Anxiety disorder is a major symptom of autism spectrum disorder (ASD) with a comorbidity rate of ~40%. However, the neural mechanisms of the emergence of anxiety in ASD remain unclear. In our study, we found that hyperactivity of basolateral amygdala (BLA) pyramidal neurons (PNs) in Shank3 InsG3680 knock-in (InsG3680^+/+) mice is involved in the development of anxiety. Electrophysiological results also showed increased excitatory input and decreased inhibitory input in BLA PNs. Chemogenetic inhibition of the excitability of PNs in the BLA rescued the anxiety phenotype of InsG3680^+/+ mice. Further study found that the diminished control of the BLA by medial prefrontal cortex (mPFC) and optogenetic activation of the mPFC-BLA pathway also had a rescue effect, which increased the feedforward inhibition of the BLA. Taken together, our results suggest that hyperactivity of the BLA and alteration of the mPFC-BLA circuitry are involved in anxiety in InsG3680^+/+ mice.
Animals ; Prefrontal Cortex/metabolism* ; Basolateral Nuclear Complex/metabolism* ; Mice ; Anxiety/metabolism* ; Nerve Tissue Proteins/genetics* ; Male ; Gene Knock-In Techniques ; Pyramidal Cells/physiology* ; Mice, Transgenic ; Neural Pathways/physiopathology* ; Mice, Inbred C57BL ; Microfilament Proteins

Microbial communities such as those residing in the human gut are highly diverse and complex, and many with important implications for health and diseases. The effects and functions of these microbial communities are determined not only by their species compositions and diversities but also by the dynamic intra- and inter-cellular states at the transcriptional level. Powerful and scalable technologies capable of acquiring single-microbe-resolution RNA sequencing information in order to achieve a comprehensive understanding of complex microbial communities together with their hosts are therefore utterly needed. Here we report the development and utilization of a droplet-based smRNA-seq (single-microbe RNA sequencing) method capable of identifying large species varieties in human samples, which we name smRandom-seq2. Together with a triple-module computational pipeline designed for the bacteria and bacteriophage sequencing data by smRandom-seq2 in four human gut samples, we established a single-cell level bacterial transcriptional landscape of human gut microbiome, which included 29,742 single microbes and 329 unique species. Distinct adaptive response states among species in Prevotella and Roseburia genera and intrinsic adaptive strategy heterogeneity in Phascolarctobacterium succinatutens were uncovered. Additionally, we identified hundreds of novel host-phage transcriptional activity associations in the human gut microbiome. Our results indicated that smRandom-seq2 is a high-throughput and high-resolution smRNA-seq technique that is highly adaptable to complex microbial communities in real-world situations and promises new perspectives in the understanding of human microbiomes.
Humans ; Gastrointestinal Microbiome/genetics* ; Bacteriophages/physiology* ; High-Throughput Nucleotide Sequencing ; Sequence Analysis, RNA/methods* ; Bacteria/virology*

Although widely applied in treating hematopoietic malignancies, transplantation of hematopoietic stem/progenitor cells (HSPCs) is impeded by HSPC shortage. Whether circulating HSPCs (cHSPCs) in steady-state blood could be used as an alternative source remains largely elusive. Here we develop a three-dimensional culture system (3DCS) including arginine, glycine, aspartate, and a series of factors. Fourteen-day culture of peripheral blood mononuclear cells (PBMNCs) in 3DCS led to 125- and 70-fold increase of the frequency and number of CD34⁺ cells. Further, 3DCS-expanded cHSPCs exhibited the similar reconstitution rate compared to CD34⁺ HSPCs in bone marrow. Mechanistically, 3DCS fabricated an immunomodulatory niche, secreting cytokines as TNF to support cHSPC survival and proliferation. Finally, 3DCS could also promote the expansion of cHSPCs in patients who failed in HSPC mobilization. Our 3DCS successfully expands rare cHSPCs, providing an alternative source for the HSPC therapy, particularly for the patients/donors who have failed in HSPC mobilization.
Antigens, CD34/metabolism* ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; Humans ; Leukocytes, Mononuclear/metabolism* ; Peptides/metabolism*

In recent years,studies have shown that inflammation is an important factor in secondaryischemic injury.The exploration of the related mechanisms of the occurrence of inflammation has become ahot spot in the field of cerebral ischemia.Toll-like receptor 4 (TLR4) signaling pathway is one of theclassical inflammatory pathways.Studies have shown that TLR4 is involved in the early inflammatoryresponse after cerebral ischemic injury and the late nerve repair.This article reviews the research progress ofthis signaling pathway in cerebral ischemia injury,so as to seek a therapeutic target against inflammatoryinjury caused by cerebral ischemia through the analysis of its potential research direction.

