Isolation, biological and whole genome characteristics of a Proteus mirabilis bacteriophage strain.

Proteus mirabilis, a naturally resistant zoonotic bacterium belonging to the Enterobacteriaceae family, has exhibited an alarming increase in drug resistance. Consequently, there is an urgent need to explore alternative antimicrobial agents. Bacteriophages, viruses that selectively target bacteria, are abundant in the natural environment and have demonstrated potential as a promising alternative to antibiotics. In this study, we successfully isolated four strains of Proteus mirabilis phages from sewage obtained from a chicken farm in Sichuan, China. Subsequently, we characterized one of the most potent lytic phages, Q29, by examining its biological and genomic features. Comparative genomic analysis revealed the functional genes and phylogenetic evolution of Q29 phages. Our findings revealed that Proteus mirabilis bacteriophage Q29 possesses an icosahedral symmetrical head with a diameter of 95 nm and a tail length of 240 nm. Moreover, phage Q29 exhibited stability within a temperature range of 37 ℃ to 55 ℃ and under pH conditions ranging from 4 to 9. The optimal multiplicity of infection (MOI) for this phage was determined to be 0.001. Furthermore, the one-step growth curve results indicated an incubation period of approximately 15 min, an outbreak period of approximately 35 min, and an average cleavage quantity of approximately 60 plaque-forming units (PFU) per cell. The genome of phage Q29 was found to have a total length of 58,664 base pairs and encoded 335 open reading frames (ORFs) without carrying any antibiotic resistance genes. Additionally, genetic evolutionary analysis classified phage Q29 within the family Caudalidae and the genus Myotail. This study provides valuable research material for further development of Proteus mirabilis bacteriophage biologics as promising alternatives to antibiotics, particularly in light of the growing challenge of antibiotic resistance posed by this bacterium.

Research Note: Isolation and complete genome analysis of a genotype II goose astrovirus in Sichuan Province, China.

The emergence of Goose astrovirus (GoAstV) has led to the gout in geese. This study aimed to isolate and identify the GoAstV from diseased goslings in Sichuan Province, China, followed by performing whole genome phylogenetic analysis of the isolate. The GoAstV was successfully isolated by inoculating the diseased gosling liver and kidney homogenate into the 11-day-old goose embryo allantoic cavity for 3 passages, and the isolate was named as GoAstV-C2 strain. The virus particles were spherical, without capsule, and the size was about 28 nm under transmission electronic microscope. The complete genome length of GoAstV-C2 was 7.035 nt, and the whole genome sequence phylogenetic analysis revealed that it belongs to the GoAstV genotype II (GoAstV-II) subgenotype IIc. The isolated GoAstV-C2 strain was able to be stably passaged in the goose embryo and uric acid sedimentation was observed. The complete genome bioinformation of GoAstV-C2 determined the evolutionary characteristics of the GoAstV isolated from Sichuan, China. This finding lays a foundation for the development of preventive measures, effective vaccines, and therapeutic drugs.

A Type IIb, but Not Type IIa, GnRH Receptor Mediates GnRH-Induced Release of Growth Hormone in the Ricefield Eel.

Multiple gonadotropin-releasing hormone receptors (GnRHRs) are present in vertebrates, but their differential physiological relevances remain to be clarified. In the present study, we identified three GnRH ligands GnRH1 (pjGnRH), GnRH2 (cGnRH-II), and GnRH3 (sGnRH) from the brain, and two GnRH receptors GnRHR1 (GnRHR IIa) and GnRHR2 (GnRHR IIb) from the pituitary of the ricefield eel Monopterus albus. GnRH1 and GnRH3 but not GnRH2 immunoreactive neurons were detected in the pre-optic area, hypothalamus, and pituitary, suggesting that GnRH1 and GnRH3 may exert hypophysiotropic roles in ricefield eels. gnrhr1 mRNA was mainly detected in the pituitary, whereas gnrhr2 mRNA broadly in tissues of both females and males. In the pituitary, GnRHR1 and GnRHR2 immunoreactive cells were differentially distributed, with GnRHR1 immunoreactive cells mainly in peripheral areas of the adenohypophysis whereas GnRHR2 immunoreactive cells in the multicellular layers of adenohypophysis adjacent to the neurohypophysis. Dual-label fluorescent immunostaining showed that GnRHR2 but not GnRHR1 was localized to somatotropes, and all somatotropes are GnRHR2-positive cells and vice versa at all stages examined. GnRH1 and GnRH3 were shown to stimulate growth hormone (Gh) release from primary culture of pituitary cells, and to decrease Gh contents in the pituitary of ricefield eels 12 h post injection. GnRH1 and GnRH3 stimulated Gh release probably via PLC/IP3/PKC and Ca2+ pathways. These results, as a whole, suggested that GnRHs may bind to GnRHR2 but not GnRHR1 to trigger Gh release in ricefield eels, and provided novel information on differential roles of multiple GnRH receptors in vertebrates.

