Suppression of histone deacetylases by SAHA relieves bone cancer pain in rats via inhibiting activation of glial cells in spinal dorsal horn and dorsal root ganglia.

BACKGROUND: Robust activation of glial cells has been reported to occur particularly during the pathogenesis of bone cancer pain (BCP). Researchers from our group and others have shown that histone deacetylases (HDACs) play a significant role in modulating glia-mediated immune responses; however, it still remains unclear whether HDACs are involved in the activation of glial cells during the development of BCP. METHODS: BCP model was established by intra-tibia tumor cell inoculation (TCI). The expression levels and distribution sites of histone deacetylases (HDACs) in the spinal dorsal horn and dorsal root ganglia were evaluated by Western blot and immunofluorescent staining, respectively. Suberoylanilide hydroxamic acid (SAHA), a clinically used HDAC inhibitor, was then intraperitoneally and intrathecally injected to rescue the increased expression levels of HDAC1 and HDAC2. The analgesic effects of SAHA administration on BCP were then evaluated by measuring the paw withdrawal thresholds (PWTs). The effects of SAHA on activation of glial cells and expression of proinflammatory cytokines (TNF-α, IL-1ß, and IL-6) in the spinal dorsal horn and dorsal root ganglia of TCI rats were further evaluated by immunofluorescent staining and Western blot analysis. Subsequently, the effects of SAHA administration on tumor growth and cancer cell-induced bone destruction were analyzed by hematoxylin and eosin (HE) staining and micro-CT scanning. RESULTS: TCI caused rapid and long-lasting increased expression of HDAC1/HDAC2 in glial cells of the spinal dorsal horn and dorsal root ganglia. Inhibiting HDACs by SAHA not only reversed TCI-induced upregulation of HDACs but also inhibited the activation of glial cells in the spinal dorsal horn and dorsal root ganglia, and relieved TCI-induced mechanical allodynia. Further, we found that SAHA administration could not prevent cancer infiltration or bone destruction in the tibia, which indicated that the analgesic effects of SAHA were not due to its anti-tumor effects. Moreover, we found that SAHA administration could inhibit GSK3ß activity in the spinal dorsal horn and dorsal root ganglia, which might contributed to the relief of BCP. CONCLUSION: Our findings suggest that HDAC1 and HDAC2 are involved in the glia-mediated neuroinflammation in the spinal dorsal horn and dorsal root ganglia underlying the pathogenesis of BCP, which indicated that inhibiting HDACs by SAHA might be a potential strategy for pain relief of BCP.

Efficient CRISPR/Cas9-mediated gene editing in Guangdong small-ear spotted pig cells using an optimized electrotransfection method.

OBJECTIVES: Guangdong Small-ear Spotted (GDSS) pigs are a pig breed native to China that possesses unfortunate disadvantages, such as slow growth rate, low lean-meat percentage, and reduced feed utilization. In contrast to traditional genetic breeding methods with long cycle time and high cost, CRISPR/Cas9-mediated gene editing for the modification of the pig genome can quickly improve production traits, and therefore this technique exhibits important potential in the genetic improvement and resource development of GDSS pigs. In the present study, we aimed to establish an efficient CRISPR/Cas9-mediated gene-editing system for GDSS pig cells by optimizing the electrotransfection parameters, and to realize efficient CRISPR/Cas9-mediated gene editing of GDSS pig cells. RESULTS: After optimization of electrotransfection parameters for the transfection of GDSS pig cells, we demonstrated that a voltage of 150 V and a single pulse with a pulse duration of 20 ms were the optimal electrotransfection parameters for gene editing in these cells. In addition, our study generated GDSS pig single-cell colonies with biallelic mutations in the myostatin (MSTN) gene and insulin-like growth factor 2 (IGF2) intron-3 locus, which play an important role in pig muscle growth and muscle development. The single-cell colonies showed no foreign gene integration or off-target effects, and maintained normal cell morphology and viability. These gene-edited, single-cell colonies can in the future be used as donor cells to generate MSTN- and IGF2-edited GDSS pigs using somatic cell nuclear transfer (SCNT). CONCLUSIONS: This study establishes the foundation for genetic improvement and resource development of GDSS pigs using CRISPR/Cas9-mediated gene editing combined with SCNT.

CRISPR/Cas9-mediated MSTN disruption accelerates the growth of Chinese Bama pigs.

