Effect of salinity stress and surfactant treatment with zinc and boron on morpho-physiological and biochemical indices of fenugreek (Trigonella foenum-graecum).

Micronutrient application has a crucial role in mitigating salinity stress in crop plants. This study was carried out to investigate the effect of zinc (Zn) and boron (B) as foliar applications on fenugreek growth and physiology under salt stress (0 and 120 mM). After 35 days of salt treatments, three levels of zinc (0, 50, and 100 ppm) and two levels of boron (0 and 2 ppm) were applied as a foliar application. Salinity significantly reduced root length (72.7%) and shoot length (33.9%), plant height (36%), leaf area (37%), root fresh weight (48%) and shoot fresh weight (75%), root dry weight (80%) and shoot dry weight (67%), photosynthetic pigments (78%), number of branches (50%), and seeds per pod (56%). Fenugreek's growth and physiology were improved by foliar spray of zinc and boron, which increased the length of the shoot (6%) and root length (2%), fresh root weight (18%), and dry root weight (8%), and chlorophyll a (1%), chlorophyll b (25%), total soluble protein content (3%), shoot calcium (9%) and potassium (5%) contents by significantly decreasing sodium ion (11%) content. Moreover, 100 ppm of Zn and 2 ppm of B enhanced the growth and physiology of fenugreek by reducing the effect of salt stress. Overall, boron and zinc foliar spray is suggested for improvement in fenugreek growth under salinity stress.

Evaluation of Antimicrobial and Antioxidant Potential of Oxalis corymbosa Extracts.

This work aimed to assess the antioxidant and antimicrobial properties of Oxalis corymbosa extracts. Biochemical analyses were conducted on various plant parts, utilizing enzymatic and non-enzymatic assays. Parameters such as total soluble protein, chlorophyll, and carotenoid contents were also evaluated to elucidate the role of bioactive chemical compounds. The antimicrobial screening of extracts was performed against the bacterial and fungal strains Escherichia coli, Staphylococcus aureus, and Candida albicans, respectively. Results indicated that chlorophyll a, chlorophyll b, total chlorophyll, carotenoid content, anthocyanin content, catalase, peroxidase, and superoxide dismutase were most abundant in the O. corymbosa leaves. Moreover, total ascorbate peroxidase content, total phenolic content, and total flavonoid content were found to be higher in the roots compared to other parts. High-performance liquid chromatography analysis identified chlorogenic acid as the major component, followed by gallic acid, caffeic acid, quercetin, and salicylic acid. Regarding antibacterial potential, each extract exhibited significant activity, with methanolic and ethyl acetate extracts demonstrating the maximum inhibition zone against S. aureus and E. coli, respectively. These findings highlight the substantial antioxidant and antibacterial potential of different parts of O. corymbosa, suggesting their promising applications as ingredients in various nutraceutical products.

The Effects of Artificial Diets on the Expression of Molecular Marker Genes Related to Honey Bee Health.

Honey bees are commonly used to study metabolic processes, yet the molecular mechanisms underlying nutrient transformation, particularly proteins and their effects on development, health, and diseases, still evoke varying opinions among researchers. To address this gap, we investigated the digestibility and transformation of water-soluble proteins from four artificial diets in long-lived honey bee populations (Apis mellifera ligustica), alongside their impact on metabolism and DWV relative expression ratio, using transcriptomic and protein quantification methods. Diet 2, characterized by its high protein content and digestibility, was selected for further analysis from the other studied diets. Subsequently, machine learning was employed to identify six diet-related molecular markers: SOD1, Trxr1, defensin2, JHAMT, TOR1, and vg. The expression levels of these markers were found to resemble those of honey bees who were fed with Diet 2 and bee bread, renowned as the best natural food. Notably, honey bees exhibiting chalkbrood symptoms (Control-N) responded differently to the diet, underscoring the unique nutritional effects on health-deficient bees. Additionally, we proposed a molecular model to elucidate the transition of long-lived honey bees from diapause to development, induced by nutrition. These findings carry implications for nutritional research and beekeeping, underscoring the vital role of honey bees in agriculture.

Low on-treatment levels of serum soluble CD8 (sCD8) predict better outcomes in advanced non-small cell lung cancer patients treated with atezolizumab.

