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Harnessing mitochondria is considered as a promising method for biosynthesis of terpenes due to the adequate supply of acetyl-CoA and redox equivalents in mitochondria. However, mitochondrial engineering often causes serious metabolic burden indicated by poor cell growth. Here, we systematically analyzed the metabolic burden caused by the compartmentalization of the MVA pathway in yeast mitochondria for squalene synthesis. The phosphorylated intermediates of the MVA pathway, especially mevalonate-5-P and mevalonate-5-PP, conferred serious toxicity within mitochondria, which significantly compromised its possible advantages for squalene synthesis and was difficult to be significantly improved by routine pathway optimization. These phosphorylated intermediates were converted into ATP analogues, which strongly inhibited ATP-related cell function, such as mitochondrial oxidative respiration. Fortunately, the introduction of a partial MVA pathway from acetyl-CoA to mevalonate in mitochondria as well as the augmentation of the synthesis of mevalonate in cytosol could significantly promote the growth of yeasts. Accordingly, a combinatorial strategy of cytoplasmic and mitochondrial engineering was proposed to alleviate the metabolic burden caused by the compartmentalized MVA pathway in mitochondria and improve cell growth. The strategy also displayed the superimposed effect of cytoplasmic engineering and mitochondrial engineering on squalene production. Through a two-stage fermentation process, the squalene titer reached 21.1 g/L with a specific squalene titer of 437.1 mg/g dcw, which was the highest at present. This provides new insight into the production of squalene and other terpenes in yeasts based on the advantages of mitochondrial engineering.

Grinding is the main processing method for the particle-reinforced composites, where removal mechanism is of great significance to surface usability performance. In this study, Hopkinson pressure bar tests of SiCp/Al composite were carried out, and the constitutive model of SiCp/Al composite was obtained. Based on the finite element model of Hopkinson pressure bar tests, the interfacial constitute model of SiCp/Al composite was obtained. Single abrasive grain grinding experiments were designed and conducted to obtain the grinding force. Based on the interfacial constitute model, a new integrated grinding simulation model for the high volume fraction SiCp/Al composite was established and verified by the results of the single abrasive grain experiments, then the single abrasive grain grinding force prediction model was established and verified. Several grinding mechanism models for SiCp/Al composites were proposed, and the debonding between the interface of silicon carbides and aluminum alloy was observed. Finally, the multi-abrasive grain grinding forces were measured under different grinding process parameters and the influence of the grinding process parameters on the grinding force was discussed. This research about the micro-grinding mechanism of SiCp/Al composites provides a theoretical support for the study of surface quality.

Opioid analgesics remain the mainstay for managing intractable chronic pain, but their use is limited by detrimental side effects such as analgesic tolerance and hyperalgesia. Calcium-dependent synaptic plasticity is a key determinant in opiates tolerance and hyperalgesia. However, the exact substrates for this calcium-dependent synaptic plasticity in mediating these maladaptive processes are largely unknown. Canonical transient receptor potential 1, 4, and 5 (TRPC1, 4, 5) proteins assemble into heteromultimeric nonselective cation channels with high Ca2+ permeability and influence various neuronal functions. However, whether and how TRPC1/4/5 channels contribute to the development of opiates tolerance and hyperalgesia remains elusive. Here, we show that TRPC1/4/5 channels contribute to the generation of morphine tolerance and hyperalgesia. Chronic morphine exposure leads to upregulation of TRPC1/4/5 channels in the spinal cord. Spinally expressed TRPC1, TPRC4, and TRPC5 are required for chronic morphine-induced synaptic long-term potentiation (LTP) as well as remodeling of synaptic spines in the dorsal horn, thereby orchestrating functional and structural plasticity during the course of morphine-induced hyperalgesia and tolerance. These effects are attributed to TRPC1/4/5-mediated Ca2+ elevation in the spinal dorsal horn induced by chronic morphine treatment. This study identifies TRPC1/4/5 channels as a promising novel target to prevent the unwanted morphine tolerance and hyperalgesia.

