[Biological characteristics and genomic information of a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient and its effects on bacterial biofilm].

Objective: To isolate a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient, and to study its biological characteristics, genomic information, and effects on bacterial biofilm. Methods: (1) In 2018, pan-drug resistant Klebsiella pneumoniae UA168 (hereinafter referred to as the host bacteria) solution isolated from the blood of a burn patient in Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (hereinafter referred to as Ruijin Hospital) was used to isolate and purify the bacteriophage against pan-drug resistant Klebsiella pneumoniae from the sewage of Ruijin Hospital with sewage co-culture method, drip plate method, and double-agar plate method. The bacteriophage was named as phage KP168 and the plaque morphology was observed. (2) The phage KP168 solution was taken for cesium chloride density gradient centrifugation and dialysis, and then the morphology of phage KP168 was observed through transmission electron microscope after phosphotungstic acid negative staining. (3) The phage KP168 solution was taken to determine the lytic ability of the phage KP168 against 20 strains of pan-drug resistant Klebsiella pneumoniae isolated from the burned patients' blood in Ruijin Hospital by the drip plate method, and then the lysis rate was calculated. (4) The phage KP168 solution at a initial titer of 9.3×10(11) plaque-forming unit (PFU)/mL (400 µL per tube) and the host bacteria solution at a concentration of 1×10(9) colony-forming unit (CFU)/mL (4 mL per tube) were conventionally shaking cultured together for 4 hours at multiplicity of infection (MOI) of 10.000, 1.000, 0.100, 0.010, or 0.001, respectively (1 tube per MOI). The titer of phage KP168 was measured by the double-agar plate method (the measurement method was the same below) to select the optimal MOI. The experiment was repeated three times. (5) The host bacteria solution at a concentration of 1×10(9) CFU/mL (4 mL per tube) and the phage KP168 solution at an adjusted titer of 5×10(7) PFU/mL (400 µL per tube) were mixed at the MOI of 0.005. The plaques were counted 0 (immediately), 1, 2, 3, 4, 5, 15, and 30 minutes (1 tube at each time point) after mixing by the double-agar plate method (the counting method was the same below), and the percentage of adsorbed phages was calculated to screen for the optimal adsorption time. The experiment was repeated three times. (6) The host bacteria solution at a concentration of 1×10(9) CFU/mL (300 µL per tube) and the phage KP168 solution at a titer of 5×10(8) PFU/mL (60 µL per tube) were mixed at MOI of 0.005 and conventionally shaking cultured after standing for the optimal adsorption time. The phage KP168 titer was measured 0 (immediately), 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 minutes after culture, and a one-step growth curve was drawn. The experiment was repeated three times. (7) The phage KP168 solution at a titer of 2.5×10(10) PFU/mL was left to stand for 1 hour at 37, 40, 50, 60, or 70 ℃ (3 tubes at each time point, 1 mL per tube) for counting the plaques, and then the thermal stability curve was drawn. SM buffer at a pH values of 5.0, 6.0, 7.0, 7.4, 8.0, 9.0, or 10.0 were added to the phage KP168 solution at a titer of 3.0×10(10) PFU/mL, respectively. The mixed solution was left to stand for 1 hour at 37 ℃ (3 tubes of each pH, each tube containing 100 µL phage KP168 solution and 900 µL SM buffer), and then the plaques were counted, and an acid-base stability curve was drawn. (8) The phage KP168 solution was taken for DNA extraction and sequencing after dialysis as in experiment (2). The whole genome was annotated with Prokka to obtain the coding sequence of phage KP168. Nucleotide's BLAST function was used to proceed nucleic acid sequence alignment for finding a known phage with the highest similarity to the phage KP168 nucleic acid sequence, and Blastx function was used to translate the coding sequence into protein for its function prediction. The comparison with Antibiotic Resistance Genes Database and Virulence Factors Database was proceeded. (9) In a 96-well plate, at a MOI of 1.000, 0.100, 0.010 or 0.001 (3 wells per MOI), 20 µL phage KP168 solution at a initial titer of 5.8×10(10) PFU/mL was added to 200 µL host bacteria solution at a concentration of 1.5×10(8) CFU/mL (the same concentration below) for co-cultivation for 48 hours. After 200 µL host bacteria solution was left to stand for 48 hours, 20 µL phage KP168 solution at a titer of 1×10(6,) 1×10(7,) 1×10(8,) 1×10(9,) or 1×10(10) PFU/mL (3 wells per titer) was added respectively for action for 4 hours. In both experiments, 200 µL host bacteria solution added with 20 µL SM buffer (3 wells) acted as a negative control, and 220 µL LB culture medium (3 wells) acted as a blank control. Absorbance values were measured by a microplate reader, and inhibition/destruction rates of biofilm were calculated. The experiments were both repeated three times. Results: (1) The plaques of phage KP168 successfully isolated and purified were transparent and round, and its diameter was approximately 1.5 mm. (2) The phage KP168 has a regular polyhedron structure with a diameter of about 50 nm and without a tail. (3) The phage KP168 could lyse 13 of 20 strains of Klebsiella pneumoniae from burned patients, with a lysis rate of 65.0%. (4) When MOI was 1.000, the titer was the highest after co-culturing the phage KP168 with the host bacteria for 4 hours, which was the optimal MOI. (5) After the mixing of the phage KP168 with the host bacteria for 4 minutes, the percentage of the adsorbed phage reached the highest, which was the optimal adsorption time. (6) The one-step growth curve showed that during the lysis of the host bacteria by phage KP168, the incubation period was about 10 minutes, and the lysis period was about 40 minutes. (7) With the condition of 40 ℃ or pH 7.4, the number of plaques and the activity of phage KP168 reached the highest. (8) The genome of phage KP168 was a linear double-stranded DNA with a length of 40 114 bp. There were 48 possible coding sequences. It had the highest similarity to Klebsiella phage_vB_Kp1. The most similar known proteins corresponding to the translated proteins of coding sequences contained 23 hypothetical proteins and 25 proteins with known functions. No resistance genes or virulence factor genes were found. The GeneBank accession number was KT367885. (9) After 48 hours of co-cultivation of the phage KP168 and the host bacteria at each MOI, the inhibition rates of biofilm were similar, with an average of about 45%. After the phage KP168 with a titer of 1×10(9) PFU/mL acted on the biofilm formed by the host bacteria for 4 h, the destruction rate of biofilm was the highest, reaching an average of 42%. Conclusions: In this study, a bacteriophage against pan-drug resistant Klebsiella pneumoniae from a burn patient, phage KP168, is isolated from sewage, which belongs to the tailless phage. It has a wide host spectrum, short adsorption time, and short incubation period, with certain thermal and acid-base stability. Its genomic information is clear, and it does not contain resistance genes or virulence factor genes. It also has an inhibitory effect on the formation of bacterial biofilm and a destructive effect on the formed bacterial biofilm.