BACKGROUND: Immunotherapy has changed the paradigm of treating non-small cell lung cancer (NSCLC). But, selecting patients who will achieve long-term benefits from treatment remains unsatisfactory. Here, we investigated the possible use of the soluble form of CD8 antigen (sCD8) in predicting durable disease control after PD-1/PD-L1 blockade. CD8 is a marker of the cytotoxic T lymphocytes. Its soluble form (sCD8) is secreted under activation of the immune system but also has immunosuppressive properties. The data about serum sCD8 in patients dosed with anti-PD-1/PD-L1 drugs are lacking. METHODS AND RESULTS: We included 42 NSCLC patients and collected samples at baseline and for the first 3 months of atezolizumab immunotherapy. The serum sCD8 concentrations were measured with the ELISA kit and correlated with treatment outcomes. Patients with durable (≥ 12 months) disease control presented lower serum sCD8 than those without long-term benefits. The sCD8 levels measured at the end of cycle 2 (sCD8.2) were the earliest time point that successfully differentiated patients (3.76 vs. 9.68 ng/mL, respectively, p < 0.001). Individuals with low sCD8.2 (≤ 4.09 ng/mL) presented longer progression-free survival (HR = 0.061, p < 0.001) and overall survival (HR = 0.104, p < 0.05) compared to individuals with high sCD8.2 (median values unreached vs. 4.4 months and 14.4 months for PFS and OS, respectively). CONCLUSIONS: Serum sCD8 could be an early biomarker of durable disease control after anti-PD-L1 treatment. Higher sCD8 in patients with worse outcomes could suggest the inhibitory effect of sCD8 on cytotoxic T-cells activation.

Optimized rapid production of recombinant secreted proteins in CHO cells grown in suspension: The case of RBD.

Chinese Hamster Ovary cells (CHO) have become the most common workhorse for the commercial production of therapeutic proteins, as well as for the production of recombinant proteins for biomedical research. The ability to grow at high density in suspension, the adaptability to serum free media, and the ease transfection and scale up, made CHO cell line highly productive and robust for large-scale production. Here, we present an optimized workflow used to successfully express and purify a number of human proteins with a yield up to 5 mg/L of culture. The entire protocol, from the synthetic gene design to the assessment of purified protein quality, can be completed in 2 weeks. The established cell culture platform has been efficiently adapted to rapidly produce the receptor-binding domain (RBD) in SARS-CoV-2 S protein, a protein required by many laboratories in 2020 to better understand the initial step of infection related to COVID-19 pandemic. An overall yield of 2 mg of high quality soluble RBD per liter of culture was obtained, a production 10-times cheaper than commercial preparations, this representing an intriguing strategy for future challenges.

Limitations to photosynthesis in bryophytes: certainties and uncertainties regarding methodology.

Bryophytes are the group of land plants with the lowest photosynthetic rates, which was considered to be a consequence of their higher anatomical CO2 diffusional limitation compared with tracheophytes. However, the most recent studies assessing limitations due to biochemistry and mesophyll conductance in bryophytes reveal discrepancies based on the methodology used. In this study, we compared data calculated from two different methodologies for estimating mesophyll conductance: variable J and the curve-fitting method. Although correlated, mesophyll conductance estimated by the curve-fitting method was on average 4-fold higher than the conductance obtained by the variable J method; a large enough difference to account for the scale of differences previously shown between the biochemical and diffusional limitations to photosynthesis. Biochemical limitations were predominant when the curve-fitting method was used. We also demonstrated that variations in bryophyte relative water content during measurements can also introduce errors in the estimation of mesophyll conductance, especially for samples which are overly desiccated. Furthermore, total chlorophyll concentration and soluble proteins were significantly lower in bryophytes than in tracheophytes, and the percentage of proteins quantified as Rubisco was also significantly lower in bryophytes (<6.3% in all studied species) than in angiosperms (>16% in all non-stressed cases). Photosynthetic rates normalized by Rubisco were not significantly different between bryophytes and angiosperms. Our data suggest that the biochemical limitation to photosynthesis in bryophytes is more relevant than so far assumed.

Small Prokaryotic DNA-Binding Proteins Protect Genome Integrity throughout the Life Cycle.

