Structural Analysis of an Evolved Transketolase Reveals Divergent Binding Modes.

The S385Y/D469T/R520Q variant of E. coli transketolase was evolved previously with three successive smart libraries, each guided by different structural, bioinformatical or computational methods. Substrate-walking progressively shifted the target acceptor substrate from phosphorylated aldehydes, towards a non-phosphorylated polar aldehyde, a non-polar aliphatic aldehyde, and finally a non-polar aromatic aldehyde. Kinetic evaluations on three benzaldehyde derivatives, suggested that their active-site binding was differentially sensitive to the S385Y mutation. Docking into mutants generated in silico from the wild-type crystal structure was not wholly satisfactory, as errors accumulated with successive mutations, and hampered further smart-library designs. Here we report the crystal structure of the S385Y/D469T/R520Q variant, and molecular docking of three substrates. This now supports our original hypothesis that directed-evolution had generated an evolutionary intermediate with divergent binding modes for the three aromatic aldehydes tested. The new active site contained two binding pockets supporting π-π stacking interactions, sterically separated by the D469T mutation. While 3-formylbenzoic acid (3-FBA) preferred one pocket, and 4-FBA the other, the less well-accepted substrate 3-hydroxybenzaldehyde (3-HBA) was caught in limbo with equal preference for the two pockets. This work highlights the value of obtaining crystal structures of evolved enzyme variants, for continued and reliable use of smart library strategies

Single active-site mutants are sufficient to enhance serine:pyruvate α-transaminase activity in an ω-transaminase

We have analysed the natural evolution of transaminase structure and sequence between an α-transaminase serine-pyruvate aminotransferase, and an ω-transaminase from Chromobacterium violaceum with <20% sequence identity, and identified the active-site regions which are least conserved structurally. We also show that these structural changes correlate strongly with transaminase substrate specificity during evolution and therefore might normally be presumed to be essential determinants of substrate specificity. However, key residues are often conserved spatially during evolution and yet come from within a different region of the sequence via structural reorganisations. Here we also show that α-transaminase-type serine-pyruvate aminotransferase activity, can be engineered into the CV2025 ω-transaminase scaffold with any one of many possible single point mutations at three key positions, without the requirement for significant backbone remodeling, or repositioning of the residue from a different region of sequence. This finding has significant implications for enzyme redesign in which solutions to substrate specificity changes may be found that are significantly more efficient than by engineering in all sequence and structure determinants identified by correlation to substrate specificity. This article is protected by copyright. All rights reserved

Prognostic value of donor cytotoxic T-lymphocyte precursor frequencies for acute graft-versus-host disease in hematopoietic stem cell transplantation from HLA-matched siblings: A single center experience in a cohort of 92 patients

We investigated the prognostic value of cytotoxic T-lymphocyte precursor frequencies (CTL-p-f) for the development of graft-versus-host disease (GvHD) in a cohort of 92 recipients of a hematopoietic stem cell transplantation from HLA-matched sibling donors. CTL-p-f and clinical variables were correlated with acute GvHD and chronic GvHD in univariate and multivariate analyses. CTL-p-f resulted an independent risk factor for severe acute GvHD. Moreover, a trend towards a correlation between CTL-p-f and chronic GvHD was observed. In summary CTL-p-f may be considered as a functional assay useful for identifying patients at high risk of severe GVHD. ©2006 Ferrata Storti Foundation

X-FaCT: Xenopus-Fast Clearing Technique

Accessibility and imaging of cell compartments in big specimens are crucial for cellular biological research but also a matter of contention. Confocal imaging and tissue clearing on whole organs allow for 3D imaging of cellular structures after being subjected to in-toto immunohistochemistry. Lately, the passive CLARITY technique (PACT) has been adapted to clear and immunolabel large specimens or individual organs of several aquatic species. We recently demonstrated tissue clearing on one-week old tadpole brain (Fini et al., Sci Rep 7:43786, 2017). We here describe a further simplified version with clearing of small tissue samples (thickness inferior to 500 μm)) carried out by immersion in a fructose-based high-refractive index solution (fbHRI). By refining steps of the protocol, we were able to reduce the overall procedure time by two thirds. This offers the advantages of reducing the time of experimentation to a week and minimizes procedure-induced tissue deformations. This protocol can be easily adapted to be performed on thick section. We present an example of immunohistochemistry performed on NF45 Xenopus laevis brains with anti-pH 3 (phosphorylated histone H3) antibody used to stain chromatin condensation commonly associated with proliferation

A comparison of cytotoxic T lymphocyte precursor frequencies in responder/stimulator pairs with increasing degrees of mismatch