The antitumor immune response involves a cascade of cancer-immunity cycles. Developing a combination therapy aimed at the cancer-immunity cycle is of great importance. In this research, we designed and tested a combined therapeutic-Au nanorod (AuNR)/doxorubicin (DOX) gel (AuNR/DOX gel)-in which the sustained release of DOX was controlled by Pluronic gel. DOX served as an immunogenic tumor cell death (ICD) inducer, triggering the production of damage-associated molecular patterns (DAMPs). Mild photothermal therapy (Mild PTT) produced by 880 nm laser-irradiated AuNRs also generated tumor-associated antigens. Maleimide-modified liposomes (L-Mals), as antigen capturing agents, promoted tumor antigen uptake by DCs. Ultimately, more CD8+ T cells and fewer regulatory T cells (Tregs) infiltrated the tumor, eliciting antitumor responses from the PD-L1 antibody. Our results indicate that this combination strategy promotes a positive shift in the cancer-immunity cycle and holds much promise for combination strategy will lead to development of an antitumor drug delivery system. STATEMENT OF SIGNIFICANCE: Developing a combination therapy for cancer-immunity cycle is of great importance due to antitumor immune response involving a cascade of cancer-immunity cycles. Cancer-immunity cycle usually includes tumor antigen release, antigen presentation, immune activation, trafficking, infiltration, specific recognition of tumor cells by T cells, and finally cancer cell killing. In this research, we designed a combination strategy based on Au nanorod/doxorubicin gel via mild photothermal therapy combined with antigen-capturing liposomes and anti-PD-L1 agent promoting a positive shift in the cancer-immunity cycle. Our results indicate that this combination strategy promotes a positive shift in the cancer-immunity cycle and holds much promise for combination strategy will lead to development of an antitumor drug delivery system.

The purpose of the study was to evaluate urinary citrate/creatinine (UCi/UCr) and urinary calcium/citrate (UCa/UCi) ratios for distinguishing stone formers (SF) from non-stone formers (NSF) in an at-risk population. This was a retrospective study that included all pediatric patients who underwent urinary citrate testing from April 2017 to March 2018. The urinary levels of citrate, calcium, sodium, potassium, creatinine, oxalate, urate, pH, and specific gravity (SG) were measured in our clinical laboratory. Diagnosis of kidney stones was obtained through chart review.A total of 97 patients were included (46 NSF and 51 SF). The UCi/UCr ratio was not significantly different between NSF and SF. Median UCa/UCr ratio was higher in SF (0.67) compared with NSF (0.21, p The study has demonstrated significantly higher UCa/UCi and UCa/UCr in SF compared with NSF, while the use of urinary oxalate, urate, pH, and SG did not differentiate between SF from NSF. We also confirmed a positive correlation between UNa/UCr and UCa/UCr. While the utility of UCa/UCr is well established, our data suggest that UCa/UCi rather than UCi/UCr may be more predictive in the clinical setting when evaluating for nephrolithiasis.

Xiu-Chai Zheng,1,2,* Wei Ren,1,2,* Shuang Zhang,1,2 Ting Zhong,1,2 Xiao-Chuan Duan,1,2 Yi-Fan Yin,1,2 Mei-Qi Xu,1,2 Yan-Li Hao,1,2 Zhan-Tao Li,1,2 Hui Li,1,2 Man Liu,1,2 Zhuo-Yue Li,1,2 Xuan Zhang1,2 1Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing, People’s Republic of China; 2Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing, People’s Republic of China *These authors contributed equally to this work Background: In the present study, the tumor-specific, pH-responsive peptide H7K(R2)2 -modified, theranostic liposome-containing paclitaxel (PTX) and superparamagnetic iron oxide nanoparticles (SPIO NPs), PTX/SPIO-SSL-H7K(R2)2, was prepared by using H7K(R2)2 as the targeting ligand, SPIO NPs as the magnetic resonance imaging (MRI) agent, PTX as antitumor drug. Methods: The PTX/SPIO-SSL-H7K(R2)2 was prepared by a thin film hydration method. The characteristics of PTX/SPIO-SSL-H7K(R2)2 were evaluated. The targeting effect, MRI, and antitumor activity of PTX/SPIO-SSL-H7K(R2)2 were investigated detail in vitro and in vivo in human breast carcinoma MDA-MB-231 cell models. Results: Our results of in vitro flow cytometry, in vivo imaging, and in vivo MR imaging confirmed the pH-responsive characteristic of H7K(R2)2 in MDA-MB-231 cell line in vitro and invivo. The results of in vivo MRI and in vivo antitumor activity confirmed the theranostic effect of PTX/SPIO-SSL-H7K(R2)2 in MDA-MB-231 tumor-bearing model. Conclusion: Considering all our in vitro and in vivo results, we conclude that we developed targeting modified theranostic liposome which could achieve both role of antitumor and MRI. Keywords: tumor-specific pH-responsive peptide, paclitaxel, superparamagnetic iron oxide nanoparticles, liposome, theranostic efficiency

