Controlled expression of lysis gene E by a mutant of the promoter pL of the thermo-inducible λcI857-pL system.

AIMS: To identify a lambda promoter pL mutant that could extend the thermal stability of the thermo-inducible λcI857-pR/pL system and to evaluate the effects of the modified system for the controlled expression of lysis gene E during the production of bacterial ghosts (BGs). METHODS AND RESULTS: The promoter pL mutant was identified by random mutagenesis and site-directed mutagenesis. The results showed that a T â†’ 35C mutation in the pL promoter was responsible for the phenotype alteration. Under the same induction conditions, the lysis rates of the modified lytic system on Escherichia coli and Salmonella enteritidis were significantly lower than that of the control, while the lysis rates of Escherichia coli with the thermo-inducible lytic system were significantly higher than that of S. enteritidis with the corresponding plasmid (P < 0·05). CONCLUSIONS: Increasing the heat stability of the thermo-inducible lytic systems decreased lysis efficiency during the production of BGs. There exist differences in the lysis efficiency of thermo-inducible lytic systems between different bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings enrich current knowledge about modifications to thermo-inducible systems and provide a reference for the application of these modified systems for the production of BGs and controlled gene expression in bacteria.

[Retrospective analysis of the effectiveness of treating multi-space infection combined with descending necrotizing mediastinitis at oral maxillofacial and cervical region via multidisciplinary team collaboration].

Objective: To explore the strategy and experience for treating maxillofacial and cervical multi-space infection combined with descending necrotizing mediastinitis (DNM) via multidisciplinary team (MDT) collaboration. Methods: A total of 36 patients with maxillofacial and cervical multi-space infection complicated with DNM admitted to the First Affiliated Hospital of Zhengzhou University from July 2011 to July 2019 were included in the study. The clinical data of the patients were retrospectively analyzed, including gender, age, symptoms at admission, source of infection, preoperative and postoperative evaluation indicators, MDT strategy and prognosis. Results: There were 26 males and 10 females with an average age of (51.6±17.6) years (8-80 years). The course of disease before admission was (8.9±8.4) days (2-30 days). All patients were admitted with maxillofacial and neck swelling and pain as the main complaints. Odontogenic infection accounted for 39% (14/36), throat floor swelling and pain accounted for 25% (9/36) and unknown maxillofacial swelling accounted for 36% (13/36). There were 28 cases receiving surgical treatment, 26 cases were cured and discharged (72%), 10 cases died (28%). In the patients treated with multidisciplinary therapy (mainly by surgery), the white blood cell count, neutrophil percentage, C-reactive protein and procalcitonin levels were significantly improved compared with those at admission at each observation time point after operation (P<0.05). The length of stay was positively correlated with the levels of C-reactive protein (r=0.545, P<0.05) and procalcitonin (r=0.504, P<0.05). The prognosis of patients treated with surgery (26/28) was better than that of patients without surgery (0/8) (P<0.01). Conclusions: The patients with maxillofacial and cervical multi-space infection combined with DNM might be in critical condition. The surgical based MDT strategy has an important impact on the prognosis of patients. White blood cell count and other inflammatory indicators monitoring can effectively observe the changes of the patient's condition.

[Clinical features and imaging analysis of primary bone lymphoma of the jaw].