Genomes of all organisms are persistently threatened by endogenous and exogenous assaults. Bacterial mechanisms of genome maintenance must provide protection throughout the physiologically distinct phases of the life cycle. Spore-forming bacteria must also maintain genome integrity within the dormant endospore. The nucleoid-associated proteins (NAPs) influence nucleoid organization and may alter DNA topology to protect DNA or to alter gene expression patterns. NAPs are characteristically multifunctional; nevertheless, Dps, HU and CbpA are most strongly associated with DNA protection. Archaea display great variety in genome organization and many inhabit extreme environments. As of yet, only MC1, an archaeal NAP, has been shown to protect DNA against thermal denaturation and radiolysis. ssDNA are intermediates in vital cellular processes, such as DNA replication and recombination. Single-stranded binding proteins (SSBs) prevent the formation of secondary structures but also protect the hypersensitive ssDNA against chemical and nuclease degradation. Ionizing radiation upregulates SSBs in the extremophile Deinococcus radiodurans.

Biochemical characterization of solid-state fermented cassava roots (Manihot esculenta Crantz) and its application in broiler feed formulation.

The biochemical parameters of solid-state fermented peeled and unpeeled cassava roots (Manihot esculenta Crantz) and their application in broiler feed formulations were investigated. Fermentation occurred at room temperature for 72 h (pH 3-9). The samples utilized for five (5) broiler starter feeds were labeled: control, unfermented unpeeled cassava (UUC), unfermented peeled cassava (UPC), fermented unpeeled cassava (FUC), and fermented peeled cassava (FPC). Formulations were made by substituting fermented/non-fermented cassava roots at pH 7 for maize (w/w%). Fermentation-induced changes included increased soluble and total protein concentrations (69.3 and 334.5 mg/g) and (9.6 and 10.8%), respectively, in cultures prepared with peeled and unpeeled cassava at pH 7 compared to the control (p < 0.05), and a reduction (p < 0.01) in cyanide concentration from 44.4 to 78.7 mg/kg in the control to 8.5 and 13.7 mg/kg in fermented cassava at pH 7. Birds fed FUC and FPC meal (0.6 and 0.5 kg) gained significantly more weight (p < 0.05) than those fed the control (0.3 kg). The biochemical parameters aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, and urea levels in broiler serum did not differ significantly (p > 0.05) for birds fed with fermented peeled and unpeeled cassava. Conversely, serum albumin and calcium levels were significantly lower (p < 0.05) for birds fed with the control feed compared to birds fed with fermented feeds. The results imply that fermented peeled and unpeeled cassava roots could be a safe and nutritionally beneficial replacement for maize in broiler diet.

Preliminary Evidence for a Relationship between Elevated Plasma TNFα and Smaller Subcortical White Matter Volume in HCV Infection Irrespective of HIV or AUD Comorbidity.

Classical inflammation in response to bacterial, parasitic, or viral infections such as HIV includes local recruitment of neutrophils and macrophages and the production of proinflammatory cytokines and chemokines. Proposed biomarkers of organ integrity in Alcohol Use Disorders (AUD) include elevations in peripheral plasma levels of proinflammatory proteins. In testing this proposal, previous work included a group of human immunodeficiency virus (HIV)-infected individuals as positive controls and identified elevations in the soluble proteins TNFα and IP10; these cytokines were only elevated in AUD individuals seropositive for hepatitis C infection (HCV). The current observational, cross-sectional study evaluated whether higher levels of these proinflammatory cytokines would be associated with compromised brain integrity. Soluble protein levels were quantified in 86 healthy controls, 132 individuals with AUD, 54 individuals seropositive for HIV, and 49 individuals with AUD and HIV. Among the patient groups, HCV was present in 24 of the individuals with AUD, 13 individuals with HIV, and 20 of the individuals in the comorbid AUD and HIV group. Soluble protein levels were correlated to regional brain volumes as quantified with structural magnetic resonance imaging (MRI). In addition to higher levels of TNFα and IP10 in the 2 HIV groups and the HCV-seropositive AUD group, this study identified lower levels of IL1ß in the 3 patient groups relative to the control group. Only TNFα, however, showed a relationship with brain integrity: in HCV or HIV infection, higher peripheral levels of TNFα correlated with smaller subcortical white matter volume. These preliminary results highlight the privileged status of TNFα on brain integrity in the context of infection.

