Infection prevention and early warning in neonatal intensive care unit based on physiological sensor monitoring.

The infection rate in the Neonatal Intensive Care Unit (NICU) is very high, which is also one of the important causes of morbidity and even death in critically ill neonates and premature infants. At present, the monitoring system of the Neonatal Intensive Care Unit is not very complete, and it is difficult to provide early warning of neonatal illness. Coupled with the untimely response measures, it has brought certain difficulties to the ward's infection prevention and control work. The rapid development of the Internet of Things (IoT) in recent years has made the application fields of various sensor devices more and more extensive. This paper studied infection prevention and early warning in the Neonatal Intensive Care Unit based on physiological sensors. Combined with a wireless network and physiological sensors, this paper built an intelligent monitoring system for the Neonatal Intensive Care Unit, which aimed to monitor various physiological data of newborns in real-time and dynamically, and gave early warning signals, so that medical staff could take preventive measures in time. The experiments showed that the monitoring system proposed in this paper could obtain the physiological information of neonates in time, which brought convenience to prevention and early warning work, and reduced the infection rate of neonatal wards by 7.39%.

A Cu(II)-based coordination polymer: catalytic properties and treatment activity on stroke.

A fresh Cu(II) coordination polymer, i.e., [Cu3(Hmbdc)2(mbdc)2(dmphen)2]n (1, H2mbdc = isophthalic acid, dmphen = 4,7-dimethyl-1,10-phenanthroline), has been generated with the hydrothermal reactions between Cu salts and the mixed ligands of 4,7-dimethyl-1,10-phenanthroline and isophthalic acid. Moreover, the catalytic activity of 1 was evaluated via degrading the Congo red with a method of Fenton with an excellent degradation efficiency of 95.8% at 100 min. Next, the application value of compound on stroke was assessed, and the related mechanisms were explored at the same time. First of all, the tumor necrosis factor-α and recombinant rat IL-1ß content released into the plasma were determined with enzyme-linked immunosorbent assay detection kit. Besides, the activation of the HMGB1/TLR4 signaling pathway activation in cerebral vascular endothelial cells was also determined with real-time reverse transcription-polymerase chain reaction assay.

Protective effects of quercetin and taraxasterol against H2O2-induced human umbilical vein endothelial cell injury in vitro.

Due to the association between inflammation and endothelial dysfunction in atherosclerosis, the blockage of the inflammatory process that occurs on the endothelial cells may be a useful way of preventing atherosclerosis. In the present study, human umbilical vein endothelial cells (HUVECs) were used to investigate the protective effects of quercetin and taraxasterol against H2O2-induced oxidative damage and inflammation. HUVECs were pretreated with quercetin or taraxasterol at concentrations ranging between 0 and 210 µM for 12 h, prior to being administered different concentrations of H2O2 for 4 h. Cell viability and levels of apoptosis were assessed through cell counting kit-8 (CCK-8) and terminal deoxynucleotidyl transferase dUTP nick end labeling assays, respectively, to determine the injury to the HUVECs. The viability loss in the H2O2-induced HUVECs was markedly restored in a concentration-dependent manner by pretreatment with quercetin or taraxasterol. This effect was accompanied by significantly decreased expression of vascular cell adhesion molecule 1 (VCAM-1) and cluster of differentiation (CD)80 for taraxasterol and that of CD80 for quercetin. In conclusion, the present study showed the protective effects of quercetin and taraxasterol against cell injury and inflammation in HUVECs and indicated that the effects were mediated via the downregulation of VCAM-1 and CD80 expression. This study has therefore served as a preliminary investigation on the anti-atherosclerotic and cardiovascular protective effects of quercetin and taraxasterol as dietary supplements.

The Vibrio cholerae flagellar regulatory hierarchy controls expression of virulence factors.

