Plasma and tissue transferrin and ferritin, and gene expression of ferritin, transferrin, and transferrin receptors I and II in channel catfish Ictalurus punctatus fed diets with different concentrations of inorganic or organic iron.

Ferritin, transferrin, and transferrin receptors I and II play a vital role in iron metabolism, health, and indication of iron deficiency anaemia in fish. To evaluate the use of high-iron diets to prevent or reverse channel catfish (Ictalurus punctatus) anaemia of unknown causes, we investigated the expression of these iron-regulatory genes and proteins in channel catfish fed plant-based diets. Catfish fingerlings were fed five diets supplemented with 0 (basal), 125, and 250 mg/kg of either inorganic iron or organic iron for 2 weeks. Ferritin, transferrin, and transferrin receptor I and II mRNA and protein expression levels in fish tissues (liver, intestine, trunk kidney, and head kidney) and plasma were determined. Transferrin (iron transporter) and TfR (I and II) genes were generally highly expressed in fish fed the basal diet compared to those fed the iron-supplemented diets. In contrast, ferritin (iron storage) genes were more expressed in the trunk kidney of fish fed the iron-supplemented diets than in those fed the basal diet. Our results demonstrate that supplementing channel catfish plant-based diets with iron from either organic or inorganic iron sources affected the expression of the iron-regulatory genes and increased body iron status in the fish.

Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry.

Human CLCA2 regulates store-operated calcium entry (SOCE) by interacting with Orai1 and STIM1. It is expressed as a 943aa type I transmembrane protein that is cleaved at amino acid 708 to produce a diffusible 100 kDa product. The N-terminal ectodomain contains a hydrolase-like subdomain with a conserved HEXXH zinc-binding motif that is proposed to cleave the precursor autoproteolytically. Here, we tested this hypothesis and its link to SOCE. We first studied the conditions for autocleavage in isolated membranes and then in a purified protein system. Cleavage was zinc-dependent and abolished by mutation of the E in the HEXXH motif to Q, E165Q. Cleavage efficiency increased with CLCA2 concentration, implying that it occurs in trans. Accordingly, the E165Q mutant was cleaved by co-transfected wildtype CLCA2. Moreover, CLCA2 precursors with different epitope tags co-immunoprecipitated. In a membrane-free system utilizing immunopurified protease and target, no cleavage occurred unless the target was first denatured, implying that membranes provide essential structural or conformational cues. Unexpectedly, cleavage caused a conformational shift: an N-terminal antibody that immunoprecipitated the precursor failed to precipitate the N-terminal product unless the product was first denatured with an ionic detergent. The E165Q mutation abolished the stimulation of SOCE caused by wildtype CLCA2, establishing that the metalloprotease activity is required for this regulatory function.

Effects of microplastics and nanoplastics in shrimp: Mechanisms of plastic particle and contaminant distribution and subsequent effects after uptake.

To date, previous studies have reported the adverse effects of microplastics (MPs) and nanoplastics (NPs) on both freshwater and marine organisms. However, the information on MPs' and NPs' effects on shrimp species is scarce. In addition, the factors influencing the distribution of these particles in aquatic systems have been explained, yet the mechanisms behind MPs and NPs distribution and consumption, specifically to crustaceans and shrimp, have not been elucidated in detail. The effects of MPs and NPs as well as plastic-carried contaminants and pathogens on shrimp are critical to shrimp production and subsequent human consumption. Recent findings are required to review and discuss to open up new avenues for emerging Shrimp and crustacean research for sustainability. This review summarizes the distribution and fate of MPs and NPs along with contaminants and pathogens and identifies potential risks to shrimp health. The transport of MPs and NPs is influenced by their plastic properties, hydrodynamics, and water properties. Additionally, the fate of these particles on a plastic surface (plastisphere) is regulated by contaminant properties. Pathogens thriving on plastic surfaces and contaminants adsorbed can reach aquatic organisms directly with plastic particles or indirectly after release to an aquatic environment. MPs and NPs can be absorbed by shrimp through their gills and mouth and accumulate in their internal organs. Innate immunity influenced the degree of survival rate, tissue damage, alteration of gut microbiota, and increased oxidative stress caused by MPs and NPs accumulation. The studies on the effects of MPs and NPs are still not sufficient to understand how these particles are absorbed from various parts of the shrimp body and the fate of these plastics inside the body.

Elevated concentrations of organic and inorganic forms of iron in plant-based diets for channel catfish prevent anemia but damage liver and intestine, respectively, without impacting growth performance.