Tracking Drug-Induced Epithelial-Mesenchymal Transition in Breast Cancer by a Microfluidic Surface-Enhanced Raman Spectroscopy Immunoassay.

Epithelial-mesenchymal transition (EMT) is a primary mechanism for cancer metastasis. Detecting the activation of EMT can potentially convey signs of metastasis to guide treatment management and improve patient survival. One of the classic signatures of EMT is characterized by dynamic changes in cellular expression levels of E-cadherin and N-cadherin, whose soluble active fragments have recently been reported to be biomarkers for cancer diagnosis and prognosis. Herein, a microfluidic immunoassay (termed "SERS immunoassay") based on sensitive and simultaneous detection of soluble E-cadherin (sE-cadherin) and soluble N-cadherin (sN-cadherin) for EMT monitoring in patients' plasma is presented. The SERS immunoassay integrates in situ nanomixing and surface-enhanced Raman scattering readout to enable accurate detection of sE-cadherin and sN-cadherin from as low as 10 cells mL-1 . This assay enables tracking of a concurrent decrease in sE-cadherin and increase in sN-cadherin in breast cancer cells undergoing drug-induced mesenchymal transformation. The clinical potential of the SERS immunoassay is further demonstrated by successful detection of sE-cadherin and sN-cadherin in metastatic stage IV breast cancer patient plasma samples. The SERS immunoassay can potentially sense the activation of EMT to provide early indications of cancer invasions or metastasis.

A proposed sample handling of ovine cotyledon for proteomic studies.

Ovine trophoblast is a suitable material for placental studies. However, a universal protocol for handling ruminant cotyledon samples has still not been reported. Considering the villous structure of ovine cotyledon, we suggest procedures to prepare cotyledons with limited inherent contamination, using semi-dry conditions to avoid freezing damage and sample errors. The cytosolic water-soluble proteins were physically extracted from the frozen cotyledons. High homogeneity was demonstrated between the replicates of both tissue and extract samples. Importantly, the chemical lysis of placental crude extracts was necessary for protein separation and immunoreaction. The integrity of stored tissues was histologically validated using a formalin-fixed paraffin-embedded technique. Using label-free proteomics, we detected 388 Ovis protein-groups in at least two of three biological replicates of either the tissue or extract. Although the water-soluble proteins were dominated by hemoglobin subunits, ten proteins were identified exclusively in all extract replicates. The physical extraction selectively reduced the membrane, extracellular matrix, and cytoskeleton proteins. The hydrolase enzymes, in the extract, hindered the identification of some specific proteins, such as histone H2A. In summary, the proposed workflow may guide further proteomic investigations of ovine cotyledon biology. Furthermore, our proteomic data have inferred some potential mechanisms of ovine trophoblast at parturition.

Purified Splenic amastigotes of Leishmania donovani-Immunoproteomic approach for exploring Th1 stimulatory polyproteins.

Visceral leishmaniasis (VL) represents one of the most challenging infectious diseases worldwide. The reason that once infected, patient develops immunity against Leishmania parasite has paved way to develop prophylactic vaccines against disease, but only some of these have moved ahead for clinical trials. Herein, the study to explore novel and potential vaccine candidates was extended to pathogenic form of parasite, that is, amastigote form which is less explored due to complexity of its purification process. Methods and results. Classical protocol of purification of splenic amastigotes was modified to obtain highly pure amastigotes which was confirmed by Western blotting in support with proteomics studies. Fractionation and sub-fractionation of purified splenic amastigotes revealed four sub-fractions, belonging to 97 to 68 kDa and 68 to 43 kDa ranges, which showed long-lasting protection with remarkable Th1-type cellular responses in hamsters vaccinated with these sub-fractions (LTT, NO, QRT-PCR). Further proteomics analysis, to identify and understand the precise nature and function of these protective protein sub-fractions, identified a total of 47 proteins including twenty-five hypothetical proteins/unknowns. Amastigote stage has potential Th1-stimulatory vaccine candidates, notably, among identified proteins, major were uncharacterized proteins/hypothetical proteins, which once characterized may serve as novel and potential vaccine candidates/drug targets.