CRISPR/Cas9-mediated genome editing technology is a simple and highly efficient and specific genome modification approach with wide applications in the animal industry. CRISPR/Cas9-mediated genome editing combined with somatic cell nuclear transfer rapidly constructs gene-edited somatic cell-cloned pigs for the genetic improvement of traits or simulation of human diseases. Chinese Bama pigs are an excellent indigenous minipig breed from Bama County of China. Research on genome editing of Chinese Bama pigs is of great significance in protecting its genetic resource, improving genetic traits and in creating disease models. This study aimed to address the disadvantages of slow growth and low percentage of lean meat in Chinese Bama pigs and to knock out the myostatin gene (MSTN) by genome editing to promote growth and increase lean meat production. We first used CRISPR/Cas9-mediated genome editing to conduct biallelic knockout of the MSTN, followed by somatic cell nuclear transfer to successfully generate MSTN biallelic knockout Chinese Bama pigs, which was confirmed to have significantly faster growth rate and showed myofibre hyperplasia when they reached sexual maturity. This study lays the foundation for the rapid improvement of production traits of Chinese Bama pigs and the generation of gene-edited disease models in this breed.

[Meta-analysis on effect of compound Danshen injection in treating neonatal hypoxic-ischemic encephalopathy].

To systematically evaluate the clinical efficacy and safety of compound Danshen injection in treating hypoxic-ischemic encephalopathy (HIE) of newborns. Computer retrievals were made in PubMed, Embase, Cochrane Library, CBM, CNKI, VIP and China info (before May 2014) and relevant literature references, and manual retrievals were made for journals and conference papers, in order to collect randomized or semi-randomized controlled trials concerning compound Danshen injection in the treatment of neonatal HIE. The quality of included references was evaluated according to literatures recommended by Cochrane Handbook. RevMan 5. 3 software was applied in the statistical treatment. Finally, a total of 13 randomized controlled trials were included, covering 1,211 patients (including 639 patients in the compound Danshen injection-treated group and 572 patients in the control group). Meta-analysis results showed that the routine treatment combined with compound Danshen injection can improve the treatment efficiency of neonatal HIE [RR = 1.28; 95% CI (1.21-1.36)], reduce the mortality rate [RR = 0.42; 95% CI (0.23-0.75)] and the incidence of long-term neurological sequelae [RR = 0.48; 95% CI (0.35-0.65)], with statistical differences. No fatal side effect was observed in all of included trials. So far, limited evidences in this study proved that the application of compound Danshen injection in the treatment of neonatal HIE can enhance the clinical efficiency. However, because of the low quality of the included trials, more well-designed and large-scale multi-center randomized controlled trials shall be made in the future.

Connections between EM2- and SP-containing terminals and GABAergic neurons in the mouse spinal dorsal horn.

Endomorphin-2 (EM2) demonstrates a potent antinociceptive effect in pain modulation. To investigate the potential interactions of EM2- and substance P (SP)-containing primary afferents and γ-amino butyric acid (GABA)-containing interneurons in lamina II in nociceptive transmission, connections between EM2- and SP-containing terminals and GABAergic neurons in the spinal dorsal horn were studied. Double-immunofluorescent labeling showed that approximately 62.3 % of EM2-immunoreactive neurons exhibited SP-immunostaining, and 76.9 % of SP-immunoreactive neurons demonstrated EM2-immunoreactivities in the dorsal root ganglion (DRG). Dense double-labeled EM2- and SP-immunoreactivities were mainly observed in lamina II of the lumbar dorsal horn. Furthermore, triple-immunofluorescent labeling results revealed that EM2 and SP double-labeled terminals overlapped with GABAergic neurons. Immuno-electron microscopy confirmed that the EM2- or SP-immunoreactive terminals formed synapses with GABA-immunoreactive dendrites in lamina II of the lumbar dorsal horn. During noxious information transmission induced by formalin plantar injection, GABAergic neurons expressing FOS in their nuclei were contacted with EM2- or SP-immunoreactive terminals. These results suggest that the interactions between EM2- and SP-containing terminals and GABAergic interneurons in the lamina II influence pain transmission and modulation in the spinal dorsal horn.

Differential iron-bioavailability with relation to nutrient compositions in polished rice among selected Chinese genotypes using Caco-2 cell culture model.

