Spider silk proteins undergo a complex series of molecular events before being converted into an outstanding hierarchically organized fiber. Recent literature has underlined the crucial role of the C-terminal domain in silk protein stability and fiber formation. However, the effect of pH remains to be clarified. We have thus developed an efficient purification protocol to obtain stable native-like recombinant MaSp1 C-terminal domain of Nephila clavipes (NCCTD). Its structure was investigated as a function of pH using circular dichroism, fluorescence and solution NMR spectroscopy. The results show that the NCCTD structure is very sensitive to pH and suggest that a molten globule state occurs at pH 5.0 and below. Electronic microscopy images also indicate fiber formation at low pH and coarser globular particles at more basic pH. The results are consistent with a spinning process model where the NCCTD acts as an aggregation nucleus favoring the β-aggregation of the hydrophobic polyalanine repeats upon spinning.

We have investigated the effect of pH, salts and shear on the hydrodynamical diameter of recombinant major ampullate (MA) rMaSpI silk proteins in solution as a function of time using (1) H solution NMR spectroscopy. The results indicate that the silk proteins in solution are composed of two diffusing populations, a high proportion of "native" solubilized proteins and a small amount of high molecular weight oligomers. Similar results are observed with the MA gland content. Salts help maintaining the proteins in a compact form in solution over time and inhibit aggregation, the absence of salts triggering protein assembly leading to a gel state. Moreover, the aggregation kinetics of rMaSpI at low salt concentration accelerates as the pH is close to the isoelectric point of the proteins, suggesting that the pH decrease tends to slow down aggregation. The data also support the strong impact of shear on the spinning process and suggest that the assembly is driven by a nucleation conformational conversion mechanism. Thus, the adjustment of the physicochemical conditions in the ampulla seems to promote a stable, long term storage. In addition, the optimization of protein conformation as well as their unfolding and aggregation propensity in the duct leads to a specifically organized structure.

The RNA-binding protein p54(nrb) is involved in many nuclear processes including transcription, RNA processing, and retention of hyperedited RNAs. In interphase cells, p54(nrb) localizes to the nucleoplasm and concentrates with protein partners in the paraspeckles via an interaction with the non-coding RNA Neat1. During mitosis, p54(nrb) becomes multiphosphorylated and the effects of this modification are not known. In the present study, we show that p54(nrb) phosphorylation does not affect the interactions with its protein partners but rather diminishes its general RNA-binding ability. Biochemical assays indicate that in vitro phosphorylation of a GST-p54(nrb) construct by CDK1 abolishes the interaction with 5' splice site RNA sequence. Site-directed mutagenesis shows that the threonine 15 residue, located N-terminal to the RRM tandem domains of p54(nrb), is involved in this inhibition. In vivo analysis reveals that Neat1 ncRNA co-immunoprecipitates with p54(nrb) in either interphase or mitotic cells, suggesting that p54(nrb)-Neat1 interaction is not modulated by phosphorylation. Accordingly, in vitro phosphorylated GST-p54(nrb) still interacts with PIR-1 RNA, a G-rich Neat1 sequence known to interact with p54(nrb). In vitro RNA binding assays show that CDK1-phosphorylation of a GST-p54(nrb) construct abolishes its interaction with homoribopolymers poly(A), poly(C), and poly(U) but not with poly(G). These data suggest that p54(nrb) interaction with RNA could be selectively modulated by phosphorylation during mitosis.

Feeding practices have been found to influence gut microbiota which play a major role in immunity of poultry. In the present study, changes in cecal microbiota and humoral responses resulting in the 55 ppm bacitracin (BACI), 1% each of cranberry (CP1) and wild blueberry (BP1) pomace alone or in combination (CP+BP) feeding in broiler Cobb 500 vaccinated or not against coccidiosis were investigated. In the non-vaccinated group, no significant treatment effects were observed on performance parameters. Vaccination significantly affected bird’s performance parameters particularly during the growing phase from 10 to 20 days of age. In general, the prevalence of coccidiosis and necrotic enteritis (NE) was reduced by vaccination (P < 0.05). BACI-treated birds showed low intestinal lesion scores, and both CP1 and BP1 feed supplementations reduced Eimeria acervulina and Clostridium perfringens incidences similar to BACI. Vaccination induced change in serum enzymes, minerals, and lipid levels in 21-day old birds while, levels of triglyceride (TRIG) and non-esterified fatty acids (NEFA) were higher (P < 0.05) in CP1 treated non–vaccinated group than in the control. The levels of NEFA were lower in BACI- and CP1-fed birds than in the control in non-vaccinated day 28 old birds. The highest levels of all estimated three immunoglobulins (IgY, IgM, and IgA) were found in the vaccinated birds. Metagenomics analysis of the cecal bacterial community in 21-day old birds showed the presence of Firmicutes (90%), Proteobacteria (5%), Actinobacteria (2%), and Bacteroidetes (2%). In the vaccinated group, an effect of BACI was noted on Proteobacteria (P = 0.03). Vaccination and/or dietary treatments influenced the population of Lactobacillaceae, Enterobacteriaceae, Clostridiaceae, and Streptococcaceae which were among the most abundant families. Overall, this study revealed that besides their beneficial effects on performance, alike bacitracin, berry pomaces in poultry feed have profound impacts on the chicken cecal microbiota and blood metabolites that could be influenced by vaccination against coccidiosis.