Spatial transcriptome uncovers rich coordination of metabolism in E. coli K12 biofilm.

Microbial communities often display region-specific properties, which give rise to complex interactions and emergent behaviors that are critical to the homeostasis and stress response of the communities. However, systems-level understanding of these properties still remains elusive. In this study, we established RAINBOW-seq and profiled the transcriptome of Escherichia coli biofilm communities with high spatial resolution and high gene coverage. We uncovered three modes of community-level coordination, including cross-regional resource allocation, local cycling and feedback signaling, which were mediated by strengthened transmembrane transport and spatially specific activation of metabolism. As a consequence of such coordination, the nutrient-limited region of the community maintained an unexpectedly high level of metabolism, enabling it to express many signaling genes and functionally unknown genes with potential sociality functions. Our work provides an extended understanding of the metabolic interplay in biofilms and presents a new approach of investigating complex interactions in bacterial communities on the systems level.

Virological evaluation of natural and modified attapulgite against porcine epidemic diarrhoea virus.

BACKGROUND: The Porcine Epidemic Diarrhea Virus (PEDV) has caused significant economic losses in the global swine industry. As a potential drug for treating diarrhea, the antiviral properties of attapulgite deserve further study. METHODS: In this study, various methods such as RT-qPCR, Western blot, viral titer assay, Cytopathic Effect, immunofluorescence analysis and transmission electron microscopy were used to detect the antiviral activity of attapulgite and to assess its inhibitory effect on PEDV. RESULTS: When exposed to the same amount of virus, there was a significant decrease in the expression of the S protein, resulting in a viral titer reduction from 10-5.613 TCID50/mL to 10-2.90 TCID50/mL, which represents a decrease of approximately 102.6 folds. Results of cytopathic effect and indirect immunofluorescence also indicate a notable decrease in viral infectivity after attapulgite treatment. Additionally, it was observed that modified materials after acidification had weaker antiviral efficacy compared to powdered samples that underwent ultrasonic disintegration, which showed the strongest antiviral effects. CONCLUSION: As a result, Attapulgite powders can trap and adsorb viruses to inhibit PEDV in vitro, leading to loss of viral infectivity. This study provides new materials for the development of novel disinfectants and antiviral additives.

A strategy to distinguish similar traditional Chinese medicines by liquid chromatography-mass spectrometry, electronic senses, and gas chromatography-ion mobility spectrometry: Marsdeniae tenacissimae Caulis and Paederiae scandens Caulis as examples.

INTRODUCTION: Marsdeniae tenacissimae Caulis (MTC), a popular traditional Chinese medicine, has been widely used in the treatment of tumor diseases. Paederiae scandens Caulis (PSC), which is similar in appearance to MTC, is a common counterfeit product. It is difficult for traditional methods to effectively distinguish between MTC and PSC. Therefore, there is an urgent need for a rapid and accurate method to identify MTC and PSC. OBJECTIVES: The aim is to distinguish between MTC and PSC by analyzing the differences in nonvolatile organic compounds (NVOCs), taste, odor, and volatile organic compounds (VOCs). METHODS: Liquid chromatography-mass spectrometry (LC-MS) was utilized to analyze the NVOCs of MTC and PSC. Electronic tongue (E-tongue) and electronic nose (E-nose) were used to analyze their taste and odor respectively. Gas chromatography-ion mobility spectrometry (GC-IMS) was applied to analyze VOCs. Finally, multivariate statistical analyses were conducted to further investigate the differences between MTC and PSC, including principal component analysis, orthogonal partial least squares discriminant analysis, discriminant factor analysis, and soft independent modeling of class analysis. RESULTS: The results of this study indicate that the integrated strategy of LC-MS, E-tongue, E-nose, GC-IMS, and multivariate statistical analysis can be effectively applied to distinguish between MTC and PSC. Using LC-MS, 25 NVOCs were identified in MTC, while 18 NVOCs were identified in PSC. The major compounds in MTC are steroids, while the major compounds in PSC are iridoid glycosides. Similarly, the distinct taste difference between MTC and PSC was precisely revealed by the E-tongue. Specifically, the pronounced bitterness in PSC was proven to stem from iridoid glycosides, whereas the bitterness evident in MTC was intimately tied to steroids. The E-nose detected eight odor components in MTC and six in PSC, respectively. The subsequent statistical analysis uncovered notable differences in their odor profiles. GC-IMS provided a visual representation of the differences in VOCs between MTC and PSC. The results indicated a relatively high relative content of 82 VOCs in MTC, contrasted with 32 VOCs exhibiting a similarly high relative content in PSC. CONCLUSION: In this study, for the first time, the combined use of LC-MS, E-tongue, E-nose, GC-IMS, and multivariate statistical analysis has proven to be an effective method for distinguishing between MTC and PSC from multiple perspectives. This approach provides a valuable reference for the identification of other visually similar traditional Chinese medicines.

