A novel co-target of ACY1 governing plasma membrane translocation of SphK1 contributes to inflammatory and neuropathic pain.

Previous studies validate that inhibiting sodium channel 1.8 (Nav1.8) effectively relieves inflammatory and neuropathic pain. However, Nav1.8 blockers have cardiac side effects in addition to analgesic effects. Here, we constructed a spinal differential protein expression profile using Nav1.8 knockout mice to screen common downstream proteins of Nav1.8 in inflammatory and neuropathic pain. We found that aminoacylase 1 (ACY1) expression was increased in wild-type mice compared to Nav1.8 knockout mice in both pain models. Moreover, spinal ACY1 overexpression induced mechanical allodynia in naive mice, while ACY1 suppression alleviated inflammatory and neuropathic pain. Further, ACY1 could interact with sphingosine kinase 1 and promote its membrane translocation, resulting in sphingosine-1-phosphate upregulation and the activation of glutamatergic neurons and astrocytes. In conclusion, ACY1 acts as a common downstream effector protein of Nav1.8 in inflammatory and neuropathic pain and could be a new and precise therapeutic target for chronic pain.

Pharmacological inhibition of the cGAS-STING signaling pathway suppresses microglial M1-polarization in the spinal cord and attenuates neuropathic pain.

Neuroinflammation plays a vital role in the development of neuropathic pain and is mediated mainly by microglia. Suppressing microglial M1-polarization attenuates neuropathic pain. Recently, the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway has emerged as a key mediator of inflammation and shows potential in modulating microglial polarization. In this study, we evaluated whether cGAS-STING is a potential therapeutic target. Spared nerve injury (SNI) surgery was conducted in adult male rats to establish a neuropathic pain model. We showed that SNI promoted microglial M1-polarization and induced cGAS-STING pathway activation in the spinal cord. Double-label immunofluorescence assays showed that cGAS-STING activation mainly occurred in neurons and microglia but not astrocytes. We further conducted in vitro experiments using BV-2 microglial cells. The results showed that LPS-induced microglial M1-polarization was accompanied by cGAS-STING pathway activation, but cGAS-STING inhibition by antagonists suppressed LPS-induced microglial M1-polarization. In vivo, we also showed that a cGAS antagonist and a STING antagonist suppressed the microglial M1-polarization and ameliorated the mechanical allodynia induced by SNI. These findings suggested that the cGAS-STING pathway might be a potential therapeutic target for treating neuropathic pain. However, further research is warranted to verify our findings in female rodents.

Identification of Differentially Expressed Plasma lncRNAs As Potential Biomarkers for Breast Cancer.

BACKGROUND: Breast cancer is the most common malignant tumor in women and is not easy to diagnose. Increasing evidence has underscored that long non-coding RNAs (lncRNAs) play important regulatory roles in the occurrence and progression of many cancers, including breast cancer. We aimed to identify lncRNAs in plasma as potential biomarkers for breast cancer. PATIENTS AND METHODS: We analyzed the Gene Expression Omnibus (GEO) datasets GSE22820, GSE42568, and GSE65194 to identify the common differential genes between cancer tissues and adjacent tissues. Then 14 lncRNAs were identified among the common differential genes and validated by using real-time quantitative polymerase chain reaction in 92 patients with breast cancer and 100 healthy controls. Receiver operating characteristic (ROC) curves were constructed to evaluate their diagnostic value for breast cancer. RESULTS: Integrated analysis of the GEO datasets identified three significantly upregulated and 11 downregulated lncRNAs in breast cancer tissues. Compared with healthy controls, MIAT was significantly upregulated in breast cancer patient plasma, and LINC00968 and LINC01140 were significantly downregulated. ROC curve analysis suggested that these three lncRNAs can discriminate breast cancer from healthy individual with high specificity and sensitivity. CONCLUSION: This research identified three differentially expressed lncRNAs in breast cancer patient plasma. Our data suggest that these three lncRNAs can be used as potential diagnostic biomarkers of breast cancer.

Pivotal role of the gut microbiota in congenital insensitivity to pain with anhidrosis.

