Circulating tumour cells demonstrate an altered response to hypoxia and an aggressive phenotype

BACKGROUND: Tumours contain hypoxic regions that select for an aggressive cell phenotype; tumour hypoxia induces metastasis-associated genes. Treatment refractory patients with metastatic cancer show increased numbers of circulating tumour cells (CTCs), which are also associated with disease progression. The aim of this study was to examine the as yet unknown relationship between hypoxia and CTCs. METHODS: We generated human MDA-MB-231 orthotopic xenografts and, using a new technology, isolated viable human CTCs from murine blood. The CTCs and parental MDA-MB-231 cells were incubated at 21 and 0.2% (hypoxia) oxygen, respectively. Colony formation was assayed and levels of hypoxia- and anoxia-inducible factors were measured. Xenografts generated from CTCs and parental cells were compared. RESULTS: MDA-MB-231 xenografts used to generate CTCs were hypoxic, expressing hypoxia factors: hypoxia-inducible factor1 alpha (HIF1alpha) and glucose transporter protein type 1 (GLUT1), and anoxia-induced factors: activating transcription factor 3 and 4 (ATF3 and ATF4). Parental MDA-MB-231 cells induced ATF3 in hypoxia, whereas CTCs expressed it constitutively. Asparagine synthetase (ASNS) expression was also higher in CTCs. Hypoxia induced ATF4 and the HIF1alpha target gene apelin in CTCs, but not in parental cells. Hypoxia induced lower levels of carbonic anhydrase IX (CAIX), GLUT1 and BCL2/adenovirus E1B 19-KD protein-interacting protein 3 (BNIP3) proteins in CTCs than in parental cells, supporting an altered hypoxia response. In chronic hypoxia, CTCs demonstrated greater colony formation than parental cells. Xenografts generated from CTCs were larger and heavier, and metastasised faster than MDA-MB-231 xenografts. CONCLUSION: CTCs show an altered hypoxia response and an enhanced aggressive phenotype in vitro and in vivo

Regulation of TFEB and V-ATPases by mTORC1

TORC1 is a key regulator of cell growth in response to nutrients and acts at the surface of the late endosome. This study identifies V-ATPase genes as transcriptional targets of TORC1 and implicates the transcription factor TFEB as an important mediator of TORC1-dependent gene expression and TORC1-regulated endocytosis

Rat-bite Fever–A Rare Diagnosis for a Common Pediatric Presentation: Case Report

Introduction: Fever and rash is a common pediatric presentation to the emergency department but can present a diagnostic challenge to the clinician. Here we report the successful identification and treatment of a rare zoonotic exanthem that was facilitated by a thorough history and physical exam.Case Report: Rat-bite fever is a potentially fatal systemic illness characterized by relapsing fever, rash, and migratory polyarthralgias. Treatment includes antibiotics for Streptobacillus moniliformis, the most common pathogen, as well as appropriate hygiene education and prevention strategies. We report a case of S. moniliformis in the absence of an actual rodent bite.Conclusion: Due to the generally non-specific presentation of the illness, as well as the growing trend of caring for domestic rodents, it is crucial that clinicians ask details related to zoonotic and other exposures while obtaining medical histories

Trends in cannabis use in New Jersey: Effects of COVID‐19 and cannabis legalization

Abstract Objectives With the legalization of cannabis in New Jersey on April 21, 2022, including the licensing of cannabis dispensaries, concerns have arisen about potential adverse events related to cannabis use. Here, we explore temporal trends and risk factors for cannabis‐related harm in both adult and pediatric cannabis‐related visits at a tertiary care academic institution. Methods We performed a retrospective chart review and temporal trend analysis via the electronic health record from May 1, 2019 to October 31, 2022, covering 2 years before, and 6 months after, cannabis legalization in New Jersey. The pediatric charts identified were analyzed for root causes of adverse events, and changes in the frequency of specific unsafe practices since cannabis legalization were tracked. Results We found that adult cannabis ED‐related visits significantly increased during the COVID‐19 pandemic and remained higher than pre‐pandemic levels for the remainder of the study periods, without a significant change upon legalization. Pediatric rates of cannabis‐related ED visits did not vary significantly during the study period. The vast majority of visits for children aged 0–12 years were related to accidental cannabis exposures—often a household member's edibles—whereas most visits for older children stemmed from intentional cannabis use. Conclusion This project highlights the unintended consequences of wider cannabis access in New Jersey. Notably, cannabis use increased even before its legalization, presumably in response to the COVID‐19 pandemic and its attendant mental health effects. Rates of cannabis use disorder and its highlight of other concurrent psychiatric disorders are important topics for both clinicians and lawmakers to consider

Inhibition by stabilization: Targeting the Plasmodium falciparum aldolase-TRAP complex