Guide-target mismatch effects on dCas9-sgRNA binding activity in living bacterial cells.

As an effective programmable DNA targeting tool, CRISPR-Cas9 system has been adopted in varieties of biotechnological applications. However, the off-target effects, derived from the tolerance towards guide-target mismatches, are regarded as the major problems in engineering CRISPR systems. To understand this, we constructed two sgRNA libraries carrying saturated single- and double-nucleotide mismatches in living bacteria cells, and profiled the comprehensive landscape of in vivo binding affinity of dCas9 toward DNA target guided by each individual sgRNA with particular mismatches. We observed a synergistic effect in seed, where combinatorial double mutations caused more severe activity loss compared with the two corresponding single mutations. Moreover, we found that a particular mismatch type, dDrG (D = A, T, G), only showed moderate impairment on binding. To quantitatively understand the causal relationship between mismatch and binding behaviour of dCas9, we further established a biophysical model, and found that the thermodynamic properties of base-pairing coupled with strand invasion process, to a large extent, can account for the observed mismatch-activity landscape. Finally, we repurposed this model, together with a convolutional neural network constructed based on the same mechanism, as a predictive tool to guide the rational design of sgRNA in bacterial CRISPR interference.

[Qualitative and quantitative study of flavonoids in Notoginseng Radix et Rhizoma based on UPLC-Q-TOF-MS and HPLC-DAD].

The flavonoids in Panax notoginseng were qualitatively analyzed by ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF-MS), and the content of three main flavonoids in P. notoginseng of different specifications and grades collected from different habitats was determined by HPLC-DAD. Flavonoids and anthocyanins were analyzed by UPLC-Q-TOF-MS/MS in the positive and negative ion modes, respectively. Twelve flavonoid glycosides and one anthocyanin glycoside in P. notoginseng were identified, but no flavonoid aglycones were detected. Among them, 12 compounds were identified in the underground part of P. notoginseng for the first time and eight compounds were first reported in this plant. Moreover, six and four compounds were identified in the Panax genus and the Araliaceae family for the first time, respectively. A method for simultaneous determination of three flavonoids in P. notoginseng was established by HPLC-DAD. The content of flavonoids in 721 P. notoginseng samples of 124 specifications and grades collected from 20 different habitats was simultaneously determined. Among three flavonoids determined, the content of quercetin-3-O-(2″-ß-D-xylosyl)-ß-D-galactoside was the highest with the average content in the tested samples of 161.0 µg·g~(-1). The content of compounds quercetin-3-O-hexosyl-hexoside and kaempferol-3-O-pentosyl-hexoside was relatively low, with the average content of 18.5 µg·g~(-1)(calculated as quercetin-3-O-sophoroside) and 49.4 µg·g~(-1)(calculated as kaempferol-3-O-sangbu diglycoside). There were significant differences in flavonoids content of samples from different production area. The content of flavonoids in spring P. notoginseng was significantly lower than that in winter P. notoginseng when the other influencing factors such as production areas, germplasm resources, and cultivation conditions were fixed. As for P. notoginseng of different specifications, the flavonoid content in the part connecting the taproot and the aboveground stem was significantly higher than that in other parts. The results of large-scale data showed that the flavonoid content gradually increased with the increase in the number of heads. There were significant differences between the flavonoid content in most specifications and grades, especially the 20-head P. notoginseng and countless head P. notoginseng, whose content was significantly lower and significantly higher than that of other specifications and grades, respectively. This study provides a scientific basis for the study of the effective components and quality control of P. notoginseng from the perspective of flavonoids.

