveniles.     

Dietary Nisin Modulates the Gastrointestinal Microbial Ecology and Enhances Growth Performance of the Broiler Chickens

Due to antimicrobial properties, nisin is one of the most commonly used and investigated bacteriocins for food preservation. Surprisingly, nisin has had limited use in animal feed as well as there are only few reports on its influence on microbial ecology of the gastrointestinal tract (GIT). The present study therefore aimed at investigating effects of dietary nisin on broiler chicken GIT microbial ecology and performance in comparison to salinomycin, the widely used ionophore coccidiostat. In total, 720 one-day-old male Ross 308 chicks were randomly distributed to six experimental groups. The positive control (PC) diet was supplemented with salinomycin (60 mg/kg). The nisin (NI) diets were supplemented with increasing levels (100, 300, 900 and 2700 IU nisin/g, respectively) of the bacteriocin. The negative control (NC) diet contained no additives. At slaughter (35 days of age), activity of specific bacterial enzymes (α- and β-glucosidases, α-galactosidases and β-glucuronidase) in crop, ileum and caeca were significantly higher (P<0.05) in the NC group, and nisin supplementation decreased the enzyme activities to levels observed for the PC group. A similar inhibitory influence on bacterial activity was reflected in the levels of short-chain fatty acids (SCFA) and putrefactive SCFA (PSCFA) in digesta from crop and ileum; no effect was observed in caeca. Counts of Bacteroides and Enterobacteriacae in ileum digesta were significantly (P<0.001) decreased by nisin and salinomycin, but no effects were observed on the counts of Clostridium perfringens, Lactobacillus/Enterococcus and total bacteria. Like salinomycin, nisin supplementation improved broiler growth performance in a dose-dependent manner; compared to the NC group, the body weight gain of the NI₉₀₀ and NI₂₇₀₀ groups was improved by 4.7 and 8.7%, respectively. Our findings suggest that dietary nisin exerts a mode of action similar to salinomycin and could be considered as a dietary supplement for broiler chickens.     

An engineered ultra‐high affinity Fab‐Protein G pair enables a modular antibody platform with multifunctional capability

Engineered recombinant antibody‐based reagents are rapidly supplanting traditionally derived antibodies in many cell biological applications. A particularly powerful aspect of these engineered reagents is that other modules having myriad functions can be attached to them either chemically or through molecular fusions. However, these processes can be cumbersome and do not lend themselves to high throughput applications. Consequently, we have endeavored to develop a platform that can introduce multiple functionalities into a class of Fab‐based affinity reagents in a “plug and play” fashion. This platform exploits the ultra‐tight binding interaction between affinity matured variants of a Fab scaffold (Fab^S) and a domain of an immunoglobulin binding protein, protein G (GA1). GA1 is easily genetically manipulatable facilitating the ability to link these modules together like beads on a string with adjustable spacing to produce multivalent and bi‐specific entities. GA1 can also be fused to other proteins or be chemically modified to engage other types of functional components. To demonstrate the utility for the Fab‐GA1 platform, we applied it to a detection proximity assay based on the β‐lactamase (BL) split enzyme system. We also show the bi‐specific capabilities of the module by using it in context of a Bi‐specific T‐cell engager (BiTE), which is a therapeutic assemblage that induces cell killing by crosslinking T‐cells to cancer cells. We show that GA1‐Fab modules are easily engineered into potent cell‐killing BiTE‐like assemblages and have the advantage of interchanging Fabs directed against different cell surface cancer‐related targets in a plug and play fashion.     

The influence of caffeine on the biomechanical properties of bone tissue during pregnancy in a population of rats

The influence of pregnancy on bone tissue metabolism is not completely understood. Caffeine also has a potentially negative influence on bones. The aim of this study was the evaluation of changes in the bones of pregnant rats under the influence of caffeine. The experiment was carried out on Wistar rats. The evaluation of rats' bone tissue quality was performed based on bone density measurements and resistance examinations. It analyzed the impact of caffeine on the degree of bone tissue mineralization and the composition of the bones. The mean value of pelvises 'wet' and 'dry' densities in a group of pregnant rats with caffeine intake was lower compared to the control group. The deformation in maximal load point of the femur shaft in the experimental group was significantly higher than in the control group. In the experimental group, the percentage of water in the bones was significantly higher, while the content of inorganic phase was significantly lower compared to the control group. The changes of biomechanical parameters in the group of pregnant rats with caffeine intake indicate its negative influence on the bone. Our results show higher plasticization of the bone shafts of the animals under the influence of caffeine. Higher deformation of bone shafts may have an effect on the statics of the skeleton. The administration of caffeine significantly affected the quantitative composition of the bone.     