Lipid metabolism plays an important role in the energy economy of ruminants. However, its interactions of fat, rumen fermentation, gas emission, and microorganisms are not yet clear. This study evaluated the effect of adding raw oilseeds to high-forage diets on in vitro ruminal fermentation, gas composition, and microbial profile. Three isoenergetic and isoproteic experimental diets were designed and used as fermentation substrate: control treatment (CON group) was the basal diet lacking oilseeds, the other two treatments were the basal diet supplemented by 100 g/kg dry matter (DM) raw whole soybean (S group) and 50 g/kg DM raw flaxseed (F group), respectively. Data showed that the acetate, butyrate, and total VFA concentration of culture fluids in the S group were lower (p &lt, 0.05) than in the F group. There was a tendency to a higher level (p = 0.094) of propionate concentration in the F group compared with the other two groups. The gas production in the F group was higher (p &lt, 0.05) than in the control group. There was a lower abundance of Sutterella (p &lt, 0.05) and a greater abundance of Butyrivibrio (p &lt, 0.05) in both of the two oilseed treatments. Methanobrevibacter (p = 0.078) in the F group was the lowest. Our results suggested that CH4 emission could be inhibited with flaxseed supplementation by propionate production metabolism, biohydrogenation of unsaturated fatty acid (FA), and toxicity to Methanobrevibacter, while regarding soybean seed supplementation, the emission of CH4 was more likely to be reduced through biohydrogenation of unsaturated FA modulated by Butyrivibrio.

Xiao-Chuan Duan,1,2,* Xin Yao,1,2,* Shuang Zhang,1,2 Mei-Qi Xu,1,2 Yan-Li Hao,1,2 Zhan-Tao Li,1,2 Xiu-Chai Zheng,2 Man Liu,1,2 Zhuo-Yue Li,1,2 Hui Li,1 Jing-Ru Wang,1,2 Zhen-Han Feng,1 Xuan Zhang1,2 1Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People’s Republic of China; 2Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People’s Republic of China *These authors contributed equally to this work Background: 3-(2-Nitrophenyl) propionic acid-paclitaxel (NPPA-PTX) is a paclitaxel (PTX) bioreductive prodrug synthesized by our lab. We hypothesize that NPPA-PTX can self-assemble to form nanoparticles (NPs). Materials and methods: In the present research, the theoretical partition coefficient (XlogP) and Hansen solubility parameters of NPPA-PTX were calculated. NPPA-PTX nanoparticles prepared by NPPA-PTX and DSPE-PEG (NPPA-PTX:DSPE-PEG =1:0.1, w/w) (NPPA-PTX@PEG NPs) were prepared and characterized. The cellular uptake, in vitro antitumor activity, in vivo targeting effect, tumor distribution, in vivo antitumor activity, and safety of NPPA-PTX@PEG NPs were investigated. Results: Our results indicate that NPPA-PTX can self-assemble to form NPPA-PTX@PEG NPs. Both the cellular uptake and safety of NPPA-PTX@PEG NPs were higher than those of Taxol. NPPA-PTX@PEG NPs could target tumor tissues by a passive targeting effect. In tumor tissues, NPPA-PTX@PEG NPs could completely transform into active PTX. The in vivo antitumor activity of NPPA-PTX@PEG NPs was confirmed in MDA-MB-231 tumor-bearing nude mice. Conclusion: The bioreductive prodrug NPPA-PTX could self-assemble to form NPs. The safety and antitumor activity of NPPA-PTX@PEG were confirmed in our in vitro and in vivo experiments. The NPPA-PTX@PEG NPs developed in this study could offer a new way of preparing bioreductive prodrug, self-assembled NPs suitable for antitumor therapy. Keywords: bioreductive prodrug, 3-(2-nitrophenyl) propionic acid-paclitaxel, nanoparticles, antitumor activity, in vitro, in vivo