BACKGROUND: Increasing evidence has shown that the occurrence and development of various human diseases are closely related to the gut microbiota. We compared the gut microbial communities of human subjects with congenital insensitivity to pain with anhidrosis (CIPA) and healthy controls (HCs) to assess whether fecal microbiota transplantation (FMT) into germ-free mice and mice in acute pain influenced the behaviors of the host. METHODS: We utilized 16 s rRNA analysis to compare the gut microbial communities of CIPA subjects and HCs and assessed whether FMT into germ-free mice and mice in acute pain influenced the behaviors of the host. RESULTS: In a 16 s RNA analysis, the CIPA group had significant decreases in the relative abundance of 11 bacteria, whereas 7 bacteria were significantly increased. In further animal experiments, the transplantation of fecal samples from CIPA patients to healthy mice significantly increased their scores on both the mechanical withdrawal test and the tail flick test; in an acute plantar incision model, scores were also significantly increased on the mechanical withdrawal test at 4 and 5 days after the operation. Moreover, pseudo-germ-free mice receiving fecal bacteria from patients with CIPA took significantly longer to escape and had a significantly longer path length on training days 1, 2, and 5 and also had fewer platform crossings and spent less time in the target quadrant in the probe trial. CONCLUSIONS: Our results suggest that the gut microbiota in CIPA subjects plays a key role in behaviors. Therapeutic strategies for improving the gut microbiota might alleviate CIPA symptoms.

Angiotensin II type 1 receptor blockade attenuates posttraumatic stress disorder-related chronic pain by inhibiting glial activation in the spinal cord.

Clinically, posttraumatic stress disorder (PTSD) and chronic pain are highly comorbid conditions, but the underlying mechanisms of and therapeutic strategies against PTSD-related pain remain unclear. Our previous studies suggested that dysregulation of neuroinflammation contributes to the development of stress-induced hyperalgesia. Recent studies reported that angiotensin II was a 'stress-related hormone', and could induce glial activation by stimulating the type 1 receptor (AT1R). In the present study, we aimed to investigate whether AT1R blockade could attenuate mechanical allodynia induced by PTSD-like stress. Adult male rats were exposed to single prolonged stress (SPS) to establish a model of PTSD-pain comorbidity. Our results showed that SPS exposure increased the levels of angiotensin II in the hippocampus, prefrontal cortex (PFC) and spinal cord; intraperitoneal injection of losartan attenuated SPS-induced mechanical allodynia, and suppressed SPS-induced glial activation (both microglia and astrocytes) and proinflammatory cytokine expression in the PFC and spinal cord, but not in the hippocampus. We further showed that intrathecal injection of losartan also exerted anti-hyperalgesic effect and suppressed SPS-induced glial activation and proinflammatory cytokine expression in the spinal cord. These results indicated that AT1R blockade by losartan attenuated mechanical allodynia induced by PTSD-like stress, and this may be attributed to the suppression of glial activation and proinflammatory cytokine expression in the spinal cord. Although further research is warranted to verify our findings in female rodents and to assess pharmacological effects of AT1R blockade in PFC and hippocampus, our study suggested the therapeutic potential of targeting AT1R in the treatment of PTSD-related chronic pain.

NGF-Induced Nav1.7 Upregulation Contributes to Chronic Post-surgical Pain by Activating SGK1-Dependent Nedd4-2 Phosphorylation.

At present, chronic post-surgical pain (CPSP) is difficult to prevent and cure clinically because of our lack of understanding of its mechanisms. Surgical injury induces the upregulation of voltage-gated sodium channel Nav1.7 in dorsal root ganglion (DRG) neurons, suggesting that Nav1.7 is involved in the development of CPSP. However, the mechanism leading to persistent dysregulation of Nav1.7 is largely unknown. Given that nerve growth factor (NGF) induces a long-term increase in the neuronal hyperexcitability after injury, we hypothesized that NGF might cause the long-term dysregulation of Nav1.7. In this study, we aimed to investigate whether Nav1.7 regulation by NGF is involved in CPSP and thus contributes to the specific mechanisms involved in the development of CPSP. Using conditional nociceptor-specific Nav1.7 knockout mice, we confirmed the involvement of Nav1.7 in NGF-induced pain and identified its role in the maintenance of pain behavior during long-term observations (up to 14 days). Using western blot analyses and immunostaining, we showed that NGF could trigger the upregulation of Nav1.7 expression and thus support the development of CPSP in rats. Using pharmacological approaches, we showed that the increase of Nav1.7 might be partly regulated by an NGF/TrkA-SGK1-Nedd4-2-mediated pathway. Furthermore, reversing the upregulation of Nav1.7 in DRG could alleviate spinal sensitization. Our results suggest that the maintained upregulation of Nav1.7 triggered by NGF contributes to the development of CPSP. Attenuating the dysregulation of Nav1.7 in peripheral nociceptors may be a strategy to prevent the transition from acute post-surgical pain to CPSP.

