The Species Identification and Genomic Analysis of Haemobacillus shengwangii: A Novel Pathogenic Bacterium Isolated From a Critically Ill Patient With Bloodstream Infection.

Since the first strain related to Thermicanaceae was reported in 1999, almost no literature on Thermicanaceae is available, particularly its genomics. We recently isolated a novel pathogenic bacterium, the △ strain DYY3, from the blood sample of a critically ill patient. The morphological, physiological, and biochemical characteristics of △ strain DYY3 were presented in this study, and the virulence factor genes and antibiotic resistance of DYY3 were also determined. Interestingly, the average nucleotide identity (ANI) and core-genes average amino acid identity (cAAI) analysis indicated that △ strain DYY3 was genus novel and species novel. Moreover, phylogenetic analysis based on both 16S rRNA gene and whole genomic core gene sequences suggested that △ strain DYY3 belonged to the family Thermicanaceae, and this novel taxon was thus named Haemobacillus shengwangii gen. nov., sp. nov. Besides, both the whole genome-based phylogenetic tree and amino acid identity analysis indicated that Thermicanus aegyptius, Hydrogenibacillus schlegelii, Brockia lithotrophica, and the newly discovered species H. shengwangii should belong to Thermicanaceae at the family level, and T. aegyptius was the closest species to H. shengwangii. We also constructed the first high-quality genome in the family Thermicanaceae using the next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing technologies, which certainly contributed to further genomics studies and metagenomic-based pathogenic detection in the future.

Role of hsa_circ_0066966 in proliferation and migration of hepatitis B virus-related liver cancer cells.

A large proportion of liver cancer cases is caused by hepatitis B virus (HBV) infection. In recent years, an increasing number of reports have indicated that circular RNAs (circRNAs) exert regulatory effects in cancer development, whereas the role of circRNAs in HBV-positive liver cancer requires further investigation. In the present study, abnormally expressed circRNAs were identified in HBV-positive liver cancer cells through microarray analysis. A total of 1,493 differentially expressed circRNAs [absolute fold-change (FC) ≥2] in HBV-positive liver cancer cells were detected, of which 171 were upregulated and 1,322 were downregulated. Subsequently, Gene Ontology enrichment analysis indicated that the genes of dysregulated circRNAs were mainly involved in regulating Sertoli cell differentiation and development, as well as telomeric DNA binding. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that most of these genes were enriched in cancer-related signaling pathways, including the MAPK and Hippo signaling pathways. Next, the expression levels of the top-10 dysregulated circRNAs were verified in HBV-positive liver cancer cells through reverse transcription-quantitative PCR. Among them, hsa_circ_0066966 had the highest absolute Log2FC value and was abnormally increased in HBV-positive liver cancer cells. Functional experiments further verified that knockdown of hsa_circ_0066966 had a significant inhibitory effect on the proliferation and migration of HBV-positive liver cancer cells. By contrast, overexpression of hsa_circ_0066966 in HBV-negative liver cancer cells resulted in the opposite effect. In conclusion, in the present study, comprehensive circRNA profiling in HBV-positive liver cancer cells indicated that hsa_circ_0066966 may regulate the progression of HBV-positive liver cancer.

The Genomic Characterization of KPC-Producing Klebsiella pneumoniae from the ICU of a Teaching Hospital in Shanghai, China.

