[Electroacupuncture improves limb motor function by down-regulating cytosolic phospholi-pase A2 and reducing apoptosis of nerve cells in rats with spinal cord injury].

OBJECTIVE: To observe the effect of electroacupuncture（EA） on the expression of cytosolic phospholipase A2 （cPLA2） and apoptosis of nerve cells in rats with spinal cord injury （SCI）, so as to explore its mechanisms underlying improvement of SCI. METHODS: Seventy-two female SD rats were randomly divided into model, EA, antagonist and EA+antagonist groups, with 18 rats in each group and other 18 rats were used as the sham operation （sham） group. The SCI model was established by referring to modified Allen's method with a weight impactor. The hindlimb motor function was assessed by using Basso-Beattie-Bresnahan （BBB） score. Rats of the EA group were subjected to EA stimulation at "Dazhui"（GV14）, "Yaoyangguan"（GV3）, bilateral "Ciliao"（BL32） and "Zusanli"（ST36） for 20 min, once a day for 14 days. Rats of the antagonist group received intravenous injection followed by intraperitoneal injection of arachidonyl trifluoromethyl ketone （AACOCF3, antagonist of cPLA2）, once every other day. Rats of the EA+antagonist group received EA treatment combined with antagonist injection. After the treatment, the rats were sacrificed and the spinal cord tissue was collected for detecting the protein expression of cPLA2, p-cPLA2, Bcl-2, Bax and Caspase-3 by Western blot, and the mRNA expression of cPLA2, Bcl-2, Bax and Caspase-3 using qRT-PCR. The morphological changes of the spinal cord were detected by Nissl staining. RESULTS: In comparison with the sham group, the BBB score, expression of Bcl-2 protein and mRNA were significantly down-regulated （P<0.01）, whereas the expression levels of Bax, Caspase-3 and p-cPLA2 proteins and mRNAs were considerably up-regulated in the model group （P<0.01）. Compared with the model group, the BBB score, expression levels of Bcl-2 protein and mRNA were significantly up-regulated （P<0.01, P<0.05）, while the expression levels of Bax, Caspase-3 and p-cPLA2 proteins in the EA, antagonist and EA+antagonist groups, Bax and cPLA2 mRNAs in both antagonist and EA+antagonist groups, and Caspase-3 mRNA in the EA+antagonist group were obviously down-regulated （P<0.01, P<0.05）. The effect of EA+antagonist was significantly superior to EA in increasing BBB score and in lowering expression of Bax and cPLA2 mRNAs （P<0.01, P<0.05）. Nissl staining showed reduced number of nerve cells and Nissl bodies, and striped dark blue cells in the model group, which was milder in the EA and antagonist groups, particularly in the EA+antagonist group. CONCLUSION: EA may improve the limb motor function of SCI rats, which may be related to its functions in down-regulating the expression of p-cPLA2, Bax and Caspase-3 and up-regulating Bcl-2 to reduce the apoptosis of nerve cells in the regional spinal cord.

Knockin of the G275E mutation of the nicotinic acetylcholine receptor (nAChR) α6 confers high levels of resistance to spinosyns in Spodoptera exigua.

Spinosyns, including spinosad and spinetoram, act on the insect central nervous system, gradually paralyzing or destroying the target insect. Spinosad resistance is associated with loss-of-function mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit in a number of agricultural pests. Using gene editing, nAChR α6 has been verified as a target for spinosyns in five insect species. Recently, a point mutation (G275E) in exon 9 of nAChR α6 was identified in spinosad-resistant strains of Thrips palmi and Tuta absoluta. To date, no in vivo functional evidence has been obtained to support that this mutation is involved in spinosyn resistance in lepidopteran pests. In this study, the G275E mutation was introduced into the nAChR of Spodoptera exigua using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) gene-editing technology. Reverse transcriptase-polymerase chain reaction and sequencing confirmed that this mutation was present in exon 9 of the nAChR transcripts in the edited 275E strain. The results of bioassays showed that the 275E strain was highly resistant to spinosad (230-fold) and spinetoram (792-fold) compared to the unedited background strain, directly confirming that the G275E mutation of the nAChR α6 subunit confers high levels of spinosyn resistance in S. exigua. Inheritance analysis showed that the resistance trait is autosomal and incompletely recessive. This study employs a reverse genetics approach to validate the functional role played by the G275E mutation in nAChR α6 of S. exigua in spinosyns resistance and provides another example of the use of CRISPR/Cas9 gene-editing technology to confirm the role played by candidate target site mutations in insecticide resistance.

