An engineered T7 RNA polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis.

Messenger RNA (mRNA) therapies have recently gained tremendous traction with the approval of mRNA vaccines for the prevention of SARS-CoV-2 infection. However, manufacturing challenges have complicated large scale mRNA production, which is necessary for the clinical viability of these therapies. Not only can the incorporation of the required 5' 7-methylguanosine cap analog be inefficient and costly, in vitro transcription (IVT) using wild-type T7 RNA polymerase generates undesirable double-stranded RNA (dsRNA) byproducts that elicit adverse host immune responses and are difficult to remove at large scale. To overcome these challenges, we have engineered a novel RNA polymerase, T7-68, that co-transcriptionally incorporates both di- and tri-nucleotide cap analogs with high efficiency, even at reduced cap analog concentrations. We also demonstrate that IVT products generated with T7-68 have reduced dsRNA content.

[Telemedicine in mobile geriatric rehabilitation : Results of the VITAAL study]. / Telemedizin in der mobilen geriatrischen Rehabilitation : Ergebnisse der VITAAL-Studie.

BACKGROUND: In order to implement the principle of rehabilitation before care, adaptive concepts for geriatric patients are required. Patients with visual impairments, impaired communication skills, mental illnesses or cognitive deficits are often not or only insufficiently treatable in a rehabilitation clinic. Mobile geriatric rehabilitation (MoGeRe) closes this gap in the care system, but its scope is limited. With the 22 locations in Germany, it is not possible to make a comprehensive MoGeRe possible so far. Telemedicine offers solutions here. OBJECTIVE: Telemedicine supplements to MoGeRe in the form of video visits and video recording were examined with respect to their feasibility and acceptance in a very old target group. METHOD: A total of 101 video visits and 26 diagnostic video recordings were carried out with 25 patients. Interviews with patients and team members were evaluated with the help of a qualitative content analysis. RESULTS AND DISCUSSION: In particular, the acceptance of the video visit was high among all those involved. Its potential lies in the adaptation of the individual treatment, motivation, medical guidance and supervision of the team. The video recording can offer the opportunity to enrich the interdisciplinary exchange and to evaluate and adapt the therapeutic procedure. Specific strategies such as accompanying relatives, explaining the procedure and good timing are necessary for cognitively impaired patients. Our results prove that older people should also be taken into account as users of digital media.

Circular permutation of a bacterial tyrosinase enables efficient polyphenol-specific oxidation and quantitative preparation of orobol.

Tyrosinase is a type 3 copper oxygenase that catalyzes a phenol moiety into ortho-diphenol, and subsequently to ortho-quinone. Diverse tyrosinases have been observed across the kingdom including Animalia, Bacteria, Plantae, and Fungi. Among the tyrosinases, bacterial, and mushroom tyrosinases have been extensively exploited to prepare melanin, ortho-hydroxy-polyphenols, or novel plant secondary metabolites during the past decade. And their use as a biocatalyst to prepare various functional biocompounds have drawn great attention worldwide. Herein, we tailored a bacterial tyrosinase from Bacillus megaterium (BmTy) using circular permutation (CP) engineering technique which is a novel enzyme engineering technique to covalently link original N and C termini and create new termini on the middle of its polypeptide. To construct a smart rationally-designed CP library, we introduced 18 new termini at the edge of each nine loops that link α-helical secondary structure in BmTy. Among the small library, seven functional CP variants were successfully identified and they represented dramatic change in their enzyme characteristics including kinetic properties and substrate specificity. Especially, cp48, 102, and 245 showed dramatically decreased tyrosine hydroxylase activity, behaving like a catechol oxidase. Exploiting the dramatic increased polyphenol oxidation activity of cp48, orobol (3'-hydroxy-genistein) was quantitatively synthesized with 1.48 g/L, which was a 6-fold higher yield of truncated wild-type. We examined their kinetic characters through structural speculation, and suggest a strategy to solubilize the insoluble artificial variants effectively.

Asymmetric Ketone Reduction by Imine Reductases.

