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This work identifies the protein "macrophage infectivity potentiator" of Trypanosoma cruzi trypomastigotes, as supporting a new property, namely a pro-type 1 immunostimulatory activity on neonatal cells. In its recombinant form (rTcMIP), this protein triggers the secretion of the chemokines CCL2 and CCL3 by human umbilical cord blood cells from healthy newborns, after 24h in vitro culture. Further stimulation for 72h results in secretion of IFN-γ, provided cultures are supplemented with IL-2 and IL-18. rTcMIP activity is totally abolished by protease treatment and is not associated with its peptidyl-prolyl cis-trans isomerase enzymatic activity. The ability of rTcMIP to act as adjuvant was studied in vivo in neonatal mouse immunization models, using acellular diphtheria-tetanus-pertussis-vaccine (DTPa) or ovalbumin, and compared to the classical alum adjuvant. As compared to the latter, rTcMIP increases the IgG antibody response towards several antigens meanwhile skewing antibody production towards the Th-1 dependent IgG2a isotype. The amplitude of the rTcMIP adjuvant effect varied depending on the antigen and the co-presence of alum. rTcMIP did by contrast not increase the IgE response to OVA combined with alum. The discovery of the rTcMIP immunostimulatory effect on neonatal cells opens new possibilities for potential use as pro-type 1 adjuvant for neonatal vaccines. This, in turn, may facilitate the development of more efficient vaccines that can be given at birth, reducing infection associated morbidity and mortality which are the highest in the first weeks after birth., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Radwanska, de Lemos Esteves, Linsen, Coltel, Cencig, Widart, Massart, Colson, Di Paolo, Percier, Ait Djebbara, Guillonneau, Flamand, De Pauw, Frère, Carlier and Truyens.)

Aim: To perform a systematic review to establish whether blind injections of the gleno-humeral (GHJ) joint may be an accurate alternative to injections performed imaging guidance, considering multiple anatomical approaches., Materials and Methods: Our search strategy yielded 478 articles for Scopus, 815 articles for MEDLINE, 128 articles for Cochrane Central Register of Controlled Trials and 555 articles for Embase until May 2016. One hundred and sixty-seven abstracts were retrieved after duplicates removal. Two readers independently reviewed all the 1067 abstracts. They selected for the full-text analysis only the abstracts in which the accuracy of intra-articular position of the needle was confirmed on imaging (humans) or by a surgical dissection (cadavers). Thirty-eight studies were eventually selected for the full-text reading and data extraction. The selected studies included a total of 2309 patients (2690 shoulders) and 195 cadavers (299 shoulders). To objectively assess the methodological quality of the present systematic review, "Assessment of Multiple Systematic Review" (AMSTAR) tool was used., Results: The overall accuracy of the intra-articular injection in GHJ varied from 42 to 100% in the 38 selected studies. Imaging guidance was used in 65% of articles and the overall accuracy of guided GHJ injections was higher than blind injection. However, five articles in which blind injection the GHJ was used (159 shoulders) reported accuracy as high as 100%., Conclusion: A comprehensive review of the literature confirms that guided injections of the GHJ have overall accuracy higher compared to blind injection. Nevertheless, in some studies, including a relatively large number of shoulders, blind injections have been proven to be 100% accurate. Hence, blind injections of GHJ could be proposed a cost-effective alternative to imaging-guided injection. A large prospective randomized study is needed to gauge this hypothesis and compare the cost-effectiveness of these two techniques for the most common anatomical approaches.

Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion), in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes) were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA) and human serum immunoglobulins G (hIgGs). Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine, and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing.

Background: Clear information is still lacking on the safety of corticosteroids (GCs) therapy in RA despite six decades of clinical experience., Scope: We performed a literature search in Ovid MEDLINE from January 2000 to December 2012. Our Population Intervention Comparator Outcomes (PICO) strategy search was: rheumatoid arthritis [Population], corticosteroids or glucocorticoids [Intervention], any comparison [Comparator], adverse effects [Outcome]. Studies were selected if they reported any measure of association between GCs intake and potential adverse effects in RA patients., Findings: We identified 1030 papers and selected for analysis 26 observational studies and six systematic reviews. The major side effects of GCs in RA are bone loss, risk of cardiovascular events and risk of infections as evidenced by large observational studies and not necessarily RCTs. Others associations were reported with herpes zoster, tuberculosis, hyperglycemia, cutaneous abnormalities, gastrointestinal perforation, respiratory infection and self-reported health problems such as cushingoid phenotype, ecchymosis, parchment-like skin, epistaxis, weight gain and sleep disturbance. Other potential adverse effects of GCs were studied but no association was found. These included psychological disorders, dermatophytosis, brain diseases, interstitial lung disease, memory deficit, metabolic syndrome, lymphoma, non-Hodgkin's lymphoma, renal function and cerebrovascular accidents. Most of the evidence emanates from observational researches and the inherent limitations of such data should be kept in mind., Conclusion: Recent observational data and systematic reviews suggest that GCs can lead to relatively alarming and burdensome side effects in RA. This is particularly true for patients who have longer term and higher dose therapies. GCs are largely used in RA and knowing their safety profile is essential to improve patients care. The design of new therapeutic strategies intended to minimize the daily dosing of GCs while conserving their beneficial effect should be encouraged.