Genotypic variation of iron bioavailability and the relationship between iron bioavailability and nutrient composition in polished rice among 11 rice genotypes were assessed using an in vitro digestion/Caco-2 cell model. The results indicated that significant differences in iron bioavailability were detected among tested rice genotypes, with a 3-fold range, suggesting a possibility of selecting high bioavailable iron by plant breeding. Although iron bioavailability was not significantly correlated with Fe concentration in polished rice among tested rice genotypes, the results also indicated that most of the iron dense genotypes showed relatively high ferritin formation in Caco-2 cell and transported iron. Additionally, iron bioavailability in polished rice was enhanced by addition of ascorbic acid, with a much wider range of Fe bioavailability variation in polished rice with addition of ascorbic acid than that without addition of ascorbic acid. The positive relationship between iron bioavailability in polished rice and cysteine concentration (R = 0.669) or sulfur (S) concentration (R = 0.744) among tested rice genotypes, suggests that cysteine and sulfur concentration in polished rice could be used as an indicator for high iron bioavailability.

Tripartite motif containing 69 elicits ERK2-dependent EYA4 turnover to impart pancreatic tumorigenesis.

Eyes absent homologue 4 (EYA4) is silenced in pancreatic ductal adenocarcinoma (PDAC) and functions as a tumor suppressor to restrain PDAC development, albeit the molecular mechanism underlying its downregulation remains enigmatic. Methods: Functional studies were determined by immunohistochemistry of PDAC samples from patients and Pdx1-Cre; LSL-KrasG12D/+; Trp53fl/+ (KPC) mice, three-dimensional spheroid culture, flow cytometry, MTT and subcutaneous xenograft experiments. Mechanistical studies were examined by cellular ubiquitination, cycloheximide (CHX) pulse-chase, co-immunoprecipitation, chromatin immunoprecipitation, GST-pulldown, in vitro protein kinase assay, immunofluorescence and luciferase reporter assays. Results: We screen E3 ligase that is negatively correlated with EYA4 and uncover a mutually exclusive interaction of tripartite motif containing 69 (TRIM69) with EYA4 in human PDAC. TRIM69 elicits EYA4 polyubiquitylation and turnover independent of P53 and impedes the EYA4-driven deactivation of ß-catenin/ID2 cascade, fueling PDAC cell proliferation in vitro and tumor development in mice. Expression of TRIM69 is upregulated in PDAC samples from independent cohorts of patients and the Pdx1-Cre; LSL-KrasG12D/+; Trp53fl/+ (KPC) mice, and associated with unfavorable prognosis. Depleting TRIM69 preferentially induces lethality in the EYA4-deficient PDAC cells. We further unearth that ERK2 directly binds to the D-site of mitogen-activated protein kinase (MAPK) docking groove in EYA4 Leu512/514 and phosphorylates EYA4 at Ser37, which is instrumental for EYA4 polyubiquitylation and turnover by TRIM69. Conclusion: Our results define a previously unappreciated role of TRIM69-EYA4 axis in pancreatic tumorigenesis and underscore that targeting TRIM69 might be an effective therapeutic approach for PDAC harboring EYA4 deficiency.

Spatial and temporal distribution patterns of enkephalinergic neurons in adult and developing retinas of the preproenkephalin-green fluorescent protein transgenic mouse.

Enkephalin (ENK) peptides are present in the retina of several vertebrate species and play a crucial role in establishing specific circuits during retinal development. However, there is no information available concerning the development of ENKergic neurons in the mouse retina. To address this question, we used preproenkephalin-enhanced green fluorescent protein (GFP) transgenic mice, in which ENKergic neurons are revealed by GFP. Our results showed that most GFP-positive cells were located in the proximal part of the inner nuclear layer with a scattering of GFP-immunoreactive cells in the ganglion cell layer (GCL) in the adult retina. Double immunostaining with syntaxin indicates that GFP expression was restricted to a population of amacrine cells. The proportions of glycine transporter-1 and γ-aminobutyric acid-positive cells among ENKergic neurons were 57.3 ± 2.4% and 10.1 ± 1.8%, respectively. We then injected retrograde tracer into the superior colliculus and observed that none of the ENKergic neurons in the GCL were retrogradely labeled with the tracer. GFP-positive cells were first observed at embryonic day (E) 15 in the inner neuroblastic layer at only very low levels, which gradually increased until E18. After birth, there was a steep rise in GFP expression levels, reaching maximal activity by postnatal day (P) 7. The distribution and intensity of GFP-positive cells at P15 were similar to those of adult retina. It was found that immunoreactive processes in the inner plexiform layer formed strongly stained patches. The present results provide detailed morphological evidence of the cell type and spatial and temporal distribution of ENKergic neurons in the retina.