Profiling of bacterial transcriptome from ultra-low input with MiniBac-seq.

There is a lack of appropriate methods for preparing bacterial RNA-seq library with ultra-low amount of RNA. To address this issue, we developed miniBac-seq, a strand-specific method for high-quality library construction from sub-nanogram of total RNA, which is 100-fold lower than the current benchmark kit and dramatically reduces preparation cost ($28 + $15 × samples). We further demonstrated the high sensitivity of miniBac-seq via detecting more than 500 genes from amount of total RNA equivalent to that of a single bacterial cell. Finally, we profiled the transcriptome of growth-arrested bacteria in isogenic culture of Escherichia coli. This subpopulation of bacteria is generally low in abundance but is a potent reservoir of antibiotic persistence, and their gene expression has been largely unknown due to technical limitations. Using miniBac-seq, we identified potential molecular driver towards arrested growth as well as antibiotic tolerance. Our method thus expands the capacity to quantify bacterial transcriptome in situ, which is useful to the understanding of bacterial physiology and regulation in their native contexts.

Dynamics of transcription-translation coordination tune bacterial indole signaling.

Indole signaling is an important cross-species communication pathway in the mammalian gut. In bacteria, upon induction by tryptophan, the molecular sensor (tnaC) controls indole biosynthesis by precisely coordinating dynamics of the corresponding macromolecular machineries during its transcription and translation. Our understanding of this regulatory program is still limited owing to its rapid dynamic nature. To address this shortcoming, we adopted a massively parallel profiling method to quantify the responses of 1,450 synthetic tnaC variants in the presence of three concentrations of tryptophan in living bacterial cells. The resultant dataset enabled us to comprehensively probe the key intermediate states of macromolecular machineries during the transcription and translation of tnaC. We also used modeling to provide a systems-level understanding of how these critical states collectively shape the output of this regulatory program quantitatively. A similar methodology will likely apply to other poorly understood dynamics-dependent cis-regulatory elements.

Integrated metabolomic and transcriptomic analysis reveals variation in the metabolites and genes of Platycodon grandiflorus roots from different regions.

INTRODUCTION: Platycodon grandiflorum root (PG), a popular traditional Chinese medicine, contains considerable chemical components with broad pharmacological activities. The complexity and diversity of the chemical components of PG from different origins contribute to its broad biological activities. The quality of southern PG is superior to that of northern PG, but the mechanisms underlying these differences remain unclear. OBJECTIVES: In order to study variation in the differentially accumulated metabolites (DAMs), differentially expressed genes (DEGs), as well as their interactions and signalling pathways among PG from Anhui and Liaoning. METHODS: The metabolomes based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the transcriptome based on high-throughput sequencing technology were combined to comprehensively analyse PGn and PGb. RESULTS: A total of 6515 DEGs and 83 DAMs from the comparison of PG from Anhui and Liaoning were detected. Integrated analysis of metabolomic and transcriptomic data revealed that 215 DEGs and 57 DAMs were significantly enriched in 48 pathways according to KEGG pathway enrichment analysis, and 15 DEGs and 10 DAMs significantly enriched in the main pathway sesquiterpenoid and triterpenoid and phenylpropanoid biosynthesis might play a key role in complex response or regulatory processes. CONCLUSION: Differences in PG from southern and northern China might thus stem from differences in environmental factors, such as precipitation, light duration, and humidity. The results of our study provide new insight into geographic variation in gene expression and metabolite accumulation and will enhance the utilisation of PG resources.

Establishment of CRISPR interference in Methylorubrum extorquens and application of rapidly mining a new phytoene desaturase involved in carotenoid biosynthesis.