CTLA4 enhances the osteogenic differentiation of allogeneic human mesenchymal stem cells in a model of immune activation

Allogeneic mesenchymal stem cells (allo-MSCs) have recently garnered increasing interest for their broad clinical therapy applications. Despite this, many studies have shown that allo-MSCs are associated with a high rate of graft rejection unless immunosuppressive therapy is administered to control allo-immune responses. Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is a co-inhibitory molecule expressed on T cells that mediates the inhibition of T-cell function. Here, we investigated the osteogenic differentiation potency of allo-MSCs in an activated immune system that mimics the in vivo allo-MSC grafting microenvironment and explored the immunomodulatory role of the helper T cell receptor CTLA4 in this process. We found that MSC osteogenic differentiation was inhibited in the presence of the activated immune response and that overexpression of CTLA4 in allo-MSCs suppressed the immune response and promoted osteogenic differentiation. Our results support the application of CTLA4-overexpressing allo-MSCs in bone tissue engineering.

CTLA4 enhances the osteogenic differentiation of allogeneic human mesenchymal stem cells in a model of immune activation.

Allogeneic mesenchymal stem cells (allo-MSCs) have recently garnered increasing interest for their broad clinical therapy applications. Despite this, many studies have shown that allo-MSCs are associated with a high rate of graft rejection unless immunosuppressive therapy is administered to control allo-immune responses. Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is a co-inhibitory molecule expressed on T cells that mediates the inhibition of T-cell function. Here, we investigated the osteogenic differentiation potency of allo-MSCs in an activated immune system that mimics the in vivo allo-MSC grafting microenvironment and explored the immunomodulatory role of the helper T cell receptor CTLA4 in this process. We found that MSC osteogenic differentiation was inhibited in the presence of the activated immune response and that overexpression of CTLA4 in allo-MSCs suppressed the immune response and promoted osteogenic differentiation. Our results support the application of CTLA4-overexpressing allo-MSCs in bone tissue engineering.

Trichosanthes kirilowii: A New Host of Cucurbit mild mosaic virus in China.