Vibrio cholerae is a motile bacterium responsible for the disease cholera, and motility has been hypothesized to be inversely regulated with virulence. We examined the transcription profiles of V. cholerae strains containing mutations in flagellar regulatory genes (rpoN, flrA, flrC, and fliA) by utilizing whole-genome microarrays. Results revealed that flagellar transcription is organized into a four-tiered hierarchy. Additionally, genes with proven or putative roles in virulence (e.g., ctx, tcp, hemolysin, and type VI secretion genes) were upregulated in flagellar regulatory mutants, which was confirmed by quantitative reverse transcription-PCR. Flagellar regulatory mutants exhibit increased hemolysis of human erythrocytes, which was due to increased transcription of the thermolabile hemolysin (tlh). The flagellar regulatory system positively regulates transcription of a diguanylate cyclase, CdgD, which in turn regulates transcription of a novel hemagglutinin (frhA) that mediates adherence to chitin and epithelial cells and enhances biofilm formation and intestinal colonization in infant mice. Our results demonstrate that the flagellar regulatory system modulates the expression of nonflagellar genes, with induction of an adhesin that facilitates colonization within the intestine and repression of virulence factors maximally induced following colonization. These results suggest that the flagellar regulatory hierarchy facilitates correct spatiotemporal expression patterns for optimal V. cholerae colonization and disease progression.

The Francisella tularensis pathogenicity island encodes a secretion system that is required for phagosome escape and virulence.

Francisella tularensis causes the human disease tularemia. F. tularensis is able to survive and replicate within macrophages, a trait that has been correlated with its high virulence, but it is unclear the exact mechanism(s) this organism uses to escape killing within this hostile environment. F. tularensis virulence is dependent upon the Francisella pathogenicity island (FPI), a cluster of genes that we show here shares homology with type VI secretion gene clusters in Vibrio cholerae and Pseudomonas aeruginosa. We demonstrate that two FPI proteins, VgrG and IglI, are secreted into the cytosol of infected macrophages. VgrG and IglI are required for F. tularensis phagosomal escape, intramacrophage growth, inflammasome activation and virulence in mice. Interestingly, VgrG secretion does not require the other FPI genes. However, VgrG and other FPI genes, including PdpB (an IcmF homologue), are required for the secretion of IglI into the macrophage cytosol, suggesting that VgrG and other FPI factors are components of a secretion system. This is the first report of F. tularensis FPI virulence proteins required for intramacrophage growth that are translocated into the macrophage.

Characterization of the Francisella tularensis subsp. novicida type IV pilus.

Francisella tularensis causes the disease tularaemia. Type IV pili (Tfp) genes are present in the genomes of all F. tularensis subspecies. We show that the wild-type F. tularensis subsp. novicida expresses pilus fibres on its surface, and mutations in the Tfp genes pilF and pilT disrupt pilus biogenesis. Mutations in other Tfp genes (pilQ and pilG) do not eliminate pilus expression. A mutation in pilE4 eliminates pilus expression, whereas mutations in the other pilin subunits pilE1-3 and pilE5 do not, suggesting that pilE4 is the major pilus structural subunit. The virulence regulator MglA is required for pilus expression, and it regulates the transcription of a putative Tfp glycosylation gene (FTN0431). However, MglA does not regulate transcription of pilF, pilT or pilE4, and a strain lacking FTN0431 still expresses pili; thus, it is unclear how MglA regulates pilus expression. Only pilF was also required for protein secretion, while pilE4 and pilT were not, indicating that there is very little overlap of the protein secretion/Tfp functions of the pil genes. The protein secretion component pilE1 was more important for in vitro intramacrophage growth and mouse virulence than the Tfp component pilE4. Our results provide the first genetic characterization of the novel Tfp system of F. tularensis.

Combined deletion of four Francisella novicida acid phosphatases attenuates virulence and macrophage vacuolar escape.

Francisella tularensis is a facultative intracellular pathogen and the etiologic agent of tularemia. It is capable of escape from macrophage phagosomes and replicates in the host cell cytosol. Bacterial acid phosphatases are thought to play a major role in the virulence and intracellular survival of a number of intracellular pathogens. The goal of this study was to delete the four primary acid phosphatases (Acps) from Francisella novicida and examine the interactions of mutant strains with macrophages, as well as the virulence of these strains in mice. We constructed F. novicida mutants with various combinations of acp deletions and showed that loss of the four Acps (AcpA, AcpB, AcpC, and histidine acid phosphatase [Hap]) in an F. novicida strain (DeltaABCH) resulted in a 90% reduction in acid phosphatase activity. The DeltaABCH mutant was defective for survival/growth within human and murine macrophage cell lines and was unable to escape from phagosome vacuoles. With accumulation of Acp deletions, a progressive loss of virulence in the mouse model was observed. The DeltaABCH strain was dramatically attenuated and was an effective single-dose vaccine against homologous challenge. Furthermore, both acpA and hap were induced when the bacteria were within host macrophages. Thus, the Francisella acid phosphatases cumulatively play an important role in intracellular trafficking and virulence.

