Per/Polyfluoroalkyl Substances (PFASs) in a Marine Apex Predator (White Shark, Carcharodon carcharias) in the Northwest Atlantic Ocean.

Per/polyfluoroalkyl substances (PFASs) are ubiquitous, highly persistent anthropogenic chemicals that bioaccumulate and biomagnify in aquatic food webs and are associated with adverse health effects, including liver and kidney diseases, cancers, and immunosuppression. We investigated the accumulation of PFASs in a marine apex predator, the white shark (Carcharodon carcharias). Muscle (N = 12) and blood plasma (N = 27) samples were collected from 27 sharks during 2018-2021 OCEARCH expeditions along the eastern coast of North America from Nova Scotia to Florida. Samples were analyzed for 47 (plasma) and 43 (muscle) targeted PFASs and screened for >2600 known and novel PFASs using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). Perfluoroalkyl carboxylates with carbon chain-length C11 to C14 were frequently detected above the method reporting limits in plasma samples, along with perfluorooctanesulfonate and perfluorodecanesulfonate. Perfluoropentadecanoate was also detected in 100% of plasma samples and concentrations were estimated semiquantitatively as no analytical standard was available. Total concentrations of frequently detected PFASs in plasma ranged from 0.56 to 2.9 ng mL-1 (median of 1.4 ng mL-1). In muscle tissue, nine targeted PFASs were frequently detected, with total concentration ranging from 0.20 to 0.84 ng g-1 ww. For all frequently detected PFASs, concentrations were greater in plasma than in muscle collected from the same organism. In both matrices, perfluorotridecanoic acid was the most abundant PFAS, consistent with several other studies. PFASs with similar chain-lengths correlated significantly among the plasma samples, suggesting similar sources. Total concentrations of PFASs in plasma were significantly greater in sharks sampled off of Nova Scotia than all sharks from other locations, potentially due to differences in diet. HRMS suspect screening tentatively identified 13 additional PFASs in plasma, though identification confidence was low, as no MS/MS fragmentation was collected due to low intensities. The widespread detection of long-chain PFASs in plasma and muscle of white sharks highlights the prevalence and potential biomagnification of these compounds in marine apex predators.

TRIM33 loss in multiple myeloma is associated with genomic instability and sensitivity to PARP inhibitors.

Deletions of chromosome 1p (del(1p)) are a recurrent genomic aberration associated with poor outcome in Multiple myeloma (MM.) TRIM33, an E3 ligase and transcriptional co-repressor, is located within a commonly deleted region at 1p13.2. TRIM33 is reported to play a role in the regulation of mitosis and PARP-dependent DNA damage response (DDR), both of which are important for maintenance of genome stability. Here, we demonstrate that MM patients with loss of TRIM33 exhibit increased chromosomal instability and poor outcome. Through knockdown studies, we show that TRIM33 loss induces a DDR defect, leading to accumulation of DNA double strand breaks (DSBs) and slower DNA repair kinetics, along with reduced efficiency of non-homologous end joining (NHEJ). Furthermore, TRIM33 loss results in dysregulated ubiquitination of ALC1, an important regulator of response to PARP inhibition. We show that TRIM33 knockdown sensitizes MM cells to the PARP inhibitor Olaparib, and this is synergistic with the standard of care therapy bortezomib, even in co-culture with bone marrow stromal cells (BMSCs). These findings suggest that TRIM33 loss contributes to the pathogenesis of high-risk MM and that this may be therapeutically exploited through the use of PARP inhibitors.

Whole-body galactose oxidation as a robust functional assay to assess the efficacy of gene-based therapies in a mouse model of Galactosemia.

Despite the implementation of lifesaving newborn screening programs and a galactose-restricted diet, many patients with classic galactosemia develop long-term debilitating neurological deficits and primary ovarian insufficiency. Previously, we showed that the administration of human GALT mRNA predominantly expressed in the GalT gene-trapped mouse liver augmented the expression of hepatic GALT activity, which decreased not only galactose-1 phosphate (gal-1P) in the liver but also peripheral tissues. Since each peripheral tissue requires distinct methods to examine the biomarker and/or GALT effect, this highlights the necessity for alternative strategies to evaluate the overall impact of therapies. In this study, we established that whole-body galactose oxidation (WBGO) as a robust, noninvasive, and specific method to assess the in vivo pharmacokinetic and pharmacodynamic parameters of two experimental gene-based therapies that aimed to restore GALT activity in a mouse model of galactosemia. Although our results illustrated the long-lasting efficacy of AAVrh10-mediated GALT gene transfer, we found that GALT mRNA therapy that targets the liver predominantly is sufficient to sustain WBGO. The latter could have important implications in the design of novel targeted therapy to ensure optimal efficacy and safety.

