Comparative Transcriptomics Revealed Physalis floridana Rydb. Influences on the Immune System of the 28-Spotted Ladybird Beetle (Henosepilachna vigintioctopunctata).

Physalis floridana Rydb., a member of the Solanaceae family, is renowned for its diverse secondary metabolites, including physalins and withanolides. The 28-spotted ladybird beetle (Henosepilachna vigintioctopunctata) is a notorious pest severely damaging Solanaceous crops. This study demonstrates that P. floridana Rydb. significantly impacts on the development and reproductive suppression of H. vigintioctopunctata. A comparative transcriptome analysis was performed by feeding H. vigintioctopunctata larvae on P. floridana Rydb., Solanum nigrum L., Solanum tuberosum L., and Solanum lycopersicum L. The results reveal that larvae fed on P. floridana Rydb. exhibit numerous differentially expressed genes, which are notably enriched in pathways related to energy metabolism, immunity, and detoxification. These functions and pathways are less enriched in larvae fed by other hosts. Weighted Gene Co-expression Network Analysis (WGCNA) indicates that feeding on P. floridana Rydb. influences the expression of specific genes involved in the Toll and IMD signaling pathways, impacting the immune system of H. vigintioctopunctata larvae. This study provides transcriptomic insights into larval responses to different diets and suggests that the effect of P. floridana Rydb. on the immune system of H. vigintioctopunctata is a key defense mechanism against herbivores.

High Salt-Resistant Urethanase Degrades Ethyl Carbamate in Soy Sauce.

Urethanase is a promising biocatalyst for degrading carcinogen ethyl carbamate (EC) in fermented foods. However, their vulnerability to high ethanol and/or salt and acidic conditions severely limits their applications. In this study, a novel urethanase from Alicyclobacillus pomorum (ApUH) was successfully discovered using a database search. ApUH shares 49.4% sequence identity with the reported amino acid sequences. It belongs to the Amidase Signature family and has a conserved "K-S-S" catalytic triad and the characteristic "GGSS" motif. The purified enzyme overexpressed in Escherichia coli exhibits a high EC affinity (Km, 0.306 mM) and broad pH tolerance (pH 4.0-9.0), with an optimum pH 7.0. Enzyme activity remained at 58% in 12% (w/v) NaCl, and 80% in 10% (v/v) ethanol or after 1 h treatment with the same ethanol solution at 37 °C. ApUH has no hydrolytic activity toward urea. Under 30 °C, the purified enzyme (200 U/L) degraded about 15.4 and 43.1% of the EC in soy sauce samples (pH 5.0, 6.0), respectively, in 5 h. Furthermore, the enzyme also showed high activity toward the class 2A carcinogen acrylamide in foods. These attractive properties indicate their potential applications in the food industry.

Dynamics design of a non-natural transcription factor responding to androst-4-ene-3,17-dione.

The production of androst-4-ene-3,17-dione (AD) by the steroidal microbial cell factory requires transcription factors (TFs) to participate in metabolic regulation. However, microbial cell factory lacks effective TFs that can respond to AD in its metabolic pathway. Additionally, finding and obtaining natural TFs that specifically respond to AD is a complex and onerous task. In this study, we devised an artificial TF that responds to AD, termed AdT, based on structure-guided molecular dynamics (MD) simulation. According to MD analysis of the conformational changes of AdT after binding to AD, an LBD in which the N- and C-termini exhibited convergence tendencies was used as a microswitch to guide the assembly of a DNA-binding domain lexA, a linker (GGGGS)2, and a transcription activation domain B42 into an artificial TF. As a proof of design, a AD biosensor was designed and constructed in yeast on the basis of the ligand-binding domain (LBD) of hormone receptor. In addition, the transcription factor activity of AdT was increased by 1.44-fold for its variant F320Y. Overall, we created non-natural TF elements for AD microbial cell factory, and expected that the design TF strategy will be applied to running in parallel to the signaling machinery of the host cell.

Preparation and performance of semiconductor device bonding joints based on Cu@Sn@Ag preform.

