Concentration In Cells Research Articles

BMP-7 Upregulates Id2 Through the MAPK Signaling Pathway to Improve Diabetic Tubulointerstitial Fibrosis and the Intervention of Oxymatrine.

Diabetic kidney disease is one of the most serious microvascular complications of diabetes. It progresses irreversibly to end-stage renal disease if left untreated. Bone morphogenetic protein (BMP)-7 is a negative regulator of organ fibrosis and may also play an essential role in tubulointerstitial fibrosis. This study aimed to investigate the precise role and potential molecular mechanisms of BMP-7 in the progression of diabetic nephropathy. In this study, BMP-7 was overexpressed in vivo after the replication of the diabetic rat model using streptozotocin. The results showed that BMP-7 inhibited the phosphorylation of related mitogen-activated protein kinase (MAPK) pathways while upregulating the inhibitor of differentiation (Id2) expression and effectively ameliorated pathological renal injury. Further in vitro validation showed that the inhibition of the phosphorylation of MAPKs at a high glucose concentration in renal tubular epithelial cells was followed by the upregulation of Id2 protein expression, suggesting that BMP-7 could improve diabetic nephropathy by upregulating Id2 protein levels through the BMP-7–MAPK signaling pathway. Previous laboratory studies found that oxymatrine improved renal fibrotic lesions. However, the exact mechanism is unclear. The present study showed that oxymatrine treatment in a diabetic rat model upregulated BMP-7 protein expression and inhibited MAPK pathway protein phosphorylation levels. These results suggested that oxymatrine improved the epithelial-to-mesenchymal transition process in the early stage of diabetic kidney disease by regulating the BMP-7–MAPK pathway and ameliorated renal tubulointerstitial fibrosis.

Identification and expression analysis of receptors that mediate MIP regulating larval settlement in Urechis unicinctus.

Larval attachment and metamorphosis are important processes during the development of some marine invertebrates. Myoinhibitory peptides (MIPs), a class of small molecular neuropeptides, have been revealed to be involved in regulating the larval settlement. In this paper, we identified two types of MIP membrane receptors, G-protein coupled receptor SPR and MIP-gated ion channel receptors MGIC1 and MGIC2 based on sequence homology with other species in the transcriptome database of Echiuroidea Urechis unicinctus (Xenopneusta, Urechidae). The results of in situ hybridization showed that positive signals of these receptors were obviously located in the apex of the segmentation larvae, a critical stage of U. unicinctus larval settlement. Further, these receptors were determined on the membrane of HEK293 cells by immunohistochemistry. Also, we verified that U. unicinctus MIP can activate its SPR receptor based on the results of the significantly decreased cAMP concentration in HEK293 cells. Our data will provide scientific reference for elucidating mechanism of neuropeptide regulating the larval attachment and metamorphosis in marine invertebrates.

New oxovanadium(IV) complexes overcame drug resistance and increased in vitro cytotoxicity by an apoptotic pathway in breast cancer cells

In order to examine the anticancer potential of oxovanadium(IV) complexes with thiosemicarbazone, two new complexes were prepared starting from 2-thenoyltrifluoroacetone-S-methylthiosemicarbazone. The complexes with tetradentate thiosemicarbazone ligand were characterized by elemental analysis, IR, ESI MS, and single-crystal X-ray diffraction analysis. Cytotoxicity on breast cancer cells, MDA-MB-231 and MCF-7, was determined by MTT assay. Cisplatin was positive control and the results were compared with those of the normal cells, HUVEC and 3T3. The complexes exhibited greater activity on cancer cells than cisplatin, but they were cytotoxic at several times higher concentrations in the healthy cells. In our study, the presence of thiophene and fluoro groups in the oxovanadium(IV) complexes with thiosemicarbazone increased greatly the cytotoxic activity of the complexes on breast cancer cells. Moreover, the complexes induced apoptosis-mediated cell death and also reduced the expression of MDR-1 or P-glycoprotein and ABCG2. As a result, the findings indicated that the complexes have selective cytotoxicity on breast cancer cells and can overcome multidrug resistance. These properties of the complexes make it possible to be a potential anticancer drug candidate for breast cancer treatment.