Activation of a hippocampal CREB-pCREB-miRNA-MEF2 axis modulates individual variation of spatial learning and memory capability.

Phenotypic variation is a fundamental prerequisite for cell and organism evolution by natural selection. Whereas the role of stochastic gene expression in phenotypic diversity of genetically identical cells is well studied, not much is known regarding the relationship between stochastic gene expression and individual behavioral variation in animals. We demonstrate that a specific miRNA (miR-466f-3p) is upregulated in the hippocampus of a portion of individual inbred mice upon a Morris water maze task. Significantly, miR-466f-3p positively regulates the neuron morphology, function and spatial learning, and memory capability of mice. Mechanistically, miR-466f-3p represses translation of MEF2A, a negative regulator of learning/memory. Finally, we show that varied upregulation of hippocampal miR-466f-3p results from randomized phosphorylation of hippocampal cyclic AMP (cAMP)-response element binding (CREB) in individuals. This finding of modulation of spatial learning and memory via a randomized hippocampal signaling axis upon neuronal stimulation represents a demonstration of how variation in tissue gene expression lead to varied animal behavior.

Neurobiological changes in striatal glutamate are associated with trait impulsivity of differential reinforcement of low-rate-response behavior in male rats.

Impulsive action can be measured using rat's responses on a differential reinforcement of low-rate-response (DRL) task in which performance may be varied between rats. Nevertheless, neurobiological profiles underlying the trait impulsivity of DRL behavior remain largely unknown. Here, in vivo non-invasive proton magnetic resonance spectroscopy (1H-MRS) and Western blot assay were performed to assess neurobiological changes in the dorsal striatum (DS) and nucleus accumbens (NAc) in relation to individual differences in DRL behavior. A cohort of rats was subjected to acquire a DRL task over 14 daily sessions. High impulsive (HI) and low impulsive (LI) rats were screened by behavioral measures displaying a lower response efficiency and performing more nonreinforced responses in HI rats and vice versa. MRS measurements indicated that the HI group had a lower NAc glutamate (Glu) level than did the LI group, whereas no such difference was found in the other five metabolites in this area. Moreover, no intergroup difference was observed in any metabolite in the DS. The results of Western blot assay revealed that protein expressions of GluN1 (but not GluN2B) subunit of N-methyl-D-aspartate receptors in the DS and NAc were higher in the HI group than in the LI group. This inherent timing impulsivity was not attributed to risky behavioral propensity because both Hl and LI rats could acquire a risk-dependent choice. The findings of this study, supported by certain correlations among behavioral, brain imaging, and neuroreceptor indices, provide evidence of the neurobiological changes of striatal Glu underlying trait impulsive action of DRL behavior.

Hydrogen Sulfide Relaxes Human Uterine Artery via Activating Smooth Muscle BKCa Channels.

Opening of large conductance calcium-activated and voltage-dependent potassium (BKCa) channels hyperpolarizes plasma membranes of smooth muscle (SM) to cause vasodilation, underling a key mechanism for mediating uterine artery (UA) dilation in pregnancy. Hydrogen sulfide (H2S) has been recently identified as a new UA vasodilator, yet the mechanism underlying H2S-induced UA dilation is unknown. Here, we tested whether H2S activated BKCa channels in human UA smooth muscle cells (hUASMC) to mediate UA relaxation. Multiple BKCa subunits were found in human UA in vitro and hUASMC in vitro, and high ß1 and γ1 proteins were localized in SM cells in human UA. Baseline outward currents, recorded by whole-cell and single-channel patch clamps, were significantly inhibited by specific BKCa blockers iberiotoxin (IBTX) or tetraethylammonium, showing specific BKCa activity in hUASMC. H2S dose (NaHS, 1-1000 µM)-dependently potentiated BKCa currents and open probability. Co-incubation with a Ca2+ blocker nifedipine (5 µM) or a chelator (ethylene glycol-bis (ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 5 mM) did not alter H2S-potentiated BKCa currents and open probability. NaHS also dose-dependently relaxed phenylephrine pre-constricted freshly prepared human UA rings, which was inhibited by IBTX. Thus, H2S stimulated human UA relaxation at least partially via activating SM BKCa channels independent of extracellular Ca2+.

