Noninvasive longitudinal PET/CT imaging of CAR T cells using PSMA reporter gene.

PURPOSE: Chimeric antigen receptor (CAR) T cell therapy has achieved great success in treating hematologic malignancies. However, it is yet to prove effective in the treatment of solid tumors. Thus, it is necessary to develop appropriate methodology for the long-term, accurate, and quantitative evaluation of the distribution and activities of CAR T cells in solid tumors. In the present study, we engineered TfR ΔPSMA CAR (CAR-ΔPSMA) T cells, which targeted the transferrin receptor (TfR) expressed by tumor cells and could be tracked in vivo via a reporter gene encoding the truncated prostate specific membrane antigen (ΔPSMA). We then quantitatively monitored these CAR T cells in vitro and in vivo using [68Ga]Ga-PSMA-617 positron emission tomography (PET)/computed tomography (CT). METHODS: The CAR-ΔPSMA T cells were genetically engineered by transducing T cells with a lentiviral vector encoding TfR41BBζ-T2A-ΔPSMA. Firstly, the target expression, activation, and cytotoxicity of CAR-ΔPSMA T cells were validated in vitro. Secondly, the minimum thresholds of CAR-ΔPSMA T cells detection for [68Ga]Ga-PSMA-617 PET/CT were also determined in vitro and in vivo respectively. Lastly, the feasibility of monitoring the biodistribution and infiltration of CAR-ΔPSMA T cells after systematic administration was evaluated in the breast cancer subcutaneous xenograft model. RESULTS: The CAR-ΔPSMA T cells retained activation and tumor killing capacity after transduction of the ΔPSMA-encoding reporter gene. Next, the CAR-ΔPSMA T cells could be reliably tracked by [68Ga]Ga-PSMA-617 PET/CT, the detection sensitivity of which was 250 cells/mm3 in vitro and 100 cells/mm3 in vivo. Next, the sequential imaging assays revealed that [68Ga]Ga-PSMA-617 PET/CT could be used to specifically visualize ΔPSMA+ CAR T cells at the tumor site. The increase in the [68Ga]Ga-PSMA-617 signal intensity over time allowed us to effectively detect CAR T cells in vivo. CONCLUSION: Our findings preliminarily confirmed that [68Ga]Ga-PSMA-617 PET/CT could reliably detect CAR-ΔPSMA T cells in vitro and in vivo in solid tumors, laying the foundation for the monitoring CAR T cell therapy in the future.

Development and evaluation of albumin binder-conjugated heterodimeric radiopharmaceuticals targeting integrin αvß3 and CD13 for cancer therapy.

PURPOSE: The advancement of heterodimeric tracers, renowned for their high sensitivity, marks a significant trend in the development of radiotracers for cancer diagnosis. Our prior work on [68Ga]Ga-HX01, a heterodimeric tracer targeting CD13 and integrin αvß3, led to its approval for phase I clinical trials by the China National Medical Production Administration (NMPA). However, its fast clearance and limited tumor retention pose challenges for broader clinical application in cancer treatment. This study aims to develop a new radiopharmaceutical with increased tumor uptake and prolonged retention, rendering it a potential therapeutic candidate. METHODS: New albumin binder-conjugated compounds were synthesized based on the structure of HX01. In vitro and in vivo evaluation of these new compounds were performed after labelling with 68Ga. Small-animal PET/CT imaging were conducted at different time points at 0.5-6 h post injection (p.i.) using BxPC-3 xenograft mice models. The one with the best imaging performance was further radiolabeled with 177Lu for small-animal SPECT/CT and ex vivo biodistribution investigation. RESULTS: We have synthesized novel albumin binder-conjugated compounds, building upon the structure of HX01. When radiolabeled with 68Ga, all compounds demonstrated improved pharmacokinetics (PK). Small-animal PET/CT studies revealed that these new albumin binder-conjugated compounds, particularly [68Ga]Ga-L6, exhibited significantly enhanced tumor accumulation and retention compared with [68Ga]Ga-L0 without an albumin binder. [68Ga]Ga-L6 outperformed [68Ga]Ga-L7, a compound developed using a previously reported albumin binder. Furthermore, [177Lu]Lu-L6 demonstrated rapid clearance from normal tissues, high tumor uptake, and prolonged retention in small-animal SPECT/CT and biodistribution studies, positioning it as an ideal candidate for radiotherapeutic applications. CONCLUSION: A new integrin αvß3 and CD13 targeting compound was screened out. This compound bears a novel albumin binder and exhibits increased tumor uptake and prolonged tumor retention in BxPC-3 tumors and low background in normal organs, making it a perfect candidate for radiotherapy when radiolabeled with 177Lu.