Construction of targeted insertion mutations in Francisella tularensis subsp. novicida.

Francisella tularensis is one of the most deadly bacterial agents, yet most of the genetic determinants of pathogenesis are still unknown. We have developed an efficient targeted mutagenesis strategy in the model organism F. tularensis subsp. novicida by utilizing universal priming of optimized antibiotic resistance cassettes and splicing by overlap extension (SOE). This process enables fast and efficient construction of targeted insertion mutations in F. tularensis subsp. novicida that have characteristics of nonpolar mutations; optimized targeted mutagenesis strategies will promote the study of this mysterious bacterium and facilitate vaccine development against tularemia. Moreover the general strategy of gene disruption by PCR-based antibiotic resistance cassette insertion is broadly applicable to many bacterial species.

[NEMO Delta 4-10 deletion of NEMO gene in Chinese incontinentia pigmenti cases].

OBJECTIVE: Incontinentia pigmenti (IP) is a rare X-linked dominant disorder that affects ectodermal tissues. In IP, mutations in NEMO lead to the complete loss of NF-kB activation creating a susceptibility to cellular apoptosis in response to TNF-alpha. Recently, a second nonfunctional copy of the gene, Delta NEMO was identified, opposite in direction to NEMO. Almost 90% of IP whose gene mutation type had been recognized have a recurrent genomic deletion of exons 4-10 of the NEMO (IKK gamma) gene, called NEMO Delta 4-10, which is necessary to activate the NF-kB pathway. Therefore, PCR-based detection of the NEMO deletion is a diagnostic measurement for IP. This study sought to analyze the NEMO Delta 4-10 deletion in NEMO gene of Chinese IP cases. METHODS: Seven IP cases and part of their families totally 15 persons were enrolled in this study. The 7 IP cases were aged 41 days to 8 years. Among them 1 was male and 6 were female. Four cases had family history of IP, the other 3 were sporadic cases. Fifty healthy children without any congenital diseases were taken as normal control group. According to the gene characteristics of IP, by PCR measurement NEMO Delta 4-10 deletion in NEMO gene was tested with specific primers In2/JF3R, and NEMO Delta 4-10 deletion in pseudogene Delta NEMO was checked out by primers Rev-2/JF3R. RESULTS: Five out of the 7 tested cases (case 1, 2, 3, 4, and 6) showed NEMO Delta 4-10 deletion in NEMO gene. The mothers of case 1 and case 6, 1a and 6a, also suffered from this disease, and their results were just the same as their daughters. For pseudogene Delta NEMO only case 2 and case 4 were proved having NEMO Delta 4-10 deletion, while other cases and families had negative results. For normal control group, NEMO Delta 4-10 deletion was not found either in NEMO gene or in their pseudogene Delta NEMO. CONCLUSION: Incontinentia pigmenti in most cases were caused by NEMO Delta 4-10 deletion in NEMO gene.

Clofibrate and perfluorodecanoate both upregulate the expression of the pregnane X receptor but oppositely affect its ligand-dependent induction on cytochrome P450 3A23.

The pregnane X receptor (PXR) interacts with a vast array of structurally dissimilar chemicals and confers induction of several major types of drug metabolizing enzymes such as cytochrome P450s (CYP). We previously reported that the expression of PXR was markedly increased in rats treated with clofibrate and perfluorodecanoic acid (PFDA). The present study was undertaken to test the hypothesis that induced expression of PXR increases PXR ligand-dependent induction on CYP3A23. Rat hepatocytes were treated with clofibrate or PFDA individually, or along with PXR ligand pregnenolone 16alpha-carbonitrile (PCN), and the levels of PXR and CYP3A23 were determined by Western blots. Both clofibrate and PFDA markedly increased the expression of PXR with PFDA being more potent, and the induction was abolished by actinomycin D, an inhibitor for mRNA synthesis. As expected, PCN alone markedly induced the expression of CYP3A23. Interestingly, co-treatment with clofibrate enhanced the induction, whereas co-treatment with PFDA suppressed it. Clofibrate and PFDA represent multi-classes of chemicals called peroxisome proliferators including many therapeutic agents and industrial pollutants. The opposing effects of clofibrate and PFDA on the PCN-induced expression of CYP3A23 suggest that peroxisome proliferators likely increase the expression of PXR but differentially alter its ligand-dependent induction. The interaction between PXR inducer and ligand provides a novel mechanism on how functionally and structurally distinct chemicals cooperatively regulate the expression of xenobiotic-metabolizing enzymes and transporters.