TIGIT marks exhausted T cells and serves as a target for immune restoration in patients with chronic HBV infection.

BACKGROUND: Chronic HBV infection is a serious worldwide health problem that mainly causes liver cirrhosis and hepatocellular carcinoma (HCC). Few studies have explored how T cell exhaustion helps HBV avoid immune system attack and how to reverse that exhaustion. Recently, T cell immunoglobulin and immune receptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) have been identified as coinhibitory receptors, similar to PD-1. This study explores the expression of TIGIT and the T cell function changes in patients with chronic HBV infection. RESULTS: In this study, we found that the expression of TIGIT on T cells increased significantly in patients with chronic HBV infection. High expression of TIGIT on T cells is associated with functional exhaustion. Importantly, this study demonstrates that blocking TIGIT can reverse T cell exhaustion and restore function in patients with chronic HBV infection. CONCLUSIONS: HBV induces T cell exhaustion by up-regulating the expression of TIGIT. Blocking the TIGIT/PVR signaling pathway can reverse T cell exhaustion, so this discovery provides an immunotherapy target to battle chronic HBV infection.

Relationship Between Affective Temperaments and Suicide Risk in Patients With First-Onset Major Depressive Disorder.

Background: People may endorse suicidal behavior during a major depressive episode. Affective temperaments may play a role in this risk. We explored the relationship between affective temperaments and suicide and identified some traits that can predict suicide risk in depression. Materials and Methods: We analyzed the results of the Temperament Evaluation of the Memphis, Pisa, Paris, and San Diego Auto-questionnaire (TEMPS-A) in 284 participants recruited from a psychiatric clinic and the community in Beijing and compared the subscale scores (temperaments of cyclothymic, dysthymic, anxious, irritable, and hyperthymic) among major depressive disorders (MDDs) vs. the general population as well as depressive patients with vs. without suicide risk, using Student's test, chi-square test, rank-sum test, and multivariable regression modeling. Results: The incidence of suicidal risk in depressive subjects was 47.62% (80/168). Being unmarried (p < 0.001), unemployed (p = 0.007), and temperaments of dysthymic, cyclothymic, anxious, and irritable scores (all p < 0.001) were significantly more prevalent in patients with depression than in the general population. Young age (p < 0.001), female sex (p = 0.037), unmarried (p = 0.001), more severe depression (p < 0.001), and dysthymic, anxious, and cyclothymic temperament (all p < 0.05) were significantly more prevalent in patients with depressive disorder than those without suicide risk. The logistic regression analysis showed that younger age (odds ratio [OR] = 0.937, 95% CI 0.905∼0.970), female sex (OR = 2.606, 95% CI 1.142∼5.948), more severe depression (OR = 1.145, 95% CI 1.063∼1.234), cyclothymic temperament (OR = 1.275, 95% CI 1.102∼1.475), and dysthymic temperament (OR = 1.265, 95% CI 1.037∼1.542) were all independently associated with high suicidal risk in patients with first-onset major depression (p < 0.05). Conclusion: Temperament traits differ between the general population and people suffering from MDD. Subjects with MDD who have much more severe depressive symptoms and a cyclothymic or dysthymic temperament were at a high risk of suicide.

Inflammation-Related Gene Signature: An Individualized Risk Prediction Model for Kidney Renal Clear Cell Carcinoma.