The methylotrophic bacterium Methylorubrum extorquens AM1 holds a great potential of a microbial cell factory in producing high value chemicals with methanol as the sole carbon and energy source. However, many gene functions remain unknown, hampering further rewiring of metabolic networks. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been demonstrated to be a robust tool for gene knockdown in diverse organisms. In this study, we developed an efficient CRISPRi system through optimizing the promoter strength of Streptococcus pyogenes-derived deactivated cas9 (dcas9). When the dcas9 and sgRNA were respectively controlled by medium PR/tetO and strong PmxaF-g promoters, dynamic repression efficacy of cell growth through disturbing a central metabolism gene glyA was achieved from 41.9 to 96.6% dependent on the sgRNA targeting sites. Furthermore, the optimized CRISPRi system was shown to effectively decrease the abundance of exogenous fluorescent protein gene mCherry over 50% and to reduce the expression of phytoene desaturase gene crtI by 97.7%. We then used CRISPRi technology combined with 26 sgRNAs pool to rapidly discover a new phytoene desaturase gene META1_3670 from 2470 recombinant mutants. The gene function was further verified through gene deletion and complementation as well as phylogenetic tree analysis. In addition, we applied CRISPRi to repress the transcriptional level of squalene-hopene cyclase gene shc involved in hopanoid biosynthesis by 64.9%, which resulted in enhancing 1.9-fold higher of carotenoid production without defective cell growth. Thus, the CRISPRi system developed here provides a useful tool in mining functional gene of M. extorquens as well as in biotechnology for producing high-valued chemicals from methanol. KEY POINTS: Developing an efficient CRISPRi to knockdown gene expression in C1-utilizing bacteria CRISPRi combined with sgRNAs pool to rapidly discover a new phytoene desaturase gene Improvement of carotenoid production by repressing a competitive pathway.

Improved sgRNA design in bacteria via genome-wide activity profiling.

CRISPR/Cas9 is a promising tool in prokaryotic genome engineering, but its success is limited by the widely varying on-target activity of single guide RNAs (sgRNAs). Based on the association of CRISPR/Cas9-induced DNA cleavage with cellular lethality, we systematically profiled sgRNA activity by co-expressing a genome-scale library (∼70 000 sgRNAs) with Cas9 or its specificity-improved mutant in Escherichia coli. Based on this large-scale dataset, we constructed a comprehensive and high-density sgRNA activity map, which enables selecting highly active sgRNAs for any locus across the genome in this model organism. We also identified 'resistant' genomic loci with respect to CRISPR/Cas9 activity, notwithstanding the highly accessible DNA in bacterial cells. Moreover, we found that previous sgRNA activity prediction models that were trained on mammalian cell datasets were inadequate when coping with our results, highlighting the key limitations and biases of previous models. We hence developed an integrated algorithm to accurately predict highly effective sgRNAs, aiming to facilitate CRISPR/Cas9-based genome engineering, screenings and antimicrobials design in bacteria. We also isolated the important sgRNA features that contribute to DNA cleavage and characterized their key differences among wild type Cas9 and its mutant, shedding light on the biophysical mechanisms of the CRISPR/Cas9 system.

Genome-wide screening identifies promiscuous phosphatases impairing terpenoid biosynthesis in Escherichia coli.

Terpenoids are a large family of natural compounds that are important for both biotechnological applications and basic microorganism physiology. Inspired by the current literature, we hypothesized that recently deciphered phosphatase promiscuity may be an unexplored factor that negatively affects terpenoid biosynthesis by redirecting carbon flux away from the pathway via unrecognized catalytic activities on the phosphorylated intermediates. We used lycopene as a proof-of-concept to test this hypothesis. Based on an extensive bioinformatics analysis, we selected 56 phosphatase-encoding genes in Escherichia coli and constructed a knockdown library for these genes in a lycopene overproducer via CRISPR interference (CRISPRi). We screened this phosphatase knockdown library and observed enrichment (28 of 56) for genes that impair lycopene biosynthesis. Further scaled-up cultivation, combinatorial knockdown, and knockout assays in strains that overproduce lycopene or another terpenoid (ß-carotene) confirmed the proposed relationship between promiscuous phosphatases and impaired terpenoid biosynthesis. This study hence suggests the necessity of reconsidering the interactions of promiscuous phosphatases with ubiquitous phosphorylated components of metabolic networks with respect to engineering metabolism.

