Loss of p73 Expression Contributes to Chronic Obstructive Pulmonary Disease.

Rationale: Multiciliated cell (MCC) loss and/or dysfunction is common in the small airways of patients with chronic obstructive pulmonary disease (COPD), but it is unclear if this contributes to COPD lung pathology. Objectives: To determine if loss of p73 causes a COPD-like phenotype in mice and explore whether smoking or COPD impact p73 expression. Methods: p73floxE7-E9 mice were crossed with Shh-Cre mice to generate mice lacking MCCs in the airway epithelium. The resulting p73Δairway mice were analyzed using electron microscopy, flow cytometry, morphometry, forced oscillation technique, and single-cell RNA sequencing. Furthermore, the effects of cigarette smoke on p73 transcript and protein expression were examined using in vitro and in vivo models and in studies including airway epithelium from smokers and patients with COPD. Measurements and Main Results: Loss of functional p73 in the respiratory epithelium resulted in a near-complete absence of MCCs in p73Δairway mice. In adulthood, these mice spontaneously developed neutrophilic inflammation and emphysema-like lung remodeling and had progressive loss of secretory cells. Exposure of normal airway epithelium cells to cigarette smoke rapidly and durably suppressed p73 expression in vitro and in vivo. Furthermore, tumor protein 73 mRNA expression was reduced in the airways of current smokers (n = 82) compared with former smokers (n = 69), and p73-expressing MCCs were reduced in the small airways of patients with COPD (n = 11) compared with control subjects without COPD (n = 12). Conclusions: Loss of functional p73 in murine airway epithelium results in the absence of MCCs and promotes COPD-like lung pathology. In smokers and patients with COPD, loss of p73 may contribute to MCC loss or dysfunction.

Metabolic trade-offs in Neonatal sepsis triggered by TLR4 and TLR1/2 ligands result in unique dysfunctions in neural breathing circuits.

Neonatal sepsis remains one of the leading causes of mortality in newborns. Several brainstem-regulated physiological processes undergo disruption during neonatal sepsis. Mechanistic knowledge gaps exist at the interplay between metabolism and immune activation to brainstem neural circuits and pertinent physiological functions in neonates. To delineate this association, we induced systemic inflammation either by TLR4 (LPS) or TLR1/2 (PAM3CSK4) ligand administration in postnatal day 5 mice (PD5). Our findings show that LPS and PAM3CSK4 evoke substantial changes in respiration and metabolism. Physiological trade-offs led to hypometabolic-hypothermic responses due to LPS, but not PAM3CSK4, whereas to both TLR ligands blunted respiratory chemoreflexes. Neuroinflammatory pathways modulation in brainstem showed more robust effects in LPS than PAM3CSK4. Brainstem neurons, microglia, and astrocyte gene expression analyses showed unique responses to TLR ligands. PAM3CSK4 did not significantly modulate gene expression changes in GLAST-1 positive brainstem astrocytes. PD5 pups receiving PAM3CSK4 failed to maintain a prolonged metabolic state repression, which correlated to enhanced gasping latency and impaired autoresuscitation during anoxic chemoreflex challenges. In contrast, LPS administered pups showed no significant changes in anoxic chemoreflex. Electrophysiological studies from brainstem slices prepared from pups exposed to either TLR4 or PAM3CSK4 showed compromised transmission between preBötzinger complex and Hypoglossal as an exclusive response to the TLR1/2 ligand. Spatial gene expression analysis demonstrated a region-specific modulation of PAM3CSK4 within the raphe nucleus relative to other anatomical sites evaluated. Our findings suggest that metabolic changes due to inflammation might be a crucial tolerance mechanism for neonatal sepsis preserving neural control of breathing.

Proteoglycan synthesis in conserved oligomeric Golgi subunit deficient HEK293T cells is affected differently, depending on the lacking subunit.

