Long-term sciatic nerve block led by a supramolecular arrangement of self-delivery local anesthetic nano systems.

Classical local anesthetics are unsuitable to treat regional pain lasting several days due to their limited duration and systemic toxicity. Self-delivery nano systems without excipients were designed for long-term sensory blocks. 1a self-assembled into different vehicles with different fractions of intermolecular π-π stacking, transported itself into nerve cells, and released single molecules slowly to achieve long-term duration for rats' sciatic nerve block for 11.6 h in water, 12.1 h in water with CO2 and 3.4 h in NS (normal saline). After the counter ions were changed to SO42-, 1e can self-assemble into vesicles and prolong the duration to 43.2 h, which was much longer than the 3.8 h led by (s)-bupivacaine hydrocloride (0.75%). This was mainly caused by the enhancement of self-release and counter ion exchange inside nerve cells, which were affected by the gemini surfactant structure, pKa of the counter ions and π-π stacking interactions.

A novel homozygous TUB mutation associated with autosomal recessive retinitis pigmentosa in a consanguineous Chinese family.

BACKGROUND: Retinitis pigmentosa (RP) is the most common type of inherited retinopathy. At least 69 genes for RP have been identified. A significant proportion of RP, however, remains genetically unsolved. In this study, the genetic basis of a Chinese consanguineous family with presumed autosomal recessive retinitis pigmentosa (arRP) was investigated. METHODS: Overall ophthalmic examinations, including funduscopy, decimal best-corrected visual acuity, axial length and electroretinography (ERG) were performed for the family. Genomic DNA from peripheral blood of the proband was subjected to whole exome sequencing. In silico predictions, structural modelling, and minigene assays were conducted to evaluate the pathogenicity of the variant. RESULTS: A novel homozygous variant (NM_003320.4: c.1379A > G) in the TUB gene was identified as a candidate pathogenic variant in this parental consanguineous pedigree. This variant co-segregated with the disease in this pedigree and was absent in 118 ethnically matched healthy controls. It's an extremely rare variant that is neither deposited in population databases (1000 Genomes, ExAC, GnomAD, or Exome Variant Server) nor reported in the literature. Phylogenetic analysis indicated that the Asn residue at codon 460 of TUB is highly conserved across diverse species from tropicalis to humans. It was also completely conserved among the TUB, TULP1, TULP2, and TULP3 family proteins. Multiple bioinformatic algorithms predicted that this variant was deleterious. CONCLUSIONS: A novel missense variant in TUB was identified, which was probably the pathogenic basis for arRP in this consanguineous family. This is the first report of a homozygous missense variant in TUB for RP.

Lido-OH, a Hydroxyl Derivative of Lidocaine, Produced a Similar Local Anesthesia Profile as Lidocaine With Reduced Systemic Toxicities.

Background: lidocaine is one of the most commonly used local anesthetics for the treatment of pain and arrhythmia. However, it could cause systemic toxicities when plasma concentration is raised. To reduce lidocaine's toxicity, we designed a hydroxyl derivative of lidocaine (lido-OH), and its local anesthesia effects and systemic toxicity in vivo were quantitively investigated. Method: the effectiveness for lido-OH was studied using mouse tail nerve block, rat dorsal subcutaneous infiltration, and rat sciatic nerve block models. The systemic toxicities for lido-OH were evaluated with altered state of consciousness (ASC), arrhythmia, and death in mice. Lidocaine and saline were used as positive and negative control, respectively. The dose-effect relationships were analyzed. Results: the half effective-concentration for lido-OH were 2.1 mg/ml with 95% confident interval (CI95) 1.6-3.1 (lidocaine: 3.1 mg/ml with CI95 2.6-4.3) in tail nerve block, 8.2 mg/ml with CI95 8.0-9.4 (lidocaine: 6.9 mg/ml, CI95 6.8-7.1) in sciatic nerve block, and 5.9 mg/ml with CI95 5.8-6.0 (lidocaine: 3.1 mg/ml, CI95 2.4-4.0) in dorsal subcutaneous anesthesia, respectively. The magnitude and duration of lido-OH were similar with lidocaine. The half effective doses (ED50) of lido-OH for ACS was 45.4 mg/kg with CI95 41.6-48.3 (lidocaine: 3.1 mg/kg, CI95 1.9-2.9), for arrhythmia was 16.0 mg/kg with CI95 15.4-16.8 (lidocaine: 3.0 mg/kg, CI95 2.7-3.3), and for death was 99.4 mg/kg with CI95 75.7-124.1 (lidocaine: 23.1 mg/kg, CI95 22.8-23.4). The therapeutic index for lido-OH and lidocaine were 35.5 and 5.6, respectively. Conclusion: compared with lidocaine, lido-OH produced local anesthesia at similar potency and efficacy, but with significantly reduced systemic toxicities.