The Role of Voltage-Gated Sodium Channel 1.8 in the Effect of Atropine on Heart Rate: Evidence From a Retrospective Clinical Study and Mouse Model.

Atropine is commonly used to counter the effects of the parasympathetic neurotransmitter acetylcholine on heart rate in clinical practice, such as in the perioperative period; however, individual differences in the response to atropine are huge. The association between SCN10A/voltage-gated sodium channel 1.8 (NaV1.8) and cardiac conduction has been demonstrated; however, the exact role of SCN10A/NaV1.8 in the heart rate response to atropine remains unclear. To identify the role of SCN10A variants that influence the heart rate responses to atropine, we carried out a retrospective study in 1,005 Han Chinese subjects. Our results showed that rs6795970 was associated with the heart rate response to atropine. The heart rate responses to atropine and methoctramine in NaV1.8 knockout mice were lower, whereas the heart rate response to isoproterenol was like those in wild type mice. Furthermore, we observed that the NaV1.8 blocker A-803467 alleviated the heart rate response to atropine in wild type mice. The retrospective study revealed a previously unknown role of NaV1.8 in controlling the heart rate response to atropine, as shown by the animal study, a speculative mechanism that may involve the cardiac muscarinic acetylcholine receptor M2.

Smart Material Hydrogel Transfer Devices Fabricated with Stimuli-Responsive Silk-Elastin-Like Proteins.

Three-dimensional organoid tissue culture models are a promising approach for the study of biological processes including diseases. Advances in these tissue culture technologies improve in vitro analysis compared to standard 2D cellular approaches and are more representative of the physiological environment. However, a major challenge associated with organoid systems stems from the laborious processing involved in the analysis of large numbers of organoids. Here the design, characterization, and application of silk-elastin-like protein-based smart carrier arrays for processing organoids is presented. Fabrication of hydrogel-based carrier systems at room temperature result in organized arrays of organoids that maintain tissue culture plate orientation and could be processed simultaneously for histology. The system works by transfer of the organoids to the hydrogel arrays after which the material is subjected to 65 °C to induce hydrogel contraction to secure the organoids, resulting in multisample constructs and allowing for placement on a microscope slide. Histological processing and immunostaining of these arrayed cerebral organoids analyzed within the contracted silk-elastin-like proteins (SELP) show retention of native organoid features compared to controls without the hydrogel carrier system, thus avoiding any artifacts. These SELP carriers present a useful approach for improving efficiency of scaled organoid screening and processing.

Upregulation of transcription factor 4 downregulates NaV1.8 expression in DRG neurons and prevents the development of rat inflammatory and neuropathic hypersensitivity.

The voltage sodium channel 1.8 (NaV1.8) in the dorsal root ganglion (DRG) neurons contributes to the initiation and development of chronic inflammatory and neuropathic pain. However, an effective intervention on NaV1.8 remains to be studied in pre-clinical research and clinical trials. In this study, we aimed to investigate whether transcription factor 4 (TCF4) overexpression represses NaV1.8 expression in DRG neurons, thus preventing the development of chronic pain. Using chromatin immunoprecipitation (CHIP), we verified the interaction of TCF4 and sodium voltage-gated channel alpha subunit 10A (SCN10A) enhancer in HEK293 cells and rat DRG neurons. Using a dual luciferase reporter assay, we confirmed the transcriptional inhibition of TCF4 on SCN10A promoter in vitro. To investigate the regulation of TCF4 on Nav1.8, we then upregulated TCF4 expression by intrathecally delivering an overexpression of recombinant adeno-associated virus (rAAV) in the Complete Freund's adjuvant (CFA)-induced inflammatory pain model and spared nerve injury (SNI)-induced neuropathic pain model. By using a quantitative polymerase chain reaction (qPCR), western blot, and immunostaining, we evaluated NaV1.8 expression after a noxious stimulation and the application of the TCF4 overexpression virus. We showed that the intrathecal delivery of TCF4 overexpression virus significantly repressed the increase of NaV1.8 and prevented the development of hyperalgesia in rats. Moreover, we confirmed the efficient role of an overexpressed TCF4 in preventing the CFA- and SNI-induced neuronal hyperexcitability by calcium imaging. Our results suggest that attenuating the dysregulation of NaV1.8 by targeting TCF4 may be a novel therapeutic strategy for chronic inflammatory and neuropathic pain.

