Search

Summary: Background: Metabolic reprogramming plays a pivotal role in cancer progression, contributing to substantial intratumour heterogeneity and influencing tumour behaviour. However, a systematic characterization of metabolic heterogeneity across multiple cancer types at the single-cell level remains limited. Methods: We integrated 296 tumour and normal samples spanning six common cancer types to construct a single-cell compendium of metabolic gene expression profiles and identify cell type-specific metabolic properties and reprogramming patterns. A computational approach based on non-negative matrix factorization (NMF) was utilised to identify metabolic meta-programs (MMPs) showing intratumour heterogeneity. In-vitro cell experiments were conducted to confirm the associations between MMPs and chemotherapy resistance, as well as the function of key metabolic regulators. Survival analyses were performed to assess clinical relevance of cellular metabolic properties. Findings: Our analysis revealed shared glycolysis upregulation and divergent regulation of citric acid cycle across different cell types. In malignant cells, we identified a colorectal cancer-specific MMP associated with resistance to the cuproptosis inducer elesclomol, validated through in-vitro cell experiments. Furthermore, our findings enabled the stratification of patients into distinct prognostic subtypes based on metabolic properties of specific cell types, such as myeloid cells. Interpretation: This study presents a nuanced understanding of multilayered metabolic heterogeneity, offering valuable insights into potential personalized therapies targeting tumour metabolism. Funding: National Key Research and Development Program of China (2021YFA1300601). National Natural Science Foundation of China (key grants 82030081 and 81874235). The Shenzhen High-level Hospital Construction Fund and Shenzhen Basic Research Key Project (JCYJ20220818102811024). The Lam Chung Nin Foundation for Systems Biomedicine.

Summary: Reliable blood-based tests for identifying early-stage breast cancer remain elusive. Employing single-cell transcriptomic sequencing analysis, we illustrate a close correlation between nucleotide metabolism in the breast cancer and activation of regulatory T cells (Tregs) in the tumor microenvironment, which shows distinctions between subtypes of patients with triple-negative breast cancer (TNBC) and non-TNBC, and is likely to impact cancer prognosis through the A2AR-Treg pathway. Combining machine learning with absolute quantitative metabolomics, we have established an effective approach to the early detection of breast cancer, utilizing a four-metabolite panel including inosine and uridine. This metabolomics study, involving 1111 participants, demonstrates high accuracy across the training, test, and independent validation cohorts. Inosine and uridine prove predictive of the response to neoadjuvant chemotherapy (NAC) in patients with TNBC. This study deepens our understanding of nucleotide metabolism in breast cancer development and introduces a promising non-invasive method for early breast cancer detection and predicting NAC response in patients with TNBC.

Abstract Anion exchanger 2 (AE2) is an electroneutral Na+-independent Cl-/HCO3 - exchanger belongs to the SLC4 transporter family. The widely expressed AE2 participates in a variety of physiological processes, including transepithelial acid-base secretion and osteoclastogenesis. Both the transmembrane domains (TMDs) and the N-terminal cytoplasmic domain (NTD) are involved in regulation of AE2 activity. However, the regulatory mechanism remains unclear. Here, we report a 3.2 Å cryo-EM structure of the AE2 TMDs in complex with PIP2 and a 3.3 Å full-length mutant AE2 structure in the resting state without PIP2. We demonstrate that PIP2 at the TMD dimer interface is involved in the substrate exchange process. Mutation in the PIP2 binding site leads to the displacement of TM7 and further stabilizes the interaction between the TMD and the NTD. Reduced substrate transport activity and conformation similar to AE2 in acidic pH indicating the central contribution of PIP2 to the function of AE2.