Effects of EGF and bFGF on expression of microtubule-associated protein tau and MAP-2 mRNA in human umbilical cord mononuclear cells.

The aim of this study was to explore the regulatory effects of cytokines, such as EGF and bFGF, on expression of the neural-specific molecules tau and MAP2 mRNA in mononuclear cells (MNCs) derived from human umbilical cord blood (UCB). Phenotypic changes were monitored by inverse phase-contrast microscopy. Tau and MAP2 mRNA were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Tau and MAP2-positive cells were determined by immunocytochemistry. The expression of tau mRNA was negative in uncultured cells, but MAP2 mRNA was positive; in cultured cells, tau protein mRNA expression was positive, MAP2 mRNA expression was upregulated by EGF+bFGF, EGF and bFGF compared to the control group (no cytokines). EGF+bFGF had a greater effect on MAP2 mRNA expression than EGF or bFGF alone. The same upregulatory tendency was noted for tau mRNA expression. It is concluded that MNCs derived from human UCB cells may express some neural specific molecules that can be upregulated by cytokines, especially EGF and bFGF together.

Simultaneous substitution of phenylalanine-305 and aspartate-318 of rat pregnane X receptor with the corresponding human residues abolishes the ability to transactivate the CYP3A23 promoter.

The pregnane X receptor (PXR) is a key regulator on the expression of genes involved in the elimination of chemicals. As one of the most divergent members in the nuclear receptor family, PXR is activated in a highly species-dependent manner by certain chemicals. Pregnenolone 16alpha-carbonitrile (PCN), a glucocorticoid antagonist, efficaciously activates rodent but not human PXR. This study was undertaken to investigate the structural basis for PCN-mediated activation of rat PXR. A series of rat-human chimeric PXRs were prepared to gradually replace the ligand-binding domain of human PXR with the corresponding rat sequence at an increasing length of 20 residues. Cotransfection experiments established that region(306-326) acted as a transitional conjunction from none to full PCN responsive status. Site-directed mutagenesis study identified two residues (Phe-305 and Asp-318) that were critical in supporting PCN-mediated activation, and simultaneous substitution of both residues abolished the ability of rat PXR to transactivate the CYP3A23 promoter. In addition, substitutions on Phe-305, Asp-318, or both markedly reduced the basal transcriptional activity, and the reduction occurred with the CYP3A4 but not CYP3A23 promoter. Further study with CYP3A4 and CYP3A23 hybrid reporters demonstrated that the region harboring the distal PXR element in the CYP3A4 promoter mediated the repressive activity. PXR has been shown to interact with corepressors in the absence of ligand. The decreased responsiveness toward PCN and reduced basal transcriptional activity suggest that Phe-305 and Asp-318 are involved in both ligand-binding and corepressor interactions.

Desmethoxyyangonin and dihydromethysticin are two major pharmacological kavalactones with marked activity on the induction of CYP3A23.

Kava kava (Piper methysticum), an herbal remedy, is widely used for the treatment of mild to moderate cases of anxiety. The therapeutic activity is presumably achieved through multiple constituents called kavalactones. Recently, kava extracts were shown to induce CYP3A4 and activate human pregnane X receptor (PXR). This study was undertaken to test the ability of purified kavalactones to induce CYP3A23 and activate PXR. Rat hepatocytes were treated with desmethoxyyangonin, dihydrokawain, dihydromethysticin, kawain, methysticin, or yangonin, and the expression of CYP3A23 was monitored. Among the kavalactones, only desmethoxyyangonin and dihydromethysticin markedly induced the expression of CYP3A23 (approximately 7-fold). A similar magnitude of induction was detected with combined six kavalactones at a noninductive concentration when individually used. The induced expression, however, was markedly reduced or completely abolished if dihydromethysticin, desmethoxyyangonin, or both were excluded from the mixtures. Interestingly, regardless of whether dihydromethysticin or desmethoxyyangonin was used alone or together with other kavalactones, similar amounts of total kavalactones were needed to produce comparable induction, suggesting that the inductive activity of dihydromethysticin and desmethoxyyangonin is additively/synergistically enhanced by other kavalactones. In addition, treatment with dihydromethysticin, desmethoxyyangonin, or pregnenolone 16alpha-carbonitrile (PCN) markedly increased the levels of CYP3A23 mRNA, and inhibition of mRNA synthesis abolished the induction. In contrast to PCN, dihydromethysticin and desmethoxyyangonin only slightly activated rat or human PXR. These findings suggest that the induction of CYP3A23 by dihydromethysticin and desmethoxyyangonin involves transcription activation, probably through a PXR-independent or PXR-involved indirect mechanism.