Objective: To investigate the clinical characteristics and imaging manifestations of primary bone lymphoma (PBL) located in the jaw. Methods: Clinical data of 14 PBL patients admitted to The First Affiliated Hospital of Zhengzhou University from January 2014 to April 2019 were retrospectively analyzed, which including age, gender, location, chief complaint, laboratory test, imaging manifestations and so on. Results: Among the 14 PBL patients, 10 were male and 4 were female. Age range from 4 to 79 years, median age was 56 years old. There were 6 cases involved maxillary, 7 cases involved mandible and 1 case involved mandible and maxillary simultaneously. Twelve patients were admitted with local mass as the chief complaint, 3 had a history of toothache, 4 had loosen tooth and 2 had numbness of lips and cheeks. Two cases of oral mass with mucosal surface ulcer or abscess formation. One case complained of fever, night sweats and other systemic symptoms. The imaging findings showed 8 cases of osteolytic lesions, 1 case of sclerosis, 4 cases of mixed lesions and 1 case of cystic lesions. One case had periosteum reaction. In 12 cases, there was an obvious mass, which was characterized by the large soft-tissue mass and marrow changes were associated with surprisingly little cortical destruction. Conclusions: PBL is more common in middle aged male. The upper and lower jaw bones can be involved. Clinical and imaging manifestations lack characteristics, and common dental or periodontal symptoms such as toothache, tooth loosening, and paresthesia may occur. However, the imaging characteristics of large soft tissue masses with small degree of bone destruction can be used as an important basis for the differential diagnosis of osteomyelitis, squamous cell carcinoma, osteosarcoma and other diseases.

Retraction Note: Structural simulation of adenosine phosphate via plumbagin and zoledronic acid competitively targets JNK/Erk to synergistically attenuate osteoclastogenesis in a breast cancer model.

This article has been retracted at the request of the authors. After publication, the authors found that in Figure 2B-a the first two images in the third row partly overlapped and that there is also overlap between the fourth and fifth image in the second row. The two images were taken from two adjacent wells, treated by ZA 0.3uM-CM or ZA 0.75uM-CM, with or without PL 1.25uM. This overlap may have been caused by mishandling in the imaging process when the authors made microscope observations and so the findings are no longer reliable. All authors agree to this retraction.

Current progress in black phosphorus materials and their applications in electrochemical energy storage.

Recently, a new two-dimensional material, single- or few-layered black phosphorus (BP), has attracted considerable attention for applications in electronics, optoelectronics, and batteries due to its unique properties, including large specific surface area, anisotropy, and tunable and direct band gaps. In particular, contributions to electrochemical energy storage devices, such as lithium and sodium ion batteries and supercapacitors, have emerged. However, critical issues remain to be explored before scaled-up commercial production of BP, such as preparation, stability, and performance. Herein, we present the first review of recent progress in BP-based electrochemical energy storage device. The preparation and electrochemical properties of black phosphorus, recent advances, potential challenges, and relevant perspectives in electrochemical energy storage, and the potential of BP are discussed in this work.

Regulatory effect of estrogen receptor-α-mediated Wnt/ß-catenin signaling pathway on osteoblast proliferation.

This study was designed to investigate the regulatory effect of estrogen receptor-α (ERα)-mediated Wnt/ß-catenin signaling pathway on osteoblast proliferation. Mc3T3-El cells were infected by ERα and ERß small interfering ribose nucleic acid (siRNA) viruses and treated with estradiol 2 (E2) for 120 min after 24-h infection. Western blot was used to detect expressions of ß-catenin, Gsk 3ß, p-Gsk3ß (Ser9) and CyclinDl; and methyl thiazolyl tetrazolium (MTT) was applied to detect osteoblast proliferation after interference by different ERs. Western blot results indicated that the expressions of ß-catenin, p-Gsk3ß (Ser9) and CyclinDl decreased after ERß interference and ERα + ERß joint interference, and a more obvious decrease was found after the joint interference. After ERß interference, ß-catenin, p-Gsk3ß (Ser9) and CyclinDl were strongly expressed compared with expressions in the blank control group. MTT results demonstrated that the proliferation rate of osteoblast was lower after the joint interference than after ERß interference, while a slight increase was found in the proliferation rate after ERß interference in comparison with the blank control group. It can be concluded that estradiol is able to promote the proliferation of osteoblasts in mice by ERα-mediated Wnt/ß-catenin signaling pathway.

Structural simulation of adenosine phosphate via plumbagin and zoledronic acid competitively targets JNK/Erk to synergistically attenuate osteoclastogenesis in a breast cancer model.