Enhanced Stromal Cell CBS-H2S Production Promotes Estrogen-Stimulated Human Endometrial Angiogenesis.

Angiogenesis is a physiological process for endometrial regeneration in the menstrual cycle and remodeling during pregnancy. Endogenous hydrogen sulfide (H2S), produced by cystathionine-ß synthase (CBS) and cystathionine-γ lyase (CSE), is a potent proangiogenic factor; yet, whether the H2S system is expressed in the endometrium and whether H2S plays a role in endometrial angiogenesis are unknown. This study was to test whether estrogens stimulate endometrial H2S biosynthesis to promote endometrial microvascular endothelial cell (EMEC) angiogenesis. CBS messenger RNA/protein and H2S production significantly differed among endometria from postmenopausal (POM), premenopausal secretory (sPRM), and proliferative (pPRM) nonpregnant (NP) and pregnant (Preg) women (P < .05) in a rank order of POM approximately equal to sPRM is less than pPRM is less than Preg, positively correlating with angiogenesis indices and endogenous estrogens and with no difference in CSE expression. CBS and CSE proteins were localized to stroma, glands, and vessels in endometrium, and greater stromal CBS protein was observed in the pPRM and Preg states. Estradiol-17ß (E2) (but not progesterone) stimulated CBS (but not CSE) expression and H2S production in pPRM endometrial stromal cells (ESCs) in vitro, which were attenuated by ICI 182 780. The H2S donor sodium hydrosulfide promoted in vitro EMEC angiogenesis. Co-culture with sPRM, pPRM, and Preg ESCs all stimulated EMEC migration with a rank order of sPRM less than pPRM approximately equal to Preg. CBS (but not CSE) inhibition attenuated ESC-stimulated EMEC migration. E2 did not affect EMEC migration but potentiated ESC-stimulated EMEC migration. Altogether, estrogens stimulate specific receptor-dependent stromal CBS-H2S production to promote endometrial EMEC angiogenesis in women.

Knockout of three aminopeptidase N genes does not affect susceptibility of Helicoverpa armigera larvae to Bacillus thuringiensis Cry1A and Cry2A toxins.

Bacillus thuringiensis (Bt) insecticidal toxins have been globally utilized for control of agricultural insects through spraying or transgenic crops. Binding of Bt toxins to special receptors on midgut epithelial cells of target insects is a key step in the mode of action. Previous studies suggested aminopeptidase N1 (APN1) as a receptor or putative receptor in several lepidopteran insects including Helicoverpa armigera through evidence from RNA interefence-based gene silencing approaches. In the current study we tested the role of APNs in the mode of action of Bt toxins using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated gene knockout. Three APN genes (HaAPN1, HaAPN2 and HaAPN5) were individually knocked out in a susceptible strain (SCD) of H. armigera to establish three homozygous knockout strains. Qualitative in vitro binding studies indicated binding of Cry1Ac or Cry2Ab to midgut brush border membrane vesicles was not obviously affected by APN knockout. Bioassay results showed that none of the three knockouts had significant changes in susceptibility to Cry1A or Cry2A toxins when compared with the SCD strain. This suggests that the three HaAPN genes we tested may not be critical in the mode of action of Cry1A or Cry2A toxins in H. armigera.

Identification of the ryanodine receptor mutation I4743M and its contribution to diamide insecticide resistance in Spodoptera exigua (Lepidoptera: Noctuidae).