BACKGROUND: There is much evidence that confirms the inextricable link between inflammation and malignancy. Inflammation-related regulators were involved in the progression of kidney renal clear cell carcinoma (KIRC). However, the predictive role of single gene biomarkers is inadequate, and more accurate prognostic models are necessary. We undertook the current research to construct a robust inflammation-related gene signature that could stratify patients with KIRC. METHODS: The transcriptome sequencing data along with clinicopathologic information of KIRC were obtained from TCGA. A list of inflammation-related genes was acquired from the Molecular Signatures Database. Using the RNA-seq and survival time data from the TCGA training cohort, an inflammation-related gene signature was built using bioinformatic methods, and its performance in predicting patient prognosis was assessed by Kaplan-Meier and ROC curve analyses. Furthermore, we explored the association of risk score with immune score, stromal score, tumor immune-infiltrating cells (TIICs), immunosuppressive molecules, m6A regulators, and autophagy-related biomarkers. RESULTS: Herein, nine inflammation-related hub genes (ROS1, PLAUR, ACVR2A, KLF6, GABBR1, APLNR, SPHK1, PDPN, and ADORA2B) were determined and used to build a predictive model. All sets, including training set, four testing sets, and the entire TCGA group, were divided into two groups (low and high risk), and Kaplan-Meier curves all showed an adverse prognosis for patients in the high-risk group. ESTIMATE algorithm revealed a higher immune score in the high-risk subgroup. CIBERSORT algorithm illustrated that the high-risk group showed higher-level immune infiltrates. Furthermore, LAG3, TIGIT, and CTLA4 were overexpressed in the high-risk subgroup and positively associated with risk scores. Moreover, except for METTL3 and ALKBH5, the other m6A regulators decreased in the high-risk subgroup. CONCLUSIONS: In conclusion, a novel inflammation-related gene signature comprehensively constructed in the current study may help stratify patients with KIRC.

Imbalance Model of Heart Rate Variability and Pulse Wave Velocity in Psychotic and Nonpsychotic Disorders.

OBJECTIVES: Patients with psychiatric disorders have an increased risk of cardiovascular pathologies. A bidirectional feedback model between the brain and heart exists widely in both psychotic and nonpsychotic disorders. The aim of this study was to compare heart rate variability (HRV) and pulse wave velocity (PWV) functions between patients with psychotic and nonpsychotic disorders and to investigate whether subgroups defined by HRV and PWV features improve the transdiagnostic psychopathology of psychiatric classification. METHODS: In total, 3448 consecutive patients who visited psychiatric or psychological health services with psychotic (N = 1839) and nonpsychotic disorders (N = 1609) and were drug-free for at least 2 weeks were selected. HRV and PWV indicators were measured via finger photoplethysmography during a 5-minute period of rest. Canonical variates were generated through HRV and PWV indicators by canonical correlation analysis (CCA). RESULTS: All HRV indicators but none of the PWV indicators were significantly reduced in the psychotic group relative to those in the nonpsychotic group. After adjusting for age, gender, and body mass index, many indices of HRV were significantly reduced in the psychotic group compared with those in the nonpsychotic group. CCA analysis revealed 2 subgroups defined by distinct and relatively homogeneous patterns along HRV and PWV dimensions and comprising 19.0% (subgroup 1, n = 655) and 80.9% (subgroup 2, n = 2781) of the sample, each with distinctive features of HRV and PWV functions. CONCLUSIONS: HRV functions are significantly impaired among psychiatric patients, especially in those with psychosis. Our results highlight important subgroups of psychiatric patients that have distinct features of HRV and PWV which transcend current diagnostic boundaries.

Inhibition of spinal astrocytic c-Jun N-terminal kinase (JNK) activation correlates with the analgesic effects of ketamine in neuropathic pain.

BACKGROUND: We have previously reported that inhibition of astrocytic activation contributes to the analgesic effects of intrathecal ketamine on spinal nerve ligation (SNL)-induced neuropathic pain. However, the underlying mechanisms are still unclear. c-Jun N-terminal kinase (JNK), a member of mitogen-activated protein kinase (MAPK) family, has been reported to be critical for spinal astrocytic activation and neuropathic pain development after SNL. Ketamine can decrease lipopolysaccharide (LPS)-induced phosphorylated JNK (pJNK) expression and could thus exert its anti-inflammatory effect. We hypothesized that inhibition of astrocytic JNK activation might be involved in the suppressive effect of ketamine on SNL-induced spinal astrocytic activation. METHODS: Immunofluorescence histochemical staining was used to detect SNL-induced spinal pJNK expression and localization. The effects of ketamine on SNL-induced mechanical allodynia were confirmed by behavioral testing. Immunofluorescence histochemistry and Western blot were used to quantify the SNL-induced spinal pJNK expression after ketamine administration. RESULTS: The present study showed that SNL induced ipsilateral pJNK up-regulation in astrocytes but not microglia or neurons within the spinal dorsal horn. Intrathecal ketamine relieved SNL-induced mechanical allodynia without interfering with motor performance. Additionally, intrathecal administration of ketamine attenuated SNL-induced spinal astrocytic JNK activation in a dose-dependent manner, but not JNK protein expression. CONCLUSIONS: The present results suggest that inhibition of JNK activation may be involved in the suppressive effects of ketamine on SNL-induced spinal astrocyte activation. Therefore, inhibition of spinal JNK activation may be involved in the analgesic effects of ketamine on SNL-induced neuropathic pain.

