Triple Combination of Amantadine,Ribavirin, and Oseltamivir Is Highly Active and Synergistic against Drug Resistant Influenza Virus Strains In Vitro

The rapid emergence and subsequent spread of the novel 2009 Influenza A/H1N1 virus (2009 H1N1) has prompted the World Health Organization to declare the first pandemic of the 21^st century, highlighting the threat of influenza to public health and healthcare systems. Widespread resistance to both classes of influenza antivirals (adamantanes and neuraminidase inhibitors) occurs in both pandemic and seasonal viruses, rendering these drugs to be of marginal utility in the treatment modality. Worldwide, virtually all 2009 H1N1 and seasonal H3N2 strains are resistant to the adamantanes (rimantadine and amantadine), and the majority of seasonal H1N1 strains are resistant to oseltamivir, the most widely prescribed neuraminidase inhibitor (NAI). To address the need for more effective therapy, we evaluated the in vitro activity of a triple combination antiviral drug (TCAD) regimen composed of drugs with different mechanisms of action against drug-resistant seasonal and 2009 H1N1 influenza viruses. Amantadine, ribavirin, and oseltamivir, alone and in combination, were tested against amantadine- and oseltamivir-resistant influenza A viruses using an in vitro infection model in MDCK cells. Our data show that the triple combination was highly synergistic against drug-resistant viruses, and the synergy of the triple combination was significantly greater than the synergy of any double combination tested (P<0.05), including the combination of two NAIs. Surprisingly, amantadine and oseltamivir contributed to the antiviral activity of the TCAD regimen against amantadine- and oseltamivir-resistant viruses, respectively, at concentrations where they had no activity as single agents, and at concentrations that were clinically achievable. Our data demonstrate that the TCAD regimen composed of amantadine, ribavirin, and oseltamivir is highly synergistic against resistant viruses, including 2009 H1N1. The TCAD regimen overcomes baseline drug resistance to both classes of approved influenza antivirals, and thus may represent a highly active antiviral therapy for seasonal and pandemic influenza.     

A Novel and Efficient Gene Transfer Strategy Reduces Glial Reactivity and Improves Neuronal Survival and Axonal Growth In Vitro

Background

The lack of axonal regeneration in the central nervous system is attributed among other factors to the formation of a glial scar. This cellular structure is mainly composed of reactive astrocytes that overexpress two intermediate filament proteins, the glial fibrillary acidic protein (GFAP) and vimentin. Indeed, in vitro, astrocytes lacking GFAP or both GFAP and vimentin were shown to be the substrate for increased neuronal plasticity. Moreover, double knockout mice lacking both GFAP and vimentin presented lower levels of glial reactivity in vivo, significant axonal regrowth and improved functional recovery in comparison with wild-type mice after spinal cord hemisection. From these results, our objective was to develop a novel therapeutic strategy for axonal regeneration, based on the targeted suppression of astroglial reactivity and scarring by lentiviral-mediated RNA-interference (RNAi).

Methods and Findings

In this study, we constructed two lentiviral vectors, Lv-shGFAP and Lv-shVIM, which allow efficient and stable RNAi-mediated silencing of endogenous GFAP or vimentin in vitro. In cultured cortical and spinal reactive astrocytes, the use of these vectors resulted in a specific, stable and highly significant decrease in the corresponding protein levels. In a second model — scratched primary cultured astrocytes — Lv-shGFAP, alone or associated with Lv-shVIM, decreased astrocytic reactivity and glial scarring. Finally, in a heterotopic coculture model, cortical neurons displayed higher survival rates and increased neurite growth when cultured with astrocytes in which GFAP and vimentin had been invalidated by lentiviral-mediated RNAi.

Conclusions

Lentiviral-mediated knockdown of GFAP and vimentin in astrocytes show that GFAP is a key target for modulating reactive gliosis and monitoring neuron/glia interactions. Thus, manipulation of reactive astrocytes with the Lv-shGFAP vector constitutes a promising therapeutic strategy for increasing glial permissiveness and permitting axonal regeneration after central nervous system lesions.     

