High-risk ARGs (HRA) Chip: A high-throughput qPCR-based array for assessment of high-risk ARGs from the environment.

The global surge in antibiotic resistance genes (ARGs) presents a serious public health challenge. While methods like metagenomic analysis and qPCR arrays have been instrumental in investigating ARG distributions and dynamics, the vast diversity of ARGs often complicates effective monitoring and risk assessment. Here, we developed a High-Risk ARGs (HRA) chip based on high-capacity quantitative PCR array targeting previously identified high-risk ARGs. These ARGs are known to be prevalent in human-related environments, exhibit gene mobility, and are present in ESKAPE pathogens. The HRA chip include 101 primer sets and the 16S rRNA gene as a reference. These primer sets consist of 34 obtained from previous studies, and 67 newly designed primer sets which were validated in silico and experimentally. Absolute quantification of targeted ARGs is accomplished by generating standard curves for all ARGs with serially ten-fold diluted mixed plasmids containing targeted ARG sequences. The amplification efficiencies of all ARGs exceed 99% via plasmid template dilution tests, suggesting high reliability in quantification. The performance of HRA chip is further evaluated by practical applications in environmental samples from wastewater treatment plants (WWTPs) and soils with various land use types and fertilization regimes. The results indicate the dynamics of high-risk ARGs during wastewater treatment process, and reveal the profiles of soil high-risk ARGs which is distinct from those derived via metagenomic approaches. These findings highlight the potentials of HRA Chip in the evaluation of anthropogenic impacts on the environmental resistome with a more focused spectrum of high-risk ARGs. Overall, the HRA Chip emerges as a powerful and efficient high-throughput tool for rapid detection and quantification of high-risk ARGs, facilitating comprehensive profiling of high-risk resistomes in environmental samples which is essential for human health risk assessment of ARGs.

Preparation of sodium alginate modified silver-metal organic framework and application in citric acid/PVA antimicrobial packaging.

Silver-metal organic framework (Ag@MOF) has exhibited outstanding antimicrobial activity in antimicrobial applications, and reducing the biotoxicity associated with silver has become a research priority. In this study, Ag@MOF was initially modified with sodium alginate (SA) to form SA-Ag@MOF. The results showed that SA could control the release of Ag+, reducing the release by about 8% at 24 h, and the biotoxicity was significantly reduced. Finally, SA-Ag@MOF was applied as an antimicrobial agent in citric acid-modified PVA film to develop a novel composite antimicrobial film. When added at 2 MIC, the CA3-M2 film can effectively inhibit the growth of E. coli and S. aureus, and the inhibition rate has reached 98%. For white radish packaging applications, CA3-M2 film inhibited the growth of surface microorganisms, while ensuring moisture and tissue hardness to extend shelf-life up to 7 days. Overall, the strategy conceived here can be a theoretical basis for novel antimicrobial packaging.

Integrated bacterial transcriptome and host metabolome analysis reveals insights into "Candidatus Liberibacter asiaticus" population dynamics in the fruit pith of three citrus cultivars with different tolerance.