The aim of the study was to prepare enrofloxacin nanosuspension injection and evaluate its pharmacokinetics after giving a single intramuscular injection.The high pressure homogeneous technique was used to prepare enrofloxacin nanosuspension injection and preliminary evaluation of the quality was done.The high performance liquid chromatography (HPLC) method was used to determinate content of enrofloxacin in pig plasma.And the pharmacokinetic characteristics of enrofloxacin nanosuspension injection were compared with Baytril injection.The content of enrofloxacin in this preparation is 97.9％.The average particle size of enrofloxacin nanosuspension injection was (613.21±5.78) nm,PDI was (0.22±0.02) and the potential was-2.02 mV.Maximal plasma concentrations were (0.32±0.12) and (0.67 ± 0.09) mg/L after i.m administration with enrofloxacin nanosuspension injection and Baytril injection.The peak times were (2.88 ±0.96) and (0.79±0.26) hours,respectively.Mean elimination half-lifes were (5.99± 1.37) and (4.49 ± 1.25) hours,respectively.Areas under concentration-time curve were (4.63±1.30) and (4.40±0.45) mg/L · h,respectively.Mean residence times were (9.59±2.34) and (5.41±1.10) hours,respectively.The relative bioavailability of enrofloxacin nanosuspension injection was 105.2％.The preparation method of high pressure homogeneous was simple and good reproducibility.Enrofloxacin nanosuspension injection was characterized by non-sedimentation,easy-redispersion,relatively stable.Comparing with Baytril injection,enrofloxacin nanosuspension injection had a certain slowrelease effect,showing slower elimination than enrofloxacin injeetion.

Objective To investigate the effects of compound Danshen injection on stress-induced gastric mucosal damage and its mechanism.Methods Sprague Dawley rats were used to establish immersion restraint cold stress model.50 SD rats were randomly divided into five groups(n=10 per group):① the normal control group; ② the stress model group; ③ the group treated with low dose of compound Danshen injection(50 mg/kg) ; ④ the group treated with medial dose of compound Danshen injection(100 mg/kg) ; ⑤the group treated with high dose of compound Danshen injection (200 mg/kg).The gastric mucosal ulcer index (UI),the level of malondialdehyde (MDA) in gastric mucosa,the activity of superoxide dismutase (SOD),and plasma intedeukin-1beta (IL-1β)content were detected.Results Compared with the normal control group [(0.7± 0.4) mm,(0.25 ± 0.03)nmol/mg,and (246.5 ± 45.4)Nu/mg],the stress model group exhibited a markedly increase in gastric mucosal UI[(28.4±4.5)mm](P＜0.01) and MDA level [(0.60±0.08)nmol/mg](P＜0.01) and a decrease in the activity of gastric mucosal SOD [(122.5 ±14.2) Nu/mg] (P＜0.01).In addition,an elevated level of plasma IL-1β[(31.6±8.4) pg/ml vs (8.5±3.1) pg/ml] (P＜0.01) was observed in the stress model group.Pretreatment with compound Danshen injection dose-dependently attenuated the gastric mucosal damage,characterized by a markedly decrease in gastric mucosal UI [(22.8 ± 3.7)mm、(12.2 ±3.5)mm,(6.2 ± 1.6)mm] and MDA level [(0.52 ± 0.07)nmol/mg,(0.32 ± 0.06)nmol/mg,(0.28 ±0.03)nmol/mg] and an increase in the activity of gastric mucosal SOD[(135.2± 13.6)Nu/mg,(220.7±33.5)Nu/mg,(251.2±23.7)Nu/mg] (P＜0.01).Meanwhile,compound Danshen injection reduced the content of plasma IL-1β[(27.2±7.5)pg/ml,(13.5±5.3)pg/ml,(9.3±4.4)pg/ml] (P＜0.01).Conclusion Compound Danshen injection exhibits preventive protection on the stress-induced gastric mucosal damage by water immersion restraint cold stress in SD rats,the mechanism of which might be linked to its inhibition of gastric mucosal oxidation stress and the reduced release of inflammatory mediator IL-1β.

