Machine learning and statistical models for analyzing multilevel patent data.

A recent surge of patent applications among public hospitals in China has aroused significant research interest. A country's healthcare innovation capacity can be measured by its number of patents. This paper explores the link between the number of patents and ten independent variables. Multicollinearity was carefully detected and removed by using the variable selection method and LASSO regression, respectively. The Poisson model and the negative binomial model were proposed to analyze the patent data. Three goodness of fit tests, the Pearson test, the deviance test, and the DHARMa non-parametric dispersion test, were conducted to investigate if the model has a good fit. After discovering four clusters by conducting agglomerative hierarchical clustering, these two models were replaced by the negative binomial mixed model. The likelihood ratio test was used to determine which model is more appropriate and the results reveal that the negative binomial mixed model outperforms both the Poisson model and the negative binomial model. Three variables, number of health technicians per 10,000 people, financial expenditure on science and technology as well as number of patent applications per 10,000 health personnel, have a significantly positive relationship with the number of patents in Chinese tertiary public hospitals.

Genomic Analysis of a Novel Phage Infecting the Turkey Pathogen Escherichia coli APEC O78 and Its Endolysin Activity.

Due to the increasing spread of multidrug-resistant (MDR) bacteria, phage therapy is considered one of the most promising methods for addressing MDR bacteria. Escherichia coli lives symbiotically in the intestines of humans and some animals, and most strains are beneficial in terms of maintaining a healthy digestive tract. However, some E. coli strains can cause serious zoonotic diseases, including diarrhea, pneumonia, urinary tract infections, and hemolytic uremic syndrome. In this study, we characterized a newly isolated Myoviridae phage, vB_EcoM_APEC. The phage vB_EcoM_APEC was able to infect E. coli APEC O78, which is the most common MDR E. coli serotype in turkeys. Additionally, the phage's host range included Klebsiella pneumoniae and other E. coli strains. The genome of phage vB_EcoM_APEC (GenBank accession number MT664721) was 35,832 bp in length, with 52 putative open reading frames (ORFs) and a GC content of 41.3%. The genome of vB_EcoM_APEC exhibited low similarity (79.1% identity and 4.0% coverage) to the genome of Acinetobacter phage vB_AbaM_IME284 (GenBank no. MH853787.1) according to the nucleotide Basic Local Alignment Search Tool (BLASTn). Phylogenetic analysis revealed that vB_EcoM_APEC was a novel phage, and its genome sequence showed low similarity to other available phage genomes. Gene annotation indicated that the protein encoded by orf11 was an endolysin designated as LysO78, which exhibited 64.7% identity (91.0% coverage) with the putative endolysin of Acinetobacter baumannii phage vB_AbaM_B9. The LysO78 protein belongs to glycoside hydrolase family 19, and was described as being a chitinase class I protein. LysO78 is a helical protein with 12 α-helices containing a large domain and a small domain in terms of the predicted three-dimensional structure. The results of site-directed mutagenesis indicated that LysO78 contained the catalytic residues E54 and E64. The purified endolysin exhibited broad-spectrum bacteriolytic activity against Gram-negative strains, including the genera Klebsiella, Salmonella, Shigella, Burkholderia, Yersinia, and Pseudomonas, as well as the species Chitinimonas arctica, E. coli, Ralstonia solanacearum, and A. baumannii. An enzymatic assay showed that LysO78 had highly lytic peptidoglycan hydrolases activity (64,620,000 units/mg) against E. coli APEC O78, and that LysO78 had lytic activity in the temperature range of 4-85 °C, with an optimal temperature of 28 °C and optimal pH of 8.0, and was active at pH 3.0-12.0. Overall, the results suggested that LysO78 might be a promising therapeutic agent for controlling MDR E. coli APEC O78 and nosocomial infections caused by multidrug-resistant bacteria.

Poly(indole-5-carboxylic acid)/reduced graphene oxide/gold nanoparticles/phage-based electrochemical biosensor for highly specific detection of Yersinia pseudotuberculosis.