"Candidatus Liberibacter asiaticus" (CLas), the causal agent of citrus Huanglongbing (HLB), is able to multiply to a high abundance in citrus fruit pith. However, little is known about the biological processes and phytochemical substances that are vital for CLas colonization and growth in fruit pith. In this study, CLas-infected fruit pith of three citrus cultivars ("Shatangju" mandarin, "Guanxi" pomelo, and "Shatian" pomelo) exhibiting different tolerance to CLas were collected and used for dual RNA-Seq and untargeted metabolome analysis. Comparative transcriptome analysis found that the activation of the CLas noncyclic TCA pathway and pathogenic-related effectors could contribute to the colonization and growth of CLas in fruit pith. The pre-established Type 2 prophage in the CLas genome and the induction of its CRISPR/cas system could enhance the phage resistance of CLas and, in turn, facilitate CLas population growth in fruit pith. CLas infection caused the accumulation of amino acids that were correlated with tolerance to CLas. The accumulation of most sugars and organic acids in CLas-infected fruit pith, which could be due to the phloem blockage caused by CLas infection, was thought to be beneficial for CLas growth in localized phloem tissue. The higher levels of flavonoids and terpenoids in the fruit pith of CLas-tolerant cultivars, particularly those known for their antimicrobial properties, could hinder the growth of CLas. This study advances our understanding of CLas multiplication in fruit pith and offers novel insight into metabolites that could be responsible for tolerance to CLas or essential to CLas population growth.IMPORTANCECitrus Huanglongbing (HLB, also called citrus greening disease) is a highly destructive disease currently threatening citrus production worldwide. HLB is caused by an unculturable bacterial pathogen, "Candidatus Liberibacter asiaticus" (CLas). However, the mechanism of CLas colonization and growth in citrus hosts is poorly understood. In this study, we utilized the fruit pith tissue, which was able to maintain the CLas at a high abundance, as the materials for dual RNA-Seq and untargeted metabolome analysis, aiming to reveal the biological processes and phytochemical substances that are vital for CLas colonization and growth. We provided a genome-wide CLas transcriptome landscape in the fruit pith of three citrus cultivars with different tolerance and identified the important genes/pathways that contribute to CLas colonization and growth in the fruit pith. Metabolome profiling identified the key metabolites, which were mainly affected by CLas infection and influenced the population dynamic of CLas in fruit pith.

Metabolomic Profiling Reveals the Quality Variations in Citri Reticulatae Pericarpium (Citrus reticulata Blanco cv. Chachiensis) with Different Storage Ages in Response to "Candidatus Liberibacter Asiaticus" Infection.

Citri Reticulatae Pericarpium, especially the pericarp of Citrus reticulata Blanco cv. Chachiensis (PCRC), is an important edible and medicinal ingredient for health and pharmacological properties. Citrus Huanglongbing, a devastating disease that currently threatens the citrus industry worldwide, is caused by a phloem-limited alpha-proteobacterium, "Candidatus Liberibacter asiaticus" (CLas). The industry of cultivar Chachiensis has been suffering from HLB. Although HLB affected the quality of citrus fruit, whether the quality of PCRC was affected by HLB remains unclear. In this study, we compared the metabolite profiles between HLB-affected and healthy PCRC from three sources: fresh, 6-month-old, and 9-year-old PCRC, through the untargeted LC-MS method. Compared to healthy controls, various types of bioactive compounds, mainly flavonoids, terpenoids, alkaloids, coumarins, polysaccharides, and phenolic acids, accumulated in HLB-affected PCRC, especially in the HLB-affected 9-year PCRC. In particular, isorhamnetin, isoliquiritigenin, luteolin 7-O-beta-D-glucoside, limonin, geniposide, pyrimidodiazepine, scoparone, chitobiose, m-coumaric acid, malonate, and pantothenic acid, which contributed to the pharmacological activity and health care effects of PCRC, were highly accumulated in HLB-affected 9-year-old PCRC compared to the healthy control. Multibioassay analyses revealed that HLB-affected 9-year-old PCRC had a higher content of total flavonoids and total polyphenols and exhibited similar antioxidant capacity as compared to healthy controls. The results of this study provided detailed information on the quality of HLB-affected PCRC.

Report of Citrus tristeza virus in Diaphorina citri (Hemiotera: Liviidae) insects of different sexes, color morphs, and developmental stages.

