Insights into the cellular trafficking of splice redirecting oligonucleotides complexed with chemically modified cell-penetrating peptides

Conjugates of cell-penetrating peptides (CPP) and splice redirecting oligonucleotides (ON) display clinical potential as attested by in vivo experimentation in murine models of Duchenne muscular dystrophy. However, micromolar concentrations of these conjugates are required to obtain biologically relevant responses as a consequence of extensive endosomal sequestration following endocytosis. Recent work from our group has demonstrated that appending stearic acid to CPPs increases their efficiency and that the inclusion of pH titrable entities leads to further improvement. Moreover, these modified CPPs form non covalent complexes with charged ON analogs or siRNAs, which allows decreasing the concentrations of ONs by nearly one log.These modified CPPs and the parent peptides have been compared here in the same in vitro model in terms of cell uptake, trafficking and splicing redirection activity. The increased splicing redirection activity of our modified CPPs cannot be explained by differences in cell uptake but rather by their enhanced ability to escape from endocytic vesicles. Accordingly, a clear correlation between membrane destabilizing activity and splicing redirection was observed using a liposome leakage assay.Studies of cellular trafficking for the most active PF6:ON complexes indicate uptake by clathrin-mediated endocytosis using either FACS cell uptake or a splicing redirection functional assay. Acidification of intracellular vesicles and membrane potential were found important for splicing redirection but not for cell uptake. These results do confirm that the increased potency of PF6:ON complexes is not due to the use of a non endocytic route of cell internalization as proposed for some CPPs.     

CD8 epitope escape and reversion in acute HCV infection

In the setting of acute hepatitis C virus (HCV) infection, robust HCV-specific CD8+ cytotoxic T lymphocyte (CTL) responses are associated with initial control of viremia. Despite these responses, 70-80% of individuals develop persistent infection. Although viral escape from CD8 responses has been illustrated in the chimpanzee model of HCV infection, the effect of CD8 selection pressure on viral evolution and containment in acute HCV infection in humans remains unclear. Here, we examined viral evolution in an immunodominant human histocompatibility leukocyte antigen (HLA)-B8-restricted NS3 epitope in subjects with acute HCV infection. Development of mutations within the epitope coincided with loss of strong ex vivo tetramer and interferon gamma enzyme-linked immunospot responses, and endogenous expression of variant NS3 sequences suggested that the selected mutations altered processing and presentation of the variant epitope. Analysis of NS3 sequences from 30 additional chronic HCV-infected subjects revealed a strong association between sequence variation within this region and expression of HLA-B8, supporting reproducible allele-specific selection pressures at the population level. Interestingly, transmission of an HLA-B8-associated escape mutation to an HLA-B8 negative subject resulted in rapid reversion of the mutation. Together, these data indicate that viral escape from CD8+ T cell responses occurs during human HCV infection and that acute immune selection pressure is of sufficient magnitude to influence HCV evolution.     

Escaping the professional silo: an escape room implemented in an interprofessional education curriculum

ABSTRACT

Communication skills among healthcare professionals are a necessary component in ensuring quality outcomes for patients. This report describes the design and curricular implementation of an interprofessional escape room, an innovative way to promote communication and positive team dynamics among students. In this interactive, serious game, teams of approximately eight interprofessional participants were provided with a fictitious patient case in a simulated hospital environment. Within a 45-minute time limit, students needed to use objects in the room to solve a series of puzzles to successfully complete the room by addressing all the patient’s needs. A facilitated debrief following the activity allowed participants to reflect on their communication skills and teamwork during the experience. A total of thirty students across seven professions piloted the activity, and 181 students across five professions participated in the activity as part of an academic course. Feedback from students was collected on a seven-point Likert scale and revealed the value of an interprofessional escape room in academia. This report, which describes what appears to be the first interprofessional health care escape room within an IPE curriculum, demonstrates the value of the escape room in encouraging teamwork, facilitating communication, and promoting interprofessionalism.     

LncRNA IFITM4P promotes immune escape by up-regulating PD-L1 via dual mechanism in oral carcinogenesis

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肝细胞癌患者肿瘤细胞和肿瘤浸润淋巴细胞Fas抗原与Fas配体表达的研究

为研究肝细胞癌患者肿瘤细胞、肿瘤浸润淋巴细胞Ｆａｓ抗原与ＦａｓＬ配体的表达水平,探讨Ｆａｓ系统在肝细胞癌免疫逃逸及免疫反击中的作用,采用间接免疫荧光流式细胞术检测２０例肝细胞癌患者肿瘤细胞、肿瘤浸润淋巴细胞和外周血淋巴细胞Ｆａｓ和ＦａｓＬ的表达。结果显示,肝细胞癌患者肿瘤细胞Ｆａｓ与ＦａｓＬ表达水平较自身非肿瘤肝细胞上调;肿瘤浸润淋巴细胞与自身外周血淋巴细胞相比,Ｆａｓ表达上调而ＦａｓＬ表达下调。     

人类疱疹病毒逃逸宿主抗病毒固有免疫应答机制的研究进展

人类疱疹病毒(Human herpesvirus,HHV)作为一类可引起人体蔓延性皮疹的病毒,其在宿主细胞中呈潜伏感染且被感染者可终生带毒.在长期进化过程中,宿主细胞通过各种方式以抑制该病毒的扩散,同时,疱疹病毒也通过其病毒蛋白及基因组miRNAs等影响机体固有免疫应答过程中关键蛋白及细胞因子的表达等方式干扰宿主细胞对...     

