Impairment of Methotrexate Transport Is Common in Osteosarcoma Tumor Samples

Osteosarcoma does not respond well to conventional dose methotrexate but does respond to high-dose methotrexate. Previous work has indicated that this resistance may be due to impaired transport of methotrexate across the cell membrane. In this study, the PT430 competitive displacement assay was adapted to evaluate methotrexate transport in 69 high-grade osteosarcoma tumor samples. All samples studied were shown to have relatively impaired methotrexate transport by PT430 assay. Ninety-nine percent of the samples had less than 20% PT430 displacement by methotrexate. Eighty-eight percent exhibited displacement by methotrexate at less than 50% of the displacement by trimetrexate. The high frequency of impaired transport suggests the presence of decreased functionality of the reduced folate carrier protein. The overwhelming presence of impaired transport may explain why methotrexate needs to be given in high doses to be effective in osteosarcoma therapy and suggests that reduced folate carrier-independent antifolates should be explored

ASIC1A in the bed nucleus of the stria terminalis mediates TMT-evoked freezing

Mice display an unconditioned freezing response to TMT, a predator odor isolated from fox feces. Here we found that in addition to freezing, TMT caused mice to decrease breathing rate, perhaps because of the aversive smell. Consistent with this possibility, olfactory bulb lesions attenuated this effect of TMT, as well as freezing. Interestingly, butyric acid, another foul odor, also caused mice to reduce breathing rate. However, unlike TMT, butyric acid did not induce freezing. Thus, although these aversive odors may affect breathing, the unpleasant smell and suppression of breathing by themselves are insufficient to cause freezing. Because the acid-sensing ion channel-1A (ASIC1A) has been previously implicated in TMT-evoked freezing, we tested whether Asic1a disruption also altered breathing. We found that TMT reduced breathing rate in both Asic1a+/+ and Asic1a–/– mice, suggesting that ASIC1A is not required for TMT to inhibit breathing and that the absence of TMT-evoked freezing in the Asic1a–/– mice is not due to an inability to detect TMT. These observations further indicate that ASIC1A must affect TMT freezing in another way. Because the bed nucleus of the stria terminalis (BNST) has been critically implicated in TMT-evoked freezing and robustly expresses ASIC1A, we tested whether ASIC1A in the BNST plays a role in TMT-evoked freezing. We disrupted ASIC1A in the BNST of Asic1aloxP/loxP mice by delivering Cre recombinase to the BNST with an adeno-associated virus (AAV) vector. We found that disrupting ASIC1A in the BNST reduced TMT-evoked freezing relative to control mice in which a virus expressing eGFP was injected. To test whether ASIC1A in the BNST was sufficient to increase TMT-evoked freezing, we used another AAV vector to express ASIC1A in the BNST of Asic1a–/– mice. We found region-restricted expression of ASIC1A in the BNST increased TMT-elicited freezing. Together, these data suggest that the BNST is a key site of ASIC1A action in TMT-evoked freezing

When will it be over? U.S. childrens questions and parents responses about the COVID-19 pandemic.

Parent-child conversations are important for childrens cognitive development, childrens ability to cope with stressful events, and can shape childrens beliefs about the causes of illness. In the context of a global pandemic, families have faced a multitude of challenges, including changes to their routines, that they need to convey to their children. Thus, parent-child conversations about the coronavirus pandemic might convey information about the causes of illness, but also about how and why it is necessary for children to modify their behaviors to comply with new social norms and medical guidance. The main goal of this study was to examine the questions children ask about the COVID-19 pandemic and how parents answer them. This survey included responses from a national sample of 349 predominantly white parents of children between the ages of 3 and 12 recruited through Amazons Mechanical Turk in United States. Parents reported that although children asked about COVID-19 and its causes (17.3%), children asked primarily about lifestyle changes that occurred as a result of the pandemic (24.0%) and safety (18.4%). Parents reported answering these questions by emphasizing that the purpose of different preventative measures was to protect the child (11.8%) or the family (42.7%) and providing reassurance (13.3%). Many parents discussed how it was their social responsibility to slow the spread of the virus (38.4%). Parents of younger children tended to shield them from information about COVID-19 (p = .038), while parents with more knowledge were more likely to provide explanations (p < .001). Our analysis shows that families not only discuss information about the virus but also information about changes to their lifestyle, preventative measures, and social norms