We compared the effects of using inorganic and organic forms of iron in plant-based diets on catfish performance in a feeding trial with 6-g catfish fingerlings. The objective was to determine whether dietary iron in excess of known requirements negatively affected the fish. Five diets supplemented with 0 (basal), 125, 250 mg Fe/kg of either FeSO4 or iron methionine were formulated. Weight gain, feed conversion ratio, hepatosomatic index, and survival were similar among diets. Plasma and intestine iron concentration was similar among diets. Whole-body total lipid, protein, and dry matter were similar among diets, while ash content was higher in fish fed the basal diet. Total liver iron concentration was higher in fish fed diets supplemented with 250 mg Fe/kg in both iron forms than other diets. Hematological parameters were similar among treatments. Liver necrosis, inflammation, and vacuolization were highest in fish fed the diet supplemented with 250 mg Fe/kg from organic iron, followed by those fed diets with 250 mg Fe/kg from inorganic iron. Inorganic iron-supplemented diets caused more intestinal inflammation (increased inflammatory cells, villi swelling, thicker lamina propria) than the organic iron-supplemented diets or basal diet. Organic iron at 250 mg/kg resulted in a $0.143/kg increase in feed cost. Latent iron deficiency and initial signs of anemia developed in catfish fed the basal diet. Supplemental iron from either form prevented iron deficiency. Organic iron at 125 mg/kg optimized fish performance at a cost comparable to that of fish fed other diets, but without overt negative effects.

Advances in Intracellular Calcium Signaling Reveal Untapped Targets for Cancer Therapy.

Intracellular Ca2+ distribution is a tightly regulated process. Numerous Ca2+ chelating, storage, and transport mechanisms are required to maintain normal cellular physiology. Ca2+-binding proteins, mainly calmodulin and calbindins, sequester free intracellular Ca2+ ions and apportion or transport them to signaling hubs needing the cations. Ca2+ channels, ATP-driven pumps, and exchangers assist the binding proteins in transferring the ions to and from appropriate cellular compartments. Some, such as the endoplasmic reticulum, mitochondria, and lysosomes, act as Ca2+ repositories. Cellular Ca2+ homeostasis is inefficient without the active contribution of these organelles. Moreover, certain key cellular processes also rely on inter-organellar Ca2+ signaling. This review attempts to encapsulate the structure, function, and regulation of major intracellular Ca2+ buffers, sensors, channels, and signaling molecules before highlighting how cancer cells manipulate them to survive and thrive. The spotlight is then shifted to the slow pace of translating such research findings into anticancer therapeutics. We use the PubMed database to highlight current clinical studies that target intracellular Ca2+ signaling. Drug repurposing and improving the delivery of small molecule therapeutics are further discussed as promising strategies for speeding therapeutic development in this area.

CLCA2 is a positive regulator of store-operated calcium entry and TMEM16A.

The Chloride Channel Accessory (CLCA) protein family was first characterized as regulators of calcium-activated chloride channel (CaCC) currents (ICaCC), but the mechanism has not been fully established. We hypothesized that CLCAs might regulate ICaCC by modulating intracellular calcium levels. In cells stably expressing human CLCA2 or vector, we found by calcium imaging that CLCA2 moderately enhanced intracellular-store release but dramatically increased store-operated entry of calcium upon cytosolic depletion. Moreover, another family member, CLCA1, produced similar effects on intracellular calcium mobilization. Co-immunoprecipitation revealed that CLCA2 interacted with the plasma membrane store-operated calcium channel ORAI-1 and the ER calcium sensor STIM-1. The effect of CLCA2 on ICaCC was tested in HEK293 stably expressing calcium-activated chloride channel TMEM16A. Co-expression of CLCA2 nearly doubled ICaCC in response to a calcium ionophore. These results unveil a new mechanism by which CLCA family members activate ICaCC and suggest a broader role in calcium-dependent processes.

CLCA2 Interactor EVA1 Is Required for Mammary Epithelial Cell Differentiation.

CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1) and confirmed it by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 and p63. It is thought to mediate homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Moreover, knockdown of EVA1 in immortalized human mammary epithelial cells (HMEC) caused EMT, implying that EVA1 is essential for epithelial differentiation. Both EVA1 and CLCA2 co-localized with E-cadherin at cell-cell junctions. The interacting domains were delimited by deletion analysis, revealing the site of interaction to be the transmembrane segment (TMS). The primary sequence of the CLCA2 TMS was found to be conserved in CLCA2 orthologs throughout mammals, suggesting that its interaction with EVA1 co-evolved with the mammary gland. A screen for other junctional interactors revealed that CLCA2 was involved in two different complexes, one with EVA1 and ZO-1, the other with beta catenin. Overexpression of CLCA2 caused downregulation of beta catenin and beta catenin-activated genes. Thus, CLCA2 links a junctional adhesion molecule to cytosolic signaling proteins that modulate proliferation and differentiation. These results may explain how attenuation of CLCA2 causes EMT and why CLCA2 and EVA1 are frequently downregulated in metastatic breast cancer cell lines.

The role of cancer stem cells in relapse of solid tumors.

Recurrence at secondary locations, often years after removal of the primary tumor, accounts for most of the mortality associated with solid tumors. Metastasis, resistance to chemo- and radiotherapy, and eventual relapse have been attributed to a distinct tumor subpopulation known as cancer stem cells (CSCs). In this review, we consider the properties of CSCs that lead to these outcomes, in particular the relation between epithelial-to-mesenchymal transition, stemness, and tumor initiation. We compare recent clinical and laboratory studies of breast cancer, glioblastoma, and melanoma that illustrate how most current anticancer regimens select for cells with mesenchymal and CSC properties and therefore sow the seeds of relapse. Finally, we discuss the emerging paradigm of combined therapy that targets both CSC and non-CSC tumor components.