A yeast surface display platform for screening of non-enzymatic protein secretion in Kluyveromyces lactis.

Enhancing the secretion of recombinant proteins, particularly non-enzymatic proteins that predominate in food and pharmaceutic protein products, remains a significant challenge due to limitations in high-throughput screening methods. This study addresses this bottleneck by establishing a yeast surface display system in the food-grade microorganism Kluyveromyces lactis, enabling efficient display of model target proteins on the yeast cell surface. To assess its potential as a universal high-throughput screening tool for enhanced non-enzymatic protein secretion, we evaluated the consistency between protein display levels and secretion efficiency under the influence of various genetic factors. Our results revealed a strong correlation between these two properties. Furthermore, screening in a random mutagenesis library successfully identified a mutant with improved secretion. These findings demonstrate the potential of the K. lactis surface display system as a powerful and universal tool for high-throughput screening of strains with superior non-enzymatic protein secretion capacity. We believe this study could pave the way for efficient large-scale production of heterologous food and therapeutic proteins in industries. KEY POINTS: • A YSD (yeast surface display) system was established in Kluyveromyces lactis • This system enables high-throughput screening of non-enzymatic protein secretion • This technology assists industrial production of food and therapeutic proteins.

Intravenous infusion of small umbilical cord mesenchymal stem cells could enhance safety and delay retinal degeneration in RCS rats.

BACKGROUND: Human umbilical cord mesenchymal stem cells (UCMSCs) transplantation is a promising therapy for the treatment of retinitis pigmentosa (RP). However, intravenously infused cells may be blocked in the lung, increasing the risk of vascular obstruction, which needs to be optimized to further improve safety and efficacy. METHODS: We derived small UCMSCs (S-UCMSCs) from filtering UCMSCs with a 10-µm filter, and compared with UCMSCs by flow cytometry, directional differentiation culture and transcriptome sequencing. Then the S-UCMSCs and UCMSCs were intravenously infused in the Royal College Surgeons (RCS) rats to evaluate the safety and the efficacy. RESULTS: The diameter of S-UCMSCs ranged from 5.568 to 17.231 µm, with an average diameter of 8.636 ± 2.256 µm, which was significantly smaller than that of UCMSCs. Flow cytometry, immunofluorescence and transcriptome sequencing demonstrated that the S-UCMSCs and UCMSCs were the same kind of MSCs, and the S-UCMSCs were more proliferative. After the S-UCMSCs and UCMSCs were intravenously infused into the Royal College of Surgeons (RCS) rats at a dose of 1 × 106 cells/rat, the S-UCMSCs blocked in the lungs were significantly fewer and disappeared more quickly than UCMSCs. The b wave of the flash electroretinogram was improved at 7 d, and the retinal outer nuclear layer thickness was thicker at 7 d and 14 d. The expression level of inflammation was inhibited, and the expression level of neurotrophic factors was upregulated in the retina and serum after transplantation. CONCLUSIONS: S-UCMSCs intravenous infusion was safer than UCMSCs and could delay retinal degeneration and protect visual function in RCS rats, which may be a preferable therapeutic approach for RP.

Evaluation of activated carbon synthesized by one-stage and two-stage co-pyrolysis from sludge and coconut shell.

Waste biomass and sewage sludge were used to obtain an adsorbent material with excellent performance qualities by adopting a KOH activation process via one-stage (ACone) or two-stage (ACtwo) co-pyrolysis. The main purpose of this work was to investigate the effects of both methods in terms of the physicochemical properties and adsorption capacities for methylene blue (MB). Textural analyses revealed that the surface area (Stot= 683.82 m2/g) and total pore volume (Vtot= 0.72 cm3/g) of ACtwo were more than two-fold compared with ACone (Stot= 285.33 m2/g; Vtot= 0.35 cm3/g). Thus, two-stage co-pyrolysis produced activated carbon with increased porosity, which was favorable for MB adsorption. Nevertheless, the intensity of the surface functional groups of ACtwo was weaker than for ACone, which could be due to the pore-forming mechanism. Two-stage co-pyrolysis increased the yield and aromaticity of activated carbon, but sufficient activation caused more functional groups to decompose. For the adsorbate MB, the maximum adsorption capacity of ACtwo (602.80 mg/g) was more than five-fold greater than that of ACone (101.88 mg/g), due to its excellent porosity properties. Furthermore, the interactions of MB molecules with activated carbon were via hydrogen bonds and electrostatic attraction. The adsorption process of MB onto activated carbon was accurately described by the pseudo-second-order kinetic model. Adsorption equilibrium evaluated Langmuir isotherms demonstrated that MB formed a monolayer by adsorption onto the activated carbon. Adsorption thermodynamics was used to investigate the influence of temperature on the adsorption process. Thermodynamic parameters indicated that MB adsorption onto activated carbon was spontaneous and endothermic. In conclusion, our results showed that two-stage co-pyrolysis improves the adsorption capabilities of activated carbon, so achieving better economic value from waste materials.

Intravenous Infusion of Umbilical Cord Mesenchymal Stem Cells Maintains and Partially Improves Visual Function in Patients with Advanced Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a hereditary retinal degeneration disease with no effective therapeutic approaches. Inflammatory and immune disorders are thought to play an important role in the pathogenesis of RP. Human umbilical cord mesenchymal stem cells (UCMSCs), with multiple biological functions such as anti-inflammation and immunoregulation, have been applied in different systemic diseases. We conducted a phase I/II clinical trial aiming to evaluate the safety and efficacy of intravenous administration of UCMSCs in advanced RP patients. All 32 subjects were intravenously infused with one dose of 108 UCMSCs and were followed up for 12 months. No serious local or systemic adverse effects occurred in the whole follow-up. Most patients improved their best corrected visual acuity (BCVA) in the first 3 months. The proportions of patients with improved or maintained BCVA were 96.9%, 95.3%, 93.8%, 95.4%, 90.6%, and 90.6% at the 1st, 2nd, 3rd, 6th, 9th, and 12th month follow-up, respectively. Most of the patients (81.3%) maintained or improved their visual acuities for 12 months. The average NEI VFQ-25 questionnaire scores were significantly improved at the third month (P < 0.05). The average visual field sensitivity and flash visual evoked potential showed no significant difference (P = 0.185, P = 0.711). Our results indicated that the intravenous infusion of UCMSCs was safe for advanced RP patients. Most of the patients improved or maintained their visual functions in a long term. The life qualities were improved significantly in the first 3 months, suggesting that the intravenous infusion of UCMSCs may be a promising therapeutic approach for advanced RP patients.

Evaluation of migration of heavy metals and performance of product during co-pyrolysis process of municipal sewage sludge and walnut shell.

A feasible and efficient type of biological sludge-activated carbon (BSAC) was produced by co-pyrolysis of municipal sewage sludge (MSS) and walnut shell (4:1, w/w) at 500 °C. It was found that BSAC was typical mesoporous material with favorable pore structure and abundant surface functional groups, whose performance was improved compared with conventional sludge-activated carbon (CSAC), combined with walnut shell-activated carbon (WSAC). The migration and transformation behavior of heavy metals (Zn, Cu, Ni, Cd, and Cr) in raw material after co-pyrolysis process were investigated. The results indicated that co-pyrolysis could promote mobile fraction (acid soluble/exchangeable and reducible fractions) of heavy metals to stable fraction (oxidizable and residual fractions). The leaching concentrations Cu, Ni, Cd, Cr, and Zn were lower than restrictive standards in China, and the environmental risk assessment results showed that after co-pyrolysis, the risk levels of Cu, Ni, and Cd were decreased to low risk, especially Cr in product was confirmed to no risk.