The treatment of breast cancer-induced osteolysis remains a challenge in clinical settings. Here, we explored the effect and mechanism of combined treatment with zoledronic acid (ZA) and plumbagin (PL), a widely investigated component derived from Plumbago zeylanica, against breast cancer-induced osteoclastogenesis. We found that the combined treatment with PL and ZA suppressed cell viability of precursor osteoclasts and synergistically inhibited MDA-MB-231-induced osteoclast formation (combination index=0.28) with the abrogation of recombinant mouse receptor activator of nuclear factor-κB ligand (RANKL)-induced activation of NF-κB/MAPK (nuclear factor-κB/mitogen-activated protein kinase) pathways. Molecular docking suggested a putative binding area within c-Jun N-terminal kinase/extracellular signal-regulated kinase (JNK/Erk) protease active sites through the structural mimicking of adenosine phosphate (ANP) by the spatial combination of PL with ZA. A homogeneous time-resolved fluorescence assay further illustrated the direct competitiveness of the dual drugs against ANP docking to phosphorylated JNK/Erk, contributing to the inhibited downstream expression of c-Jun/c-Fos/NFATc-1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1). Then, in vivo testing demonstrated that the combined administration of PL and ZA attenuated breast cancer growth in the bone microenvironment. Additionally, these molecules prevented the destruction of proximal tibia, with significant reduction of tartrate-resistant acid phosphatase (TRAcP)-positive osteoclast cells and potentiation of apoptotic cancer cells, to a greater extent when combined than when the drugs were applied independently. Altogether, the combination treatment with PL and ZA could significantly and synergistically suppress osteoclastogenesis and inhibit tumorigenesis both in vitro and in vivo by simulating the spatial structure of ANP to inhibit competitively phosphorylation of c-Jun N-terminal kinase/extracellular signal-regulated kinase (JNK/Erk).

Bone morphogenetic protein 2 and decorin expression in old fracture fragments and surrounding tissues.

Bone morphogenetic protein 2 (BMP-2) can promote fracture healing. Although the complex role BMP-2 in bone formation is increasingly understood, the role of endogenous BMP-2 in nonunion remains unclear. Decorin (DCN) can promote the formation of bone matrix and calcium deposition to control bone morphogenesis. In this study, tissue composition and expression of BMP-2 and DCN were detected in different parts of old fracture zones to explore inherent anti-fibrotic ability and osteogenesis. Twenty-three patients were selected, including eight cases of delayed union and 15 cases of nonunion. Average duration of delayed union or nonunion was 15 months. Fracture fragments and surrounding tissues, including bone grafts, marrow cavity contents, and sticking scars, were categorically sampled during surgery. Through observation and histological testing, component comparisons were made between fracture fragments and surrounding tissue. The expression levels of DCN and BMP-2 in different tissues were detected by immunohistochemical staining and real-time polymerase chain reaction. The expression of DCN and BMP- 2 in different parts of the nonunion area showed that, compared with bone graft and marrow cavity contents, sticking scars had the highest expression of BMP-2. Compared with the marrow cavity contents and sticking scars, bone grafts had the highest expression of DCN. The low antifibrotic and osteogenic activity of the nonunion area was associated with non-co-expression of BMP-2 and DCN. Therefore, the co-injection of osteogenic factor BMP and DCN into the nonunion area can improve the induction of bone formation and enhance the conversion of the old scar, thereby achieving better nonunion treatment.

GABAB1 and GABAB2 receptor subunits co-expressed in cultured human RPE cells regulate intracellular Ca2+ via Gi/o-protein and phospholipase C pathways.