Abstract CD8+ T cells are critical for host antitumor responses, whereas persistent antigenic stimulation and excessive inflammatory signals lead to T cell dysfunction or exhaustion. Increasing early memory T cells can improve T cell persistence and empower T cell‐mediated tumor eradication, especially for adoptive cancer immunotherapy. Here, it is reported that tumor‐associated monocytes (TAMos) are highly correlated with the accumulation of CD8+ memory T cells in human cancers. Further analysis identifies that TAMos selectively reprogram CD8+ T cells into T central memory‐like (TCM‐like) cells with enhanced recall responses. L‐NMMA, a pan nitric oxide synthase inhibitor, can mitigate TAMo‐mediated inhibition of T cell proliferation without affecting TCM‐like cell generation. Moreover, the modified T cells by TAMo exposure and L‐NMMA treatment exhibit long‐term persistence and elicit superior antitumor effects in vivo. Mechanistically, the transmembrane protein CD300LG is involved in TAMo‐mediated TCM‐like cell polarization in a cell‐cell contact‐dependent manner. Thus, the terminally differentiated TAMo subset (CD300LGhighACElow) mainly contributes to TCM‐like cell development. Taken together, these findings establish the significance of TAMos in boosting T‐cell antitumor immunity.

Abstract Cancer exerts a multitude of effects on metabolism, including the reprogramming of cellular metabolic pathways and alterations in metabolites that facilitate inappropriate proliferation of cancer cells and adaptation to the tumor microenvironment. There is a growing body of evidence suggesting that aberrant metabolites play pivotal roles in tumorigenesis and metastasis, and have the potential to serve as biomarkers for personalized cancer therapy. Importantly, high-throughput metabolomics detection techniques and machine learning approaches offer tremendous potential for clinical oncology by enabling the identification of cancer-specific metabolites. Emerging research indicates that circulating metabolites have great promise as noninvasive biomarkers for cancer detection. Therefore, this review summarizes reported abnormal cancer-related metabolites in the last decade and highlights the application of metabolomics in liquid biopsy, including detection specimens, technologies, methods, and challenges. The review provides insights into cancer metabolites as a promising tool for clinical applications.

Abstract Background Real-world data of Palbociclib are insufficient in China. This study aimed to investigate the treatment pattern and real-world outcomes in hormone receptor positive and human epidermal growth factor 2 receptor negative (HR+/HER2-) metastatic breast cancer (MBC) patients treated with Palbociclib in the northwest of China. Methods HR+/HER2- MBC patients who received Palbociclib in 8 centers from July 2017 to September 2019 were retrospectively included in this study. Real-world objective response rate (ORR), progression-free survival (PFS) and safety profiles were analyzed. The survival curves were plotted by the Kaplan-Meier method to analyze PFS, which was verified by the log-rank test. Results In total, 211 women were eligible for the analysis. A total of 85 patients (40.3%), 78 (37.0%), and 48 (22.7%) received Palbociclib in the first-, second-, third- or later-line setting, respectively. 46 patients achieved partial response and 145 patients experienced stable disease, with an ORR of 21.8% and a disease control rate of 90.5%. Following a median follow-up period of 14.2 months, the median PFS was 12.2 months (95% confidence interval, 10.1-14.3 m), and the median overall survival was not reached. Early Palbociclib initiation, sensitivity or acquired resistance to endocrine therapy, estrogen receptor and progesterone receptor double positivity, less than 3 metastatic sites, without visceral metastasis, bone metastasis only, without prior chemotherapy or endocrine therapy were associated with a prolonged PFS in MBC (All P

Background: Previous clinical studies have suggested that Toll-like receptor (TLR)2 had predictive function for endocrine resistance in HER2-positive breast cancer (BCa). Nevertheless, it remains unclear whether TLR2 would relate to development of endocrine therapy resistance in triple-positive breast cancer (TPBC). Methods: Bioinformatic analysis of TLR2 was carried out through a database. Ten tumor tissues were obtained from TPBC patients who underwent surgery, with five patients displaying primary resistance to tamoxifen (TAM) with the remaining 5 being sensitive. Different levels of proteins were identified through mass spectrometry analysis and confirmed through reverse transcription polymerase chain reaction (RT-PCR) and western blot. TAM-resistant cell lines (BT474-TAM) were established by continuous exposure to TAM, and TAM resistance was assessed via IC50. Additionally, TLR2 mRNA was analyzed through western blot and RT-PCR in BT474, BT474-TAM, MCF-7, and MCF10A cells. Furthermore, TLR2-specific interference sequences were utilized to downregulate TLR2 expression in BT474-TAM cells to elucidate its role in TAM resistance. Results: TLR2 had a correlation with decreased relapse-free survival in BCa patients from the GSE1456-GPL96 cohort, and it was involved in cancer development predominantly mediated by MAPK and PI3K pathways. TLR2 protein expression ranked in the top 5 proteins within the TAM-resistant group, and was 1.9 times greater than that in the sensitive group. Additionally, TLR2 mRNA and protein expression increased significantly in the established TAM-resistant BT474/TAM cell lines. The sensitivity of TAM was restored upon TLR2 downregulation in BT474/TAM cells. Conclusions: TLR2 might have a therapeutic value as it was involved in the TAM resistance in TPBC, with potential to be a marker for primary endocrine resistance.