Biosensor-assisted transcriptional regulator engineering for Methylobacterium extorquens AM1 to improve mevalonate synthesis by increasing the acetyl-CoA supply.

Acetyl-CoA is not only an important intermediate metabolite for cells but also a significant precursor for production of industrially interesting metabolites. Methylobacterium extorquens AM1, a model strain of methylotrophic cell factories using methanol as carbon source, is of interest because it produces abundant coenzyme A compounds capable of directing to synthesis of different useful compounds from methanol. However, acetyl-CoA is not always efficiently accumulated in M. extorquens AM1, as it is located in the center of three cyclic central metabolic pathways. Here we successfully demonstrated a strategy for sensor-assisted transcriptional regulator engineering (SATRE) to control metabolic flux re-distribution to increase acetyl-CoA flux from methanol for mevalonate production in M. extorquens AM1 with introduction of mevalonate synthesis pathway. A mevalonate biosensor was constructed and we succeeded in isolating a mutated strain (Q49) with a 60% increase in mevalonate concentration (an acetyl-CoA-derived product) following sensor-based high-throughput screening of a QscR transcriptional regulator library. The mutated QscR-49 regulator (Q8*,T61S,N72Y,E160V) lost an N-terminal α-helix and underwent a change in the secondary structure of the RD-I domain at the C terminus, two regions that are related to its interaction with DNA. 13C labeling analysis revealed that acetyl-CoA flux was improved by 7% and transcriptional analysis revealed that QscR had global effects and that two key points, NADPH generation and fumC overexpression, might contribute to the carbon flux re-distribution. A fed-batch fermentation in a 5-L bioreactor for QscR-49 mutant yielded a mevalonate concentration of 2.67g/L, which was equivalent to an overall yield of 0.055mol acetyl-CoA/mol methanol, the highest yield among engineered strains of M. extorquens AM1. This work was the first attempt to regulate M. extorquens AM1 on transcriptional level and provided molecular insights into the mechanism of carbon flux regulation.

Multiconstituent identification in root, branch, and leaf extracts of Juglans mandshurica using ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry.

As a traditional medicinal plant, Juglans mandshurica has been used for the treatment of cancer. Different organs of this plant showed anti-tumor activity in clinic and laboratory. Comparative identification of constituents in different plant organs is essential for investigation of the relationship between chemical constituents and pharmacological activities. For this aim, the roots, branches, and leaves of J. mandshurica were extracted with 50% v/v methanol and then subjected to ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry analysis conducted under low and high energy. As a result, we have to date identified 111 compounds consisting of 56 tannins, 29 flavonoids, 13 organic acids, 8 naphthalene derivatives, and 5 anthracenes. Five compounds, namely, diquercetin trihydroxy-truxinoyl-glucoside, two quercetin kaempferol dihydroxy-truxinoyl-glucosides, syringoyl-tri-galloyl-O-glucose, and dihydroxy-naphthalene syringoyl-glucoside, were tentatively identified as new compounds. Of the compounds identified, 76 were found in the root extract, 67 in the branch extract, and 37 in the leaf extract. Only six compounds including four organic acids and two tannins were found in all three extracts. We developed a rapid and sensitive ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry approach to identify multiple constituents of complex extracts without separation and ion selection. The results presented provide useful information on further research of the bioactive compounds of J. mandshurica.

Ginseng Protein Reverses Amyloid Beta Peptide and H2 O2 Cytotoxicity in Neurons, and Ameliorates Cognitive Impairment in AD Rats Induced by a Combination of D-Galactose and AlCl3.

Ginseng (Panax ginseng C.A. Meyer) is one of the most widely used herbal medicines worldwide. The present study evaluated the neuroprotective effects of ginseng protein (GP) and its possible mechanisms in a cellular and animal model of AD. The results demonstrated that GP (10-100 µg/mL) significantly improved the survival rate of neurons and reduced the cells' apoptosis and the mRNA expression of caspase-3 and Bax/Bcl-2. In addition, GP (0.1 g/kg) significantly shortened the escape latency, prolonged the crossing times and the percentage of residence time; reduced the level of Aß1-42 and p-tau, the activity of T-NOS and iNOS, and the content of MDA and NO, improved the activity of SOD, the concentration of cAMP and the protein expression of p-PKA/PKA and -CREB/CREB. The results demonstrated that GP significantly inhibited Alzheimer-like pathophysiological changes induced by Aß25-35 or H2 O2 in cells or those induced by D-gal/ Al in animals. These neuroprotective effects of GP may be associated with the cAMP/PKA/CREB pathway. Also, in combination with our previous studies, these results indicate that the anti-AD mechanism of GP was likely to activate the CREB pathway through multiple channels. Copyright © 2016 John Wiley & Sons, Ltd.