GABAB receptors associate with Gi/o-proteins that regulate voltage-gated Ca(2+) channels and thus the intracellular Ca(2+) concentration ([Ca(2+)]i), there is also reported cross-regulation of phospholipase C. These associations have been studied extensively in the brain and also shown to occur in non-neural cells (e.g. human airway smooth muscle). More recently GABAB receptors have been observed in chick retinal pigment epithelium (RPE). The aims were to investigate whether the GABAB receptor subunits, GABAB1 and GABAB2, are co-expressed in cultured human RPE cells, and then determine if the GABAB receptor similarly regulates the [Ca(2+)]i of RPE cells and if phospholipase C is involved. Human RPE cells were cultured from five donor eye cups. Evidence for GABAB1 and GABAB2 mRNAs and proteins in the RPE cell cultures was investigated using real time polymerase chain reaction, western blots and immunofluorescence. The effects of the GABAB receptor agonist baclofen, antagonist CGP46381, a Gi/o-protein inhibitor pertussis toxin, and the phospholipase C inhibitor U73122 on [Ca(2+)]i in cultured human RPE were demonstrated using Fluo-3. Both GABAB1 and GABAB2 mRNA and protein were identified in cell cultures of human RPE; antibody staining was co-localized to the cell membrane and cytoplasm. One-hundred micromolars of baclofen caused a transient increase in the [Ca(2+)]i of RPE cells regardless of whether Ca(2+) was added to the buffer. Baclofen-induced increases in the [Ca(2+)]i were attenuated by pre-treatment with CGP46381, pertussis toxin, and U73122. GABAB1 and GABAB2 are co-expressed in cell cultures of human RPE. GABAB receptors in RPE regulate the [Ca(2+)]i via a Gi/o-protein and phospholipase C pathway.

Detection of autoinducer-2 and analysis of the profile of luxS and pfs transcription in Streptococcus suis serotype 2.

The only quorum sensing (QS) system shared by Gram-positive and Gram-negative bacteria involves the production of autoinducer-2 (AI-2). However, there have been no reports concerning the AI-2 in Streptococcus suis serotype 2 (SS2). In this study, we investigated AI-2 production and analyzed the relationship between the transcription level of luxS and pfs and AI-2 production. A homologue of luxS, the gene required for AI-2 synthesis in Vibrio harveyi, was isolated from the SS2 genome. V. harveyi BB170 bioassay demonstrated luxS functionality in SS2 and its ability to complement the luxS-negative phenotype of Escherichia coli DH5alpha. Further studies showed that AI-2 activity peaked in the late exponential phase, and sodium chloride or glucose increased the production of AI-2. Real-time PCR showed that the level of transcription of pfs is highly correlated with the level of AI-2 production, while the level of transcription of luxS does not correlate with the level of AI-2 production. The results presented here demonstrate that SS2 can secrete AI-2 and that the profile of pfs transcription is highly correlated with the level of AI-2 production.

Lifetime of anode polymer in magnetically insulated ion diodes for high-intensity pulsed ion beam generation.

Generation of high-intensity pulsed ion beam (HIPIB) has been studied experimentally using polyethylene as the anode polymer in magnetically insulated ion diodes (MIDs) with an external magnetic field. The HIPIB is extracted from the anode plasma produced during the surface discharging process on polyethylene under the electrical and magnetic fields in MIDs, i.e., high-voltage surface breakdown (flashover) with bombardments by electrons. The surface morphology and the microstructure of the anode polymer are characterized using scanning electron microscopy and differential scanning calorimetry, respectively. The surface roughening of the anode polymer results from the explosive release of trapped gases or newly formed gases under the high-voltage discharging, leaving fractured surfaces with bubble formation. The polyethylene in the surface layer degrades into low-molecular-weight polymers such as polyethylene wax and paraffin under the discharging process. Both the surface roughness and the fraction of low molecular polymers apparently increase as the discharging times are prolonged for multipulse HIPIB generation. The changes in the surface morphology and the composition of anode polymer lead to a noticeable decrease in the output of ion beam intensity, i.e., ion current density and diode voltage, accompanied with an increase in instability of the parameters with the prolonged discharge times. The diode voltage (or surface breakdown voltage of polymer) mainly depends on the surface morphology (or roughness) of anode polymers, and the ion current density on the composition of anode polymers, which account for the two stages of anode polymer degradation observed experimentally, i.e., stage I which has a steady decrease of the two parameters and stage II which shows a slow decrease, but with an enhanced fluctuation of the two parameters with increasing pulses of HIPIB generation.