Background: Following traumatic bone loss or removal of bone tumors, the failure of bone allograft transplantation for large bone defect repair remains a significant problem in orthopedics. Therefore, new strategies that can efficiently enhance allograft healing and long-term incorporation are critically needed. Method: In this study, we first injected Notch-activating Jagged1 peptide to mice and then isolated bone marrow tissues and cells for proliferation and differentiation assays. Femur bone allograft surgery was also performed in Jagged1 pre-treated mice, and bone defect healing process were monitored by histology, Micro-CT and biomechanical testing. Result: Our results showed that Jagged1 therapeutic injection is sufficient to maximally activate Notch and promote bone marrow stromal cell proliferation in vivo, while no effects on bone structure were observed. More importantly, Jagged1 pre-treatment significantly promoted bone callus formation and increased bone mechanical strength during allograft healing in a femur bone defect mouse model. Conclusion: This study reveals that Notch in vivo activation can be induced by injection of Jagged1 peptide for expansion of local native stromal cells that will significantly enhance bone callus formation. The Translational potential of this Article: The clinical uses of this therapeutic strategy would be immediately applicable for chronic long bone defect repair. More importantly, this devised strategy for expansion of endogenous BMSCs can also be applied to enhance other tissue and organ repair.

Summary: Background: Most malignant brain gliomas (MBGs) are associated with dismal outcomes, mainly due to their late diagnosis. Current diagnostic methods for MBGs are based on imaging and histological examination, which limits their early detection. Here, we aimed to identify reliable plasma lipid biomarkers for non-invasive diagnosis for MBGs. Methods: Untargeted lipidomic analysis was firstly performed using a discovery cohort (n=107). The data were processed by a support vector machine (SVM)-based discriminating model to retrieve a panel of candidate biomarkers. Then, a targeted quantification method was developed, and the SVM-based diagnostic model was constructed using a training cohort (n=750) and tested using a test cohort (n=225). Finally, the performance of the diagnostic model was further evaluated in an independent validation cohort (n=920) enrolled from multiple medical centers. Findings: A panel of 11 plasma lipids was identified as candidate biomarkers with an accuracy of 0.999. The diagnostic model developed achieved a high performance in distinguishing MBGs patients from normal controls with an area under the receiver-operating characteristic curve (AUC) of 0.9877 and 0.9869 in the training and test cohorts, respectively. In the validation cohort, the 11 lipid panel still achieved an accuracy of 0.9641 and an AUC of 0.9866. Interpretation: The present study demonstrates the applicability and robustness of utilizing a machine learning algorithm to analyze lipidomic data for efficient and reliable biomarker screening. The 11 lipid biomarkers show great potential for the non-invasive diagnosis of MBGs with high throughput. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgments section.

Heteroresistance can lead to treatment failure and is difficult to detect by the methods currently employed by clinical laboratories. The aim of this study was to investigate the prevalence of the amikacin-heteroresistant Klebsiella pneumoniae strains and explore potential amikacin heteroresistance mechanism through whole-genome sequencing (WGS) and quantitative reverse-transcription PCR (qRT-PCR). In this study, 13 isolates (8.39%) were considered as amikacin-heteroresistant K. pneumoniae strains among a total of 155 K. pneumoniae strains. The majority of the heterogeneous phenotypes (11/13, 84.61%) was unstable and the minimal inhibitory concentrations (MICs) fully or partially reverted back to the level of susceptibility of the parental isolate. The frequency of heteroresistant subpopulation ranged from 2.94×10−7 to 5.59×10−6. Whole-genome sequencing and single-nucleotide variants (SNVs) analysis showed that there were different nucleotide and resultant amino acid alterations among an amikacin-heteroresistant strain S38 and the resistant subpopulation S38L in several genes. Quantitative reverse-transcription PCR analysis revealed that the increased expression of aminoglycoside resistance genes detected in amikacin-heteroresistant K. pneumoniae strains might be associated with amikacin heteroresistance. The findings raise concerns for the emergence of amikacin-heteroresistant K. pneumoniae strains and the use of amikacin as therapy for the treatment of multidrug-resistant K. pneumoniae strains.