Molecular biology of the SARs-CoV-2 spike protein: A review of current knowledge.

The global coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to an unprecedented worldwide public health emergency. Despite the concerted efforts of the scientific field, by April 25, 2021, SARS-CoV-2 had spread to over 192 countries/regions, causing more than 146 million confirmed cases including 31 million deaths. For now, an established treatment for patients with COVID-19 remains unavailable. The key to tackling this pandemic is to understand the mechanisms underlying its infectivity and pathogenicity. As a predominant focus, the coronavirus spike (S) protein is the key determinant of host range, infectivity, and pathogenesis. Thereby comprehensive understanding of the sophisticated structure of SARS-CoV-2 S protein may provide insights into possible intervention strategies to fight this ongoing global pandemic. Herein, we summarize the current knowledge of the molecular structural and functional features of SARS-CoV-2 S protein as well as recent updates on the cell entry mechanism of the SARS-CoV-2, paving the way for exploring more structure-guided strategies against SARS-CoV-2.

The Bioequivalence of Emulsified Isoflurane With a New Formulation of Emulsion: A Single-Center, Single-Dose, Double-Blinded, Randomized, Two-Period Crossover Study.

Background: Emulsified isoflurane is a novel intravenous general anesthetic obtained by encapsulating isoflurane molecules into emulsion. The formulation of emulsion has been improved according to the latest regulations of the China Food and Drug Administration. This study was designed to compare the bioequivalence of the new and previous formulation emulsion of isoflurane. Methods: In a single-center, single-dose, double-blinded, randomized, two-period crossover study, healthy volunteers received intravenous injection of 30 mg/kg of isoflurane with either previous formulation of emulsion isoflurane (PFEI) or new formulation of emulsion isoflurane (NFEI). Arterial and venous blood samples were obtained for geometric mean test/reference ratios of Cmax, AUC0-t, and AUC0-∞, as well as their 90% confidence interval (CI90) as the primary outcome. The secondary outcomes were safety measurements such as vital signs, 12-lead electrocardiography, adverse effects, and laboratory tests; and anesthesia efficacy was assessed by Modified Observer's Assessment of Alertness/Sedation (MOAA/S) score, bispectral index (BIS), and loss/recovery of eyelash reflex. Results: 24 subjects were eligible, of which 21 completed the whole experiment (NFEI n = 21, PFEI n = 23). Arterial geometric mean test/reference ratios of Cmax, AUC0-t, and AUC0-∞ were 104.50% (CI90 92.81%-117.65%), 108.23% (94.51%-123.96%), and 106.53% (93.94%∼120.80%), respectively. The most commonly seen adverse effects for NFEI and PFEI were injection pain (38.1% vs. 34.8%), hypotension (19.0% vs. 13.0%), apnea (14.3% vs. 17.4%), and upper airway obstruction (14.3% vs. 13.0%). No severe adverse effect was observed. The effectiveness of general anesthesia was similar between the two formulations. Conclusion: The CI90 of Cmax, AUC0-t, AUC0-∞, NFEI, and PFEI were within the range of 80%-125%, suggesting bioequivalence between NFEI and PFEI. The safety and anesthesia effectiveness were also similar.