Chinese cucumber (Trichosanthes kirilowii Maxim.) is a type of perennial liana plant of the Cucurbitaceae family that is mainly distributed in East Asia and northern Australia. It is an important medicinal plant and commonly used in Chinese herbalism, where it is considered to be one of the 50 fundamental herbs (2). During the summer and autumn of 2012, T. kirilowii plants showing symptoms of mild mosaic on the upper leaves and bright yellow color on the lower leaves were observed in the Haidian district of Beijing, China. Recently similar symptoms induced by Cucurbit mild mosaic virus (CuMMV) on squash have been reported. CuMMV is a new member of the genus Fabavirus in the Comovirinae subfamily, discovered in China in 2006 (1). Total RNA was extracted from five leaf samples of independent plants and used for reverse transcription with an oligo (dT)18 primer, followed by PCR with a pair of CuMMV virus-specific primers FaR13012F (5'-CGAGTGCGAGTTAGAAATTGGGATG-3') and FaR15783R (5'-TCACTTTGAGGTGATAAAACAATCC-3') to amplify a 2,772-bp fragment including RNA-dependent RNA polymerase (RdRp) coding region. The expected target fragment was obtained in all symptomatic plant samples but not from an asymptomatic plant. Nucleotide sequence comparison analysis showed that the virus isolated from T. kirilowii (GenBank Accession No. KC959843) had 95.33% nucleotide identity and 99.15% amino acid identity in the RdRp sequence with a CuMMV isolate from squash (GenBank Accession No. FJ194941) (1). In addition, symptomatic samples tested positive for CuMMV by Western blot using CuMMV small coat protein (SCP) specific polyclonal antibody (1). To our knowledge, this is the first report of T. kirilowii as natural host of CuMMV in China. The impact of CuMMV on T. kirilowii production remains to be determined; however, the extended host range for this virus suggests a potential threat of CuMMV to cucurbit crops in China. References: (1) S. W. Dong et al. Arch. Virol.157:597, 2012. (2) J. H. Hong et al. China Pharmacist 7:561, 2004.

Autologous platelet-rich clot releasate stimulates proliferation and inhibits differentiation of adult rat tendon stem cells towards nontenocyte lineages.

OBJECTIVE: To explore the effects of autologous platelet-rich clot releasate (PRCR) on proliferation and differentiation of adult rat tendon stem cells (TSCs) in vitro, following intense mechanical stretching. METHODS: TSCs were subjected to 8% mechanical stretching and subsequently incubated in control medium or medium supplemented with 2% or 10% PRCR. Collagen types I and III, peroxisome proliferator-activated receptor-γ (PPARγ), sex determining region Y-box 9 (SOX-9) and runt-related transcription factor 2 (RUNX2) concentrations were assessed via Western blotting and flow cytometry. Transforming growth factor (TGF)-ß1 and vascular endothelial growth factor concentrations were measured using enzyme-linked immunosorbent assay. Treated TSCs were also cultured in adipogenic, chondrogenic or osteogenic culture media. RESULTS: PRCR increased the number of TSCs, and the concentrations of collagen types I and III and TGF-ß1. In contrast, PRCR significantly reduced PPARγ, SOX-9 and RUNX2-positive cell numbers, and significantly reduced the numbers of TSC-derived adipocytes, chondrocytes and osteocytes. CONCLUSION: PRCR induced tenocyte differentiation while suppressing the adipocyte, chondrocyte and osteocyte lineages believed to impede tendon healing.

Hypersensitivity reactions during anesthesia. Results from the ninth French survey (2005-2007).

BACKGROUND: Hypersensitivity reactions occurring during anesthesia remain a major cause of concern for anesthesiologists. We report the results of the ninth consecutive survey of hypersensitivity reactions observed during anesthesia in France. This report will be used as an epidemiologic reference prior to this intervention. METHODS: Between January 1, 2005 to December 31, 2007, 1253 patients who experienced an immune-mediated (IgE-mediated) or non-immune-mediated (non-IgE-mediated) hypersensitivity reaction were referred to one of the 40 participating centers. Diagnosis was established on the basis of clinical history, skin tests and/or specific IgE assay. RESULTS: An IgE-mediated or non-IgE-mediated reaction was diagnosed in 786 cases (63%) and 467 cases (37%), respectively. The most common causes of anaphylaxis were neuromuscular blocking agents (NMBA) (N.=373, 47.4%), latex (N.=158, 20%), and antibiotics (N.=141, 18.1%). Succinylcholine (N.=226, 60.6%) was the most frequently incriminated NMBA, whereas the low frequency of reactions involving cis-atracurium was confirmed (N.=22, 5.9%) when market shares of each NMBA were taken into account. An increased number of reactions involving vital dyes was recorded (N.=34, 4.4%). CONCLUSION: These changes in the epidemiology of allergic reactions confirm the need for regular epidemiologic surveys of anaphylaxis in the perioperative period.

Wnt/beta-catenin is involved in the proliferation of hippocampal neural stem cells induced by hypoxia.

