Embryonic Lethality in Homozygous Human Her-2 Transgenic Mice Due to Disruption of the Pds5b Gene

The development of antigen-targeted therapeutics is dependent on the preferential expression of tumor-associated antigens (TAA) at targetable levels on the tumor. Tumor-associated antigens can be generated de novo or can arise from altered expression of normal basal proteins, such as the up-regulation of human epidermal growth factor receptor 2 (Her2/ErbB2). To properly assess the development of Her2 therapeutics in an immune tolerant model, we previously generated a transgenic mouse model in which expression of the human Her2 protein was present in both the brain and mammary tissue. This mouse model has facilitated the development of Her2 targeted therapies in a clinically relevant and suitable model. While heterozygous Her2^+/- mice appear to develop in a similar manner to wild type mice (Her2^-/-), it has proven difficult to generate homozygous Her2^+/+ mice, potentially due to embryonic lethality. In this study, we performed whole genome sequencing to determine if the integration site of the Her2 transgene was responsible for this lethality. Indeed, we report that the Her2 transgene had integrated into the Pds5b (precocious dissociation of sisters) gene on chromosome 5, as a 162 copy concatemer. Furthermore, our findings demonstrate that Her2^+/+ mice, similar to Pds5b^-/- mice, are embryonic lethal and confirm the necessity for Pds5b in embryonic development. This study confirms the value of whole genome sequencing in determining the integration site of transgenes to gain insight into associated phenotypes.     

Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2’-O-Methylation Mutant

Dengue virus (DENV) is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN) response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2’-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2’-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.     

A hidden deadly venomous insect: First eco-epidemiological assessment and risk mapping of lonomism in Argentina

BackgroundEnvenomation by the South American Lonomia saturniid caterpillars, named lonomism, constitutes an emerging and somewhat neglected public health issue in Argentina and neighboring countries. Considering that there is an intricate relationship between environment and human health in such cases, this study aimed to analyze the eco-epidemiological profile of 40 accidents and 33 occurrences of Lonomia spp. in Misiones (Argentina) between January 2014 and May 2020.Methodology/Principal findingsWe described the eco-epidemiological variables and characterized the abiotic scenario of such cases. Additionally, we obtained a density map that shows the punctual intensity of Lonomia records throughout Misiones. Most of the accidents occurred in the Department of Guaraní and involved male victims younger than 20 years old. The accidental/occasional occurrence of Lonomia spp. (considering both adult and caterpillar stages together) was significantly higher in the rural area, whereas only adult specimens were found in urban areas. We determined that the presence of this insect in Misiones is positively related to higher temperatures and solar radiation, and larger precipitation and evapotranspiration throughout the year.Conclusion/SignificanceThis study represents an initial step towards the global understanding of lonomism as a public health problem in Argentina. It provides a map of the risk level for this envenomation in Misiones, which could help authorities address public health policy efforts to implement sustainable strategies for prevention and response to this threat in Northeastern Argentina and neighboring regions.     

Intraskeletal isotopic compositions (δ13C, δ15N) of bone collagen: Nonpathological and pathological variation

Paleodiet research traditionally interprets differences in collagen isotopic compositions (δ¹³C, δ¹⁵N) as indicators of dietary distinction even though physiological processes likely play some role in creating variation. This research investigates the degree to which bone collagen δ¹³C and δ¹⁵N values normally vary within the skeleton and examines the influence of several diseases common to ancient populations on these isotopic compositions. The samples derive from two medieval German cemeteries and one Swiss reference collection and include examples of metabolic disease (rickets/osteomalacia), degenerative joint disease (osteoarthritis), trauma (fracture), infection (osteomyelitis), and inflammation (periostitis). A separate subset of visibly nonpathological skeletal elements from the German collections established normal intraindividual variation. For each disease type, tests compared bone lesion samples to those near and distant to the lesions sites. Results show that normal (nonpathological) skeletons exhibit limited intraskeletal variation in carbon‐ and nitrogen‐isotope ratios, suggesting that sampling of distinct elements is appropriate for paleodiet studies. In contrast, individuals with osteomyelitis, healed fractures, and osteoarthritis exhibit significant intraskeletal differences in isotope values, depending on whether one is comparing lesions to near or to distant sites. Skeletons with periostitis result in significant intraskeletal differences in nitrogen isotope values only, while those with rickets/osteomalacia do not exhibit significant intraskeletal differences. Based on these results, we suggest that paleodiet researchers avoid sampling collagen at or close to lesion sites because the isotope values may be reflecting both altered metabolic processes and differences in diet relative to others in the population. Am J Phys Anthropol 153:598–604, 2014. © 2013 Wiley Periodicals, Inc.     

Tissue slice cultures from humans or rodents: a new tool to evaluate biological effects of heavy ions

The aim of this interdisciplinary project is to establish slice culture preparations from rodents and humans as a new model system for studying effects of X-rays and heavy ions within normal and tumor tissues. The advantage of such slice cultures relies on the conservation of an organotypic environment, the easy treatment and observation by live-imaging microscopy, and the independence from genetic immortalization strategies used to generate cell lines. Rat brains as well as human tumors were cut into 300-μm-thick sections and cultivated in an incubator in a humidified atmosphere at 37°C. This is realized by a membrane-based culture system with a liquid–air interface. With this system, it is possible to keep rodent slices viable for several months. Human brain tumor slices remained vital for at least 21 days. Slices were irradiated with X-rays at the radiation facility of the University Hospital in Frankfurt/Main at doses up to 40 Gy. Heavy ion irradiations were performed at GSI (Darmstadt) with different ions, energies, and doses. The irradiated slices were analyzed by 3D-confocal microscopy following immunostaining for DNA damage, microglia, and proliferation markers. The phosphorylated histone γH2AX proved to be suitable for the detection of ion traversals in this system.     

