Production of ρ-Hydroxyacetophenone by Engineered Escherichia coli Heterologously Expressing 1-(4-Hydroxyphenyl)-Ethanol Dehydrogenase.

ρ-Hydroxyacetophenone is an important and versatile compound that has been widely used in medicine, cosmetics, new materials, and other fields. At present, there are two ways to obtain ρ-hydroxyacetophenone. One is to extract it from plants, such as Artemisia capillaris Thunb and Cynanchum otophyllum Schneid, and the other is to synthesize it by using chemical methods. Of these two methods, the second is the main one, although it has problems, such as flammable and explosive reagents, difficult separation of by-products, and harsh reaction conditions. To solve these issues, we adopted genetic engineering in this study to construct engineered Escherichia coli containing Hped gene or EbA309 gene. Whole-cell biotransformation was conducted under the same conditions to select the engineered E. coli with the higher activity. Orthogonal tests were conducted to determine the optimal biotransformation condition of the engineered E. coli. The results showed that the optimal condition was as follows: substrate concentration of 40 mmol/l, IPTG concentration of 0.1 mmol/l, an induction temperature of 25°C, and a transformation temperature of 35°C. Under this condition, the effects of transformation time on the ρ-hydroxyacetophenone concentration and cell growth were further studied. We found that as the transformation time extended, the ρ-hydroxyacetophenone concentration showed a gradually increasing trend. However, when the ρ-hydroxyacetophenone concentration increased to 1583.19 ± 44.34 mg/l in 24 h, cell growth was inhibited and then entered a plateau. In this research, we realized the synthesis of ρ-hydroxyacetophenone by biotransformation, and our findings lay a preliminary foundation for further improving and developing this method.

Type-I PtS2/MoS2 van der Waals heterojunctions with tunable photovoltaic effects and high photosensitivity.

Recent advances in two-dimensional (2D) materials play an essential role in boosting modern electronics and optoelectronics. Thus far, transition metal dichalcogenides (TMDs) as emerging members of 2D materials, and the van der Waals heterostructures (vdWHs) based on TMDs have been extensively investigated owing to their prominent capabilities and unique crystal structures. In this work, an original vdWH composed of molybdenum disulfide (MoS2) and platinum disulfide (PtS2) was comprehensively studied as a field-effect transistor (FET) and photodetector. A gate-tunable rectifying behavior was obtained, stemming from the band design of PtS2/MoS2 vdWH. Upon 685 nm laser illumination, it also exhibited a superior photodetection performance with a distinctly high photoresponsivity of 403 A W-1, a comparable detectivity of 1.07 × 1011 Jones, and an excellent external quantum efficiency of 7.32 × 104%. More importantly, fast rise (24 ms) and decay (21 ms) times were obtained under 685 nm light illumination attributed to the unilateral depletion region structure. Further, the photovoltaic effect and photocurrent of the heterojunction could be modulated by a back gate voltage. All these results indicated that such 2D-TMD-based vdWHs provide a new idea for realizing high-performance electronic and optoelectronic devices.

Type-I Heterostructure Based on WS2/PtS2 for High-Performance Photodetectors.

van der Waals (vdW) heterodiodes composed of two-dimensional (2D) layered materials led to a new prospect in photoelectron diodes and photovoltaic devices. Existing studies have shown that Type-I heterostructures have great potential to be used as photodetectors; however, the tunneling phenomena in Type-I heterostructures have not been fully revealed. Herein, a highly efficient nn+ WS2/PtS2 Type-I vdW heterostructure photodiode is constructed. The device shows an ultrahigh reverse rectification ratio of 105 owing to the transmission barrier-induced low reverse current. A unilateral depletion region is formed on WS2, which inhibits the recombination of carriers at the interface and makes the external quantum efficiency (EQE) of the device reach 67%. Due to the tunneling mechanism of the device, which allows the co-existence of a large photocurrent and a low dark current, this device achieves a light on/off ratio of over 105. In addition, this band design allows the device to maintain a high detectivity of 4.53 × 1010 Jones. Our work provides some new ideas for exploring new high-efficiency photodiodes.

