Engineering the Substrate Specificity of a P450 Dimerase Enables the Collective Biosynthesis of Heterodimeric Tryptophan-Containing Diketopiperazines.

Heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are an important class of bioactive secondary metabolites. Biosynthesis offers a practical opportunity to access their bioactive structural diversity, however, it is restricted by the limited substrate scopes of the HTDKPs-forming P450 dimerases. Herein, by genome mining and investigation of the sequence-product relationships, we unveiled three important residues (F387, F388 and E73) in these P450s that are pivotal for selecting different diketopiperazine (DKP) substrates in the upper binding pocket. Engineering these residues in NasF5053 significantly expanded its substrate specificity and enabled the collective biosynthesis, including 12 self-dimerized and at least 81 cross-dimerized HTDKPs. Structural and molecular dynamics analysis of F387G and E73S revealed that they control the substrate specificity via reducing steric hindrance and regulating substrate tunnels, respectively.

Laser lift-off mechanism and optical-electric characteristics of red Micro-LED devices.

The removal of a sapphire substrate by laser lift-off, photoluminescence detection technology, and the luminous efficiency of size-dependent devices are very hot issues for the Micro-LED display, which is thoroughly studied in this paper. The mechanism of thermal decomposition of the organic adhesive layer after laser irradiation is analyzed in detail, and the thermal decomposition temperature of 450 °C solved by the established one-dimensional model is highly consistent with the inherent decomposition temperature of the PI material. The spectral intensity of PL is higher, and the peak wavelength is red-shifted by about 2 nm compared to EL under the same excitation condition. The results of size-dependent device optical-electric characteristics show that the smaller the device size, the lower the luminous efficiency under the same display resolution and PPI conditions, and the higher corresponding display power consumption.

Biosynthesis of Tetronates by a Nonribosomal Peptide Synthetase-Polyketide Synthase System.

A cryptic tetronate biosynthetic pathway was identified in Kitasatospora niigatensis DSM 44781 via heterologous expression. Distinct from the currently known biosynthetic pathways, this system utilizes a partially functional nonribosomal peptide synthetase and a broadly selective polyketide synthase to direct the assembly and lactonization of the tetronate scaffold. By employing a permissive crotonyl-CoA reductase/carboxylase to provide different extender units, seven new tetronates (kitaniitetronins A-G) were obtained via precursor-directed biosynthesis.

Optical-electrical characteristic of green based on GaN micro-LED arrays.

A detailed theoretical derivation and calculation method of the difference coefficient between a light distribution pattern of a 30×20µm2 green micro-LED array and Lambert source is proposed first in this paper, to the best of our knowledge, which establishes an accurate relationship between external quantum efficiency and current efficiency (cd/A). The variation of capacitance with voltage and wavelength blueshift is illustrated by a carrier recombination mechanism. The current efficiency reaches 132.5 cd/A for the 60×50µm2 and 121.7 cd/A for the 25×15µm2 arrays, and the mechanism caused by size dependence is analyzed in detail combined with the classical ABC model.

An Atypical Acyl-CoA Synthetase Enables Efficient Biosynthesis of Extender Units for Engineering a Polyketide Carbon Scaffold.

Acyl-CoAs are key precursors of primary and secondary metabolism. Their efficient biosynthesis is often impeded by the limited substrate specificity and low in vivo activity of acyl-CoA synthetases (ACSs) due to regulatory acylation of the catalytically important lysine residue in motif A10 (Lys-A10). In this study, we identified an unusual ACS (UkaQ) from the UK-2A biosynthetic pathway that naturally lacks the Lys-A10 residue and exhibits extraordinarily broad substrate specificity. Protein engineering significantly improved its stability and catalytic activity, enabling it to synthesize a large variety of acyl-CoAs with highly robust activity. By combining it with permissive carboxylases, we produced a large array of polyketide extender units and obtained six novel halobenzyl-containing antimycin analogues through an engineered biosynthetic pathway. This study significantly expands the catalytic mode of ACSs and provides a potent tool for the biosynthesis of acyl-CoA-derived natural products.

A thermostable and CBM2-linked GH10 xylanase from Thermobifida fusca for paper bleaching.