DNA binding, but not interaction with Bmal1, is responsible for DEC1-mediated transcription regulation of the circadian gene mPer1.

DEC1 (differentially expressed in chondrocytes 1) and DEC2 are E-box-binding transcription factors and exhibit a circadian expression pattern. Recently, both proteins were found to repress the Clock/Bmal1-activated E-box promoters (e.g. mPer1). Yeast two-hybrid assay detected interactions between Bmal1 and DECs. It was hypothesized that DEC-mediated repression on the mPer1 promoter is achieved by binding to E-box elements and interacting with Bmal1. In the present study, we report that E-box binding rather than Bmal1 interaction is responsible for the observed repression. In the absence of Clock/Bmal1, both DEC1 and DEC2 markedly repressed the mPer1 promoter reporter; however, DNA-binding mutants showed no repressive activity. Similarly, DEC1, but not its DNA-binding mutants, repressed the Clock/Bmal1-induced activation. In addition, DEC1(R58P), a DNA-binding mutant with Bmal1 interactivity, repressed neither the mPer1 reporter directly nor the Clock/Bmal1-induced activation, providing direct evidence that DNA binding, rather than Bmal1 interactions, is responsible for the repression on the mPer1 promoter. Furthermore, disruption of the Sp1 site in the proximal promoter of mPer1 increased the repression of DEC1 proteins. Previous studies with mouse DEC2 showed that this factor interacts with Sp1. These findings suggest that DEC proteins regulate the expression of mPer1 through E-box binding and Sp1 interaction. Alterations on circadian systems are increasingly recognized as important risk factors for disease initiation and progression, and the expression of Dec genes is rapidly induced by environmental stimuli and is highly increased in tumour tissues. Therefore de-regulated expression of DEC genes probably alters normal circadian rhythms and contributes significantly to the pathogenesis of many diseases including cancer.

Intramolecular disulfide bonds are required for folding hydrolase B into a catalytically active conformation but not for maintaining it during catalysis.

Carboxylesterases represent a large class of hydrolytic enzymes that are involved in lipid metabolism, pharmacological determination, and detoxication of organophosphorus pesticides. These enzymes have several notable structural features including two intramolecular disulfide bonds. This study was undertaken to test the hypothesis that the disulfide bonds are required during catalysis by stabilizing the catalytically active conformation. Hydrolase B, a rat liver microsomal carboxylesterase, was reduced by dithiothreitol, electrophoretically separated and assayed for hydrolysis. Contrary to the hypothesis, reduced hydrolase B was as active as the native enzyme on the hydrolysis of 1-naphthylacetate, and sulfhydryl alkylation following reduction caused no changes in the hydrolytic activity. Interestingly, substitution of a disulfide bond-forming cysteine with an alanine caused marked reduction or complete loss of the catalytic activity, suggesting that disulfide bond formation plays a role in the biosynthetic process of hydrolase B. In support of this notion, refolding experiments restored a significant amount of hydrolytic activity when hydrolase B was unfolded with urea alone. In contrast, little activity was restored when unfolding was performed in the presence of reducing agent dithiothreitol. These results suggest that formation of the disulfide bonds plays a critical role in folding hydrolase B into the catalytically active conformation, and that the disulfide bonds play little role or function redundantly in maintaining this conformation during catalysis.

Campylobacter jejuni from patients with Guillain-Barré syndrome preferentially expresses a GD(1a)-like epitope.

GM(1)- and GD(1a)-like ganglioside mimicry in Campylobacter jejuni lipooligosaccharide (LOS) is considered to be involved in the pathogenesis of Campylobacter-induced Guillain-Barré syndrome (GBS). Compared with gastroenteritis-related isolates, GBS-related C. jejuni isolates were strongly associated with the expression of GD(1a)-like mimicry. The presence of a few genes involved in LOS ganglioside mimicry, cst-II, cgtA, and cgtB, was also associated with GBS-related strains. GD(1a)-like epitope expression may be an important virulence phenotype associated with the risk of developing GBS following campylobacter infection.