Herein, a 110 A commercial, Si continuous current diode with high heat dissipation power is attached to Cu@Sn@Ag preform, formed by electroplating and physical vapor deposition, and pressed into a preformed sheet under a pressure of 5-10 MPa. The prepared dense three-dimensional network of Cu/Cu3Sn/Ag3Sn joint, based on Cu@Sn@Ag preform obtained using transient liquid-phase diffusion soldering technology, can withstand high temperatures up to 600 °C in power device applications. The mechanical and thermal performance and power cycle reliability of the Cu@Sn@Ag joint are investigated and comparatively analyzed with PbSn5Ag2.5 joints. The results show that the average shear strength of the Cu@Sn@Ag joint is ∼35 MPa, which exceeds that of PbSn5Ag2.5 solder joint, and is similar to that of sintered nano-silver solder joint; the minimum thermal resistance of the corresponding device is ∼0.18 K W-1, near to that of PbSn5Ag2.5 joint. The growth rate of the forward voltage drops below 2% following 150 000 active power cycles, with a junction temperature difference below 60 °C, meeting the requirements of reliability test standards for vehicle specifications. It is concluded that the performance and power cycle reliability of the Cu@Sn@Ag joint are better than those of the PbSn5Ag2.5 joint.

Synthesis, physicochemical characteristics, cytocompatibility, and antibacterial properties of iron-doped biphasic calcium phosphate nanoparticles with incorporation of silver.

The application of biphasic calcium phosphate (BCP) in tissue engineering and regenerative medicine has been widely explored due to its extensively documented multi-functionality. The present study attempts to synthesize a new type of BCP nanoparticles, characterised with favourable cytocompatibility and antibacterial properties via modifications in their structure, functionality and assemblage, using dopants. In this regard, this study initially synthesized iron-doped BCP (FB) nanoparticles with silver subsequently incorporated into FB nanoparticles to create a nanostructured composite (FBAg). The FB and FBAgnanoparticles were then characterized using Fourier transform infrared spectroscopy, x-ray diffraction, ultraviolet-visible spectroscopy, and x-ray photoelectron spectroscopy. The results showed that silver was present in the FBAgnanoparticles, with a positive correlation observed between increasing AgNO3concentrations and increasing shape irregularity and reduced particle size distribution. Additionally, cell culture tests revealed that both FB and FBAgnanoparticles were compatible with bone marrow-derived mesenchymal stem cells (hBMSCs). The antibacterial activity of the FBAgnanoparticles was also tested using Gram-negativeE. coliand Gram-positiveS. aureus, and was found to be effective against both bacteria. The inhibition rates of FBAgnanoparticles againstE. coliandS. aureuswere 33.78 ± 1.69-59.03 ± 2.95%, and 68.48 ± 4.11-89.09 ± 5.35%, respectively. These findings suggest that the FBAgnanoparticles have potential use in future biomedical applications.

Multiple Functions of Compatible Solute Ectoine and Strategies for Constructing Overproducers for Biobased Production.

Ectoine and its derivative 5-hydroxyectoine are compatible solutes initially found in the hyperhalophilic bacterium Ectothiorhodospira halochloris, which inhabits the desert in Egypt. The habitat of ectoine producers implies the primary function of ectoine as a cytoprotectant against harsh conditions such as high salinity, drought, and high radiation. More extensive and in-depth studies have revealed the multiple functions of ectoine in its native producer bacterial cells and other types of cells and its biomolecular components (such as proteins and DNA) as a general protective agent. Its chemical properties as a bio-based amino acid derivative make it attractive for basic scientific research and related industries, such as the food/agricultural industry, cosmetic manufacturing, biologics, and therapeutic agent preparation. This article first discusses the functions and applications of ectoine and 5-hydroxyectoine. Subsequently, more emphasis was placed on advances in bio-based ectoine and/or 5-hydroxyectoine production. Strategies for developing more robust cell factories for highly efficient ectoine and/or 5-hydroxyectoine production are further discussed. We hope this review will provide a valuable reference for studies on the bio-based production of ectoine and 5-hydroxyectoine.

Bioinformatics-aided Protein Sequence Analysis and Engineering.