Traditional agro-ecosystems have sustained diverse aquatic plants, but have been greatly damaged by modern agricultural practices. Ottelia alismoides is a submerged macrophyte occurring in and around paddy fields, and a vulnerable species in Japan. Using four primer pairs of amplified fragment length polymorphism (AFLP) markers, genetic variation was estimated in 12 O. alismoides populations from Honshu, Shikoku and Kyushu. O. alismoides demonstrated low intra-population genetic variation (Pp = 15.7%, HE = 0.052 and I = 0.080), which was explained by its annual life history, high level of autogamy and repeated genetic bottlenecks. These factors, combined with limited gene flows (Nm = 0.113–0.127, M = 0.091), also resulted in the high genetic differentiation among populations, contributing about 70% of the total genetic variation. The gene flow might be hindered by geographic distance, mountains within islands and the strait between islands. It is, therefore, recommended that conservation of all extant populations be prioritized to maximize gene representation.

Objectives: The aim of this study was to characterise a high biofilm-forming capacity, hypermucoviscous, blaKPC and blaNDM co-producing Klebsiella pneumoniae strain (KSH203). Methods: Antimicrobial susceptibility, biofilm formation and hypermucoviscous phenotype were determined by the disk diffusion method, crystal violet staining and positive string test, respectively. Whole-genome sequencing was performed using a PacBio RS II Sequencer. High-quality reads were de novo assembled using Celera Assembler v.8.0. Genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP), and the genome characteristics were analysed by bioinformatics methods. Results: Klebsiella pneumoniae strain KSH203 was resistant to all antibiotics tested but was only intermediate-resistant to polymyxin B. This strain showed high biofilm-forming ability and a hypermucoviscous phenotype with serotype K25 belonging to the ST11 clone. KSH203 consists of a 5 464 059-bp single chromosome and four plasmids including pKSH203-NDM (53 144 bp), pKSH203-KPC (159 467 bp), pKSH203-CTX-M-3 (156 910 bp) and pKSH203-qnrS (253 705 bp). A total of 44 antimicrobial resistance genes and a large number virulence-associated genes were identified in the genome of strain KSH203. Conclusion: In this study, we illustrate the whole genome sequence of high biofilm-forming capacity, hypermucoviscous K. pneumoniae isolate KSH203 with capsular serotype K25 belonging to ST11 isolated from a patient in China, which carried a large number of antimicrobial resistance genes and virulence-associated genes. Future studies are needed to be aware of dissemination of this type of strain among environmental, animal and human isolates.

Abstract Background A carbapenem-resistant hypermucoviscous Klebsiella pneumoniae isolate was recovered from human sputum. Methods Whole genome sequencing of this isolate was carried out to reveal its clonal background, antimicrobial resistance determinants and virulence factors. Virulence assays were performed using wax moth larvae. The transfer of bla NDM-5 between bacterial strains was tested using conjugation. 59 genome assemblies of ST29 K. pneumoniae and 230 IncX3 plasmids regardless of the carriage of resistance gene were employed for phylogenetic analysis, respectively. Results The strain carried a virulence plasmid pVir-SCNJ1 bearing the virulence gene rmpA and exhibited a high virulence in wax moth. This hypervirulent strain belongs to sequence type 29 and carries bla NDM-5, which is located on a conjugative plasmid, designated pNDM5-SCNJ1, belonging to type IncX3. pNDM5-SCNJ1 was fully sequenced and shows high similarity with pNDM_MGR194, except some deletion inside the ISAba125 region. Phylogenetic analysis of IncX3 plasmids revealed that although bla NDM-5 can be evolved from bla NDM-1 via point mutations within some IncX3 plasmids, most of bla NDM-5-carrying IncX3 plasmids probably have acquired bla NDM-5 in multiple events. Conclusions In this study, we characterized a bla NDM-5-positive hypervirulent K. pneumoniae of sequence type 29 in China. Our results highlight the need for active surveillance on this lineage of carbapenem-resistant K. pneumoniae.

