Gene Therapy for Pediatric Cancer: State of the Art and Future Perspectives

While modern treatments have led to a dramatic improvement in survival for pediatric malignancy, toxicities are high and a significant proportion of patients remain resistant. Gene transfer offers the prospect of highly specific therapies for childhood cancer. “Corrective” genes may be transferred to overcome the genetic abnormalities present in the precancerous cell. Alternatively, genes can be introduced to render the malignant cell sensitive to therapeutic drugs. The tumor can also be attacked by decreasing its blood supply with genes that inhibit vascular growth. Another possible approach is to modify normal tissues with genes that make them more resistant to conventional drugs and/or radiation, thereby increasing the therapeutic index. Finally, it may be possible to attack the tumor indirectly by using genes that modify the behavior of the immune system, either by making the tumor more immunogenic, or by rendering host effector cells more efficient. Several gene therapy applications have already been reported for pediatric cancer patients in preliminary Phase 1 studies. Although no major clinical success has yet been achieved, improvements in gene delivery technologies and a better understanding of mechanisms of tumor progression and immune escape have opened new perspectives for the cure of pediatric cancer by combining gene therapy with standard therapeutic available treatments

HAsh-MaP-ERadicator: Filtering Non-Target Sequences from Next Generation Sequencing Reads

Contemporary DNA sequencing technologies are continuously increasing throughput at ever decreasing costs. Moreover, due to recent advances in sequencing technology new platforms are emerging. As such computational challenges persist. The average read length possible has taken a giant leap forward with the PacBio and Nanopore solutions. Regardless of the platform used, impurities within the DNA preparation of the sample - be it from unintentional contaminants or pervasive symbiots - remains an issue. We have developed a new tool, HAsh-MaP-ERadicator (HAMPER), for the detection and removal of non-target, contaminating DNA sequences. Integrating hash-based and mapping-based strategies, HAMPER is both memory and time efficient while maintaining a high level of sensitivity. Moreover, HAMPER was designed for flexibility: reads of any size can be efficiently examined and the user can set parameters specific for the analysis of reads produced by a particular sequencer. To evaluate our method, mock sequencing runs were generated including various contaminating species and with variable rates of mutation revealing a high level of sensitivity and specificity. Reads that are not of interest can quickly be removed using HAMPER thus improving downstream analyses

Mitoparans: mitochondriotoxic cell penetrating peptides and novel inducers of apoptosis.

Acknowledgments The authors would like to thank Keith Holding at the University of Wolverhampton for his outstanding technical support. This work was supported in part by Samantha Dickson Brain Tumour Trust.Introduction: The amphipathic helical peptide mastoparan (MP; H-INLKALAALAKKIL-NH2) inserts into biological membranes to modulate the activity of heterotrimeric G proteins and other targets. Moreover, whilst cell free models of apoptosis demonstrate MP to facilitate mitochondrial permeability transition and release of apoptogenic cytochrome c, MP-induced death of intact cells has been attributed to its non-specific membrane destabilising properties (necrotic mechanisms). However, MP and related peptides are known to activate other signalling systems, including p42/p44 MAP kinases and could therefore, also modulate cell fate and specific apoptotic events. The ability of MP to facilitate mitochondrial permeability in cell free systems has lead to proposals that MP could be of utility in tumour therapeutics provided that it conferred features of cellular penetration and mitochondrial localization. We have recently reported that our highly potent amphipathic MP analogue mitoparan (mitP; [Lys5,8Aib10]MP; Aib = -aminoisobutyric acid) specifically promotes apoptosis of human cancer cells, as was confirmed by in situ TUNEL staining and activation of caspase-3. Moreover, we have also demonstrated that mitP penetrates plasma membranes and redistributes to co-localize with mitochondria. Complementary studies, using isolated mitochondria, further demonstrated that mitP, through co-operation with a protein of the permeability transition pore complex voltage-dependent anion channel (VDAC), induced swelling and permeabilization of mitochondria, leading to the release of the apoptogenic factor cytochrome c. An expanding field of peptide and cell penetrating peptide (CPP) research has focussed on the selective targeting of tumours by engineering constructs that incorporate cell-specific or tissue–specific address motifs. Peptidyl address motifs could enhance the selectivity of drug delivery whilst the improved cellular uptake offered by CPP enhances bioavailability. Thus and as a potential therapeutic strategy, we extended our findings to design target-specific mitP analogues. The integrin-specific address motif RGD and a Fas ligand mimetic WEWT were incorporated by N-terminal acylation of mitP to produce novel tandem-linked chimeric peptides

Exploring the Diversity of Bacillus Whole Genome Sequencing Projects Using Peasant, the Prokaryotic Assembly and Annotation Tool

