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.

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.

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.

[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.

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.

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.

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.

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.

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.

Sex-Specific Associations of Testosterone With Metabolic Traits.

Background: Testosterone levels are differentially linked with diabetes risk in men and women: lower testosterone levels in men and higher testosterone levels in women are associated with type 2 diabetes, though, the mechanisms are not fully clear. We addressed sex-specific links between testosterone and major pathogenetic mechanisms of diabetes. Methods: We analyzed data of 623 subjects (202 male, 345 female without, and 76 female with oral contraceptive therapy [OCT]) for whom insulin sensitivity and insulin secretion were assessed by oral glucose tolerance test. Body fat percentage was assessed by bioelectrical impedance. Testosterone was measured by enzyme-linked immunoassay; free testosterone and Framingham risk score were calculated. Results: There were significant interactions between testosterone and sex for all tested metabolic traits. Increasing testosterone was associated with less body fat, elevated insulin sensitivity, and reduced glycemia, independent of adiposity in men. In women without OCT, testosterone correlated with more body fat, insulin resistance, and higher glucose concentrations. Testosterone was not associated with insulin secretion in either sex, but with lower Framingham risk score in men and higher Framingham risk score in women. Conclusions: Similar to diabetes risk, insulin resistance has different association directions with testosterone levels in males and females. Insulin resistance could therefore constitute the best biological candidate linking testosterone levels and diabetes prevalence. The question of antiandrogen therapy being able to improve metabolism, glucose tolerance and cardiovascular risk in women was not clarified in our study but should be reviewed with higher numbers in a carefully matched study to reduce the influence of confounding variables.

Gene x Gene Interactions Highlight the Role of Incretin Resistance for Insulin Secretion.

Introduction: Genetic polymorphisms in TCF7L2 are the strongest common risk variants for type 2 diabetes mellitus (T2D). We and others have shown that genetic variation in TCF7L2 and WFS1 affect incretin-stimulated insulin secretion. A recent genome-wide association study discovered genetic variants associated with incretin levels. We hypothesized that these SNPs (single nucleotide polymorphisms) interact with the well-known TCF7L2 variant rs7903146 on insulin secretion due to their incretin altering effect. Methods: In this retrospective analysis, we used data from the cross-sectional TUEF-cohort (n = 2929) and a hyperglycemic clamp study using additional GLP-1 infusion at the end of the clamp (n = 76). Insulin secretion was measured by evaluating OGTT-derived indexes of insulin secretion and insulin/C-peptide levels during clamp. We genotyped rs7903146 in TCF7L2, rs10010131 in WFS1, and six SNPs associated with GLP-1 and GIP levels. Results: One of the six incretin-associated SNPs, rs17681684 in GLP2R, exhibited significant SNP x SNP interactions with rs7903146 in TCF7L2 on insulin secretion (p = 0.0024) after correction for multiple testing. Three further SNP's showed nominally significant interactions (p < 0.05). In the hyperglycemic clamp study, rs7903146 in TCF7L2 also interacted with rs17681684 on AUC C-peptide during the GLP-1 stimulation phase, thereby replicating the above finding. Conclusion: The findings exemplify the role of SNP x SNP interactions in the genetics of type 2 diabetes mellitus and corroborate the existence of clinically relevant differences in incretin sensitivity.

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.

In Patients We Trust: Reliability of Self-Reported Weight and Height in Nuclear Medicine Patients.

The aim of the study was to assess the reliability of the self-reported weight and height of nuclear medicine patients in view of recommendations for weight-dependent tracer application for imaging and therapy. Methods: In total, 824 patients (334 men and 490 women) were asked to report their weight and height before imaging or therapy and their level of confidence. Subsequently, the weight and height of each patient were measured, and body mass index, body surface area, and lean body mass were calculated. Differences between reported and true values were compared for statistically significant differences. Results: The average patient age was 60 ± 14 y (range, 17-91 y). An over- or underestimation of weight by at least 10% was observed in 2% of patients, and height was overestimated by 1% by the patients. The BMI calculation was affected by incorrect self-reported values. Conclusion: Most self-reported weights and heights of nuclear medicine patients are accurate. However, since over- and underestimation of weight and height lead to incorrect body mass index, body surface area, and lean body mass values, patient weights should be measured at least for patients receiving a weight-adapted therapy or if quantification in PET/CT is needed.

