Bactericidal Efficacy of the Combination of Maresin-like Proresolving Mediators and Carbenicillin Action on Biofilm-Forming Burn Trauma Infection-Related Bacteria.

Biofilm-associated bacterial infections are the major reason for treatment failure in many diseases including burn trauma infections. Uncontrolled inflammation induced by bacteria leads to materiality, tissue damage, and chronic diseases. Specialized proresolving mediators (SPMs), including maresin-like lipid mediators (MarLs), are enzymatically biosynthesized from omega-3 essential long-chain polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), by macrophages and other leukocytes. SPMs exhibit strong inflammation-resolving activities, especially inflammation provoked by bacterial infection. In this study, we explored the potential direct inhibitory activities of three MarLs on Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa and Escherichia coli) bacteria in their biofilms that are leading bacteria in burn trauma-related infections. We also examined the effects of MarLs on the bactericidal activities of a typical broad-spectrum antibiotic, carbenicillin (carb), on these bacteria in their preformed biofilms. The results revealed that MarLs combined with carbenicillin can inhibit the survival of Gram-positive and Gram-negative bacteria in their biofilms although MarLs alone did not exhibit bactericidal activity. Thus, our findings suggest that the combination of MarLs and carbenicillin can lower the antibiotic requirements to kill the bacteria in preformed biofilms.

Thrombospondin 4, a mediator and candidate indicator of pain.

Pain is the most common symptom for which patients seek medical attention. Existing treatments for pain control are largely ineffective due to the lack of an accurate way to objectively measure pain intensity and a poor understanding of the etiology of pain. Thrombospondin 4(TSP4), a member of the thrombospondin gene family, is expressed in neurons and astrocytes and induces pain by interacting with the calcium channel alpha-2-delta-1 subunit (Cavα2δ1). In the present study we show that TSP4 expression level correlates positively with pain intensity, suggesting that TSP4 could be a novel candidate of pain indicator. Using RNAi-lentivirus (RNAi-LV) to knock down TSP4 both in vivo and in vitro, together with electrophysiological experiments involving paired patch-clamp recordings of evoked action potentials and post-synaptic currents in cultured neurons, we found that TSP4 contributes to the development of bone cancer pain, neuropathic pain, and inflammatory pain. This effect is mediated by regulation of neuron excitability via inhibition of synapsin I (Syn I) and modulation of excitatory and inhibitory presynaptic transmission via regulation of vesicular glutamate transporter 2(Vglut2), vesicular GABA transporter (VGAT), and glutamate decarboxylase (GAD) expression. The present study provides a replicable, predictive, valid indicator of pain and demonstrated the underlying molecular and electrophysiological mechanisms by which TSP4 contributes to pain.

SLC17A9 as a prognostic biomarker correlated with immune infiltrates in human non-small cell lung cancer.

The vesicular nucleotide transporter (SLC17A9) has been overexpressed in various cancers. Nonetheless, little is known about its influence on non-small cell lung cancer (NSCLC), including human lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Integrative bioinformatics analysis was performed to investigate the prognostic significance and underlying mechanisms of SLC17A9 in patients with NSCLC. Here, we found that SLC17A9 up-regulation was significantly correlated with overall survival in LUAD and LUSC (P < 0.05). Gene set enrichment analysis and protein-protein interaction results revealed that SLC17A9 up-regulation was linked to metabolic process, the hallmark of MYC targets, DNA repair, coagulation and complement. SLC17A9 expression was negatively associated with overall survival and positively related to most LUSC immune cells and immunoinhibitor (20/23), particularly immuno A2aR, PD-1, and CTLA-4 (P < 0.001). High SLC17A9 was associated with infiltrating levels of B cells, CD4+ T cells, M1 macrophages, and T cell exhaustion checkpoints such as PD-1, CTLA4, and LAG3 in LUAD. Moreover, Real-time PCR, MTS assay, EdU assay, ATP production assays and cell cycle analysis were performed to validate SLC17A9 knockdown in LUAD cells. SLC17A9 knockdown significantly inhibited cell proliferation and ATP levels by affecting P2X1, Cytochrome C, and STAT3 expression in lung cancer cells. In conclusion, the present study suggested that SLC17A9 could potentially serve as a prognostic biomarker and correlated with immune infiltrates in LUAD and LUSC.