Modulation of growth and key physiobiochemical attributes after foliar application of zinc sulphate (ZnSO4) on wheat (Triticum aestivum L.) under cadmium (Cd) stress.

A pot experiment was conducted to examine the effect of foliar application of various levels of ZnSO4 on wheat (Triticum aestivum L.) under cadmium (Cd) stress. Seeds of two wheat varieties i.e., Ujala-2016 and Anaj-2017 were sown in sand filled plastic pots. Cadmium (CdCl2) stress i.e., 0 and 0.5 mM CdCl2 was applied in full strength Hoagland's nutrient solution after 4 weeks of seed germination. Foliar spray of varying ZnSO4 levels i.e., 0, 2, 4, 6 and 8 mM was applied after 2 weeks of CdCl2 stress induction (of 6 week old plants). After 3 weeks of foliar treatment leaf samples of 9 week old wheat plants were collected for the determination of changes in various growth and physiobiochemical attributes. Results obtained showed that cadmium stress (0.5 mM CdCl2) significantly decreased shoot and root fresh and dry weights, shoot and root lengths, yield attributes, chlorophyll a contents and total phenolics, while increased hydrogen peroxide (H2O2), total soluble proteins, free proline, glycinebetaine (GB) contents, and activities of antioxidant enzymes i.e., catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD). Foliar application of varying ZnSO4 levels significantly increased various growth attributes, chlorophyll b contents, H2O2, free proline, GB and activities of antioxidant enzymes i.e., CAT, POD and APX, while decreased relative water contents and total phenolics under Cd stress or non stress conditions. Furthermore, both wheat varieties showed differential response under Cd stress and towards foliar application of ZnSO4 e.g., wheat var. Ujala-2016 was higher in shoot dry weight, root length, root fresh and dry weights, total leaf area per plant, 100 grains weight, number of tillers per plant, chlorophyll b, hydrogen peroxide (H2O2), activities of APX, POD, glycinebetaine and leaf free proline contents, while var. Anaj-2017 exhibited high shoot fresh weight, grain yield per plant, no. of grains per plant, chlorophyll contents, chlorophyll a/b ratio, total phenolics, MDA and total soluble protein contents under cadmium stress or non stress conditions.

Physiological and yield responses of purple coneflower (Echinacea purpurea (L.) Moench) to nitrogen sources at different levels of irrigation.

Echinacea purpurea (L.) is one of the most important medicinal plants in the world showing different biochemical reactions as affected by drought stress and nitrogen fertilizer. The purpose of this study was to determine the effect of nitrogen on soluble protein, enzyme activities, carotenoids metabolism, greenness and biological yield of the Echinacea purpurea under different levels of irrigation. The experiment was conducted in a research field in Iran during 2013-2015. Irrigation treatments included irrigation after 25, 50 and 75% soil water depletion, and nitrogen sources were no nitrogen (N), nitroxin, 40 kg N ha-1, 40 kg N ha-1 + nitroxin and 80 kg N ha-1. Nitroxin is a biofertilizer including Azotobacter and Azospirillum. The activity of catalase (11.9-21.3 mmol g-1fw min-1), peroxidase (40.1-48.9 µmol g-1fw min-1), polyphenoloxidase (0.79-3.63 mmol g-1fw min-1) increased in both years under drought stress conditions. The lowest greenness (50.1-45.9) was achieved from no-application of nitrogen in the irrigation treatment after 75% water depletion. The results demonstrated the beneficial effects of nitrogen sources on physiological reactions, especially peroxidase, polyphenol oxidase and carotenoids metabolism. It is concluded that irrigation of E. purpurea based on 50% water depletion and 80 kg nitrogen as well as the combination of nitroxin and 40 kg nitrogen treatments should be an appropriate choice for 2 years.

The Disordered Landscape of the 20S Proteasome Substrates Reveals Tight Association with Phase Separated Granules.