Susceptibility of insulin-secreting hepatocytes to the toxicity of pro-inflammatory cytokines.

The liver has been suggested as a suitable target organ for reversing type I diabetes by gene therapy. Whilst gene delivery systems to the hepatocyte have yet to be optimized in vivo, whether insulin-secreting hepatocytes are resistant to the autoimmune process that kills pancreatic beta-cells has never been addressed. One of the mechanisms by which beta-cells are killed in type I diabetes is by the release of the cytokines interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) by immune cells. To test the effect of the cytokines on insulin-secreting hepatocytes in vitro we exposed the betacyte, also called the HEP G2ins/g cell which possesses cytokine receptors and can synthesize, store and secrete insulin in a regulated fashion to a glucose stimulus, to the above mentioned cytokines for 14 days. Viability of the HEP G2ins/g cells was similar to that of other liver cell lines/primary cells which were more resistant to the cytokines than the beta-cell line NIT-1. The cytokines had no adverse effect for the first six days on insulin secretion, content and mRNA levels of the HEP G2ins/g cells and insulin secretion in response to 1-h exposure to 20 mM glucose was enhanced 14-fold. Our results indicate that genetically engineered hepatocytes and primary liver cells are more resistant than pancreatic beta-cells to the adverse effects of cytokines offering hope that insulin secreting hepatocytes in vivo made by gene therapy are less likely to be destroyed by cytokines released during autoimmune destruction.

Secretion of pancreatic icosapeptide from porcine pancreas.

The pancreatic polypeptide cell, the only mature endocrine cell in the fetal pig pancreas, produces equimolar amounts of two peptides, pancreatic polypeptide and pancreatic icosapeptide, from the same precursor. The amino acid sequence of pancreatic polypeptide is more homogeneous among species, whereas pancreatic icosapeptide is heterogeneous. We determined the 19-amino acid sequence of porcine pancreatic icosapeptide, which is markedly different from that of known sequences (e.g. 47% homology with human). We developed an ELISA that can measure porcine pancreatic icosapeptide levels in the range of 7.2-480 pmol/liter. Actual levels of pancreatic icosapeptide in pig sera were 9.6-25 pmol/liter. The assay requires relatively small amounts of nonextracted samples, and human and mouse sera do not cross-react. Levels of pancreatic icosapeptide rose in response to hypoglycemia in pigs and to carbachol in fetal porcine pancreatic cells in vitro. When fetal porcine pancreatic tissue was transplanted into nonobese diabetic-severe combined immune deficiency mice, porcine pancreatic icosapeptide (but not C peptide) was detectable in mouse sera for up to 3 wk after transplantation, with levels highest on d 4. Porcine pancreatic icosapeptide and insulin were detectable in grafts removed from the mice. Therefore, porcine pancreatic icosapeptide may be used as a marker of the viability of xenotransplanted fetal pig pancreatic tissue in the immediate posttransplant period.

Expression of the gene for cytochrome P-450 17 alpha-hydroxylase/C17-20 lyase (CYP17) in porcine Leydig cells: identification of a DNA sequence that mediates cAMP response.