Abstract Background Drought and its resulting oxidative damage are the major yield limiting factors for crops in arid and semi-arid regions. Recent studies have found that endophytic fungi coexisting in plants can alleviate biotic or abiotic damage to plant growth and development. In order to screen for the endophytes associated with drought stress, 12 strains of endophytic fungi with high antioxidant activity isolated from riparian plants Myricaria laxiflora were evaluated for their effects in rice by the crude extracts. Results Of the 12 endophytic fungi, Aspergillus fumigatus SG-17 functioned most effectively, with the crude extract exhibiting relatively higher antioxidant capacity both in vivo and in vitro. The subsequent MS and NMR analysis showed that the primary substance responsible for the antioxidant activity in the extract was (Z)-N-(4-hydroxystyryl) formamide (NFA), an analogue of coumarin. Enzyme activity assay in nerve cells SH-SY5Y showed that NFA could maintain the membrane integrity and regulate the antioxidase activity under oxidative stress. In rice suffering drought stress, NFA effectively alleviated the harm by regulating the contents of NADPH oxidases, antioxidants and heat shock proteins, all of which are closely related with the reactive oxygen species pathway. Conclusion These findings indicated that some endophytes from plants often subjected to flooding and oxidative stress could enhance drought resistance by producing compounds such as NFA to regulate the oxidative pathway.

An effective method for separating and purifying critical saponins (polyphyllin II and polyphyllin VII) from a Paris polyphylla var. yunnanensis extract was developed in this study which was environmentally friendly and economical. Static adsorption kinetics, thermodynamics, and the dynamic adsorption-desorption of macroporous resins were investigated, and then the conditions of purification and separation were optimized by fitting with an adsorption thermodynamics equation and a kinetic equation. Effective NKA-9 resin from seven macroporous resins was screened out to separate and purify the two saponins. The static adsorption and dynamic adsorption were chemical and physical adsorption dual-processes on the NKA-9 resin. Under the optimum parameters, the contents of polyphyllin II and polyphyllin VII in the product were 17.3-fold and 28.6-fold those in plant extracts, respectively. The total yields of the two saponins were 93.16%. This research thus provides a theoretical foundation for the large-scale industrial production of the natural drugs polyphyllin II and polyphyllin VII.

This study proposes a new method of detecting tungsten inclusions in nuclear fuel rod upper-end plug welds using energy-dispersive X-ray fluorescence (EDXRF) analysis. The Monte Carlo simulation method was used to simulate the process of detecting tungsten inclusions in nuclear fuel rod upper-end plug welds by the EDXRF. The detectable tungsten particle diameters in the zirconium alloy at different depths in welds and the detection limits of the trace tungsten dispersed in welds were obtained. Then, we constructed an experimental device that uses a CdTe detector with an X-ray tube. The results showed that the relative standard deviation of the net count rate of tungsten K-series characteristic X-rays [W (Kα)] was 1.46%, and the optimum parameters are a tube voltage of 150 kV and current of 0.5 mA. These values were used to perform energy-dispersive X-ray fluorescence analysis. These results were compared to the X-ray radiographic results, which were broadly similar. Furthermore, the results of EDXRF analysis were more legible and reliable than those from X-ray radiographic inspections. This study demonstrates the feasibility of applying EDXRF analysis to detect tungsten inclusions.