The Conserved Oligomeric Golgi (COG) complex is an eight subunit protein complex associated with Golgi membranes. Genetic defects affecting individual COG subunits cause congenital disorders of glycosylation (CDGs), due to mislocalization of Golgi proteins involved in glycosylation mechanisms. While the resulting defects in N-and O-glycosylation have been extensively studied, no corresponding study of proteoglycan (PG) synthesis has been undertaken. We here show that glycosaminoglycan (GAG) modification of PGs is significantly reduced, regardless which COG subunit that is missing in HEK293T cells. Least reduction was observed for cells lacking COG1 and COG8 subunits, that bridge the A and B lobes of the complex. Lack of these subunits did not reduce GAG chain lengths of secreted PGs, which was reduced in cells lacking any other subunit (COG2-7). COG3 knock out (KO) cells had particularly reduced ability to polymerize GAG chains. For cell-associated GAGs, the mutant cell lines, except COG4 and COG7 KO, displayed longer GAG chains than wild-type cells, indicating that COG subunits play a role in cellular turnover of PGs. In light of the important roles PGs play in animal development, the effects KO of individual COG subunits have on GAG synthesis could explain the variable severity of COG associated CDGs.

An update in club cell biology and its potential relevance to chronic obstructive pulmonary disease.

Club cells are found in human small airways where they play an important role in immune defense, xenobiotic metabolism, and repair after injury. Over the past few years, data from single-cell RNA sequencing (scRNA-seq) studies has generated new insights into club cell heterogeneity and function. In this review, we integrate findings from scRNA-seq experiments with earlier in vitro, in vivo, and microscopy studies and highlight the many ways club cells contribute to airway homeostasis. We then discuss evidence for loss of club cells or club cell products in the airways of patients with chronic obstructive pulmonary disease (COPD) and discuss potential mechanisms through which this might occur.

Triarylphosphine-Coordinated Bipyridyl Ru(II) Complexes Induce Mitochondrial Dysfunction.

While cancer cells rely heavily upon glycolysis to meet their energetic needs, reducing the importance of mitochondrial oxidative respiration processes, more recent studies have shown that their mitochondria still play an active role in the bioenergetics of metastases. This feature, in combination with the regulatory role of mitochondria in cell death, has made this organelle an attractive anticancer target. Here, we report the synthesis and biological characterization of triarylphosphine-containing bipyridyl ruthenium (Ru(II)) compounds and found distinct differences as a function of the substituents on the bipyridine and phosphine ligands. 4,4'-Dimethylbipyridyl-substituted compound 3 exhibited especially high depolarizing capabilities, and this depolarization was selective for the mitochondrial membrane and occurred within minutes of treatment in cancer cells. The Ru(II) complex 3 exhibited an 8-fold increase in depolarized mitochondrial membranes, as determined by flow cytometry, which compares favorably to the 2-fold increase observed by carbonyl cyanide chlorophenylhydrazone (CCCP), a proton ionophore that shuttles protons across membranes, depositing them into the mitochondrial matrix. Fluorination of the triphenylphosphine ligand provided a scaffold that maintained potency against a range of cancer cells but avoided inducing toxicity in zebrafish embryos at higher concentrations, displaying the potential of these Ru(II) compounds for anticancer applications. This study provides essential information regarding the role of ancillary ligands for the anticancer activity of Ru(II) coordination compounds that induce mitochondrial dysfunction.

Around the world in electrochemistry: a review of the electrochemistry curriculum in high schools.