[Metabolic engineering study on biosynthesis of 4-hydroxybenzyl alcohol from L-tyrosine in Escherichia coli].

As an important active ingredient in the rare Chinese herb Gastrodiae Rhizoma and also the main precursor for gastrodin biosynthesis, 4-hydroxybenzyl alcohol has multiple pharmacological activities such as anti-inflammation, anti-tumor, and anti-cerebral ischemia. The pharmaceutical products with 4-hydroxybenzyl alcohol as the main component have been increasingly favored. At present, 4-hydroxybenzyl alcohol is mainly obtained by natural extraction and chemical synthesis, both of which, however, exhibit some shortcomings that limit the long-term application of 4-hydroxybenzyl alcohol. The wild and cultivated Gastrodia elata resources are limited. The chemical synthesis requires many steps, long time, and harsh reaction conditions. Besides, the resulting by-products are massive and three reaction wastes are difficult to treat. Therefore, how to artificially prepare 4-hydroxybenzyl alcohol with high yield and purity has become an urgent problem facing the medical researchers. Guided by the theory of microbial metabolic engineering, this study employed the genetic engineering technologies to introduce three genes ThiH, pchF and pchC into Escherichia coli for synthesizing 4-hydroxybenzyl alcohol with L-tyrosine. And the fermentation conditions of engineering strain for producing 4-hydroxybenzyl alcohol in shake flask were also discussed. The experimental results showed that under the conditions of 0.5 mmol·L~(-1) IPTG, 15 ℃ induction temperature, and 40 ℃ transformation temperature, M9 Y medium containing 200 mg·L~(-1) L-tyrosine could be transformed into(69±5)mg·L~(-1) 4-hydroxybenzyl alcohol, which has laid a foundation for producing 4-hydroxybenzyl alcohol economically and efficiently by further expanding the fermentation scale in the future.

The Role of TMEM16A/ERK/NK-1 Signaling in Dorsal Root Ganglia Neurons in the Development of Neuropathic Pain Induced by Spared Nerve Injury (SNI).

Increasing evidence suggests that transmembrane protein 16A (TMEM16A) in nociceptive neurons is an important molecular component contributing to peripheral pain transduction. The present study aimed to evaluate the role and mechanism of TMEM16A in chronic nociceptive responses elicited by spared nerve injury (SNI). In this study, SNI was used to induce neuropathic pain. Drugs were administered intrathecally. The expression and cellular localization of TMEM16A, the ERK pathway, and NK-1 in the dorsal root ganglion (DRG) were detected by western blot and immunofluorescence. Behavioral tests were used to evaluate the role of TMEM16A and p-ERK in SNI-induced persistent pain and hypersensitivity. The role of TMEM16A in the hyperexcitability of primary nociceptor neurons was assessed by electrophysiological recording. The results show that TMEM16A, p-ERK, and NK-1 are predominantly expressed in small neurons associated with nociceptive sensation. TMEM16A is colocalized with p-ERK/NK-1 in DRG. TMEM16A, the MEK/ERK pathway, and NK-1 are activated in DRG after SNI. ERK inhibitor or TMEM16A antagonist prevents SNI-induced allodynia. ERK and NK-1 are downstream of TMEM16A activation. Electrophysiological recording showed that CaCC current increases and intrathecal application of T16Ainh-A01, a selective TMEM16A inhibitor, reverses the hyperexcitability of DRG neurons harvested from rats after SNI. We conclude that TMEM16A activation in DRG leads to a positive interaction of the ERK pathway with activation of NK-1 production and is involved in the development of neuropathic pain after SNI. Also, the blockade of TMEM16A or inhibition of the downstream ERK pathway or NK-1 upregulation may prevent the development of neuropathic pain.

Broken-Gap PtS2/WSe2 van der Waals Heterojunction with Ultrahigh Reverse Rectification and Fast Photoresponse.