Neuroligins facilitate the development of bone cancer pain via regulating synaptic transmission: an experimental study.

BACKGROUND: The underlying mechanism of chronic pain involves the plasticity in synaptic receptors and neurotransmitters. This study aimed to investigate potential roles of Neuroligins (NLs) within the spinal dorsal horn of rats in a newly established Bone Cancer Pain (BCP) model. The objective was to explore the mechanism of neuroligin involved in the occurrence and development of bone cancer pain. METHODS: Using our rat BCP model, we assessed pain hypersensitivity over time. Quantitative real-time polymerase chain reaction and Western blot analysis were performed to investigate NL expression, and NLs were overexpressed in the rat spinal cord using lentiviral vectors. Immunofluorescence staining and whole-cell patch-clamp recordings were deployed to investigate the role of NLs in the development of BCP. RESULTS: We observed reduced expression levels of NL1 and NL2, but not of NL3, within the rat spinal cord, which were found to be associated with and essential for the development of BCP in our model. Accordingly, NL1 or NL2 overexpression in the spinal cord alleviated mechanical hypersensitivity of rats. Electrophysiological experiments indicated that NL1 and NL2 are involved in BCP via regulating γ-aminobutyric acid-ergic interneuronal synapses and the activity of glutamatergic interneuronal synapses, respectively. CONCLUSIONS: Our observations unravel the role of NLs in cancer-related chronic pain and further suggest that inhibitory mechanisms are central features of BCP in the spinal dorsal horn. These results provide a new perspective and basis for subsequent studies elucidating the onset and progression of BCP.

Differential epitranscriptome and proteome modulation in the brain of neonatal mice exposed to isoflurane or sevoflurane.

BACKGROUND: Repeated neonatal exposure to anesthetics may disturb neurodevelopment and cause neuropsychological disorders. The m6A modification participates in the gene regulation of neurodevelopment in mouse fetuses exposed to anesthetics. This study aims to explore the underlying molecular mechanisms of neurotoxicity after early-life anesthesia exposure. METHODS: Mice were exposed to isoflurane (1.5%) or sevoflurane (2.3%) for 2 h daily during postnatal days (PND) 7-9. Sociability, spatial working memory, and anxiety-like behavior were assessed on PND 30-35. Synaptogenesis, epitranscriptome m6A, and the proteome of brain regions were evaluated on PND 21. RESULTS: Both isoflurane and sevoflurane produced abnormal social behaviors at the juvenile age, with different sociality patterns in each group. Synaptogenesis in the hippocampal area CA3 was increased in the sevoflurane-exposed mice. Both anesthetics led to numerous persistent m6A-induced alterations in the brain, associated with critical metabolic, developmental, and immune functions. The proteins altered by isoflurane exposure were mainly associated with epilepsy, ataxia, and brain development. As for sevoflurane, the altered proteins were involved in social behavior. CONCLUSIONS: Social interaction, the modulation patterns of the m6A modification, and protein expression were altered in an isoflurane or sevoflurane-specific way. Possible molecular pathways involved in brain impairment were revealed, as well as the mechanism underlying behavioral deficits following repeated exposure to anesthetics in newborns.

Schnurri-2 Promotes the Expression of Excitatory Glutamate Receptors and Contributes to Neuropathic Pain.

Neuropathic pain is a type of chronic pain with complex mechanisms, and current treatments have shown limited success in treating patients suffering from chronic pain. Accumulating evidence has shown that the pathogenesis of neuropathic pain is mediated by the plasticity of excitatory neurons in the dorsal horn of the spinal cord, which provides insights into the treatment of hyperalgesia. In this study, we found that Schnurri-2 (Shn2) was significantly upregulated in the L4-L6 segments of the spinal cord of C57 mice with spared nerve injury, which was accompanied by an increase in GluN2D subunit and glutamate receptor subunit 1 (GluR1) levels. Knocking down the expression of Shn2 using a lentivirus in the spinal cord decreased the GluN2D subunit and GluR1 levels in spared nerve injury mice and eventually alleviated mechanical allodynia. In summary, Shn2 regulates neuropathic pain, promotes the upregulation of GluN2D in glutamatergic neurons and increases the accumulation of GluR1 in excitatory neurons. Taken together, our study provides a new underlying mechanism for the development of neuropathic pain.