PURPOSE: This study retrospectively analyzed the genome characteristics of blaKPC-2 in multidrug-resistant Klebsiella pneumoniae collected from the ICU of a teaching hospital in Shanghai, China. METHODS: From February 2018 to December 2019, 36 strains of multidrug-resistant Klebsiella pneumoniae were collected from the bronchoalveolar lavage fluid of critically ill patients. The genome of all isolates was obtained through the Illumina sequence, and single nucleotide polymorphisms of the blaKPC-2 gene were analyzed to explore blaKPC-2's evolutionary characteristics. Different strains' genetic relationships and homology were studied by constructing an evolutionary tree on a single copy orthologue. Pacbio combined Illumina sequence was conducted to evaluate the structure and potential mobility of drug-resistant plasmids of the strain KP-s26. RESULTS: The distribution of resistance and virulence genes had little difference, but most strains had significant differences in the plasmid-encoded region. Most strains (31/36) carried the carbapenemase gene blaKPC-2, with no single nucleotide polymorphism in different strains. Extended-spectrum ß-lactamase resistance genes, such as blaCTX-M and blaSHV, were found in the isolates, but no metallo-ß-lactamases were detected. All strains with blaKPC-2 coexisted with chromosomal-associated fosfomycin resistance genes fosA6, and the coexistence of blaKPC-2 and blaCTX variants (blaCTX-M-15, blaCTX-M-65, and blaCTX-M-27) was also detected in 29/31 strains. The isolate KP-s26 carried five circular plasmids. pA and pB were conjugate plasmids, as they carried drug resistance genes and contained a complete IV secretion system. CONCLUSION: The blaKPC-2 carbapenemase gene is relatively conservative in the process of evolution; drug-resistant plasmids containing conjugated transfer elements contribute to the spreading of drug resistance. The coexistence of blaKPC-2 with fosA6 or blaCTX-M variants was associated with increased fosfomycin resistance and broad-spectrum ß-lactam resistance, respectively. CLINICAL TRIALS REGISTRATION: Clinical Trials.gov Identifier: NCT03950544.

Low Molecular Pectin Inhibited the Lipid Accumulation by Upregulation of METTL7B.

Inhibition of lipid accumulation is the key step to prevent nonalcoholic fatty liver (NAFL) progressing to nonalcoholic steatohepatitis. We aimed to study the effect of low-molecular-weight citrus pectin (LCP) against lipid accumulation and the underlying mechanism. Oleic acid (OA)-induced lipid deposition in HepG2 cells was applied to mimic in vitro model of lipid accumulation. Oil Red O (ORO) stain result showed lipid accumulation was significantly reduced, and levels of adipose triglyceride lipase (ATGL) and carnitine palmitoyltransferase-1 (CPT-1), involved in triacylglycerol catabolism and fatty acid ß-oxidation, detected by RT-qPCR were increased after OA-stimulated HepG2 cells treated with LCP. RNA sequencing analysis identified 740 differentially expressed genes (DEGs) in OA-stimulated HepG2 cells treated with the LCP group (OA+LCP group), and bioinformatics analysis indicated that some DEGs were enriched in lipid metabolism-related processes and pathways. The expression of the top 8 known DEGs in the OA+LCP group was then verified by RT-qPCR, which showed that fold change (abs) of METTL7B was the highest among the 8 candidates. In addition, overexpression of METTL7B in HepG2 cells significantly inhibited the lipid accumulation and enhanced levels of ATGL and CPT-1. In conclusion, LCP inhibited lipid accumulation through the upregulation of METTL7B, and further enhancement of ATGL and CPT-1 levels. LCP is expected to develop as a promising agent to ameliorate fat accumulation in NAFL.

Clustered precursors in bone marrow sections predict early relapse in patients with acute myeloid leukemia within hematologic remission.

BACKGROUND: Bone marrow (BM) aspiration is largely used for relapse assessment in acute myeloid leukemia (AML). It remains unclear what roles that BM trephine biopsy plays on relapse assessment. METHODS: Bone marrow (BM) sections during complete remission (CR) from 60 acute myeloid leukemia (AML) patients were retrospectively analyzed. Computer image processing technology was performed for detection of the distance between precursors and endosteum, and density of precursors was also calculated under light microscopic image. Immunohistochemistry was used to identify the immunophenotype of clustered precursors. RESULTS: Except for single and double precursors, there existed clustered precursors of 3-5 cells during CR. Here, we demonstrated that clustered precursors, but not single and double precursors, were useful in risk factor of relapse. Area under the receiving operator curve (ROC) was of 0.007 (CI 95%, from 0.572 to 0.851). Using a standard cut-off value of >4.0/mm² for cluster density, early relapse was detected with a sensitivity of 51.5% and a specificity of 85.7%.Multivariate Cox regression analysis revealed that clustered precursor is an independent risk factor for early relapse (Adjusted HR: 0.325, 95% CI: 0.156-0.679, p = 0.003). CONCLUSIONS: Cumulatively, clustered precursors in BM sections during CR may serve as an independent risk factor of early relapse and poor outcome for AML patients in cluster density > 4.0/mm² in sections. Early aggressive interventions are needed to prevent hematologic relapse.