Nucleus-targeting drug delivery systems (NTDDs) deliver chemotherapeutic agents to nuclei in order to improve the efficacy of anti-tumour therapy. Histone H1 (H1) plays a key role in establishing and maintaining higher order chromatin structures and could bind to cell membranes. In the present study, we selected H1 as a target to prepare a novel H1-mediated NTDD. Low molecular weight heparin (LMHP) and doxorubicin (DOX) were combined to form LMHP-DOX. Then, a novel NTDD consisting of LMHP-DOX nanoparticles (LMHP-DOX NPs) was prepared by self-assembly. The characteristics of LMHP-DOX and LMHP-DOX NPs were investigated. Histone H1 high-expressive prostate cancer PC-3M cell line was selected as the cell model. Cellular uptake, and the in vitro and in vivo anti-tumour activity of LMHP-DOX NPs were evaluated on H1 high-expressive human prostate cancer PC-3M cells. Our results indicated that intact LMHP-DOX NPs mediated by H1 could be absorbed by H1 high-expressive PC-3M cells, escape from the lysosomes to the cytoplasm, and localize in the perinuclear region via H1-mediated, whereby DOX could directly enter the cell nucleus and quickly increase the concentration of DOX in the nuclei of H1 high-expressive PC-3M cells to enhance the apoptotic activity of cancer cells. The anti-coagulant activity of LMHP-DOX NPs was almost completely diminished in rat blood compared with that of LMHP, indicating the safety of LMHP-DOX NPs. Compared to traditional NTDD strategies, LMHP-DOX NPs avoid the complicated modification of nucleus-targeting ligands and provide a compelling solution for the substantially enhanced nuclear uptake of chemotherapeutic agents for the development of more intelligent NTDDs.

Small molecule modified anticancer drug conjugates (SMMDCs) can self-assemble into nanoparticles (NPs) as therapeutic NP platforms for cancer treatment. Here we demonstrate that the XlogP and Hansen solubility parameters of paclitaxel (PTX) SMMDCs is essential for SMMDCs self-assembling into NPs. The amorphous state of PTX SMMDCs will also affect SMMDCs self-assembling into NPs. However, the antitumor activity of these PTX SMMDCs NPs decreased along with their XlogP values, indicating that a suitable XlogP value for designing the SMMDCs is important for self-assembling into NPs and for possessing antitumor activity. For higher level XlogP SMMDCs, a degradable linker should be considered in the design of SMMDCs to overcome the problem of lower antitumor activity. It is preferable that the hydrophilic groups in the SMMDCs should be present on the surface of self-assembling NPs.

The growing use of nanoscale zero-valent iron (NZVI) in the remediation of contaminated groundwater raises concerns regarding its transport in aquifers. Laboratory-scale sand-packed column experiments were conducted with bare and sucrose-modified NZVI (SM-NZVI) to improve our understanding of the transport of the nanoparticles in saturated porous media, as well as the role of media size, suspension injection rate and concentration on the nanoparticle behavior. As the main indicative parameters, the normalized effluent concentration was measured and the deposition rate coefficient (k) was calculated for different simulated conditions. Overall, compared to the high retention of bare NZVI in the saturated silica column, SM-NZVI suspension could travel through the coarse sand column easily. However, the transport of SM-NZVI particles was not very satisfactory in a smaller size granular matrix especially in fine silica sand. Furthermore, the value of k regularly decreased with the increasing injection rate of suspension but increased with suspension concentration, which could reflect the role of these factors in the SM-NZVI travel process. The calculation of k-value at the tests condition adequately described the experimental results from the point of deposition dynamics, which meant the assumption of first-order deposition kinetics for the transport of NZVI particles was reasonable and feasible.