Non-invasive visualization of liver fibrosis with [68Ga]Ga-DOTA-FAPI-04 PET from preclinical insights to clinical translation.

PURPOSE: The reversibility of early liver fibrosis highlights the need for improved early detection and monitoring techniques. Fibroblast activation protein (FAP) is a promising theranostics target significantly upregulated during fibrosis. This preclinical and preliminary clinical study investigated a FAP-targeted probe, gallium-68-labeled FAP inhibitor 04 ([68Ga]Ga-DOTA-FAPI-04), for its capability to visualize liver fibrosis. METHODS: The preclinical study employed [68Ga]Ga-DOTA-FAPI-04 micro-positron emission tomography (PET)/computed tomography (CT) on carbon tetrachloride-induced mice model (n = 34) and olive oil-treated control group (n = 26), followed by validation of the probe's biodistribution. Hepatic uptake was correlated with fibrosis and inflammation levels, quantified through histology and serum assays. FAP and α-smooth muscle actin expression were determined by immunohistochemistry, as well as immunofluorescence. The subsequent clinical trial enrolled 26 patients with suspected or confirmed liver fibrosis to undergo [68Ga]Ga-DOTA-FAPI-04 PET/magnetic resonance imaging or PET/CT. Key endpoints included correlating [68Ga]Ga-DOTA-FAPI-04 uptake with histological inflammation grades and fibrosis stages, and evaluating its diagnostic and differential efficacy compared to established serum markers and liver stiffness measurement (LSM). RESULTS: [68Ga]Ga-DOTA-FAPI-04 mean uptake in mice livers was notably higher than in control mice, increasing from week 6 [0.70 ± 0.11 percentage injected dose per cubic centimeter (%ID/cc)], peaking at week 10 (0.97 ± 0.15%ID/cc) and slightly reducing at week 12 (0.89 ± 0.28%ID/cc). The hepatic biodistribution and FAP expression showed a consistent trend. In the patient cohort, hepatic [68Ga]Ga-DOTA-FAPI-04 uptake presented moderate correlations with inflammation grades (r = 0.517 to 0.584, all P < 0.05) and fibrosis stages (r = 0.653 to 0.698, all P < 0.01). The average SUVmax to background ratio in the liver showed superior discriminative ability, especially between stage 0 and stage 1, outperforming LSM (area under curve 0.984 vs. 0.865). CONCLUSION: [68Ga]Ga-DOTA-FAPI-04 PET shows significant potential for non-invasive visualization and dynamic monitoring of liver fibrosis in both preclinical experiment and preliminary clinical trial, especially outperforming other common clinical indicators in the early stage. TRIAL REGISTRATION: NCT04605939. Registered October 25, 2020, https://clinicaltrials.gov/study/NCT04605939.

Detection and phenotype analysis of a novel Ael blood group allele.

BACKGROUND AND OBJECTIVES: The presence of blood subtypes may lead to difficulties in blood group identification; however, third-generation sequencing (TGS) can help in accurately identifying difficult blood groups, and study the serological characteristics and molecular mechanism of Ael subtypes. MATERIALS AND METHODS: ABO blood group was identified by the standard serological technique, weak blood group antigen was identified by adsorption-elution experiments, ABH substance in the saliva was determined and glycosyltransferase activity of A and B was detected. The ABO gene full-length sequence and promoter region were amplified by specific primers using single-molecule real-time sequencing, with the amplified products being sequenced directly and analysed in real time. RESULTS: The patient was serologically identified as Ael subtype, and TGS analysis revealed new intron mutations in Ael patients (c.467C>T; c.29-10T>A). CONCLUSION: The discovery of the new allele and the identification of ABO subtypes can be combined with serological characterization and molecular biological methods.

