Modern metagenomic environmental DNA studies are almost completely reliant on next-generation sequencing, making evaluations of these methods critical. We compare two next-generation sequencing techniques – amplicon and shotgun – on water samples across four of Brazil’s major river floodplain systems (Amazon, Araguaia, Paraná, and Pantanal). Less than 50% of phyla identified via amplicon sequencing were recovered from shotgun sequencing, clearly challenging the dogma that mid-depth shotgun recovers more diversity than amplicon-based approaches. Amplicon sequencing also revealed ~27% more families. Overall the amplicon data were more robust across both biodiversity and community ecology analyses at different taxonomic scales. Our work doubles the sampling size in similar environmental studies, and novelly integrates environmental data (e.g., pH, temperature, nutrients) from each site, revealing divergent correlations depending on which data are used. While myriad variants on NGS techniques and bioinformatic pipelines are available, our results point to core differences that have not been highlighted in any studies to date. Given the low number of taxa identified when coupling shotgun data with clade-based taxonomic algorithms, previous studies that quantified biodiversity using such bioinformatic tools should be viewed cautiously or re-analyzed. Nonetheless, shotgun has complementary advantages that should be weighed when designing projects.

Glioblastoma multiforme (GBM) is the most common brain malignancies in adults. Most GBM patients succumb to the disease less than 1 year after diagnosis due to the highly invasive nature of the tumor, which prevents complete surgical resection and gives rise to tumor recurrence. The invasive phenotype also confers radioresistant and chemoresistant properties to the tumor cells; therefore, there is a critical need to develop new therapeutics that target drivers of GBM invasion. Amplification of EGFR is observed in over 50% of GBM tumors, of which half concurrently overexpress the variant EGFRvIII, and expression of both receptors confers a worse prognosis. EGFR and EGFRvIII cooperate to promote tumor progression and invasion, in part, through activation of the Stat signaling pathway. Here, it is reported that EGFRvIII activates Stat5 and GBM invasion by inducing the expression of a previously established mediator of glioma cell invasion and survival: fibroblast growth factor-inducible 14 (Fn14). EGFRvIII-mediated induction of Fn14 expression is Stat5 dependent and requires activation of Src, whereas EGFR regulation of Fn14 is dependent upon Src-MEK/ERK-Stat3 activation. Notably, treatment of EGFRvIII-expressing GBM cells with the FDA-approved Stat5 inhibitor pimozide blocked Stat5 phosphorylation, Fn14 expression, and cell migration and survival. Because EGFR inhibitors display limited therapeutic efficacy in GBM patients, the EGFRvIII-Stat5-Fn14 signaling pathway represents a node of vulnerability in the invasive GBM cell populations. Targeting critical effectors in the EGFRvIII-Stat5-Fn14 pathway may limit GBM tumor dispersion, mitigate therapeutic resistance, and increase survival. .

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An ideal in vivo gene therapy platform provides safe, reprogrammable, and precise strategies which modulate cell and tissue gene regulatory networks with a high temporal and spatial resolution. Clustered regularly interspaced short palindromic repeats (CRISPR), a bacterial adoptive immune system, and its CRISPR-associated protein 9 (Cas9), have gained attention for the ability to target and modify DNA sequences on demand with unprecedented flexibility and precision. The precision and programmability of Cas9 is derived from its complexation with a guide-RNA (gRNA) that is complementary to a desired genomic sequence. CRISPR systems open-up widespread applications including genetic disease modeling, functional screens, and synthetic gene regulation. The plausibility of in vivo genetic engineering using CRISPR has garnered significant traction as a next generation in vivo therapeutic. However, there are hurdles that need to be addressed before CRISPR-based strategies are fully implemented. Some key issues center on the controllability of the CRISPR platform, including minimizing genomic-off target effects and maximizing in vivo gene editing efficiency, in vivo cellular delivery, and spatial-temporal regulation. The modifiable components of CRISPR systems: Cas9 protein, gRNA, delivery platform, and the form of CRISPR system delivered (DNA, RNA, or ribonucleoprotein) have recently been engineered independently to design a better genome engineering toolbox. This review focuses on evaluating CRISPR potential as a next generation in vivo gene therapy platform and discusses bioengineering advancements that can address challenges associated with clinical translation of this emerging technology.

Abstracts iii53NEURO-ONCOLOGY • MAY 2017 given via the ommaya reservoir on day 3 and 5 to complete one cycle of the therapy. This 7-day cycle was repeated at the next week. Each two-cycle constituted one full course of therapy. The treatment consisted of two 1-week cycles of therapy over 8-12 weeks period. Side-reaction were observed, such as headache,fever,and so on. Besides side-reaction, The efficacy was assessed by MRI scans retreated at 2-or 3-month interval and the survival of the patient summarized according to the follow-up also. RESULTS: From September 2002 to May 2004, six malignant glioma patients were treated with local CIK cells therapy for ten courses, all the patients were followed up from 7 to 24 months by the end of March 2005. The Side-reaction included fever in two cases, headache in three cases and transient aphasia in one case and limb weakness in one patients, they were all controlled and the patient could tolerate. For the six patients in our study, with two out of 6 patients were still alive by the end of May 2016, survival time without recurrence was 13 and 12 years and the Karnofsky Permanence Scale were 100 and 70, respectively. Of the four patients with tumor recurrence, disease-free survival time was twenty months without other treatment after immunotherapy in one case, the rest three patients died. The efficacy was assessed: CR was in two cases;PR and ST were each in one case and PR in two cases. CONCLUSION: CIK cells can be administered intracavitary into CNS to treat primary malignant gliomas,the side-reaction was well tolerated; According to the eligibility criteria strictly, local cytokine-induced killer cell therapy for malignant gliomas appears to be safe and effective, it should be considered an option for patients with high-grade gliomas refractive to standard therapeutic approaches. INTRODUCTION: Therapies targeting T-cell inhibitory checkpoint signalling pathways are redefining cancer survival in many solid malignancies. Unfortunately only a minority of patients respond, with mounting evidence that tumour response and patient survival are significantly associated with the presence of tumour PD-L1 expression, which prevents immune activation in situ within the tumour. We hypothesised that pre-conditioning of the tumour immune microenvironment using targeted, virus-mediated interferon stimulation, would upregulate PD-L1 protein expression and improve the efficacy of sequential checkpoint blockade. Oncolytic viruses (OV) stimulate anti-tumour immunity, and represent a promising novel form of cancer immunotherapy. However almost all brain tumour studies to date administer OV via intra-lesion injection, following a largely untested belief that intravenous (IV) administration will not deliver virus to this site, due to exclusion by the blood brain barrier. MATERIALS AND METHODS: In this windowof-opportunity clinical study 9 patients were injected IV once with 1x10 10 TCID 50 of oncolytic human Orthoreovirus (reovirus) ahead of planned surgical resection of their brain tumour....