Diaphorina citri, also known as the Asian citrus psyllid, is the main vector of 'Candidatus Liberibacter asiaticus' (CLas) associated with citrus Huanglongbing. It has been reported that D. citri could also be infected by Citrus tristeza virus (CTV), a virus that has been previously reported to be vectored by certain aphid species. In this study, the CTV and CLas profiles in different organs, color variants, developmental stages, or sexes of D. citri insects were analyzed. Although no significant differences were found between nymphs and adults in CTV titers, we found that the third instar nymph of D. citri was more efficient in CTV and CLas acquisition compared to the fourth and fifth instars and adults. With the instars of D. citri development, the relationship between the acquiring of CTV and CLas by D. citri seemed to follow an inverse trend, with the titer of CLas increased and the titer of CTV decreased. No significant differences were observed between the 2 sexes of D. citri in acquiring either CTV or CLas titers in the field. However, no differences were drawn among the 3 color morph variants for CTV titers. CTV titers in the midguts of adult D. citri were significantly higher than those in the salivary glands. Both CTV-positive incidence and CTV titers in the midguts of adult D. citri increased with increasing exposure periods. This study provides new data to deepen our understanding of the CTV-involved interaction between D. citri and CLas.

Antibacterial photodynamic properties of silver nanoparticles-loaded curcumin composite material in chitosan-based films.

In order to cope with the increasingly severe food contamination and safety problems, a powerful sterilization of food packaging material is urgently needed. Chitosan (CS) has potential applications in food packaging due to its good film-forming properties, but its antibacterial activity is not sufficient to meet the needs in practical applications. Silver nanoparticles (AgNPs) have the problem of weak immediate antibacterial activity as a broad-spectrum antibacterial agent. Therefore, in this study, AgNPs@GA@Cur-POTS (AGCP) composite antibacterial system was prepared by combining AgNPs with antibacterial photodynamic therapy using gallic acid (GA) as a reducing agent, curcumin (Cur) as a photosensitizer and perfluorosilane (POTS) for surface modification. The results showed that AGCP could produce a large number of reactive oxygen species under blue light irradiation, killing >90 % of E. coli and S. aureus within 2 h. Subsequently, the composite film of CS loaded with AGCP (CS/AGCP) was prepared by the flow-delay method. The CS/AGCP composite film exhibited excellent barrier properties and antioxidant activity, while its antibacterial rates against E. coli and S. aureus reached 98.44 ± 1.27 % and 99.11 ± 0.24 %, respectively, while the OD630 values of the two groups of bacteria treated with it showed no significant increase in incubation for up to 132 h, exhibiting remarkable and sustained antibacterial effects. Taken together, this work will provide a new strategy for antibacterial food packaging.

Variation of endosymbiont and citrus tristeza virus (CTV) titers in the Huanglongbing insect vector, Diaphorina citri, on CTV-infected plants.

"Candidatus Liberibacter asiaticus" (CLas) is a notorious agent that causes Citrus Huanglongbing (HLB), which is transmitted by Diaphorina citri (D. citri). We recently found that the acquisition and transmission of CLas by D. citri was facilitated by Citrus tristeza virus (CTV), a widely distributed virus in the field. In this study, we further studied whether different CTV strains manipulate the host preference of D. citri, and whether endosymbionts variation is related to CTV strains in D. citri. The results showed that the non-viruliferous D. citri preferred to select the shoots infected with CTV, without strain differences was observed in the selection. However, the viruliferous D. citri prefered to select the mixed strain that is similar to the field's. Furthermore, D. citri effectively acquired the CTV within 2-12 h depending on the strains of the virus. The persistence period of CTV in D. citri was longer than 24 days, without reduction of the CTV titers being observed. These results provide a foundation for understanding the transmission mode of D. citri on CTV. During the process of CTV acquisition and persistence, the titers of main endosymbionts in D. citri showed similar variation trend, but their relative titers were different at different time points. The titers of the "Candidatus Profftella armatura" and CTV tended to be positively correlated, and the titers of Wolbachia and "Candidatus Carsonella ruddii" were mostly negatively related with titers of CT31. These results showed the relationship among D. citri, endosymbionts, and CTV and provided useful information for further research on the interactions between D. citri and CLas, which may benefit the development of approaches for the prevention of CLas transmission and control of citrus HLB.

Local environment, surface characteristics and stochastic processes shape the dynamics of urban dustbin surface microbiome.