A Novel Mutation in the RPE65 Gene Causing Leber Congenital Amaurosis and Its Transcriptional Expression In Vitro

The retinal pigment epithelium-specific 65 kDa protein is an isomerase encoded by the RPE65 gene (MIM 180069) that is responsible for an essential enzymatic step required for the function of the visual cycle. Mutations in the RPE65 gene cause not only subtype II of Leber congenital amaurosis (LCA) but also early-onset severe retinal dystrophy (EOSRD). This study aims to investigate a Chinese case diagnosed as EOSRD and to characterize the polymorphisms of the RPE65 gene. A seven-year-old girl with clinical symptoms of EOSRD and her parents were recruited into this study. Ophthalmologic examinations, including best-corrected visual acuity, slit-lamp, Optical coherence tomography (OCT), and fundus examination with dilated pupils, were performed to determine the clinical characteristics of the whole family. We amplified and sequenced the entire coding region and adjacent intronic sequences of the coding regions of the RPE65 gene for the whole family to explore the possible mutation. Our results demonstrate that the patient exhibited the typical clinically features of EOSRD. Her bilateral decimal visual acuity was 0.3 and 0.4 in the left and right eyes, respectively. Spectral-domain optical coherence tomography (SD-OCT) was used to assess the retinal stratification for the whole family. All together, we identified four mutations within the RPE65 gene (c.1056G>A, c.1243+2T>A, c.1338+20A>C and c.1590C>A) in the patient. Among the four mutations, c.1056G>A and c.1338+20A>C had been reported previously and another two were found for the first time in this study. Her mother also carried the novel mutation (c.1243+2T>A). Either a single or a compound heterozygous or a homozygous one mutation is expected to cause EOSRD because mutations of RPE65 gene usually cause an autosomal recessive disease. Therefore, we speculate that the c.1590C>A mutation together with the c.1243+2T>A mutation may cause the patient’s phenotype.     

A Novel Compound from the Mushroom Cryptoporus volvatus Inhibits Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) In Vitro

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a serious contagious disease in the swine industry. At present, there are no effective control strategies against PRRSV. Thus, there is an urgent need for new treatment regimens that have efficacious antiviral activity to compensate for vaccines. The anti-infective effect of Cryptoporus volvatus has previously been demonstrated in Tradational Chinese Medicine. In this report, we expected to identify a new anti-PRRSV agent in the aqueous extract of C. volvatus, by employing a combination of modern chromatographic purification techniques and indirect immunofluorescence assay (IFA). Our results showed that C. volvatus extracts from every separation step differed in their inhibitory potency on PRRSV. One anti-PRRSV component designated as C_M-H-L-5 was isolated from water-soluble fraction of C. volvatus. The inhibition induced by C_M-H-L-5 occurred in a dose-dependent manner. C_M-H-L-5 appeared to be a low-molecular-weight polyol fragment with amide groups and carboxylic acid groups. Collectively, our findings imply that C_M-H-L-5 from the aqueous extract of C. volvatus has the potential to be used for anti-PRRSV therapy.     

Contribution of Baicalin on the Plasma Protein Binding Displacement and CYP3A Activity Inhibition to the Pharmacokinetic Changes of Nifedipine in Rats In Vivo and In Vitro

Baicalin purified from the root of Radix scutellariae is widely used in clinical practices. This study aimed to evaluate the effect of baicalin on the pharmacokinetics of nifedipine, a CYP3A probe substrate, in rats in vivo and in vitro. In a randomised, three-period crossover study, significant changes in the pharmacokinetics of nifedipine (2 mg/kg) were observed after treatment with a low (0.225 g/kg) or high (0.45 g/kg) dose of baicalin in rats. In the low- and high-dose groups of baicalin-treated rats, C _max of total nifedipine decreased by 40%±14% (P<0.01) and 65%±14% (P<0.01), AUC_0–∞ decreased by 41%±8% (P<0.01) and 63%±7% (P<0.01), V_d increased by 85%±43% (P<0.01) and 224%±231% (P<0.01), and CL increased by 97%±78% (P<0.01) and 242%±135% (P<0.01), respectively. Plasma protein binding experiments in vivo showed that C _max of unbound nifedipine significantly increased by 25%±19% (P<0.01) and 44%±29% (P<0.01), respectively, and there was a good correlation between the unbound nifedipine (%) and baicalin concentrations (P<0.01). Furthermore, in vitro results revealed that baicalin was a competitive displacer of nifedipine from plasma proteins. In vitro incubation experiments demonstrated that baicalin could also competitively inhibit CYP3A activity in rat liver microsomes in a concentration-dependent manner. In conclusion, the pharmacokinetic changes of nifedipine may be modulated by the inhibitory effects of baicalin on plasma protein binding and CYP3A–mediated metabolism.     

