American Burn Association Burn Prevention Framework.

In February 2020, burn prevention experts from a variety of professional backgrounds gathered for a national Burn Prevention Summit. Through lively discussion and debate, this group came to a consensus on several core burn prevention concepts in order to create a framework for burn prevention program planning. The resulting document includes components of a successful program, a five-step process for program planning, best practices in messaging, and general advice from the summit attendees. This framework is designed for both novice professionals who are new to burn prevention programming development and experienced professionals who would like to strengthen existing programming.

Signalling pathways and transcriptional regulators orchestrating liver development and cancer.

Liver development is controlled by key signals and transcription factors that drive cell proliferation, migration, differentiation and functional maturation. In the adult liver, cell maturity can be perturbed by genetic and environmental factors that disrupt hepatic identity and function. Developmental signals and fetal genetic programmes are often dysregulated or reactivated, leading to dedifferentiation and disease. Here, we highlight signalling pathways and transcriptional regulators that drive liver cell development and primary liver cancers. We also discuss emerging models derived from pluripotent stem cells, 3D organoids and bioengineering for improved studies of signalling pathways in liver cancer and regenerative medicine.

H3K4 Trimethylation Is Required for Postnatal Pancreatic Endocrine Cell Functional Maturation.

During pancreas development, endocrine progenitors differentiate into the islet cell subtypes, which undergo further functional maturation in postnatal islet development. In islet ß-cells, genes involved in glucose-stimulated insulin secretion are activated, and glucose exposure increases the insulin response as ß-cells mature. We investigated the role of H3K4 trimethylation in endocrine cell differentiation and functional maturation by disrupting TrxG complex histone methyltransferase activity in mouse endocrine progenitors. In the embryo, genetic inactivation of TrxG component Dpy30 in NEUROG3+ cells did not affect the number of endocrine progenitors or endocrine cell differentiation. H3K4 trimethylation was progressively lost in postnatal islets, and the mice displayed elevated nonfasting and fasting glycemia as well as impaired glucose tolerance by postnatal day 24. Although postnatal endocrine cell proportions were equivalent to controls, islet RNA sequencing revealed a downregulation of genes involved in glucose-stimulated insulin secretion and an upregulation of immature ß-cell genes. Comparison of histone modification enrichment profiles in NEUROG3+ endocrine progenitors and mature islets suggested that genes downregulated by loss of H3K4 trimethylation more frequently acquire active histone modifications during maturation. Taken together, these findings suggest that H3K4 trimethylation is required for the activation of genes involved in the functional maturation of pancreatic islet endocrine cells.

TrxG Complex Catalytic and Non-catalytic Activity Play Distinct Roles in Pancreas Progenitor Specification and Differentiation.

Appropriate regulation of genes that coordinate pancreas progenitor proliferation and differentiation is required for pancreas development. Here, we explore the role of H3K4 methylation and the Trithorax group (TrxG) complexes in mediating gene expression during pancreas development. Disruption of TrxG complex assembly, but not catalytic activity, prevented endocrine cell differentiation in pancreas progenitor spheroids. In vivo loss of TrxG catalytic activity in PDX1+ cells increased apoptosis and the fraction of progenitors in the G1 phase of the cell cycle. Pancreas progenitors were reallocated to the acinar lineage, primarily at the expense of NEUROG3+ endocrine progenitors. Later in development, acinar and endocrine cell numbers were decreased, and increased gene expression variance and reduced terminal marker activation in acinar cells led to their incomplete differentiation. These findings demonstrate that TrxG co-activator activity is required for gene induction, whereas TrxG catalytic activity and H3K4 methylation help maintain transcriptional stability.

Chromatin Regulators in Pancreas Development and Diabetes.

The chromatin landscape of a cell is dynamic and can be altered by chromatin regulators that control nucleosome placement and DNA or histone modifications. Together with transcription factors, these complexes help dictate the transcriptional output of a cell and, thus, balance cell proliferation and differentiation while restricting tissue-specific gene expression. In this review, we describe current research on chromatin regulators and their roles in pancreas development and the maintenance of mature ß cell function, which, once elucidated, will help us better understand how ß cell differentiation occurs and is maintained. These studies have so far implicated proteins from several complexes that regulate DNA methylation, nucleosome remodeling, and histone acetylation and methylation that could become promising targets for diabetes therapy and stem cell differentiation.

A 22-mer segment in the structurally pliable regulatory domain of metazoan CTP: phosphocholine cytidylyltransferase facilitates both silencing and activating functions.