Dustbins function as critical infrastructures for urban sanitation, creating a distinct breeding ground for microbial assemblages. However, there is no information regarding the dynamics of microbial communities and the underlying mechanism for community assembly on dustbin surfaces. Here, surface samples were collected from three sampling zones (business building, commercial street and residential community) with different types (kitchen waste, harmful waste, recyclables, and others) and materials (metallic and plastic); and distribution pattern and assembly of microbial communities were investigated by high-throughput sequencing. Bacterial and fungal communities showed the distinct community variations across sampling zones and waste sorting. Core community and biomarker species were significantly correlated with the spatial distribution of overall community. The detection of pathogens highlighted the potential risk of surface microbiome. Human skin, human feces and soil biomes were the potential source environments of the surface microbiomes. Neutral model prediction suggested that microbial community assembly was significantly driven by stochastic processes. Co-association patterns varied with sampling zones and waste types, and neutral amplicon sequence variants (ASVs) that fall within the 95 % confidence intervals of neutral model were largely involved in the stability of microbial networks. These findings improve our understanding of the distribution pattern and the underlying assembly of microbial community on the dustbin surface, thus enabling prospective prediction and assessment of urban microbiomes and their impacts on human health.

Citrus tristeza virus Promotes the Acquisition and Transmission of 'Candidatus Liberibacter Asiaticus' by Diaphorina citri.

Diaphorina citri Kuwayama (D. citri) is an insect vector of phloem-limited 'Candidatus Liberibacter asiatus' (CLas), the presumed pathogen of citrus Huanglongbing (HLB). Recently, our lab has preliminarily found it acquired and transmitted Citrus tristeza virus (CTV), which was previously suggested to be vectored by species of aphids. However, the influences of one of the pathogens on the acquisition and transmission efficiency of the other pathogen remain unknown. In this study, CLas and CTV acquisition and transmission by D. citri at different development stages under field and laboratory conditions were determined. CTV could be detected from the nymphs, adults, and honeydew of D. citri but not from the eggs and exuviates of them. CLas in plants might inhibit CTV acquisition by D. citri as lower CTV-positive rates and CTV titers were detected in D. citri collected from HLB-affected trees compared to those from CLas-free trees. D. citri were more likely to obtain CTV than CLas from host plants co-infected with the two pathogens. Intriguingly, CTV in D. citri facilitated the acquisition and transmission of CLas, but CLas carried by D. citri had no significant effect on the transmission of CTV by the same vector. Molecular detection and microscopy methods confirmed the enrichment of CTV in the midgut after a 72-h acquisition access period. Collectively, these results raise essential scientific questions for further research on the molecular mechanism of pathogen transmission by D. citri and provide new ideas for the comprehensive prevention and control of HLB and CTV.

Current Epidemic Situation and Control Status of Citrus Huanglongbing in Guangdong China: The Space-Time Pattern Analysis of Specific Orchards.

Huanglongbing (HLB) is the most harmful bacterial disease in citrus production in the world, and has been seriously ravaging the citrus groves of South China since the 1930s. The surveillance of the epidemiological characteristics of HLB is of utmost priority for citrus production in this region. In order to explore the effects of disease control measures, analyses on the space-time statistical features of the HLB epidemic, from 2019 to 2021, within six orchards in the Guangdong province are presented. Overall, the number of citrus plants in the orchards usually slightly decreased year by year. The reduction was mainly related to the level of plant susceptibility, which is correlated with citrus varieties. The maximum disease severity (incidence and race increment) was correlated with the awareness of this disease and the management intensity applied by the manager. A higher disease index was found in the conventional management orchards than in the comprehensive prevention and control orchards. Proper insect-protective screen houses can effectively prevent the epidemic of HLB, without affecting the fruit quality, and can also aid with higher yields. A high correlation was found between the geometry and topography of orchards and the HLB epidemic due to the wind direction from May to September and the Asia citrus psyllid activity characteristics. For flat orchards, the incidence of HLB in the north and entrance areas was higher than that in the southwest. In the mountain area, the incidence of the windward side in the south was higher than that of the leeward side in the north. Diseased trees tended to have an edge effect in the grove, whereas the trees of the same disease scale were found clustered in their distribution. These results allow a better understanding of HLB epidemiology and provide guidance for the early warning of HLB in new groves in areas that are severely affected by this disease. Furthermore, they also provide a scientific basis for the comprehensive prevention and control of HLB in old groves.