"This Has Changed Everything": The Impact of the COVID-19 Pandemic on School Nursing Practice in New Mexico.

As state and local government implemented school closures in response to the COVID-19 pandemic, school health services delivery experienced a fundamental change. School nurses were confronted with significant challenges to care for their students, while responding to the avalanche of public health responsibilities thrust upon them without training or resources. This qualitative study, conducted by a community-based participatory research partnership explored school nurses' experiences and perspectives in urban and rural communities across New Mexico. Thirty-four school nurses participated in semi-structured qualitative interviews identifying 3 distinct pandemic stages and the following themes: 1) change/confusion of school nurse identity; 2) mental health challenges and stressors; and 3) lessons learned. These study results provide contextual depth to challenges that both urban and rural school nurses in New Mexico experienced during the pandemic and outline the important role school nurses have during public health emergencies in school settings.

Associations between total mercury, trace minerals, and blood health markers in Northwest Atlantic white sharks (Carcharodon carcharias).

The ecology and life-histories of white sharks make this species susceptible to mercury bioaccumulation; however, the health consequences of mercury exposure are understudied. We measured muscle and plasma total mercury (THg), health markers, and trace minerals in Northwest Atlantic white sharks. THg in muscle tissue averaged 10.0 mg/kg dry weight, while THg in blood plasma averaged 533 µg/L. THg levels in plasma and muscle were positively correlated with shark precaudal length (153-419 cm), and THg was bioaccumulated proportionally in muscle and plasma. Nine sharks had selenium:mercury molar ratios in blood plasma >1.0, indicating that for certain individuals the potential protective effects of the trace mineral were diminished, whereas excess selenium may have protected other individuals. No relationships between plasma THg and any trace minerals or health markers were identified. Thus, we found no evidence of negative effects of Hg bioaccumulation, even in sharks with very high THg.

DNA metabarcoding of cloacal swabs provides insight into diets of highly migratory sharks in the Mid-Atlantic Bight.

The abundances of migratory shark species observed throughout the Mid-Atlantic Bight (MAB) during productive summer months suggest that this region provides critical habitat and prey resources to these taxa. However, the principal prey assemblages sustaining migratory shark biomass in this region are poorly defined. We applied high-throughput DNA metabarcoding to shark feces derived from cloacal swabs across nine species of Carcharhinid and Lamnid sharks to (1) quantify the contribution of broad taxa (e.g., invertebrates, fishes) supporting shark biomass during seasonal residency in the MAB and (2) determine whether the species displayed distinct dietary preference indicative of resource partitioning. DNA metabarcoding resulted in high taxonomic (species-level) resolution of shark diets with actinopterygian and elasmobranch fishes as the dominant prey categories across the species. DNA metabarcoding identified several key prey groups consistent across shark taxa that are likely integral for sustaining their biomass in this region, including Atlantic menhaden (Brevoortia tyrannus), Atlantic mackerel (Scomber scombrus), and benthic elasmobranchs, including skates. Our results are consistent with previously published stomach content data for the shark species of similar size range in the Northwest Atlantic Ocean, supporting the efficacy of cloacal swab DNA metabarcoding as a minimally invasive diet reconstruction technique. The high reliance of several shark species on Atlantic menhaden could imply wasp-waist food-web conditions during the summer months, whereby high abundances of forage fishes sustain a diverse suite of migratory sharks within a complex, seasonal food web.

Metabolic Alterations in Multiple Myeloma: From Oncogenesis to Proteasome Inhibitor Resistance.

Despite significant improvements in treatment strategies over the past couple of decades, multiple myeloma (MM) remains an incurable disease due to the development of drug resistance. Metabolic reprogramming is a key feature of cancer cells, including MM, and acts to fuel increased proliferation, create a permissive tumour microenvironment, and promote drug resistance. This review presents an overview of the key metabolic adaptations that occur in MM pathogenesis and in the development of resistance to proteasome inhibitors, the backbone of current MM therapy, and considers the potential for therapeutic targeting of key metabolic pathways to improve outcomes.