Yersinia pseudotuberculosis is an enteric bacterium causing yersiniosis in humans. The existing Yersinia pseudotuberculosis detection methods are time-consuming, requiring a sample pretreatment step, and are unable to discriminate live/dead cells. The current work reports a phage-based electrochemical biosensor for rapid and specific detection of Yersinia pseudotuberculosis. The conductive poly(indole-5-carboxylic acid), reduced graphene oxide, and gold nanoparticles are applied for surface modification of the electrode. They possess ultra-high redox stability and retain 97.7% of current response after performing 50 consecutive cycles of cyclic voltammetry.The specific bacteriophages vB_YepM_ZN18 we isolated from hospital sewage water were immobilized on modified electrodes by Au-NH2 bond between gold nanoparticles and phages. The biosensor fabricated with nanomaterials and phages were utilized to detect Yersinia pseudotuberculosis successfully with detection range of 5.30 × 102 to 1.05 × 107 CFU mL-1, detection limit of 3 CFU mL-1, and assay time of 35 min. Moreover, the biosensor can specifically detect live Yersinia pseudotuberculosis without responding to phage-non-host bacteria and dead Yersinia pseudotuberculosis cells. These results suggest that the proposed biosensor is a promising tool for the rapid and selective detection of Yersinia pseudotuberculosis in food, water, and clinical samples.

Genomic, Antimicrobial, and Aphicidal Traits of Bacillus velezensis ATR2, and Its Biocontrol Potential against Ginger Rhizome Rot Disease Caused by Bacillus pumilus.

Ginger rhizome rot disease, caused by the pathogen Bacilluspumilus GR8, could result in severe rot of ginger rhizomes and heavily threaten ginger production. In this study, we identified and characterized a new Bacillus velezensis strain, designated ATR2. Genome analysis revealed B. velezensis ATR2 harbored a series of genes closely related to promoting plant growth and triggering plant immunity. Meanwhile, ten gene clusters involved in the biosynthesis of various secondary metabolites (surfactin, bacillomycin, fengycin, bacillibactin, bacilysin, difficidin, macrolactin, bacillaene, plantazolicin, and amylocyclicin) and two clusters encoding a putative lipopeptide and a putative phosphonate which might be explored as novel bioactive compounds were also present in the ATR2 genome. Moreover, B. velezensis ATR2 showed excellent antagonistic activities against multiple plant pathogenic bacteria, plant pathogenic fungi, human pathogenic bacteria, and human pathogenic fungus. B. velezensis ATR2 was also efficacious in control of aphids. The antagonistic compound from B. velezensis ATR2 against B.pumilus GR8 was purified and identified as bacillomycin D. In addition, B. velezensis ATR2 exhibited excellent biocontrol efficacy against ginger rhizome rot disease on ginger slices. These findings showed the potential of further applications of B. velezensis ATR2 as a biocontrol agent in agricultural diseases and pests management.

Inter-rater Reliability of 4-Item Arterial Doppler Waveform Classification System for Description of Arterial Doppler Waveforms.

Background: Non-invasive Doppler waveform (DW) analysis is a widely adopted method for detecting and evaluating lower extremity peripheral artery disease (PAD). Previous investigations have reported that broad heterogeneity in the description of Doppler waveforms is reduced by using a classification method. The reliability of arterial Doppler classification, however, is unknown. Purpose: The purpose of this study is to assess the reliability of a 4-category arterial DW classification method among Chinese sonographers. Methods: During a national ultrasound conference in China attendees were invited to classify thirty arterial Doppler waveforms. After viewing a 4-category (triphasic, biphasic, monophasic, and other) arterial Doppler waveform descriptor presentation, attendees were asked to classify 15 continuous wave (CW) and 15 pulsed wave (PW) Doppler waveforms. Their responses were recorded via mobile phone and the reliability of this 4-category classification was estimated by Fleiss' Kappa inter-rater statistical analysis. Results: One hundred and seventy-eight attendees participated in the analysis. The Kappa coefficient of Fleiss (κ) for all attendees was 0.522 (p < 0.005) with 95% confidence interval (CI): 0.520-0.523. The reliability of the waveform descriptor triphasic was the highest (κ = 0.621, p < 0.005), and other was the lowest (κ = 0.341, p < 0.005). Conclusion: The inter-rater reliability of a 4-category arterial Doppler waveform classification by Chinese sonographers is considered weak (κ = 0.522, CI95%: 0.520-0.523, p < 0.005). This study reinforces the importance of assessing DW classification reliability and the development of DW descriptors that are more accurately predictive of clinical hemodynamic events.