The 14-day repeated-dose toxicity study of a fixed-dose combination, QXOH/levobupivacaine, via subcutaneous injection in beagle dogs.

QXOH-Levobupivacaine (LB) is a fixed-dose combination of 35-mM QXOH and 10-mM LB. It was developed for perioperative analgesia because of its long-acting analgesic effect. The purpose of this study was to evaluate the potential toxicity of QXOH-LB in beagle dogs in accordance with the Guidance on the repeated-dose toxicity published by the China Food and Drug Administration. Groups of five male and five female beagle dogs received normal saline, QXOH-LB (2, 4, and 8 mg/kg, calculated as QXOH), QXOH (2, 4, and 8 mg/kg), or LB (2 mg/kg, equals the concentration of LB in 8-mg/kg QXOH-LB group) at the volume of 1 mL/kg once per day for 14 days through subcutaneous injection. No mortality was observed. Dogs in the control group as well as animals treated with 2-mg/kg QXOH or QXOH-LB exhibited normal behaviors. Clinical signs of toxicity in dogs treated with 4 and 8 mg/kg of QXOH or QXOH-LB included decreased activity, unsteady gait, jerks, tremors, vocalization, emesis, ataxia, lateral/sternal recumbency, deep/rapid respiration, and gasping. Additionally, neurological function was found to be affected by QXOH and QXOH-LB at the doses of 4 and 8 mg/kg. All clinical signs were recovered within 24 h. The no-observed-adverse-effect level of QXOH and QXOH-LB was considered to be 2 mg/kg. Toxicokinetic data showed that exposure to QXOH and LB increased as QXOH-LB doses were increased from 4 to 8 mg/kg. There was no evidence of drug accumulation or any effect of gender.

Novel compound LL-a produces long and nociceptive-selective regional anesthesia via TRPV1 channels in rodents sciatic nerve block model.

BACKGROUND AND OBJECTIVE: Long-acting nociceptive-selective regional anesthesia has remained an elusive clinical goal. We aspired to identify a novel compound that would produce nociceptive-selective regional anesthesia through the transient receptor potential vanilloid 1 (TRPV1) channels. METHODS: We designed and synthesized a novel compound (LL-a) that penetrates the cell membrane through TRPV1 channels and binds to voltage-gated sodium channels. The regional anesthetic effect of LL-a was evaluated in a rodent sciatic nerve block model. Electrophysiological recording was applied to test the inhibition of LL-a on voltage-gated sodium channel currents. RESULTS: LL-a inhibited sodium channel currents on the dorsal root ganglion neurons of mice and this action was diminished by TRPV1 channel knockout. In a sciatic nerve block model of a rat, 0.2% and 0.4% (w/v) LL-a produced selective sensory block with median (IQR) durations of 42.0 (24.0, 48.0) and 72.0 (69.0, 78.0) hours, respectively. No motor block was found for 0.2% LL-a. 0.4% LL-a produced a motor block with a median (IQR) duration of 3.0 (0.0, 6.0) hours. This selective sensory block was not observed on TRPV1 knockout mice. As a positive control, 0.5% and 0.75% levobupivacaine produced a non-selective sciatic nerve block with median (IQR) durations of 2.8 (2.6, 2.8) and 3.8 (3.8, 4.8) hours, respectively. No systemic or local irritation was observed during injection of LL-a and sensory and motor function completely recovered for all the animals. CONCLUSIONS: LL-a is a potential novel local anesthetic for long-lasting nociceptive-selective analgesia.

The Preclinical Pharmacological Study of a Novel Long-Acting Local Anesthetic, a Fixed-Dose Combination of QX-OH/Levobupivacaine, in Rats.