Xylanases have the potential to be used as bio-deinking and bio-bleaching materials and their application will decrease the consumption of the chlorine-based chemicals currently used for this purpose. However, xylanases with specific properties could act effectively, such as having significant thermostability and alkali resistance, etc. In this study, we found that TfXyl10A, a xylanase from Thermobifida fusca, was greatly induced to transcript by microcrystalline cellulose (MCC) substrate. Biochemical characterization showed that TfXyl10A is optimally effective at temperature of 80 °C and pH of 9.0. After removing the carbohydrate-binding module (CBM) and linker regions, the optimum temperature of TfXyl10A-CD was reduced by 10°C (to 70°C), at which the enzyme's temperature tolerance was also weakened. While truncating only the CBM domain (TfXyl10AdC) had no significant effect on its thermostability. Importantly, polysaccharide-binding experiment showed that the auxiliary domain CBM2 could specifically bind to cellulose substrates, which endowed xylanase TfXyl10A with the ability to degrade xylan surrounding cellulose. These results indicated that TfXyl10A might be an excellent candidate in bio-bleaching processes of paper industry. In addition, the features of active-site architecture of TfXyl10A in GH10 family were further analyzed. By mutating each residue at the -2 and -1 subsites to alanine, the binding force and enzyme activity of mutants were observably decreased. Interestingly, the mutant E51A, locating at the distal -3 subsite, exhibited 90% increase in relative activity compared with wild-type (WT) enzyme TfXyl10A-CD (the catalytic domain of TfXyl110A). This study explored the function of a GH10 xylanase containing a CBM2 domain and the contribution of amino acids in active-site architecture to catalytic activity. The results obtained provide guidance for the rational design of xylanases for industrial applications under high heat and alkali-based operating conditions, such as paper bleaching.

Incentives Promoting Contracted Family Doctor Service Policy to Improve Continuity and Coordination in Diabetes Patient Management Care in China.

Background: As the first step toward building a gatekeeping system in China, the governments have introduced a contracted family doctor service (CFDS) policy in primary healthcare (PHC) facilities. This study was to examine the association between apply of incentive to improve the implementation of CFDS and the performance on diabetes management care. Methods: We conducted a cross-sectional study in 72 PHC facilities in 6 cities that piloted the CFDS. Multivariate regression models were applied, based on a sample of 827 PHC providers and 420 diabetic patients. Results: PHC providers who reported the performance being linked with increased income were 168.1 and 78.0% more likely to have good continuity and coordination of diabetes patient management care, respectively. Additional one-point percentage of PHC providers whose performance on CFDS was assessed was associated with 7.192 times higher probability of patients with control of blood glucose. Discussion: Inclusion of incentives rewarding better performance on CFDS were associated with better delivery process and outcome performance on diabetes management care. Conclusion: Design and implementation of the incentive should be accompanied with the policy of CFDS, in order to increase the proportion of performance-related income of PHC providers, thereby improving the quality of diabetes management care.

Biosynthesis of pyrroloindoline-containing natural products.

Covering: up to 2022Pyrroloindoline is a privileged tricyclic indoline motif widely present in many biologically active and medicinally valuable natural products. Thus, understanding the biosynthesis of this molecule is critical for developing convenient synthetic routes, which is highly challenging for its chemical synthesis due to the presence of rich chiral centers in this molecule, especially the fully substituted chiral carbon center at the C3-position of its rigid tricyclic structure. In recent years, progress has been made in elucidating the biosynthetic pathways and enzymatic mechanisms of pyrroloindoline-containing natural products (PiNPs). This article reviews the main advances in the past few decades based on the different substitutions on the C3 position of PiNPs, especially the various key enzymatic mechanisms involved in the biosynthesis of different types of PiNPs.

A Pair of Atypical KAS III Homologues with Initiation and Elongation Functions Program the Polyketide Biosynthesis in Asukamycin.

ß-Ketoacyl-ACP synthase III (KAS III) is a class of important C-C bond-forming enzymes that mostly catalyze the initiation of polyketide and fatty acid biosynthesis. In this study, we elucidated an unusual polyketide synthase (PKS) system that involves two unique KAS IIIs (AsuC3 and C4) in the biosynthesis of the upper triene chain of asukamycin. Significantly, AsuC3 and C4 have both initiation and iterative elongation activity, while being functionally biased toward the elongation and initiation steps, respectively. Mutational analysis revealed that their catalytic activities rely on the catalytic triad Cys-His-Asn. Unlike other KAS IIIs, AsuC3 and C4 are very promiscuous and can accept various lengths of acyl-CoAs with either cyclic, branched or linear acyl moieties. By cooperation with the permissive ketoreductase (AsuC7) and dehydratase (AsuC8/C9), a large variety of polyenes can be efficiently synthesized. This study significantly broadens the understanding of KAS IIIs and polyketide biosynthesis.