P-Rex2 mediation of synaptic plasticity contributes to bone cancer pain.

Bone cancer pain (BCP) seriously affects the quality of life; however, due to its complex mechanism, the clinical treatment was unsatisfactory. Recent studies have showed several Rac-specific guanine nucleotide exchange factors (GEFs) that affect development and structure of neuronal processes play a vital role in the regulation of chronic pain. P-Rex2 is one of GEFs that regulate spine density, and the present study was performed to examine the effect of P-Rex2 on the development of BCP. Tumor cells implantation induced the mechanical hyperalgesia, which was accompanied by an increase in spinal protein P-Rex2, phosphorylated Rac1 (p-Rac1) and phosphorylated GluR1 (p-GluR1), and number of spines. Intrathecal injection a P-Rex2-targeting RNAi lentivirus relieved BCP and reduced the expression of P-Rex2, p-Rac1, p-GluR1, and number of spines in the BCP mice. Meanwhile, P-Rex2 knockdown reversed BCP-enhanced AMPA receptor (AMPAR)-induced current in dorsal horn neurons. In summary, this study suggested that P-Rex2 regulated GluR1-containing AMPAR trafficking and spine morphology via Rac1/pGluR1 pathway is a fundamental pathogenesis of BCP. Our findings provide a better understanding of the function of P-Rex2 as a possible therapeutic target for relieving BCP.

NWD1 facilitates synaptic transmission and contributes to neuropathic pain.

Neuropathic pain is the most common symptom for which patients seek medical attention. Existing treatments to control pain are largely ineffective because of poor understanding the underlying mechanisms. Synaptic plasticity is fundamental to the spinal sensitivity of neuropathic pain. In the present study, we showed that SNL induced significant allodynia and hyperalgesia as well as upregulation of Nwd1 and GluN2B, which were reversed by knockdown of NWD1. Electrophysiological experiments demonstrated that SNL enhanced synaptic transmission, which was prevented by knockdown of NWD1. In vitro experiments showed that knockdown of NWD1 inhibited dendritic growth and synaptogenesis. Taken together, our results suggest that NWD1 enhances synaptic transmission and contributes to the development of neuropathic pain by enhancing GluN2B synaptic expression and anchor and promoting excitatory synaptogenesis.

Neuroligin1 Contributes to Neuropathic Pain by Promoting Phosphorylation of Cofilin in Excitatory Neurons.

Neuropathic pain is a kind of chronic pain that remains difficult to treat due to its complicated underlying mechanisms. Accumulating evidence has indicated that enhanced synaptic plasticity of nociceptive interneurons in the superficial spinal dorsal horn contributes to the development of neuropathic pain. Neuroligin1 (NL1) is a type of excitatory postsynaptic adhesion molecule, which can mediate excitatory synaptic activity, hence promoting neuronal activation. Vglut2 is the most common marker of excitatory glutamatergic neurons. To explore the role of NL1 in excitatory neurons in nociceptive regulation, we used transgenic mice with cre recombinase expression driven by the Vglut2 promoter combined with viral vectors to knockdown the expression of NL1 in excitatory neurons in the spinal dorsal horn. We found that NL1 was upregulated in the L4-L6 spinal dorsal horn in Vglut2-cre+/- mouse subjected to spared nerve injury (SNI). Meanwhile, the expression of phosphorylated cofilin (p-cofilin) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit 1 (GluR1) was also increased. Spinal microinjection of a cre-dependent NL1-targeting RNAi in Vglut2-cre+/- mouse alleviated the neuropathic pain-induced mechanical hypersensitivity and reduced the increase in p-cofilin and GluR1 caused by SNI. Taken together, NL1 in excitatory neurons regulates neuropathic pain by promoting the SNI-dependent increase in p-cofilin and GluR1 in the spinal dorsal horn. Our study provides a better understanding of the role of NL1 in excitatory neurons, which might represent a possible therapeutic target for alleviating neuropathic pain.

Shank3 contributes to neuropathic pain by facilitating the SNI-dependent increase of HCN2 and the expression of PSD95.