Significance of Specific Oxidoreductases in the Design of Hypoxia-Activated Prodrugs and Fluorescent Turn off-on Probes for Hypoxia Imaging.

Simple SummaryHypoxia-activated prodrugs (HAPs), selectively reduced by specific oxidoreductases under hypoxic conditions, form cytotoxic agents damaging the local cancer cells. On the basis of the reported clinical data concerning several HAPs, one can draw conclusions regarding their preclinical attractiveness and, regrettably, the low efficacy of Phase III clinical trials. Clinical failure may be explained, inter alia, by the lack of screening of patients on the basis of tumor hypoxia and low availability of specific oxidoreductases involved in HAP activation. There is surprisingly little information on the quantification of these enzymes in cells or tissues, compared to the advanced research associated with the use of HAPs. Our knowledge about the expression and activity of these enzymes in various cancer cell lines under hypoxic conditions is inadequate. Only in a few cases were researchers able to demonstrate the differences in the expression or activity of selected oxidoreductases, depending on the oxygen concentration. Additionally, it was cell line dependent. More systematic studies are required. The optical probes, based on turning on the fluorescence emission upon irreversible reduction catalyzed by the overexpressed oxidoreductases, can be helpful in this type of research. Ultimately, such sensors can estimate both the oxidoreductase activity and the degree of oxygenation in one step. To achieve this goal, their response must be correlated with the expression or activity of enzymes potentially involved in turning on their emissions, as determined by biochemical methods. In conclusion, the incorporation of biomarkers to identify hypoxia is a prerequisite for successful HAP therapies. However, it is equally important to assess the level of specific oxidoreductases required for their activation.Hypoxia is one of the hallmarks of the tumor microenvironment and can be used in the design of targeted therapies. Cellular adaptation to hypoxic stress is regulated by hypoxia-inducible factor 1 (HIF-1). Hypoxia is responsible for the modification of cellular metabolism that can result in the development of more aggressive tumor phenotypes. Reduced oxygen concentration in hypoxic tumor cells leads to an increase in oxidoreductase activity that, in turn, leads to the activation of hypoxia-activated prodrugs (HAPs). The same conditions can convert a non-fluorescent compound into a fluorescent one (fluorescent turn off–on probes), and such probes can be designed to specifically image hypoxic cancer cells. This review focuses on the current knowledge about the expression and activity of oxidoreductases, which are relevant in the activation of HAPs and fluorescent imaging probes. The current clinical status of HAPs, their limitations, and ways to improve their efficacy are briefly discussed. The fluorescence probes triggered by reduction with specific oxidoreductase are briefly presented, with particular emphasis placed on those for which the correlation between the signal and enzyme expression determined with biochemical methods is achievable.

Functionalized Mesoporous Silica as Doxorubicin Carriers and Cytotoxicity Boosters.

Mesoporous silica nanoparticles (MSNs) bearing methyl, thiol or glucose groups were synthesized, and their encapsulation and release behaviors for the anticancer drug Doxorubicin (Dox) were investigated in comparison with nonporous homologous materials. The chemical modification of thiol-functional silica with a double bond glucoside was completed for the first time, by green thiol-ene photoaddition. The MSNs were characterized in terms of structure (FT-IR, Raman), morphology (TEM), porosity (nitrogen sorption–desorption) and Zeta potential measurements. The physical interactions responsible for the Dox encapsulation were investigated by analytic methods and MD simulations, and were correlated with the high loading efficiency of MSNs with thiol and glucose groups. High release at pH 5 was observed in most cases, with thiol-MSN exhibiting 98.25% cumulative release in sustained profile. At pH 7.4, the glucose-MSN showed 75.4% cumulative release, while the methyl-MSN exhibited a sustained release trend. The in vitro cytotoxicity was evaluated on NDHF, MeWo and HeLa cell lines by CellTiter-Glo assay, revealing strong cytotoxic effects in all of the loaded silica at low equivalent Dox concentration and selectivity for cancer cells. Atypical applications of each MSN as intravaginal, topical or oral Dox administration route could be proposed.