The rapidly growing area of asymmetric imine reduction by imine reductases (IREDs) has provided alternative routes to chiral amines. Here we report the expansion of the reaction scope of IREDs by showing the stereoselective reduction of 2,2,2-trifluoroacetophenone. Assisted by an in silico analysis of energy barriers, we evaluated asymmetric hydrogenations of carbonyls and imines while considering the influence of substrate reactivity on the chemoselectivity of this novel class of reductases. We report the asymmetric reduction of C=N as well as C=O bonds catalysed by members of the IRED enzyme family.

Engineering strictosidine synthase: rational design of a small, focused circular permutation library of the ß-propeller fold enzyme.

Strictosidine synthases catalyze the formation of strictosidine, a key intermediate in the biosynthesis of a large variety of monoterpenoid indole alkaloids. Efforts to utilize these biocatalysts for the preparation of strictosidine analogs have however been of limited success due to the high substrate specificity of these enzymes. We have explored the impact of a protein engineering approach called circular permutation on the activity of strictosidine synthase from the Indian medicinal plant Rauvolfia serpentina. To expedite the discovery process, our study departs from the usual process of creating a random protein library, followed by extensive screening. Instead, a small, focused library of circular permutated variants of the six bladed ß-propeller protein was prepared, specifically probing two regions which cover the enzyme active site. The observed activity changes suggest important roles of both regions in protein folding, stability and catalysis.

Improved biocatalysts from a synthetic circular permutation library of the flavin-dependent oxidoreductase old yellow enzyme.

Members of the old yellow enzyme (OYE) family are widely used, effective biocatalysts for the stereoselective trans-hydrogenation of activated alkenes. To further expand their substrate scope and improve catalytic performance, we have applied a protein engineering strategy called circular permutation (CP) to enhance the function of OYE1 from Saccharomyces pastorianus. CP can influence a biocatalyst's function by altering protein backbone flexibility and active site accessibility, both critical performance features because the catalytic cycle for OYE1 is thought to involve rate-limiting conformational changes. To explore the impact of CP throughout the OYE1 protein sequence, we implemented a highly efficient approach for cell-free cpOYE library preparation by combining whole-gene synthesis with in vitro transcription/translation. The versatility of such an ex vivo system was further demonstrated by the rapid and reliable functional evaluation of library members under variable environmental conditions with three reference substrates ketoisophorone, cinnamaldehyde, and (S)-carvone. Library analysis identified over 70 functional OYE1 variants with several biocatalysts exhibiting over an order of magnitude improved catalytic activity. Although catalytic gains of individual cpOYE library members vary by substrate, the locations of new protein termini in functional variants for all tested substates fall within the same four distinct loop/lid regions near the active site. Our findings demonstrate the importance of these structural elements in enzyme function and support the hypothesis of conformational flexibility as a limiting factor for catalysis in wild type OYE.

Toll-like receptors 2 and 4 impair insulin-mediated brain activity by interleukin-6 and osteopontin and alter sleep architecture.

Impaired insulin action in the brain represents an early step in the progression toward type 2 diabetes, and elevated levels of saturated free fatty acids are known to impair insulin action in prediabetic subjects. One potential mediator that links fatty acids to inflammation and insulin resistance is the Toll-like receptor (TLR) family. Therefore, C3H/HeJ/TLR2-KO (TLR2/4-deficient) mice were fed a high-fat diet (HFD), and insulin action in the brain as well as cortical and locomotor activity was analyzed by using telemetric implants. TLR2/4-deficient mice were protected from HFD-induced glucose intolerance and insulin resistance in the brain and displayed an improvement in cortical and locomotor activity that was not observed in C3H/HeJ mice. Sleep recordings revealed a 42% increase in rapid eye movement sleep in the deficient mice during daytime, and these mice spent 41% more time awake during the night period. Treatment of control mice with a neutralizing IL-6 antibody improved insulin action in the brain as well as cortical activity and diminished osteopontin protein to levels of the TLR2/4-deficient mice. Together, our data suggest that the lack of functional TLR2/4 protects mice from a fat-mediated impairment in insulin action, brain activity, locomotion, and sleep architecture by an IL-6/osteopontin-dependent mechanism.