Joint repair: Growing new cartilage from placental stem cells Stem cells derived from placental tissue may help in treating damaged joints, thanks to an improved method for encouraging them to form cartilage. Placenta-derived stem cells are readily available, as the placenta is usually discarded after birth. These stem cells can easily be grown into cartilage tissue in a laboratory, but not when transplanted. Researchers led by Yufeng Dong (Louisiana State University Health Sciences Center, Shreveport, USA) and Xifu Shang (Anhui Provincial Hospital, Hefei, China) treated stem cells with a naturally occurring protein, JAG1, which blocks the signal that prevents them from forming cartilage. They found that JAG1 increased the stem cells’ capacity for cartilage formation. When JAG1-stimulated stem cells were injected into a mouse model of joint disease, cartilage formation was improved, and joint inflammation was reduced.

Abstract To determine whether cell sheets generated with long-term passaged (P10) aging human mesenchymal stromal cells (MSCs) could be used for bone tissue regeneration as tissue engineered periosteum in a femoral allograft mouse model similar to fresh passaged (P3) young MSCs. At 3 weeks after transplantation of MSC sheets, results showed more bony callus formed between allograft and host bone ends in both young P3 MSC and aged P10 MSC sheet-wrapped groups when compared to allograft alone. At 6 weeks, while both MSC sheet-wrapped allografts showed more bony callus formation when compared to allograft alone groups, the bony callus size in aged P10 MSC sheet groups was significantly less than young P3 MSC sheet groups. Biomechanical testing confirmed that P3 MSC sheet-grafted femurs had the highest biomechanical strength in the three groups. Histology sections showed that the area of the chondriod callus in the aged P10 MSC sheet groups was significantly larger than in P3 MSC sheet groups. Finally, a significant increase of chondro-osteoclast activity was observed in the P3 MSC sheet-grafted femur. Our data demonstrates that extensive long-term culture-induced MSC aging impaired their osteogenic ability and subsequent bony callus formation, and could be used to induce cartilaginous callus formation.

Abstract Oleanolic acid (OA), a pentacyclic triterpenoid, has been shown to modulate multiple signaling pathways in a variety of cell linages. But the mechanisms underlying OA-mediated mesenchymal stromal cell (MSC) osteogenic differentiation are not known. In this study, we examined effects of OA on cell viability, osteogenic differentiation in MSCs, and the involvement of Notch and BMP signaling. OA induced bone marrow derived MSC differentiation towards osteoprogenitor cells and inhibited Notch signaling in a dose dependent manner. Constitutive activation of Notch signaling fully blocked OA induced MSC osteogenic differentiation. The expression level of early osteogenic marker genes, ALP, Runx2, and type I collagen, which play a critical role in MSC to osteoblast transition and servers as a downstream target of BMP signaling, was significantly induced by OA. Furthermore, BMP2 mediated MSC osteogenic differentiation was significantly enhance by OA treatment, indicating a synergistic effect between BMP2 and OA. Our results suggest that OA is a promising bioactive agent for bone tissue regeneration, and inhibition of Notch signaling is required for its osteogenic effects on MSCs.

Abstract EG5 (KIF11) is a member of the kinesin-like protein family involved in centrosome separation and bipolar spindle formation. When a cell enters mitosis, CDK1 phosphorylates EG5 at Thr926 and promotes EG5 localization on the mitotic spindle which drives bipolar spindle formation. EG5 provides power for spindle movement and thus controls the dynamics of spindle assembly. However, little is known about EG5 regulation or how EG5 detaches from the spindle upon mitotic exit. In this study we identify EG5 as a novel substrate of PP2A phosphatase, and we show that the PP2A/B55α complex plays an important role in mitotic exit by a mechanism involving EG5. The PP2A/B55α complex physically associates with the EG5 C-terminal tail domain and dephosphorylates EG5 at Thr926 that enables mitotic exit. Conversely PP2A knockdown cells show a high level of phospho-EG5 in late metaphase, which is associated with a delay in mitotic exit. These phenotypic features are similar to those induced by EG5/T926D transfection that mimics phosphorylated EG5 status. Our results argue that PP2A controls mitotic exit through EG5 dephosphorylation. Lack of PP2A leads to abnormal EG5 activation, resulting in delay of mitotic exit.