Neuronal NR2B-containing NMDA receptor mediates spinal astrocytic c-Jun N-terminal kinase activation in a rat model of neuropathic pain.

Spinal N-methyl d-aspartate receptor (NMDAR) plays a pivotal role in nerve injury-induced central sensitization. Recent studies suggest that NMDAR also contributes to neuron-astrocyte signaling. c-Jun N-terminal kinase (JNK) is persistently and specifically activated (indicated by phosphorylation) in spinal cord astrocytes after nerve injury and thus it is considered as a dependable indicator of pain-related astrocytic activation. NMDAR-mediated JNK activation in spinal dorsal horn might be an important form of neuron-astrocyte signaling in neuropathic pain. In the present study, we observed that intrathecal injection of MK-801, a noncompetitive NMDA receptor antagonist, or Ro25-6981 and ifenprodil, which are selective antagonists of NR2B-containing NMDAR each significantly reduced nerve injury-induced JNK activation. Double immunostaining showed that NR2B was highly expressed in neurons, indicating the effect of NMDAR antagonists on JNK activation was indirect. We further observed that intrathecal injection of NMDA (twice a day for 3 days) significantly increased spinal JNK phosphorylation. Besides, NMDAR-related JNK activation could be blocked by a neuronal nitric oxide synthase (nNOS) selective inhibitor (7-nitroindazole sodium salt) but not by a nNOS sensitive guanylyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Finally, real-time RT-PCR and immunostaining showed that nerve injury-induced interleukin-1beta expression was dependent on astrocytic JNK activation. Treatments targeting NMDAR-nNOS pathway also influenced interleukin-1beta expression, which further confirmed our hypothesis. Taken together, our results suggest that neuronal NMDAR-nNOS pathway could activate astrocytic JNK pathway. Excitatory neuronal transmission initiates astrocytic activation-induced neuroinflammation in this way, which contributes to nerve injury-induced neuropathic pain.

Expression pattern of enkephalinergic neurons in the developing spinal cord revealed by preproenkephalin-green fluorescent protein transgenic mouse and its colocalization with GABA immunoreactivity.

To gain better insight into the ontogenic function of enkephalin (ENK) in the spinal cord, it is necessary to have a clear picture of the developing pattern of the ENKergic neurons. To address this question, we used transgenic mice which reveal ENKergic neurons easily by expressing enhanced green fluorescent protein (GFP) under the specific transcriptional control of the preproenkephalin (PPE) gene. GFP-positive neurons first appeared at embryonic day (E) 11.5 in the ventromedial part of the cervical ventral gray matter. At E13, they were mainly present in the intermediate zone. Thereafter, GFP-positive neurons increased progressively in number and extended from ventral to dorsal regions. Quantitative analysis showed that GFP-positive neurons peaked in number at postnatal day (P) 7 at the cervical level. The number of GFP-positive neurons reached a peak at P3 at the lumbar level. At P21, the distribution pattern of GFP immunoreactivity was similar to that in the adult spinal cord. Double-labeling results showed that about one-third of the total γ-aminobutyric acid cell population colocalized with GFP: 34.9 ± 3.5% at E16 and 32.4 ± 3.7% at P3. Double-labeled neurons accounted for nearly half of the GFP-positive neurons: 42.4 ± 2.4% at E16 and 44.1 ± 2.9% at P3. Taken together, the present results suggest that ENKergic neurons develop according to a rostrocaudal and ventrodorsal gradient. These results have broad implications for understanding the functional roles of ENKergic neurotransmission in the developing spinal cord.

Effects of intrathecal isoflurane administration on nociception and Fos expression in the rat spinal cord.