Regulation of CYP17 gene expression in porcine Leydig cells was investigated in primary culture. We previously reported the sequence of the 5' upstream and much of the pig gene. (Zhang et al. (1992) Biochim. Biophys. Acta. 1131, 345-348). DNase I footprinting assays identified a region between -193 and -174 that was bound by nuclear proteins. Examination of the DNA sequence in this region revealed putative Sp1 and AP-2 binding sites, but gel retardation assays using an oligonucleotide from -198 to -168 as a probe revealed two specific DNA-protein complexes that were not Sp1 or AP-2. The oligonucleotide was cloned into a reporter gene containing a minimal porcine CYP17 promoter and the resultant construct was transiently transfected into porcine Leydig cells. This chimeric construct had both basal and cAMP-induced transcriptional activities. Southwestern blot identified a prominent binding of a nuclear protein around 68 kDa and a weaker binding of a nuclear protein around 110 kDa. Sequences between -250/+1 are highly homologous to those sequences from human, bovine and rodent CYP17 gene, but the -193/-174 region has no homology to those genes. Other regions of the porcine CYP17 were also important for the basal and cAMP-mediated regulation. Luciferase expression vectors were prepared with 5' flanking DNA from the porcine CYP17 gene and were expressed in primary culture of porcine Leydig cells. The region between -587/-325 was important for basal transcription, and a region of DNA between -325 and -140 was important for cAMP regulation.

Binding of lipid droplets and mitochondria to intermediate filaments in rat Leydig cells.

We have examined the distribution of lipid droplets and mitochondria in relation to the cytoskeletons of Leydig cells in primary culture by using light and electron microscopy on living, intact and detergent-extracted cells. After mild extraction with Triton X-100 lipid droplets and mitochondria retained their original distribution within the cell. Double immunofluorescent microscopy showed that both structures co-localise with intermediate filaments. Transmission electron microscopy of intact (unextracted) and mildly extracted Leydig cells showed that intermediate filaments are closely associated with mitochondria and lipid droplets. By examination of stereo pairs, intermediate filaments were shown to establish direct contact with mitochondria and lipid droplets. The association of droplets and mitochondria with intermediate filaments suggests possible mechanisms by which the transport of cholesterol takes place from droplets to mitochondria where this substrate enters the steroidogenic pathway.

Cyclic AMP regulates expression of the rat gene for steroid 17 alpha-hydroxylase/C17-20 lyase P-450 (CYP17) in rat Leydig cells.

The upstream region of the rat CYP17 gene shows significant homology to the upstream regions of the bovine and human genes, 53 and 60 percent, respectively. The start site of transcription was determined by primer extension and S1 nuclease protection to be 41 base pairs (bp) upstream of the initiating methionine codon. Expression vectors were constructed by ligation of upstream sequences into promoterless chloramphenicol acetyl transferase (CAT) vectors. Transient transfection studies using primary cultures of rat Leydig cells indicate a strong cAMP-responsive element located within the -26/+65 region. Stimulation by cyclic AMP was abolished when sequences upstream of -264 were included in expression vectors. No significant expression was seen in Leydig cells in the absence of dbcAMP nor was there any expression in the presence or absence of dbcAMP in rat skin fibroblasts or in mouse adrenal (Y-1) cells in which CYP17 is not normally expressed. Three possible regulatory elements were found in the 5' upstream region: a CRE/ATF consensus sequence (GACGTCA) starting at position -57; a GRE consensus sequence (TGTTCT) starting at position -501; and a consensus sequence for AP-1 binding (TTAGTCA) starting at position -659. It was concluded that the CRE/ATF at -57 is not responsible for increased transcription in the presence of cyclic AMP.

Gene for 17 alpha-hydroxylase/C (17-20) lyase P-450: complete nucleotide sequence of the porcine gene and 5' upstream sequence of the rat gene.

We describe the isolation and characterization of a full-length clone for the porcine 17 alpha-hydroxylase/C(17-20) lyase (CYP17) gene. The complete exon and partial intron sequences are presented including approx. 1000 bp of the 5' upstream sequence. In addition we describe the isolation and characterization of the 5' upstream region of the rat CYP17 gene. The sequences of the first exon, part of the first intron, and approx. 3.5 kb of the 5' upstream region are presented.