Broken-gap van der Waals (vdW) heterojunctions based on 2D materials are promising structures to fabricate high-speed switching and low-power multifunctional devices thanks to its charge transport versus quantum tunneling mechanism. However, the tunneling current is usually generated under both positive and negative bias voltage, resulting in small rectification and photocurrent on/off ratio. In this paper, we report a broken-gap vdW heterojunction PtS2/WSe2 with a bilateral accumulation region design and a big band offset by utilizing thick PtS2 as an effective carrier-selective contact, which exhibits an ultrahigh reverser rectification ratio approaching 108 and on/off ratio over 108 at room temperature. We also find excellent photodetection properties in such a heterodiode with a large photocurrent on/off ratio over 105 due to its ultralow forward current and a comparable photodetectivity of 3.8 × 1010 Jones. In addition, the response time of such a photodetector reaches 8 µs owing to the photoinduced tunneling mechanism and reduced interface trapping effect. The proposed heterojunction not only demonstrates the high-performance broken-gap heterodiode but also provides in-depth understanding of the tunneling mechanism in the development of future electronic and optoelectronic applications.

2D Silicon-Based Semiconductor Si2 Te3 toward Broadband Photodetection.

Silicon-based semiconductor materials dominate modern technology for more than half a century with extraordinary electrical-optical performance and mutual processing compatibility. Now, 2D materials have rapidly established themselves as prospective candidates for the next-generation semiconductor industry because of their novel properties. Considering chemical and processing compatibility, silicon-based 2D materials possess significant advantages in integrating with silicon. Here, a systematic study is reported on the structural, electrical, and optical performance of silicon telluride (Si2 Te3 ) 2D material, a IV-VI silicon-based semiconductor with a layered structure. The ultrawide photoluminescence (PL) spectra in the range of 550-1050 nm reveals the intrinsic defects in Si2 Te3 . The Si2 Te3 -based field-effect transistors (FETs) and photodetectors show a typical p-type behavior and a remarkable broadband spectral response in the range of 405-1064 nm. Notably, the photoresponsivity and detectivity of the photodetector device with 13.5 nm in thickness and upon 405 nm illumination can reach up to 65 A W-1 and 2.81 × 1012 Jones, respectively, outperforming many traditional broadband photodetectors. It is believed this work will excite interests in further exploring the practical application of 2D silicon-based materials in the field of optoelectronics.

[Expression of ANO1 during cardiac fibroblasts differentiation and its electrophysiological characteristics as calcium-activated chloride channel protein].

OBJECTIVE: To investigate the expression and electrophysiological characteristics of calcium-activated chlorine channel anoctamin-1 (ANO1) protein during the differentiation of cardiac fibroblasts (CFs) into myofibroblasts (MFs), and to elucidate the role of ANO1 in myocardial fibrosis. METHODS: The primary CFs from neonatal rats were isolated and the cells differentiated into MFs by subculture. The Ca2+-activated Cl- current (ICl(Ca)) in CFs and MFs were measured by whole-cell patch clamp, and the expressions of ANO1, α-smooth muscle actin(α-SMA)and vimentin in CFs and MFs were detected by immunofluorescence assay and Western blot, respectively. RESULTS: The current density in the early adherent CFs was stronger than that in MFs. ANO1 was expressed preferentially within and around the nuclei, and a small amount of ANO1 was expressed on the cell membrane. Moreover, ANO1 expression was weak in the early adherent CFs and displayed stronger expression in the MFs with proliferation tendency. CONCLUSION: The expression of ANO1 is closely related to the differentiation of MFs and it may be involved in modulation myocardial fibrosis.

A Self-Powered Photovoltaic Photodetector Based on a Lateral WSe2-WSe2 Homojunction.