BACKGROUND AND OBJECTIVE: Isoflurane has been used as an inhaled anaesthetic for nearly 30 years. Isoflurane inhalation during anaesthesia also produces an anti-nociceptive effect. Whether this occurs at the spinal or supraspinal level remains unknown. With a novel type of liquid isoflurane, the present study examined the effects of intrathecal isoflurane on the nociceptive response and Fos expression in the rat spinal cord. METHODS: Thirty-six rats were randomly assigned to three groups as follows: group A (n = 6), intrathecal physiological saline 50 µl kg⁻¹; and group B and C (n = 6 each), intrathecal isoflurane at doses of 25 µl kg⁻¹ or 50 µl kg⁻¹, respectively. Noxious thermal (Hargreaves test), mechanical (von Frey test) and chemical (formalin 5%, 50 µl) stimuli were applied to a hind paw after intrathecal isoflurane injection to study its anti-nociceptive effect. In addition, the expression of Fos protein and c-fos mRNA in the spinal dorsal horns was detected by immunohistochemistry and real-time reverse transcriptase PCR, respectively. RESULTS: Compared with the physiological saline control, intrathecal isoflurane significantly suppressed spontaneous paw flinches in rats induced by formalin injection and paw withdrawal induced by thermal and mechanical stimuli in a dose-dependent manner. Immunohistochemistry and real-time reverse transcriptase PCR revealed that isoflurane administration inhibited formalin injection-induced c-fos expression in the spinal cord. CONCLUSIONS: These data suggest that isoflurane can exert anti-nociceptive effects at the spinal level by preventing neuronal activation.

Crosstalk between hypoxia-sensing ULK1/2 and YAP-driven glycolysis fuels pancreatic ductal adenocarcinoma development.

Autophagy and glycolysis are two catabolic processes that manipulate pancreatic ductal adenocarcinoma (PDAC) development in response to hypoxia sensing, yet the underlying mechanism of how they are interlinked remain elusive. Methods: The functional roles of Unc-51 like kinase 1 and 2 (ULK1/2) in pyruvate kinase M2 (PKM2) transcription and glycolysis under hypoxia were assessed by chromatin immunoprecipitation, luciferase reporter, glucose consumption and lactate production assay. Co-immunoprecipitation, cellular ubiquitination, His-pulldown, in vitro protein kinase assay, immunofluorescence, immunohistochemistry, CRISPR technology, in silico studies were adopted to determine the molecular mechanism. Correlation analyses were performed in KPC (Pdx1-Cre; LSL-KrasG12D/+; Trp53fl/+) mice and clinical samples from PDAC patients. Therapeutic potential of ULK1/2 inhibitor and 2-deoxyglucose (2-DG) or 3-bromopyruvate (3-BP) was evaluated in cell-derived xenograft (CDX) and the patient-derived xenograft (PDX) models of nude mice. Results: ULK1/2, but not ULK3, augments hypoxic glycolysis in PDAC cells mediated by PKM2 independent of BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3). Mechanistically, hypoxia stimulates ULK1 to translocate into nucleus, where it interacts with and phosphorylates yes-associated protein (YAP) at Ser227, resulting in YAP stabilization through blockade of ubiquitin-proteasome system (UPS), which in turn facilitates PKM2 transcription, glycolysis, cell proliferation in vitro as well as PDAC growth in mice. ULK1/2 is positively correlated with YAP and PKM2 in tumor tissues from KPC mice and clinical samples from PDAC patients. Pharmacological deactivation of ULK1/2 potentiates the antineoplastic efficacy of 2-DG and 3-BP in CDX and PDX models. Conclusion: Our findings underscore the Ser227 autophosphorylation-dependent nuclear YAP stabilization as a central node that couples ULK1/2-initiated autophagy to hypoxic glycolysis during PDAC development and propose that targeting ULK1/2 combined with 2-DG or 3-BP might be a feasible therapeutic strategy against PDAC.

Neurochemical properties of enkephalinergic neurons in lumbar spinal dorsal horn revealed by preproenkephalin-green fluorescent protein transgenic mice.