Visualizing the effects of salt concentration in planar polymer light-emitting electrochemical cells

The effects of salt concentration have been visualized in bilayer, planar polymer light-emitting electrochemical cells (PLECs) with a gap size of 2 mm. Electrochemical doping and junction electroluminescence (EL) have been observed in a luminescent conjugated polymer (CP) top layer with a different solid polymer electrolyte (SPE) underlayer. By varying the salt concentration and thickness of the SPE layer, the initial cell current, peak cell current, doping propagation speed and junction formation time can all be altered/controlled. This is explained by a limiting ionic resistance in the SPE layer which is both salt concentration and thickness dependent. This study establishes the role of electrolyte ions to support doping current in PLECs. The unexpected observation of a systematic junction shift towards the cathode suggests the electrolyte ions, when functioning as counter ions for doping, are also affected by the salt concentration. At high salt concentrations, the light-emitting junction overlaps with the cathode edges which is undesirable. Thus, the bilayer PLECs allowed for the study and visualization of functions of mobile ions in PLEC operation. Moreover, the bilayer PLECs can potentially be used to study the SPE layer itself using highly sensitive imaging techniques.

Linalool Activates Oxidative and Calcium Burst and CAM3-ACA8 Participates in Calcium Recovery in Arabidopsis Leaves.

Plants produce linalool to respond to biotic stress, but the linalool-induced early signal remains unclear. In wild-type Arabidopsis, plant resistance to diamondback moth (Plutella xylostella) increased more strongly in a linalool-treated group than in an untreated control group. H2O2 and Ca2+, two important early signals that participated in biotic stress, burst after being treated with linalool in Arabidopsis mesophyll cells. Linalool treatment increased H2O2 and intracellular calcium concentrations in mesophyll cells, observed using a confocal microscope with laser scanning, and H2O2 signaling functions upstream of Ca2+ signaling by using inhibitors and mutants. Ca2+ efflux was detected using non-invasive micro-test technology (NMT), and Ca2+ efflux was also inhibited by NADPH oxidase inhibitor DPI (diphenyleneiodonium chloride) and in cells of the NADPH oxidase mutant rbohd. To restore intracellular calcium levels, Ca2+-ATPase was activated, and calmodulin 3 (CAM3) participated in Ca2+-ATPase activation. This result is consistent with the interaction between CAM7 and Ca2+-ATPase isoform 8 (ACA8). In addition, a yeast two-hybrid assay, firefly luciferase complementation imaging assay, and an in vitro pulldown assay showed that CAM3 interacts with the N-terminus of ACA8, and qRT-PCR showed that some JA-related genes and defense genes expressions were enhanced when treated with linalool in Arabidopsis leaves. This study reveals that linalool enhances H2O2 and intracellular calcium concentrations in Arabidopsis mesophyll cells; CAM3-ACA8 reduces intracellular calcium concentrations, allowing cells to resume their resting state. Additionally, JA-related genes and defense genes’ expression may enhance plants’ defense when treated with linalool.

Effects of long-acting muscarinic antagonists on promoting ciliary function in airway epithelium