Identification of Polar Constituents in the Decoction of Juglans mandshurica and in the Medicated Egg Prepared with the Decoction by HPLC-Q-TOF MS².

As a folk medicinal plant, Juglans mandshurica has been used for the treatment of cancer in China and Korea. Traditionally, J. mandshurica is decocted together with chicken eggs. Both the decoction and medicated eggs possess anti-tumor properties. Clarifying the constituents of the decoction and absorbed by the medicated eggs is essential for the investigation of the active principles of J. mandshurica. Herein, the medicated eggs were prepared by decocting raw chicken eggs, having unbroken shells, with the decoction of J. mandshurica. A systematic investigation of the chemical profile of the J. mandshurica decoction and the medicated egg extraction was conducted by HPLC-Q-TOF-MS². In total, 93 peaks, including 45 tannins, 14 naphthalene derivatives, 17 organic acids, 3 diarylheptanoids, 4 lignans, 3 anthraquinones, 1 flavonoid glycoside, 3 amino acids, and 3 nitrogenous compounds, were tentatively identified in the decoction. In the medicated egg extraction, 44 peaks including 11 organic acids, 3 amino acids, 3 nitrogenous compounds, 8 naphthalene derivatives, 3 diarylheptanoids, 15 tannins, and 1 lignan were tentatively identified. The chemical profile presented provided a detailed overview of the polar chemical constituents in J. mandshurica and useful information for the research of bioactive compounds of this plant.

Intermediate-sensor assisted push-pull strategy and its application in heterologous deoxyviolacein production in Escherichia coli.

Because high-throughput screening tools are typically unavailable when using the pathway-engineering approach, we developed a new strategy, named intermediate sensor-assisted push-pull strategy, which enables sequential pathway optimization by incorporating a biosensor targeting a key pathway intermediate. As proof of concept, we constructed an L-Trp biosensor and used it to optimize the deoxyviolacein biosynthetic pathway, which we divided into two modules with L-Trp being the product of the upstream and the substrate of the downstream module for deoxyviolacein synthesis. Using the biosensor and fluorescence-activated cell sorting, the activities of the two modules were sequentially and independently optimized in Escherichia coli to achieve the desired phenotypes. By this means, we increased the deoxyviolacein titer 4.4-fold (1.92 g/L), which represents the greatest deoxyviolacein production reported. This work suggests that a biosynthetic pathway can be enhanced to produce a value-added secondary metabolite(s) without available end-product screening method by using a central metabolic junction molecule biosensor(s).

DomSign: a top-down annotation pipeline to enlarge enzyme space in the protein universe.

BACKGROUND: Computational predictions of catalytic function are vital for in-depth understanding of enzymes. Because several novel approaches performing better than the common BLAST tool are rarely applied in research, we hypothesized that there is a large gap between the number of known annotated enzymes and the actual number in the protein universe, which significantly limits our ability to extract additional biologically relevant functional information from the available sequencing data. To reliably expand the enzyme space, we developed DomSign, a highly accurate domain signature-based enzyme functional prediction tool to assign Enzyme Commission (EC) digits. RESULTS: DomSign is a top-down prediction engine that yields results comparable, or superior, to those from many benchmark EC number prediction tools, including BLASTP, when a homolog with an identity >30% is not available in the database. Performance tests showed that DomSign is a highly reliable enzyme EC number annotation tool. After multiple tests, the accuracy is thought to be greater than 90%. Thus, DomSign can be applied to large-scale datasets, with the goal of expanding the enzyme space with high fidelity. Using DomSign, we successfully increased the percentage of EC-tagged enzymes from 12% to 30% in UniProt-TrEMBL. In the Kyoto Encyclopedia of Genes and Genomes bacterial database, the percentage of EC-tagged enzymes for each bacterial genome could be increased from 26.0% to 33.2% on average. Metagenomic mining was also efficient, as exemplified by the application of DomSign to the Human Microbiome Project dataset, recovering nearly one million new EC-labeled enzymes. CONCLUSIONS: Our results offer preliminarily confirmation of the existence of the hypothesized huge number of "hidden enzymes" in the protein universe, the identification of which could substantially further our understanding of the metabolisms of diverse organisms and also facilitate bioengineering by providing a richer enzyme resource. Furthermore, our results highlight the necessity of using more advanced computational tools than BLAST in protein database annotations to extract additional biologically relevant functional information from the available biological sequences.