[Enhanced Antibiotic Resistant Bacteria Removal from Wastewater Treatment Plant by Different Disinfection Technologies].

Based on the removal of total heterotrophic bacteria (HPC) and antibiotic resistant bacteria (ARB), including the ampicillin resistant bacteria (AMP), erythromycin resistant bacteria (ERY), tetracycline resistant bacteria (TET), kanamycin resistant bacteria (KAN), and ciprofloxacin resistant bacteria (CIP), this study investigates the enhanced removal performance of ARB by different disinfection technologies. The experimental results showed that ARB removal by ultraviolet (UV) disinfection from the wastewater treatment plant (WWTP) was only 18.2%-40.9% and AMP was the highest in content. ERY could be selectively removed by different disinfection technologies; however, there was no distinguished selective removal performance for other four types of ARB (P<0.05). For ARB, COD and NH4+-N removal, the optimal ozone, chlorination, and UV concentration or dosage were 5.0 mg·L-1, 25.0 mg·L-1, and 45.0 mJ·cm-2, respectively, and the corresponding ARB removal efficiencies were 45.5%-74.5%, 66.1%-85.5%, and 68.6%-85.5%. Furthermore, the combined UV and chlorine treatment could achieve better ARB removal performance.

[The Role of Hydrogen Sulfide in Acute Liver Injury Induced by Traumatic Stress in Rats].

OBJECTIVE: To explore the role of hydrogen sulfide (H2S) in acute liver injury induced by crushing hind limbs of rats. METHODS: The rats were randomly divided into the following groups: control, crushing, H2S donor sodium hydrosulfide (NaHS) + crushing, H2S inhibitor propargylglycine (PAG) + crushing group. The acute liver injury model was established by 'crushing the hind limbs of rats with standard weight. Rats were sacrificed at 30 min and 120 min after the crush. The activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by colorimetric method, and the content of H2S in plasma and the contents of malondialdehyde (MDA), protein carbonyl, glutathione (GSH) in the liver and the activity of H2S generating enzyme (cystathionine y-lyase, CSE) were determined by chemical method. The expression of CSE mRNA in liver was detected by RT-PCR. RESULTS: For crush injury group, the levels of AST and ALT in serum, MDA and protein carbonyl in liver increased. The levels of GSH, CSE, CSE mRNA in liver and H2S in serum decreased. The administration of NaHS before limbs crush could attenuate the changes of liver injury, but the pre-treatment with PAG could exacerbate the changes. CONCLUSION: The decrease of H2S production could involve in mediating the acute liver injury induced by traumatic stress in rats.

[Effects of restraint position on changes of diaphragmatic mechanical characteristic in rats].

OBJECTIVE: To observe effects of restraint position on the changes of diaphragmatic mechanical characteristic in rats, and try to explore the role of nitric oxide (NO). METHODS: Rat model of restraint position was established. Rats were divided into control group, restraint position 12h and 24h groups. The markers of respiratory functions in vivo and the biomechanical markers of diaphragmatic characteristic ex vivo were evaluated. Serum NO levels were measured with spectrophotometry. The expressions of nNOS and iNOS mRNA in diaphragm were detected using RT-PCR. RESULTS: Compared with control group, respiratory rate, tidal volume and minute ventilation were significantly decreased in the restraint position 12h and 24h groups. Pt of diaphragm significantly decreased and force-generating capacity reduced at low frequency stimulation in 12h group. Force-generating capacity over the full range reduced at low and high frequency stimulation in 24h group. Pt of diaphragm in control and restraint position groups increased after L-NNA pre-incubation. Force-frequency relationship after L-NNA pre-incubation reduced in 24h group. NO level in serum increased significantly in the restraint position groups. Diaphragmatic nNOS mRNA expression was upregulated significantly in the restraint position groups. CONCLUSION: Restraint position induces the decreasement of diaphragmatic contractility and the decreasement is mediated by NO from diaphragm or circulation blood.