Novel spore lytic enzyme from a Bacillus phage leading to spore killing.

Bacterial spores maintain metabolic dormancy and have high resistance to external pressure. Germination requires degradation of the spore cortex and the participation of germination-specific cortex-lytic enzymes (GSLEs). Previously reported GSLEs have been identified in bacteria and facilitate germination. In this study, we have characterized a novel spore lytic enzyme, Ply67, from Bacillus pumilus phage vB_BpuM_BpSp. Ply67 had a similar cortex-lytic activity to GSLEs but disrupted the inner membranes (IMs) of spores, leading to spore killing rather than germination. The amino acid sequence of the complete protein, Ply67FL, exhibited 40% homology to the GSLE SleB. Domain prediction showed that Ply67FL was composed of three domains: a signal peptide, N-terminal domain protein and C-terminal domain protein. Ply67FL rapidly caused E. coli cells lysis when it was expressed in E. coli. The protein containing the C-terminal domain protein, Ply67C, could kill B. pumilus spores. The protein containing the N-terminal domain protein, Ply67N, could combine with the decoated B. pumilus spores, indicating that N-terminal was the binding domain and C-terminal was the hydrolase domain. The protein lacking the signal peptide but containing the N-terminal and C-terminal domain proteins, Ply67, had activity against spores of various Bacillus species. The surface of spores treated with Ply67 shrank and the permeability barrier was disrupted, and the inner contents leaked out. Immunoelectron microscopic observation showed that Ply67 was mainly acted on the spore cortex. Overall, Ply67 is a novel spore lytic enzyme that differs from other GSLEs not only in amino acid sequence but also in activity against spores, and Ply67 might have the potential to kill spores of pathogenic Bacillus species, e.g., B. cereus and B. anthracis.

Diagnostic efficacy of intravascular ultrasound combined with Gd2O3-EPL contrast agent for patients with atherosclerosis.

Atherosclerosis is a cardiovascular disease that is pathologically associated with the growth of atherosclerotic plaques and vascular vulnerability. Intravascular ultrasound (IVUS) has been used to evaluate and treat cardiovascular diseases. Accumulating evidence has demonstrated that Gd2O3-doped nanoparticles contrast can be applied for the diagnosis of human diseases. In the present study, eplerenone (EPL), a mineralocorticoid receptor antagonist, was first doped with Gd2O3 nanoparticles (Gd2O3-EPL), following which its diagnostic efficacy for use in IVUS measurements (Gd2O3-EPL-IVUS) was evaluated for patients suspected with atherosclerosis. Gd2O3-EPL-IVUS presented with higher accuracy and sensitivity compared with IVUS in diagnosing 188 patients with suspected atherosclerosis. Gd2O3-EPL-IVUS exhibited stronger signals associated with plaque morphology compared with aloe IVUS for patients with atherosclerosis. In addition, Gd2O3-EPL-IVUS application resulted in clearer arterial plaque images compared with IVUS by binding mineralocorticoid receptors. Atherosclerosis was subsequently confirmed in all patients using computerized tomography-coronary angiography. Gd2O3-EPL-IVUS showed more accuracy in measuring vessel size, plaque burden and minimal lumen area compared with IVUS analysis alone. In conclusion, these outcomes suggest that Gd2O3-EPL-IVUS is a reliable tool for the evaluation of coronary lesions in patients with atherosclerosis.

Reliability and Validity Study of the Children's Eating Behavior Questionnaire in Chinese School-Age Children.