The Gly385(388)Arg Polymorphism of the FGFR4 Receptor Regulates Hepatic Lipogenesis Under Healthy Diet.

CONTEXT: The effect of a lifestyle intervention to reduce liver fat content in nonalcoholic fatty liver disease in humans is influenced by genetics. We hypothesized that the amino acid exchange in human Gly388Arg (mouse homolog: Gly385Arg) in fibroblast growth factor receptor 4 (FGFR4), which regulates bile acid, lipid, and glucose metabolism, could determine hepatic lipid accumulation and insulin sensitivity. Mechanisms of this substitution were studied in mice under normal chow and high-fat diets. DESIGN: In humans, the Gly388Arg polymorphism was studied for its relationship with changes in liver fat content and insulin sensitivity during 9 months of a lifestyle intervention. We also studied a knock-in mouse strain with an Arg385 allele introduced into the murine FGFR4 gene under normal chow and high-fat diets. RESULTS: In humans, the FGFR4 Arg388 allele was not associated with liver fat content or insulin sensitivity in subjects who were overweight and obese before lifestyle intervention. However, it was associated with less decrease in liver fat content and less increase in insulin sensitivity during the intervention. In mice receiving normal chow, the FGFR4 Arg385 allele was associated with elevated hepatic triglyceride content, altered hepatic lipid composition, and increased hepatic expression of genes inducing de novo lipogenesis and glycolysis. Body fat mass and distribution, glucose tolerance, and insulin sensitivity were unaltered. The FGFR4 Arg385 allele had no effect on glucose or lipid metabolism under the high-fat diet. CONCLUSION: Our data indicate that the FGFR4 Arg388(385) allele affects hepatic lipid and glucose metabolism specifically during healthy caloric intake.

Pore Engineering for Enhanced Mass Transport in Encapsulin Nanocompartments.

Encapsulins are robust and engineerable proteins that form hollow, nanosized, icosahedral capsids, making them attractive vehicles for drug delivery, scaffolds for synthetic bionanoreactors, and artificial organelles. A major limitation of native encapsulins is the small size of pores in the protein shell. At 3 Å diameter, these pores impose significant restrictions on the molecular weight and diffusion rate of potential substrates. By redesigning the pore-forming loop region in encapsulin from Thermotoga maritima, we successfully enlarged pore diameter up to an estimated 11 Å and increased mass transport rates by 7-fold as measured by lanthanide ion diffusion assay. Our study demonstrates the high tolerance of encapsulin for protein engineering and has created a set of novel, functionally improved scaffolds for applications as bionanoreactors.

cGMP-dependent protein kinase I (cGKI) modulates human hepatic stellate cell activation.

BACKGROUND: The activation of hepatic stellate cells (HSCs) plays a crucial role in liver fibrosis, however the role of HSCs is less understood in hepatic insulin resistance. Since in the liver cGMP-dependent protein kinase I (cGKI) was detected in HSC but not in hepatocytes, and cGKI-deficient mice that express cGKI selectively in smooth muscle but not in other cell types (cGKI-SM mice) displayed hepatic insulin resistance, we hypothesized that cGKI modulates HSC activation and insulin sensitivity. MATERIALS AND METHODS: To study stellate cell activation in cGKI-SM mice, retinol storage and gene expression were studied. Moreover, in the human stellate cell line LX2, the consequences of cGKI-silencing on gene expression were investigated. Finally, cGKI expression was examined in human liver biopsies covering a wide range of liver fat content. RESULTS: Retinyl-ester concentrations in the liver of cGKI-SM mice were lower compared to wild-type animals, which was associated with disturbed expression of genes involved in retinol metabolism and inflammation. cGKI-silenced LX2 cells showed an mRNA expression profile of stellate cell activation, altered matrix degradation and activated chemokine expression. On the other hand, activation of LX2 cells suppressed cGKI expression. In accordance with this finding, in human liver biopsies, we observed a negative correlation between cGKI mRNA and liver fat content. CONCLUSIONS: These results suggest that the lack of cGKI possibly leads to stellate cell activation, which stimulates chemokine expression and activates inflammatory processes, which could disturb hepatic insulin sensitivity.