Most of the currently available knowledge about protein structure and function has been obtained from laboratory experiments. As a complement to this classical knowledge discovery activity, bioinformatics-assisted sequence analysis, which relies primarily on biological data manipulation, is becoming an indispensable option for the modern discovery of new knowledge, especially when large amounts of protein-encoding sequences can be easily identified from the annotation of highthroughput genomic data. Here, we review the advances in bioinformatics-assisted protein sequence analysis to highlight how bioinformatics analysis will aid in understanding protein structure and function. We first discuss the analyses with individual protein sequences as input, from which some basic parameters of proteins (e.g., amino acid composition, MW and PTM) can be predicted. In addition to these basic parameters that can be directly predicted by analyzing a protein sequence alone, many predictions are based on principles drawn from knowledge of many well-studied proteins, with multiple sequence comparisons as input. Identification of conserved sites by comparing multiple homologous sequences, prediction of the folding, structure or function of uncharacterized proteins, construction of phylogenies of related sequences, analysis of the contribution of conserved related sites to protein function by SCA or DCA, elucidation of the significance of codon usage, and extraction of functional units from protein sequences and coding spaces belong to this category. We then discuss the revolutionary invention of the "QTY code" that can be applied to convert membrane proteins into water- soluble proteins but at the cost of marginal introduced structural and functional changes. As machine learning has been done in other scientific fields, machine learning has profoundly impacted protein sequence analysis. In summary, we have highlighted the relevance of the bioinformatics-assisted analysis for protein research as a valuable guide for laboratory experiments.

Understanding Protein Functions in the Biological Context.

Proteins are essential biomacromolecules in all living systems because they are the prominent ultimate executives of the genetic information stored in DNA. Thus, studying protein is one of the central tasks in biological sciences. The complexity, diversity, and dynamics of a protein's structure, function, and structure-function relationship, the inherent structural fragility and thus the requirements on handling proteins to maintain protein's structural and functional orderliness make it a rather tricky task to work with protein. The approach to understanding the functions of a protein has been progressing steadily. In this paper, we reviewed the progress on the approach to the functional study of proteins that tremendously contributed to understanding their biological significance. Emphasis was put on the advances in the age in which high-throughput DNA sequencing and bioinformatics analysis are revolutionizing biological study.

Mutations in the regulatory regions result in increased streptomycin resistance and keratinase synthesis in Bacillus thuringiensis.

Keratinases are a group of proteases of great industrial significance. To take full advantage of Bacillus species as an inherent superior microbial producer of proteases, we performed the ribosome engineering to improve the keratinase synthesis capacity of the wild-type Bacillus thuringiensis by inducing streptomycin resistance. Mutant Bt(Str-O) was identified as a stable keratinase overproducer. Comparative characterization of the two strains revealed that, although the resistance to Streptomycin increased by eight-fold in MIC, the mutant's resistance to other commonly used antibiotics was not affected. Furthermore, the mutant exhibited an enhanced keratinase synthesis (1.5-fold) when cultured in a liquid LB medium. In the whole feather degradation experiment, the mutant could secret twofold keratinase into the medium, reaching 640 U/mL per 107 CFU. By contrast, no significant differences were found in the scanning electron microscopic analysis and spore formation experiment. To understand the genetic factors causing these phenotypic changes, we cloned and analyzed the rpsL gene. No mutation was observed. We subsequently determined the genome sequences of the two strains. Comparing the rpsL gene revealed that the emergence of streptomycin resistance was not necessarily dependent on the mutation(s) in the generally recognized "hotspot." Genome-wide analysis showed that the phenotypic changes of the mutant were the collective consequence of the genetic variations occurring in the regulatory regions and the non-coding RNA genes. This study demonstrated the importance of genetic changes in regulatory regions and the effectiveness of irrational ribosome engineering in creating prokaryotic microbial mutants without sufficient genetic information.

The Establishment of Quantitatively Regulating Expression Cassette with sgRNA Targeting BIRC5 to Elucidate the Synergistic Pathway of Survivin with P-Glycoprotein in Cancer Multi-Drug Resistance.