Mechanical stability of multidomain proteins and novel mechanical clamps

We estimate the size of mechanostability for 318 multidomain proteins which are single-chain and contain up to 1021 amino acids. We predict existence of novel types of mechanical clamps in which interdomain contacts play an essential role. Mechanical clamps are structural regions which are the primary source of a protein's resistance to pulling. Among these clamps there is one that opposes tensile stress due to two domains swinging apart. This movement strains and then ruptures the contacts that hold the two domains together. Another clamp also involves tensile stress but it originates from an immobilization of a structural region by a surrounding knot-loop (without involving any disulfide bonds). Still another mechanism involves shear between helical regions belonging to two domains. We also consider the amyloid-prone cystatin C which provides an example of a two-chain 3D domain-swapped protein. We predict that this protein should withstand remarkably large stress, perhaps of order 800 pN, when inducing a shearing strain. The survey is generated through molecular dynamics simulations performed within a structure-based coarse grained model.     

Synthesis and antiproliferative activity of novel α- and β-dialkoxyphosphoryl isothiocyanates

A series of 15 mostly new dialkoxyphosphoryl alkyl and aralkyl isothiocyanates were synthesized using two alternative strategies, and their in vitro antiproliferative activity against several cancer cell lines (including drug resistant) is here demonstrated. The IC₅₀ values measured for the new compounds are within the range of 6.3-21.5 μM, and they are quite similar to the activity of two best and most extensively investigated natural benzyl isothiocyanate (A) and phenethyl isothiocyanate (B). Preliminary studies utilizing the cell cycle and reduced glutathione level analysis performed on A549 lung cancer cell line using representative compounds revealed important differences in the mechanism of action possibly correlated with their chemical properties. Hydrophobic compounds react mainly with the cytosolic glutathione reduced leading to its depletion, causing an oxidative stress and cell cycle arrest in G0/G1 phase. On the other hand, hydrophilic compounds cause moderate cell cycle arrest and massive cell death associated with moderate reduced glutathione depletion. These suggest that significant changes in the chemical structure of isothiocyanates, which do not lead to the significant changes in antiproliferative activity, but simultaneously cause a differences in the mechanism of action are possible.     

WNT protein-independent constitutive nuclear localization of beta-catenin protein and its low degradation rate in thalamic neurons

Nuclear localization of β-catenin is a hallmark of canonical Wnt signaling, a pathway that plays a crucial role in brain development and the neurogenesis of the adult brain. We recently showed that β-catenin accumulates specifically in mature thalamic neurons, where it regulates the expression of the Ca(v)3.1 voltage-gated calcium channel gene. Here, we investigated the mechanisms underlying β-catenin accumulation in thalamic neurons. We report that a lack of soluble factors produced either by glia or cortical neurons does not impair nuclear β-catenin accumulation in thalamic neurons. We next found that the number of thalamic neurons with β-catenin nuclear localization did not change when the Wnt/Dishevelled signaling pathway was inhibited by Dickkopf1 or a dominant negative mutant of Dishevelled3. These results suggest a WNT-independent cell-autonomous mechanism. We found that the protein levels of APC, AXIN1, and GSK3β, components of the β-catenin degradation complex, were lower in the thalamus than in the cortex of the adult rat brain. Reduced levels of these proteins were also observed in cultured thalamic neurons compared with cortical cultures. Finally, pulse-chase experiments confirmed that cytoplasmic β-catenin turnover was slower in thalamic neurons than in cortical neurons. Altogether, our data indicate that the nuclear localization of β-catenin in thalamic neurons is their cell-intrinsic feature, which was WNT-independent but associated with low levels of proteins involved in β-catenin labeling for ubiquitination and subsequent degradation.     

TGF superfamily and MMP2, MMP9, TIMP1 genes expression in the endometrium of women with impaired reproduction

During the putative "implantation window", a period of maximal endometrial receptivity that spans 7-9 days after ovulation, a series of changes on the structural and molecular level occur that render the endometrium susceptible to implantation for the human embryo. Many members of the TGFbetas are expressed by human endometrium at different stages of menstrual cycle. Also studies regarding the MMP2 gene expression and activity of MMP2 in the implantation window have shown a higher expression and activity of MMP2 in women with impaired fertility. We have examined by RT-PCR the expression of TGFbeta2 and MMP2, MMP9 and TIMP1 in 28 patients with idiopathic infertility, 16 patients with unexplained recurrent miscarriage and 16 control women were enrolled in this study. Seven to nine days after ovulation endometrial biopsy by Pipelle or hysteroscopy was performed to assess the expression of TGFbeta2 , MMP2, MMP9 and TIMP1. We found that in endometria from women with idiopathic infertility TGFbeta2 expression was 2.8 fold higher than in endometria from control group and 2.1 fold higher in endometrial samples from women with unexplained recurrent miscarriage compared to the control group. The MMP2, MMP9 and TIMP1 expression in endometrial samples revealed no significant differences between the study groups and control group. There was a statistically significant negative correlation between TGFbeta2 and MMP9 expression in endometria from women in control group. The present investigations suggest that dysregulated TGFbeta2, MMP2, MMP9 and TIMP1 expression are associated with infertility and early pregnancy loss. However the exact mechanism of how overexpression of endometrial TGFbetaand MMPs interferes with implantation may be more complex.