Adeno-associated viral vectors (AAV) are one of the most efficient engineered tools for delivering genetic material into host cells. The commercialization of AAV-based drugs goes hand in hand with the need to increase manufacturing capacities and to develop appropriate quality controls. In particular, accurate methods to assess the level of residual DNA in AAV vector stocks are needed, considering the potential risk of co-transferring oncogenic or immunogenic sequences with the therapeutic vectors. Our laboratory has developed an assay based on high-throughput sequencing (HTS) to exhaustively identify and quantify DNA species in recombinant AAV batches. Compiled with a computational analysis of the single nucleotide variants (SNV), Single-Stranded Virus Sequencing (SSV-Seq) also provides information regarding rAAV genome identity. In this study, we show that the PCR amplification of regions with high GC content and including mononucleotide stretches can be biased during the DNA library preparation, leading to drops in the sequencing coverage along the AAV vector genome. To circumvent this problem, we have developed a PCR-free protocol, named SSV-Seq 2.0, that is optimized for the sequencing of rAAV genomes that contain sequences with a high percentage of GC and homopolymers, such as the CAG promoter. HTS-based assays are indispensable to provide accurate data to the regulatory agencies regarding nucleic acids content in AAV vector batches and to improve the safety and efficacy of these viral vectors.

Graphene quantum dots (GQDs), the latest member of graphene family, have attracted outstanding interest in the last few years, due to their outstanding physical, chemical, electrical, optical and biological properties. Their strong size-dependent photoluminescence (PL) and the presence of many reactive groups on the graphene surface allow their multimodal conjugation with therapeutic agents, targeting ligands, polymers, light responsive agents, fluorescent dyes, and functional nanoparticles, making them valuable agents for cancer diagnosis and treatment. In this review, the very recent advances covering the last three years on the applications of GQDs as drug delivery systems (DDS) and theranostic tools for anticancer therapy are discussed, highlighting the relevant factors which regulate their biocompatibility. Among these factors, the size, kind and degree of surface functionalization have shown to greatly affect their use in biological systems. Toxicity issues, which still represent an open challenge for the clinical development of GQDs based therapeutic agents, are also discussed at cellular and animal levels.

Substituted alkyl pyrazines - other than being extracted from various natural sources such as coffee beans, cocoa beans, nuts and vegetables - can be synthesized by the use of traditional chemical methods or by the help of certain microorganisms. The importance of pyrazines for food industry is expected to grow in the upcoming years due to the higher demand for ready meals, coffee and chocolate drinks; the roasty, nutty and earthy smell is reminiscent for coffee and cocoa depending on substitution and concentration of pyrazines. The growing awareness of people about the ingredients and the origin of their daily food has strongly influenced the market with labels like ’organic‘ and ’natural‘. Many flavor ingredients prepared by biotechnology methods have conquered the market in recent years and are destined to replace and optimize the ineffective (0.01% pyrazine/kg biomass) extraction from plants or animal sources. This overview focuses on the achievements and the upcoming challenges in pyrazine synthesis. Major parts deal with the extraction of natural products from sugar molasses, the chemical synthesis, fermentation by microorganisms and preparative methods by biocatalysis. The different types of production are decisive for the declaration and value of the final product and can span from 200-3500 $/kg for the synthetically produced or the naturally extracted 2,5-dimethylpyrazine, respectively.

Synthetic biology has promoted the development of biosensors as tools for detecting trace substances. In the past, biosensors based on synthetic biology have been designed on living cells, but the development of cell biosensors has been greatly limited by defects such as the obstruction of cell membrane. However, the advent of cell-free synthetic biology addresses these limitations. Biosensors based on the cell-free protein synthesis system have the advantages of higher safety, higher sensitivity, and faster response time over cell biosensors, which makes cell-free biosensors have a broader application prospect. This review summarizes the workflow of various cell-free biosensors, including the identification of analytes and signal output. The detection range of cell-free biosensors is greatly enlarged by different recognition mechanisms and output methods. In addition, the review also discusses the applications of cell-free biosensors in environmental monitoring and health diagnosis, as well as existing deficiencies and aspects that should be improved. In the future, through continuous improvement and optimization, the potential of cell-free biosensors will be stimulated, and their application fields will be expanded.

Despite rapid progress in the field, scalable high-yield production of AAV is still one of the critical bottlenecks the manufacturing sector is facing. The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as a mainstream platform for a scalable production of recombinant proteins with clinically approved products for human use. In this review, we provide a detailed overview on advancements in IC-BEVS for rAAV production. Since the first report of baculovirus induced production of rAAV in insect cells in 2002, this platform has undergone significant evolutions. The original three baculovirus system was improved to enhance expression stability and further streamlined to reduce the number of baculoviruses to two and eventually one. The one baculovirus system consisting of an inducible packaging insect cell line was further improved to enhance the AAV vector quality and potency. In parallel, implementation of advanced manufacturing approaches and control of critical processing parameters have demonstrated promising results with process validation in large scale bioreactor runs. Moreover, optimization of molecular design of vectors to enable higher cell-specific yield of functional AAV particles in combination with bioprocess intensification strategies may further contribute to addressing current and future manufacturing challenges.

The pandemic outbreaks of coronavirus disease 2019 (COVID-19) was first discovered in Wuhan, Hubei, China in December 2019. The COVID-19 was caused by the novel coronavirus, namely severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It took 30 days to spread to all provinces of China [1]. Recently, the confirmed cases of COVID-19 have been reported from about 200 countries or regions on March 30, 2020, and killed almost 30 thousand people [2]. Efficient identification of the infection by SARS-CoV-2 has been one of the most important tasks to facilitate all the following counter measurements in dealing with infectious disease. In Taiwan, a COVID-19 Open Science Platform adhering to the spirit of open science: sharing sources, data, and methods to promote progress in academic research while corroborating findings from various disciplines has established in mid-February 2020, for collaborative research in support of the development of detection methods, therapeutics, and a vaccine for COVID-19. Research priorities include infection control, epidemiology, clinical characterization and management, detection methods (including viral RNA detection, viral antigen detection, and serum antibody detection), therapeutics (neutralizing antibody and small molecule drugs), vaccines, and SARS-CoV-2 pathogenesis. In addition, research on social ethics and the law are included to take full account of the impact of the COVID-19 virus.

There is a vast number of biomaterials ranging from drug-eluting stents, coated implants, drug delivery devices and artificial organs, among others, that have been developed in recent years. However, translation of many of these biomaterials to clinic is often plagued by biocompatibility challenges. This review focuses on strategies implemented in some of the recently developed biomaterials -- particularly for soft and hard tissue regeneration, organ manufacturing and disease remediation -- to overcome potential foreign body response to the incorporation of the biomaterials in the host.