Static spacers play a crucial role in the treatment of complex periprosthetic joint infections of the knee.

PURPOSE: To determine the superior spacer design, a growing number of studies are comparing treatment results between patients having been treated with articulating and static knee spacers in the setting of two-stage revision for periprosthetic joint infection (PJI). In contrast, the primary objective of this study was to compare preoperative characteristics between patients from both spacer groups and examine whether significant differences were present prior to spacer implantation. METHODS: This retrospective, single-centre, cohort study examined the preoperative situation of 80 consecutive knee PJIs between 2017 and 2020. All patients underwent two-stage revision, with 35 (44%) receiving an articulating and 45 (56%) a static spacer. RESULTS: No significant differences were observed in terms of patient gender (p = 0.083), age (p = 0.666), comorbidity (p = 0.1) and preoperative clinical function (p = 0.246). Static spacers were significantly more often used in the presence of a periarticular fistula (p = 0.033), infection of a revision implant (p < 0.001), higher degree of bone loss (p < 0.001) and infection caused by a difficult-to-treat pathogen (p = 0.038). Complication and revision rates were similar for both spacer types during the interim period, while patients with articulating spacers demonstrated a superior clinical function (p < 0.001) during the interim period and after reimplantation. CONCLUSION: Static spacers are being utilised in significantly more complex and unfavourable preoperative scenarios. Therefore, a preoperative selection bias may be at least partially accountable for any disparities observed in postoperative outcomes. To achieve the best possible results, surgeons should know and respect the distinct indications of static and articulating spacers and consequently understand and use them as complementary surgical options. LEVEL OF EVIDENCE: Level III.

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LibGENiE - A bioinformatic pipeline for the design of information-enriched enzyme libraries.

Enzymes are potent catalysts with high specificity and selectivity. To leverage nature's synthetic potential for industrial applications, various protein engineering techniques have emerged which allow to tailor the catalytic, biophysical, and molecular recognition properties of enzymes. However, the many possible ways a protein can be altered forces researchers to carefully balance between the exhaustiveness of an enzyme screening campaign and the required resources. Consequently, the optimal engineering strategy is often defined on a case-by-case basis. Strikingly, while predicting mutations that lead to an improved target function is challenging, here we show that the prediction and exclusion of deleterious mutations is a much more straightforward task as analyzed for an engineered carbonic acid anhydrase, a transaminase, a squalene-hopene cyclase and a Kemp eliminase. Combining such a pre-selection of allowed residues with advanced gene synthesis methods opens a path toward an efficient and generalizable library construction approach for protein engineering. To give researchers easy access to this methodology, we provide the website LibGENiE containing the bioinformatic tools for the library design workflow.

Impact and Modification of the New PJI-TNM Classification for Periprosthetic Joint Infections.

The comprehensive "PJI-TNM classification" for the description of periprosthetic joint infections (PJI) was introduced in 2020. Its structure is based on the well-known oncological TNM classification to appreciate the complexity, severity, and diversity of PJIs. The main goal of this study is to implement the new PJI-TNM classification into the clinical setting to determine its therapeutic and prognostic value and suggest modifications to further improve the classification for clinical routine use. A retrospective cohort study was conducted at our institution between 2017 and 2020. A total of 80 consecutive patients treated with a two-stage revision for periprosthetic knee joint infection were included. We retrospectively assessed correlations between patients' preoperative PJI-TNM classification and their therapy and outcome and identified several statistically significant correlations for both classifications, the original and our modified version. We have demonstrated that both classifications provide reliable predictions already at the time of diagnosis regarding the invasiveness of surgery (duration of surgery, blood and bone loss during surgery), likelihood of reimplantation, and patient mortality during the first 12 months after diagnosis. Orthopedic surgeons can use the classification system preoperatively as an objective and comprehensive tool for therapeutic decisions and patient information (informed consent). In the future, comparisons between different treatment options for truly similar preoperative baseline situations can be obtained for the first time. Clinicians and researchers should be familiar with the new PJI-TNM classification and start implementing it into their routine practice. Our adjusted and simplified version ("PJI-pTNM") might be a more convenient alternative for the clinical setting.