Efficacy of sensory-based static balance training on the balance ability, aging attitude, and perceived stress of older adults in the community: a randomized controlled trial.

BACKGROUND: This study explores the effect of sensory-based static balance training on the balance ability, aging attitude, and perceived stress of older adults in the community. It provides a practical basis for the in-depth implementation and revision of the community health management model. METHODS: A randomized controlled intervention study was conducted from 2022 to 2023. A total of 72 older adults were recruited and randomly divided into an intervention group (36 individuals) and a control group (36 individuals). Balance ability (measured by the Short Physical Performance Battery and One Leg Stand Test), aging attitudes, and perceived stress were assessed at baseline and at the 12-week and 24-week follow-ups. Repeated-measures ANOVA and generalized estimating equations were used to compare outcome measures. RESULTS: Sensory-based static balance training was beneficial for balance ability and aging attitude among participants in the intervention group. At the end of the intervention, participants in the intervention group showed significant improvements in SPPB scores and OLST scores compared with the control group (FSPPB = 12.347, P = 0.001, Waldχ2OLST = 45.530, P < 0.001), as well as significant differences in aging attitudes (FAAQ = 18.549, P < 0.001). Multiple comparisons at different time points in the intervention group reveal a significant intervention effect (FSPPB = 29.211, Waldχ2OLST = 80.428, FAAQ = 45.981, all P < 0.05). However, the difference in perceived stress before and after the intervention was not significant (FCPSS = 2.876, P = 0.095). CONCLUSIONS: Sensory-based static balance training significantly improved balance ability and aging attitudes among older adults in the community. The effect on perceived stress among older adults in the community was not significant. TRIAL REGISTRATION: Registered in the Chinese Clinic on 04/06/2022. The registration number is ChiCTR2200060541.

Data-independent acquisition mass spectrometry reveals comprehensive plasma protein profiles in the natural history of patients with hereditary transthyretin amyloidosis (ATTRv).

OBJECTIVES: Hereditary transthyretin amyloidosis (ATTRv) is a rare, fatal, autosomal dominant disease with more than 140 mutations discovered. Three phenotypes of amyloid infiltration are neuropathy (ATTRv-PN), cardiopathy (ATTRv-CM), and neuropathy + cardiopathy (ATTRv-MIX). The lack of ATTR-specific biomarkers, difficulties in biopsy evidence, and limited known pathogenic mechanisms have made diagnosis difficult. Newly emerging noninvasive measures for monitoring progression and disease-modifying therapies have improved early diagnosis and patient management. METHODS: Our research applies the latest technology, Data-Independent Acquisition-Based Quantitative Proteomics (DIA), to reveal comprehensive plasma protein profiles in the natural history of Chinese patients with hereditary transthyretin amyloidosis (ATTRv). We analyzed differentially expressed proteins (DEPs) in three phenotypes (ATTRv-PN, ATTRv-CM, and ATTRv-MIX). RESULTS: Serum samples were collected from a total of 18 patients (6 ATTRv-PN, 5 ATTRv-CM, and 7 ATTRv-MIX patients) and 20 healthy participants as a control group. Combined with the results of the proteomic and bioinformatic analyses, we found 30 DEPs and protein interaction networks clustered in KRT family proteins and DSC3 between ATTRv-PN and the control, which were rich in the estrogen signaling pathway and the cell adhesion molecule (CAM) pathway. CONCLUSION: This study demonstrates a global and significant proteomic profile in different stages of ATTRv.

Preclinical evaluation of a dual-receptor targeted tracer [68Ga]Ga-HX01 in 10 different subcutaneous and orthotopic tumor models.