The Ubiquitin Proteasome System in Genome Stability and Cancer.

Faithful DNA replication during cellular division is essential to maintain genome stability and cells have developed a sophisticated network of regulatory systems to ensure its integrity. Disruption of these control mechanisms can lead to loss of genomic stability, a key hallmark of cancer. Ubiquitination is one of the most abundant regulatory post-translational modifications and plays a pivotal role in controlling replication progression, repair of DNA and genome stability. Dysregulation of the ubiquitin proteasome system (UPS) can contribute to the initiation and progression of neoplastic transformation. In this review we provide an overview of the UPS and summarize its involvement in replication and replicative stress, along with DNA damage repair. Finally, we discuss how the UPS presents as an emerging source for novel therapeutic interventions aimed at targeting genomic instability, which could be utilized in the treatment and management of cancer.

TIF1 Proteins in Genome Stability and Cancer.

Genomic instability is a hallmark of cancer cells which results in excessive DNA damage. To counteract this, cells have evolved a tightly regulated DNA damage response (DDR) to rapidly sense DNA damage and promote its repair whilst halting cell cycle progression. The DDR functions predominantly within the context of chromatin and requires the action of chromatin-binding proteins to coordinate the appropriate response. TRIM24, TRIM28, TRIM33 and TRIM66 make up the transcriptional intermediary factor 1 (TIF1) family of chromatin-binding proteins, a subfamily of the large tripartite motif (TRIM) family of E3 ligases. All four TIF1 proteins are aberrantly expressed across numerous cancer types, and increasing evidence suggests that TIF1 family members can function to maintain genome stability by mediating chromatin-based responses to DNA damage. This review provides an overview of the TIF1 family in cancer, focusing on their roles in DNA repair, chromatin regulation and cell cycle regulation.

The E3 ligase HUWE1 inhibition as a therapeutic strategy to target MYC in multiple myeloma.

Proteasome inhibitors have provided a significant advance in the treatment of multiple myeloma (MM). Consequently, there is increasing interest in developing strategies to target E3 ligases, de-ubiquitinases, and/or ubiquitin receptors within the ubiquitin proteasome pathway, with an aim to achieve more specificity and reduced side-effects. Previous studies have shown a role for the E3 ligase HUWE1 in modulating c-MYC, an oncogene frequently dysregulated in MM. Here we investigated HUWE1 in MM. We identified elevated expression of HUWE1 in MM compared with normal cells. Small molecule-mediated inhibition of HUWE1 resulted in growth arrest of MM cell lines without significantly effecting the growth of normal bone marrow cells, suggesting a favorable therapeutic index. Studies using a HUWE1 knockdown model showed similar growth inhibition. HUWE1 expression positively correlated with MYC expression in MM bone marrow cells and correspondingly, genetic knockdown and biochemical inhibition of HUWE1 reduced MYC expression in MM cell lines. Proteomic identification of HUWE1 substrates revealed a strong association of HUWE1 with metabolic processes in MM cells. Intracellular glutamine levels are decreased in the absence of HUWE1 and may contribute to MYC degradation. Finally, HUWE1 depletion in combination with lenalidomide resulted in synergistic anti-MM activity in both in vitro and in vivo models. Taken together, our data demonstrate an important role of HUWE1 in MM cell growth and provides preclinical rationale for therapeutic strategies targeting HUWE1 in MM.

Diverse compounds from pleuromutilin lead to a thioredoxin inhibitor and inducer of ferroptosis.

The chemical diversification of natural products provides a robust and general method for the creation of stereochemically rich and structurally diverse small molecules. The resulting compounds have physicochemical traits different from those in most screening collections, and as such are an excellent source for biological discovery. Herein, we subject the diterpene natural product pleuromutilin to reaction sequences focused on creating ring system diversity in few synthetic steps. This effort resulted in a collection of compounds with previously unreported ring systems, providing a novel set of structurally diverse and highly complex compounds suitable for screening in a variety of different settings. Biological evaluation identified the novel compound ferroptocide, a small molecule that rapidly and robustly induces ferroptotic death of cancer cells. Target identification efforts and CRISPR knockout studies reveal that ferroptocide is an inhibitor of thioredoxin, a key component of the antioxidant system in the cell. Ferroptocide positively modulates the immune system in a murine model of breast cancer and will be a useful tool to study the utility of pro-ferroptotic agents for treatment of cancer.

