High-Throughput Roll-to-Roll Processed Large-Area Perovskite Solar Cells Using Rapid Radiation Annealing Technique.

The development of a stable roll-to-roll (R2R) process for flexible large-area perovskite solar cells (PSCs) and modules is a pressing challenge. In this study, we introduced a new R2R PSC manufacturing system that employs a two-step deposition method for coating perovskite and uses intensive pulsed light (IPL) for annealing. This system has successfully fabricated small-sized cells and the first-ever large-sized, R2R-processed flexible modules. A key focus of our work was to accelerate the conversion of PbI2 to perovskite. To this end, we utilized IPL annealing and incorporated additives into the PbI2 layer. With these modifications, the R2R-processed perovskite films achieved a power conversion efficiency (PCE) of 16.87%, representing the highest reported value for R2R two-step processed PSCs. However, these cells exhibited hysteresis in reverse and forward PCE measurements. To address this, we introduced a dual-annealing process consisting of IPL followed by a 2-min thermal heating step. This approach successfully reduced hysteresis, resulting in low-hysteresis, R2R-processed flexible PSCs. Moreover, we fabricated large-scale flexible modules (10 × 10 cm2) with a PCE of 11.25% using the dual-annealing system, marking a significant milestone in this field.

Bio-specific immobilization of enzymes on electrospun PHB nanofibers.

Enzyme immobilization provides substantial advantages in terms of improving the efficiency of enzymatic process as well as enhancing the reusability of enzymes. Phasins (PhaPs) are naturally occurring polyhydroxyalkanoate (PHA)-binding proteins, and thus can potentially be used as a fusion partner for oriented immobilization of enzymes onto PHA supports. However, presently available granular PHA supports have low surface-area-to-volume ratio and limited configurational flexibility of enzymatic reactions. In this study, we explored the use of electrospun polyhydroxybutyrate (PHB) nanofibers as an alternative support for high density immobilization of a PhaP-fused lipase. As envisioned, the electrospun PHB nanofibers could anchor 120-fold more enzyme than PHB granules of the same weight. Furthermore, the enzymes immobilized onto the PHB nanofibers exhibited markedly higher stability and activity compared to when immobilized on conventional immobilization supports. Our approach combines the advantageous features of nanofibrous material and specificity of biomolecular interaction for the efficient use of enzymes, which can be widely adopted in the development of various enzymatic processes.

Loss of abutment teeth for double crown-retained removable partial dentures compared to clasp-retained removable partial dentures: a systematic review and meta-analysis.

Objective: To analyze the loss of abutment teeth for double crown-retained removable partial dentures (DC-RPDs) compared to clasp-retained removable partial dentures (C-RPDs). Method and materials: A search was conducted in the Ovid MEDLINE, Embase, Web of Science databases, and a manual search. The search was conducted based on the PICO framework with inclusion and exclusion criteria. After extracting the data of selected studies, a meta-analysis was performed to estimate abutment loss with 95% confidence interval (CI). The statistical significance was defined as P < .05, and the heterogeneity of the data was assessed based on the chi-squared test and I2 statistics. Risk of bias assessment was conducted using Cochrane Risk of Bias tool and Newcastle-Ottawa Scale. Results: A total of 4,692 records were identified from electronic databases and 38 studies were included for quantitative synthesis of 3,393 subjects with 13,552 abutment teeth. A total of 1,226 abutment teeth were lost with > 4,016 RPDs. Seven studies were compounded for > 668 C-RPDs (mean follow-up time ≤ 5 years) and six studies for 893 C-RPDs (mean follow-up time < 5 years), where the estimates of abutment loss were 5% (95% CI 2% to 8%) and 8% (95% CI 5% to 13%), respectively. The data were not significantly different (P = .1), and were heterogenous between the studies (τ2 ≥ 0.34, I2 ≥ 87.38%). Thirteen studies were compounded for 1,223 DC-RPDs (mean follow-up time ≤ 5 years) and eight studies for 1,033 DC-RPDs (mean follow-up time > 5 years), where the estimates of abutment loss were 6% (95% CI 5% to 8%) and 12% (95% CI 8% to 18%), respectively. The data were heterogenous (τ2 ≥ 0.17, I2 ≥ 75.86%), and were significantly different between the studies (P = .005). Overall, C-RPDs were not significantly different from DC-RPDs in abutment loss (P ≥ .3). A significant predictor for abutment loss was follow-up time with DC-RPDs (P = .005), where the risk of abutment loss per year was 18% (P = .0001). In contrast, follow-up time was not a significant factor for C-RPDs (P = .1). None of the included studies were at high risk of bias. Conclusion: Within the limitations of the current systematic review and meta-analysis, abutment loss was not significantly different between C-RPDs and DC-RPDs. A significant predictor was follow-up time for DC-RPDs, whereas this factor was not significant for C-RPDs. Further research is needed to investigate critical factors for abutment loss with RPDs.