[Influence of pre-ALIP number and its distance from trabeculae on AML relapse].

This study was purposed to detect the abnormal quantity and localization of pre-ALIP in bone marrow of acute myelocytic leukemia patients (AML) during the complete remission (CR) and investigate their correlation with AML relapse. The bone marrow biopsy and prognosis of 62 patients with CR were retrospectively analyzed. The bone marrow was divided into the pre-relapse group and the no-relapse group according to prognosis of patients. In order to clarify the correlation of abnormal quantity and localization of pre-ALIP with AML relapse, the number of single and double-cluster precursor cells and the sum of both were calculated, and their distance from bone trabeculae was surveyed with the computer image segment method. The results showed that the number of pre-ALIP in pre-relapse group (11 ± 11.71/mm(2)) and no-relapse group (8.33 ± 9.17/mm(2)) were more than that in normal control group (5.29 ± 4.00) (P < 0.01). The number of pre-ALIP more than 11/mm(2) was observed in 17 among all AML patients, and out of them 12 patients with pre-ALIP number >11/mm(2) (70.6) were found in the pre-relapse group, which was higher than that in no-relapse group (P < 0.05). While the distance between pre-ALIP and trabeculae [(341.31 ± 266.16) µm] in pre-relapse group showed the tendency of migrating to the intermediate zone of bone trabeculae, compared with that in no-relapse group [(242.41 ± 174.65) µm, P < 0.01]. Moreover, about 77.8 of 18 patients showed the distance of pre-ALIP from trabeculae was more than 341 µm in the pre-relapse group, and significantly higher than that in no-relapse group (P < 0.01). It is concluded that the average number of "pre-ALIP" more than 11/mm(2) or the average distance from trabeculae longer than 341 µm in bone marrow sections during CR may be the indicators for early relapse of AML.

[Characteristics of "pre-ALIP" in bone marrow sections of patients with acute myeloid leukemia].

To detect the characteristics of "pre-ALIP" and to investigate their relevance with the development of acute myeloid leukemia (AML) by computer image procession technology, bone marrow (BM) was collected by aspiration/trephine biopsy from AML patients during the complete remission (CR). BM sections were stained by HGF (haematoxylin-Giemsa-acid fuchsin) and photographed by optical microscope imaging system. 4 kinds of computer image segmentation technologies were compared to select the best one for detecting the localization and quantitation of the precursor cells. Planimetry was combined with morphology to segment bone trabeculae. The number of single and double-cluster precursor cells and their distance from bone trabeculae was detected with Euclidean distance change method in BM images of AML patients, and compared with the normal controls. Moreover, the morphological characteristics of "pre-ALIP" were investigated, and the correlation with the development of AML was analyzed. The results showed that the computer image segmentation method based on morphology could identify the precursor cells and bone trabeculae more exactly in BM image, as compared with the methods of 8-Sobel operater. Canny operator and watershed algorithm. Bone trabeculae could be segmented with combinative methods of morphology and planimetry. The number of single precursor cells (19.27 ± 11.60)/mm(2) and double-cluster precursor cells (1.77 ± 1.76)/mm(2) in CR group were higher than that in normal controls (p < 0.05). The distance of single precursor cells from bone trabeculae in CR group were closer to bone trabeculae than that in controls [(230.12 ± 97.68) µm vs (260.92 ± 99.88 µm)] (p < 0.05), but the distance of double-cluster precursor cells from bone trabeculae in AML patients was (274.56 ± 139.48) µm, which showed no statistically significant different from controls (p > 0.05), while the double-cluster precursor cells showed the tendency of migrating to the intermediate zone of bone trabeculae compared with the single precursor cells in CR group (p < 0.05). It is concluded that the structure of "pre-ALIP" in BM tissue exists before the occurrence of ALIP. The characteristics of "pre-ALIP" are single and double-cluster precursor cells with abnormal localization or quantitation, which showed correlation with the development of AML.

[Earlier hematopoietic reconstitution by mouse cord blood transplantation combined with bone marrow c-kit(+) hematopoietic progenitor cells].