Neuropathic pain is a very complex chronic pain state, the detailed molecular mechanisms of which remain unclear. In the present study, Shank3 was found to play an important role in neuropathic pain in rats following spared nerve injury (SNI). Shank3 was upregulated in the spinal dorsal horn of rats subjected to SNI, and mechanical hypersensitivity to noxious stimuli in these rats could be alleviated by knock down of Shank3. Shank3 also interacted with hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) and promoted the expression of HCN2 in central neurons of the spinal dorsal. Together with the SNI-dependent increase of HCN2, we also found that the postsynaptic protein of excitatory synapse (PSD95) was increased in rats following SNI. Taken together, our results showed that Shank3 modulated neuropathic pain by facilitating the SNI-dependent increase of HCN2 and the expression of PSD95 in spinal dorsal horn neurons. Our findings revealed new synaptic remodeling mechanisms linking Shank3 with neuropathic pain.

Virus Injection to the Pituitary via Transsphenoidal Approach and the Innervation of Anterior and Posterior Pituitary of Rat.

The theory holds that the anterior pituitary in mammals receives humoral regulation. Previous studies have reported that the pars distalis of the anterior pituitary of several mammalian species contains substance P-, calcitonin gene-related peptide (CGRP)-, and galanin-like immunoreactive nerve fibers, but the origins of these nerve fibers are unclear. Removal of the pituitary gland, also called hypophysectomy, involves methods that access the pituitary gland via the transauricular or parapharyngeal pathways. However, these methods are not applicable for viral tracer injection to investigate the innervation of the anterior pituitary. The transauricular technique leads to inaccuracies in locating the pituitary gland, while the parapharyngeal approach causes high mortality in animals. Here, we introduce a protocol that accesses the pituitary gland in the rat via the transsphenoidal pathway. This method imitates surgical manipulations such as endotracheal intubation and sphenoid bone drilling, which involve the use of custom-made devices. Using the transsphenoidal pathway greatly improves the survival rate of rats because no additional dissection of blood vessels and nerves is required. Moreover, the pituitary gland can be viewed clearly and directly during the operation, making it possible to accurately inject pseudorabies virus (PRV) 152-expressing enhanced green fluorescent protein (EGFP) into the anterior or posterior pituitary, respectively. After injecting PRV 152 into the anterior pituitary, we found no evidence of direct innervation of the anterior pituitary in the rat brain. However, PRV 152 injection into the posterior pituitary revealed retrograde transneuronal cell bodies in many brain areas, including the CA1 field of the hippocampus, the basolateral amygdaloid nucleus, posterior part (BLP), the arcuate hypothalamic nucleus (Arc), the dorsal portion of the dorsomedial hypothalamic nucleus (DMD), the suprachiasmatic nucleus (SCh), and the subfornical organ (SFO). In the present study, we provide a description of a possible model of hypophysectomy or pituitary injection, and identify brain regions involved in regulating the rat pituitary gland using transneuronal retrograde cell body labeling with PRV.

Visualization of gene therapy with a liver cancer-targeted adeno-associated virus 3 vector.

Background: To evaluate the feasibility of a self-complementing recombinant adeno-associated virus 3 (scrAAV3) vector targeting liver cancer and non-invasively monitor gene therapy of liver cancer. Materials and methods: An scrAAV3-HSV1-TK-kallistatin (ATK) gene drug was constructed, which contained the herpes virus thymidine kinase (HSV1-TK) reporter gene and human endogenous angiogenesis inhibitor (kallistatin) gene for non-invasive imaging of gene expression. Subcutaneous xenografted tumors of hepatoma in nude mice were generated for positron emission tomography/computed tomography (PET/CT) imaging. The ATK group was injected with the ATK gene through the tail vein, and an imaging agent was injected 2 weeks later. PET/CT imaging was performed at 1 hour after injection of the imaging agent. The control group was injected with phosphate-buffered saline at the same volume as the ATK gene drug. HE staining is used for pathological observation of tumor sections. HSV1-TK and kallistatin expression was identified by immunofluorescence, real-time quantitative PCR, and western blotting. Results: Radioactivity on PET/CT images was significantly higher in the ATK group compared with the control group. 18F-FHBG uptake values of left forelegs in ATK and control groups were 0.591±0.151% and 0.017 ± 0.011% ID/g (n=5), respectively (P<0.05). After injection of the ATK gene drug, mRNA and protein expression of HSV1-TK and kallistatin in subcutaneous xenograft tumors was detected successfully. In vitro analysis demonstrated significant differences in the expression of HSV1-TK and kallistatin between ATK and control groups (P<0.05). Conclusions: The scrAAV3 vector has a strong liver cancer-targeting ability, and the ATK gene drug can be used for targeted and non-invasive monitoring of liver cancer gene therapy.