Enkephalin (ENK) has been implicated in nociceptive transmission in the spinal cord while its functional role is not clear because of difficulties in ideally visualizing ENKergic neurons. We thus developed preproenkephalin-green fluorescent protein transgenic mice to better identify ENKergic neurons. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) together with immunohistochemistry and in situ hybridization were first employed to confirm the successful transgenic manipulation and its application in showing spinal ENKergic neurons. The proportions of ENKergic neurons in the spinal cord laminae I, II, III and IV-VI among dorsal horn neurons were 15.8 +/- 3.1%, 39.5 +/- 3.3%, 11.8 +/- 1.9% and 10.7 +/- 2.1%, respectively. Double labeling with other molecules was then performed to further clarify the neurochemical properties of spinal ENKergic neurons. GABA was found to exist in 42.9 +/- 2.8% of ENKergic neurons that were mainly located in lamina I-III. The proportions of parvalbumin-, calretinin-, calbindin- and neuronal nitric oxide synthase-positive cells among the ENKergic neurons were 5.2 +/- 0.7%, 42.6 +/- 2.3%, 25.8 +/- 2.2% and 11.1 +/- 1.6%, respectively. Compared with previously findings obtained with ENK antibody labeling, this line of newly generated mice can be a reliable tool for the study of specific spinal ENKergic neuronal population.

[Preliminary Screening for the Urban Forest Against Combined Air Pollution].

Combined air pollution has become one of the most important city diseases in China. The construction of an urban forest not only needs landscape aesthetics, but also requires selecting a plant of high comprehensive tolerance threshold based on the needs of the ecological environment of each city, which has become a standard to maintain the sustainable development of the urban forest ecological function under environmental pollution. According to the comprehensive factor analysis of the sorption and absorption capacity of 537 plants to six air pollutants (i.e., sulfur dioxide, nitrogen dioxide, hydrogen fluoride, chlorine, ozone, and particulate matters), the results showed that the tree species with strong comprehensive tolerance ability to six air pollutants were Morus alba, Platycladus orientalis, and Ailanthus altissima; the tree species with medium comprehensive tolerance ability were Populus tomentosa, Acer truncatum, Sabina chinensis, Amygdalus davidiana, Salix babylonica, Paulownia fortunei, and Pinus tabulaeformis; the trees species with relatively weak comprehensive tolerance ability were Robinia pseudoacacia, Populus×canadensis, Ginkgo biloba, Juglans regia, Platanus acerifolia, Koelreuteria paniculata, Lagerstroemia indica, and Forsythia suspensa. According to the characteristics of climate, economic structure, and air pollutants of the cities in the north and south of China, the urban forest should be constructed using selected species with a strong comprehensive tolerance ability to achieve maximum purification effect of the urban forest ecological service function.

Impact of the Family Environment on the Emotional State of Medical Staff During the COVID-19 Outbreak: The Mediating Effect of Self-Efficacy.

During the outbreak of the coronavirus disease 2019 (COVID-19), the medical staff was facing severe work pressure, which led to a negative emotional state. The purpose of this study was to explore the relationship between the family environment and the emotional state of the medical staff members during the COVID-19 outbreak. Due to the importance of self-efficacy in regulating mental health, the mediating role of self-efficacy in the association between family environment and emotional state was also explored. A cross-sectional survey was performed, using an online questionnaire, on 645 medical staff who participated in the epidemic prevention and control tasks during the COVID-19 outbreak in Beijing. Family environment, self-efficacy, anxiety, and depressive symptoms were measured by the Family Environment Scale-Chinese Version (FES-CV), the General Self-Efficacy Scale (GSES), the Generalized Anxiety Disorder Scale-7 (GAD-7), and the Patient Health Questionnaire-9 (PHQ-9), respectively. Correlation analysis and mediating effect analysis were used to explore the relationships between them. First, a higher prevalence of anxiety (39%) and depressive (33%) symptoms were confirmed among the medical staff. Second, the symptoms of anxiety and depression were negatively correlated with the dimensions of cohesion and expressiveness and positively correlated with the dimensions of conflict in the FES-CV scale. Third, self-efficacy significantly mediated the association between the family environment and anxiety symptoms (P < 0.001) as well as the family environment and depressive symptoms (P < 0.001). These findings show that a negative family environment was the main predictor of symptoms of anxiety and depression in the medical staff during the COVID-19 outbreak. Furthermore, we found that self-efficacy played a critical mediating role between the family environment and the symptoms of anxiety and depression. Our study also indicates that improvements in the family environment benefit the mental health care of the medical staff, and high self-efficacy enhances this effect.