Loss of nonvital abutment teeth for removable partial dentures: a systematic review and meta-analysis.

OBJECTIVE: To analyze the loss of nonvital abutment teeth compared to vital abutment teeth supporting removable partial dentures (RPDs). METHOD AND MATERIALS: An electronic search was conducted in the Ovid MEDLINE, Embase, and Web of Science databases, and supplemented by a manual search. The search was done to identify clinical studies reporting the loss of nonvital abutment teeth compared to vital abutment teeth for RPDs. The data were extracted from each selected article and meta-analysis was performed using a random effects model to estimate adjusted proportion of abutment loss with RPDs along with 95% confidence interval (CI). Risk of bias assessment was conducted using Cochrane Risk of Bias tools and Newcastle-Ottawa Scale. The statistical significance was set as P value < .05. RESULTS: A total of 3,898 records were identified from electronic databases and 8 studies were included for quantitative synthesis of 834 subjects with 1,036 RPDs. No additional records were identified through manual search. Among 1,152 nonvital abutment teeth, 123 teeth were lost. The estimate of nonvital abutment loss was 13% (95% CI 9-18%]. The data were statistically significant (P < .0001), and were heterogenous between the studies (χ2 [df = 7] = 35.9, P < .0001; τ2 = 0.25, I2 = 76.46%). A total of 2,186 vital abutment teeth were compounded where 114 teeth were lost. The estimate of vital abutment loss was 4% (95% CI 2-7%). The data were statistically significant (P < .0001), and were heterogenous between the studies (χ2 [df = 7] = 23.7, P = .01; τ2 = 0.56, I2 = 86.31%). The risk of abutment loss was approximately three times greater with the loss of vitality (odds ratio = 3.04, 95% CI 1.53-6.05; P = .001). In addition, abutment loss was significantly greater with increasing follow-up time (P = .01). None of the included studies were considered to be at high risk of bias. CONCLUSION: Within the limitations of this systematic review and meta-analysis, the loss of nonvital abutment teeth was significantly greater than that of vital abutment teeth for RPDs. Further research is needed to identify critical factors associated with the loss of nonvital abutment teeth.

High-Level Production of Bacteriotoxic Phospholipase A1 in Bacterial Host Pseudomonas fluorescens Via ABC Transporter-Mediated Secretion and Inducible Expression.

: Bacterial phospholipase A1 (PLA1) is used in various industrial fields because it can catalyze the hydrolysis, esterification, and transesterification of phospholipids to their functional derivatives. It also has a role in the degumming process of crude plant oils. However, bacterial expression of the foreign PLA1-encoding gene was generally hampered because intracellularly expressed PLA1 is inherently toxic and damages the phospholipid membrane. In this study, we report that secretion-based production of recombinant PlaA, a bacterial PLA1 gene, or co-expression of PlaS, an accessory gene, minimizes this harmful effect. We were able to achieve high-level PlaA production via secretion-based protein production. Here, TliD/TliE/TliF, an ABC transporter complex of Pseudomonas fluorescens SIK-W1, was used to secrete recombinant proteins to the extracellular medium. In order to control the protein expression with induction, a new strain of P. fluorescens, which had the lac operon repressor gene lacI, was constructed and named ZYAI strain. The bacteriotoxic PlaA protein was successfully produced in a bacterial host, with help from ABC transporter-mediated secretion, induction-controlled protein expression, and fermentation. The final protein product is capable of degumming oil efficiently, signifying its application potential.

Isolation and characterization of a novel metagenomic enzyme capable of degrading bacterial phytotoxin toxoflavin.

