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A computer-guided design tool to increase the efficiency of cellular conversions.

ABSTRACT: Human cell conversion technology has become an important tool for devising new cell transplantation therapies, generating disease models and testing gene therapies. However, while transcription factor over-expression-based methods have shown great promise in generating cell types in vitro, they often endure low conversion efficiency. In this context, great effort has been devoted to increasing the efficiency of current protocols and the development of computational approaches can be of great help in this endeavor. Here we introduce a computer-guided design tool that combines a computational framework for prioritizing more efficient combinations of instructive factors (IFs) of cellular conversions, called IRENE, with a transposon-based genomic integration system for efficient delivery. Particularly, IRENE relies on a stochastic gene regulatory network model that systematically prioritizes more efficient IFs by maximizing the agreement of the transcriptional and epigenetic landscapes between the converted and target cells. Our predictions substantially increased the efficiency of two established iPSC-differentiation protocols (natural killer cells and melanocytes) and established the first protocol for iPSC-derived mammary epithelial cells with high efficiency.

SUBMITTER: Jung S

PROVIDER: S-EPMC7954801 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The level of adoption of herd management information system veterinary automated management and production control program (VAMPP) Bovine and its impact on productivity of 912 specialized dairy systems with at least 5 years of information recorded was studied. Herds were classified as low (n = 389), medium (n = 343), or high (n = 180) adoption level on the basis of extent and consistency of record keeping for variables related to production, reproduction, and health. For each herd, within-year averages were obtained for six performance traits: age at first calving (AFC), days open (DO), daily milk yield (DMY), productive life (PL), incidence of clinical mastitis (MAST), and incidence of lameness (LAM). These variables were investigated with a generalized linear mixed model that included the fixed effects of the adoption level, follow-up year, and their interaction, adjusted by the fixed effects of herd size, agroecological zone, calendar period, breed group, and the random effects of variation between/within herds. A significant effect of the adoption level over all the variables was observed, except DMY and PL. The follow-up year was significant for all the variables except LAM. There were marked reductions for AFC and DO in the first 4 years of follow-up. AFC was higher and DO shorter in the low compared to medium and high level of adoption herds (p < 0.001). DMY showed a significant increasing trend (p < 0.001), regardless of the adoption level. There was higher MAST and LAM incidence in the higher adoption level (p < 0.05). The economic benefit was estimated through a stochastic simulation model using an approach based in partial budget analysis. For a herd with a medium level of adoption, the change in gross margin (GMMIS $USD) and marginal return rate (MRRMIS %) for the first 5 years of use of the system was estimated. Under these conditions, there was a GMMIS of $6890 and MRRMIS of 163%. Variation of ±10% in DMY and DO caused changes in the GMMIS of ±$1000 and ±$110, respectively, and in MRRMIS ±24% and ±4%, respectively. The trends suggest a positive influence of VAMPP in productive and reproductive traits during the first years of implementation, with less benefit for the low adoption levels.

Project description:Cytochrome P450 enzymes are promising biocatalysts for industrial use because they catalyze site-selective C-H oxidation and have diverse catalytic reactions and a broad substrate range. In this study, the 2α-hydroxylation activity of CYP154C2 from Streptomyces avermitilis MA-4680T toward androstenedione (ASD) was identified by an in vitro conversion assay. The testosterone (TES)-bound structure of CYP154C2 was solved at 1.42 Å, and this structure was used to design eight mutants, including single, double, and triple mutants, to improve the conversion efficiency. Mutants L88F/M191F and M191F/V285L were found to enhance the conversion rates significantly (i.e., 8.9-fold and 7.4-fold for TES, 46.5-fold and 19.5-fold for ASD, respectively) compared with the wild-type (WT) enzyme while retaining high 2α-position selectivity. The substrate binding affinity of the L88F/M191F mutant toward TES and ASD was enhanced compared with that of WT CYP154C2, supporting the measured increase in the conversion efficiencies. Moreover, the total turnover number and kcat/Km of the L88F/M191F and M191F/V285L mutants increased significantly. Interestingly, all mutants containing L88F generated 16α-hydroxylation products, suggesting that L88 in CYP154C2 plays a vital role in substrate selectivity and that the amino acid corresponding to L88 in the 154C subfamily affects the orientation of steroid binding and substrate selectivity. IMPORTANCE Hydroxylated derivatives of steroids play essential roles in medicine. Cytochrome P450 enzymes selectively hydroxylate methyne groups on steroids, which can dramatically change their polarity, biological activity and toxicity. There is a paucity of reports on the 2α-hydroxylation of steroids, and documented 2α-hydroxylate P450s show extremely low conversion efficiency and/or low regio- and stereoselectivity. This study conducted crystal structure analysis and structure-guided rational engineering of CYP154C2 and efficiently enhanced the conversion efficiency of TES and ASD with high regio- and stereoselectivity. Our results provide an effective strategy and theoretical basis for the 2α-hydroxylation of steroids, and the structure-guided rational design of P450s should facilitate P450 applications in the biosynthesis of steroid drugs.

Project description:BackgroundTuberculosis screening in psoriasis patients is complex due to the immunological alterations associated with psoriasis, the presence of comorbidities, and the effect of immunosuppressive treatment. However, it is not established whether the results of screening tests are affected by these factors in psoriasis patients.ObjectivesTo determine whether there is a change in the results of the tuberculin skin test (TST) or the interferon-gamma release assay (IGRA) in psoriasis patients living in tuberculosis (TB)-endemic area after 12 weeks of methotrexate (MTX) treatment and to investigate the association of the test results with clinical and inflammatory markers.MethodsForty-five patients were selected for a prospective single-arm self-controlled study and followed for at least 18 months. The TST, IGRA, Psoriasis Area and Severity Index (PASI), and inflammatory factors (erythrocyte sedimentation rate (ESR), C-reactive protein, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha levels), were determined before and after 12 weeks of oral 15 mg per week MTX administration and compared. The associations between the IGRA and TST results were verified before and after treatment according to inflammatory factors and clinical characteristics (age, blood glucose, weight, body mass index, disease duration, and PASI).ResultsWe collected data on 25 patients who completed the full course of therapy and the follow-up. None of the patients developed TB. TST positivity was significantly elevated at week 12 (25% baseline vs 44% at week 12, P < 0.037). Three IGRAs followed the TST conversions. There was no difference between TST and IGRA pre- or posttreatment. Serum IFN-γ increased significantly in week 12 (15.95 pg/ml baseline vs 18.82 pg/ml at week 12, P < 0.005) and tended to be higher among TST-positive patients (P = 0.072). The baseline IGRA was associated with a higher ESR (P = 0.038). None of the test results were associated with clinical characteristics.ConclusionsIn addition to the classic booster effect, TST conversions in patients using MTX can occur due to an increase in IFN-γ. However, it is not possible to exclude true TST conversions. Therefore, other diagnostic methods, like IGRA or chest tomography, should be used when the TST has intermediate results.

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A computer-guided design tool to increase the efficiency of cellular conversions.

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