Proteasomal degradation is the main route of regulated proteostasis. The 20S proteasome is the core particle (CP) responsible for the catalytic activity of all proteasome complexes. Structural constraints mean that only unfolded, extended polypeptide chains may enter the catalytic core of the 20S proteasome. It has been previously shown that the 20S CP is active in degradation of certain intrinsically disordered proteins (IDP) lacking structural constrains. Here, a comprehensive analysis of the 20S CP substrates in vitro is conducted. It is revealed that the 20S CP substrates are highly disordered. However, not all the IDPs are 20S CP substrates. The group of the IDPs that are 20S CP substrates, termed 20S-IDPome are characterized by having significantly more protein binding partners, more posttranslational modification sites, and are highly enriched for RNA binding proteins. The vast majority of them are involved in splicing, mRNA processing, and translation. Remarkably, it is found that low complexity proteins with prion-like domain (PrLD), which interact with GR or PR di-peptide repeats, are the most preferential 20S CP substrates. The finding suggests roles of the 20S CP in gene transcription and formation of phase-separated granules.

Extremely stable high molecular mass soluble multiprotein complex from eggs of sea urchin Strongylocentrotus intermedius with phosphatase activity.

It was proposed that most biological processes are performed by different protein complexes. In contrast to individual proteins and enzymes, their complexes usually have other biological functions, and their formation may be important system process for the expansion of diversity and biological functions of different molecules. Identification and characterization of embryonic components including proteins and their multiprotein complexes seem to be very important for an understanding of embryo function. We have isolated and analyzed for the first time a very stable multiprotein complex (SPC; approximately 1100 kDa) from the soluble fraction of extracts of the sea urchin embryos. By fast protein liquid chromatography (FPLC) gel filtration the SPC was well separated from other extract proteins. Stable multiprotein complex is stable in different drastic conditions but dissociates moderately in the presence of 8M urea + 1.0M NaCl. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis data, this complex contains many major, moderate and minor proteins with molecular masses from 10 to 95 kDa. The SPC was destroyed by 8M urea or SDS, and its components were separated using thin layer chromatography, ion-exchange chromatography, gel filtration, and reverse phase chromatography. Using matrix-assisted laser desorption/ionization mass spectrometry of partially dissociated SPC, it was shown that the complex contains not only proteins (10-95 kDa) but also few dozens of peptides with molecular masses from 2 to 9.5 kDa. Short peptides form very strong complexes, which at the treatment of SPC with urea or SDS can be partially break down into smaller complexes having different peptide compositions. Reverse phase chromatography of these complexes after all type of abovementioned chromatographies led to detection from 6 to 11 distinct peaks corresponding to new complexes containing up to a few dozens of peptides. The SPCs possess alkaline phosphatase activity. Progress in the study of embryos protein complexes can help to understand their biological functions.

Identification of a perchloric acid-soluble protein (PSP)-like ribonuclease from Trichomonas vaginalis.

A perchloric acid-soluble protein (PSP), named here tv-psp1, was identified in Trichomonas vaginalis. It is expressed under normal culture conditions according to expressed sequence tag (EST) analysis. On the other hand, Tv-PSP1 protein was identified by mass spectrometry with a 40% of identity to human PSP (p14.1). Polyclonal antibodies against recombinant Tv-PSP1 (rTv-PSP1) recognized a single band at 13.5 kDa in total protein parasite extract by SDS-PAGE and a high molecular weight band analyzed by native PAGE. Structural analysis of Tv-PSP1, using dynamic light scattering, size exclusion chromatography, and circular dichroism spectroscopy, showed a trimeric structure stable at 7 M urea with 38% α-helix and 14% ß-sheet in solution and a molecular weight of 40.5 kD. Tv-PSP1 models were used to perform dynamic simulations over 100 ns suggesting a stable homotrimeric structure. Tv-PSP1 was located in the nucleus, cytoplasm, and hydrogenosomes of T. vaginalis, and the in silico analysis by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) showed interactions with RNA binding proteins. The preliminary results of RNA degradation analysis with the recombinant Tv-PSP1 showed RNA partial deterioration suggesting a possible putative ribonuclease function.

Molecular cloning and expression analysis of cytochrome c oxidase subunit II from Sitophilus zeamais.