Algorithm-aided engineering of aliphatic halogenase WelO5* for the asymmetric late-stage functionalization of soraphens.

Late-stage functionalization of natural products offers an elegant route to create novel entities in a relevant biological target space. In this context, enzymes capable of halogenating sp3 carbons with high stereo- and regiocontrol under benign conditions have attracted particular attention. Enabled by a combination of smart library design and machine learning, we engineer the iron/α-ketoglutarate dependent halogenase WelO5* for the late-stage functionalization of the complex and chemically difficult to derivatize macrolides soraphen A and C, potent anti-fungal agents. While the wild type enzyme WelO5* does not accept the macrolide substrates, our engineering strategy leads to active halogenase variants and improves upon their apparent kcat and total turnover number by more than 90-fold and 300-fold, respectively. Notably, our machine-learning guided engineering approach is capable of predicting more active variants and allows us to switch the regio-selectivity of the halogenases facilitating the targeted analysis of the derivatized macrolides' structure-function activity in biological assays.

Whole-Cell Photoenzymatic Cascades to Synthesize Long-Chain Aliphatic Amines and Esters from Renewable Fatty Acids.

Long-chain aliphatic amines such as (S,Z)-heptadec-9-en-7-amine and 9-aminoheptadecane were synthesized from ricinoleic acid and oleic acid, respectively, by whole-cell cascade reactions using the combination of an alcohol dehydrogenase (ADH) from Micrococcus luteus, an engineered amine transaminase from Vibrio fluvialis (Vf-ATA), and a photoactivated decarboxylase from Chlorella variabilis NC64A (Cv-FAP) in a one-pot process. In addition, long chain aliphatic esters such as 10-(heptanoyloxy)dec-8-ene and octylnonanoate were prepared from ricinoleic acid and oleic acid, respectively, by using the combination of the ADH, a Baeyer-Villiger monooxygenase variant from Pseudomonas putida KT2440, and the Cv-FAP. The target compounds were produced at rates of up to 37 U g-1 dry cells with conversions up to 90 %. Therefore, this study contributes to the preparation of industrially relevant long-chain aliphatic chiral amines and esters from renewable fatty acid resources.

Creation of (R)-Amine Transaminase Activity within an α-Amino Acid Transaminase Scaffold.

The enzymatic transamination of ketones into (R)-amines represents an important route for accessing a range of pharmaceuticals or building blocks. Although many publications have dealt with enzyme discovery, protein engineering, and the application of (R)-selective amine transaminases [(R)-ATA] in biocatalysis, little is known about the actual in vivo role and how these enzymes have evolved from the ubiquitous α-amino acid transaminases (α-AATs). Here, we show the successful introduction of an (R)-transaminase activity in an α-amino acid aminotransferase with one to six amino acid substitutions in the enzyme's active site. Bioinformatic analysis combined with computational redesign of the d-amino acid aminotransferase (DATA) led to the identification of a sextuple variant having a specific activity of 326 milliunits mg-1 in the conversion of (R)-phenylethylamine and pyruvate to acetophenone and d-alanine. This value is similar to those of natural (R)-ATAs, which typically are in the range of 250 milliunits mg-1. These results demonstrate that (R)-ATAs can evolve from α-AAT as shown here for the DATA scaffold.

Exploring the Biocatalytic Potential of Fe/α-Ketoglutarate-Dependent Halogenases.

Freestanding Fe/α-ketoglutarate-dependent halogenases are oxidoreductases that catalyze the installation of halogen atoms into unactivated sp3 -hybridized carbon centers with high stereo- and regioselectivity. Since their discovery in 2014, a small number of indole alkaloid and amino acid halogenases have been identified and characterized. First enzyme engineering examples suggest that the accessible substrate range of these enzymes may be expanded through the use of rational enzyme design and directed evolution. Structural investigations of non-heme iron halogenases acting on freestanding as well as tethered substrates are beginning to inform about the principles of the underlying halogenation mechanism.

Evolved Aliphatic Halogenases Enable Regiocomplementary C-H Functionalization of a Pharmaceutically Relevant Compound.