Introduction: Previous studies demonstrated that 35 mM QX-OH/10 mM Levobupivacaine (LL-1), a fixed-dose combination, produced a long-acting effect in rat local anesthesia models. All preclinical pharmacodynamic results indicated that LL-1 had potential for postsurgical pain treatment. The objective of this study was to investigate the pharmacokinetics of LL-1. Then, the possible mechanism of the extended duration by the combination was examined. Methods and Results: All experiments were examined and approved by the Committee of Animal Care of the West China Hospital Sichuan University (Ethical approval number, 2015014A). The compound action potentials were recorded to verify the pharmacodynamic result in ex vivo. In frog sciatic nerve, LL-1 produced an effective inhibition with rapid onset time. The concentration-time profiles of LL-1 were determined in plasma and local tissues after sciatic nerve block. The maximum concentration of QX-OH and levobupivacaine were 727.22 ± 43.38 µg/g and 256.02 ± 28.52 µg/g in muscle, 634.26 ± 36.04 µg/g and 429.63 ± 48.64 µg/g in sciatic nerve, and 711.71 ± 25.14 ng/ml and 114.40 ± 10.19 ng/ml in plasma, respectively. The absorption of QX-OH into circulation was very rapid at 0.71 ± 0.06 h, which was faster than that of levobupivacaine (4.11 ± 0.39 h, p = 0.003). The half-time of QX-OH in plasma and local tissues had no significant difference (p = 0.329), with the values of 2.64 h, 3.20 h, and 3.79 h in plasma, muscle, and sciatic nerve, respectively. The elimination profile of levobupivacaine differed from that of QX-OH, which was slower eliminated from plasma (4.89 ± 1.77 h, p = 0.036) than from muscle (1.38 ± 0.60 h) or sciatic nerve (1.28 ± 0.74 h). When levobupivacaine was used alone, the Tmax in plasma was 1.07 ± 0.16 h. Interestingly, the Tmax of levobupivacaine in the plasma was increased by four times in combination with QX-OH (4.11 ± 0.39 h). Levobupivacaine promotes cellular QX-OH uptake. Conclusion: The preclinical pharmacokinetic study of LL-1 in the rat plasma, muscle, and sciatic nerve was accomplished. Then, the possible mechanism of the prolonged duration was that QX-OH delayed the absorption of levobupivacaine from the injection site into circulation, and levobupivacaine accelerated QX-OH to accumulate into cells.

A Fixed-Dose Combination, QXOH/Levobupivacaine, Produces Long-Acting Local Anesthesia in Rats Without Additional Toxicity.

QXOH, a QX314 derivative with longer duration and lesser local toxicity, is a novel local anesthetic in preclinical drug development. Previous studies demonstrated that bupivacaine can prolong the effects of QX314. So, we attempted to combine QXOH with levobupivacaine to shorten the onset time and lengthen the duration. In this study, we investigated the efficacy, local and systemic toxicity in rats. In subcutaneous infiltration anesthesia, the inhibition of cutaneous trunci muscle reflex for QXOH-LB was greater than QXOH and levobupivacaine in the first 8 h (QXOH-LB vs. QXOH, P = 0.004; QXOH-LB vs. LB, P = 0.004). The completely recovery time for QXOH-LB (17.5 ± 2.5 h) was significantly longer than levobupivacaine (9.0 ± 1.3 h, P = 0.034) and QXOH (9.8 ± 0.9 h, P = 0.049). In sciatic nerve block, QXOH-LB produced a rapid onset time, which was obviously shorter than QXOH. For sensory, the time to recovery for QXOH-LB was 17.3 ± 2.6 h, which was statistically longer than 6.0 ± 1.8 h for QXOH (P = 0.027), and 4 h for levobupivacaine (P = 0.001). Meanwhile, the time to motor recovery for QXOH-LB was 7.9 ± 2.8 h, significantly longer than 4 h for levobupivacaine (P = 0.003) but similar to 6.0 ± 1.7 h for QXOH (P = 0.061). In local toxicity, there was no significant difference of histological score regarding muscle and sciatic nerve in QXOH-LB, QXOH, levobupivacaine and saline (P < 0.01). In the combination, the interaction index of LD50 was 1.39, indicating antagonistic interaction between QXOH and levobupivacaine in terms of systemic toxicity. In this study, we demonstrated that QXOH-LB produced cutaneous anesthesia which was 2-fold greater than that produced by QXOH or LB alone, and elicited sciatic nerve block with a potency that was 5- and 3-fold that of LB and QXOH, respectively. Local tissue inflammation by QXOH-LB was mild, similar to that induced by LB. This fixed-dose combination led to an antagonistic interaction between QXOH and LB in terms of systemic toxicity. These results suggested that QXOH-LB induced a long-lasting local anesthesia, likely, avoiding clinically important local and systemic toxicities.