Long-term outcomes in adults with leukemia treated with transplantation of two unrelated umbilical cord blood units.

BACKGROUND: Wide application of umbilical cord blood transplantation (UCBT) in adult patients is limited by low cell-dose available in one umbilical cord blood (UCB) unit. The aim of this study was to investigate the safety and long-term outcomes of UCBT from unrelated donors in adult and adolescent patients with leukemia. METHODS: Thirteen patients with leukemia received double-unit UCBT with human leukocyte antigen (HLA) mismatched at 0 - 2 loci. We analyzed the engraftment, graft-versus-host disease (GVHD) and survival. RESULTS: Twelve evaluable patients (92.3%) had neutrophil and platelet engraftment at a median of 21 days (range, 16-38 days) and 34 days (range, 25 - 51 days), respectively. At day 30, engraftment was derived from one donor in 8 patients (66.7%, 95%CI 40.0% - 93.4%), and from both donors in 4 patients (33.3%, 95%CI 6.7% - 60.0%) with 1 unit predominated. Unit with larger nucleated cell (NC) dose would predominate in engraftment (P = 0.039), whereas CD34(+) cell dose or HLA-match failed to demonstrate any relationship with unit predominance. Only one patient developed grade II acute graft-versus-host disease (aGVHD). Chronic GVHD (cGVHD) was observed in 2 of 11 patients who survived more than 100 days, and both were limited. The median follow-up after transplantation for the 13 patients was 45 months (range 1.5 - 121.0 months) and 72 months (range 41.0 - 121.0 months) for the 8 alive and with full donor chimerism. The 5-year cumulative disease free survival (DFS) was (61.5 ± 13.5)%. Of the 13 patients, 5 patients died in 1 year and 1-year transplantation related mortality (TRM) was 23.1% (95%CI 0.2% - 46.0%). CONCLUSION: Double-unit UCBT from unrelated donors with HLA-mismatched at 0-2 loci may overcome the cell-dose barrier and be feasible for adults and adolescents with leukemia.

[Mechanism of acute liver injury induced by crushing hindlimbs in rabbits].

OBJECTIVE: To investigate the role of oxidative stress in acute liver injury during crushing hindlimbs in rabbit. METHODS: The crushing injury model in rabbit was established by intermittent crushing the hind limbs of rabbit with standard weight. The ALT and AST activities were spectrophotometrically measured. The weight ratio (wet/dry,W/D) of livers was measured with scale, and the pathologic changes were observed. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and total anti-oxidant capacity (T-AOC) as well as malondialdehyde (MDA) level were spectrophotometrically measured. RESULTS: As compared with control rabbits, crushing hindlimbs of rabbits induced acute liver injury with the increase in ALT and AST activities in serum,which were 4.31 (P < 0.01) and 10.54 times (P < 0.01) of control group respectively, there were cellular swellings and slight congestion of hepatic sinuses. In addition,crushing hind-limbs elicited significant decrease in SOD,CAT,GSH-Px activity and T-AOC to 17%, 29%, 24% and 21% (P < 0.01) compared with control group respectively, whereas MDA level markedly enhanced. CONCLUSION: Crushing hindlimbs of rabbits induced acute liver injury and significant decrease in anti-oxidant capacity, the latter maybe play an important role in crushing hind-limbs of rabbits-elicited the acute liver injury.

[Comparative genomic hybridization analysis of nasopharyngeal carcinoma drug-resistant cell CNE2/DDP and its parent cell CNE2].