[Studies on optimizing extraction technics of flowers of Polygonum orientale by response surface methodology].

OBJECTIVE: To analyze and optimize extraction technics of Polygonum orientale flowers by response surface methodology. METHODS: With the index for the content of taxifolin in flowers of Polygonum orientale, the effect of three factors such as concentration of alcohol, extraction time and solvent-solid ratio was designed by Box-Behnken central composite. Extraction technic parameters of Polygonum orientale flowers was optimized by response surface methodology. RESULTS: The optimizing extraction conditions of Polygonum orientale flowers were as follows: ethanol concentration was 65%, extracting time was 129 min and solvent-solid ratio was 18. Under the conditions, the average yield of taxifolin in 3 validation experiments was 2.79 mg/g. CONCLUSION: Optimizing extraction technics by response surface methodology is reasonable, simple, and has good predictability.

RNA polymerase stalling-derived genome instability underlies ribosomal antibiotic efficacy and resistance evolution.

Bacteria often evolve antibiotic resistance through mutagenesis. However, the processes causing the mutagenesis have not been fully resolved. Here, we find that a broad range of ribosome-targeting antibiotics cause mutations through an underexplored pathway. Focusing on the clinically important aminoglycoside gentamicin, we find that the translation inhibitor causes genome-wide premature stalling of RNA polymerase (RNAP) in a loci-dependent manner. Further analysis shows that the stalling is caused by the disruption of transcription-translation coupling. Anti-intuitively, the stalled RNAPs subsequently induce lesions to the DNA via transcription-coupled repair. While most of the bacteria are killed by genotoxicity, a small subpopulation acquires mutations via SOS-induced mutagenesis. Given that these processes are triggered shortly after antibiotic addition, resistance rapidly emerges in the population. Our work reveals a mechanism of action of ribosomal antibiotics, illustrates the importance of dissecting the complex interplay between multiple molecular processes in understanding antibiotic efficacy, and suggests new strategies for countering the development of resistance.

Novel strategy for Poxviridae prevention: Thermostable combined subunit vaccine patch with intense immune response.

Poxviruses gained international attention due to the sharp rise in monkeypox cases in recent years, highlighting the urgent need for the development of a secure and reliable vaccine. This study involved the development of an innovative combined subunit vaccine (CSV) targeting poxviruses, with lumpy skin disease virus (LSDV) serving as the model virus. To this end, the potential sites for poxvirus vaccines were fully evaluated to develop and purify four recombinant proteins. These proteins were then successfully delivered to the dermis in a mouse model by utilizing dissolvable microneedle patches (DMPs). This approach simplified the vaccination procedure and significantly mitigated the associated risk. CSV-loaded DMPs contained four recombinant proteins and a novel adjuvant, CpG, which allowed DMPs to elicit the same intensity of humoral and cellular immunity as subcutaneous injection. Following immunization with SC and DMP, the mice exhibited notable levels of neutralizing antibodies, albeit at a low concentration. It is noteworthy that the CSV loaded into DMPs remained stable for at least 4 months at room temperature, effectively addressing the storage and transportation challenges. Based on the study findings, CSV-loaded DMPs are expected to be utilized worldwide as an innovative technique for poxvirus inoculation, especially in underdeveloped regions. This novel strategy is crucial for the development of future poxvirus vaccines.