Dynamics of Microbial Community and Potential Microbial Pollutants in Shopping Malls.

Shopping malls offer various niches for microbial populations, potentially serving as sources and reservoirs for the spread of microorganisms of public health concern. However, knowledge about the microbiome and the distribution of human pathogens in malls is largely unknown. Here, we examine the microbial community dynamics and genotypes of potential pathogens from floor and escalator surfaces in shopping malls and adjacent road dusts and greenbelt soils. The distribution pattern of microbial communities is driven primarily by habitats and seasons. A significant enrichment of human-associated microbiota in the indoor environment indicates that human interactions with surfaces might be another strong driver for mall microbiomes. Neutral community models suggest that the microbial community assembly is strongly driven by stochastic processes. Distinct performances of microbial taxonomic signatures for environmental classifications indicate the consistent differences of microbial communities of different seasons/habitats and the strong anthropogenic effect on homogenizing microbial communities of shopping malls. Indoor environments harbored higher concentrations of human pathogens than outdoor samples, also carrying a high proportion of antimicrobial resistance-associated multidrug efflux genes and virulence genes. These findings enhanced the understanding of the microbiome in the built environment and the interactions between humans and the built environment, providing a basis for tracking biothreats and communicable diseases and developing sophisticated early warning systems. IMPORTANCE Shopping malls are distinct microbial environments which can facilitate a constant transmission of microorganisms of public health concern between humans and the built environment or between human and human. Despite extensive investigation of the natural environmental microbiome, no comprehensive profile of microbial ecology has been reported in malls. Characterizing microbial distribution, potential pathogens, and antimicrobial resistance will enhance our understanding of how these microbial communities are formed, maintained, and transferred and help establish a baseline for biosurveillance of potential public health threats in malls.

Physiological Variables Influenced by 'Candidatus Liberibacter asiaticus' Infection in Two Citrus Species.

'Candidatus Liberibacter asiaticus' is the bacterium associated with the citrus disease known as huanglongbing (HLB). This study evaluated the influence of 'Ca. L. asiaticus' infection on a number of key plant physiological variables concerning photosynthesis, cell integrity, reactive oxygen species scavengers' activity, and osmoregulation of two different species of citrus-the pomelo Citrus maxima and the mandarin C. reticulata 'Tankan'-relative to their measured 'Ca. L. asiaticus' infection load. Results indicated that all measured physiological variables except soluble sugar were affected by increased 'Ca. L. asiaticus' infection titers, wherein the variety C. maxima proved overall more resistant than C. reticulata. 'Ca. L. asiaticus' infection was linked in both plants to decrease in chlorophyll concentration, cell membrane permeability, and malondialdehyde, as well as increased free proline and starch contents. Chlorophyll fluorescence measurements taken 9 months after grafting the mandarin C. reticulata with 'Ca. L. asiaticus' scions revealed a significant decrease in the photosynthesis variables maximum photochemical quantum yield of photosystem II (PSII), effective photochemical quantum yield of PSII, and coefficient of photochemical fluorescence quenching assuming interconnected PSII antennae, whereas nonphotochemical fluorescence quenching increased significantly; C. maxima plants, on the other hand, did not show significant differences until the 12th month from infection exposure. The variables superoxide dismutase, catalase, peroxidase, and soluble protein initially increased and later decreased. In addition, progression of 'Ca. L. asiaticus' replication in both citrus species was accompanied by rapid changes in three reactive oxygen species scavenging enzymes in C. maxima, while the pattern was different in C. reticulata. We hypothesize that the observed interspecific differences in physiological change are related to their relative resistance against 'Ca. L. asiaticus' infection. These results provide a scaffold for better describing the pathogenesis, selecting the most resistant breeds, or even validating pertaining omics research; ultimately, these detailed observations can facilitate the diagnosis of 'Ca. L. asiaticus' infection.