Insect ryanodine receptors (RyRs) are the targets of diamide insecticides. Two point mutations G4946E and I4790M (numbering according to Plutella xylostella, PxRyR) in the transmembrane domain of the insect RyRs associated with diamide resistance have so far been identified in three lepidopteran pests, P. xylostella, Tuta absoluta and Chilo suppressalis. In this study, we identified one of the known RyR target site resistance mutations (I4790M) in a field-collected population of Spodoptera exigua. The field-collected WF population of S. exigua exhibited 154 fold resistance to chlorantraniliprole when compared with the susceptible WH-S strain. Sequencing the transmembrane domains of S. exigua RyR (SeRyR) revealed that the resistant WF strain was homozygous for the I4743M mutation (corresponding to I4790M in PxRyR), whereas the G4900E allele (corresponding to G4946E of PxRyR) was not detected. The 4743M allele was introgressed into the susceptible WH-S strain by crossing WF with WH-S, followed by three rounds of backcrossing with WH-S. The introgressed strain 4743M was homozygous for the mutant 4743M allele and shared about 94% of its genetic background with that of the recipient WH-S strain. Compared with WH-S, the near-isogenic 4743M strain showed moderate levels of resistance to chlorantraniliprole (21 fold), cyantraniliprole (25 fold) and flubendiamide (22 fold), suggesting that the I4743M mutation confers medium levels of resistance to all three diamides. Genetic analysis showed diamide resistance in the 4743M strain was inherited as an autosomal and recessive trait. Results from this study have direct implications for the design of appropriate resistance monitoring and management practices to sustainably control S. exigua.

Synthesis of Epoxides from Alkyl Bromides and Alcohols with in Situ Generation of Dimethyl Sulfonium Ylide in DMSO Oxidations.

Direct conversion of the readily available alkyl bromides and alcohols to value-added epoxides using dimethyl sulfoxide (DMSO) under mild reaction conditions has been developed. Benzyl and allyl bromides, and activated and unactivated alcohols all proceeded smoothly to give epoxides in high to excellent yield. Dimethyl sulfide, generated by DMSO oxidations, was in situ elaborated to form the substituted dimethyl sulfonium ylide species that participates in the Corey-Chaykovsky epoxidation in a domino and one-pot fashion, respectively.

Genetic analysis and molecular detection of resistance to chlorpyrifos mediated by the A216S substitution in acetylcholinesterase-1 in the plant bug Apolygus lucorum.

The green plant bug Apolygus lucorum is a major pest of Bacillus thuringiensis cotton in China. Previously, we reported that chlorpyrifos resistance in a laboratory-selected strain of A. lucorum (BZ-R) is associated with the homozygosis of an allele in the ace-1 gene encoding an alanine to serine substitution at position 216 of acetylcholinesterase-1. Here we describe the results of crosses between the resistant BZ-R strain (41-fold to chlorpyrifos) and the unselected susceptible BZ-S strain homozygous for the wild type alanine allele at position 216. Resistance to chlorpyrifos was inherited as a semi-dominant trait mainly controlled by a single autosomal gene and co-segregates strongly but not completely with the serine substitution in ace-1. Synergism bioassays and enzyme assays showed that minor contributions to resistance are also made by enhanced cytochrome P450 and carboxylesterase activities. A survey of 25 field populations from five Chinese provinces showed strong positive correlations between 50% lethal concentration against chlorpyrifos and S216 allele and genotype frequencies, although the most tolerant populations still only show 40%-50% S216 allele frequencies. The results above provide important information for designing effective resistance monitoring and management strategies for A. lucorum in China.

Task-Dependent Differences in Operant Behaviors of Rats With Acute Exposure to High Ambient Temperature: A Potential Role of Hippocampal Dopamine Reuptake Transporters.