Electrochemistry education of future researchers and citizens is crucial if we are to decarbonise economies and reach targets for net zero. In this paper, we take an overview of electrochemistry within school education. We used curriculum documents obtained from national and state education department websites and from local teachers, examples of assessments and insights from the chemistry education literature to evaluate the extent of electrochemistry education around the world. We found that there is a great deal of electrochemistry included in the intended curriculum for high schools although there is variability depending on how early students are able to specialise in a smaller number of subjects. A range of contexts are used to illustrate the key ideas including galvanic and electrolytic cells, electrolysis and analysis. There is generally constructive alignment between assessment items and the intended curriculum although in some cases assessment was more simplistic than the intended curriculum would suggest. The effectiveness of the taught curriculum is undermined by low teacher confidence in teaching electrochemistry especially more advanced concepts. Additionally, there are a number of misconceptions generated when students learn electrochemistry with some of these potentially arising from published resources such as textbooks. Supplementary Information: The online version contains supplementary material available at 10.1007/s10008-023-05548-0.

Zebrafish Cancer Avatars: A Translational Platform for Analyzing Tumor Heterogeneity and Predicting Patient Outcomes.

The increasing number of available anti-cancer drugs presents a challenge for oncologists, who must choose the most effective treatment for the patient. Precision cancer medicine relies on matching a drug with a tumor's molecular profile to optimize the therapeutic benefit. However, current precision medicine approaches do not fully account for intra-tumoral heterogeneity. Different mutation profiles and cell behaviors within a single heterogeneous tumor can significantly impact therapy response and patient outcomes. Patient-derived avatar models recapitulate a patient's tumor in an animal or dish and provide the means to functionally assess heterogeneity's impact on drug response. Mouse xenograft and organoid avatars are well-established, but the time required to generate these models is not practical for clinical decision-making. Zebrafish are emerging as a time-efficient and cost-effective cancer avatar model. In this review, we highlight recent developments in zebrafish cancer avatar models and discuss the unique features of zebrafish that make them ideal for the interrogation of cancer heterogeneity and as part of precision cancer medicine pipelines.

Secretory Cells Are the Primary Source of pIgR in Small Airways.

Loss of secretory IgA (SIgA) is common in chronic obstructive pulmonary disease (COPD) small airways and likely contributes to disease progression. We hypothesized that loss of SIgA results from reduced expression of pIgR (polymeric immunoglobulin receptor), a chaperone protein needed for SIgA transcytosis, in the COPD small airway epithelium. pIgR-expressing cells were defined and quantified at single-cell resolution in human airways using RNA in situ hybridization, immunostaining, and single-cell RNA sequencing. Complementary studies in mice used immunostaining, primary murine tracheal epithelial cell culture, and transgenic mice with secretory or ciliated cell-specific knockout of pIgR. SIgA degradation by human neutrophil elastase or secreted bacterial proteases from nontypeable Haemophilus influenzae was evaluated in vitro. We found that secretory cells are the predominant cell type responsible for pIgR expression in human and murine airways. Loss of SIgA in small airways was not associated with a reduction in secretory cells but rather a reduction in pIgR protein expression despite intact PIGR mRNA expression. Neutrophil elastase and nontypeable H. influenzae-secreted proteases are both capable of degrading SIgA in vitro and may also contribute to a deficient SIgA immunobarrier in COPD. Loss of the SIgA immunobarrier in small airways of patients with severe COPD is complex and likely results from both pIgR-dependent defects in IgA transcytosis and SIgA degradation.

Long-read sequencing of the zebrafish genome reorganizes genomic architecture.

BACKGROUND: Nanopore sequencing technology has revolutionized the field of genome biology with its ability to generate extra-long reads that can resolve regions of the genome that were previously inaccessible to short-read sequencing platforms. Over 50% of the zebrafish genome consists of difficult to map, highly repetitive, low complexity elements that pose inherent problems for short-read sequencers and assemblers. RESULTS: We used long-read nanopore sequencing to generate a de novo assembly of the zebrafish genome and compared our assembly to the current reference genome, GRCz11. The new assembly identified 1697 novel insertions and deletions over one kilobase in length and placed 106 previously unlocalized scaffolds. We also discovered additional sites of retrotransposon integration previously unreported in GRCz11 and observed the expression of these transposable elements in adult zebrafish under physiologic conditions, implying they have active mobility in the zebrafish genome and contribute to the ever-changing genomic landscape. CONCLUSIONS: We used nanopore sequencing to improve upon and resolve the issues plaguing the current zebrafish reference assembly, GRCz11. Zebrafish is a prominent model of human disease, and our corrected assembly will be useful for studies relying on interspecies comparisons and precise linkage of genetic events to disease phenotypes.