Integrated Transcriptome and Metabolome Analysis Reveals Phenylpropanoid Biosynthesis and Phytohormone Signaling Contribute to "Candidatus Liberibacter asiaticus" Accumulation in Citrus Fruit Piths (Fluffy Albedo).

"Candidatus Liberibacter asiaticus" (CLas) is a phloem-restricted α-proteobacterium that is associated with citrus huanglongbing (HLB), which is the most destructive disease that affects all varieties of citrus. Although midrib is usually used as a material for CLas detection, we recently found that the bacterium was enriched in fruits, especially in the fruit pith. However, no study has revealed the molecular basis of these two parts in responding to CLas infection. Therefore, we performed transcriptome and UHPLC-MS-based targeted and untargeted metabolomics analyses in order to organize the essential genes and metabolites that are involved. Transcriptome and metabolome characterized 4834 differentially expressed genes (DEGs) and 383 differentially accumulated metabolites (DAMs) between the two materials, wherein 179 DEGs and 44 DAMs were affected by HLB in both of the tissues, involving the pathways of phenylpropanoid biosynthesis, phytohormone signaling transduction, starch and sucrose metabolism, and photosynthesis. Notably, we discovered that the gene expression that is related to beta-glucosidase and endoglucanase was up-regulated in fruits. In addition, defense-related gene expression and metabolite accumulation were significantly down-regulated in infected fruits. Taken together, the decreased amount of jasmonic acid, coupled with the reduced accumulation of phenylpropanoid and the increased proliferation of indole-3-acetic acid, salicylic acid, and abscisic acid, compared to leaf midribs, may contribute largely to the enrichment of CLas in fruit piths, resulting in disorders of photosynthesis and starch and sucrose metabolism.

Uncovering the diversity and contents of gene cassettes in class 1 integrons from the endophytes of raw vegetables.

Rapid spread of antibiotic resistance genes (ARGs) in pathogens is threatening human health. Integrons allow bacteria to integrate and express foreign genes, facilitating horizontal transfer of ARGs in environments. Consumption of raw vegetables represents a pathway for human exposure to environmental ARGs. However, few studies have focused on integron-associated ARGs in the endophytes of raw vegetables. Here, based on the approach of qPCR and clone library, we quantified the abundance of integrase genes and analyzed the diversity and contents of resistance gene cassettes in class 1 integrons from the endophytes of six common raw vegetables. The results revealed that integrase genes for class 1 integron were most prevalent compared with class 2 and class 3 integron integrase genes (1-2 order magnitude, P < 0.05). The cucumber endophytes harbored a higher absolute abundance of integrase genes than other vegetables, while the highest bacterial abundance was detected in cabbage and cucumber endophytes. Thirty-two unique resistance gene cassettes were detected, the majority of which were associated with the genes encoding resistance to beta-lactam and aminoglycoside. Antibiotic resistance gene cassettes accounted for 52.5 % of the functionally annotated gene cassettes, and blaTEM-157 and aadA2 were the most frequently detected resistance cassettes. Additionally, carrot endophytes harbored the highest proportion of antibiotic resistance gene cassettes in the class 1 integrons. Collectively, these results provide an in-depth view of acquired resistance genes by integrons in the raw vegetable endophytes and highlight the potential health risk of the transmission of ARGs via the food chain.

Gallic acid functionalized chitosan immobilized nanosilver for modified chitosan/Poly (vinyl alcohol) composite film.