新型蛋白酶体抑制剂YSY-01A对肿瘤细胞促血管生成作用的抑制及其机制初探

YSY-01A是一种新型蛋白酶体抑制剂,前期研究已经证实其对肿瘤细胞的增殖有抑制作用．但是它对肿瘤血管生成是否有影响尚不明确．本研究旨在探明YSY-01A阻碍肿瘤细胞促进血管生成的作用及机制．我们首先将磺酰罗丹明B(sulforhodamine B,SRB)法与细胞共培养(Transwell)模型相结合,探讨YSY-01A抑制人结肠癌细胞(HT-29 cells)对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)的增殖促进作用;运用高内涵筛选(high content screening,HCS)法研究YSY-01A对HT-29细胞中NF-κB核转位的影响;利用Western blot法检测YSY-01A对HT-29细胞中缺氧诱导因子-1α(hypoxia- inducible factor-1α,HIF-1α)及血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达调控．为了观察YSY-01A对HUVEC增殖和运动有无直接抑制作用,我们采用SRB法观察YSY-01A对HUVEC的增殖抑制作用;运用HCS法分别考察YSY-01A对HUVEC的运动抑制和细胞毒作用．结果证实,YSY-01A可以阻碍HT-29细胞对HUVEC的增殖促进作用并具有浓度依赖性．YSY-01A还可抑制HT-29细胞中NF-κB的核转位,下调HIF-1α及VEGF的表达．进一步研究证实,YSY-01A能够浓度依赖地抑制HUVECs的增殖和运动,而不伴有明显的细胞毒作用．上述结果表明,YSY-01A可以通过抑制蛋白酶体活性下调肿瘤细胞中促血管生成因子的表达,进而在血管内皮细胞中发挥抗血管生成作用．     

The Marine-Derived Oligosaccharide Sulfate (MdOS), a Novel Multiple Tyrosine Kinase Inhibitor,Combats Tumor Angiogenesis both In Vitro and In Vivo

Despite the emerging success of multi-targeted protein tyrosine kinase (PTK) inhibitors in cancer therapy, significant side effects and resistance concerns seems to be avoided unlikely. The aim of the present study was to identify novel multi-targeting PTK inhibitors. The kinase enzymatic activities were measured by enzyme-linked immunosorbent assay (ELISA). The antiproliferative activities in human microvascular endothelial cells (HMECs) were evaluated by sulforhodamine (SRB) assay. The phosphorylation of kinases and their downstream molecules was probed by western blot analysis. The binding mode between MdOS and PTKs was profiled by surface plasmon resonance (SPR) approach and molecular simulation. Tube formation assay, rat aortic ring method and chicken chorioallantoic membrane assay were combined to illustrate the in vitro and in vivo anti-angiogenic effects. Results indicated that MdOS, a novel marine-derived oligosaccharide sulfate, exhibited a broad-spectrum PTK inhibitory action. At an enzymatic level, MdOS inhibited HER2, EGFR, VEGFR, PDGFR, c-Kit, FGFR1 and c-Src, with little impact on FGFR2. In cellular settings, MdOS inhibited phosphorylation of PTKs, exemplified by HER2, EGFR and VEGFR2, and downstream molecules of Erk1/2 and AKT. Further studies demonstrated that MdOS acted as an ATP-competitive inhibitor via directly binding to the residues of entrance rather than those of the ATP-binding pocket. Furthermore, MdOS inhibited proliferation and tube formation of HMECs, arrested microvessel outgrowth of rat aortic rings and hindered the neovascularization of chick allantoic membrane. Taken together, results presented here indicated that MdOS exhibited anti-angiogenic activity in a PTK-dependent manner and make it a promising agent for further evaluation in PTK-associated cancer therapy.     