BackgroundThe Gram-negative bacterium Kerstersia gyiorum, a potential etiological agent of clinical infections, was isolated from several human patients presenting clinical symptoms. Its significance as a possible pathogen has been previously overlooked as no disease has thus far been definitively associated with this bacterium. To better understand how the organism contributes to the infectious disease, we determined the complete genomic sequence of K. gyiorum SWMUKG01, the first clinical isolate from southwest China.ResultsThe genomic data obtained displayed a single circular chromosome of 3, 945, 801 base pairs in length, which contains 3, 441 protein-coding genes, 55 tRNA genes and 9 rRNA genes. Analysis on the full spectrum of protein coding genes for cellular structures, two-component regulatory systems and iron uptake pathways that may be important for the success of the bacterial survival, colonization and establishment in the host conferred new insights into the virulence characteristics of K. gyiorum. Phylogenomic comparisons with Alcaligenaceae species indicated that K. gyiorum SWMUKG01 had a close evolutionary relationships with Alcaligenes aquatilis and Alcaligenes faecalis.ConclusionsThe comprehensive analysis presented in this work determinates for the first time a complete genome sequence of K. gyiorum, which is expected to provide useful information for subsequent studies on pathogenesis of this species.

Carbapenem-resistant Enterobacteriaceae have been a global public health issue in recent years. Here, a carbapenem-resistant Kluyvera cryocrescens strain SCW13 was isolated from hospital sewage, and was then subjected to whole-genome sequencing (WGS). Based on WGS data, antimicrobial resistance genes were identified. Resistance plasmids were completely circularized and further bioinformatics analyses of plasmids were performed. A conjugation assay was performed to identify a self-transmissible plasmid mediating carbapenem resistance. A phylogenetic tree was constructed based on the core genome of publicly available Kluyvera strains. The isolate SCW13 exhibited resistance to cephalosporin and carbapenem. blaNDM-1 was found to be located on a ~53-kb self-transmissible IncX3 plasmid, which exhibited high similarity to the previously reported pNDM-HN380, which is an epidemic blaNDM-1-carrying IncX3 plasmid. Further, we found that SCW13 contained a chromosomal blaKLUC-2 gene, which was the probable origin of the plasmid-born blaKLUC-2 found in Enterobacter cloacae. Phylogenetic analysis showed that K. cryocrescens SCW13 exhibited a close relationship with K. cryocrescens NCTC10483. These findings highlight the further dissemination of blaNDM through clonal IncX3 plasmids related to pNDM-HN380 among uncommon Enterobacteriaceae strains, including Kluyvera in this case.

BACKGROUND: Paragonimiasis is a food-borne trematodiasis, a serious public health issue and a neglected tropical disease. Paragonimus skrjabini is a unique species found in China. Unlike paragonimiasis westermani, it is nearly impossible to make a definitive diagnosis for paragonimiasis skrjabini by finding eggs in sputum or feces. Immunodiagnosis is the best choice to detect paragonimiasis skrjabini. There is an urgent need to develop a novel, rapid and simple immunoassay for large-scale screening patients in endemic areas. METHODOLOGY/PRINCIPAL FINDINGS: To develop a rapid, simple immunodiagnostic assay for paragonimiasis, rabbit anti-human IgG was conjugated to colloidal gold particles and used to detect antibodies in the sera of paragonimiasis patients. The synthesis and identification of colloidal gold particles and antibody-colloidal gold conjugates were performed. The size of colloidal gold particles was examined using a transmission electron microscope (TEM). The average diameter of colloidal gold particles was 17.46 nm with a range of 14.32-21.80 nm according to the TEM images. The formation of antibody-colloidal gold conjugates was monitored by UV/Vis spectroscopy. Excretory-secretory (ES) antigen of Paragonimus skrjabini was coated on nitrocellulose membrane as the capture line. Recombinant Staphylococcus protein A was used to prepare the control line. This rapid gold immunochromatographic strip was assembled in regular sequence through different accessories sticked on PVC board. The relative sensitivity and specificity of the strip was 94.4% (51/54) and 94.1% (32/34) respectively using ELISA as the standard method. Its stability and reproducibility were quite excellent after storage of the strip at 4°C for 6 months. CONCLUSIONS/SIGNIFICANCE: Immunochromatographic strip prepared in this study can be used in a rapid one-step immunochromatographic assay, which is instantaneous and convenient.