Toxoflavin, a 7-azapteridine phytotoxin produced by the bacterial pathogens such as Burkholderia glumae and Burkholderia gladioli, has been known as one of the key virulence factors in crop diseases. Because the toxoflavin had an antibacterial activity, a metagenomic E. coli clone capable of growing well in the presence of toxoflavin (30 µg/ml) was isolated and the first metagenome-derived toxoflavin-degrading enzyme, TxeA of 140 amino acid residues, was identified from the positive E. coli clone. The conserved amino acids for metal-binding and extradiol dioxygenase activity, Glu-12, His-8 and Glu-130, were revealed by the sequence analysis of TxeA. The optimum conditions for toxoflavin degradation were evaluated with the TxeA purified in E. coli. Toxoflavin was totally degraded at an initial toxoflavin concentration of 100 µg/ml and at pH 5.0 in the presence of Mn2+, dithiothreitol and oxygen. The final degradation products of toxoflavin and methyltoxoflavin were fully identified by MS and NMR as triazines. Therefore, we suggested that the new metagenomic enzyme, TxeA, provided the clue to applying the new metagenomic enzyme to resistance development of crop plants to toxoflavin-mediated disease as well as to biocatalysis for Baeyer-Villiger type oxidation.

Cell-free synthesis of functional phospholipase A1 from Serratia sp.

BACKGROUND: Phospholipase A1 is an enzyme that hydrolyzes phospholipids at the sn-1 position. It has potential applications across diverse fields including food, pharmaceutical, and biofuel industries. Although there has been increasing interest in the use of phospholipase A1 for degumming of plant oils during biodiesel production, production of recombinant phospholipase A1 has been hampered by low efficiency of gene expression and its toxicity to the host cell. RESULTS: While expression of phospholipase A1 in Escherichia coli resulted in extremely low productivity associated with inhibition of transformed cell growth, drastically higher production of functional phospholipase A1 was achieved in a cell-free protein synthesis system where enzyme expression is decoupled from cell physiology. Compared with expression in E. coli, cell-free synthesis resulted in an over 1000-fold higher titer of functional phospholipase A1. Cell-free produced phospholipase A1 was also used for successfully degumming crude plant oil. CONCLUSIONS: We demonstrate successful production of Serratia sp. phospholipase A1 in a cell-free protein synthesis system. Including the phospholipase A1 investigated in this study, many industrial enzymes can interfere with the regular physiology of cells, making cellular production of them problematic. With the experimental results presented herewith, we believe that cell-free protein synthesis will provide a viable option for rapid production of important industrial biocatalysts.

Alleviation of temperature-sensitive secretion defect of Pseudomonas fluorescens ATP-binding cassette (ABC) transporter, TliDEF, by a change of single amino acid in the ABC protein, TliD.

An ABC transporter, TliDEF, from Pseudomonas fluorescens SIK W1, mediates the secretion of its cognate lipase, TliA, in a temperature-dependent secretion manner; the TliDEF-mediated secretion of TliA was impossible at the temperatures over 33°C. To isolate a mutant TliDEF capable of secreting TliA at 35°C, the mutagenesis of ABC protein (TliD) was performed. The mutated tliD library where a random point mutation was introduced by error-prone PCR was coexpressed with the wild-type tliE, tliF and tliA in Escherichia coli. Among approximately 10,000 colonies of the tliD library, we selected one colony that formed transparent halo on LB-tributyrin plates at 35°C. At the growth temperature of 35°C, the selected mutant TliD showed 1.75 U/ml of the extracellular lipase activity, while the wild-type TliDEF did not show any detectable lipase activity in the culture supernatant of E. coli. Moreover, the mutant TliD also showed higher level of TliA secretion than the wild-type TliDEF at other culture temperatures, 20°C, 25°C and 30°C. The mutant TliD had a single amino acid change (Ser287Pro) in the predicted transmembrane region in the membrane domain of TliD, implying that the corresponding region of TliD was important for causing the temperature-dependent secretion of TliDEF. These results suggested that the property of ABC transporter could be changed by the change of amino acid in the ABC protein.

In situ immobilized lipase on the surface of intracellular polyhydroxybutyrate granules: preparation, characterization, and its promising use for the synthesis of fatty acid alkyl esters.