Quantitative analysis and regulating gene expression in cancer cells is an innovative method to study key genes in tumors, which conduces to analyze the biological function of the specific gene. In this study, we found the expression levels of Survivin protein (BIRC5) and P-glycoprotein (MDR1) in MCF-7/doxorubicin (DOX) cells (drug-resistant cells) were significantly higher than MCF-7 cells (wild-type cells). In order to explore the specific functions of BIRC5 gene in multi-drug resistance (MDR), a CRISPR/Cas9-mediated knocking-in tetracycline (Tet)-off regulatory system cell line was established, which enabled us to regulate the expression levels of Survivin quantitatively (clone 8 named MCF-7/Survivin was selected for further studies). Subsequently, the determination results of doxycycline-induced DOX efflux in MCF-7/Survivin cells implied that Survivin expression level was opposite to DOX accumulation in the cells. For example, when Survivin expression was down-regulated, DOX accumulation inside the MCF-7/Survivin cells was up-regulated, inducing strong apoptosis of cells (reversal index 118.07) by weakening the release of intracellular drug from MCF-7/Survivin cells. Also, down-regulation of Survivin resulted in reduced phosphorylation of PI3K, Akt, and mTOR in MCF-7/Survivin cells and significantly decreased P-gp expression. Previous studies had shown that PI3K/Akt/mTOR could regulate P-gp expression. Therefore, we speculated that Survivin might affect the expression of P-gp through PI3K/Akt/mTOR pathway. In summary, this quantitative method is not only valuable for studying the gene itself, but also can better analyze the biological phenomena related to it.

A Thermostable Aluminum-Tolerant Protease Produced by Feather-Degrading Bacillus thuringiensis Isolated from Tea Plantation.

BACKGROUND: Proteases with keratinolytic activity are widely used in biotechnologies. The feather-degrading Bacillus thuringensis isolated from soil sample of a tea plantation produced high level of extracellular keratinase. OBJECTIVE: This study aimed to analyze the properties by biochemical and enzymological methods to gain information for better utilization of the enzyme. METHODS: The enzyme was purified with ion exchange and size exclusion chromatography. The substrate preference, optimal pH and temperature, and the effects of organic solvents and ions were checked. Circular dichroism was performed to compare the secondary structures of the native and apo-enzyme. RESULTS: The enzyme worked best at 50°C, and it was an acidic serine protease with an optimal pH of 6.2. Ions Ca2+ and Mg2+ were essential for its activity. Organic solvents and other metal ions generally deactivated the enzyme in a concentration-dependent manner. However, Mn2+ and DMSO, which were frequently reported as inhibitors of protease, could activate the enzyme at low concentration (0.01 to 2 mmol/L of Mn2+; DMSO <2%, v/v). The enzyme exhibited high resistance to Al3+, which might be explained by the soil properties of its host's residence. Circular dichroism confirmed the contribution of ions to the structure and activity. CONCLUSION: The enzyme was a thermostable aluminum-tolerant serine protease with unique biochemical properties.

Transcriptomic analysis of tea plant (Camellia sinensis) revealed the co-expression network of 4111 paralogous genes and biosynthesis of quality-related key metabolites under multiple stresses.

The tea plant is an essential economic plant in many countries. However, its growing season renders them vulnerable to stresses. To understand the transcriptomic influences of these stresses on tea plants, we sequenced and analyzed the transcriptomes under drought, high-temperature, and pest. Paralogs were identified by comparing 14 evolutionarily close genomes. The differentially expressed paralog (DEPs) genes were analyzed regarding single or multiple stresses, and 1075 of the 4111 DEPs were commonly found in all the stresses. The co-expression network of the DEPs and TFs indicated that genes of catechin biosynthesis were associated with most transcription factors specific to each stress. The genes playing a significant role in the late response to drought and pest stress mainly functioned in the early response to high-temperature. This study revealed the relationship between stress and regulation of QRM synthesis and the role of QRMs in response to these (a)biotic stresses.

Polyvinyl Alcohol/Sodium Alginate Hydrogels Incorporated with Silver Nanoclusters via Green Tea Extract for Antibacterial Applications.