Socioeconomic status and depressive symptoms in older people with the mediation role of social support: A population-based longitudinal study.

BACKGROUND: Depressive symptoms has become an increasingly important public health issue, contributing to disability and disease burden around the world. Higher socioeconomic status (SES) has been found to be associated with lower prevalence of depression, but there are few studies about the older Chinese adults with long-term follow up and rigorous prospective design. Meanwhile, there is little conclusive evidence about the mechanisms through which SES influences the onset of depressive symptoms. OBJECTIVE: To prospectively examine the association of baseline socioeconomic factors with the risks of developing depressive symptoms during 7-year follow up in older Chinese population, and to study the mechanism by which SES impacts the prevalence of depressive symptoms. METHODS: A total of 5677 individuals over 45 years who participated in an ongoing nationally representative prospective cohort study, China Health and Retirement Longitudinal Study, were free from depressive symptoms at baseline, and completed 7-year follow-up were included. Depressive symptoms were assessed using the 10-item Center for Epidemiological Studies Depression Scale score. Cox proportional hazards regression models were used to examine the association of SES and the incidence of depressive symptoms in 2011 to 2018. Generalized structural equation model was used to analyze the mediation effects of social support on the relation between SES and depressive symptoms. RESULTS: During the 7-year follow-up, 2398 (42.2%) cases were identified as depressive symptoms. Compared with the lowest level of household income, participants with the highest level of household income had a 20% reduction in risk (95% CI, 0.70-0.92, P < 0.001). Participants who had junior high school or above education had a 41% lower risk of depressive disorders compared with illiterate participants (95% CI, 0.52-0.69, P < 0.001). The relationship between SES and depressive symptoms was partially mediated by the social support, where higher social support was negatively associated with depressive symptoms. The proportion of mediation effect was even larger for women compared with men. CONCLUSION: Socioeconomic factors were independently associated with the development of depressive symptoms, and the relationship was partially mediated by social support. Social support could be an effective intervention to alleviate the negative effects of lower SES on mental health. Multiple-level policies should precisely target low-SES groups, and timely intervention to promote social support for this group should be used to reduce the influence of depression on individuals, family as well as the whole society.

Production of Heterodimeric Diketopiperazines Employing a Mycobacterium-Based Whole-Cell Biocatalysis System.

Heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are an important class of bioactive secondary metabolites. P450-mediated biocatalysis offers a practical avenue to access their structural diversity; however, many of these enzymes are insoluble in Escherichia coli and difficult to operate in Streptomyces. Through validation of the functions of two pairs Mycobacterium smegmatis sourced redox partners in vitro, and comparing the efficiency of different biocatalytic systems with tricky P450s in vivo, we herein demonstrated that M. smegmatis is much more efficient, robust, and cleaner in metabolites background than the regularly used E. coli or Streptomyces systems. The M. smegmatis-based system can completely convert 1 g L-1 of cyclodipeptide into HTDKPs within 18 h with minimal background metabolites. On the basis of this efficient system, 12 novel HTDPKs were readily obtained by using two HTDKP-forming P450s (NasbB and NASS1868). Among them, five compounds have neuroprotective properties. Our study significantly expands the bioactive chemical scope of HTDKPs and provides an excellent biocatalysis platform for dealing with problematic enzymes from Actinomycetes.

Full-color micro-LED displays with cadmium-free quantum dots patterned by photolithography technology: retraction.

The referenced article [Appl. Opt.59, 11112 (2020)APOPAI0003-693510.1364/AO.412267] has been retracted by the author.

Quantity and Quality of Healthcare Professionals, Transfer Delay and In-hospital Mortality Among ST-Segment Elevation Myocardial Infarction: A Mixed-Method Cross-Sectional Study of 89 Emergency Medical Stations in China.