BackgroundMucociliary clearance (MCC) is an essential defense mechanism in airway epithelia for removing pathogens from the respiratory tract. Impaired ciliary functions and MCC have been demonstrated in asthma and chronic obstructive pulmonary disease (COPD). Long-acting muscarinic antagonists (LAMAs) are a major class of inhaled bronchodilators, which are used for treating asthma and COPD; however, the effects of LAMAs on ciliary function remain unclear. This study aimed to identify the effects of LAMAs on airway ciliary functions.MethodsWild-type BALB/c mice were treated with daily intranasal administrations of glycopyrronium for 7 days, and tracheal samples were collected. Cilia-driven flow and ciliary activity, including ciliary beat frequency (CBF), ciliary beating amplitude, effective stroke velocity, recovery stroke velocity and the ratio of effective stroke velocity to recovery stroke velocity, were analyzed by imaging techniques. Using in vitro murine models, tracheal tissues were transiently cultured in media with/without LAMAs, glycopyrronium or tiotropium, for 60 min. Cilia-driven flow and ciliary activity were then analyzed. Well-differentiated normal human bronchial epithelial (NHBE) cells were treated with glycopyrronium, tiotropium, or vehicle for 60 min, and CBF was evaluated. Several mechanistic analyses were performed.ResultsIntranasal glycopyrronium administration for 7 days significantly increased cilia-driven flow and ciliary activity in murine airway epithelium. In the murine tracheal organ culture models, treatment with glycopyrronium or tiotropium for 60 min significantly increased cilia-driven flow and ciliary activity in airway epithelium. Further, we confirmed that 60-min treatment with glycopyrronium or tiotropium directly increased CBF in well-differentiated NHBE cells. In the mechanistic analyses, neither treatment with glycopyrronium nor tiotropium affected intracellular calcium ion concentrations in well-differentiated NHBE cells. Glycopyrronium did not increase protein kinase A activity in well-differentiated NHBE cells. Moreover, glycopyrronium had no effect on extracellular adenosine triphosphate concentration.ConclusionsLAMAs exert a direct effect on airway epithelium to enhance ciliary function, which may improve impaired MCC in asthma and COPD. Further investigations are warranted to elucidate the underlying mechanisms of the effects of LAMAs on the promotion of airway ciliary function.

Novel Chemical Probes targeting the Polyamine Regulatory Circuit

Polyamines are vital for cellular life. Polyamines such as putrescine, spermidine, and spermine regulate the essential cellular process, including gene expression and cell proliferation. Given its cellular role, polyamine concentration in cells is tightly controlled. However, aberrant polyamine metabolism is associated with numerous diseases, including cancer. Growing evidence suggests that elevated polyamine levels are the absolute requirement for tumor growth and progression. The concentrations of cellular polyamines are controlled by the polyamine regulatory circuit comprising three different proteins: Ornithine Decarboxylase (ODC), Ornithine Decarboxylase Antizyme (OAZ), and Antizyme Inhibitor (AZIN). While ODC is directly involved in polyamine biosynthesis, OAZ and AZIN regulate the ODC activity via protein‐protein interactions. The dysregulation of ODC, OAZ, and AZIN leads to elevated polyamines in numerous pathologies, making them attractive targets to control polyamine levels. Besides regulating polyamine synthesis, OAZ is believed to modulate polyamine transport via multiple pathways. However, the precise mechanism of OAZ‐mediated polyamine regulation outside of the polyamine biosynthetic pathway remains elusive. We hypothesize that OAZ sequestration results in increased polyamine uptake. To provide evidence to our hypothesis, we generated covalent OAZ‐mimetic inactivators targeting both ODC and AZIN. Using biochemical experiments, we demonstrate that these OAZ‐mimetics inhibit the activity of ODC and prevent the sequestration of OAZ by AZIN. Using Mass spectrometric experiments, we determine the covalent binding of OAZ‐mimetics to both ODC and AZIN. We employ a novel gel assay to assess the rate of covalent modification of ODC and AZIN. Finally, using computational modeling, we determine the mode of binding of OAZ‐mimetics to both ODC and AZIN to provide insights into inhibition mechanisms.

Perspective on fluorescence cell imaging with ionophore-based ion-selective nano-optodes.