Burden of subclinical cardiovascular disease in “Metabolically Benign” and “At-Risk” overweight and obese women: the study of women\u27s health across the nation (SWAN)

Background: Metabolically benign obese individuals have a 10-year cardiovascular disease (CVD) risk comparable to healthy normal weight individuals. However, the burden of subclinical CVD among metabolically benign obese is not well known.Methods: In cross-sectional analyses of 475 mid-life women, we compared common carotid artery intima media thickness (CCA-IMT), aortic pulse wave velocity (aPWV) and coronary (CAC) and aortic calcification (AC) among three groups: healthy normal weight, metabolically benign overweight/obese (/elevated CRP), and at-risk overweight/obese (≥3 metabolic syndrome components/elevated CRP).Results: The mean (SD) CCA-IMT and aPWV were lowest in the normal weight group (n=145), followed by the benign overweight/obese (n=260) and at-risk overweight/obese (n=70) groups [CCA-IMT: 0.64 (0.08) vs. 0.68 (0.09) vs. 0.73 (0.13) mm, pConclusions: Metabolically benign overweight/obese women have a significantly greater subclinical CVD burden than normal weight women, despite published data finding similar CVD event rates between the two groups. Prospective studies tracking the progression of subclinical atherosclerosis to clinical CVD in these women are needed

The protein kinase PERK/EIF2AK3 regulates proinsulin processing not via protein synthesis but by controlling endoplasmic reticulum chaperones

International audienceLoss-of-function mutations of the protein kinase PERK (EIF2AK3) in humans and mice cause permanent neonatal diabetes and severe proinsulin aggregation in the endoplasmic reticulum (ER), highlighting the essential role of PERK in insulin production in pancreatic cells. As PERK is generally known as a translational regulator of the unfolded protein response (UPR), the underlying cause of these cell defects has often been attributed to derepression of proinsulin synthesis, resulting in proinsulin overload in the ER. Using high-resolution imaging and standard protein fractionation and immunological methods we have examined the PERK-dependent phenotype more closely. We found that whereas proinsulin aggregation requires new protein synthesis, global protein and proinsulin synthesis are down-regulated in PERK-inhibited cells, strongly arguing against proinsulin overproduction being the root cause of their aberrant ER phenotype. Furthermore, we show that PERK regulates proinsulin proteostasis by modulating ER chaperones, including BiP and ERp72. Transgenic overexpression of BiP and BiP knockdown (KD) both promoted proinsulin aggregation, whereas ERp72 overexpression and knockdown rescued it. These findings underscore the importance of ER chaperones working in concert to achieve control of insulin production and identify a role for PERK in maintaining a functional balance among these chaperones

Interlaboratory Comparison of δ13C and δD Measurements of Atmospheric CH4 for Combined Use of Data Sets from Different Laboratories

We report results from a worldwide interlaboratory comparison of samples among laboratories that measure (or measured) stable carbon and hydrogen isotope ratios of atmospheric CH4 (δ13C-CH4 and δD-CH4). The offsets among the laboratories are larger than the measurement reproducibility of individual laboratories. To disentangle plausible measurement offsets, we evaluated and critically assessed a large number of intercomparison results, some of which have been documented previously in the literature. The results indicate significant offsets of δ13C-CH4 and δD- CH4 measurements among data sets reported from different laboratories; the differences among laboratories at modern atmospheric CH4 level spread over ranges of 0.5 ‰ for δ13C-CH4 and 13 ‰ for δD-CH4. The intercomparison results summarized in this study may be of help in future at tempts to harmonize δ13C-CH4 and δD-CH4 data sets from different laboratories in order to jointly incorporate them into modelling studies. However, establishing a merged data set, which includes δ13C-CH4 and δD-CH4 data from multiple laboratories with desirable compatibility, is still challenging due to differences among laboratories in instrument settings, correction methods, traceability to reference materials and long-term data management. Further efforts are needed to identify causes of the interlaboratory measurement offsets and to decrease those to move towards the best use of available δ13C-CH4 and δD-CH4 data sets