BACKGROUND: Transfer delay provokes prolongation of prehospital time, which contributes to treatment delay that endangers patients with ST-segment elevation myocardial infarction (STEMI). A key constraint in reducing transfer delay is the shortage of emergency healthcare workers. This study was to explore the influence of the quality and quantity of healthcare professionals at emergency medical stations on transfer delay and in-hospital mortality among STEMI patients. METHODS: A cross-sectional study using mixed methods was conducted at 89 emergency stations in 9 districts in China's Shenzhen province. Based on a sample of 31 hospitals, 1,255 healthcare professionals, and 3,131 patients with STEMI, a generalized linear model was used to explore the associations between the quality and quantity of healthcare professionals and transfer delay and in-hospital mortality among STEMI patients. Qualitative data were collected and analyzed to explore the reasons for the lack of qualified healthcare professionals at emergency medical stations. RESULTS: The analysis of the quantity of healthcare professionals showed that an increase of one physician per 100,000 individuals was associated with decreased transfer delay for patients with STEMI by 5.087 min (95% CI -6.722, -3.452; P < 0.001). An increase of one nurse per 100,000 individuals was associated with decreased transfer delay by 1.471 min (95% CI -2.943, 0.002; P=0.050). Analysis of the quality of healthcare professionals showed that an increase of one physician with an undergraduate degree per 100,000 individuals was associated with decreased transfer delay for patients with STEMI by 8.508 min (95% CI -10.457, -6.558; P < 0.001). An increase of one nurse with an undergraduate degree per 100,000 individuals was associated with decreased transfer delay by 6.645 min (95% CI -8.218, -5.072; P < 0.001). Qualitative analysis illustrated that the main reasons for low satisfaction of healthcare professionals at emergency medical stations included low income, limited promotion opportunities, and poor working environment. CONCLUSIONS: The quantity and quality of emergency healthcare professionals are key factors influencing transfer delay in STEMI patients. The government should increase the quantity of healthcare professionals at emergency medical stations, strengthen the training, and improve their performance by linking with clinical pathways to enhance job enthusiasm among emergency healthcare professionals.

Full-color micro-LED displays with cadmium-free quantum dots patterned by photolithography technology.

A full-color display consisting of red and green photoluminescence cadmium-free quantum dots (QDs) as the color conversion material and excited by a 68×68 blue micro-LED flip chip array mounted on an active-matrix driving board was completed in this study. The QD photoresist (QDPR) lithography technology was reported in detail, and it has been proven to be a stable process route. The suitable thickness of 12±1µm of the QDPR and black matrix was proposed to reduce the light cross talk between different sub-pixels. The thickness of the common color filter of 1-2 µm was made successfully between the quantum dot film and the cover glass, which can greatly reduce the leakage of blue backlight and decrease the quantum dots excitation by the ambient light, as well as improve the color gamut and color purity of the display panel. In addition, the high red and green light conversion efficiency reaches up to 78.1% and 296.5%, respectively, and the screen display can reach 98.8% NTSC on the CIE 1931 chromaticity. Representative RGB monochromatic pictures were displayed successfully and ≤0.04 viewing angle deviation of the display was measured precisely.

Molecular basis of regio- and stereo-specificity in biosynthesis of bacterial heterodimeric diketopiperazines.

Bacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are a growing family of bioactive natural products. They are challenging to prepare by chemical routes due to the polycyclic and densely functionalized backbone. Through functional characterization and investigation, we herein identify a family of three related HTDKP-forming cytochrome P450s (NasbB, NasS1868 and NasF5053) and reveal four critical residues (Qln65, Ala86, Ser284 and Val288) that control their regio- and stereo-selectivity to generate diverse dimeric DKP frameworks. Engineering these residues can alter the specificities of the enzymes to produce diverse frameworks. Determining the crystal structures (1.70-1.47 Å) of NasF5053 (ligand-free and substrate-bound NasF5053 and its Q65I-A86G and S284A-V288A mutants) and molecular dynamics simulation finally elucidate the specificity-conferring mechanism of these residues. Our results provide a clear molecular and mechanistic basis into this family of HTDKP-forming P450s, laying a solid foundation for rapid access to the molecular diversity of HTDKP frameworks through rational engineering of the P450s.

Size-dependent optical-electrical characteristics of blue GaN/InGaN micro-light-emitting diodes.

To derive the impact of chip size reduction on optical efficiency in micro-LED array panels, blue InGaN/GaN LEDs, which consist of 21×7 arrays (60 ppi display) with different mesa sizes on sapphire substrates, are designed and fabricated in this study. Changing the mesa area of the chip is first proposed to investigate the luminous efficiency (cd/A) of the screen. The current efficiency with a peak wavelength of 450 nm reaches up to 14.29 cd/A for the biggest pixel 50µm×60µm and to 12.25 cd/A for the 15µm×25µm chip, delivering high-level efficiencies to the current LED research field. The mechanisms of size-dependent efficiency variation trends and efficiency droops of blue LEDs are investigated experimentally, confirming that the current efficiency is more efficient at high injection current density while exhibiting poorer performance at the low current density region for smaller chips. The peak efficiency corresponds to higher current density with a decrease in chip size according to the carrier recombination ABC model. Moreover, the characteristic curve of the spectrum and the changes in the yellow light band under different incident light conditions (i.e., 355 nm and 375 nm) are analyzed by photoluminescence.