Inceptor correlates with markers of prostate cancer progression and modulates insulin/IGF1 signaling and cancer cell migration.

OBJECTIVE: The insulin/insulin-like growth factor 1 (IGF1) pathway is emerging as a crucial component of prostate cancer progression. Therefore, we investigated the role of the novel insulin/IGF1 signaling modulator inceptor in prostate cancer. METHODS: We analyzed the expression of inceptor in human samples of benign prostate epithelium and prostate cancer. Further, we performed signaling and functional assays using prostate cancer cell lines. RESULTS: We found that inceptor was expressed in human benign and malignant prostate tissue and its expression positively correlated with various genes of interest, including genes involved in androgen signaling. In vitro, total levels of inceptor were increased upon androgen deprivation and correlated with high levels of androgen receptor in the nucleus. Inceptor overexpression was associated with increased cell migration, altered IGF1R trafficking and higher IGF1R activation. CONCLUSIONS: Our in vitro results showed that inceptor expression was associated with androgen status, increased migration, and IGF1R signaling. In human samples, inceptor expression was significantly correlated with markers of prostate cancer progression. Taken together, these data provide a basis for investigation of inceptor in the context of prostate cancer.

Novel protease inhibitors via computational redesign of subtilisin BPN' propeptide.

The propeptide domain of subtilisin BPN' functions as a molecular chaperone for its cognate protease yet quickly assumes a predominantly unfolded structure following cleavage by the mature protease. In contrast, structural stabilization of the propeptide domain has been proposed to competitively inhibit protease self-cleavage, suggesting the possibility for the generation of novel proteinaceous subtilisin inhibitors. Using a Rosetta fixed backbone design, we have redesigned the subtilisin BPN' propeptide structure to generate synthetic peptide sequences with increased and tunable structural stability. Molecular dynamics simulations provide supporting evidence that the artificial sequences retain structure without its protease cognate unlike the inherently disordered wild-type propeptide. Experimental evaluation of two designer domains by spectroscopic methods verified their structural integrity. Furthermore, the novel propeptide domains were shown to possess significantly enhanced thermostability. Nevertheless, their modest functional performance as protease inhibitors raises doubt that propeptide stability alone is sufficient for effective inhibitor design.

Diabetes and the Prostate: Elevated Fasting Glucose, Insulin Resistance and Higher Levels of Adrenal Steroids in Prostate Cancer.

Although epidemiological studies suggest a lower prostate cancer incidence rate in patients with type 2 diabetes, cancer survival is markedly reduced. Underlying mechanisms that connect the two diseases are still unclear. Potential links between type 2 diabetes and prostate cancer are hallmarks of the metabolic syndrome, such as hyperglycemia and dyslipidemia. Therefore, we explored the systemic metabolism of 103 prostate cancer patients with newly diagnosed and yet untreated prostate cancer compared to 107 healthy controls, who were carefully matched for age and BMI. Here, we report that patients with prostate cancer display higher fasting blood glucose levels and insulin resistance, without changes in insulin secretion. With respect to lipid metabolism, serum triglyceride levels were lower in patients with prostate cancer. In addition, we report increased adrenal steroid biosynthesis in these patients. Our results indicate that higher fasting glucose levels in patients with prostate cancer may be explained at least in part by insulin resistance, due to the enhanced synthesis of adrenal steroids.

Directed evolution of an orthogonal nucleoside analog kinase via fluorescence-activated cell sorting.