HER‐2 expression is not prognostic in osteosarcoma; a Children's Oncology Group prospective biology study

BackgroundSince the initial reports of human epidermal growth factor receptor 2 (HER-2) expression as being prognostic in osteosarcoma, numerous small studies varying in the interpretation of the immunohistochemical (IHC) staining patterns have produced conflicting results. The Children's Oncology Group therefore embarked on a prospective biology study in a larger sample of patients to define in osteosarcoma the prognostic value of HER-2 expression using the methodology employed in the initial North American study describing an association between HER-2 expression and outcome.ProcedureThe analytic patient population was comprised of 149 patients with newly diagnosed osteosarcoma, 135 with localized disease and 14 with metastatic disease, all of whom had follow up clinical data. Paraffin embedded material from the diagnostic biopsy was stained with CB11 antibody and scored by two independent observers. Correlation of HER-2 IHC score and demographic variables was analyzed using a Fisher's exact test and correlation with survival using a Kaplan-Meier analysis.ResultsNo association was found with HER-2 status and any of the demographic variables tested including the presence or absence of metastatic disease at diagnosis. No association was found between HER-2 status and either event free survival or overall survival in the patients with localized disease.ConclusionHER-2 expression is not prognostic in osteosarcoma in the context of this large prospective study. HER-2 expression cannot be used as a basis for stratification of therapy. Identification of potential prognostic factors should occur in the context of large multi-institutional biology studies

Insulin-Like Growth Factor 1 Receptor and Response to Anti-IGF1R Antibody Therapy in Osteosarcoma

<div><p>Background</p><p>Survival outcomes for patients with osteosarcoma (OS) have remained stagnant over the past three decades. Insulin-like growth factor 1 receptor (IGF1R) is over-expressed in a number of malignancies, and anti-IGF1R antibodies have and are currently being studied in clinical trials. Understanding the molecular aberrations which result in increased tumor response to anti-IGF1R therapy could allow for the selection of patients most likely to benefit from IGF1R targeted therapy.</p><p>Methods</p><p><i>IGF1R</i> mRNA expression was assessed by RT PCR in OS patient primary tumors, cell lines, and xenograft tumors. <i>IGF1R</i> copy number was assessed by 3 approaches: PCR, FISH, and dot blot analysis. Exons 1–20 of <i>IGF1R</i> were sequenced in xenograft tumors and 87 primary OS tumors, and surface expression of <i>IGF1R</i> was assessed by flow cytometry. Levels of mRNA and protein expression, copy number, and mutation status were compared with tumor response to anti-<i>IGF1R</i> antibody therapy in 4 OS xenograft models.</p><p>Results</p><p><i>IGF1R</i> mRNA is expressed in OS. Primary patient samples and xenograft samples had higher mRNA expression and copy number compared with corresponding cell lines. <i>IGF1R</i> mRNA expression, cell surface expression, copy number, and mutation status were not associated with tumor responsiveness to anti-IGF1R antibody therapy.</p><p>Conclusions</p><p><i>IGF1R</i> is expressed in OS, however, no clear molecular markers predict response to IGF1R antibody-mediated therapy. Additional pre-clinical studies assessing potential predictive biomarkers and investigating targetable molecular pathways critical to the proliferation of OS cells are needed.</p></div