Design of Silk-Elastin-Like Protein Nanoparticle Systems with Mucoadhesive Properties.

Transmucosal drug delivery is a promising avenue to improve therapeutic efficacy through localized therapeutic administration. Drug delivery systems that increase retention in the mucosal layer are needed to improve efficiency of such transmucosal platforms. However, the applicability of such systems is often limited by the range of chemistries and properties that can be achieved. Here we present the design and implementation of silk-elastin-like proteins (SELPs) with mucoadhesive properties. SELP-based micellar-like nanoparticles provide a system to tailor chemical and physical properties through genetic engineering of the SELP sequence, which enables the fabrication of nanoparticles with specific chemical and physical features. Analysis of the adhesion of four different SELP-based nanoparticle systems in an artificial mucus system, as well as in in vitro cellular assays indicates that addition of mucoadhesive chemical features on the SELP systems increases retention of the particles in mucosal environments. The results indicated that SELP-based nanoparticles provide a useful approach to study and develop transmucosal protein drug delivery system with unique mucoadhesive properties. Future studies will serve to further expand the range of achievable properties, as well as the utilization of SELPs to fabricate mucoadhesive materials for in vivo testing.

Molecular Epidemiology and Mechanisms of 43 Low-Level Linezolid-Resistant Enterococcus faecalis Strains in Chongqing, China.

BACKGROUND: Enterococcus faecalis strains with low-level resistance to linezolid (an oxazolidinone antibiotic) have become common. No large-scale study has examined the underlying mechanisms in linezolid-resistant E. faecalis (LRE) strains. We investigated these mechanisms and molecular characteristics in Chongqing, China. METHODS: A total of 1,120 non-duplicated E. faecalis strains collected from August 2014 to June 2017 underwent drug susceptibility testing. LRE strains were screened for optrA, cfr, and mutations in the 23S rRNA and ribosomal proteins L3 and L4 by PCR amplification and sequencing. Multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used for epidemiological analysis. RESULTS: All 43 low-level LRE strains (minimum inhibitory concentration: 8-16 mg/L) harbored optrA; cfr and 23S rRNA mutations were not detected. Novel mutations in the ribosomal proteins L3 and L4-one deletion (Q103del) and four substitutions (S113L, T35A, I98V, and N79D)-were identified. Novel amino acid substitutions at positions E60K, G197D, and T285P of the OptrA protein were observed. MLST revealed 20 types of LRE strains; the most common type was ST16 (32.6%). PFGE showed 14 strains of ST16 with unique banding patterns. Eight novel sequence types (ST823 to ST830) and one allele (gki95) were identified for the first time in China. CONCLUSIONS: optrA plays an important role in linezolid resistance and may serve as a marker for resistance screening. Since the L3 and L4 mutations did not simultaneously occur in the same strain, they play a negligible role in linezolid resistance. Epidemiological investigation suggested that the LRE cases were sporadic.

Naringin protects against perfluorooctane sulfonate-induced liver injury by modulating NRF2 and NF-κB in mice.