Lateral homojunctions made of two-dimensional (2D) layered materials are promising for optoelectronic and electronic applications. Here, we report the lateral WSe2-WSe2 homojunction photodiodes formed spontaneously by thickness modulation in which there are unique band structures of a unilateral depletion region. The electrically tunable junctions can be switched from n-n to p-p diodes, and the corresponding rectification ratio increases from about 1 to 1.2 × 104. In addition, an obvious photovoltaic behavior is observed at zero gate voltage, which exhibits a large open voltage of 0.49 V and a short-circuit current of 0.125 nA under visible light irradiation. In addition, due to the unilateral depletion region, the diode can achieve a high detectivity of 4.4 × 1010 Jones and a fast photoresponse speed of 0.18 ms at Vg = 0 and Vds = 0. The studies not only demonstrated the great potential of the lateral homojunction photodiodes for a self-power photodetector but also allowed for the development of other functional devices, such as a nonvolatile programmable diode for logic rectifiers.

Upregulation of Nav1.7 by endogenous hydrogen sulfide contributes to maintenance of neuropathic pain.

Nav1.7 is closely associated with neuropathic pain. Hydrogen sulfide (H2S) has recently been reported to be involved in numerous biological functions, and it has been shown that H2S can enhance the sodium current density, and inhibiting the endogenous production of H2S mediated by cystathionine ß­synthetase (CBS) using O­(carboxymethyl)hydroxylamine hemihydrochloride (AOAA) can significantly reduce the expression of Nav1.7 and thus the sodium current density in rat dorsal root ganglion (DRG) neurons. In the present study, it was shown that the fluorescence intensity of H2S was increased in a spared nerve injury (SNI) model and AOAA inhibited this increase. Nav1.7 is expressed in DRG neurons, and the expression of CBS and Nav1.7 were increased in DRG neurons 7, 14 and 21 days post­operation. AOAA inhibited the increase in the expression of CBS, phosphorylated (p)­MEK1/2, p­ERK1/2 and Nav1.7 induced by SNI, and U0126 (a MEK blocker) was able to inhibit the increase in p­MEK1/2, p­ERK1/2 and Nav1.7 expression. However, PF­04856264 did not inhibit the increase in CBS, p­MEK1/2, p­ERK1/2 or Nav1.7 expression induced by SNI surgery. The current density of Nav1.7 was significantly increased in the SNI model and administration of AOAA and U0126 both significantly decreased the density. In addition, AOAA, U0126 and PF­04856264 inhibited the decrease in rheobase, and the increase in action potential induced by SNI in DRG neurons. There was no significant difference in thermal withdrawal latency among each group. However, the time the animals spent with their paw lifted increased significantly following SNI, and the time the animals spent with their paw lifted decreased significantly following the administration of AOAA, U0126 and PF­04856264. In conclusion, these data show that Nav1.7 expression in DRG neurons is upregulated by CBS­derived endogenous H2S in an SNI model, contributing to the maintenance of neuropathic pain.

Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury.

Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain. We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons. To this aim, rats with persistent hyperalgesia were randomly divided into four groups. Rats in the control group received no treatment, and the rat sciatic nerve was only exposed in the sham group. Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide, an inhibitor of NKCC1, based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group. In the experiment measuring thermal withdrawal latency, bumetanide (15 mg/kg) was intravenously administered. In the patch clamp experiment, bumetanide (10 µg/µL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour, or bumetanide (5 µg/µL) was intrathecally injected. The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats. We found that the thermal withdrawal latency of rats was significantly decreased on days 7, 14, and 21 after model establishment. After intravenous injection of bumetanide, the reduction in thermal retraction latency caused by model establishment was significantly inhibited. Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7, 14, and 21 after model establishment. No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment. The Cl- (chloride ion) fluorescent probe technique was used to evaluate the change of Cl- concentration in dorsal root ganglion neurons of chronic constriction injury model rats. We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl- fluorescent probe whose fluorescence intensity decreases as Cl- concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment. The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased, and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment. After bumetanide administration, the above indicators were significantly suppressed. These results confirm that CCI can induce abnormal overexpression of NKCC1, thereby increasing the Cl- concentration in dorsal root ganglion neurons; this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia. In addition, bumetanide can achieve analgesic effects. All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital, College of Medicine, Shihezi University, China on February 22, 2017 (approval No. A2017-169-01).