The integrated mechanisms of dynamic signaling of sodium channels involved in clinical pain are still not yet clear. In this study, a new rat inflammatory pain model was developed by using the unilateral intraplantar injection of BmK I, a receptor site 3-specific modulator of sodium channels from the venom of scorpion Buthus martensi Karsch (BmK). It was found that BmK I could induce several kinds of inflammatory pain-related behaviors including spontaneous pain companied with unique episodic paroxysms, primary thermal hypersensitivity, and mirror-image mechanical hypersensitivity with different time course of development, which could be suppressed by morphine, indomethacin, or bupivacaine to a different extent. The dramatic attenuation by pretreatment with resiniferatoxin (RTX), an ultrapotent analog of capsaicin, on BmK I-induced pain-related behaviors, paw edema, and spinal L4-L5 c-Fos expression demonstrated that capsaicin-sensitive primary afferent neurons played important roles in pain induced by BmK I. Furthermore, the electrophysiological recordings showed that BmK I persistently increased whole-cell and tetrodotoxin-resistant (TTX-R) peak sodium currents and significantly delayed the inactivation phase of whole-cell sodium currents but could not enhance capsaicin-evoked inward currents, in acute isolated small dorsal root ganglion neurons of rat. The results strongly suggested that the dynamic modulation of BmK I on sodium channels located in peripheral primary afferent neurons, especially in capsaicin-sensitive neurons, mediated pain sensation. Thus, BmK I may be a valuable pharmacological tool to understand the sodium channel-involved pain mechanisms.

This study showed that rat unilateral intracerebroventricular injection of BmK alphaIV, a sodium channel modulator derived from scorpion Buthus martensi Karsch, induced clusters of spikes, epileptic discharges and convulsion-related behavioral changes. BmK alphaIV potently promoted the release of endogenous glutamate from rat cerebrocortical synaptosomes. In vitro examination of the effect of BmK alphaIV on intrasynaptosomal free calcium concentration [Ca(2+)](i) and sodium concentration [Na(+)](i) revealed that BmK alphaIV-evoked glutamate release from synaptosomes was associated with an increase in Ca(2+) and Na(+) influx. Moreover, BmK alphaIV-mediated glutamate release and ion influx was completely blocked by tetrodotoxin, a blocker of sodium channel. Together, these results suggest that the induction of BmK alphaIV-evoked epileptic seizures may be involved in the modulation of BmK alphaIV on tetrodotoxin-sensitive sodium channels located on the nerve terminal, which subsequently enhances the Ca(2+) influx to cause an increase of glutamate release. These findings may provide some insight regarding the mechanism of neuronal action of BmK alphaIV in the central nervous system for understanding epileptogenesis involved in sodium channels.

In the present study, we investigated the anti-nociceptive effect and the underlying mechanism of BmK AS, an active peptide purified from scorpion Buthus martensi Karsch. The results showed that BmK AS can significantly relieve formalin-induced two-phase spontaneous flinching response and carrageenan-induced mechanical hyperalgesia. Using the whole-cell patch-clamp recording, exposure of acutely isolated sensory neurons to 500 nM BmK AS produced a one-fold decrease in the number of action potentials (APs) evoked by a ramp of depolarizing current. To investigate the mechanism of action of BmK AS, isolated membrane current and Ca2+ influx on rat primary sensory neurons were examined. BmK AS produced insignificant effect on voltage-dependent I(K) and KCl or caffeine-induced Ca2+ influx, but caused remarkable suppressive effect on tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) I(Na). Further experiments showed that BmK AS reduced the peak TTX-R and TTX-S Na+ conductance in a dose-dependent manner, prompted the voltage-dependent activation, and caused a negative shift of the steady-state inactivation of TTX-R and TTX-S I(Na). Thus, the present results indicate the anti-nociceptive response of BmK AS may be ascribed to its specific modulation of voltage-gated Na+ channels of sensory neurons.