Non-heme iron halogenases are synthetically valuable biocatalysts that are capable of halogenating unactivated sp3 -hybridized carbon centers with high stereo- and regioselectivity. The reported substrate scope of these enzymes, however, is limited primarily to the natural substrates and their analogues. We engineered the halogenase WelO5* for chlorination of a martinelline-derived fragment. Using structure-guided evolution, a halogenase variant with a more than 290-fold higher total turnover number and a 400-fold higher apparent kcat compared to the wildtype enzyme was generated. Moreover, we identified key positions in the active site that allow direction of the halogen to different positions in the target substrate. This is the first example of enzyme engineering to expand the substrate scope of a non-heme iron halogenase beyond the native indole-alkaloid-type substrates. The highly evolvable nature of WelO5* underscores the usefulness of this enzyme family for late-stage halogenation.

Biocatalytic Production of Amino Carbohydrates through Oxidoreductase and Transaminase Cascades.

Plant-derived carbohydrates are an abundant renewable resource. Transformation of carbohydrates into new products, including amine-functionalized building blocks for biomaterials applications, can lower reliance on fossil resources. Herein, biocatalytic production routes to amino carbohydrates, including oligosaccharides, are demonstrated. In each case, two-step biocatalysis was performed to functionalize d-galactose-containing carbohydrates by employing the galactose oxidase from Fusarium graminearum or a pyranose dehydrogenase from Agaricus bisporus followed by the ω-transaminase from Chromobacterium violaceum (Cvi-ω-TA). Formation of 6-amino-6-deoxy-d-galactose, 2-amino-2-deoxy-d-galactose, and 2-amino-2-deoxy-6-aldo-d-galactose was confirmed by mass spectrometry. The activity of Cvi-ω-TA was highest towards 6-aldo-d-galactose, for which the highest yield of 6-amino-6-deoxy-d-galactose (67 %) was achieved in reactions permitting simultaneous oxidation of d-galactose and transamination of the resulting 6-aldo-d-galactose.

[A Slightly Different Gastroenteritis]. / Die etwas andere Gastroenteritis.

A Slightly Different Gastroenteritis Abstract. We report the case of a 81-year-old female presenting with gastroenteritic symptoms and laboratory findings of a hemolytic anemia, thrombocytopenia and acute renal failure with fatal outcome. A hemolytic-uremic syndrome caused by an infection with EHEC was diagnosed. As a further complication, a urinary tract infection with K. pneumoniae was diagnosed and treated.

ß-Phenylalanine Ester Synthesis from Stable ß-Keto Ester Substrate Using Engineered ω-Transaminases.

The successful synthesis of chiral amines from ketones using ω-transaminases has been shown in many cases in the last two decades. In contrast, the amination of ß-keto acids is a special and relatively new challenge, as they decompose easily in aqueous solution. To avoid this, transamination of the more stable ß-keto esters would be an interesting alternative. For this reason, ω-transaminases were tested in this study, which enabled the transamination of the ß-keto ester substrate ethyl benzoylacetate. Therefore, a ω-transaminase library was screened using a coloring o-xylylenediamine assay. The ω-transaminase mutants 3FCR_4M and ATA117 11Rd show great potential for further engineering experiments aiming at the synthesis of chiral (S)- and (R)-ß-phenylalanine esters. This alternative approach resulted in the conversion of 32% and 13% for the (S)- and (R)-enantiomer, respectively. Furthermore, the (S)-ß-phenylalanine ethyl ester was isolated by performing a semi-preparative synthesis.

Fully automatized high-throughput enzyme library screening using a robotic platform.

A fully automatized robotic platform has been established to facilitate high-throughput screening for protein engineering purposes. This platform enables proper monitoring and control of growth conditions in the microtiter plate format to ensure precise enzyme production for the interrogation of enzyme mutant libraries, protein stability tests and multiple assay screenings. The performance of this system has been exemplified for four enzyme classes important for biocatalysis such as Baeyer-Villiger monooxygenase, transaminase, dehalogenase and acylase in the high-throughput screening of various mutant libraries. This allowed the identification of novel enzyme variants in a sophisticated and highly reliable manner. Furthermore, the detailed optimization protocols should enable other researchers to adapt and improve their methods. Biotechnol. Bioeng. 2016;113: 1421-1432. © 2016 Wiley Periodicals, Inc.