Background: Emerging resistance of the malaria parasite Plasmodium to current therapies underscores the critical importance of exploring novel strategies for disease eradication. Plasmodium species are obligate intracellular protozoan parasites. They rely on an unusual form of substrate-dependent motility for their migration on and across host-cell membranes and for host cell invasion. This peculiar motility mechanism is driven by the 'glideosome', an actin-myosin associated, macromolecular complex anchored to the inner membrane complex of the parasite. Myosin A, actin, aldolase, and thrombospondin-related anonymous protein (TRAP) constitute the molecular core of the glideosome in the sporozoite, the mosquito stage that brings the infection into mammals. Methods: Virtual library screening of a large compound library against the PfAldolase-TRAP complex was used to identify candidate compounds that stabilize and prevent the disassembly of the glideosome. The mechanism of these compounds was confirmed by biochemical, biophysical and parasitological methods. Results: A novel inhibitory effect on the parasite was achieved by stabilizing a protein-protein interaction within the glideosome components. Compound 24 disrupts the gliding and invasive capabilities of Plasmodium parasites in in vitro parasite assays. A high-resolution, ternary X-ray crystal structure of PfAldolase-TRAP in complex with compound 24 confirms the mode of interaction and serves as a platform for future ligand optimization. Conclusion: This proof-of-concept study presents a novel approach to anti-malarial drug discovery and design. By strengthening a protein-protein interaction within the parasite, an avenue towards inhibiting a previously "undruggable" target is revealed and the motility motor responsible for successful invasion of host cells is rendered inactive. This study provides new insights into the malaria parasite cell invasion machinery and convincingly demonstrates that liver cell invasion is dramatically reduced by 95 % in the presence of the small molecule stabilizer compound 24.Fil: Nemetski, Sondra Maureen. New York University School of Medicine; Estados Unidos. New York-Presbyterian Hospital-Weill Cornell Medical College; Estados UnidosFil: Cardozo, Timothy J.. New York University School of Medicine; Estados UnidosFil: Bosch, Gundula. Johns Hopkins University Bloomberg School of Public Health; Estados Unidos. Johns Hopkins Malaria Research Institute ; Estados UnidosFil: Weltzer, Ryan. Johns Hopkins University Bloomberg School of Public Health; Estados Unidos. Johns Hopkins Malaria Research Institute ; Estados UnidosFil: O'Malley, Kevin. Johns Hopkins University Bloomberg School of Public Health; Estados Unidos. Johns Hopkins Malaria Research Institute ; Estados UnidosFil: Ejigiri, Ijeoma. New York University School of Medicine; Estados UnidosFil: Kumar, Kota Arun. New York University School of Medicine; Estados Unidos. University of Hyderabad; Estados UnidosFil: Buscaglia, Carlos Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Nussenzweig, Victor. New York University School of Medicine; Estados UnidosFil: Sinnis, Photini. Johns Hopkins University Bloomberg School of Public Health; Estados Unidos. New York University School of Medicine; Estados Unidos. Johns Hopkins Malaria Research Institute; Estados UnidosFil: Levitskaya, Jelena. Johns Hopkins University Bloomberg School of Public Health; Estados Unidos. Johns Hopkins Malaria Research Institute; Estados UnidosFil: Bosch, Jürgen. Johns Hopkins University Bloomberg School of Public Health; Estados Unidos. Johns Hopkins Malaria Research Institute; Estados Unido

Regulation of the Unfolded Protein Response by eIF2Bδ Isoforms*

Cells respond to a variety of stresses, including unfolded proteins in the endoplasmic reticulum (ER), by phosphorylating a subunit of translation initiation factor eIF2, eIF2α. eIF2α phosphorylation inactivates the eIF2B complex. The inactivation of eIF2B not only suppresses the initiation of protein translation but paradoxically up-regulates the translation and expression of transcription factor ATF-4. Both of these processes are important for the cellular response to ER stress, also termed the unfolded protein response. Here we demonstrate that cellular response resulting from eIF2α phosphorylation is attenuated in several cancer cell lines. The deficiency of the unfolded protein response in these cells correlates with the expression of a specific isoform of a regulatory eIF2B subunit, eIF2Bδ variant 1 (V1). Replacement of total eIF2Bδ with V1 renders cells insensitive to eIF2α phosphorylation; specifically, they neither up-regulate ATF-4 and ATF-4 targets nor suppress protein translation. Expression of variant 2 eIF2Bδ in ER stress response-deficient cells restores the stress response. Our data suggest that V1 does not interact with the eIF2 complex, a requisite for eIF2B inhibition by eIF2α phosphorylation. Together, these data delineate a novel physiological mechanism to regulate the ER stress response with a large potential impact on a variety of diseases that result in ER stress

Inhibition of Nonsense-Mediated RNA Decay by the Tumor Microenvironment Promotes Tumorigenesis ▿ †

While nonsense-mediated RNA decay (NMD) is an established mechanism to rapidly degrade select transcripts, the physiological regulation and biological significance of NMD are not well characterized. We previously demonstrated that NMD is inhibited in hypoxic cells. Here we show that the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) translation initiation factor by a variety of cellular stresses leads to the inhibition of NMD and that eIF2α phosphorylation and NMD inhibition occur in tumors. To explore the significance of this NMD regulation, we used an unbiased approach to identify approximately 750 NMD-targeted mRNAs and found that these mRNAs are overrepresented in stress response and tumor-promoting pathways. Consistent with these findings, the inhibition of NMD promotes cellular resistance to endoplasmic reticulum stress and encourages tumor formation. The transcriptional and translational regulations of gene expression by the microenvironment are established mechanisms by which tumor cells adapt to stress. These data indicate that NMD inhibition by the tumor microenvironment is also an important mechanism to dynamically regulate genes critical for the response to cellular stress and tumorigenesis