17ß-Estradiol Attenuates Neuropathic Pain Caused by Spared Nerve Injury by Upregulating CIC-3 in the Dorsal Root Ganglion of Ovariectomized Rats.

17ß-estradiol plays a role in pain sensitivity, analgesic drug efficacy, and neuropathic pain prevalence, but the underlying mechanisms remain unclear. Here, we investigated whether voltage-gated chloride channel-3 (ClC-3) impacts the effects of 17ß-estradiol (E2) on spared nerve injury (SNI)-induced neuropathic pain in ovariectomized (OVX) female Sprague Dawley rats that were divided into OVX, OVX + SNI, OVX + SNI + E2, OVX + SNI + E2 + DMSO (vehicle, dimethyl sulfoxide), or OVX + SNI + E2+Cltx (ClC-3-blocker chlorotoxin) groups. Changes in ClC-3 protein expression were monitored by western blot analysis. Behavioral testing used the paw withdrawal threshold to acetone irritation and paw withdrawal thermal latency (PWTL) to thermal stimulation. Immunofluorescence indicated the localization and protein expression levels of ClC-3. OVX + SNI + E2 rats were subcutaneously injected with 17ß-estradiol once daily for 7 days; a sheathed tube was implanted, and chlorotoxin was injected for 4 days. Intrathecal Cltx to OVX and OVX + SNI rats was administered for 4 consecutive days (days 7-10 after SNI) to further determine the contribution of ClC-3 to neuropathic pain. Patch clamp technology in current clamp mode was used to measure the current threshold (rheobase) dorsal root ganglion (DRG) neurons and the minimal current that evoked action potentials (APs) as excitability parameters. The mean number of APs at double-strength rheobase verified neuronal excitability. There was no difference in behaviors and ClC-3 expression after OVX. Compared with OVX + SNI rats, OVX + SNI + E2 rats showed a lower paw withdrawal threshold to the acetone stimulus, but the PWTL was not significantly different, indicating increased sensitivity to cold but not to thermal pain. Co-immunofluorescent data revealed that ClC-3 was mainly distributed in A- and C-type nociceptive neurons, especially in medium/small-sized neurons. 17ß-estradiol administration was associated with increased expression of ClC-3. 17ß-estradiol-induced increase in ClC-3 expression was blocked by co-administration of Cltx. Cltx causes hyperalgesia and decreased expression of ClC-3 in OVX rats. Patch clamp results suggested that 17ß-estradiol attenuated the excitability of neurons induced by SNI by up-regulating the expression of ClC-3 in the DRG of OVX rats. 17ß-estradiol administration significantly improved cold allodynia thresholds in OVX rats with SNI. The mechanism for this decreased sensitivity may be related to the upregulation of ClC-3 expression in the DRG.

Age­associated variation in the expression and function of TMEM16A calcium­activated chloride channels in the cochlear stria vascularis of guinea pigs.