Emerging,Non-PCV13 Serotypes 11A and 35B of Streptococcus pneumoniae Show High Potential for Biofilm Formation In Vitro

Background

Since the use of pneumococcal conjugate vaccines PCV7 and PCV13 in children became widespread, invasive pneumococcal disease (IPD) has dramatically decreased. Nevertheless, there has been a rise in incidence of Streptococcus pneumoniae non-vaccine serotypes (NVT) colonising the human nasopharynx. Nasopharyngeal colonisation, an essential step in the development of S. pneumoniae-induced IPD, is associated with biofilm formation. Although the capsule is the main pneumococcal virulence factor, the formation of pneumococcal biofilms might, in fact, be limited by the presence of capsular polysaccharide (CPS).

Methodology/Principal Findings

We used clinical isolates of 16 emerging, non-PCV13 serotypes as well as isogenic transformants of the same serotypes. The biofilm formation capacity of isogenic transformants expressing CPSs from NVT was evaluated in vitro to ascertain whether this trait can be used to predict the emergence of NVT. Fourteen out of 16 NVT analysed were not good biofilm formers, presumably because of the presence of CPS. In contrast, serotypes 11A and 35B formed ≥45% of the biofilm produced by the non-encapsulated M11 strain.

Conclusions/Significance

This study suggest that emerging, NVT serotypes 11A and 35B deserve a close surveillance.     

The Rickettsia prowazekii ExoU Homologue Possesses Phospholipase A1 (PLA1), PLA2, and Lyso-PLA2 Activities and Can Function in the Absence of Any Eukaryotic Cofactors In Vitro

Here we have characterized the Rickettsia prowazekii RP534 protein, a homologue of the Pseudomonas aeruginosa ExoU phospholipase A (PLA) secreted cytotoxin. Our studies showed that purified recombinant RP534 PLA possessed the predicted PLA₂ and lyso-PLA₂ activities based on what has been published for P. aeruginosa ExoU. RP534 also displayed PLA₁ activity under the conditions tested, whereas ExoU did not. In addition, recombinant RP534 displayed a basal PLA activity that could hydrolyze phosphatidylcholine in the absence of any eukaryotic cofactors. Interestingly, the addition of bovine liver superoxide dismutase 1 (SOD1), a known activator of P. aeruginosa ExoU, resulted in an increased rate of RP534-catalyzed phospholipid hydrolysis, indicating that mechanisms of activation of the ExoU family of PLAs may be evolutionarily conserved. The mechanism of SOD1-dependent stimulation of RP534 was further examined using active site mutants and a fluorogenic phospholipid substrate whose hydrolysis by RP534 over a short time course is measureable only in the presence of SOD1. These studies suggest a mechanism by which SOD1 stimulates RP534 activity once it has bound to the substrate. We also show that antibody raised against RP534 was useful for immunoprecipitating active RP534 from R. prowazekii lysed cell extracts, thus verifying that this protein is expressed and active in rickettsiae isolated from embryonated hen egg yolk sacs.     

Exploring the Effect of Hesperetin–HSPC Complex—A Novel Drug Delivery System on the In Vitro Release, Therapeutic Efficacy and Pharmacokinetics

