Rapid discrimination of Shigella spp. and Escherichia coli via label-free surface enhanced Raman spectroscopy coupled with machine learning algorithms.

Shigella and enterotoxigenic Escherichia coli (ETEC) are major bacterial pathogens of diarrheal disease that is the second leading cause of childhood mortality globally. Currently, it is well known that Shigella spp., and E. coli are very closely related with many common characteristics. Evolutionarily speaking, Shigella spp., are positioned within the phylogenetic tree of E. coli. Therefore, discrimination of Shigella spp., from E. coli is very difficult. Many methods have been developed with the aim of differentiating the two species, which include but not limited to biochemical tests, nucleic acids amplification, and mass spectrometry, etc. However, these methods suffer from high false positive rates and complicated operation procedures, which requires the development of novel methods for accurate and rapid identification of Shigella spp., and E. coli. As a low-cost and non-invasive method, surface enhanced Raman spectroscopy (SERS) is currently under intensive study for its diagnostic potential in bacterial pathogens, which is worthy of further investigation for its application in bacterial discrimination. In this study, we focused on clinically isolated E. coli strains and Shigella species (spp.), that is, S. dysenteriae, S. boydii, S. flexneri, and S. sonnei, based on which SERS spectra were generated and characteristic peaks for Shigella spp., and E. coli were identified, revealing unique molecular components in the two bacterial groups. Further comparative analysis of machine learning algorithms showed that, the Convolutional Neural Network (CNN) achieved the best performance and robustness in bacterial discrimination capacity when compared with Random Forest (RF) and Support Vector Machine (SVM) algorithms. Taken together, this study confirmed that SERS paired with machine learning could achieve high accuracy in discriminating Shigella spp., from E. coli, which facilitated its application potential for diarrheal prevention and control in clinical settings. Graphical abstract.

Effect of CXCL12/CXCR4 signaling on neuropathic pain after chronic compression of dorsal root ganglion.

Neuropathic pain is a complex, chronic pain state that often accompanies tissue damage, inflammation or injury of the nervous system. However the underlying molecular mechanisms still remain unclear. Here, we showed that CXCL12 and CXCR4 were upregulated in the dorsal root ganglion (DRG) after chronic compression of DRG (CCD), and some CXCR4 immunopositive neurons were also immunopositive for the nociceptive neuronal markers IB4, TRPV1, CGRP, and substance P. The incidence and amplitude of CXCL12-induced Ca2+ response in primary sensory neurons from CCD mice was significantly increased compared to those from control animals. CXCL12 depolarized the resting membrane potential, decreased the rheobase, and increased the number of action potentials evoked by a depolarizing current at 2X rheobase in neurons from CCD mice. The mechanical and thermal hypernociception after CCD was attenuated by administration of a CXCR4 antagonist AMD3100. These findings suggest that CXCL12/CXCR4 signaling contributes to hypernociception after CCD, and targeting CXCL12/CXCR4 signaling pathway may alleviate neuropathic pain.

Development and application of a novel reverse transcription real-time PCR method for miR-499 quantification.

OBJECTIVES: MicroRNAs (miRNAs) are endogenous small RNAs of 21-25 nucleotides that can pair with sites in 3' untranslated regions in mRNAs of protein-coding genes to downregulate their expression. Recently, miR-499 and other miRNAs released in circulating blood have been reported as promising biomarkers for acute myocardial infarction (AMI). In the present study, we developed a novel reverse-transcription real-time PCR assay for human miR-499 quantification. DESIGN AND METHODS: miR-499 was reverse-transcribed with a 3' portion-specific primer into cDNAs. The cDNAs were further extended with overlap PCR. The extended cDNAs were determined by quantitative, real-time PCR. Synthetic miR-499 was put into the RT reaction over an optimal range to generate standard curves for absolute quantification of miR-499. RESULTS: In the presence of 0.0001 amol/µL to 1.0×106 amol/µL of synthetic miR-499, we observed a linear correlation (R²=0.999) between the logarithm of the amount of input RNA and the CT value. The miR-499 was reliably measured at a detection limit of 0.0001 amol/µL. The miR-499 measurements in spiked plasma samples indicated excellent correlation between the novel qRT PCR and classic stem loop qRT PCR. The qRT PCR analysis demonstrated that miR-499 was detected with higher levels in plasma from the patient with AMI in acute phase (AMI) compared with those from the control groups (P<0.001). CONCLUSIONS: We developed a novel reverse-transcription real-time PCR assay for human miR-499 quantification. The good reproducibility and wide linearity range may permit more use of it in the quantification of other human miRNAs in future.

Human PIH1 associates with histone H4 to mediate the glucose-dependent enhancement of pre-rRNA synthesis.