Addition of dexmedetomidine to QX-314 enhances the onset and duration of sciatic nerve block in rats.

An experimental set-up was designed to observe whether adding dexmedetomidine to QX-314 would enhance the onset and duration of sensory and motor function in a rat sciatic nerve block model. Fifty-six Sprague-Dawley rats received unilateral sciatic nerve blocks with 0.2 mL of 35 mmol/L QX-314 alone, dexmedetomidine (5.3 µmol/L (1 µg/kg), 26.4 µmol/L (5 µg/kg), 52.8 µmol/L (10 µg/kg)) alone, or a combination of the two. Thermal nociception and motor function were assessed by an investigator blinded to the drug treatment, and sciatic nerves and perineural tissues were harvested at 14 days after injection. In addition, we examined the effects of these solutions on compound action potentials in isolated frog sciatic nerves. Dexmedetomidine added to QX-314 enhanced the onset and duration of thermal nociception block and motor block (P < 0.05) without aggravating histopathological injuries. Furthermore, 52.8 µmol/L dexmedetomidine added to 35 mmol/L QX-314 showed less inflammation than QX-314 alone at 14 days (P = 0.003). Dexmedetomidine plus QX-314 was shown to dose-dependently reduce the compound action potentials relative to QX-314 alone (P < 0.05). It was concluded that co-administration of QX-314 with a clinical dose of dexmedetomidine produced a synergistic anesthetic effect to enhance the effect of sciatic nerve block.

Janus particles self-assembled from a small organic atypical asymmetric gemini surfactant.

A series of atypical asymmetric gemini surfactants with an amphiphilic carbonate group (-O-CO-O-) have been prepared. Some of these compounds could self-assemble in water into gourd-shaped Janus particles (JPs). Initial results suggested that the formation of JPs was highly likely to be related to their atypical gemini surfactant structure. To our knowledge, this is the first report on JPs that are self-assembled from a single kind of small organic molecule. We believe that our results will be utilized in many fields.

Correction: The quaternary lidocaine derivative QX-314 in combination with bupivacaine for long-lasting nerve block: Efficacy, toxicity, and the optimal formulation in rats.

[This corrects the article DOI: 10.1371/journal.pone.0174421.].

QX-OH, a QX-314 derivative agent, produces long-acting local anesthesia in rats.

QX-314 has been shown to produce long-acting local anesthesia in vivo in animals; however, translation to humans has been impeded by concerns about toxicity. We investigated whether the newly emerged QX-OH molecule could confer long-lasting anesthesia with a low local toxicity in rats. In rat sciatic nerve block model, QX-OH 25mM produced a longer sensory block than QX-314 25mM (median [25th, 75th percentiles], 5.5 [4.25, 6] h vs. 3 [3, 4] h; P=0.03). QX-OH 35mM produced a longer sensory block than QX-314 35mM (8 [6, 12] h vs. 6 [4, 6.5] h, P=0.038). QX-OH at 35 and 45mM generated longer motor blocks than QX-314, with tissue toxicity less than that of QX-314 at the same concentration. In contrast with bupivacaine, QX-OH was clearly superior in terms of sensory and motor blockade durations after a single bolus injection. There was no significant difference in tissue toxicity between QX-OH (25 and 35mM) and bupivacaine. In rat cutaneous trunci pinprick model, the QX-OH-induced pain threshold remained significantly different from baseline at 6h (25mM, P<0.0001), 10h (35mM, P<0.0001), and 12h (45mM, P<0.0001). The time required for full recovery from the subcutaneous anesthetic effect was significantly longer for QX-OH than for QX-314 and bupivacaine. So QX-OH produced concentration-dependent, reversible, and long-acting local anesthesia in animal models with a moderate local toxicity.