Cold-induced dishabituation in rodents exposed to recurrent hypoglycaemia.

AIMS/HYPOTHESIS: Recurrent hypoglycaemia in people with diabetes leads to progressive suppression of counterregulatory hormonal responses to subsequent hypoglycaemia. Recently it has been proposed that the mechanism underpinning this is a form of adaptive memory referred to as habituation. To test this hypothesis, we use two different durations of cold exposure to examine whether rodents exposed to recurrent hypoglycaemia exhibit two characteristic features of habituation, namely stimulus generalisation and dishabituation. METHODS: In the first study (stimulus generalisation study), hyperinsulinaemic-hypoglycaemic (2.8 mmol/l) glucose clamps were performed in non-diabetic rodents exposed to prior moderate-duration cold (4°C for 3 h) or control conditions. In the second study (dishabituation study), rodents exposed to prior recurrent hypoglycaemia or saline (154 mmol/l NaCl) injections over 4 weeks underwent a longer-duration cold (4°C for 4.5 h) exposure followed 24 h later by a hyperinsulinaemic-hypoglycaemic (2.8 mmol/l) glucose clamp. Output measures were counterregulatory hormone responses during experimental hypoglycaemia. RESULTS: Moderate-duration cold exposure blunted the adrenaline (epinephrine) response (15,266 ± 1920 vs 7981 ± 1258 pmol/l, Control vs Cold; p < 0.05) to next day hypoglycaemia in healthy non-diabetic rodents. In contrast, the suppressed adrenaline response (Control 5912 ± 1417 vs recurrent hypoglycaemia 1836 ± 736 pmol/l; p < 0.05) that is associated with recurrent hypoglycaemia was restored following longer-duration cold exposure (recurrent hypoglycaemia + Cold 4756 ± 826 pmol/l; not significant vs Control). CONCLUSIONS/INTERPRETATION: Non-diabetic rodents exhibit two cardinal features of habituation, namely stimulus generalisation and dishabituation. These findings provide further support for the hypothesis that suppressed counterregulatory responses following exposure to recurrent hypoglycaemia in diabetes result from habituation.

Biological Investigations of Ru(II) Complexes With Diverse ß-diketone Ligands.

The ß-diketone scaffold is a commonly used synthetic intermediate, and is a functional group found in natural products such as curcuminoids. This core structure can also act as a chelating ligand for a variety of metals. In order to assess the potential of this scaffold for medicinal inorganic chemistry, seven different κ2-O,O'-chelating ligands were used to construct Ru(II) complexes with polypyridyl co-ligands, and their biological activity was evaluated. The complexes demonstrated promising structure-dependent cytotoxicity. Three complexes maintained high activity in a tumor spheroid model, and all complexes demonstrated low in vivo toxicity in a zebrafish model. From this series, the best compound exhibited a ~ 30-fold window between cytotoxicity in a 3-D tumor spheroid model and potential in vivo toxicity. These results suggest that κ2-O,O'-ligands can be incorporated into Ru(II)-polypyridyl complexes to create favorable candidates for future drug development.

More than just sugars: Conserved oligomeric Golgi complex deficiency causes glycosylation-independent cellular defects.