Characterization of aminopeptidase encoding gene anp-1 and its association with development in Caenorhabditis elegans.

BACKGROUND: Aminopeptidases play important roles in various biological processes in nematodes including growth, development and reproduction. Although the aminopeptidases have been shown to regulate reproduction in Caenorhabditis elegans (C. elegans), the role of aminopeptidases in development and aging has not been reported. This study focused on the function of aminopeptidase AlaNyl aminopeptidase 1 (ANP-1) on development in C. elegans. METHODS: In the present study, we reported the identification of ANP-1 in C. elegans along with sequence analysis and its functional expression and characterization. The phenotype changes were observed when anp-1 mutated. Then, differential expression genes (DEGs) between wild type strain (N2) and anp-1 deletion strain (RB804) were identified using transcriptome sequencing method. Finally, DEGs were verified by qRT-PCR assay. RESULTS: Our observations suggested that anp-1 mutation induced small body size in the L4/young adult stage of C. elegans, however, there was no difference between N2 and RB804 in adult stage. Moreover, deletion of anp-1 resulted in shortening lifespan and laying fewer eggs. DEGs (184 genes) were observed between N2 groups and RB804 groups by transcriptome sequencing. According to GO annotations and KEGG enrichment analysis, these DEGs play vital roles in development regulation in C. elegans. These data demonstrate ANP-1 participates in development and aging of C. elegans and will considerably contribute to the existing knowledge of aminopeptidase function in C. elegans.

Darkness increases the population growth rate of the poultry red mite Dermanyssus gallinae.

BACKGROUND: The poultry red mite (PRM), Dermanyssus gallinae, is one of the most economically deleterious ectoparasites affecting egg-laying hens worldwide. It may be possible to control D. gallinae populations by manipulating lighting regimes within poultry units. However, no studies have clearly shown the effects of darkness on the population growth rate of D. gallinae. METHODS: The effect of darkness on the population growth rate of D. gallinae was investigated, together with the first description of the molecular identity of the mite from China. Mite variables under two lighting regimens (1:23 h L:D and 12:12 h L:D) were compared, including number of mites and eggs, survival and feeding rates, engorgement, oviposition, hatchability and the life-cycle of D. gallinae. RESULTS: The results showed that the number of mites (13,763 ± 956) and eggs (5424 ± 317) in the rearing system with prolonged darkness of 1:23 h L:D at 4th week were 2.4- and 3.6-fold higher than those under a conventional lighting regimen of 12:12 h L:D, respectively. The feeding rates of mites under prolonged darkness ranged from 36.7 ± 1.1% to 52.0 ± 7.0%, which were significantly higher than those under conventional lighting regimen (ranging from 22.6 ± 1.9% to 37.3 ± 1.6%). The mean weight of engorged females (0.26 ± 0.01 mg) and the mean number of eggs per female (on average 5.87 ± 0.36) under prolonged darkness were significantly higher than those under conventional lighting regimen (0.22 ± 0.01 mg and 3.62 ± 0.31, respectively). However, the survival rate ranging from 98.07 ± 0.10% to 98.93 ± 0.19%, hatchability of 97.93 ± 0.01% and the life-cycle of D. gallinae (9 days) was not affected by the lighting period. CONCLUSIONS: Our findings demonstrated that prolonged darkness significantly promoted the proliferation levels of D. gallinae, resulting in increased number of mites and eggs in the rearing system. The promoted population growth of D. gallinae was found to be related to the increased feeding rate, engorgement level and oviposition level of mites under prolonged darkness. The egg hatchability, the survival rates and the duration of life-cycle of D. gallinae were not affected by the light regimes.