Silver-based nanoparticles and biomaterials have extensive biomedical applications owing to their unique antimicrobial properties. Thus, green and facile synthesis of such materials is highly desirable. This study reports an antibacterial hydrogel based on polyvinyl alcohol/sodium alginate network with the incorporation of silver nanoparticles (AgNPs), which is greenly synthesized by reductive metabolites obtained from the leaves of green tea. The 'flower-shape' AgNPs were acquired, it formed a mono-disperse system with a distinct uniform interparticle separation. The average size of AgNPs varied from 129.5 to 243.6 nm, which could be regulated by using different volumes of the green tea extract. Zeta potentials of the AgNPs were from -39.3 mV to -20.3 mV, indicating the moderate stability of the particles in water. In the next stage, the antibacterial polyvinyl alcohol/sodium alginate hydrogels were fabricated by incorporating prepared AgNPs. Scanning Electron Microscopy (SEM) images showed that the porous structure was obtained, and Energy Dispersive X-Ray (EDX) analysis confirmed that the AgNPs were uniformly dispersed in the polymer network. The hydrogels exhibited superior water absorption properties, which were characterized by a high swelling ratio (500-900%) and fast equilibrium. The hydrogels also exhibited good antimicrobial activity in assays with Gram-positive bacteria Escherichia coli and Gram-negative bacteria Staphylococcus aureus. To sum up, a process for the green preparation of antibacterial hydrogels based on AgNPs derived from tea leaves as a conveniently available cheap local agricultural product was established.

Nanostructured selenium-doped biphasic calcium phosphate with in situ incorporation of silver for antibacterial applications.

Selenium-doped nanostructure has been considered as an attractive approach to enhance the antibacterial activity of calcium phosphate (CaP) materials in diverse medical applications. In this study, the selenium-doped biphasic calcium phosphate nanoparticles (SeB-NPs) were first synthesized. Then, silver was in situ incorporated into SeB-NPs to obtain nanostructured composite nanoparticles (AgSeB-NPs). Both SeB-NPs and AgSeB-NPs were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), ultraviolet-visible spectroscopy (UV-Vis), X-ray photoelectron spectroscopy (XPS), and Raman spectra. The results confirmed that the SeO32- was doped at the PO43- position and silver nanoparticles were deposited on the surface of SeB-NPs. Next, Transmission Electron Microscopy (TEM) analysis displayed that the prepared AgSeB-NPs had a needle-cluster-like morphology. CCK-8 analysis revealed SeB-NPs and AgSeB-NPs had good cytocompatibility with osteoblasts. The antibacterial activity of the prepared AgSeB-NPs was confirmed by using Gram-negative E. coli and Gram-positive S. aureus. The above results manifested the significance of the final AgSeB-NPs for biomedical applications.

Effective isolation of antioxidant Phelligridin LA from the fermentation broth of Inonotus baumii by macroporous resin.

Phelligridin LA (PLA) is a natural product with vigorous free radical scavenging activities accumulated in the liquid fermentation of herbal medicinal fungus Inonotus baumii. Aiming to establish an efficient isolation method of PLA from the fermentation broth, we evaluated the adsorption of PLA by macroporous resins. The best resin ADS-17 was screened for six candidates with various physical properties and adsorption behaviors. Studies on the thermodynamics and kinetics of the process revealed that the adsorption reaction could take place spontaneously, which implied that the heat generated in adsorption might compensate for the decrease in entropy. The Freundlich theory could be utilized to fit the experimental data. The pseudo-second-order equation could describe the process, and the adsorption rate was primarily controlled by liquid film diffusion and pore diffusion. The influencing operation factors (temperature, pH, and the ratio of fermentation broth to resin) of the adsorption process were optimized with response surface methodology. The optimized condition (temperature 22.81 °C, pH 5.19, and the ratio of fermentation broth to resin or RLS 5.11) supported an adsorption rate of 97.03%. These findings would be indispensable for further optimization of the efficient separation of PLA from the fermentation broth, and the fermentation production of PLA in which separation would be included.

The differential expression patterns and co-expression networks of paralogs as an indicator of the TNM stages of lung adenocarcinoma and squamous cell carcinoma.

Cancers constitute a severe threat to human health. Elucidating the association between the expression patterns of the paralogous genes and transcription factors (TF) and the progression of cancers by comprehensively investigating the expression patterns and co-expression networks will contribute to the in-depth understanding of the pathogenesis of cancers. Here, we identified the paralogous gene pairs and systematically analyzed the expression patterns of these paralogs and the known TFs to elucidate the associations with Tumor, Node, Metastasis (TNM) staging information across ten cancers. We found that the expression of ~60% paralogs was cancer-dependent, and more than 50% of the differentially expressed TFs pairs showed positive expression correlations. The down-regulation patterns of paralogs and TFs were closely associated with the M and N developmental stages of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Our results will help to understand the roles of paralogs and TFs in cancer progression and to screen prognostic biomarkers for early cancer diagnosis.