Children's Eating Behavior Questionnaire (CEBQ) was widely used in western countries to measure children's eating behavior and played an important role in the research on the risk of childhood obesity, but it is rarely used in China yet. This study aimed to examine the Chinese version of CEBQ's reliability and validity in a pilot study, then applied it to bigger population. Data was collected with CEBQ in two districts of Shanghai, respectively. Using stratified cluster sampling method, a total of 2,520 children were included in the study. The questionnaires were filled out by children's parents. It was proved that the Cronbach's coefficient α of the questionnaire was greater than 0.7, indicating that the questionnaire had good internal consistency. Aside from the subscales 'Emotional undereating' and 'Food fussiness', the Pearson's correlation coefficients of the other subscales were all greater than 0.5. It showed that test-retest reliability of CEBQ was acceptable. We found that boys and girls had different eating behavior characteristics. And there was a graded association between eating behavior and nutritional status of school-age children. It suggested that appetitive traits of school-age children could be used as indicators of susceptibility to weight gain. In conclusion, the Chinese version of CEBQ was of good reliability and validity, so it is considered as a reliable tool that can be applied to Chinese school-age children's eating behavior and related studies.

Complete genome sequence of the novel phage vB_BthS-HD29phi infecting Bacillus thuringiensis.

The phage vB_BthS-HD29phi infecting Bacillus thuringiensis strain HD29 was isolated and purified. The morphology of the phage showed that it belongs to the family Siphoviridae. The phage genome was 32,181 bp in length, comprised linear double-stranded DNA with an average G + C content of 34.9%, and exhibited low similarity to known phage genomes. Genomic and phylogenetic analysis revealed that vB_BthS-HD29phi is a novel phage. In total, 50 putative ORFs were predicted in the phage genome, and only 18 ORFs encoded proteins with known functions. This article reports the genome sequence of a new tailed phage and increases the known genetic diversity of tailed phages.

Prevalence and Diversity Analysis of Candidate Prophages to Provide An Understanding on Their Roles in Bacillus Thuringiensis.

Bacillus thuringiensis (Bt) is widely used in producing biological insecticides. Phage contaminations during Bt fermentation can cause severe losses of yields. Lots of strategies have been engaged to control extrinsic phage contamination during Bt fermentation, but their effectiveness is low. In this study, the candidate endogenous prophages (prophages) in 61 Bt chromosomes that had been deposited in GenBank database were analyzed. The results revealed that all chromosomes contained prophage regions, and 398 candidate prophage regions were predicted, including 135 putative complete prophages and 263 incomplete prophage regions. These putative complete prophages showed highly diverse genetic backgrounds. The inducibility of the prophages of ten Bt strains (4AJ1, 4BD1, HD-1, HD-29, HD-73, HD-521, BMB171, 4CC1, CT-43, and HD-1011) was tested, and the results showed that seven of the ten strains' prophages were inducible. These induced phages belonged to the Siphoviridae family and exhibited a broad host spectrum against the non-original strains. The culture supernatants of the two strains (BMB171, 4CC1) could lyse Bt cells, but no virions were observed, which was speculated to be caused by lysin. The functional analysis of the putative complete prophage proteins indicated that some proteins, such as antibiotic resistance-associated proteins and restriction endonucleases, might increase the fitness of the Bt strains to different environments. The findings of this study provided understanding on the high prevalence and diversity of Bt prophages, as well as pointed out the role of prophages in the life cycle of Bt.

Characterization of rhizosphere bacterial community and berry quality of Hutai No.8 (Vitis vinifera L.) with different ages, and their relations.

BACKGROUND: Rhizosphere soil microbial communities play an important role in grapevine growth. However, the relationship of the rhizosphere soil bacterial community and berry quality of Hutai No.8 grape with different tree-ages is unclear. In this work, the Biolog-ECO technique was used to explore the functional diversity of the rhizosphere soil bacterial communities of Hutai No.8 grape with five ages (3, 5, 7, 9 and 11 years old). Meanwhile, grape berry quality indicators related to berry appearance, flavor and functional substance quality was also examined. RESULTS: Principal component analysis of grape berry quality mainly separated 3-year-old (first bear fruit) and the other tree-ages. In particular, peel weight and total soluble solid content of 3-year-old grape berry was significantly less than that of others. Furthermore, average well color development, species richness and Shannon's diversity index increased significantly with grapevine age. Moreover, the metabolic activities and functional diversity of soil microbial communities in using carbon sources were significantly increasing with grapevine age. Moreover, there were significant correlation between physicochemical indices of grape berry quality and six functional categories of carbon sources. CONCLUSION: Tree-ages could greatly affect the rhizosphere microbial community structure and richness, and then affect the grape berry quality. © 2019 Society of Chemical Industry.