Endo-beta-1,4-xylanases of the family 11 glycosyl-hydrolases are catalytically active over a wide range of pH. Xyl1 from Streptomyces sp. S38 belongs to this family, and its optimum pH for enzymatic activity is 6. Xyn11 from Bacillus agaradhaerens and XylJ from Bacillus sp. 41M-1 share 85% sequence identity and have been described as highly alkalophilic enzymes. In an attempt to better understand the alkalophilic adaptation of xylanases, the three-dimensional structures of Xyn11 and Xyl1 were compared. This comparison highlighted an increased number of salt-bridges and the presence of more charged residues in the catalytic cleft as well as an eight-residue-longer loop in the alkalophilic xylanase Xyn11. Some of these charges were introduced in the structure of Xyl1 by site-directed mutagenesis with substitutions Y16D, S18E, G50R, N92D, A135Q, E139K, and Y186E. Furthermore, the eight additional loop residues of Xyn11 were introduced in the homologous loop of Xyl1. In addition, the coding sequence of the XylJ catalytic domain was synthesized by recursive PCR, expressed in a Streptomyces host, purified, and characterized together with the Xyl1 mutants. The Y186E substitution inactivated Xyl1, but the activity was restored when this mutation was combined with the G50R or S18E substitutions. Interestingly, the E139K mutation raised the optimum pH of Xyl1 from 6 to 7.5 but had no effect when combined with the N92D substitution. Modeling studies identified the possible formation of an interaction between the introduced lysine and the substrate, which could be eliminated by the formation of a putative salt-bridge in the N92D/E139K mutant.

Xyl1 from Streptomyces sp. S38 belongs to the low molecular mass family 11 of endo-beta-1,4-xylanases. Its three-dimensional structure has been solved at 2.0 A and its optimum temperature and pH for enzymatic activity are 60 degrees C and 6.0, respectively. Aspergillus kawachii xylanase XynC belongs to the same family but is an acidophilic enzyme with an optimum pH of 2.0. Structural comparison of Xyl1 and XynC showed differences in residues surrounding the two glutamic acid side chains involved in the catalysis that could be responsible for the acidophilic adaptation of XynC. Mutations W20Y, N48D, A134E, and Y193W were introduced by site-directed mutagenesis and combined in multiple mutants. Trp 20 and Tyr 193 are involved in substrate binding. The Y193W mutation inactivated Xyl1 whereas W20Y decreased the optimum pH of Xyl1 to 5.0 and slightly increased its specific activity. The N48D mutation also decreased the optimum pH of Xyl1 by one unit. The A134E substitution did not induce any change, but when combined with N48D, a synergistic effect was observed with a 1.4 unit decrease in the optimum pH. Modeling showed that the orientations of residue 193 and of the fully conserved Arg 131 are different in acidophilic and "alkaline" xylanases whereas the introduced Tyr 20 probably modifies the pKa of the acid-base catalyst via residue Asn 48. Docking of a substrate analog in the catalytic site highlighted striking differences between Xyl1 and XynC in substrate binding. Hydrophobicity calculations showed a correlation between acidophilic adaptation and a decreased hydrophobicity around the two glutamic acid side chains involved in catalysis.

In a general approach to the understanding of protein adaptation to high temperature, molecular models of the closely related mesophilic Streptomyces sp. S38 Xyl1 and thermophilic Thermomonospora fusca TfxA family 11 xylanases were built and compared with the three-dimensional (3D) structures of homologous enzymes. Some of the structural features identified as potential contributors to the higher thermostability of TfxA were introduced in Xyl1 by site-directed mutagenesis in an attempt to improve its thermostability and thermophilicity. A new Y11-Y16 aromatic interaction, similar to that present in TfxA and created in Xyl1 by the T11Y mutation, improved both the thermophilicity and thermostability. Indeed, the optimum activity temperature (70 vs. 60 degrees C) and the apparent Tm were increased by about 9 degrees C, and the mutant was sixfold more stable at 57 degrees C. The combined mutations A82R/F168H/N169D/delta170 potentially creating a R82-D169 salt bridge homologous to that present in TfxA improved the thermostability but not the thermophilicity. Mutations R82/D170 and S33P seemed to be slightly destabilizing and devoid of influence on the optimal activity temperature of Xyl1. Structural analysis revealed that residues Y11 and Y16 were located on beta-strands B1 and B2, respectively. This interaction should increase the stability of the N-terminal part of Xyl1. Moreover, Y11 and Y16 seem to form an aromatic continuum with five other residues forming putative subsites involved in the binding of xylan (+3, +2, +1, -1, -2). Y11 and Y16 might represent two additional binding subsites (-3, -4) and the T11Y mutation could thus improve substrate binding to the enzyme at higher temperature and thus the thermophilicity of Xyl1.