The present study was designed to investigate the expression and function of transmembrane protein 16 (TMEM16A), a calcium­activated chloride channel (CaCC), in the stria vascularis (SV) of the cochlea of guinea pigs at different ages, and to understand the role of CaCCs in the pathogenesis of presbycusis (age­related hearing loss), the most common type of sensorineural hearing loss that occurs with natural aging. Guinea pigs were divided into the following groups: 2 weeks (young group), 3 months (youth group), 1 year (adult group), D­galactose intervention (D­gal group; aging model induced by subcutaneous injection of D­galactose) and T16Ainh­A01 (intraperitoneal injection of 50 µg/kg/day TMEM16A inhibitor T16Ainh­A01 for 2 weeks). Differences in the hearing of guinea pigs between the various age groups were analyzed using auditory brainstem response (ABR), and immunofluorescence staining was performed to detect TMEM16A expression in the SV and determine the distribution. Reverse transcription­quantitative PCR and western blot analyses were conducted to detect the mRNA and protein levels of TMEM16A in SV in the different age groups. Morris water maze behavior analysis demonstrated that spatial learning ability and memory were damaged in the D­gal group. Superoxide dismutase activity and malondialdehyde content assays indicated that there was oxidative stress damage in the D­gal group. The ABR thresholds gradually increased with age, and the increase in the T16Ainh­A01 group was pronounced. Immunofluorescence analysis in the cochlear SV of guinea pigs in different groups revealed that expression of TMEM16A increased with increasing age (2 weeks to 1 year); fluorescence intensity was reduced in the D­gal model of aging. As the guinea pigs continued to mature, the protein and mRNA contents of TMEM16A in the cochlea SV increased gradually, but were decreased in the D­gal group. The findings indicated that CaCCs in the cochlear SV of guinea pigs were associated with the development of hearing in guinea pigs, and that downregulation of TMEM16A may be associated with age­associated hearing loss.

[Effects of Ramipril on the expression of connexin 43 in cerebral arteries of spontaneously hypertensive rats].

The present study was designed to examine whether Ramipril (an inhibitor of angiotensin-converting enzyme) affected spontaneous hypertension-induced injury of cerebral artery by regulating connexin 43 (Cx43) expression. Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were randomly divided into WKY, WKY + Ramipril, SHR, and SHR + Ramipril groups (n = 8). The arterial pressure was monitored by the tail-cuff method, and vascular function in basilar arteries was examined by pressure myography. Hematoxylin-eosin (HE) staining was used to show vascular remodeling. The expression and distribution of Cx43 was determined by using immunofluorescence and immunohistochemistry analysis. The protein and mRNA levels of Cx43 were examined by Western blot and real-time PCR analysis, respectively. The results showed that chronic Ramipril treatment significantly attenuated blood pressure elevation (P < 0.01, n = 8) and blood vessel wall thickness in SHR (P < 0.01, n = 8). The cerebral artery contraction rate in the SHR group was higher than that in the WKY group (P < 0.05, n = 8). The cerebral artery contraction rate in the SHR + Ramipril group was lower than that in the SHR group (P < 0.05, n = 8). Pretreatment with 2-APB (Cx43 non-specific blocker) or Gap26 (Cx43 specific blocker) significantly decreased the vasoconstriction rate, while pretreatment with AAP10 (Cx43 non-specific agonist) significantly increased the vasoconstriction in the SHR + Ramipril group (P < 0.05, n = 8). In addition, the expression of Cx43 mRNA and protein in cerebral arteries of SHR group was higher than that of WKY group (P < 0.05, n = 8). The mRNA and protein expression of Cx43 in cerebral arteries of SHR + Ramipril group was significantly lower than that of SHR group (P < 0.05, n = 8). These results suggest that Ramipril can down-regulate the expression of Cx43 mRNA and protein in cerebral arterial cells of SHR, lower blood pressure, promote vasodilation, and improve arterial damage and vascular dysfunction caused by hypertension.

[Candesartan inhibits the angiotensin II-induced proliferation and migration of rat thoracic aortic smooth muscle A7r5 cells and its mechanism].

Objective To investigate the effect of candesartan on angiotensin II (Ang II)-induced proliferation and migration of vascular smooth muscle cells and its effect on connexin 43 (Cx43). Methods A7r5 cells were cultured in vitro and randomly divided into control group, AngII group and AngII combined with candesartan group. Cell viability was detected by CCK-8 assay; the migration and invasion ability of A7r5 cells were measured by wound-healing and TranswellTM assay; the expression and distribution of Cx43 on A7r5 cells were detected by immunofluorescence assay. Cx43, osteopontin (OPN), proliferating cell nuclear antigen (PCNA), p-MEK1/2 and p-ERK1/2 protein levels of A7r5 cells were detected by Western blot analysis. Results Compared with the control group, the AngII group had higher cell proliferation and migration ability, and the AngII combined with candesartan group had a lower concentration than the AngII group. Cx43 was expressed in the A7r5 cell membrane and nuclear membrane. The expression of Cx43 were enhanced in the AngII group than in the control group, and the expressions of Cx43, OPN, PCNA, p-MEK1/2 and p-ERK1/2 significantly increased compared with AngII. The expression of protein significantly decreased in AngII combined with candesartan group. Conclusion Candesartan can reduce the proliferation and migration of smooth muscle cells induced by AngII, and its mechanism may be related to Cx43 and MEK/ERK signaling pathways.