This study was purposed to investigate the accelerating effect of newborn mouse cord blood transplantation combined with hematopoietic progenitor cells of bone marrow (BM) on the early hematopoietic reconstitution after transplantation, and the long-term chimerism of cord blood-derived cells, so as to develop a combined transplantation method for accelerating the early hematopoietic reconstitution. The lin(-)sca-1(-)c-kit(+) (c-kit(+)) cells and lin(-)sca-1(+) (sca-1(+)) cells in the bone marrow of BDF1 mice were isolated by MACS method. Biological characteristics in vitro of isolated fractions were observed and compared by semisolid colony culture combined with Giemsa staining. After transplantation of cord blood (CB) alone, or together with graded numbers of either c-kit(+) or sca-1(+) cells isolated from BDF1 mice (CD45.1) bone marrow into lethally irradiated CD45.2 congenic BDF1 mice, numbers of WBC and platelet were measured within 22 days of post-transplantation. The proportion of chimerism on granulocyte, T and B cell was dynamically measured by flow cytometry within 60 weeks of post-transplantation. The results showed that the number of colony from BM c-kit(+) cells cultured in semi-solid agar medium was significantly smaller than that from BM sca-1(+) population, which showed low proliferative potential in vitro and morphological characteristics of medium- or large-sized blast-like cells. The co transplantation of CB and BM c-kit(+) cells or sca-1(+) cells at the dosages of 1 × 10(4) or 2.5 × 10(4) or 5 × 10(4) to recipient mice leads to the quantity of WBC and platelets increased to 1 × 10(9)/L and 1 × 10(12)/L at day 12, whereas the injection of CB alone resulted at day 17. When mice were transplanted with CB together with BM c-kit(+)cells, and the CB-donor type cells in the peripheral blood increased progressively, while congenic donor BM-derived stem cells decreased gradually. After cotransplantation with CB and BM c-kit(+) cells for 60 weeks, a frequency of complete chimerism in CB-derived cells was continually maintained in granulates (96.68 ± 2.68)% and B lymphocytes (92.55 ± 3.04)%, while T lymphocytes (67.96 ± 7.91)% were dominantly derived from CB. On the other hand, congenic bone marrow or residual-derived cells were the dominant population, and the ratio of CB-derived cells in the peripheral blood was less than 10% (6.19 ± 7.62)% after cotransplantation with CB and sca-1(+)cells for 60 weeks. It is concluded that the cotransplantation of CB and BM congenic c-kit(+) cells is able to accelerate early hematopoietic reconstitution of recipient mice due to congenic marrow cells. Complete or main chimerism of cord blood is formed in long-term multilineage reconstitution of granulocytes, B cells and T lymphocytes.

A subset of myeloid dendritic cells derived from peripheral blood monocytes represented a predominant subset characterized by their potential tumor-inhibiting activity.

Besides their role as potent antigen-presenting cells, myeloid dendritic cells (MDCs), but not plasmacytoid dendritic cells (PDCs), have been reported to have cytotoxic or cytostatic activity on some tumor cells. In this article, we analyzed the tumoristatic potential of a distinct peripheral blood monocyte-derived MDC subset which co-expressed PDC-specific marker CD123. CD123(+) MDCs represented a subset of small-sized DCs and accounted for 45-60% of peripheral blood monocytes cultured with granulocyte-macrophage colony-stimulating factor and interleukine-4 (IL-4) for 7 d. They exhibited more significant antiproliferative activity toward hematological tumor cell lines of Jurkat, HL60, and myelodysplastic syndromes over-leukemia than CD123(-) MDCs even at a low effecter/target ratio. Pretreatment of MDC and their supernatant with TRAIL-R2:Fc significantly reduced the tumoristatic effect of CD123(+) MDCs but not of CD123(-) MDCs and their supernatant. CD123(+) MDCs expressed higher level of cytoplasmic TNF-alpha-related apoptosis-inducing ligand (TRAIL) than CD123(-) MDCs, whereas both expressed very little surface and soluble TRAIL. These results reveal that CD123(+) cells represented a predominant subset of MDCs generated from peripheral blood monocytes in vitro, characterized by their potential tumoristic activity partially via cytoplasmic TRAIL.