Evaluation of the cardiotoxicity and resuscitation of rats of a newly developed mixture of a QX-314 analog and levobupivacaine.

OBJECTIVE: This study was designed to evaluate the cardiotoxicity of a QX-314 analog (QX-OH) and a mixture of QX-OH and levobupivacaine (LL-1) and to compare the ability to resuscitate rats after asystole induced by levobupivacaine (Levo-BUP), QX-314, QX-OH, and LL-1. METHODS: First, we used the "up-and-down" method to determine median dose resulting in appearance of cardiotoxicity (CD50C) and asystole (CD50A) of Levo-BUP, QX-314, QX-OH, and LL-1 in rats. Safety index (SI; ratio of CD50C compared with 2-fold median effective dose needed to produce sensory blockade) of the 4 drugs was calculated. Isobolograms were used for drug interaction analysis. Second, rats received 1.2-fold CD50A in the 4 groups. When asystole occurred, standard cardiopulmonary resuscitation was started and continued for 30 min or until return of spontaneous circulation (ROSC) with native rate-pressure product ≥30% baseline for 5 min. RESULTS: Ranking of CD50C was Levo-BUP < QX-314 ≈ QX-OH. Ranking of CD50A was Levo-BUP < QX-314 < QX-OH. However, the SI of Levo-BUP was significantly higher than that of QX-314 (10.60 vs. 1.20) or QX-OH (10.60 vs. 1.44). The SI of LL-1 was similar to that of Levo-BUP. Nonsynergistic interaction was observed for cardiac effects between QX-OH and Levo-BUP. ROSC was attained initially by 8 of 8 rats in the Levo-BUP group, 3 of 8 in the QX-314 group, 6 of 8 in the QX-OH group, and 8 of 8 in the LL-1 group. Sustained recovery was achieved in the Levo-BUP group but not in the other groups. CONCLUSION: Levo-BUP and LL-1 are safer than QX-314 or QX-OH. Cardiac effects between QX-OH and Levo-BUP were nonsynergistic. Initial successful resuscitation could be achieved in the QX-OH- and LL-1-induced asystole, but advanced life support might be needed.

The quaternary lidocaine derivative QX-314 in combination with bupivacaine for long-lasting nerve block: Efficacy, toxicity, and the optimal formulation in rats.

OBJECTIVE: The quaternary lidocaine derivative (QX-314) in combination with bupivacaine can produce long-lasting nerve blocks in vivo, indicating potential clinical application. The aim of the study was to investigate the efficacy, safety, and the optimal formulation of this combination. METHODS: QX-314 and bupivacaine at different concentration ratios were injected in the vicinity of the sciatic nerve in rats; bupivacaine and saline served as controls (n = 6~10). Rats were inspected for durations of effective sensory and motor nerve blocks, systemic adverse effects, and histological changes of local tissues. Mathematical models were established to reveal drug-interaction, concentration-effect relationships, and the optimal ratio of QX-314 to bupivacaine. RESULTS: 0.2~1.5% QX-314 with 0.03~0.5% bupivacaine produced 5.8~23.8 h of effective nerve block; while 0.5% bupivacaine alone was effective for 4 h. No systemic side effects were observed; local tissue reactions were similar to those caused by 0.5% bupivacaine if QX-314 were used < 1.2%. The weighted modification model was successfully established, which revealed that QX-314 was the main active ingredient while bupivacaine was the synergist. The formulation, 0.9% QX-314 plus 0.5% bupivacaine, resulted in 10.1 ± 0.8 h of effective sensory and motor nerve blocks. CONCLUSION: The combination of QX-314 and bupivacaine facilitated prolonged sciatic nerve block in rats with a satisfactory safety profile, maximizing the duration of nerve block without clinically important systemic and local tissue toxicity. It may emerge as an alternative approach to post-operative pain treatment.