Phage Reduce Stability for Regaining Infectivity during Antagonistic Coevolution with Host Bacterium.

The coevolution between phage and host bacterium is an important force that drives the evolution of the microbial community, yet the coevolution mechanisms have still not been well analyzed. Here, by analyzing the interaction between a Bacillus phage vB_BthS_BMBphi and its host bacterium, the coevolution mechanisms of the first-generation phage-resistant bacterial mutants and regained-infectivity phage mutants were studied. The phage-resistant bacterial mutants showed several conserved mutations as a potential reason for acquiring phage resistance, including the mutation in flagellum synthesis protein FlhA and cell wall polysaccharide synthesis protein DltC. All the phage-resistant bacterial mutants showed a deleted first transmembrane domain of the flagellum synthesis protein FlhA. Meanwhile, the regain-infectivity phage mutants all contained mutations in three baseplate-associated phage tail proteins by one nucleotide, respectively. A polymorphism analysis of the three mutant nucleotides in the wild-type phage revealed that the mutations existed before the interaction of the phage and the bacterium, while the wild-type phage could not infect the phage-resistant bacterial mutants, which might be because the synchronized mutations of the three nucleotides were essential for regaining infectivity. This study for the first time revealed that the synergism mutation of three phage baseplate-associated proteins were essential for the phages' regained infectivity. Although the phage mutants regained infectivity, their storage stability was decreased and the infectivity against the phage-resistant bacterial mutants was reduced, suggesting the phage realized the continuation of the species by way of "dying to survive".

Crystal structure of Bacillus thuringiensis Cry7Ca1 toxin active against Locusta migratoria manilensis.

Insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis (Bt) are widely used as environmentally friendly insecticides. As the only known Cry protein with insecticidal activity against Locusta migratoria manilensis, a locust subspecies that causes extensive destruction of crops, the Cry7Ca1 protein from Bt strain BTH-13 identified in our previous study is of particular interest to locust prevention and control. However, the three-dimensional structure of Cry7Ca1 toxin (the active form of the Cry7Ca1 protein) and the mechanisms of the Cry7Ca1 insecticidal specificity remain largely elusive. Here, we report a 2.3 Å crystal structure of the Cry7Ca1 toxin and carry out a systematic comparison of all available Cry toxins structures. A cluster of six loops in Cry toxin domain II, named Apex here, are the most variable structural elements and were documented to contribute in insecticidal specificity. The Cry7Ca1 toxin Apex loops are different from those of other Cry toxins in length, conformation, and sequence. Electrostatic potential analysis further revealed that Cry7Ca1 is the only structure-available Cry toxin that does not have a high contrast of surface electrostatic potentials in the Apex. We further suggest that the L1/L2 loops in the center of the Cry7Ca1 Apex may be worthy of attention in future efforts to unravel the Cry7Ca1 insecticidal specificity as they exhibit unique features not found in the corresponding regions of other Cry toxins. Our work highlights the uniqueness of the Apex in the Cry7Ca1 toxin and may assist exploration of the insecticidal mechanism of the Cry7Ca1 against Locusta migratoria manilensis.

Isolation of A Novel Bacillus thuringiensis Phage Representing A New Phage Lineage and Characterization of Its Endolysin.

Phages, the parasites of bacteria, are considered as a new kind of antimicrobial agent due to their ability to lyse pathogenic bacteria. Due to the increase of available phage isolates, the newly isolated phage showed increasing genomic similarities with previously isolated phages. In this study, the novel phage vB_BthS_BMBphi, infecting the Bacillus thuringiensis strain BMB171, is isolated and characterized together with its endolysin. This phage is the first tadpole-like phage infecting the Bacillus strains. Genomic analysis shows that the phage genome is dissimilar to all those of previously characterized phages, only exhibiting low similarities with partial regions of the B. thuringiensis prophages. Phylogenetic analysis revealed that the phage was distant from the other Bacillus phages in terms of evolution. The novel genome sequence, the distant evolutionary relationship, and the special virion morphology together suggest that the phage vB_BthS_BMBphi could be classified as a new phage lineage. The genome of the phage is found to contain a restriction modification system, which might endow the phage with immunity to the restriction modification system of the host bacterium. The function of the endolysin PlyBMB encoded by the phage vB_BthS_BMBphi was analyzed, and the endolysin could lyse all the tested Bacillus cereus group strains, suggesting that the endolysin might be used in controlling pathogenic B. cereus group strains. The findings of this study enrich the understanding of phage diversity and provide a resource for controlling the B. cereus group pathogenic bacteria.