Food spoilage caused by bacterial growth is a serious threat to human health. Silver nanoparticles (AgNPs) have antibacterial activity against various pathogenic microorganisms, but their rapid release often leads to cumulative toxicity. In this paper, silver nanoparticles (AgNPs@CG) were reduced and immobilized in situ using gallic acid-functionalized chitosan (CG) as a reducing and stabilizing agent to achieve a long-lasting and stable controlled release of Ag+. The AgNPs@CG was incorporated into the CG/PVA composite film. The results showed that the release of Ag+ was only 0.686 ± 0.022 mg·L-1 after seven days, which had a long-lasting antibacterial effect on E. coli and S. aureus. In addition, CG/PVA/AgNPs-2 composite film can significantly improve the freshness preservation effect in fresh-cut apple preservation applications. In conclusion, CG/PVA/AgNPs composite film has potential applications as effective and safe packaging material to extend the shelf life of food products.

Nanopore sequencing analysis of integron gene cassettes in sewages and soils.

Integrons are genetic elements that can facilitate rapid spread of antibiotic resistance by insertion and removal of genes. However, knowledge about the diversity and distribution of gene cassettes embedded in class 1 integron is still limited. In this study, we sequenced integron gene cassettes using nanopore sequencing and quantified antibiotic resistance genes (ARGs) and integrase genes in the manured soils and sewages of a bioreactor. The results showed that class 1 integron integrase genes were the most abundant in soils and sewages compared with class 2 and class 3 integrase genes. Long-term manure application exacerbated the enrichment of total ARGs, integrase genes and antibiotic resistance-associated gene cassettes, while antibiotics and heavy metals showed no impact on the overall resistome profile. Sewage treatment could efficiently remove the absolute abundance of integrase genes (~3 orders of magnitude, copies/L) and antibiotic resistance gene cassettes. The resistance gene cassettes mainly carried the ARGs conferring resistance to aminoglycoside and beta-lactams in soils and sewages, some of which were persistent during the sewage treatment. This study underlined that soil and sewage were potential reservoirs for integron-mediated ARGs transfer, indicating that anthropogenic activity played a vital role in the prevalence and diversity of resistance gene cassettes in integrons.

A Novel Microviridae Phage (CLasMV1) From "Candidatus Liberibacter asiaticus".

"Candidatus Liberibacter asiaticus" (CLas) is an unculturable phloem-limited α-proteobacterium associated with citrus Huanglongbing (HLB; yellow shoot disease). HLB is currently threatening citrus production worldwide. Understanding the CLas biology is critical for HLB management. In this study, a novel single-stranded DNA (ssDNA) phage, CLasMV1, was identified in a CLas strain GDHZ11 from Guangdong Province of China through a metagenomic analysis. The CLasMV1 phage had a circular genome of 8,869 bp with eight open reading frames (ORFs). While six ORFs remain uncharacterized, ORF6 encoded a replication initiation protein (RIP), and ORF8 encoded a major capsid protein (MCP). Based on BLASTp search against GenBank database, amino acid sequences of both MCP and RIP shared similarities (coverage > 50% and identity > 25%) to those of phages in Microviridae, an ssDNA phage family. Phylogenetic analysis revealed that CLasMV1 MCP and RIP sequences were clustered with genes from CLas and "Ca. L. solanacearum" (CLso) genomes and formed a unique phylogenetic lineage, designated as a new subfamily Libervirinae, distinct to other members in Microviridae family. No complete integration form but partial sequence (∼1.9 kb) of CLasMV1 was found in the chromosome of strain GDHZ11. Read-mapping analyses on additional 15 HiSeq data sets of CLas strains showed that eight strains harbored complete CLasMV1 sequence with variations in single-nucleotide polymorphisms (SNPs) and small sequence insertions/deletions (In/Dels). PCR tests using CLasMV1-specific primer sets detected CLasMV1 in 577 out of 1,006 CLas strains (57%) from southern China. This is the first report of Microviridae phage associated with CLas, which expands our understanding of phage diversity in CLas and facilitates current research in HLB.

Expanding control of the tumor cell cycle with a CDK2/4/6 inhibitor.