Predictors of Discharge Against Medical Advice in a Tertiary Paediatric Hospital.

BACKGROUND: Patients who discharge against medical advice (DAMA) from hospital carry a significant risk of readmission and have increased rates of morbidity and mortality. We sought to identify the demographic and clinical characteristics of DAMA patients from a tertiary paediatric hospital. METHODS: Data were extracted retrospectively from electronic medical records for all inpatient admissions over a 5-year period. Demographic characteristics (age, sex, Aboriginality, socioeconomic status and remoteness of residence) and clinical characteristics (admitting hospital site, level of urgency on admission, diagnosis and previous DAMA) were extracted and logistic regression models were used to identify predictors of DAMA with 95% confidence intervals. RESULTS: There were 246,359 admissions for 124,757 patients, of which 1871 (0.8%) admissions and 1730 patients (1.4%) DAMA. Predictors of DAMA in a given admission were hospital site (OR 4.8, CI 4.2-5.7, p < 0.01), a mental health/behavioural diagnosis (OR 3.3, CI 2.2-4.8, p < 0.01), Aboriginality (OR 1.6, CI 1.3-2.1, p < 0.01), emergency rather than elective admissions (OR 0.7ha, CI 0.6-0.8, p < 0.01), a gastrointestinal diagnosis (OR 1.5, CI 1.1-2.0, p = 0.04) and a history of previous DAMA (OR 2.0, CI 1.2-3.2, p = 0.05). CONCLUSIONS: There are clear predictors of DAMA in this tertiary hospital admission cohort and identification of these provides opportunities for intervention at a practice and policy level in order to prevent adverse outcomes.

TRAF6 Silencing Attenuates Multiple Myeloma Cell Adhesion to Bone Marrow Stromal Cells.

The bone marrow (BM) microenvironment plays an important role in supporting proliferation, survival and drug resistance of Multiple Myeloma (MM) cells. MM cells adhere to bone marrow stromal cells leading to the activation of tumour-promoting signaling pathways. Activation of the NFκB pathway, in particular, is central to the pathogenesis of MM. Tumour necrosis factor receptor-associated factor 6 (TRAF6) is a key mediator of NFκB activation and has previously been highlighted as a potential therapeutic target in MM. Here, we demonstrate that adherence of MM cell lines to stromal cells results in a reciprocal increase in TRAF6 expression. Knockdown of TRAF6 expression attenuates the ability of MM cells to bind to stromal cells and this is associated with a decrease in NFκB-induced expression of the adhesion molecules ICAM1 and VCAM1. Finally, we show that knockdown of TRAF6 sensitizes MM cells to treatment with bortezomib when co-cultured with stromal cells. Inhibiting TRAF6 represents a promising strategy to target MM cells in the BM microenvironment.

Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP).

Serine hydrolases play diverse roles in regulating host-pathogen interactions in a number of organisms, yet few have been characterized in the human pathogen Staphylococcus aureus. Here we describe a chemical proteomic screen that identified ten previously uncharacterized S. aureus serine hydrolases that mostly lack human homologs. We termed these enzymes fluorophosphonate-binding hydrolases (FphA-J). One hydrolase, FphB, can process short fatty acid esters, exhibits increased activity in response to host cell factors, is located predominantly on the bacterial cell surface in a subset of cells, and is concentrated in the division septum. Genetic disruption of fphB confirmed that the enzyme is dispensable for bacterial growth in culture but crucial for establishing infection in distinct sites in vivo. A selective small molecule inhibitor of FphB effectively reduced infectivity in vivo, suggesting that it may be a viable therapeutic target for the treatment or management of Staphylococcus infections.

TRIM proteins in blood cancers.

Post-translational modification of proteins with ubiquitin plays a central role in regulating numerous cellular processes. E3 ligases determine the specificity of ubiquitination by mediating the transfer of ubiquitin to substrate proteins. The family of tripartite motif (TRIM) proteins make up one of the largest subfamilies of E3 ligases. Accumulating evidence suggests that dysregulation of TRIM proteins is associated with a variety of diseases. In this review we focus on the involvement of TRIM proteins in blood cancers.