Combination of active behaviors and passive structures contributes to the cleanliness of housefly wing surfaces: A new insight for the design of cleaning materials.

Evolutionary pressure has pushed many extant plants and animals to develop micro/nanostructures on their surfaces to keep them clean. These structures have become ideal models for bio-inspired design. Although microstructures on biological surfaces have been widely studied, little attention has been paid to the combined role of microstructures and animal's active cleaning behaviors in keeping their surfaces clean. In this study, we explored the relationship between these micro/nanostructures and wettability as well as the role of the housefly cleaning behaviors in keeping their wings clean. Hierarchical structures consisting of microscale macrotrichias with nanoscale grooves on the wings were observed under scanning electron microscope. The wings were hydrophobic (CA = 133.3°) but with high adhesion to water (CAH = 87.5°), indicating that they were non-self-cleaning surfaces. Macroscale droplets standing on the wings could be best described as being in a transitional wetting state between Wenzel and Cassie-Baxter states due to the presence of the nanoscale grooves, which increased the resistance to water penetration. The hydrophobicity decreased (CA = 109.9°) when the nanostructures were removed by coating the wings with a thick layer of polydimethylsiloxane (PDMS). The houseflies could highly efficiently remove the microscale droplets atop the macrotrichias, and reduce bacterial contamination on their wings through grooming and flutter activities. These active cleaning behaviors could offset the absence of self-cleaning properties and play a key role in keeping the wings clean. The results indicate that housefly wings could be used as a template for the design of special functional surfaces. The present findings not only improve our understanding of the wettability and cleaning properties of natural surfaces, but also provide important insights into the design of bio-inspired materials.

Housefly larvae (Musca domestica) significantly accelerates degradation of monensin by altering the structure and abundance of the associated bacterial community.

Vermicomposting of livestock manure using housefly larvae is a promising biotechnology for waste reduction and control of antibiotic pollution. Monensin (MON), an ionophore polyether antibiotic (IPA), is widely used in broiler feed to control coccidiosis. However, MON residues in litter have become a major source of pollution in the environment. In this work, we studied the efficiency of housefly larvae (Musca domestica) on monensin attenuation during a 12-day laboratory scale vermicomposting experiment. We observed a 94.99% reduction in MON concentration after four days in treatment groups, while it took twelve days to remove more than 94.71% of MON in the control group. We found that the bacterial community composition of the substrate was reshaped by housefly larvae. From the treatment groups, three MON-degrading bacterial strains were isolated and identified as Acinetobacter sp., Stenotrophomonas sp. and Alcaligenes sp. based on 16 S rRNA gene sequence analysis. These three strains were among dominant the bacteria in treated substrates, showing between 52.80% and 89.25% degradation of MON in mineral salt medium within 28 days. Furthermore, two MON-degrading bacteria (Stenotrophomonas sp. and Alcaligenes sp.) were more abundant in treatment groups and larvae gut groups compared with those in control groups. The abundance enhancement of MON-degrading bacteria was related to the change in ambient temperature and pH in the substrates, which were affected by housefly larvae activities. Our results confirm that housefly larvae can significantly accelerate degradation of MON in chicken manure by increasing the abundance of MON-degrading bacteria.

Determination of sRNA expressions by RNA-seq in Yersinia pestis grown in vitro and during infection.