Limiting dilution assays of helper or cytotoxic T cell precursor (HTL-p and CTL-p) frequencies have been developed to measure, with high sensitivity and specificity, donor/recipient alloreactivity before bone marrow transplantation (BMT). Most studies demonstrated a significant correlation between the frequency of donor host-specific CTL-p before transplant and the incidence of severe (≥ grade II) acute graft-versus-host disease (aGvHD) in unrelated donor BMT. Recently we demonstrated that the CTL-p assay was sensitive enough to provide similar predictive information also in HLA-identical sibling BMT. In the present study, the CTL-p frequencies were compared in four groups of responder/stimulator pairs with different degree of mismatch: (1) monozygotic twins (MZ) (n=7), (2) HLA-identical sibling pairs (SIB) (n=67), (3) HLA-matched unrelated pairs (HLA-A, B serologically and HLA-DRBI allele-matched) (MUP) (n=59), (4) 2-4 antigen HLA-mismatched pairs (MM) (n=27). Logistic regression analysis showed that as mismatch increased, the probability of a high (&gt;1:100.000) CTL-p frequency (HF) significantly increased. Then, MUP group was divided in two subgroups: (3a) well-matched pairs (WM) (n=20): matched for HLA-DRB3, B4, B5 alleles and, by DNA heteroduplex analysis, for HLA-A, B genes; (3b) matched pairs (M) (n=39): with either a single HLA-A, B mismatch as revealed by DNA heteroduplex analysis, or a single mismatch in HLA-DRB3, B4, B5 alleles. The CTL-p frequencies were analysed again in the five groups, re-using a logistic regression model. The probability of a HF response was significantly higher in the M than in SIB group. Otherwise, WM and SIB groups had the same probability of a HF response. The probability of a HF response in the M group was significantly higher than in WM group, and lower, though not significantly, than in MM group. The results confirmed not only the power of CTL-p assay in the identification of functionally significant polymorphisms, but also in measuring the degree of mismatch in responder/stimulator pairs. They also showed that when unrelated pairs were more closely matched, CTL-p frequencies approached those of SIB. In conclusion, the CTL-p assay may be an important "in vitro" tool in BMT helping also to predict alloreactivity due to minor histocompatibility antigen differences, which still remain difficult to define despite the improvements in unrelated donor matching. © 2001 Blackwell Science Ltd,

Epidemiologic data about polymorphous light eruption in Italy

Aim. Polymorphous light eruption (PLE) is the most common idiopathic photodermatosis. It describes a broad clinical spectrum with chronic recurrences. It is often characterized by non scarring pruritic erythematous papules, vesicles or plaques. UV exposure is the main pathogenetic factor. The aim of this study was to evaluate the prevalence of PLE in Italy, the main clinical features and the clinical course and recurrences in a Mediterranean population. Methods. The study was carried out on 4 416 subjects in 8 Dermatological Units in Italy, distributed over the whole country. Subjects were required to fill a simple questionnaire (43 questions) exploring the following topics: phototype and phenotype, and modalities of solar exposure. In the subjects with a previous PLE another questionnaire was submitted to investigate the clinical features of PLE, number of recurrences, familiar, pathological and pharmacological anamnesis. The study was carried out in healthy volunteers, not affected by any dermatological disease. Results. Among the 4 416 apparently healthy subjects who filled out the survey, 212 gave a history consistent with a diagnosis of PLE. The PLE prevalence was 5.89% without significant differences among the Dermatological Units distributed at different latitudes in our Country. The coalescent papules type of PLE was the most common clinical picture (36.4%); the body site most frequently affected was the trunk (61.1%). On the contrary, chronically sun exposed body site (i.e. the face) is affected just in few cases. Also people chronically sun exposed developed PLE less frequently than occasionally sun exposed people. Sometimes, PLE developed after a particularly intense sun exposure (37.7% of PLE). Conclusion. No correlations with drug assumption or environmental chemical compound have been underlined

Cytotoxic T lymphocyte precursor frequency as a predictor of acute graft-versus-host disease in bone marrow transplantation from HLA-identical siblings

The measurement of precursor frequencies of donor anti-recipient cytotoxic T lymphocytes (CTL-p) has been shown to predict the incidence and the severity of acute graft-versus-host disease (aGVHD) in unrelated donor bone marrow transplantation (BMT). In HLA-identical sibling BMT, where aGVHD is most likely caused by minor histocompatibility antigen mismatches, this assay did not appear to be sensitive enough to provide similar predictive information. In this study, the CTL-p frequencies and the incidence and severity of aGVHD in 51 onco-hematological patients transplanted from HLA-identical siblings were compared. Sibling donors were selected on the basis of HLA identity using serological typing for HLA-A, B, C antigens, whereas HLA-DRB was tested by molecular analysis. Sibling identity was also confirmed by DNA heteroduplex analyses. Fifteen out of 21 (71%) patients with high precursor frequency (&gt;1:100 x 103) and 12 out of 30 (40%) with low precursor frequency (&lt;1:100 x 103) experienced clinically significant (II-IV) aGVHD. A significant correlation (P = 0.04) between CTL-p frequency and severe aGVHD was demonstrated. Moreover there was a positive trend for a high frequency response according to an increasing grade of aGVHD, which was statistically significant (P = 0.04). In our experience the CTL-p assay is a helpful predictive test for aGVHD in HLA-identical sibling BMT, indicating high risk patients possibly requiring additional prophylaxis