Single Nucleotide Polymorphisms in the G-Protein Coupled Receptor Kinase 5 (GRK5) Gene are associated with Plasma LDL-Cholesterol Levels in Humans.

Genetically modified mice models suggest an important role for G-protein-coupled receptor kinase 5 (GRK5) in the pathophysiology of obesity and related disorders. We investigated whether single nucleotide polymorphisms (SNPs) in the gene encoding GRK5 affect cardiometabolic traits in humans. We genotyped 3 common SNPs in intron 1 (rs1980030, rs10466210, rs9325562) and one SNP in intron 3 (rs10886471) of GRK5 in 2332 subjects at risk for type 2 diabetes. Total- and visceral fat mass were measured by magnetic resonance (MR) tomography and liver fat content by 1H-MR spectroscopy. Insulin secretion and sensitivity were estimated during an OGTT and measured during the euglycemic, hyperinsulinemic clamp (n = 498). Carriers of the minor allele of rs10466210 and rs1980030 had higher total- and LDL-cholesterol levels (p = 0.0018 and p = 0.0031, respectively, for rs10466210; p = 0.0035 and p = 0.0081, respectively, for rs1980030), independently of gender, age, BMI and lipid-lowering drugs. The effects of rs10466210 withstood Bonferroni correction. Similar associations were observed with apolipoprotein B levels (p = 0.0034 and p = 0.0122, respectively). Carriers of the minor allele of rs10466210 additionally displayed a trend for higher intima-media thickness of the carotid artery (p = 0.075). GRK5 may represent a novel target for strategies aiming at lowering LDL-cholesterol levels and at modifying cardiovascular risk.

Androgen receptor overexpression in prostate cancer in type 2 diabetes.

OBJECTIVE: While prostate cancer does not occur more often in men with diabetes, survival is markedly reduced in this patient group. Androgen signaling is a known and major driver for prostate cancer progression. Therefore, we analyzed major components of the androgen signaling chain and cell proliferation in relation to type 2 diabetes. METHODS: Tumor content of 70 prostate tissue samples of men with type 2 diabetes and 59 samples of patients without diabetes was quantified by an experienced pathologist, and a subset of 51 samples was immunohistochemically stained for androgen receptor (AR). mRNA expression of AR, insulin receptor isoform A (IR-A) and B (IR-B), IGF-1 receptor (IGF1R), Cyp27A1 and Cyp7B1, PSA gene KLK3, PSMA gene FOLH1, Ki-67 gene MKI67, and estrogen receptor beta (ESR2) were analyzed by RT-qPCR. RESULTS: AR mRNA and protein expression were associated with the tumor content only in men with diabetes. AR expression also correlated with downstream targets PSA (KLK3) and PSMA (FOLH1) and increased cell proliferation. Only in diabetes, AR expression was correlated to higher IR-A/IR-B ratio and lower IR-B/IGF1R ratio, thus, in favor of the mitogenic isoforms. Reduced Cyp27A1 and increased Cyp7B1 expressions in tumor suggest lower levels of protective estrogen receptor ligands in diabetes. CONCLUSIONS: We report elevated androgen receptor signaling and activity presumably due to altered insulin/IGF-1 receptors and decreased levels of protective estrogen receptor ligands in prostate cancer in men with diabetes. Our results reveal new insights why these patients have a worse prognosis. These findings provide the basis for future clinical trials to investigate treatment response in patients with prostate cancer and diabetes.