The binding of BmK IT2 to insect and mammal sodium channels was investigated by surface plasmon resonance technique. The results showed that BmK IT2 could bind not only to cockroach neuronal membranes but also to rat cerebrocortical and hippocampal synaptosomes with distinct affinity. The binding of BmK IT2 could be competed significantly by BmK AS and BmK abT, but not by AaH II, BmK I and veratridine. Furthermore, BmK alphaIV could partially inhibit the binding of BmK IT2 to rat cerebrocortical synaptosomes and cockroach neuronal membranes, but not to rat hippocampal synaptosomes. These results suggested that BmK IT2 had diverse binding properties on the mammal and insect sodium channels.

In this paper, the central neuronal activities elicited by BmK I, a specific voltage-gated Na+ channel modulator, were examined by monitoring the c-Fos expression pattern of rat spinal cord. c-Fos protein in laminae I-II, V-VI, and VII-X could be detected at 0.5 h, increased steadily at 1 h, reached a peak at 2 h, and then decreased rapidly from 4 to 24 h after Bmk I was subcutaneously injected into the rat hind paw. However, c-Fos expression in laminae III-IV was activated to a peak at 0.5 h and then declined gradually from 0.5 to 24 h. Furthermore, c-Fos expression could be induced by BmK I in a dose-dependent manner. In addition, the increase of c-Fos expression in laminae I-II, V-VI, and VII-X induced by BmK I, and not in laminae III-IV, could be partially inhibited by systemic morphine in a dose-dependent manner. The results suggested that peripheral administration of BmK I could evoke a profound change of spinal neuronal activities manifested as specific patterns of c-Fos expression, which may be partially attributed to the selective modulation of BmK I on voltage-gated Na+ channels located in peripheral nociceptors.

In the present communication, we determined the dynamic release of amino acid transmitters from spinal dorsal horn induced by scorpion Buthus martensi Karsch (BmK) venom and a neurotoxin (BmK I). The results found that glutamate and aspartate release could be evoked significantly within the initial 30 min with the applied doses of either 0.05 and 0.01 mg BmK venom or 0.01 and 0.002 mg BmK I. However, gamma-aminobutyric acid (GABA) release could be largely evoked during the second 30 min by the venom, but not by BmK I. The result suggested that nociceptive afferent fibers could be activated to induce excitatory amino acid release from spinal dorsal horn by nociceptive factors such as BmK I, but the delayed release of GABA might be attributed to the modulating role of some antinociceptive components in the venom.

This study was carried out to determine low-boiling point hydrocarbon mixtures C 1 -C 5 using gas chromatography supplied with a flame ionization detector (FID) and a low-pole capillary column. The temperature of the column reached -30 centigrade and all components eluted within 4 minutes. Sufficient resolution was available for the components with low retentions. The limits of the relative standard deviations (RSD) were from 0.19% to 2.40%. The method was found to be suitable for the determination of natural gas, petroleum, environmental quality, etc.

AFP-producing gastric carcinoma(APGC) are known to have a poorer prognosis and to show a higher incidence of liver metastasis than ordinary gastric carcinoma. Therefore, surgical resection, chemotherapy or radiotherapy is usually not satisfactory. In recent years, effects of arsenic trioxide (As 2 O 3 ) on cell proliferation and induced apoptosis of solid tumors have been widely studied, while little is known about the APGC. This study was undertaken to delineate the effect of As 2 O 3 on AFP gene expression ,the cell growth and apopotosis ,and to study the effect of the siRNA targeting AFP gene on the drug sensitivity of APGC cell line FU97 to As 2 O 3 . AFP gene sequence-specific siRNA expression vector (AFP-siRNA) and sequence-unrelated control were transfected into FU97. At 6h after transfection ,the cells were cultured with 2 mumol/L As 2 O 3 , with corresponding untreated cells serving as controls.The growth inhibition rate on FU97 was determined by MTT assay; Apoptosis of FU97 was investigated by DNA gel electrophore; The expressions of AFP,VEGF were determined by realtime-PCR, western blot, ELISA and chemistry luminescence. The combination of AFP-siRNA with arsenic trioxide resulted in significantly inhibitory action on growth of FU97 cells ,as well as descending of expression of AFP and VEGF.The effective siRNA targeting AFP can increase the drug sensitivity of the FU97 to arsenic trioxide.