Photobacterium lipolyticum M37 lipase (LipM37) was immobilized on the surface of intracellular polyhydroxybutyrate (PHB) granules in Escherichia coli. LipM37 was genetically fused to Cupriavidus necator PHA synthase (PhaC Cn ), and the engineered PHB operon containing the lip M37 -phaC Cn successfully mediated the accumulation of PHB granules (85 wt.%) inside E. coli cells. The PHB granules were isolated from the crude cell extract, and the immobilized LipM37 was comparable with the free form of LipM37 except for a favorable increase in thermostability. The immobilized LipM37 was used to synthesize oleic acid methyl ester (biodiesel) and oleic acid dodecyl ester (wax ester), and yielded 98.0 % conversion in esterification of oleic acid and dodecanol. It was suggested that the LipM37-PhaCCn fusion protein successfully exhibited bifunctional activities in E. coli and that in situ immobilization of lipase to the intracellular PHB could be a promising approach for expanding the biocatalytic toolbox for industrial chemical synthesis.

Isolation and characterization of cold-active family VIII esterases from an arctic soil metagenome.

Functional screening for lipolytic enzymes at low temperatures resulted in the isolation of the novel cold-active esterases, EstM-N1 and EstM-N2, from a metagenomic DNA library of arctic soil samples. EstM-N1 and EstM-N2 were 395 and 407 amino acids in length, respectively, and showed the highest similarity to class C ß-lactamases. However, they shared a relatively low level of sequence similarity (30%) with each other. Phylogenetic analysis of bacterial lipolytic enzymes confirmed that EstM-N1 and EstM-N2 belonged to family VIII of bacterial esterases/lipases. The (His)(6)-tagged esterases were purified to about 99% homogeneity from the soluble fraction of recombinant Escherichia coli cultures. The purified EstM-N1 and EstM-N2 retained more than 50% of maximal activity in the temperature range of 0-35 °C, with optimal temperatures of 20 °C and 30 °C, respectively. Both enzymes preferred the short acyl chains of p-nitrophenyl esters and exhibited very narrow substrate specificity, indicating that they are typical esterases. The ß-lactamase activity of EstM-N1 and EstM-N2 was also detected and reached about 31% and 13% of the positive control enzyme, Bacillus cereus ß-lactamase, respectively. These first cold-active esterases belonging to family VIII are expected to be useful for potential biotechnological applications as interesting biocatalysts.

Gene cloning, expression, and characterization of a new carboxylesterase from Serratia sp. SES-01: comparison with Escherichia coli BioHe enzyme.

The carboxylesterase-encoding gene (bioHs) of a newly isolated strain, Serratia sp. SES-01, was cloned from the genomic DNA library by detecting formation of transparent halo around the colony on LB-tributyrin agar plates. The amino acid sequence of BioHs was highly similar to the members of the BioH enzyme family involved in the biotin biosynthetic pathway; it showed the highest similarity (91%) with that of Serratia proteamaculans. To compare BioHs with other BioH enzymes, the relatively well-known bioHe gene of E. coli was cloned with PCR. After we achieved high-level expression of soluble BioHs and BioHe through the exploration of different culture conditions, the purified BioHs and BioHe enzymes were characterized in terms of specificity, activity, and stability. BioHe was generally more robust to a change in temperature and pH and an addition of organic solvents than BioHs. The two enzymes exhibited a strong preference for carboxylesterase rather than for thioesterase and were optimal at relatively low temperatures (20-40 degrees ) and alkaline pHs (7.5-9.0). The results in this study strongly suggested that both the BioHs and BioHe enzymes would be potential candidates for use as a carboxylesterase in many industrial applications.

High-level secretion of Pseudomonas fluorescens type I secretion system-dependent lipase in Serratia marcescens.

The type I secretion system-dependent lipase, TliA, of Pseudomonas fluorescens was successfully produced in quantity in Serratia marcescens by coexpressing its cognate ABC transporter, TliDEF. Compared with P. fluorescens and Escherichia coli, S. marcescens showed an outstanding capacity for the secretory production of TliA, which was done with the expression vectors available for use in E. coli, and no growth phase-dependency, which was unlike the typical feature of TOSS-mediated protein secretion. Among the S. marcescens tested, the highest amount of TliA (approximately 2600 units ml(-1)) was achieved by S. marcescens KCTC 2798 containing the expression plasmid pTliDEFA-223. Our results also suggest that strains of Serratia will provide a valuable opportunity for producing other extracellular TOSS-dependent proteins effectively as well as the TliDEF-dependent TliA in this study.