CK2 phosphorylation-dependent interaction between aprataxin and MDC1 in the DNA damage response

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1, resolves abortive DNA ligation intermediates during DNA repair. Here, we demonstrate that aprataxin localizes at sites of DNA damage induced by high LET radiation and binds to mediator of DNA-damage checkpoint protein 1 (MDC1/NFBD1) through a phosphorylation-dependent interaction. This interaction is mediated via the aprataxin FHA domain and multiple casein kinase 2 di-phosphorylated S-D-T-D motifs in MDC1. X-ray structural and mutagenic analysis of aprataxin FHA domain, combined with modelling of the pSDpTD peptide interaction suggest an unusual FHA binding mechanism mediated by a cluster of basic residues at and around the canonical pT-docking site. Mutation of aprataxin FHA Arg29 prevented its interaction with MDC1 and recruitment to sites of DNA damage. These results indicate that aprataxin is involved not only in single strand break repair but also in the processing of a subset of double strand breaks presumably through its interaction with MDC1.     

Distribution of myogenic progenitor cells and myonuclei is altered in women with vs. those without chronically painful trapezius muscle

It is hypothesized that repeated recruitment of low-threshold motor units is an underlying cause of chronic pain in trapezius myalgia. This study investigated the distribution of satellite cells (SCs), myonuclei, and macrophages in muscle biopsies from the trapezius muscle of 42 women performing repetitive manual work, diagnosed with trapezius myalgia (MYA; 44 ± 8 yr; mean ± SD) and 20 matched healthy controls (CON; 45 ± 9 yr). Our hypothesis was that muscle of MYA, in particular type I fibers, would demonstrate higher numbers of SCs, myonuclei, and macrophages compared with CON. SCs were identified on muscle cross sections by combined immunohistochemical staining for Pax7, type I myosin, and laminin, allowing the number of SCs associated with type I and II fibers to be determined. We observed a pattern of SC distribution in MYA previously only reported for individuals above 70 yr of age. Compared with CON, MYA demonstrated 19% more SCs per fiber associated with type I fibers (MYA 0.098 ± 0.039 vs. CON 0.079 ± 0.031; P < 0.05) and 40% fewer SCs associated with type II fibers (MYA 0.047 ± 0.017 vs. CON 0.066 ± 0.035; P < 0.05). The finding of similar numbers of macrophages between the two groups was not in line with our hypothesis and suggests that the elevated SC content of MYA was not due to heightened inflammatory cell contents, but rather to provide new myonuclei. The findings of greater numbers of SCs in type I fibers of muscle subjected to repeated low-intensity work support our hypothesis and provide new insight into stimuli capable of regulating SC content.     

Sequencing of 6.7 Mb of the melon genome using a BAC pooling strategy

Background  

Cucumis melo (melon) belongs to the Cucurbitaceae family, whose economic importance among horticulture crops is second only to Solanaceae. Melon has a high intra-specific genetic variation, morphologic diversity and a small genome size (454 Mb), which make it suitable for a great variety of molecular and genetic studies. A number of genetic and genomic resources have already been developed, such as several genetic maps, BAC genomic libraries, a BAC-based physical map and EST collections. Sequence information would be invaluable to complete the picture of the melon genomic landscape, furthering our understanding of this species' evolution from its relatives and providing an important genetic tool. However, to this day there is little sequence data available, only a few melon genes and genomic regions are deposited in public databases. The development of massively parallel sequencing methods allows envisaging new strategies to obtain long fragments of genomic sequence at higher speed and lower cost than previous Sanger-based methods.     

Loss of glutathione peroxidase 3 expression is correlated with epigenetic mechanisms in endometrial adenocarcinoma

Glutathione peroxidase 3 (GPX3) is one of the key enzymes in the cellular defense against oxidative stress and the hepatocyte growth factor receptor, (MET) has been suggested to be influenced by the GPX3 gene expression. In a previous microarray study performed by our group, Gpx3 was identified as a potential biomarker for rat endometrial adenocarcinoma (EAC), since the expression was highly downregulated in rat EAC tumors. Herein, we have investigated the mRNA expression and Gpx3 and Met in rat EAC by real time quantitative PCR (qPCR), and the methylation status of Gpx3. In addition we have examined the expression of GPX3 and MET in 30 human EACs of different FIGO grades and 20 benign endometrial tissues. We found that the expression of GPX3 was uniformly down regulated in both rat and human EAC, regardless of tumor grade or histopathological subtype, implying that the down-regulation is an early event in EAC. The rate of Gpx3 promoter methylation reaches 91%, where biallelic methylation was present in 90% of the methylated tumors. The expression of the Met oncogene was slightly upregulated in EACs that showed loss of expression of Gpx3, but no tumor suppressor activity of Gpx3/GPX3 was detected. Preliminary results also suggest that the production of H₂O₂ is higher in rat endometrial tumors with down-regulated Gpx3 expression. A likely consequence of loss of GPX3 protein function would be a higher amount of ROS in the cancer cell environment. Thus, the results suggest important clinical implications of the GPX3 expression in EAC, both as a molecular biomarker for EAC and as a potential target for therapeutic interventions.