Perfluorooctane sulfonate (PFOS), a persistent organic pollutant, has been demonstrated to cause multiple toxicities. In this study, we explored the role of naringin (Nar) in alleviating PFOS-caused mouse liver injury and its potential mechanisms. Male mice were intragastrically administered PFOS (10 mg/kg/day) alone or with Nar (100 mg/kg/day) for 3 weeks. Nar supplementation led to resumption of elevated serum hepatic enzyme activities and increased relative liver weight in PFOS-challenged mice. Moreover, Nar treatment increased hepatic expression of transcription factor NRF2 protein and its regulated antioxidative enzyme genes heme oxygenase­1, superoxide dismutase and catalase, with an inhibition of malondialdehyde and hydrogen peroxide production. Furthermore, simultaneous administration of Nar suppressed PFOS-induced elevation in NF-κB activity and generation of inflammatory cytokines TNF-α and IL-6 in the liver. In addition, Nar enhanced anti-apoptotic Bcl-2 expression, decreased pro-apoptotic Bax expression and inhibited caspase­3 activation in liver tissue in mice exposed to PFOS. Our results indicate that Nar protects against PFOS-induced hepatotoxicity in mice via modulating oxidative, inflammatory and apoptotic pathways.

Whole transcriptome analysis reveals potential novel mechanisms of low-level linezolid resistance in Enterococcus faecalis.

Linezolid is an oxazolidinone antibiotic commonly used to treat serious infections caused by vancomycin-resistant enterococcus. Recently, low-level linezolid resistant Enterococcus faecalis strains have emerged worldwide, but the resistant mechanisms remain undefined. Whole-transcriptome profiling was performed on an E. faecalis strain P10748 with low-level linezolid resistance in comparison with a linezolid-susceptible strain 3138 and the standard control strain ATCC29212. The functions of differentially expressed genes (DEGs) were predicted, with some DEGs potentially involved in drug resistance were validated by PCR and quantitative PCR (qPCR). RNA-Seq on three E. faecalis strains generated 1920 unigenes, with 98% of them assigned to various function groups. A total of 150 DEGs were identified in the linezolid resistant strain P10748 compared to the linezolid susceptible strains 3138 and ATCC29212. Functional analysis indicated a significant transcriptomic shift to membrane transportation and biofilm formation in strain P10748, with three significantly up-regulated DEGs predicted to be associated with drug resistance through active efflux pumps and biofilm formation. The existence of these three DEGs was further confirmed by PCR and qPCR. The significant upregulation of genes associated with efflux pumps and biofilm formation in the linezolid resistant strain suggests their roles in low-level resistance to linezolid in E. faecalis.

Development and evaluation of enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification for diagnosis of invasive aspergillosis.

Invasive aspergillosis (IA) is a life-threatening infection in immunocompromised patients, rapid and sensitive detection of Aspergillus from clinical samples has been a major challenge in the early diagnosis of IA. An enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification (NASBA-ELISA) was developed to fulfil the need for the efficient diagnosis of these infections. The primers targeting 18S rRNA were selected for the amplification of Aspergillus RNA by the isothermal digoxigenin (DIG)-labeling NASBA process. The DIG-labeled RNA amplicons were hybridized with a specific biotinylated DNA probe immobilized on streptavidin-coated microtiter plate. The hybrids were colorimetrically detected by the addition of an anti-DIG antibodies linked to ALP and substrate (disodium 4-nitrophenyl phosphate). The detection limit of the Aspergillus NABSA-ELISA system was 1 CFU and the RNA in non-target bacteria or fungus was not amplified. The performance of this NASBA-ELISA compared to RT-PCR and galactomannan (GM) was evaluated by testing blood samples from 86 patients at high risk for IA. The sensitivity of NASBA-ELISA, RT-PCR and GM-ELISA was 80.56 % (95 % CI 63.98-91.81), 72.22 % (95 % CI 54.81-85.80), 58.33 % (95 % CI 40.76-74.49), respectively, and the specificity was 80.00 % (95 % CI 66.28-89.97), 84.00 % (95 % CI 70.89-92.83), 82.00 % (95 % CI 68.56-91.42). The efficiency of the three methods in various combinations was also evaluated. Combination of NASBA-ELISA and GM-ELISA testing achieved perfect specificity (100 %; 95 % CI 92.89-100) and perfect positive predictive value (100 %; 95 % CI 83.16-100). The best sensitivity (97.22 %; 95 % CI 85.47-99.93) and the highest Youden index (0.652) were obtained by testing with both NASBA and RT-PCR in parallel. In conclusion, the NASBA-ELISA assay consists of an alternative process for large-scale samples detection with semi-quantitative results and provides good clinical performance without resorting to expensive equipment. This assay makes it possible for the NASBA based RNA diagnosis to become a routine work in laboratories in less developed countries with fewer resources.