CTP:phosphocholine cytidylyltransferase (CCT), an amphitropic enzyme that regulates phosphatidylcholine synthesis, is composed of a catalytic head domain and a regulatory tail. The tail region has dual functions as a regulator of membrane binding/enzyme activation and as an inhibitor of catalysis in the unbound form of the enzyme, suggesting conformational plasticity. These functions are well conserved in CCTs across diverse phyla, although the sequences of the tail regions are not. CCT regulatory tails of diverse origins are composed of a long membrane lipid-inducible amphipathic helix (m-AH) followed by a highly disordered segment, reminiscent of the Parkinson disease-linked protein, α-synuclein, which we show shares a novel sequence motif with vertebrate CCTs. To unravel features required for silencing, we created chimeric enzymes by fusing the catalytic domain of rat CCTα to the regulatory tail of CCTs from Drosophila, Caenorhabditis elegans, or Saccharomyces cerevisiae or to α-synuclein. Only the tail domains of the two invertebrate CCTs were competent for both suppression of catalytic activity and for activation by lipid vesicles. Thus, both silencing and activating functions of the m-AH can tolerate significant changes in length and sequence. We identified a highly amphipathic 22-residue segment in the m-AH with features conserved among animal CCTs but not yeast CCT or α-synuclein. Deletion of this segment from rat CCT increased the lipid-independent V(max) by 10-fold, equivalent to the effect of deleting the entire tail, and severely weakened membrane binding affinity. However, membrane binding was required for additional increases in catalytic efficiency. Thus, full activation of CCT may require not only loss of a silencing conformation in the m-AH but a gain of an activating conformation, promoted by membrane binding.

Respiratory syncytial virus impairs macrophage IFN-alpha/beta- and IFN-gamma-stimulated transcription by distinct mechanisms.

Macrophages are the primary lung phagocyte and are instrumental in maintenance of a sterile, noninflamed microenvironment. IFNs are produced in response to bacterial and viral infection, and activate the macrophage to efficiently counteract and remove pathogenic invaders. Respiratory syncytial virus (RSV) inhibits IFN-mediated signaling mechanisms in epithelial cells; however, the effects on IFN signaling in the macrophage are currently unknown. We investigated the effect of RSV infection on IFN-mediated signaling in macrophages. RSV infection inhibited IFN-beta- and IFN-gamma-activated transcriptional mechanisms in primary alveolar macrophages and macrophage cell lines, including the transactivation of important Nod-like receptor family genes, Nod1 and class II transactivator. RSV inhibited IFN-beta- and IFN-gamma-mediated transcriptional activation by two distinct mechanisms. RSV impaired IFN-beta-mediated signal transducer and activator of transcription (STAT)-1 phosphorylation through a mechanism that involves inhibition of tyrosine kinase 2 phosphorylation. In contrast, RSV-impaired transcriptional activation after IFN-gamma stimulation resulted from a reduction in the nuclear STAT1 interaction with the transcriptional coactivator, CBP, and was correlated with increased phosphorylation of STAT1beta, a dominant-negative STAT1 splice variant, in response to IFN-gamma. In support of this concept, overexpression of STAT1beta was sufficient to repress the IFN-gamma-mediated expression of class II transactivator. These results demonstrate that RSV inhibits IFN-mediated transcriptional activation in macrophages, and suggests that paramyxoviruses modulate an important regulatory mechanism that is critical in linking innate and adaptive immune mechanisms after infection.

Attenuation of herpes simplex virus neurovirulence with picornavirus cis-acting genetic elements.

Viral pathogenesis depends on a suitable milieu in target host cells permitting viral gene expression, propagation, and spread. In many instances, viral genomes can be manipulated to select for propagation in certain tissues or cell types. This has been achieved for the neurotropic poliovirus (PV) by exchange of the internal ribosomal entry site (IRES), which is responsible for translation of the uncapped plus-strand RNA genome. The IRES of human rhinovirus type 2 (HRV2) confers neuron-specific replication deficits to PV but has no effect on viral propagation in malignant glioma cells. We report here that placing the critical gamma(1)34.5 virulence genes of herpes simplex virus type 1 (HSV) under translation control of the HRV2 IRES results in neuroattenuation in mice. In contrast, IRES insertion permits HSV propagation in malignant glioma cell lines that do not support replication of HSV recombinants carrying gamma(1)34.5 deletions. Our observations indicate that the conditions for alternative translation initiation at the HRV2 IRES in malignant glioma cells differ from those in normal central nervous system (CNS) cells. Picornavirus regulatory sequences mediating cell type-specific gene expression in the CNS can be utilized to target cancerous cells at the level of translation regulation outside their natural context.

Targeted therapy for glioblastoma multiforme neoplastic meningitis with intrathecal delivery of an oncolytic recombinant poliovirus.