Behavioral or cognitive functions are known to be influenced by thermal stress from the change in ambient temperature (Ta). However, little is known about how increased Ta (i.e., when the weather becomes warm or hot) may affect operant conditioned behavior and the neural substrates involved. The present study thus investigated the effects of high Ta on operant behaviors maintained on a fixed-ratio 1 (FR1) and a differential reinforcement for low-rate responding 10 s (DRL 10-s) schedule of reinforcement. The rats were randomly assigned to three groups receiving acute exposure to Ta of 23°C, 28°C, and 35°C, respectively, for evaluating the effects of high Ta exposure on four behavioral tests. Behavioral responses in an elevated T-maze and locomotor activity were not affected by Ta treatment. Regarding operant tests, while the total responses of FR1 behavior were decreased only under 35°C when compared with the control group of 23°C, those of DRL 10-s behavior were significantly reduced in both groups of 28°C and 35°C. Distinct patterns of inter-response time (IRT) distribution of DRL behavior appeared among the three groups; between-group differences of behavioral changes produced by high Ta exposure were confirmed by quantitative analyses of IRT data. Western blot assays of dopamine (DA) D1 and D2 receptor, DA transporter (DAT) and brain-derived neurotrophic factor (BDNF) were conducted for the sample tissues collected in six brain areas from all the subjects after acute high Ta exposure. Significant Ta-related effects were only revealed in the dorsal hippocampus (dHIP). In which, the DAT levels were increased in a Ta-dependent fashion that was associated with operant behavior changes under high Ta exposure. And, there as an increased level of D1 receptors in the 28°C group. In summary, these data indicate that the performance of operant behavior affected by the present high Ta exposure is task-dependent, and these changes of operant behaviors cannot be attributed to gross motor function or anxiety being affected. The regulation of dHIP DAT may be involved in this operant behavioral change under high Ta exposure.

Two novel sodium channel mutations associated with resistance to indoxacarb and metaflumizone in the diamondback moth, Plutella xylostella.

Indoxacarb and metaflumizone belong to a relatively new class of sodium channel blocker insecticides (SCBIs). Due to intensive use of indoxacarb, field-evolved indoxacarb resistance has been reported in several lepidopteran pests, including the diamondback moth Plutella xylostella, a serious pest of cruciferous crops. In particular, the BY12 population of P. xylostella, collected from Baiyun, Guangdong Province of China in 2012, was 750-fold more resistant to indoxacarb and 70-fold more resistant to metaflumizone compared with the susceptible Roth strain. Comparison of complementary DNA sequences encoding the sodium channel genes of Roth and BY12 revealed two point mutations (F1845Y and V1848I) in the sixth segment of domain IV of the PxNav protein in the BY population. Both mutations are located within a highly conserved sequence region that is predicted to be involved in the binding sites of local anesthetics and SCBIs based on mammalian sodium channels. A significant correlation was observed among 10 field-collected populations between the mutant allele (Y1845 or I1848) frequencies (1.7% to 52.5%) and resistance levels to both indoxacarb (34- to 870-fold) and metaflumizone (1- to 70-fold). The two mutations were never found to co-exist in the same allele of PxNav , suggesting that they arose independently. This is the first time that sodium channel mutations have been associated with high levels of resistance to SCBIs. F1845Y and V1848I are molecular markers for resistance monitoring in the diamondback moth and possibly other insect pest species.

Early amplitude-integrated EEG monitoring 6 h after birth predicts long-term neurodevelopment of asphyxiated late preterm infants.

UNLABELLED: The present study aimed to assess the prognostic value of early amplitude-integrated electroencephalogram (aEEG) in late preterm infants who were born at a gestational age between 34 0/7 and 36 6/7 weeks for the prediction of neurobehavioral development. Late preterm infants (n = 170) with normal, mild, and severe asphyxia underwent continuous recording of aEEG for 4-6 h starting 6-8 h after delivery. The recordings were analyzed for background pattern, sleep-wake cycle (SWC), and seizures. Survivors were assessed at 18 months by neurological examination and Bayley Scales of Infant Development II. The incidence of adverse neurological outcome in the asphyxia group was significantly higher than in the normal group. For late preterm infants in the asphyxia group, abnormal aEEG pattern had a predictive potential of neurological outcomes with sensitivity of 78.57% (specificity, 87.80%; positive predictive value [PPV], 68.75%; negative predictive value [NPV], 92.31%; power, 85.45%). Non-SWC and intermediate SWC significantly were increased (25.45 and 52.73%, respectively) in the asphyxia group vs. the normal group. SWC pattern had neurological prognosis value in the asphyxia group with sensitivity of 64.29% (specificity, 87.80%; PPV, 64.29%; NPV, 87.80%; power, 81.82%). CONCLUSION: Early aEEG patterns are important determinants of long-term prognosis of neurodevelopmental outcome in asphyxiated late preterm infants.