KEAP1-modifying small molecule reveals muted NRF2 signaling responses in neural stem cells from Huntington's disease patients.

The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.

An orthogonalized platform for genetic code expansion in both bacteria and eukaryotes.

In this study, we demonstrate the feasibility of expanding the genetic code of Escherichia coli using its own tryptophanyl-tRNA synthetase and tRNA (TrpRS-tRNATrp) pair. This was made possible by first functionally replacing this endogenous pair with an E. coli-optimized counterpart from Saccharomyces cerevisiae, and then reintroducing the liberated E. coli TrpRS-tRNATrp pair into the resulting strain as a nonsense suppressor, which was then followed by its directed evolution to genetically encode several new unnatural amino acids (UAAs). These engineered TrpRS-tRNATrp variants were also able to drive efficient UAA mutagenesis in mammalian cells. Since bacteria-derived aminoacyl-tRNA synthetase (aaRS)-tRNA pairs are typically orthogonal in eukaryotes, our work provides a general strategy to develop additional aaRS-tRNA pairs that can be used for UAA mutagenesis of proteins expressed in both E. coli and eukaryotes.

Separation and Enrichment of Hematopoietic Stem Cells for CCN Studies.

The regulation of blood cell production (hematopoiesis) by CCN proteins is an area of increasing interest to hematologists. There is some discordance in the literature in this area due to the use of mixed or ill-defined cell populations for experiments. Expression of, and response to, CCN proteins is specific to both cell type and differentiation status. Here, we describe methods to prepare defined hematopoietic cell populations and associated functional assays.

Identification of the APC/C co-factor FZR1 as a novel therapeutic target for multiple myeloma.

Multiple Myeloma (MM) is a haematological neoplasm characterised by the clonal proliferation of malignant plasma cells in the bone marrow. The success of proteasome inhibitors in the treatment of MM has highlighted the importance of the ubiquitin proteasome system (UPS) in the pathogenesis of this disease. In this study, we analysed gene expression of UPS components to identify novel therapeutic targets within this pathway in MM. Here we demonstrate how this approach identified previously validated and novel therapeutic targets. In addition we show that FZR1 (Fzr), a cofactor of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C), represents a novel therapeutic target in myeloma. The APC/C associates independently with two cofactors, Fzr and Cdc20, to control cell cycle progression. We found high levels of FZR1 in MM primary cells and cell lines and demonstrate that expression is further increased on adhesion to bone marrow stromal cells (BMSCs). Specific knockdown of either FZR1 or CDC20 reduced viability and induced growth arrest of MM cell lines, and resulted in accumulation of APC/CFzr substrate Topoisomerase IIα (TOPIIα) or APC/CCdc20 substrate Cyclin B. Similar effects were observed following treatment with proTAME, an inhibitor of both APC/CFzr and APC/CCdc20. Combinations of proTAME with topoisomerase inhibitors, etoposide and doxorubicin, significantly increased cell death in MM cell lines and primary cells, particularly if TOPIIα levels were first increased through pre-treatment with proTAME. Similarly, combinations of proTAME with the microtubule inhibitor vincristine resulted in enhanced cell death. This study demonstrates the potential of targeting the APC/C and its cofactors as a therapeutic approach in MM.

The role of the CCN family of proteins in blood cancers.

Haematopoiesis is the term used to describe the production of blood cells. This is a tightly regulated hierarchical system in which mature circulating blood cells develop from a small population of haematopoietic stem (HSC) and progenitor cells within the microenvironment of the bone marrow. Molecular and genetic abnormalities arising in these stem cells lead to a block in the normal programme of proliferation and differentiation and result in the development of the blood cancers known as the leukaemias and lymphomas. Recently the regulatory role of the bone marrow microenvironment or niche has also become increasingly recognised. The interface between the bone and bone marrow (endosteum) and the region surrounding the blood vessels (perivascular) provide distinct niches harbouring quiescent HSC or proliferative HSC respectively. Current chemotherapeutic regimes can often successfully target the proliferative HSC but disease relapse occurs due to residual quiescent HSC. Understanding these developmental and regulatory processes and the associated cell communication mechanisms are thus crucial to the development of new treatment strategies. The CCN family of proteins have been recognised to play a key role in all aspects of haematopoiesis.