Effects of Liposomes Charge on Extending Sciatic Nerve Blockade of N-ethyl Bromide of Lidocaine in Rats.

N-methyl bromide of lidocaine (QX-314) is a potential local anaesthetic with compromised penetration through cell membranes due to its obligated positive charge. Liposomes have been widely used for drug delivery with promising efficacy and safety. Therefore we investigated the local anaesthetic effects and tissue reactions of QX-314 in combination with anionic, cationic or neutral liposomes in rat sciatic nerve block model, and explored the effects of these liposomes on cellular entry of QX-314 in human embryonic kidney 293 cells. The results demonstrated that anionic liposomes substantially prolonged the duration of sensory (25.7 ± 8.3 h) and motor (41.4 ± 6.1 h) blocks of QX-314, while cationic and neutral ones had little effects. Tissue reactions from QX-314 with anionic liposomes were similar to those with commonly used local anaesthetic bupivacaine. Consistent with in vivo results, the anionic liposomes produced the greatest promotion of cellular entry of QX-314 in a time-dependent manner. In conclusion, ultra-long lasting nerve blocks were achieved by a mixture of QX-314 and anionic liposomes with a satisfactory safety profile, indicating a potential approach to improve postoperative pain management. The liposome-induced enhancement in cellular uptake of QX-314 may underlie the in vivo effects.

OVA12, a novel tumor antigen, promotes cancer cell growth and inhibits 5-fluorouracil-induced apoptosis.

To achieve a better understanding of mechanisms that underlie carcinogenesis and to identify novel target molecules for diagnosis and therapy of carcinoma, we previously identified 24 distinct gene clones by immunoscreening of a cDNA library derived from an ovarian cancer patient through SEREX analysis. Among these genes we focused on a novel gene termed OVA12 and which putatively encodes a 114-amino-acid protein. In the present study, we found that OVA12 was ubiquitously overexpressed in diverse human tumor cell lines. Interestingly, we noticed that overexpression of OVA12 promoted proliferation of cancer cells in vitro and accelerated tumor growth in nude mice as compared to controls. Conversely, specific downregulation of OVA12 inhibited tumor cell proliferation and tumor growth both in vitro and in vivo. Furthermore, OVA12 inhibited 5-FU-induced apoptosis through specific upregulation of Mcl-1 and survivin. These results demonstrate that OVA12 is able to promote tumor growth, suggesting that this antigen might be a new potential target for development of cancer therapy.

OVA66, a tumor associated protein, induces oncogenic transformation of NIH3T3 cells.