The persistent decrease in cost and difficulty of whole genome sequencing of microbial organisms has led to a dramatic increase in the number of species and strains characterized from a wide variety of environments. Microbial genome sequencing can now be conducted by small laboratories and as part of undergraduate curriculum. While sequencing is routine in microbiology, assembly, annotation and downstream analyses still require computational resources and expertise, often necessitating familiarity with programming languages. To address this problem, we have created a light-weight, user-friendly tool for the assembly and annotation of microbial sequencing projects. The Prokaryotic Assembly and Annotation Tool, Peasant, automates the processes of read quality control, genome assembly, and annotation for microbial sequencing projects. High-quality assemblies and annotations can be generated by Peasant without the need of programming expertise or high-performance computing resources. Furthermore, statistics are calculated so that users can evaluate their sequencing project. To illustrate the computational speed and accuracy of Peasant, the SRA records of 322 Illumina platform whole genome sequencing assays for Bacillus species were retrieved from NCBI, assembled and annotated on a single desktop computer. From the assemblies and annotations produced, a comprehensive analysis of the diversity of over 200 high-quality samples was conducted, looking at both the 16S rRNA phylogenetic marker as well as the Bacillus core genome. Peasant provides an intuitive solution for high-quality whole genome sequence assembly and annotation for users with limited programing experience and/or computational resources. The analysis of the Bacillus whole genome sequencing projects exemplifies the utility of this tool. Furthermore, the study conducted here provides insight into the diversity of the species, the largest such comparison conducted to date

Inhibition of the Mitochondrial Permeability Transition for Cytoprotection: Direct versus Indirect Mechanisms

Mitochondria are fascinating organelles, which fulfill multiple cellular functions, as diverse as energy production, fatty acid β oxidation, reactive oxygen species (ROS) production and detoxification, and cell death regulation. The coordination of these functions relies on autonomous mitochondrial processes as well as on sustained cross-talk with other organelles and/or the cytosol. Therefore, this implies a tight regulation of mitochondrial functions to ensure cell homeostasis. In many diseases (e.g., cancer, cardiopathies, nonalcoholic fatty liver diseases, and neurodegenerative diseases), mitochondria can receive harmful signals, dysfunction and then, participate to pathogenesis. They can undergo either a decrease of their bioenergetic function or a process called mitochondrial permeability transition (MPT) that can coordinate cell death execution. Many studies present evidence that protection of mitochondria limits disease progression and severity. Here, we will review recent strategies to preserve mitochondrial functions via direct or indirect mechanisms of MPT inhibition. Thus, several mitochondrial proteins may be considered for cytoprotective-targeted therapies

Carbon monoxide prevents hepatic mitochondrial membrane permeabilization

<p>Abstract</p> <p>Background</p> <p>Low concentrations of carbon monoxide (CO) protect hepatocytes against apoptosis and confers cytoprotection in several models of liver. Mitochondria are key organelles in cell death control <it>via </it>their membrane permeabilization and the release of pro-apoptotic factors.</p> <p>Results</p> <p>Herein, we show that CO prevents mitochondrial membrane permeabilization (MMP) in liver isolated mitochondria. Direct and indirect approaches were used to evaluate MMP inhibition by CO: mitochondrial swelling, mitochondrial depolarization and inner membrane permeabilization. Additionally, CO increases mitochondrial reactive oxygen species (ROS) generation, and their scavenging, by ß-carotene addition, decreases CO protection, which reveals the key role of ROS. Interestingly, cytochrome c oxidase transiently responds to low concentrations of CO by decreasing its activity in the first 5 min, later on there is an increase of cytochrome c oxidase activity, which were detected up to 30 min.</p> <p>Conclusion</p> <p>CO directly prevents mitochondrial membrane permeabilization, which might be implicated in the hepatic apoptosis inhibition by this gaseoustransmitter.</p

Far-UVC (222 nm) efficiently inactivated an airborne pathogen in a room-sized chamber

Funding: We acknowledge the financial assistance of the United Kingdom’s Department for Health and Social Care (2020/092).Many infectious diseases, including COVID-19, are transmitted by airborne pathogens. There is a need for effective environmental control measures which, ideally, are not reliant on human behaviour. One potential solution is Krypton Chloride (KrCl) excimer lamps (often referred to as Far-UVC), which can efficiently inactivate pathogens, such as coronaviruses and influenza, in air. Research demonstrates that when KrCl lamps are filtered to remove longer-wavelength ultraviolet emissions they do not induce acute reactions in the skin or eyes, nor delayed effects such as skin cancer. While there is laboratory evidence for Far-UVC efficacy, there is limited evidence in full-sized rooms. For the first time, we show that Far-UVC deployed in a room-sized chamber effectively inactivates aerosolised Staphylococcus aureus. At a room ventilation rate of 3 air-changes-per-hour (ACH), with 5 filtered-sources the steady-state pathogen load was reduced by 98.4% providing an additional 184 equivalent air changes (eACH). This reduction was achieved using Far-UVC irradiances consistent with current American Conference of Governmental Industrial Hygienists threshold limit values for skin for a continuous 8-h exposure. Our data indicate that Far-UVC is likely to be more effective against common airborne viruses, including SARS-CoV-2, than bacteria and should thus be an effective and “hands-off” technology to reduce airborne disease transmission. The findings provide room-scale data to support the design and development of effective Far-UVC systems.Publisher PDFPeer reviewe