The CDK4/6 inhibitor, palbociclib (PAL), significantly improves progression-free survival in HR+/HER2- breast cancer when combined with anti-hormonals. We sought to discover PAL resistance mechanisms in preclinical models and through analysis of clinical transcriptome specimens, which coalesced on induction of MYC oncogene and Cyclin E/CDK2 activity. We propose that targeting the G1 kinases CDK2, CDK4, and CDK6 with a small-molecule overcomes resistance to CDK4/6 inhibition. We describe the pharmacodynamics and efficacy of PF-06873600 (PF3600), a pyridopyrimidine with potent inhibition of CDK2/4/6 activity and efficacy in multiple in vivo tumor models. Together with the clinical analysis, MYC activity predicts (PF3600) efficacy across multiple cell lineages. Finally, we find that CDK2/4/6 inhibition does not compromise tumor-specific immune checkpoint blockade responses in syngeneic models. We anticipate that (PF3600), currently in phase 1 clinical trials, offers a therapeutic option to cancer patients in whom CDK4/6 inhibition is insufficient to alter disease progression.

The Clinical Significance of Serum IL-33 and sST2 Alterations in the Post-Stroke Depression.

INTRODUCTION: This study was to test whether the serum levels of IL-33 and sST2 are correlated with the development of depression after acute ischemic stroke. METHODS: Patients diagnosed with acute ischemic stroke were selected. This study took the 24-item Hamilton Depression Rating Scale (HAMD) (score ≥20) as the diagnostic criteria for depression. On the 21st day after admission, patients who met the depression diagnostic criteria were included in the depression group, and patients who failed to meet the diagnostic criteria were included in the non-depression group. The serum levels of IL-33, sST2 and hsCRP were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: On 1st day after stroke, compared with the non-depression group, there was no significant difference in the serum IL-33, sST2 and hsCRP levels in the depression group; on 21st day after stroke, compared with the non-depression group, the serum IL-33 and hsCRP levels were significantly increased, while the sST2 level was significantly decreased in the depression group. Correlation analysis showed that IL-33 was positively correlated with the depression quantitative score and hsCRP, while sST2 was negatively correlated with the depression quantitative score and hsCRP. Regression analysis showed that IL-33 and sST2 were independent risk factors for the depression after acute ischemic stroke. DISCUSSION: The abnormal alterations of serum IL-33 and sST2 levels in the stroke patients may serve as one of the risk factors for the occurrence and exacerbation of the depression, and its mechanism may be related to the promotion of inflammatory factor production in vivo.

Preliminary Report on the Acquisition, Persistence, and Potential Transmission of Citrus tristeza virus by Diaphorina citri.

Citrus tristeza virus (CTV) is one of the most important citrus tree viruses: a graft-transmissible virus that can be vectored by several aphid species. Diaphorina citri is the insect vector of "Candidatus Liberibacter spp.", a bacterium associated with citrus Huanglongbing (HLB). However, no detailed description of the relationship between CTV and D. citri has been reported. In this study, D. citri adults collected from CTV-infected "Shatangju" mandarin, "Newhall" sweet orange, and "fingered citron" trees in different orchards yielded CTV-positive rates of 40%, 65%, and 95%, respectively, upon detection by conventional PCR. Illumina HiSeq sequencing followed by de novo assembly recovered the primary full CTV genome from the RNA of 30 D. citri adults sampled from CTV-positive citrus plants. Molting and adult emergence did not affect the presence or titers of CTV within the D. citri; however, the persistence of CTV in psyllids varied among different host plant species. Groups of 10 D. citri (from a population 85% CTV-positive) were shown to potentially transmit CTV to two citrus species, "Shatangju" mandarin and "Eureka" lemon, yielding 58.33% and 83.33% CTV-positive plants, respectively. No transmission of CTV to orange jasmine plants occurred. Thus, this study reports on the ability of D. citri to acquire and transmit CTV, making D. citri as a vector of two important citrus pathogens, warranting further attention and investigation.