PURPOSE: The integrin αvß3 and aminopeptidase N (APN/CD13) play vital roles in the tumor angiogenesis process. They are highly expressed in a variety of tumor cells and proliferating endothelial cells during angiogenesis, which have been considered as highly promising targets for tumor imaging. Arginine-glycine-aspartic (RGD) and asparagine-glycine-arginine (NGR) are two peptides specifically binding to the integrin αvß3 and CD13, respectively. In this study, we optimized our previously developed probe and preclinically evaluated the new integrin αvß3 and CD13 dual-targeted probe, NOTA-RGD-NGR (denoted as HX01) radiolabeled with 68Ga, in 10 different subcutaneous and orthotopic tumor models. METHODS: The specific activity and radiochemical purity of [68Ga]Ga-HX01 were identified. The dual-receptor targeting ability was confirmed by a series of blocking studies and partly muted tracers using BxPC-3 xenograft model. The dynamic imaging study and dose escalation study were explored to determine the optimal imaging time point and dosage in the BxPC-3 xenograft model. Next, we established a variety of subcutaneous and orthotopic tumor models including pancreas (BxPC-3), breast (MCF-7), gallbladder (NOZ), lung (HCC827), ovary (SK-OV-3), colorectal (HCT-8), liver (HuH-7), stomach (NUGC-4), and glioma (U87) cancers. All models underwent [68Ga]Ga-HX01 PET/CT imaging about 2 weeks post-inoculation, with a subset of them undergoing [18F]FDG PET/CT scan performed concurrently, and their results were compared. In addition, ex vivo biodistribution studies were also performed for verifying the semi-quantitative results of the non-invasive PET images. RESULTS: [68Ga]Ga-HX01 significantly outperformed single target probes in the BxPC-3 xenograft model. All blocking and single target groups exhibited significantly descending tumor uptake. The high tumor uptakes were found in BxPC-3, MCF-7, and NOZ subcutaneous tumors (%ID/g > 1.1), while middle uptakes were observed in HCC827, SK-OV-3, HCT-8, and HuH-7 subcutaneous tumor (%ID/g 0.7-1.0). Due to the low background, the tumor-to-muscle and tumor-to-blood ratios of [68Ga]Ga-HX01 were higher than that of [18F]FDG. CONCLUSIONS: [68Ga]Ga-HX01, as a dual target imaging agent, exhibited superior in vivo performance in different subcutaneous and orthotopic mice models of human tumors over [18F]FDG and its respectively mono-receptor targeted agents, which warrants the future clinical translation for tumor imaging.

Visualizing γδ T cells by very late antigen-4-targeted positron emission tomography.

PURPOSE: γδ T cell-based immunotherapy has been rolled out as a promising treatment strategy for malignant tumors due to their potent anti-tumor cytotoxicity, ease of expansion, and unrestricted MHC feature. However, the dynamics and outcomes of γδ T cells in tumor sites are poorly understood. Reported strategies rely on ex vivo biolabeling, significantly limiting the application of γδ T cell molecular imaging. Herein, we investigated whether VLA-4 (very late antigen-4), a crucial component in the effective trafficking of lymphocytes, could serve as a biomarker to non-invasively visualize γδ T cells. METHODS: VLA-4-targeted tracer, 68 Ga-LLP2A, was evaluated in MDA-MB-231- and A549-bearing mice with adoptive transfer of γδ T cells by longitudinal PET/CT imaging. Imaging data were verified by ex vivo biodistribution studies, and the co-localization of CD3 and VLA-4 was validated by immunohistochemistry studies. RESULTS: 68 Ga-LLP2A showed high specificity to VLA-4-expressing γδ T cells in both in vitro and tumor-bearing mice with adoptive transfer of γδ T cells. Longitudinal PET imaging of 68 Ga-LLP2A in tumor-bearing mice with adoptive transfer of γδ T cells showed an increasing tumor tracer uptake, revealing the tumor-specific homing of γδ T cells. The presence of VLA-4-expressing γδ T cells in tumors was confirmed via histological analysis. CONCLUSION: To the best of our knowledge, we reported the first molecular probe, 68 Ga-LLP2A, for in vivo imaging of γδ T cells in live tumors, which advances PET imaging of γδ T cells and supports the translation of imaging agents for immunotherapeutic monitoring.

Mapping the Minnesota Living with Heart Failure Questionnaire (MLHFQ) to SF-6Dv2 in Chinese patients with heart failure.