BACKGROUND AND AIM: Beta-catenin, as a major effector molecule in the canonical Wnt signaling pathway, could regulate adult neurogenesis. Here, the role of Wnt/ß-catenin signaling pathway in the proliferation of hippocampal neural stem cells (NSCs) induced by hypoxia was investigated. METHODS: The hippocampal NSCs of neonatal green fluorescent protein transgenic mice on day 0 were cultured in hypoxia (5% O(2)) and traditional O(2) (20% O(2)). The expression of ß-catenin, p-GSK-3ß, and cyclinD1 in NSCs was measured under hypoxia or traditional O(2) by western blotting. NSCs were electroporated with pTOPFLASH reporter in different conditions and the LEF/TCF-dependent luciferase activity was assayed. RESULTS: Hypoxia increased the proliferation and reduced the apoptosis of hippocampal NSCs. NSCs proliferation was inhibited by transfecting with pAxin, whereas promoted by transfecting with pß-catenin. CONCLUSION: Hypoxia could enhance the proliferation of hippocampal NSCs and ß-catenin contributed to this action.

Research on the reactivation of Syk expression caused by the inhibition of DNA promoter methylation in the lung cancer.

The aim of this study was to study the expression of Syk gene and methylation in its promoter region in the lung cancer and to investigate the relationship between silencing of the Syk gene and DNA methylation of the Syk promoter region in lung cancer cell lines. Real-time PCR and immunohistochemistry were used to examine the Syk expression in specimens from 3 lung cancer cell lines and 16 lung cancer patients (tumor tissues and adjacent normal tissues). MSP was used to analyze the methylation status of the Syk promoter region. We also investigated the role of restoring Syk expression by using a DNA methyltransferase inhibitor, 5-aza-CdR, in suppressing invasion of lung cancer cell lines. No expression of the Syk gene was detected in the 3 lung cancer cell lines. In the 16 lung cancer patient samples, Syk expression was significantly lower in the tumor tissues than that in their adjacent normal tissues (P<0.05). Consistently, immunohistochemistry analysis of Syk protein expression showed that in the lung cancer tissues Syk protein expression was also significantly lower than that in their adjacent normal tissues. In the two lung cancer cell lines (NL9980, YTMLC-9) that lack the endogenous Syk expression, 4uM demethylation agent 5-aza-CdR treatment was able to reactivate the Syk gene expression, but not NCI-H446. In conclusion, hypermethylation leads to silencing of the Syk gene in human lung carcinoma cell lines. Methylation of the Syk promoter and loss of Syk expression in lung cancer cell lines are independent biomarkers. Syk may be a potential tumor suppressor in human lung cancer.

Rapid gas chromatographic profiling and screening of biologically active amines.

An efficient method is described for the simultaneous determination of 57 amines including volatile aliphatic amines, nonvolatile polyamines and catecholamines present in aqueous samples. The method is based on two-phase isobutyloxycarbonylation (isoBOC) with a pH shift. In 1.0 M phosphate buffer at pH 7.5, phenolic hydroxyl groups were allowed to react with isobutyl chloroformate in the dichloromethane phase, and subsequently pH of the aqueous phase was increased to 12.0 for the reaction of basic amino functions. The resulting N(O)-isoBOC amines were recovered by solid-phase extraction using Chromosorb P in normal phase partition mode, with subsequent tert.- butyldimethylsilylation of the remaining hydroxyl groups for gas chromatographic analysis. Using this combined procedure, linear responses were obtained in the concentration range of 0.2-12 ppm, with correlation coefficients varying from 0.945 to 0.999 for most of the amines studied except for 5-methoxytryptamine (0.864). Temperature-programmed retention index (I) sets as measured on DB-5 and DB-17 dual-capillary columns of different polarity were characteristic of each amine and thus, useful in the screening for amines by computer I matching. When applied to saliva samples, the present method allowed rapid screening for each spiked amine and unspiked polyamines such as 1,3-diaminopropane, putrescine, cadaverine and spermidine.

Chronic allyl chloride poisoning. An epidemiology, clinical, toxicological and neuropathological study.

It was previously reported that chronic exposure to allyl chloride resulted in liver and kidney damage. No neurotoxic effect of allyl chloride had been noticed until two outbreaks of polyneuropathy without liver and kidney dysfunction due to exposure to allyl chloride in China in the early 1970's. Epidemiological and clinical studies done within 1973-1982 revealed that the main risk of industrial exposure to allyl chloride is damage to the peripheral nervous system. Polyneuropathy is thought to be the main clinical manifestation of chronic allyl chloride poisoning. Electroneuromyography is essential and valuable for early diagnosis and biological monitoring. Toxicological and neuropathological studies in rabbits and mice have given the evidence of a pattern of central-peripheral distal axonopathy in peripheral nervous system which has further confirmed the neurotoxicity of allyl chloride found in man. Based on the above results, the maximum allowable concentration of allyl chloride and diagnostic criteria for chronic allyl chloride poisoning are proposed.