The tumor associated antigen OVA66 has been demonstrated to be highly expressed in malignant tumors and implicated in various cellular processes. To further elucidate its oncogenic character, we established an OVA66 stably overexpressed NIH3T3 cell line and a vector transfected control, named NIH3T3-flagOVA66 and NIH3T3-mock, respectively. NIH3T3-flagOVA66 cells showed faster cell cycling, proliferation, cell migration and more resistance to 5-fluorouracil-induced apoptosis. When NIH3T3-flagOVA66 and NIH3T3-mock cells were injected into nude mice for xenograft tumorigenicity assays, the NIH3T3-flagOVA66 cells formed tumors whereas no tumors were observed in mice inoculated with NIH3T3-mock cells. Analysis of PI3K/AKT and ERK1/2 MAPK signaling pathways by serum stimulation indicated hyperactivation of AKT and ERK1/2 in NIH3T3-flagOVA66 cells compared with NIH3T3-mock cells, while a decreased level of p-AKT and p-ERK1/2 were observed in OVA66 knocked down HeLa cells. To further validate if the p-AKT or p-ERK1/2 is essential for OVA66 induced oncogenic transformation, we treated the cells with the PI3K/AKT specific inhibitor LY294002 and the ERK1/2 MAPK specific inhibitor PD98059 and found either inhibitor can attenuate the cell colony forming ability in soft agar and the cell viability of NIH3T3-flagOVA66 cells, suggesting aberrantly activated AKT and ERK1/2 signaling be indispensible of the tumorigenic role of OVA66. Our results indicate that OVA66 is important in oncogenic transformation, promoting proliferation, cell migration and reducing apoptosis via hyperactivating PI3K/AKT and ERK1/2 MAPK signaling pathway. Thus, OVA66 might be a novel target for early detection, prevention and treatment of tumors in the future.

OVA66 increases cell growth, invasion and survival via regulation of IGF-1R-MAPK signaling in human cancer cells.

Ovarian cancer-associated antigen 66 (OVA66), also known as CML66 (GenBank Accession No. AF283301), was first identified in an ovarian carcinoma complementary DNA (cDNA) expression library and was shown to play a role in tumorigenesis. Here, we find that OVA66 influences tumorigenesis by regulating the type I insulin-like growth factor receptor (IGF-1R) signaling pathway. Stable knockdown of OVA66 in cancer cells attenuated phosphorylation of IGF-1R and extracellular signal-regulated kinase 1/2 (ERK1/2)-Hsp27; similarly, a higher level of p-IGF-1R and ERK1/2-Hsp27 signaling was also detected after OVA66 overexpression in HO8910 cells. In vivo knockdown of OVA66 both reduced tumor burden in nude mice and decreased phosphorylation of IGF-1R, ERK1/2 and hsp27. We blocked IGF-1R function both by small interfering RNA (siRNA) and with the chemical inhibitor Linsitinib (OSI-906). By either method, tumorigenesis was inhibited regardless of OVA66 expression; thus, mechanistically, IGF-1R, probably, lies downstream of OVA66 in cancer cells. We also found that OVA66 regulates expression of murine double minute 2 (MDM2); this attenuates ubiquitination of IGF-1R in response to IGF-1 stimulation and promotes active ERK1/2 signaling. Thus, we propose that combined overexpression of OVA66 and MDM2 promotes oncogenesis by enhancing activation of the IGF-1R-ERK1/2 signaling pathway.

Transdermal anaesthesia with lidocaine nano-formulation pretreated with low-frequency ultrasound in rats model.

Rapid local transdermal anaesthetic is desirable in clinic. In this paper, lidocaine loaded poly(epsilon-caprolactone)-poly(ethylene glycol)-poly(epsilon-caprolactone) (PCL-PEG-PCL) nanoparticles were prepared, and a novel transdermal lidocaine formulation: lidocaine loaded PCL-PEG-PCL nanoparticles in F127 hydrogel (Nano-Lido Gel), was demonstrated. These lidocaine loaded PCL-PEG-PCL nanoparticles with mean particle size of ca. 200 nm had drug loading of about 40%. The efficiency of transdermal anaesthesia of four treatments: EMLA cream (E), Nano-Lido Gel (N), EMLA cream with brief focal ultrasound pretreatment (EU), and Nano-Lidocaine Gel with brief focal ultrasound pretreatment (NU), was evaluated by tail-flick latency test assay in rats. Results indicated that the topical anaesthesia onset time in NU was 5 times and 2.5 times shorter than that in E and EU. The efficiency of anaesthesia in NU, expressed as maximum possible effects (MPE) value, was significantly higher than that in other treatments. It provided a novel path to develop rapid transdermal anaesthesia by combination of ultrasound pretreatment and lidocaine nano-formulation based on polymeric nanoparticles.