PURPOSE: Mapping the Minnesota Living with Heart Failure Questionnaire (MLHFQ) to SF-6Dv2 in Chinese patients with chronic heart failure, and to obtain the health utility value for health economic assessment. METHODS: Four statistical algorithms, including ordinary least square method (OLS), Tobit model, robust MM estimator (MM) and censored least absolute deviations (CLAD), were used to establish the alternative model. Models were validated by using a tenfold cross-validation technique. The mean absolute error (MAE) and root mean square error (RMSE) were used to evaluate the prediction performance of the model. The Spearman correlation coefficient and Intraclass Correlation Coefficients (ICC) were used to examine the relationship between the predicted and observed SF-6Dv2 values. RESULTS: A total of 195 patients with chronic heart failure were recruited from 3 general hospitals in Beijing. The MLHFQ summary score and domain scores of the study sample were negatively correlated with SF-6Dv2 health utility value. The OLS regression model established based on the MLHFQ domain scores was the optimal fitting model and the predicted value was highly positively correlated with the observed value. CONCLUSION: The MLHFQ can be mapped to SF-6Dv2 by OLS, which can be used for health economic assessment of cardiovascular diseases such as chronic heart failure.

Electroacupuncture vs Prucalopride for Severe Chronic Constipation: A Multicenter, Randomized, Controlled, Noninferiority Trial.

INTRODUCTION: This multicenter, randomized, noninferiority trial compared electroacupuncture with prucalopride for the treatment of severe chronic constipation (SCC). METHODS: Participants with SCC (≤ 2 mean weekly complete spontaneous bowel movements [CSBMs]) were randomly assigned to receive either 28-session electroacupuncture over 8 weeks with follow-up without treatment over 24 weeks or prucalopride (2 mg/d before breakfast) over 32 weeks. The primary outcome was the proportion of participants with ≥3 mean weekly CSBMs over weeks 3-8, based on the modified intention-to-treat population, with -10% as the noninferior margin. RESULTS: Five hundred sixty participants were randomized, 280 in each group. Electroacupuncture was noninferior to prucalopride for the primary outcome (36.2% vs 37.8%, with a difference of -1.6% [95% confidence interval, -8% to 4.7%], P < 0.001 for noninferiority); almost the same results were found in the per-protocol population. The proportions of overall CSBM responders through weeks 1-8 were similar in the electroacupuncture and prucalopride groups (24.91% vs 25.54%, with a difference of -0.63% [95% confidence interval, -7.86% to 6.60%, P = 0.864]). Except during the first 2-week treatment, no between-group differences were found in outcomes of excessive straining, stool consistency, and quality of life. Adverse events occurred in 49 (17.69%) participants in the electroacupuncture group and 123 (44.24%) in the prucalopride group. One non-treatment-related serious adverse event was recorded in the electroacupuncture group. DISCUSSION: Electroacupuncture was noninferior to prucalopride in relieving SCC with a good safety profile. The effects of 8-week electroacupuncture could sustain for 24 weeks after treatment. Electroacupuncture is a promising noninferior alternative for SCC (see Visual Abstract, http://links.lww.com/AJG/B776).

Carbamazepine-induced immune thrombocytopenia confirmed by modified MASPAT test.

A 76-year-old man suffering post-herpetic neuralgia developed severe thrombocytopenia 15 days after the administration of carbamazepine. Carbamazepine-dependent platelet antibodies were proved to be present in the patient's serum by a modified Monoclonal Antibody Solid-phase Platelet Antibody Test (MASPAT), and the diagnosis of carbamazepine-induced immune thrombocytopenia was confirmed. For the patient, carbamazepine should be advised to be avoided permanently. The present report advocated the application of a modified MASPAT test for the detection of carbamazepine-dependent platelet antibodies.

Surface chemistry induces mitochondria-mediated apoptosis of breast cancer cells via PTEN/PI3K/AKT signaling pathway.