BACKGROUND: Small non-coding RNAs (sRNAs) facilitate host-microbe interactions. They have a central function in the post-transcriptional regulation during pathogenic lifestyles. Hfq, an RNA-binding protein that many sRNAs act in conjunction with, is required for Y. pestis pathogenesis. However, information on how Yersinia pestis modulates the expression of sRNAs during infection is largely unknown. METHODOLOGY AND PRINCIPAL FINDINGS: We used RNA-seq technology to identify the sRNA candidates expressed from Y. pestis grown in vitro and in the infected lungs of mice. A total of 104 sRNAs were found, including 26 previously annotated sRNAs, by searching against the Rfam database with 78 novel sRNA candidates. Approximately 89% (93/104) of these sRNAs from Y. pestis are shared with its ancestor Y. pseudotuberculosis. Ninety-seven percent of these sRNAs (101/104) are shared among more than 80 sequenced genomes of 135 Y. pestis strains. These 78 novel sRNAs include 62 intergenic and 16 antisense sRNAs. Fourteen sRNAs were selected for verification by independent Northern blot analysis. Results showed that nine selected sRNA transcripts were Hfq-dependent. Interestingly, three novel sRNAs were identified as new members of the transcription factor CRP regulon. Semi-quantitative analysis revealed that Y. pestis from the infected lungs induced the expressions of six sRNAs including RyhB1, RyhB2, CyaR/RyeE, 6S RNA, RybB and sR039 and repressed the expressions of four sRNAs, including CsrB, CsrC, 4.5S RNA and sR027. CONCLUSIONS AND SIGNIFICANCE: This study is the first attempt to subject RNA from Y. pestis-infected samples to direct high-throughput sequencing. Many novel sRNAs were identified and the expression patterns of relevant sRNAs in Y. pestis during in vitro growth and in vivo infection were revealed. The annotated sRNAs accounted for the most abundant sRNAs either expressed in bacteria grown in vitro or differentially expressed in the infected lungs. These findings suggested these sRNAs may have important functions in Y. pestis physiology or pathogenesis.

[Detection of Yersinia pestis sRNA by digoxigenin-labeled Northern blot].

OBJECTIVE: With the application of high-throughput sequencing methods, more and more sRNAs are required to be verified. In this study, we developed the digoxigenin-labeled Northern blot method for detection of Yersinia pestis sRNA. METHODS: Total RNAs extracted from Yersinia pestis grown under low-iron conditions were loaded onto 10% denaturing urea polyacrylamide gel (dPAGE), electrophoresed and transferred to nylon membranes. Northern blots were fixed to the membrane by UV cross-linking and subjected to hybridization with 3' -end digoxigenin-labeled oligonucleotides RNA probe for RyhB1 and RyhB2 overnight and followed by washing, blocking, immunological detection and finally exposed to film. RESULTS: Exposure time of digoxigenin-labelled Northern blot was 20s-3 min. The detection sensitivity of RyhB1 and RyhB2 was 0.005 microg and 0.05 microg, respectively. RyhB1 and RyhB2 probe specificity was high and no cross reaction with each other was found. Positively charged or neutral nylon membranes are applicable to the hybridization reaction. Hybridization for RNA probe can proceed within 42 to 65 degrees C with reduced non-specific probe interactions by increasing the temperature while the hybridization temperature for DNA oligonucleotide probes seemed to be determined empirically. CONCLUSION: A digoxigenin-labeled Northern blot has been developed for detection of Yersinia pestis sRNA, which was characterized by good specificity, high sensitivity, longer shelf life and short exposure times and provided an power tool for validation of bacterial sRNAs and function studies.

Two sRNA RyhB homologs from Yersinia pestis biovar microtus expressed in vivo have differential Hfq-dependent stability.

Small non-coding RNAs (sRNAs) have been shown to modulate gene expression at the post-transcriptional level. RyhB, an iron-responsive sRNA, is conserved in Escherichia coli and other Enterobacteriae, indicating the downregulation of numerous genes during iron depletion. This sRNA is tightly regulated by the ferric uptake regulator (Fur) and interacts with the RNA binding protein Hfq. Hfq is generally purported to be essential for stabilizing sRNAs and promoting sRNA-mRNA duplex formation. Maintenance of iron homeostasis is an essential step in the lifecycle of Yersinia pestis. Y. pestis encodes two RyhB homologs, RyhB1 and RyhB2. In this study, we found that as in the case of E. coli, both RyhB homologs in Y. pestis are negatively regulated by Fur and have a half-life of >30 min. In the absence of Hfq, RyhB1 is rapidly degraded, while RyhB2 retains its stability. RyhB1 stabilization is mediated by Hfq, but RyhB2 does not require Hfq for stability. Additionally, both RyhBs are upregulated in lungs infected with Y. pestis, while the ryhB mutant shows no visible effects on virulence in mice upon either subcutaneous or intranasal inoculation. Collectively, our results indicate that the two RyhB homologs have common regulatory features in Y. pestis-infected lungs and in vitro, but that stability of RyhB1 and RyhB2 is differentially dependent on Hfq.