BACKGROUND & OBJECTIVE: Developed from fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH) is a new molecular and cellular technique,used to examine the genomic imbalances,especially the loss and amplification of chromosomes,and to locate these alterations in certain chromosome regions. For a comprehensive understanding of the possible differences in genomic DNA between nasopharyngeal carcinoma drug-resistant and drug-sensitive cells, we analyzed the genomic DNA of a nasopharyngeal carcinoma drug-resistant cell line CNE2/DDP and its parent cell line CNE2 with CGH. METHODS: Genomic DNA was extracted from CNE2/DDP and CNE2 cells, as well as from normal placenta tissue. Fluorescent random primed labeling method was used to label the DNA probes (CNE2 and CNE2/DDP cells with green fluorescein-12-dUTP and normal placenta tissue with red tetramethylrhodamine-5-dUTP). The labeled DNA probes were then co-hybridized with normal lymphocyte metaphase chromosomes. Signals were taken by charge coupled device (CCD) and processed with Quips CGH Program after fluorescent hybridization. The green-to-red fluorescent ratio was calculated automatically and showed with graphs. RESULTS: There were extensive chromosome changes in CNE2, mainly the gain of 1q, 3q, 5p, 6p, 7p, 8q, 9q, 11p, 12q and 19q, and the loss of 4q, 12p, 13p, 14p, 15p, 18, 20q, 21p and 22. However, the CNE2/DDP cells, which come from the CNE2, showed a relatively normal karyotype except loss of 8p and gain of 8q and 19q. Consistent result was achieved after the CNE2/DDP cells were cultured in the medium free of any drugs for over one month. It appears that the CNE2/DDP cell has relative normal and much more stable chromosome constitution than its parent CNE2 cell. CONCLUSION: The CNE2/DDP is a single drug-resistant clone selected from CNE2,which is a blend of sub-clones that have different sorts of chromosome number and structure. It strongly suggests that the appearance of tumor drug-resistance is mainly a select process in which the would-be drug-resistant cell clone be selected from unfavorable survival condition.

[Establishment of a human nasopharyngeal carcinoma drug-resistant cell line CNE2/DDP and screening of drug-resistant genes].

BACKGROUND & OBJECTIVE: Chemotherapy constitutes one of the chief supplementary methods in the treatment of nasopharyngeal carcinoma (NPC). However, the appearance of drug resistance often causes failure of chemotherapy. For overcoming drug resistance, it is of great importance to screen drug-resistant associated genes so as to identify potential molecular targets. This study was designed to establish a drug-resistant cell line from a human nasopharyngeal carcinoma cell line CNE2, and to screen human nasopharyngeal carcinoma drug-resistant genes by a new strategy based on improved subtractive hybridization. METHODS: The drug-resistant cell line was established by a program of treating the human nasopharyngeal carcinoma cells CNE2 in the medium with repeated sharp high and then low but gradually increasing concentration of cisplatin. Drug sensitivity was measured by MTT assay. Fluorescence activated cell analysis(FACS) was employed for determining the concentration of fluorescence dye rhodamine 123 within the cells. Cell growth curve, doubling time, and cell morphology were measured and observed. The drug-resistant genes were screened by a new strategy of PCR-based subtractive hybridization. Sequencing and blast analysis were performed after the differentially expressed genes had been verified by reverse dot blotting. The result was further confirmed by RT-PCR. RESULTS: The resistance indexes of CNE2/DDP to cisplatin (DDP), 5-fluorouracil (5-FU), and vincristine (VCR) were 27.9, 227.9, and 55.5, respectively, indicating its multi-drug resistant property. FACS analysis showed that the concentration of rhodamine 123 was much lower in CNE2/DDP cells than in CNE2 cells (12.98 vs. 243.62). The CNE2/DDP cells appeared smaller, more regularly round, and longer doubling time (26 hours vs. 19 hours) than CNE2 cells. Six differentially expressed sequences were discovered using improved subtractive hybridization; all of them were found to be homologous to known genes after sequencing analysis. Three of them were highly expressed in CNE2/DDP cells. Among them, one sequence, which encodes a 79 amino acid protein,known as DC13 protein (DC13), was a function unknown gene which has certain relationship with malignancy. The other two sequences were ubiquitin C gene and NADH dehydrogenase subunit 2 (ND2) gene, respectively. The other three of the six sequences, whose expression were inhibited in CNE2/DDP cells, were cytochrome C oxidase subunit I(COX1), ribosomal protein L27(RPL27),and ribosomal protein S27 (RPS27) genes, respectively. CONCLUSION: A drug- resistant cell line CNE2/DDP, which showed a typical resistant phenotype to anti-cancer drugs was established. The PCR-based improved subtractive hybridization is an effective approach to identify differentially expressed genes. Many genes, both known and unknown, might contribute to the existence of drug-resistant phenotype, through increasing or decreasing their expression.