PURPOSE: The toxicity and antitumor activity of regional intrathecal delivery of an oncolytic recombinant poliovirus, PVS-RIPO, was evaluated in rodent models of glioblastoma multiforme neoplastic meningitis. EXPERIMENTAL DESIGN: To evaluate for toxicity, PVS-RIPO was administered into the spinal cord of transgenic mice that express the human poliovirus receptor, CD155, and into the intrathecal space of athymic rats without tumor. To evaluate efficacy, two different doses of PVS-RIPO were administered intrathecally 3 days after athymic rats were inoculated intrathecally with an aggressive human glioblastoma multiforme xenograft. RESULTS: No clinical or histologic evidence of toxicity was found. In efficacy studies, median survival was increased by 174.47% from 8.5 days in the group treated with UV light-inactivated virus to 15 days in the rats treated with 1.0 x 10(7) plaque-forming units (pfu) of PVS-RIPO (P < 0.0001). A similar increase in median survival was seen in the group receiving 1.0 x 10(9) pfu PVS-RIPO (P < 0.0001); however, there was no statistically significant dose-response relationship (P = 0.345). In addition, 1 of 10 rats in lower-dose PVS-RIPO-treated group and 3 of 10 rats in higher-dose PVS-RIPO-treated group survived >60 days after tumor cell inoculation and had no evidence of residual tumor at autopsy. CONCLUSION: These results suggest that intrathecal treatment with PVS-RIPO may be useful for treatment of neoplastic meningitis in patients with glioblastoma multiforme and provides a rationale for clinical trials in this area.

Oncolytic viruses for cancer therapy II. Cell-internal factors for conditional growth in neoplastic cells.

Recent advances in our understanding of virus-host interactions have fueled new studies in the field of oncolytic viruses. The first part of this review explained how cell-external factors, such as cellular receptors, influence tumor tropism and specificity of oncolytic virus candidates. In the second part of this review, we focus on cellinternal factors that mediate tumor-specific virus growth. An oncolytic virus must be able to replicate within cancerous cells and kill them without collateral damage to healthy surrounding cells. This desirable property is inherent to some proposed oncolytic viral agents or has been achieved by genetic manipulation in others.

Genetic determinants of cell type-specific poliovirus propagation in HEK 293 cells.

The ability of poliovirus to propagate in neuronal cells can be reduced by introducing appropriate nucleotide substitutions into the viral genome. Specific mutations scattered throughout the poliovirus genome yielded the live attenuated vaccine strains of poliovirus. Neuron-specific propagation deficits of the Sabin strains are partially encrypted within a confined region of the internal ribosomal entry site (IRES), which carries attenuating point mutations in all three serotypes. Recently, high levels of neurovirulence attenuation were achieved with genetically engineered polioviruses containing heterologous IRES elements. This is exemplified with poliovirus recombinants replicating under control of a human rhinovirus type 2 (HRV2) IRES element. We have carried out experiments delineating the genetic basis for neuronal IRES function. Neuronal dysfunction of the HRV2 IRES is determined mainly by IRES stem-loop domain V, the locus for attenuating point mutations within the Sabin strains. Neuronal incompetence associated with HRV2 IRES domain V is substantially more pronounced than that observed with the attenuating IRES point mutation of the Sabin serotype 1 vaccine strain. Mix-and-match recombination of polio and HRV2 IRES domain V suggests that the attenuation phenotype correlates with overall structural features rather than primary sequence. Our experiments have identified HEK 293 cells as a novel system for the study of neuron-specific replication phenotypes of poliovirus. This cell line, originally derived from embryonic human kidney, has recently been described to display neuronal characteristics. We report propagation properties in HEK 293 cells for poliovirus recombinants with attenuated neurovirulence in experimental animals that corroborate this observation.

Oncolytic viruses for cancer therapy I. Cell-external factors: virus entry and receptor interaction.

After being recognized for their anti-neoplastic properties at the beginning of the last century, viruses are again being considered for use as therapeutic agents against cancer. Certain virus species have a propensity to replicate within transformed cells, which are commonly rendered vulnerable due to tumor-specific defects in their defense against viral infection. Other viruses have been modified to subject them to tumor-specific growth conditions. Oncolytic viruses carry the promise to efficiently target cancer cells for destruction and spread throughout tumor tissue to reach distant neoplastic loci without causing collateral damage to healthy tissues. In contrast to conventional cancer chemotherapy, viral anti-neoplastic agents require complex interactions with the host organism to reach their target and to unfold their oncolytic activity. Recent progress in the elucidation of the molecular mechanisms of viral pathogenesis has opened up new opportunities to manipulate virus-host interactions, generating effective anti-tumor strategies. On the other hand, significant obstacles towards the application of safe and efficacious viral therapies have become apparent. These frequently relate to the lack of cell culture and animal tumor models that accurately reflect the characteristics of cancerous tissues in patients. Throughout the past century, viral therapeutics against cancer have evolved into a new class of treatment strategies characterized by unique opportunities and challenges. A growing number of oncolytic viruses has entered clinical investigation or is scheduled to do so in the near future. Great efforts are being undertaken to rekindle an old idea and, with the help of new technologies, to realize its promise of new treatment facilities for cancer.