Formal synthesis of cephalotaxine.

A formal synthesis of cephalotaxine, the parent member of the Cephalotaxus alkaloids, was achieved. It features a practical four-step assembly of the benzazepine-bearing pentacyclic ring system through two alkylation reactions, acidic hydrolysis, and aldolization.

[Effects of recombinant human erythropoietin on serum levels of neuron-specific enolase, S-100ß protein and myelin basic protein in rats following status epilepticus].

OBJECTIVE: This study examined the effect of recombinant human erythropoietin (r-HuEPO) on the serum levels of neuron-specific enolase (NSE), S-100ß protein and myelin basic protein (MBP) in young rats 24 hrs after lithium-pilocarpine-induced status epilepticus (SE) in order to study the potential role of r-HuEPO in epileptic brain damage. METHODS: Forty 19-21-day-old male Sprague-Dawley (SD) rats were randomly divided into four groups (n=10): normal control group, SE, r-HuEPO pretreated-SE and r-HuEPO. SE was induced by lithium-pilocarpine. R-HuEPO (500 IU/kg) was intraperitoneally injected in the r-HuEPO pretreated-SE and r-HuEPO groups 4 hrs before SE. Serum levels of NSE, S-100ß and MBP were determined 24 hrs after the SE event. RESULTS: Serum levels of NSE, S-100ß and MBP in the SE group increased significantly compared with those in the normal control and the r-HuEPO groups (P＜0.05). The r-HuEPO pretreated-SE group showed significantly decreased serum levels of NSE, S-100ß and MBP compared with the SE group (P＜0.05). CONCLUSIONS: r-HuEPO may reduce the expression of NSE, S-100ß and MBP and thus might provide an early protective effect against epileptic brain injury.

Changes of serum and urine neutrophil gelatinase-associated lipocalin in type-2 diabetic patients with nephropathy: one year observational follow-up study.

We initiated the present work to explore whether neutrophil gelatinase-associated lipocalin (NGAL) could be used to predict the progression of diabetic nephropathy in type-2 diabetic patients. Seventy-four type-2 diabetic patients were divided into normo-, micro- and macro-albuminuria groups according to their 24 h-urinary albumin excreting rate. Serum and urine NGAL, and other clinical parameters were detected. Patients were followed and measurements were repeated 1 year later. An increased tendency of urine NGAL and a decreased tendency of serum NGAL were detected, from normo-albuminuria group to macro-albuminuria group. Serum NGAL was found to rise after follow-up. Moreover, urine NGAL was found to be correlated positively with cystatin C, urea nitrogen, and serum creatinine (SCr), and inversely with glomerular filtration rate (GFR), while serum NGAL correlated negatively with cystatin C and urea nitrogen, at both baseline and follow-up levels. The results indicate that NGAL correlates closely with renal function. Both serum and urine NGAL are sensitive for predicting the progression of type-2 diabetic nephropathy but they may change differently. Serum NGAL may be more useful in early detection and urine NGAL may be more meaningful in renal function assessment.

Fingerprint of Hedyotis diffusa Willd. by HPLC-MS.

A HPLC-MS fingerprint method has been developed based on the consistent chromatographic features of the major chemical constituents among 10 batches of Hedyotis diffusa Willd. Chromatographic separation was conducted on a Hypersil-Keystone Hypurity C(18) column using methanol:water:acetic acid as the mobile phase. Major compounds, including oleanolic acid, ursolic acid and ferulic acid, were analysed by HPLC-MS. Their analysis was ascertained by comparison with data derived from the standard compounds. The HPLC-MS fingerprint was successfully applied to analyse and differentiate samples from different geographical origins, or processing methods. H. diffusa was well distinguished from Hedyotis chrysotricha by HPLC-MS. Therefore the establishment of fingerprint of H. diffusa is critical in assessing and controlling its overall quality.