Ribosome Hibernation as a Stress Response of Bacteria.

Ribosome is primarily regarded as the committing organelle for the translation process. Besides the expansion of its function from a translational machine for protein synthesis to a regulatory platform for protein quality control, the activity regulation and recycling of ribosome have been deepened significantly. Recent advances have confirmed a novel mechanism in the regulation of ribosome activity when a cell encounters adverse conditions. Due to the binding of certain protein factors onto a ribosome, the structural and functional change of the ribosome inside the cell will take place, thereby leading to the formation of inactive ribosomes (70S monomer or 100S dimer), or ribosome hibernation. By ribosome hibernation, the overall protein synthesis rate of a cell could be slowed down. The resistance to adverse conditions or chemicals of the host cell will be enhanced. In this paper, we discussed the phenomenon, molecular mechanism, and physiological effect of ribosome hibernation when cells are under stresses. And then, we discussed the resuscitation of a hibernating ribosome and the role of ribosome hibernation in the treatment of antimicrobial infection.

Small design from big alignment: engineering proteins with multiple sequence alignment as the starting point.

Multiple sequence alignment (MSA) is a fundamental way to gain information that cannot be obtained from the analysis of any individual sequence included in the alignment. It provides ways to investigate the relationship between sequence and function from a perspective of evolution. Thus, the MSA of proteins can be employed as a reference for protein engineering. In this paper, we reviewed the recent advances to highlight how protein engineering was benefited from the MSA of proteins. These methods include (1) engineering the thermostability or solubility of proteins by making it closer to the consensus sequence of the alignment through introducing site mutations; (2) structure-based engineering proteins with comparative modeling; (3) creating paleoenzymes featured with high thermostability and promiscuity by constructing the ancestral sequences derived from multiple sequence alignment; and (4) incorporating site-mutations targeting the evolutionarily coupled sites identified from multiple sequence alignment.

Incorporation of nonstandard amino acids into proteins: principles and applications.

The cellular ribosome shows a naturally evolved strong preference for the synthesis of proteins with standard amino acids. An in-depth understanding of the translation process enables scientists to go beyond this natural limitation and engineer translating systems capable of synthesizing proteins with artificially designed and synthesized non-standard amino acids (nsAA) featuring more bulky sidechains. The sidechains can be functional groups, with chosen biophysical or chemical activities, that enable the direct application of these proteins. Alternatively, the sidechains can be designed to contain highly reactive groups: enabling the ready formation of conjugates via a covalent bond between the sidechain and other chemicals or biomolecules. This co-translational incorporation of nsAAs into proteins allows for a vast number of possible applications. In this paper, we first systematically summarized the advances in the engineering of the translation system. Subsequently, we reviewed the extensive applications of these nsAA-containing proteins (after chemical modification) by discussing representative reports on how they can be utilized for different purposes. Finally, we discussed the direction of further studies which could be undertaken to improve the current technology utilized in incorporating nsAAs in order to use them to their full potential and improve accessibility across disciplines.

Engineering the Translational Machinery for Biotechnology Applications.

The ribosome is an essential organelle in charge of the translational processes in all kinds of cells. Currently, the scenario of its function has been significantly expanded from the classic machine for protein synthesis to a regulatory platform for quality control to maintain the protein homeostasis in a living cell. The ribosome is much more than a mechanical device with a static structure: it is inherently dynamic in structure and function, especially in response to the environmental fluctuations. Considerable effort has been made to regulate its structure and physiological function by engineering the components of a ribosome. The findings of the pioneering studies significantly deepened our understanding of a ribosome and exemplified how a ribosome could be engineered for biotechnology purposes in the era of synthetic biology. The engineering of ribosome offered highly accessible methods capable of comprehensively optimizing the performance of strains of industrial importance. In this article, the relevant recent advances were systematically reviewed.