Ribosome biogenesis is critical in the growth of eukaryotic cells, in which the synthesis of precursor ribosomal RNA is the first and rate-limiting step. Here, we show that human PIH1 domain-containing protein 1 (PIH1) interacts directly with histone H4 and recruits the Brg1-SWI/SNF complex via SNF5 to human rRNA genes. This process is likely involved in PIH1-dependent DNase I-hypersensitive chromatin remodeling at the core promoter of the rRNA genes. PIH1 mediates the occupancy of not only the Brg1 complex but also the Pol I complex at the core promoter and enhances transcription initiation of rRNA genes. Additionally, the interaction between PIH1 and H4K16 expels TIP5, a component of the silencing nucleolar remodeling complex (NoRC), from the core region, suggesting that PIH1 is involved in the derepression of NoRC-silenced rRNA genes. These data indicate that PIH1 is a positive regulator of human rRNA genes and is of great importance for the recovery of human cells from nutrient starvation and the transition to glucose-induced exponential growth in vivo.

[Prenatal diagnosis of ß-thalassaemia using cell-free fetal DNA in maternal plasma].

OBJECTIVE: To investigate the clinical feasibility of cell-free fetal DNA (cffDNA)-based noninvasive prenatal diagnosis of ß-thalassemia. METHODS: Nine samples of amniotic fluid were obtained to detect the 8 common and 9 relatively rare mutation sites of ß-thalassaemia in Guangdong Province. The maternal blood samples were also collected for extracting and purification of the cffDNA, and a duplex PCR was performed using 3 pairs of primers and the fetal ß-globin genotype was analyzed by reverse dot-blot hybridization. RESULTS: Among the 9 cases, 5 showed fetal genotypes of ß-thalassemia inherited from the father by examination of the amniotic fluid, and 2 fetuses were identified to have ß-thalassemia genes inherited from the father determined based on the cffDNA in the maternal blood. CONCLUSIONS: The cffDNA-based noninvasive prenatal diagnosis is feasible for ß-thalassemia, but the contamination of the maternal background DNA results in a low detection rate.

Homeostatic interactions between MEKK3 and TAK1 involved in NF-kappaB signaling.

Several members of the mitogen-activated protein kinase kinase kinase (MAP3K) family including MEKK3 and TGFbeta-activating kinase (TAK1) play nonredundant roles in activation of the NF-kappaB transcription factor. However, the mechanism by which MEKK3 mediates NF-kappaB signaling is not fully understood. In this report we investigate the association of murine MEKK3 with other proteins and their roles in NF-kappaB activation. Using tandem affinity purification TAK1 was identified as an endogenous protein that interacts with MEKK3. MEKK3-TAK1 interactions were confirmed by fluorescence resonance energy transfer and coimmunoprecipitation. MEKK3-TAK1 complexes contain non-phosphorylated forms of both molecules. Expression of non-phosphorylated TAK1 interferes with MEKK3 phosphorylation and NF-kappaB reporter activity induced by transient MEKK3 expression or TNFalpha stimulation. Addition of TAB1 facilitates TAK1 autophosphorylation and reverses the inhibitory effects of TAK1 on MEKK3 phosphorylation and NF-kappaB signal transduction in human 293 cells and TAK1 deficient mouse embryonic fibroblasts. The data provide insights into the homeostatic interactions that maintain basal NF-kappaB levels by holding the enzymes MEKK3 and TAK1 in their inactive state.

High-fat feeding reduces endothelium-dependent vasodilation in rats: differential mechanisms for saturated and unsaturated fatty acids?

1. Chronic feeding with a high-fat diet can cause metabolic syndrome in rodents similar to humans, but the role of saturated versus unsaturated fats in vascular tension remains unclear. 2. The present study shows that rats on a diet rich in either saturated or unsaturated fat had higher blood pressure compared with chow-fed rats (approximately 130 vs 100 mmHg, respectively), along with hyperlipidaemia and insulin resistance. Compared with responses of phenylephrine-preconstricted artery segments from chow-fed rats, vasorelaxation of isolated renal arteries from high-fat fed rats was reduced substantially (> 50%) in response to acetylcholine (0.01-10 micromol/L) and moderately to nitroprusside (>or=1 micromol/L) at low concentrations. Acetylcholine-induced vasorelaxation of arteries from high-fat fed rats was also more sensitive to inhibition by the nitric oxide (NO) synthase inhibitors NG-nitro-L-arginine and methylene blue. 3. In human umbilical vein endothelial cells, the production of NO and endothelin-1 was significantly inhibited by unsaturated fatty acids. In comparison, saturated fatty acids stimulated endothelin-1 production without altering NO production. 4. The data indicate that both saturated and unsaturated high-fat feeding may result in an increase in blood pressure owing to reduced endothelium-dependent vasorelaxation in the arterial system. The impaired endothelium-dependent vasorelaxation induced by saturated and unsaturated fatty acids may involve different mechanisms.