In the present study, using the single fiber recording technique, we found that BmK I, the main toxic component in scorpion Buthus martensi Karsch (BmK) venom, induced dramatic increase in excitability of rapidly adapting (RA) and type I slowly adapting (SAI) low threshold mechanical A fibers of rat. Five micrograms BmK I (691 nmol, in 10 microl saline) administrated to the receptive fields induced spontaneous activity in 80% of RA and SAI fibers, increased the response to 10 g-10 s stimulation at about 20 times and altered the firing pattern to burst mode with maximal NS (number of spikes in burst) averaging from all fibers studied as many as 59. The increase in the excitability of RA and SAI fibers did not recover completely in 2h. Our finding suggests that the gigantic abnormal activity in low threshold mechanical A fibers is involved in BmK scorpion sting pain, and the experimental model of BmK scorpion sting pain can be used to study A-fiber related central pathway which is important for relief of refractory neuropathic pain likewise.

The role of capsaicin-sensitive primary afferent fibers in rat pain-related behaviors and paw edema induced by scorpion Buthus martensi Karch (BmK) venom was investigated in this study. It was found that functional depletion of capsaicin-sensitive primary afferent fibers with a single systemic injection of resiniferatoxin (RTX) dramatically decreased spontaneous nociceptive behaviors, prevented the development of primary mechanical and thermal hyperalgesia as well as mirror-image mechanical hyperalgesia. RTX treatment significantly attenuated BmK venom-induced c-Fos expression in all laminaes of bilateral L4-L5 lumbar spinal cord, especially in superficial laminaes. Moreover, RTX treatment markedly reduced the early paw edema induced by BmK venom. Thus, the results indicate that capsaicin-sensitive primary afferent fibers play a critical role in various pain-related behaviors and paw edema induced by BmK venom in rats.

In the present study, it was investigated whether the degranulation of mast cells and histamine release were involved in rat pain-related behaviors and edema induced by the venom of scorpion Buthus martensi Karch (BmK) or not. It was found that the obvious degranulation of mast cells could be triggered in rat hindpaw skin by BmK venom. The chronic degranulation of mast cells using compound 48/80 relieved the spontaneous nociceptive responses, the primary thermal and bilateral mechanical hyperalgesia and the rat paw edema, as well as partially reduced c-Fos expression in superficial layers (laminae I-II) of bilateral spinal cord induced by BmK venom. In addition, individual peripheral co-administration of either 100 nmol chlorpheniramine or 100 nmol pyrilamine (histamine H(1) receptor antagonist) or 500 nmol cimetidine (histamine H(2) receptor antagonist) and BmK venom suppressed the spontaneous nociceptive responses, partially the primary thermal and bilateral mechanical hyperalgesia and rat paw edema induced by BmK venom. Thus, these results suggest that the peripheral cellular incidents of mast cells degranulation and histamine release are involved in BmK venom-induced pain-related behaviors and inflammation.

In the present study, BmK alphaIV, a novel modulator of sodium channels, was cloned from venomous glands of the Chinese scorpion (Buthus martensi Karsch) and expressed successfully in Escherichia coli. The BmK alphaIV gene is composed of two exons separated by a 503 bp intron. The mature polypeptide contains 66 amino acids. BmK alphaIV has potent toxicity in mice and cockroaches. Surface-plasmon-resonance analysis found that BmK alphaIV could bind to both rat cerebrocortical synaptosomes and cockroach neuronal membranes, and shared similar binding sites on sodium channels with classical AaH II (alpha-mammal neurotoxin from the scorpion Androctonus australis Hector), BmK AS (beta-like neurotoxin), BmK IT2 (the depressant insect-selective neurotoxin) and BmK abT (transitional neurotoxin), but not with BmK I (alpha-like neurotoxin). Two-electrode voltage clamp recordings on rNav1.2 channels expressed in Xenopus laevis oocytes revealed that BmK alphaIV increased the peak amplitude and prolonged the inactivation phase of Na+ currents. The structural and pharmacological properties compared with those of other scorpion alpha-toxins suggests that BmK alphaIV represents a novel subgroup or functional hybrid of alpha-toxins and might be an evolutionary intermediate neurotoxin for alpha-toxins.