Cytochrome c oxidase subunit II (COX II) containing a dual core CuA active site is one of the core subunits of mitochondrial Cytochrome c oxidase (Cco), which plays a significant role in the physiological process. In this report, the full-length cDNA of COXII gene was cloned from Sitophilus zeamais, which had an open reading frame (ORF) of 684 bp encoding 227 amino acids residues. The predicted COXII protein had a molecular mass of 26.2 kDa with pI value of 6.37. multiple sequence alignment and phylogenetic analysis indicated that Sitophilus zeamais COXII had high sequence identity with the COXII of other insect species. The gene was subcloned into the expression vector pET-32a, and induced by isopropyl ß-d-thiogalactopyranoside (IPTG) in E. coli Transetta (DE3) expression system. Finally the recombinant COXII with 6-His tag was purified using affinity chromatography with Ni(2+)-NTA agarose. Western Blotting (WB) showed the recombinant protein was about 44 kD, and the concentration of fusion protein was 50 µg/mL. UV-spectrophotometer and infrared spectrometer analysis showed that recombinant COXII could catalyze the oxidation of substrate Cytochrome C (Cyt c), and influenced by allyl isothiocyanate (AITC). By using molecular docking method, It was found that a sulfur atom of AITC structure could form a length of 2.9 Å hydrogen bond with Leu-31. These results suggested that tag-free COXII was functional and one of the action sites of AITC, which will be helpful to carry out a point mutation in binding sites for the future research.

A profiling approach of the natural variability of foliar N remobilization at the rosette stage gives clues to understand the limiting processes involved in the low N use efficiency of winter oilseed rape.

Oilseed rape, a crop requiring a high level of nitogen (N) fertilizers, is characterized by low N use efficiency. To identify the limiting factors involved in the N use efficiency of winter oilseed rape, the response to low N supply was investigated at the vegetative stage in 10 genotypes by using long-term pulse-chase (15)N labelling and studying the physiological processes of leaf N remobilization. Analysis of growth and components of N use efficiency allowed four profiles to be defined. Group 1 was characterized by an efficient N remobilization under low and high N conditions but by a decrease of leaf growth under N limitation. Group 2 showed a decrease in leaf growth under low N supply that was associated with a low N remobilization efficiency under both N supplies despite a high remobilization of soluble proteins. In response to N limitation, Group 3 is characterized by an increase in N use efficiency and leaf N remobilization compared with high N that is not sufficient to sustain the leaf biomass production at a similar level to non-limited plants. Genotypes of Group 4 subjected to low nitrate were able to maintain leaf growth to the same level as under high N. The profiling approach indicated that enhancement of amino acid export and soluble protein degradation was crucial for N remobilization improvement. At the whole-plant level, N fluxes revealed that Group 4 showed a high N remobilization in source leaves combined with a better N utilization in young leaves. Consequently, an enhanced N remobilization limits N loss in fallen leaves, but this remobilized N needs to be efficiently utilized in young leaves to improve N use efficiency.

Tau acts as a mediator for Alzheimer's disease-related synaptic deficits.

The two histopathological hallmarks of Alzheimer's disease (AD) are amyloid plaques containing multiple forms of amyloid beta (Aß) and neurofibrillary tangles containing phosphorylated tau proteins. As mild cognitive impairment frequently occurs long before the clinical diagnosis of AD, the scientific community has been increasingly interested in the roles of Aß and tau in earlier cellular changes that lead to functional deficits. Therefore, great progress has recently been made in understanding how Aß or tau causes synaptic dysfunction. However, the interaction between the Aß and tau-initiated intracellular cascades that lead to synaptic dysfunction remains elusive. The cornerstone of the two-decade-old hypothetical amyloid cascade model is that amyloid pathologies precede tau pathologies. Although the premise of Aß-tau pathway remains valid, the model keeps evolving as new signaling events are discovered that lead to functional deficits and neurodegeneration. Recent progress has been made in understanding Aß-PrP(C) -Fyn-mediated neurotoxicity and synaptic deficits. Although still elusive, many novel upstream and downstream signaling molecules have been found to modulate tau mislocalization and tau hyperphosphorylation. Here we will discuss the mechanistic interactions between Aß-PrP(C) -mediated neurotoxicity and tau-mediated synaptic deficits in an updated amyloid cascade model with calcium and tau as the central mediators.