Mechanism of persistent hyperalgesia in neuropathic pain caused by chronic constriction injury.

Transmembrane member 16A (TMEM16A) is involved in many physiological functions, such as epithelial secretion, sensory conduction, nociception, control of neuronal excitability, and regulation of smooth muscle contraction, and may be important in peripheral pain transmission. To explore the role of TMEM16A in the persistent hyperalgesia that results from chronic constriction injury-induced neuropathic pain, a rat model of the condition was established by ligating the left sciatic nerve. A TMEM16A selective antagonist (10 µg T16Ainh-A01) was intrathecally injected at L5-6. For measurement of thermal hyperalgesia, the drug was administered once at 14 days and thermal withdrawal latency was recorded with an analgesia meter. For measurement of other indexes, the drug was administered at 12 days, once every 6 hours, totally five times. The measurements were performed at 14 days. Western blot assay was conducted to analyze TMEM16A expression in the L4-6 dorsal root ganglion. Immunofluorescence staining was used to detect the immunoreactivity of TMEM16A in the L4-6 dorsal root ganglion on the injured side. Patch clamp was used to detect electrophysiological changes in the neurons in the L4-6 dorsal root ganglion. Our results demonstrated that thermal withdrawal latency was shortened in the model rats compared with control rats. Additionally, TMEM16A expression and the number of TMEM16A positive cells in the L4-6 dorsal root ganglion were higher in the model rats, which induced excitation of the neurons in the L4-6 dorsal root ganglion. These findings were inhibited by T16Ainh-A01 and confirm that TMEM16A plays a key role in persistent chronic constriction injury-induced hyperalgesia. Thus, inhibiting TMEM16A might be a novel pharmacological intervention for neuropathic pain. All experimental protocols were approved by the Animal Ethics Committee at the First Affiliated Hospital of Shihezi University School of Medicine, China (approval No. A2017-170-01) on February 27, 2017.

The Protective Effect of Propofol Against Ischemia-Reperfusion Injury in the Interlobar Arteries: Reduction of Abnormal Cx43 Expression as a Possible Mechanism.

BACKGROUND/AIMS: This experimental study aims to observe whether the protective effect of propofol against renal ischemia-reperfusion injury (IRI) in the rat interlobar artery occurs through altered expression of the gap junction protein connexin 43 (Cx43). METHODS: This study randomly divided male Sprague Dawley (SD) rats into an untreated control group, a sham-operated control group (sham group), an ischemia-reperfusion group (IR group), a propofol group (propofol+IR group) and a fat emulsion group (Intralipid group). The ischemia/reperfusion model was prepared through resection of the right kidney and noninvasive arterial occlusion of the left kidney. Forty-five minutes after renal ischemia-reperfusion, an automatic biochemical analyzer was employed to measure blood urea nitrogen (BUN) and serum creatinine (SCr); changes in renal tissue pathology were observed using hematoxylin and eosin (HE) staining, and the vasomotor activity of the interlobar artery was detected using a pressure mechanogram technique. The protein expression of Cx43 in renal artery cross-sections was determined through western blotting. RESULTS: The experimental study confirmed that the BUN and SCr of rats markedly increased after ischemia-reperfusion injury; additionally, we observed some coagulation necrosis and shedding of cells, some solidification of nuclear chromatin, degeneration of cytoplasmic vacuoles, high renal interstitial vascular congestion and obvious inflammatory cell infiltration, characterized by focal hemorrhages. Furthermore, the contraction activity of the renal interlobar artery greatly decreased, and the tension of the arteries in the renal lobe increased remarkably. After the gap junction blocking agents 2-APB and Gap27 were applied, the systolic velocity of blood vessels and the vascular contraction rate both decreased. In addition, the expression of Cx43 in kidney tissues increased markedly. The damage was more severe after 24 h of ischemic reperfusion than after only 4 h. However, after pretreatment with propofol, regardless of whether ischemia-reperfusion was applied for 4 h or 24 h, the previously increased expression of Cx43 decreased obviously, and all forms of renal damage were reversed. CONCLUSION: Our research suggests new ways for propofol to relieve ischemia-reperfusion injury by decreasing the abnormal expression of the gap junction protein Cx43. This study reveals a novel mechanism for the action of propofol against IRI, and we hope this finding will lead to new treatments for IRI.