Hesperetin is known to exhibit a variety of pharmacological activities in mammalian cell systems. Although it shows appreciable bioavailability when administered orally, its faster elimination from body creates the need of frequent administration to maintain effective plasma concentration. To overcome this limitation, a phospholipid complex of hesperetin was prepared and evaluated for antioxidant activity and pharmacokinetic profile. The hesperetin content of the complex was determined by a spectrophotometer and the surface characteristics of the complex were studied by means of microscope. The antioxidant activity was evaluated in carbon-tetrachloride-intoxicated rats at a dose level of 100 mg/kg body weight, p.o. The complex was studied for in vitro drug release characteristics and effect of complexation on serum concentration of hesperetin in rats was also studied along with main pharmacokinetic parameters. The results showed that the complex has a sustained release property and enhanced antioxidant activity (P < 0.05 and <0.01) as compared to free hesperetin at the same dose level. Pharmacokinetic study depicted that the complex has higher relative bioavailability and acted for a longer period of time. The study therefore suggests that phospholipid complex of hesperetin produced better antioxidant activity than free drug at the same dose level and the effect persisted for a longer period of time, which may be helpful in solving the problems of faster elimination of the molecule.     

An Accurate,Flexible and Small Optical Fiber Sensor: A Novel Technological Breakthrough for Real-Time Analysis of Dynamic Blood Flow Data In Vivo

Because of the limitations of existing methods and techniques for directly obtaining real-time blood data, no accurate microflow in vivo real-time analysis method exists. To establish a novel technical platform for real-time in vivo detection and to analyze average blood pressure and other blood flow parameters, a small, accurate, flexible, and nontoxic Fabry-Perot fiber sensor was designed. The carotid sheath was implanted through intubation of the rabbit carotid artery (n = 8), and the blood pressure and other detection data were determined directly through the veins. The fiber detection results were compared with test results obtained using color Doppler ultrasound and a physiological pressure sensor recorder. Pairwise comparisons among the blood pressure results obtained using the three methods indicated that real-time blood pressure information obtained through the fiber sensor technique exhibited better correlation than the data obtained with the other techniques. The highest correlation (correlation coefficient of 0.86) was obtained between the fiber sensor and pressure sensor. The blood pressure values were positively related to the total cholesterol level, low-density lipoprotein level, number of red blood cells, and hemoglobin level, with correlation coefficients of 0.033, 0.129, 0.358, and 0.373, respectively. The blood pressure values had no obvious relationship with the number of white blood cells and high-density lipoprotein and had a negative relationship with triglyceride levels, with a correlation coefficient of –0.031. The average ambulatory blood pressure measured by the fiber sensor exhibited a negative correlation with the quantity of blood platelets (correlation coefficient of −0.839, P<0.05). The novel fiber sensor can thus obtain in vivo blood pressure data accurately, stably, and in real time; the sensor can also determine the content and status of the blood flow to some extent. Therefore, the fiber sensor can obtain partially real-time vascular rheology information and may thus enable the early diagnosis of blood rheology disorders and diseases.     

Focus: The Science of Stress: In Times of Adversity: A Neuroscience Perspective on Stress,Health, and Implications for Society Post-pandemic

The relationship between chronic stress and chronic disease (including mental illness) is well established: HPA-axis hyperactivity leads to hormonal dysregulation of primary mediators (eg, glucocorticoids, cytokines, etc.), allostatic overload, and neurological degradation, followed by clinical manifestations of disease. Amid the largest public health crisis of the century lay a myriad of challenges pushing people beyond their limit. From experiencing loss of connection or dealing with loss of life to financial shocks of COVID-19 lockdowns or infection by the SARS-CoV-2 virus, stress is at an all-time high, threatening both brain and mental health at scale. Fortunately, there is a way forward: the neuroscience of resilience teaches us that it is possible to resist, recover, and redirect the brain from trauma to re-establish balance in the body and improve well-being. At the same time, health follows a social gradient: adverse and protective psychosocial factors are shaped by wider social and economic determinants of health. This paper argues the neurobiology of stress is not separate from health disparities linked to adverse factors (ie, stress) created by complex social and economic contexts. Therefore, the field of neuroscience is challenged to inform multi-context and multi-level approaches and engage with decision-makers to enact policies and interventions aimed at promoting the resilient element in a wider population health context. Undoubtedly, achieving such a goal for current and future generations to benefit and lead healthier lives requires a heroic effort from all key stakeholders. The cost of willful neglect to resolve these issues is too expensive.