Objective To investigate the effects of tetrandrine on myocardial hypertrophy in renohypertensive rats and its possible mechanism.Methods The myocardial hypertrophy models were established in two-kidney-one-clip (2K1C)renovascular hypertensive rats.Before renal artery constriction,all the rats were randomly divided into four groups(n=15 per group):(1) the sham-operated control group; (2) the 2K1C renohypertensive group; (3) the tetrandrine group,the two-kidney-one-clip renohypertensive rats were treated with tetrandrine at a dose of 50 ml/kg · d-1 from the post-operated 5th week; (4) the enapril group,the two-kidney-one-clip renohypertensive rats were treated with enapril at a dose of 6 ml/kg· d-1 from the post-operated 5th week.The standard tail-cuff method was used to measure blood pressure in conscious rats.After drug treatment for 8 weeks,the rats were killed and left ventricle was obtained to measure the ratio of left ventricle weight to body weight (LVW/BW),myocardial angiotensin Ⅱ content,and mRNA expressions of inflammatory cytokines tumor necrosis factor-α(TNF-α)and interleukin-1 β (IL-1β).Results Compared with shamgroup[(14.90±3.31)kPa; (1.89±0.27)mg/g; (27.38±7.10)pg/mg; (0.72±0.10)and(0.65±0.10)fold of GAPDH expression],the untreated 2K1C renohypertensive rats exhibited a significant increase in blood pressure [(23.53 ± 3.40) kPa],LVW/BW [(2.83 ± 0.40) mg/g],angiotensin Ⅱ content [(43.51 ± 7.37) pg/mg],and mRNA expressions of TNF-α and IL-1β [(1.47 ± 0.14) and (1.07 ± 0.11) fold of GAPDH expression].Treatment with tetradrine significantly attenuated the increase in blood pressure [(15.81 ± 3.12) kPa] and LVW/BW [(1.94 ±0.31) mg/g],angiotensin Ⅱ content [(31.31 ± 6.69) pg/mg],and mRNA expressions of TNF-α and IL-1β [(0.76 ±0.11) and (0.63 ± 0.09) fold of GAPDH expression].Conclusion Long-term related to its inhibition of local production or release of angiotensin Ⅱ and inflammatory cytokines TNF-α and IL-1β in the myocardium of renohypertensive rats.

OBJECTIVETo investigate the transcriptional regulation of pacemaker channel I(f) mediated by vasoactive peptide endothelin-1 (ET-1) in neonatal rat ventricular myocytes and its mechanism.

METHODSNeonatal rat ventricular myocytes were enzymatically isolated. I(f) current was recorded using the whole-cell patch-clamp technique. The expression of hyperpolarization-activated cyclic nucleotide-gated channel (HCN) isoforms HCN2 and HCN4 were measured by quantitative RT-PCR.

RESULTSET-1 increased the expression of HCN2 and HCN4 mRNA in a dose- and time-dependent manner. These effects were blocked by specific ETA receptor antagonist BQ-123 but not the ETB receptor antagonist BQ-788. The effects of ET-1 on HCN2 and HCN4 mRNA expression were not affected by the p38 mitogen-activated protein kinase (MAPK) inhibitor (SB-203580).

CONCLUSIONThese findings indicate that ET-1 stimulates the expression of pacemaker channel I(f) in cardiomyocytes via ETA receptor through a p38 MAPK-independent signaling pathway, which might be linked to the intrinsic arrhythmogenic potential of ET-1.

Animals ; Animals, Newborn ; Cyclic Nucleotide-Gated Cation Channels ; drug effects ; Endothelin-1 ; metabolism ; Imidazoles ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; Oligopeptides ; pharmacology ; Patch-Clamp Techniques ; Piperidines ; pharmacology ; Pyridines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