[Gene cloning and prokaryotic expression of glycosyltransferase from Ligustrum quihoui].

According to the previous results from transcriptome analysis of Ligustrum quihoui, a glycosyltransferase gene(xynzUGT) was cloned by rapid amplification of cDNA ends(RACE). The full length cDNA of xynzUGT was 1 598 bp, consisting of 66 bp 5'-UTR, 1 440 bp ORF and 92 bp 3'-UTR. The ORF encoded a 480 amino-acid protein(xynzUGT) with a molecular weight of 54 826.67 Da and isoelectric point of 5.82. The structure of enzyme was analyzed by using bioinformatics method, the results showed that the primary structure contained a highly conserved PSPG box of glycosyltransferase, the secondary structure included α helix(38%), ß sheet(12.1%) and random coil(49.9%), and tertiary structure was constructed by peptide chain folding to form two face-to-face α/ß/α domains(often referred to as a Rossmann domains), between which a substrate binding pocket is sandwiched. The phylogenetic tree analysis indicated that xynzUGT might catalyze glycosylation of phenylpropanoids, such as tyrosol. Further simulation experiment of molecular docking between enzyme and tyrosol showed that Gly138 and Ser285 located in the binding pocket interacted with tyrosol by hydrogen bonding. SDS-PAGE analysis exhibited that the prokaryotic expression system successfully expressed recombinant xynzUGT with molecular weight of 58 370.57 Da, but it exists in the form of non-soluble inclusion bodies. Using the molecular chaperone and enzyme co-expression method, the soluble expression was promoted to some extent. The above works laid the foundation for further studying on enzymatic reaction in vitro and clarifying the functional mechanism of enzyme.

Modulating drug release from gastric-floating microcapsules through spray-coating layers.

Floating dosage forms with prolonged gastric residence time have garnered much interest in the field of oral delivery. However, studies had shown that slow and incomplete release of hydrophobic drugs during gastric residence period would reduce drug absorption and cause drug wastage. Herein, a spray-coated floating microcapsule system was developed to encapsulate fenofibrate and piroxicam, as model hydrophobic drugs, into the coating layers with the aim of enhancing and tuning drug release rates. Incorporating fenofibrate into rubbery poly(caprolactone) (PCL) coating layer resulted in a complete and sustained release for up to 8 h, with outermost non-drug-holding PCL coating layer serving as a rate-controlling membrane. To realize a multidrug-loaded system, both hydrophilic metformin HCl and hydrophobic fenofibrate were simultaneously incorporated into these spray-coated microcapsules, with metformin HCl and fenofibrate localized within the hollow cavity of the capsule and coating layer, respectively. Both drugs were observed to be completely released from these coated microcapsules in a sustained manner. Through specific tailoring of coating polymers and their configurations, piroxicam loaded in both the outer polyethylene glycol and inner PCL coating layers was released in a double-profile manner (i.e. an immediate burst release as the loading dose, followed by a sustained release as the maintenance dose). The fabricated microcapsules exhibited excellent buoyancy in simulated gastric fluid, and provided controlled and sustained release, thus revealing its potential as a rate-controlled oral drug delivery system.