The conserved oligomeric Golgi (COG) complex controls membrane trafficking and ensures Golgi homeostasis by orchestrating retrograde vesicle trafficking within the Golgi. Human COG defects lead to severe multisystemic diseases known as COG-congenital disorders of glycosylation (COG-CDG). To gain better understanding of COG-CDGs, we compared COG knockout cells with cells deficient to 2 key enzymes, Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase and uridine diphosphate-glucose 4-epimerase (GALE), which contribute to proper N- and O-glycosylation. While all knockout cells share similar defects in glycosylation, these defects only account for a small fraction of observed COG knockout phenotypes. Glycosylation deficiencies were not associated with the fragmented Golgi, abnormal endolysosomes, defective sorting and secretion or delayed retrograde trafficking, indicating that these phenotypes are probably not due to hypoglycosylation, but to other specific interactions or roles of the COG complex. Importantly, these COG deficiency specific phenotypes were also apparent in COG7-CDG patient fibroblasts, proving the human disease relevance of our CRISPR knockout findings. The knowledge gained from this study has important implications, both for understanding the physiological role of COG complex in Golgi homeostasis in eukaryotic cells, and for better understanding human diseases associated with COG/Golgi impairment.

Optimized cell transplantation using adult rag2 mutant zebrafish.

Cell transplantation into adult zebrafish has lagged behind mouse models owing to the lack of immunocompromised strains. Here we have created rag2(E450fs) mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft muscle, blood stem cells and various cancers. rag2(E450fs) mutant zebrafish are the first immunocompromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer.

Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA).

Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe context-dependent assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA-generated ZFNs, we rapidly altered 20 genes in Danio rerio, Arabidopsis thaliana and Glycine max. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multigene pathways or genome-wide alterations.

Examining Phosphatases Through Immunofluorescent Microscopy.

Immunofluorescent microscopy enables the examination of cellular expression and localization of proteins. Cellular localization can often impact protein function, as certain molecular interactions occur in specific cellular compartments. Here we describe in detail the processes necessary for identifying phosphatases in the cell through immunofluorescent microscopy. Identification of phosphatase expression and localization could lead to the discovery of protein function in disease states along with potential substrates and binding partners.

Identification of Quiescent Cells in a Zebrafish T-Cell Acute Lymphoblastic Leukemia Model Using Cell Proliferation Staining.

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect CSCs from antiproliferative chemotherapy drugs. In T-cell acute lymphoblastic leukemia (T-ALL) patient-derived xenograft (PDX) mouse models, quiescent cells are associated with treatment resistance and stemness. Cell proliferation dyes are popular tools for the tracking of cell division. The fluorescent dye is covalently anchored into amine groups on the membrane and macromolecules inside the cell. This allows for the tracking of labeled cells for up to 10 divisions, which can be resolved by flow cytometry. Ultimately, cells with the highest proliferation rates will have low dye retention, as it will be diluted with each cell division, while dormant, slower-dividing cells will have the highest retention. The use of cell proliferation dyes to isolate dormant cells has been optimized and described in T-ALL mouse models. Complementary to the existing mouse models, the rag2:Myc-derived zebrafish T-ALL model provides an excellent venue to interrogate self-renewal in T-ALL due to the high frequency of leukemic stem cells (LSCs) and the convenience of zebrafish for large-scale transplant experiments. Here, we describe the workflow for the staining of zebrafish T-ALL cells with a cell proliferation dye, optimizing the concentration of the dye for zebrafish cells, passaging successfully stained cells in vivo, and the collection of cells with varying levels of dye retention by live cell sorting from transplanted animals. Given the absence of well-established cell surface makers for LSCs in T-ALL, this approach provides a functional means to interrogate quiescent cells in vivo. For representative results, we describe the engraftment efficiency and the LSC frequency of high and low dye-retaining cells. This method can help investigate additional properties of quiescent cells, including drug response, transcriptional profiles, and morphology.

A dynamic model of inorganic arsenic-induced carcinogenesis reveals an epigenetic mechanism for epithelial-mesenchymal plasticity.