Nucleoside analogs (NAs) represent an important category of prodrugs for the treatment of viral infections and cancer, yet the biological potency of many analogs is compromised by their inefficient activation through cellular 2'-deoxyribonucleoside kinases (dNKs). We herein report the directed evolution and characterization of an orthogonal NA kinase for 3'-deoxythymidine (ddT), using a new FACS-based screening protocol in combination with a fluorescent analog of ddT. Four rounds of random mutagenesis and DNA shuffling of Drosophila melanogaster 2'-deoxynucleoside kinase, followed by FACS analysis, yielded an orthogonal ddT kinase with a 6-fold higher activity for the NA and a 20-fold k(cat)/K(M) preference for ddT over thymidine, an overall 10,000-fold change in substrate specificity. The contributions of individual amino acid substitutions in the ddT kinase were evaluated by reverse engineering, enabling a detailed structure-function analysis to rationalize the observed changes in performance. Based on our results, kinase engineering with fluorescent NAs and FACS should prove a highly versatile method for evolving selective kinase:NA pairs and for studying fundamental aspects of the structure-function relationship in dNKs.

Encapsulin Nanocontainers as Versatile Scaffolds for the Development of Artificial Metabolons.

The construction of non-native biosynthetic pathways represents a powerful, modular strategy for the production of valuable synthons and fine chemicals. Accordingly, artificially affixing enzymes that catalyze sequential reactions onto DNAs, proteins, or synthetic scaffolds has proven to be an effective route for generating de novo metabolons with novel functionalities and superior efficiency. In recent years, nanoscale microbial compartments known as encapsulins have emerged as a class of robust and highly engineerable proteinaceous containers with myriad applications in biotechnology and synthetic biology. Herein we report the concurrent surface functionalization and internal packaging of encapsulins from Thermotoga maritima to generate a catalytically competent two-enzyme metabolon. Encapsulins were engineered to covalently sequester up to 60 copies of a dihydrofolate reductase (DHFR) enzyme variant on their exterior surfaces using the SpyCatcher bioconjugation system, while their lumens were packaged with a tetrahydrofolate-dependent demethylase enzyme using short peptide affinity tags abstracted from the encapsulin's native protein cargo. Successful cross-talk between the two colocalized enzymes was confirmed as tetrahydrofolate produced by externally tethered DHFR was capable of driving the demethylation of a lignin-derived aryl substrate by packaged demethylases, albeit slowly. The subsequent introduction of a previously reported pore-enlarging deletion in the encapsulin shell was shown to enhance metabolite exchange such that the encapsulin-based metabolon functioned at speeds equivalent to those of the two enzymes freely dispersed in solution. Our work thus further emphasizes the engineerability of encapsulins and their potential use as flexile scaffolds for biocatalytic applications.

Circular permutation of Bacillus circulans xylanase: a kinetic and structural study.

The 20 kDa Bacillus circulans Bcx is a well-studied endoxylanase with a beta-jellyroll fold that places its N- and C-termini in salt bridge contact. Initial experiments verified that Bcx could be circularly permuted by PCR methods to introduce new termini in loop regions while linking its native termini directly or via one or two glycines. Subsequently, a library of circular permutants, generated by random DNase cleavage of the circularized Bcx gene, was screened for xylanase activity on xylan in Congo Red-stained agar. Analysis of 35 unique active circular permutants revealed that, while many of the new termini were introduced in external loops as anticipated, a surprising number were also located within beta-strands. Furthermore, several permutations placed key catalytic residues at or near the new termini with minimal deleterious effects on activity and, in one case, a 4-fold increase. The structure of one permutant was determined by X-ray crystallography, whereas three others were probed by NMR spectroscopy. These studies revealed that the overall conformation of Bcx changed very little in response to circular permutation, with effects largely being limited to increased local mobility near the new and the linked old termini and to a decrease in global stability against thermal denaturation. This library of circularly permuted xylanases provides an excellent set of new start points for directed evolution of this commercially important enzyme, as well as valuable constructs for intein-mediated replacement of key catalytic residues with unnatural analogues. Such approaches should permit new insights into the mechanism of enzymatic glycoside hydrolysis.

Lesion dose in differentiated thyroid carcinoma metastases after rhTSH or thyroid hormone withdrawal: 124I PET/CT dosimetric comparisons.