Characteristics and optimised fermentation of a novel magnetotactic bacterium, Magnetospirillum sp. ME-1.

Magnetotactic bacteria (MTB) can biosynthesise magnetosomes, which have great potential for commercial applications. A new MTB strain, Magnetospirillum sp. ME-1, was isolated and cultivated from freshwater sediments of East Lake (Wuhan, China) using the limiting dilution method. ME-1 had a chain of 17 ± 4 magnetosomes in the form of cubooctahedral crystals with a shape factor of 0.89. ME-1 was closest to Magnetospirillum sp. XM-1 according to 16S rRNA gene sequence similarity. Compared with XM-1, ME-1 possessed an additional copy of mamPA and a larger mamO in magnetosome-specific genes. ME-1 had an intact citric acid cycle, and complete pathway models of ammonium assimilation and dissimilatory nitrate reduction. Potential carbon and nitrogen sources in these pathways were confirmed to be used in ME-1. Adipate was determined to be used in the fermentation medium as a new kind of dicarboxylic acid. The optimised fermentation medium was determined by orthogonal tests. The large-scale production of magnetosomes was achieved and the magnetosome yield (wet weight) reached 120 mg L-1 by fed-batch cultivation of ME-1 at 49 h in a 10-L fermenter with the optimised fermentation medium. This study may provide insights into the isolation and cultivation of other new MTB strains and the production of magnetosomes.

Complete Genome Sequence of Bacillus thuringiensis Serovar rongseni Reference Strain SCG04-02, a Strain Toxic to Plutella xylostella.

Bacillus thuringiensis (Bt) is widely used to control agricultural and forestry pests, though there are only a few available complete genome sequences of the Bt reference strain. Here, we report the complete genome sequence of B. thuringiensis serovar rongseni reference strain SCG04-02, which is toxic to Plutella xylostella.

Complete Genome Sequence of Bacillus thuringiensis subsp. jinghongiensis Reference Strain YGd22-03.

Bacillus thuringiensis is widely used in producing ecofriendly microbial agents for the purpose of controlling insect pests. In this study, we determined the complete genome sequence of B. thuringiensis subsp. jinghongiensis reference strain YGd22-03, which contains three cry genes and one cerecidin biosynthetic gene cluster.

Complete Genome Sequence of Magnetospirillum sp. ME-1, a Novel Magnetotactic Bacterium Isolated from East Lake, Wuhan, China.

A novel spiral magnetotactic bacterium, Magnetospirillum sp. ME-1, was isolated from East Lake in China. Here we report the complete genome of ME-1, which contains a 4,551,873-bp circular chromosome and a 5,222-bp circular plasmid. The magnetosome biogenesis-specific genes are located in a 97,664-bp magnetosome genomic island.

Jumbo Bacteriophages: An Overview.

Tailed bacteriophages with genomes larger than 200 kbp are classified as Jumbo phages, and are rarely isolated by conventional methods. These phages are designated "jumbo" owing to their most notable features of a large phage virion and large genome size. However, in addition to these, jumbo phages also exhibit several novel characteristics that have not been observed for phages with smaller genomes, which differentiate jumbo phages in terms of genome organization, virion structure, progeny propagation, and evolution. In this review, we summarize available reports on jumbo phages and discuss the differences between jumbo phages and small-genome phages. We also discuss data suggesting that jumbo phages might have evolved from phages with smaller genomes by acquiring additional functional genes, and that these additional genes reduce the dependence of the jumbo phages on the host bacteria.