BACKGROUND: Blood-letting puncture on 12-well points of hand is a kind of effective emergent approaches on cerebral apoplexy. It is testified in animal experiment that bleeding on 12-well points of hand can dilate cerebral vessels, enhance blood flow in brain, improve acute anoxic state in ischemic brain tissue and relieve acid toxin due to accumulation of lactate.OBJECTIVE: To explore the effects and mechanism of blood-letting puncture of 12-well points of hand nitric oxide (NO) contents and nitricoxide synthase (NOS) activity after cerebral ischemia in rats.DESIGN: Randomized controlled animal experiment was designed.SETTING: Department of Physiology of Medical Institute of Xianning College.MATERIALS: The experiment was performed in Department of Physiology of Medical Institute of Xianning College from March 2003 to February 2004. Totally 84 Wistar rats were employed in the experiment, aged of 2 or 3 months, of either sex, body weighted (230±20) g and provided from Experimental Animal Center of Medical Institute of Xianning College.METHODS: Totally 84 rats were randomized into sham operation group,ischemia group and ischemia + bleeding group, 28 rats in each one. Modified Longa method [3] was applied to prepare the model of embolism of cerebral middle artery in rat. In ischemia + bleeding group, after cerebral ischemia, blood-letting puncture was applied with three-edged needle on Shaoshang (LU11), Shangyang (LI1), Zhongchong (PC9), Guanchong (TE1),Shoochong (HT9) and Shaoze (SI1) in sequence firstly on the left foreleg,and then on the right one, corresponding to the analogy of 12-well points of hand of human. One blood drop was just required. NO content and NOS activity were assayed in 30 minutes, 1 hour, 2 hours and 4 hours of ischemia in brain tissue successively in each group.MAIN OUTCOME MEASURES: NO content and NOS activity in brain tissue in each group.RESULTS: ① NO content in 30 minutes, 1 hour, 2 hours and 4 hours of ischemia in ischemia group was (116.16±26.63), (118.94±24.47),(115.65±25.29) and (108.87±26.52) μmol/L successively and NOS ac tivity was (507.22±92.52), (502.08±92.52), (510.71±96.63) and (495.29-±88.41) μkat/L, which was higher significantly than the sham operation group (t=2.474-4.731, P ＜ 0.05 or 0.001). ② In ischemia + bleeding group,, NO content was (91.8±11.51), (93.55±13.88), (92.52±11.62) and (84.3±11.51) μmol/L successively and NOS activity was (337.6±88.41),(340.99±96.63), (344.48±84.3) and (337.6±90.46) μkat/L, indicating significant difference in comparison with ischemia group (t=2.199-3.507,P ＜ 0.05-0.01).CONCLUSION: Blood-letting puncture on 12-well points of hand inhibits the increased NO content and NOS activity in ischemic brain tissue and alleviates the injury of free radical to brain tissue so that the focal brain ischemia of rats is protected.

BACKGROUND: L-arginine (L-Arg) is the precursor of endogenous nitric oxide (NO), which joins the pain regulation of peripheral myeloid level and above.OBJECTIVE: To study the role and mechanism of NO in nucleus raphe magnus (NRM) in pain regulation and electroacupuncture analgesia.DESIGN: A random control experiment on rats.SETTING: Physiology Department of Medical College of Xianning College.MATERIALS: The experiment was done during May 2002 to March 2003in the Physiology Department of Medical College of Xianning College. The 63 Wistar rats were randomized into 7 groups with 9 in each. ①. Five groups for the experiment of L-Arg dose-effect relationship: Normal saline group and L-Arg 1, 2, 4, 8 mmol groups. ②. Two groups for the experiment of relationship between L-Arg and electroacupuncture analgesia: Normal saline + electroacupuncture group and L-Arg + electroacupuncture group.METHODS: ①Experiment of L-Arg dose-effect relationship: Normal saline group was injected normal saline. L-Arg 1, 2, 4, 8 mmol groups were injected L-Arg 1, 2, 4, 8 mmol respectively. The volume was all 1.5 μL.Then, the hot water (50±0.5) ℃ was used to stimulate the tail once every 10 minutes to cause tail flick for testing the pain threshold. The observation was carried out continuously for 90 minutes. ②. Experiment of relationship between L-Arg and electroacupuncture analgesia: The two groups were injected normal saline and L-Arg 8 mmol respectively. The volume was all 1.5 μL. Ten minutes later, Zusanli (ST 36) of both posterior legs of rats were punctured with electric stimulation. The frequency was 4-16 Hz, and the intensity in an increasing order of 1, 2, 3V given 10 minutes of each. The electroacupuncture was totally 30 minutes, during which, testing the pain threshold 3 times. After needling, the pain threshold was still tested once every 10 minutes, till 90 minutes after injecting L-Arg.MAIN OUTCOME MEASURES: Pain threshold at different time points.RESULTS: The 63 rats entered the result analysis. ① The pain threshold of L-Arg 1 mmol group at all time points was lower than that of the normal saline group, but without statistical significance (P ＞ 0.05). The pain threshold of L-Arg 2, 4, 8 mmol groups was much lower than that of the normal saline group at all time points (P ＜ 0.001), and the effect was lasting to 80 minutes after injecting L-Arg, and with the increase of L-Arg density, the amplitude of pain threshold lowering was increased. ② The pain threshold of L-Arg + electroacupuncture group was much lower than that of the normal saline + electroacupuncture group at the time points of 20-50 minutes (P ＜ 0.01).CONCLUSION: ① The L-Arg injection to NRM can lower the pain threshold and there is a dose-dependent effect in it. ② The electroacupuncture can raise the pain threshold, and the L-Arg injection can weaken the effect of electroacupuncture analgesia. ③ NO in NRM joins in the pathological and physiological processes of pain and electroacupuncture analgesia. Its increase can lower the pain threshold greatly.