Inorganic arsenic (iAs) causes cancer by initiating dynamic transitions between epithelial and mesenchymal cell phenotypes. These transitions transform normal cells into cancerous cells, and cancerous cells into metastatic cells. Most in vitro models assume that transitions between states are binary and complete, and do not consider the possibility that intermediate, stable cellular states might exist. In this paper, we describe a new, two-hit in vitro model of iAs-induced carcinogenesis that extends to 28 weeks of iAs exposure. Through week 17, the model faithfully recapitulates known and expected phenotypic, genetic, and epigenetic characteristics of iAs-induced carcinogenesis. By 28 weeks, however, exposed cells exhibit stable, intermediate phenotypes and epigenetic properties, and key transcription factor promoters (SNAI1, ZEB1) enter an epigenetically poised or bivalent state. These data suggest that key epigenetic transitions and cellular states exist during iAs-induced epithelial-to-mesenchymal transition (EMT), and that it is important for our in vitro models to encapsulate all aspects of EMT and the mesenchymal-to-epithelial transition (MET). In so doing, and by understanding the epigenetic systems controlling these transitions, we might find new, unexpected opportunities for developing targeted, cell state-specific therapeutics.

Zebrafish drug screening identifies Erlotinib as an inhibitor of Wnt/ß-catenin signaling and self-renewal in T-cell acute lymphoblastic leukemia.

The Wnt/ß-catenin pathway's significance in cancer initiation, progression, and stem cell biology underscores its therapeutic potential. However, the clinical application of Wnt inhibitors remains limited due to challenges posed by off-target effects and complex cross-talk of Wnt signaling with other pathways. In this study, we leveraged a zebrafish model to perform a robust and rapid drug screening of 773 FDA-approved compounds to identify Wnt/ß-catenin inhibitors with minimal toxicity. Utilizing zebrafish expressing a Wnt reporter, we identified several drugs that suppressed Wnt signaling without compromising zebrafish development. The efficacy of the top hit, Erlotinib, extended to human cells, where it blocked Wnt/ß-catenin signaling downstream of the destruction complex. Notably, Erlotinib treatment reduced self-renewal in human T-cell Acute Lymphoblastic Leukemia cells, which rely on active ß-catenin signaling for maintenance of leukemia-initiating cells. Erlotinib also reduced leukemia-initiating cell frequency and delayed disease formation in zebrafish models. This study underscores zebrafish's translational potential in drug discovery and repurposing and highlights a new use for Erlotinib as a Wnt inhibitor for cancers driven by aberrant Wnt/ß-catenin signaling.

Phosphatase and Pseudo-Phosphatase Functions of Phosphatase of Regenerating Liver 3 (PRL-3) Are Insensitive to Divalent Metals In Vitro.

Phosphatase of regenerating liver 3 (PRL-3) is associated with cancer metastasis and has been shown to interact with the cyclin and CBS domain divalent metal cation transport mediator (CNNM) family of proteins to regulate the intracellular concentration of magnesium and other divalent metals. Despite PRL-3's importance in cancer, factors that regulate PRL-3's phosphatase activity and its interactions with CNNM proteins remain unknown. Here, we show that divalent metal ions, including magnesium, calcium, and manganese, have no impact on PRL-3's structure, stability, phosphatase activity, or CNNM binding capacity, indicating that PRL-3 does not act as a metal sensor, despite its interaction with CNNM metal transporters. In vitro approaches found that PRL-3 is a broad but not indiscriminate phosphatase, with activity toward di- and tri-nucleotides, phosphoinositols, and NADPH but not other common metabolites. Although calcium, magnesium, manganese, and zinc-binding sites were predicted near the PRL-3 active site, these divalent metals did not specifically alter PRL-3's phosphatase activity toward a generic substrate, its transition from an inactive phospho-cysteine intermediate state, or its direct binding with the CBS domain of CNNM. PRL-3's insensitivity to metal cations negates the possibility of its role as an intracellular metal content sensor for regulating CNNM activity. Further investigation is warranted to define the regulatory mechanisms governing PRL-3's phosphatase activity and CNNM interactions, as these findings could hold potential therapeutic implications in cancer treatment.