Tumor cell can be significantly influenced by various chemical groups of the extracellular matrix proteins. However, the underlying molecular mechanisms involved in the interaction between cancer cells and functional groups in the extracellular matrix remain unknown. Using chemically modified surfaces with biological functional groups (CH3, NH2, OH), it was found that hydrophobic surfaces modified with CH3 and NH2 suppressed cell proliferation and induced the number of apoptotic cells. Mitochondrial dysfunction, cytochrome c release, Bax upregulation, cleaved caspase-3 and PARP, and Bcl-2 downregulation indicated that hydrophobic surfaces with CH3 and NH2 triggered the activation of intrinsic apoptotic signaling pathway. Cells on the CH3- and NH2-modified hydrophobic surfaces showed downregulated expression and activation of integrin ß1, with a subsequent decrease of focal adhesion kinase (FAK) activity. The RhoA/ROCK/PTEN signaling was then activated to inhibit the phosphorylation of PI3K and AKT, which are essential for cell proliferation. However, pretreatment of MDA-MB-231 cells with SF1670, a PTEN inhibitor, abolished the hydrophobic surface-induced activation of the intrinsic pathway. Taken together, the present results indicate that CH3- and NH2-modified hydrophobic surfaces induce mitochondria-mediated apoptosis by suppressing the PTEN/PI3K/AKT pathway, but not OH surfaces. These findings are helpful to understand the interaction between extracellular matrix and cancer cells, which might provide new insights into the mechanism potential intervention strategies for tumor prognosis.

Acidic pHe regulates cytoskeletal dynamics through conformational integrin ß1 activation and promotes membrane protrusion.

An acidic extracellular pH (pHe) in the tumor microenvironment has been suggested to facilitate tumor growth and metastasis. However, the molecular mechanisms by which tumor cells sense acidic signal to induce a transition to an aggressive phenotype remain elusive. Here, we showed that an acidic pHe (pH 6.5) stimulation resulted in protrusion and epithelial-mesenchymal transition (EMT) of cancer cells, which promoted migration and matrix degeneration. Using computational molecular dynamics simulations, we reported acidic pHe-induced opening of the Integrin dimers (α5ß1) headpiece which indicated the activation of integrin. Moreover, acidic pHe promoted maturation of focal adhesions, temporal activation of Rho GTPases and microfilament reorganization through integrin ß1-activated FAK signaling. Furthermore, mechanical balance of cytoskeleton (actin, tubulin and vimentin) contributed to acidic pHe-triggered protrusion and morphology change. Taken together, these findings revealed that integrin ß1 could be a novel pH-regulated sensitive molecule which confers protrusion and malignant phenotype of cancer cells.

Coupling between ATP hydrolysis and protein conformational change in maltose transporter.

As the intracellular part of maltose transporter, MalK dimer utilizes the energy of ATP hydrolysis to drive protein conformational change, which then facilitates substrate transport. Free energy evaluation of the complete conformational change before and after ATP hydrolysis is helpful to elucidate the mechanism of chemical-to-mechanical energy conversion in MalK dimer, but is lacking in previous studies. In this work, we used molecular dynamics simulations to investigate the structural transition of MalK dimer among closed, semi-open and open states. We observed spontaneous structural transition from closed to open state in the ADP-bound system and partial closure of MalK dimer from the semi-open state in the ATP-bound system. Subsequently, we calculated the reaction pathways connecting the closed and open states for the ATP- and ADP-bound systems and evaluated the free energy profiles along the paths. Our results suggested that the closed state is stable in the presence of ATP but is markedly destabilized when ATP is hydrolyzed to ADP, which thus explains the coupling between ATP hydrolysis and protein conformational change of MalK dimer in thermodynamics. Proteins 2017; 85:207-220. © 2016 Wiley Periodicals, Inc.

Lack of Association between MYO9B Gene Polymorphisms and Susceptibility to Coeliac Disease in Caucasians: Evidence from a Meta-Analysis.

OBJECTIVE: Coeliac disease (CD) is a complex disorder influenced by environmental and genetic factors. Recently, a number of studies reported MYO9B gene is associated with CD, but the results are controversial. The aim of this study is to clarify this dispute by means of a meta-analysis. METHODS: The databases of PubMed, Web of Science, and Embase updated to August 2015 were retrieved. Crude odds ratio (OR) and corresponding 95% confidence interval (95%CI) as effect size were calculated by fixed or random effect model according to the heterogeneity. RESULTS: A total of 8 studies including 2272 cases and 5419 controls were enrolled in this meta-analysis. There was no significant association both in allele and genotype comparisons between the MYO9B (rs2305764, rs2305767, rs1457092) polymorphism and CD in Caucasian populations. No publication bias was detected in this meta-analysis. CONCLUSIONS: This meta-analysis indicates that MYO9B gene polymorphisms might be not associated with CD susceptibility in Caucasians. Further studies are still needed for definitive conclusions.