Efficient biosynthesis of heterodimeric C3-aryl pyrroloindoline alkaloids.

Many natural products contain the hexahydropyrrolo[2, 3-b]indole (HPI) framework. HPI containing chemicals exhibit various biological activities and distinguishable structural arrangement. This structural complexity renders chemical synthesis very challenging. Here, through investigating the biosynthesis of a naturally occurring C3-aryl HPI, naseseazine C (NAS-C), we identify a P450 enzyme (NascB) and reveal that NascB catalyzes a radical cascade reaction to form intramolecular and intermolecular carbon-carbon bonds with both regio- and stereo-specificity. Surprisingly, the limited freedom is allowed in specificity to generate four types of C3-aryl HPI scaffolds, and two of them were not previously observed. By incorporating NascB into an engineered strain of E. coli, we develop a whole-cell biocatalysis system for efficient production of NAS-C and 30 NAS analogs. Interestingly, we find that some of these analogs exhibit potent neuroprotective properties. Thus, our biocatalytic methodology offers an efficient and simple route to generate difficult HPI framework containing chemicals.

Puromycin A, B and C, cryptic nucleosides identified from Streptomyces alboniger NRRL B-1832 by PPtase-based activation.

Natural product discovery is pivot for drug development, however, this endeavor is often challenged by the wide inactivation or silence of natural products biosynthetic pathways. We recently developed a highly efficient approach to activate cryptic/silenced biosynthetic pathways through augmentation of the phosphopantetheinylation of carrier proteins. By applying this approach in the Streptomyces alboniger NRRL B-1832, we herein identified three cryptic nucleosides products, including one known puromycin A and two new derivatives (puromycin B and C). The biosynthesis of these products doesn't require the involvement of carrier protein, indicating the phosphopantetheinyl transferase (PPtase) indeed plays a fundamental regulatory role in metabolites biosynthesis. These results demonstrate that the PPtase-based approach have a much broader effective scope than the previously assumed carrier protein-involving pathways, which will benefit future natural products discovery and biosynthetic studies.

Structural Basis of a Broadly Selective Acyltransferase from the Polyketide Synthase of Splenocin.

Polyketides are a large family of pharmaceutically important natural products, and the structural modification of their scaffolds is significant for drug development. Herein, we report high-resolution X-ray crystal structures of the broadly selective acyltransferase (AT) from the splenocin polyketide synthase (SpnD-AT) in the apo form and in complex with benzylmalonyl and pentynylmalonyl extender unit mimics. These structures revealed the molecular basis for the stereoselectivity and substrate specificity of SpnD-AT, and enabled the engineering of the industrially important Ery-AT6 to broaden its substrate scope to include three new types of extender units.

Stabilization of Multimeric Proteins via Intersubunit Cyclization.

Proteins with high catalytic efficiency and selectivity under mild conditions have long been appreciated by industrial and medicinal fields. These proteins, which are commonly multimeric, often possess low stability, impeding wider application. Currently, strategies to improve the stability of multimeric proteins concentrate on enhancing the interaction at internal interface of the subunits. In this report, we confirmed that the largely underestimated subunit terminal ends are as significant as the internal interface for protein stability. By connecting both the terminal ends and internal interface of subunits, the tetrameric Leifsonia alcohol dehydrogenase (LnADH) protein can been cyclized into a rigid form with significantly improved thermostability and resilience. The improvement in the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment (T5015) and melting temperature (Tm ) of the modified protein was 18°C and 23.3°C, respectively, which is superior to the results achieved by normal protein engineering. Our study provided a novel strategy to effectively improve the stability of multimeric proteins, which is suitable not only for the short-chain dehydrogenase/reductase (SDR) family but also other classes of proteins with close terminal ends.IMPORTANCE Industrially interesting proteins are generally multimeric proteins; however, their applications are often restricted due to low stability caused by the natural tendency of subunit disassociation. Current approaches targeting this problem mainly focus on enhancing the internal interfaces of the subunits to avoid their disassociation. In this study, we identified and confirmed the external interface to be significant for improving the stability of multimeric proteins. By connecting the terminal ends and internal interface with disulfide bonds, we found that the multimeric protein LnADH cyclized into a robust monomeric-like form, resulting in superior thermostability compared to traditional protein engineering. This intersubunit cyclization approach is efficient and easy to perform, providing a novel method for engineering many important classes of multimeric proteins.