PURPOSE: Renal radioiodine excretion is ~50% faster during euthyroidism versus hypothyroidism. We therefore sought to assess lesion dose/GBq of administered 131I activity (LDpA) in iodine-avid metastases (IAM) of differentiated thyroid carcinoma (DTC) in athyreotic patients after recombinant human thyroid-stimulating hormone (rhTSH) versus after thyroid hormone withdrawal (THW). METHODS: We retrospectively compared mean LDpA between groups of consecutive patients (N=63) receiving 124I positron emission tomography/computed tomography (124I PET/CT) aided by rhTSH (n=27) or THW (n=36); we prospectively compared LDpA after these stimulation methods within another individual. Data derived from serial PET scans and one CT scan performed 2-96 h post-124I ingestion. A mixed model analysis of covariance (ANCOVA) calculated the treatment groups' mean LDpAs adjusting for statistically significant baseline intergroup differences: non-IAM were more prevalent, median IAM count/patient lower in cervical lymph nodes and higher in distant sites, median stimulated thyroglobulin higher, mean cumulative radioiodine activity greater and prior diagnostic scintigraphy more frequent in the rhTSH patients. RESULTS: Mean LDpAs were: rhTSH group (n=71 IAM), 30.6 Gy/GBq; THW group (n=66 IAM), 51.8 Gy/GBq. The difference in group means (rhTSH less THW), -21.2 Gy/GBq, was statistically non-significant (p=0.1667). However, the 95% confidence interval of that difference (-51.4 to +9 Gy/GBq) suggested a trend favouring THW. The within-patient comparison found 2.9- to 10-fold higher LDpAs under THW. CONCLUSION: We found some suggestions, but no statistically significant evidence, that rhTSH administration results in a lower radiation dose to DTC metastases than does THW. A large, well-controlled, prospective within-patient study should resolve this issue.

Improved triglyceride transesterification by circular permuted Candida antarctica lipase B.

Lipases represent a versatile class of biocatalysts with numerous potential applications in industry including the production of biodiesel via enzyme-catalyzed transesterification. In this article, we have investigated the performance of cp283, a variant of Candida antarctica lipase B (CALB) engineered by circular permutation, with a series of esters, as well as pure and complex triglycerides. In comparison with wild-type CALB, the permutated enzyme showed consistently higher catalytic activity (2.6- to 9-fold) for trans and interesterification of the different substrates with 1-butanol and ethyl acetate as acyl acceptors. Differences in the observed rates for wild-type CALB and cp283 are believe to be related to changes in the rate-determining step of the catalytic cycle as a result of circular permutation.

Synthesis of fluorescent nucleoside analogs as probes for 2'-deoxyribonucleoside kinases.

We are reporting on the synthesis of fluorescent nucleoside analogs with modified sugar moieties (e.g., sugars other than ribose and 2'-deoxyribose). Four novel derivatives of the fluorescent thymidine analog 6-methyl-3-(beta-D-2'-deoxyribofuranosyl) furano-[2,3-d]pyrimidin-2-one were synthesized via Sonogashira reaction and subsequent copper-catalyzed cycloaddition. These compounds represent promising tools for studying nucleoside metabolism inside living cells, as well as for screening directed evolution libraries of 2'-deoxyribonucleoside kinases with new and improved activity for the corresponding nucleoside analogs.

Transcript Levels of Aldo-Keto Reductase Family 1 Subfamily C (AKR1C) Are Increased in Prostate Tissue of Patients with Type 2 Diabetes.