Survey of microbial contamination and characterization of Escherichia coli in kiwifruit orchards in Shaanxi, China, 2013.

The aim of the study was to survey three foodborne pathogens in kiwifruit orchards as a continuous monitoring program. A total of 193 samples were collected from 11 kiwifruit orchards in Shaanxi province in October 2013. Among the 193 samples, 68 Escherichia coli isolates were recovered, while no Staphylococcus aureus and Salmonella was recovered. All E. coli isolates were characterized by antimicrobial susceptibility testing, detection of virulence genes, and the ability to produce biofilm formation. The isolates were further examined by random amplified polymorphic DNA (RAPD) analysis. E. coli isolates displayed resistance most frequently to tetracycline (48.5%). Two E. coli isolates (2.9%) were positive for the eae gene (the intimin gene). All E. coli isolates lacked the ability to make biofilm formation. Multilocus sequence typing analysis demonstrated that one isolate in kiwifruit orchards shared the same sequence type with a human clinical isolate. RAPD results showed a close relationship among E. coli isolates from fresh fruit, fallen fruit, soil, air, and irrigation water. This study could provide a further understanding of microbial contamination in kiwifruit orchards based on our previous study and help growers take appropriate measures for prevention.

Punicalagin inhibits Salmonella virulence factors and has anti-quorum-sensing potential.

Punicalagin, an essential component of pomegranate rind, has been demonstrated to possess antimicrobial activity against several food-borne pathogens, but its activity on the virulence of pathogens and its anti-quorum-sensing (anti-QS) potential have been rarely reported. This study investigated the efficacy of subinhibitory concentrations of punicalagin on Salmonella virulence factors and QS systems. A broth microdilution method was used to determine the MICs of punicalagin for 10 Salmonella strains. Motility assay and quantitative reverse transcription (RT)-PCR were performed to evaluate the effects of punicalagin on the virulence attributes and QS-related genes of Salmonella. The MICs of punicalagin for several Salmonella strains ranged from 250 to 1,000 µg/ml. Motility assays showed that punicalagin, at 1/16× MIC and 1/32× MIC, significantly decreased bacterial swimming and swarming motility, which corresponded to downregulation of the motility-related genes (fliA, fliY, fljB, flhC, and fimD) in RT-PCR assays. RT-PCR also revealed that punicalagin downregulated the expression of most of the selected genes involved in Salmonella virulence. Moreover, a QS inhibition assay indicated that punicalagin dose dependently inhibited the production of violacein by Chromobacterium violaceum and repressed the expression of QS-related genes (sdiA and srgE) in Salmonella. In addition, punicalagin significantly reduced Salmonella invasion of colonic cells (P<0.01) with no impact on adhesion. These findings suggest that punicalagin has the potential to be developed as an alternative or supplemental agent for prevention of Salmonella infection.

Prevalence, distribution, and diversity of Escherichia coli, Staphylococcus aureus, and Salmonella in kiwifruit orchards and processing plants.

The aim of the study was to investigate the prevalence, distribution, and diversity of three foodborne bacteria in kiwifruit orchards and processing plants. Fourteen kiwifruit orchards and two processing plants in Shaanxi province were visited for sampling in 2012. Fruit samples and environmental samples in orchards and plants were taken for isolation of Escherichia coli, Staphylococcus aureus, and Salmonella. All isolates were characterized by antimicrobial susceptibility testing and detection of virulence genes. Selected isolates were further examined by pulsed-field gel electrophoresis (PFGE) analysis. In total, 160 E. coli isolates and 14 S. aureus isolates were recovered from 407 samples from orchards and plants, while no Salmonella was recovered. E. coli isolates displayed resistance most frequently to streptomycin (65.6%), and S. aureus isolates displayed resistance most frequently to erythromycin (21.4%). Three E. coli isolates (1.9%) were positive for stx2 and two S. aureus isolates (14.3%) were positive for both seb and seh. Seventy-seven E. coli isolates and 14 S. aureus isolates were analyzed by PFGE. PFGE results showed that both E. coli and S. aureus isolates were diverse, and blades for slicing during the processing could be an important contamination source. This study could provide useful information for kiwifruit growers and industry to establish proper management practices that help minimize the chance of microbial contamination from farm to table.