RBD Double Mutations of SARS-CoV-2 Strains Increase Transmissibility through Enhanced Interaction between RBD and ACE2 Receptor

The COVID-19 pandemic, caused by SARS-CoV-2, has led to catastrophic damage for global human health. The initial step of SARS-CoV-2 infection is the binding of the receptor-binding domain (RBD) in its spike protein to the ACE2 receptor in the host cell membrane. Constant evolution of SARS-CoV-2 generates new mutations across its genome including the coding region for the RBD in the spike protein. In addition to the well-known single mutation in the RBD, the recent new mutation strains with an RBD “double mutation” are causing new outbreaks globally, as represented by the delta strain containing RBD L452R/T478K. Although it is considered that the increased transmissibility of double-mutated strains could be attributed to the altered interaction between the RBD and ACE2 receptor, the molecular details remain to be elucidated. Using the methods of molecular dynamics simulation, superimposed structural comparison, free binding energy estimation, and antibody escaping, we investigated the relationship between the ACE2 receptor and the RBD double mutants of L452R/T478K (delta), L452R/E484Q (kappa), and E484K/N501Y (beta, gamma). The results demonstrated that each of the three RBD double mutants altered the RBD structure and enhanced the binding of the mutated RBD to ACE2 receptor. Together with the mutations in other parts of the virus genome, the double mutations increase the transmissibility of SARS-CoV-2 to host cells.     

丙型肝炎的致病机制及药物治疗

丙型肝炎（丙肝）病毒（hepatitis C virus,HCV）可以通过几种途径影响宿主免疫功能,使病毒在宿主细胞内持续复制,最终导致HCV慢性感染。目前治疗慢性丙肝的方法主要是聚乙二醇干扰素和利巴韦林联合用法,此种疗法在HCV1型患者中约50％不能产生持续病毒学应答,且有不良反应。近年来,随着对HCV复制以及病毒非结构蛋白功能的深入研究,针对减少HCV载量的特异性药物的研发取得了较大进展。本文就HCV的细胞人侵、复制、逃避宿主的固有和获得性免疫及抗HCV临床试验药物的最新进展进行综述。     

Molecular virology of hepatitis B virus and the development of antiviral drug resistance

Abstract: The active replication phases of the hepatitis B virus (HBV) are marked by a high frequency of mutational events resulting from an enormous daily viral turnover rate and an error‐prone polymerase. Because HBV is not directly cytopathic, it is the host's immune response that accounts for most of the liver disease associated with persistent infection. HBV escape mutants can be found at the transitional phases of chronic hepatitis B, such as hepatitis B e antigen (HBeAg)‐positive versus HBeAg‐negative infection, and these mutants are selected out from the diverse quasispecies pool comprising the HBV population at that time. Not surprisingly, the introduction of nucleoside and nucleotide analog therapy has also seen the emergence of drug resistance, which has become the major factor limiting the long‐term application of antiviral agents for patients with chronic hepatitis B. Thus, the prevention of resistance requires the adoption of strategies that effectively control virus replication and exploit a detailed understanding of the mechanisms and processes that drive the emergence of drug resistance.     

原发性高血压病人醛固酮逃逸现象及螺内酯的干预研究

目的探讨原发性高血压病人使用血管紧张素抑制剂(ACEI)依那普利治疗后所并发的醛固酮(Ald)逃逸现象,并观察联合使用醛固酮受体拮抗剂螺内酯后的效果.方法 65例原发性高血压病人使用依那普利治疗,分别于治疗前及治疗后1个月、3个月、6个月采血检测血管紧张素Ⅱ(AngⅡ)、醛固酮(Ald)浓度,根据治疗3个月时的Ald浓度,判断有无并发Ald逃逸,对有Ald逃逸者,联合使用螺内酯3个月,观察其治疗效果.结果 依那普利治疗后1个月AngⅡ、Ald与治疗前相比均下降,但3个月时,AngⅡ有所升高,Ald明显增高,65例中有28例并发Ald逃逸,发生率约43%,并发Ald逃逸的28例病人使用螺内酯3个月后,AngⅡ、Ald均有所下降,但并未达到有统计学意义.结论原发性高血压病人长期(3个月以上)使用ACEI后部分病人会出现Ald逃逸现象,ACEI联合使用螺内酯可能会改善Ald逃逸现象.