Bacteriophages of the Urinary Microbiome

Bacterial viruses (bacteriophages) play a significant role in microbial community dynamics. Within the human gastrointestinal tract, for instance, associations amongst bacteriophages (phages), microbiota stability, and human health have been discovered. In contrast to the gastrointestinal tract, the phages associated with the urinary microbiota are largely unknown. Preliminary metagenomic surveys of the urinary virome indicate a rich diversity of novel lytic phage sequences, at an abundance far outnumbering eukaryotic viruses. These surveys, however, exclude the lysogenic phages residing within the bacteria of the bladder. To characterize this phage population, we examined 181 genomes representative of the phylogenetic diversity of bacterial species within the female urinary microbiota and found 457 phage sequences, 226 of which were predicted with high confidence. Phages were prevalent within the bladder bacteria: 86% of the genomes examined contained at least one phage sequence. Most of these phages are novel, exhibiting no discernible sequence homology to public data repositories. The presence of phages with substantial sequence similarity within the microbiota of different women supports the existence of a core community of phages within the bladder. Furthermore, the observed variation between the phage populations of women with and without overactive bladder symptoms suggests that phages may contribute to urinary health. To complement our bioinformatic analyses, viable phages were cultivated from the bacterial isolates for characterization; a novel coliphage was isolated, which is obligately lytic in the laboratory strain E. coli C. Sequencing of bacterial genomes facilitates a comprehensive cataloguing of the urinary virome while also revealing phage-host interactions.Importance Bacteriophages are abundant within the human body. But while some niches have been well surveyed, the phage population within the urinary microbiome is largely unknown. Our study is the first survey of the lysogenic phage population within the urinary microbiota. Most notably, the abundance of prophage exceeds that of the bacteria. Furthermore, many of the prophage sequences identified exhibited no recognizable sequence homology to data repositories. This suggests a rich diversity of uncharacterized phage species present in the bladder. Additionally, we observed a variation in the abundance of phages between bacteria isolated from asymptomatic \u27healthy\u27 individuals and those with urinary symptoms thus suggesting that, like phages within the gut, phages within the bladder may contribute to urinary health

Becoming Vigilant Subjects

Becoming Vigilant Subjects argues that practices of vigilance are key to forming individual subjectivity. The book emerged from a multi-disciplinary working group at the Collaborative Research Center for ›Cultures of Vigilance‹ at LMU Munich. The authors include anthropologists, historians, and literary scholars. They draw on historically and culturally diverse case studies to examine how individuals develop their own vigilant selves in response to being observed by (often powerful) others – be they present, absent, or imagined. The authors argue that, in the interplay between this assumed observation and individual watchfulness, subjectivity emerges. However, as shown in the case studies, this is an ambivalent process. The focus of this book is therefore on the becoming – rather than being – of subjects against the backdrop of heightened attention, which is directed towards objectives beyond individual goals and tasks. The different cases, relating to the realm of religion, citizenship, and migration, show how individuals engage with, and potentially change, the social world within which they are embedded. All of these examples emphasize that subjects are not just shaped by the context of vigilance, but have agency and the ability to transform their own circumstances. Becoming Vigilant Subjects makes a valuable contribution to the as yet understudied topics of subjectivity and vigilance, by interrogating how both inform one another

Permanent Occlusion of Feeding Arteries and Draining Veins in Solid Mouse Tumors by Vascular Targeted Photodynamic Therapy (VTP) with Tookad

Antiangiogenic and anti-vascular therapies present intriguing alternatives to cancer therapy. However, despite promising preclinical results and significant delays in tumor progression, none have demonstrated long-term curative features to date. Here, we show that a single treatment session of Tookad-based vascular targeted photodynamic therapy (VTP) promotes permanent arrest of tumor blood supply by rapid occlusion of the tumor feeding arteries (FA) and draining veins (DV), leading to tumor necrosis and eradication within 24–48 h.A mouse earlobe MADB106 tumor model was subjected to Tookad-VTP and monitored by three complementary, non-invasive online imaging techniques: Fluorescent intravital microscopy, Dynamic Light Scattering Imaging and photosensitized MRI. Tookad-VTP led to prompt tumor FA vasodilatation (a mean volume increase of 70%) with a transient increase (60%) in blood-flow rate. Rapid vasoconstriction, simultaneous blood clotting, vessel permeabilization and a sharp decline in the flow rates then followed, culminating in FA occlusion at 63.2 sec±1.5SEM. This blockage was deemed irreversible after 10 minutes of VTP treatment. A decrease in DV blood flow was demonstrated, with a slight lag from FA response, accompanied by frequent changes in flow direction before reaching a complete standstill. In contrast, neighboring, healthy tissue vessels of similar sizes remained intact and functional after Tookad-VTP.Tookad-VTP selectively targets the tumor feeding and draining vessels. To the best of our knowledge, this is the first mono-therapeutic modality that primarily aims at the larger tumor vessels and leads to high cure rates, both in the preclinical and clinical arenas