Aldo-keto reductase family 1 (AKR1) enzymes play a crucial role in diabetic complications. Since type 2 diabetes (T2D) is associated with cancer progression, we investigated the impact of diabetes on AKR1 gene expression in the context of prostate cancer (PCa) development. In this study, we analyzed benign (BEN) prostate and PCa tissue of patients with and without T2D. Furthermore, to replicate hyperglycemia in vitro, we treated the prostate adenocarcinoma cell line PC3 with increasing glucose concentrations. Gene expression was quantified using real-time qPCR. In the prostate tissue of patients with T2D, AKR1C1 and AKR1C2 transcripts were higher compared to samples of patients without diabetes. In PC3 cells, high glucose treatment induced the gene expression levels of AKR1C1, C2, and C3. Furthermore, both in human tissue and in PC3 cells, the transcript levels of AKR1C1, C2, and C3 showed positive associations with oncogenes, which are involved in proliferation processes and HIF1α and NFκB pathways. These results indicate that in the prostate glands of patients with T2D, hyperglycemia could play a pivotal role by inducing the expression of AKR1C1, C2, and C3. The higher transcript level of AKR1C was furthermore associated with upregulated HIF1α and NFκB pathways, which are major drivers of PCa carcinogenesis.

Increased Expressions of Matrix Metalloproteinases (MMPs) in Prostate Cancer Tissues of Men with Type 2 Diabetes.

Type 2 diabetes (T2D) is associated with worse prognosis of prostate cancer (PCa). The molecular mechanisms behind this association are still not fully understood. The aim of this study was to identify key factors, which contribute to the more aggressive PCa phenotype in patients with concurrent T2D. Therefore, we investigated benign and PCa tissue of PCa patients with and without diabetes using real time qPCR. Compared to patients without diabetes, patients with T2D showed a decreased E-cadherin/N-cadherin (CDH1/CDH2) ratio in prostate tissue, indicating a switch of epithelial-mesenchymal transition (EMT), which is a pivotal process in carcinogenesis. In addition, the gene expression levels of matrix metalloproteinases (MMPs) and CC chemokine ligands (CCLs) were higher in prostate samples of T2D patients. Next, prostate adenocarcinoma PC3 cells were treated with increasing glucose concentrations to replicate hyperglycemia in vitro. In these cells, high glucose induced expressions of MMPs and CCLs, which showed significant positive associations with the proliferation marker proliferating cell nuclear antigen (PCNA). These results indicate that in prostate tissue of men with T2D, hyperglycemia may induce EMT, increase MMP and CCL gene expressions, which in turn activate invasion and inflammatory processes accelerating the progression of PCa.

Human Prostate Cancer is Characterized by an Increase in Urea Cycle Metabolites.

Despite it being the most common incident of cancer among men, the pathophysiological mechanisms contributing to prostate cancer (PCa) are still poorly understood. Altered mitochondrial metabolism is postulated to play a role in the development of PCa. To determine the key metabolites (which included mitochondrial oncometabolites), benign prostatic and cancer tissues of patients with PCa were analyzed using capillary electrophoresis and liquid chromatography coupled with mass spectrometry. Gene expression was studied using real-time PCR. In PCa tissues, we found reduced levels of early tricarboxylic acid cycle metabolites, whereas the contents of urea cycle metabolites including aspartate, argininosuccinate, arginine, proline, and the oncometabolite fumarate were higher than that in benign controls. Fumarate content correlated positively with the gene expression of oncogenic HIF1α and NFκB pathways, which were significantly higher in the PCa samples than in the benign controls. Furthermore, data from the TCGA database demonstrated that prostate cancer patients with activated NFκB pathway had a lower survival rate. In summary, our data showed that fumarate content was positively associated with carcinogenic genes.

Characterization of Hormone-Dependent Pathways in Six Human Prostate-Cancer Cell Lines: A Gene-Expression Study.

Prostate cancer (PCa), the most incident cancer in men, is tightly regulated by endocrine signals. A number of different PCa cell lines are commonly used for in vitro experiments, but these are of diverse origin, and have very different cell-proliferation rates and hormone-response capacities. By analyzing the gene-expression pattern of main hormone pathways, we systematically compared six PCa cell lines and parental primary cells. We compared these cell lines (i) with each other and (ii) with PCa tissue samples from 11 patients. We found major differences in the gene-expression levels of androgen, insulin, estrogen, and oxysterol signaling between PCa tissue and cell lines, and between different cell lines. Our systematic characterization gives researchers a solid basis to choose the appropriate PCa cell model for the hormone pathway of interest.