Inorganic ions are ubiquitous in all kinds of cells with highly dynamic spatial and temporal distribution. Taking advantage of different types of fluorescent probes, fluorescence microscopic imaging and quantitative analysis of ion concentrations in cells have rapidly advanced. A family of fluorescent nanoprobes based on ionophores has emerged in recent years with the potential to establish a unique platform for the analysis of common biological ions including Na+, K+, Ca2+, Cl-, and so on. This article aims at providing a retrospect and outlook of ionophore-based ion-selective nanoprobes and the applications in cell imaging.

Effect of Mild Acidosis on CA2+ Signaling in Rat Carotid Body Chemoreceptor Cells: Role of CAV, ASIC, and Task

Carotid body (CB) glomus cells serve as peripheral chemoreceptors that detect changes in arterial O2 pressure (pO2) and pH, and adjust the level of ventilation to help maintain a normal level of blood pO2 and pH. Severe levels of hypoxia and acidosis are well known to cause sustained rise in cytosolic Ca2+ concentration ([Ca2+]c) in CB glomus cells. We showed recently that isolated gloms cells exhibit spontaneous [Ca2+]c oscillations and that mild levels of hypoxia increase the frequency and amplitude of Ca2+oscillations to augment the secretory response. To understand how mild levels of acidosis regulate Ca2+signaling in glomus cells, we studied the effects of small changes in extracellular pH (pHo) on the kinetics of Ca2+oscillations and the roles of Cav, ASIC and TASK. A decrease in pHo from 7.4 to 7.3 (mild acidosis) and 7.2 (moderate acidosis) produced significant increases in the frequency and amplitude of Ca2+ oscillations without affecting the resting basal [Ca2+]c. These effects on Ca2+ oscillations were not blocked by 0.1 mM amiloride (an ASIC inhibitor) that blocked Na‐H exchange in glomus cells. Mild acidosis was associated with a small but significant inhibition of TASK current and cell depolarization. The increase in Ca2+oscillations produced by mild acidosis was strongly inhibited by nifedipine (1 µM; L‐type Cav inhibitor), as well as by TTA‐P2 (20 µM; T‐type Cav inhibitor). Mild acidosis (0.1 pH unit decrease) and mild hypoxia (~5% O2) produced similar levels of change in the kinetics of Ca2+oscillations and produced an additive effect on Ca2+oscillations. Our study shows that mild to moderate levels of acidosis inhibit TASK to increase Ca2+ influx via both L‐ and T‐type Cav, resulting in increased frequency and amplitude of Ca2+ oscillations. Our study also indicate that ASICs are not involved in the stimulatory effects of mild acidosis on Ca2+oscillations.

Toxic photochemistry and signaling repercussions of blue light in cells exposed to retinal

Age‐related visual damage by blue light and its adverse effects on quality of life for many people >55 yrs have been well documented. Eliminating blue wavelengths has been shown to reduce age‐related visual impairment and facilitate vision preservation in the elderly. The blue region of the visible spectrum is abundant in sunlight and common electronic light emitters. Many adults receive increased ultraviolet and blue light exposure upon retirement by moving closer to the tropics and spending more time outdoors. The cornea and lens of the eye are largely opaque to ultraviolet, while blue light readily permeates into the retina. We propose that all‐trans‐retinal (ATR), the light‐sensitive chromophore released from photoexcited photopigments in the retina, could induce age‐related vision impairment. Our data demonstrate that exposing cells incubated with ATR and exposed to blue light or sunlight permanently distorted the chemical properties of the crucial signaling lipid PIP2 and disrupted its subcellular location and signaling. Similar conditions permanently displaced G proteins in the phototransduction cascade and Ras family G proteins from the plasma membrane to the cell interior. Chemical evidence shows the oxidation of their farnesyl lipid anchor during this perturbation. Upon ATR and blue light, the percent positivity for TUNEL stain, indicating DNA damage, is significantly higher in primary cells isolated from mouse retina. We also show α‐Tocopherol, a lipid‐soluble antioxidant, inhibits the above cellular photodamage. Therefore, we now ask the critical question: could resident ATR concentrations in photoreceptor cells cause retina neurodegeneration upon physiologically relevant light exposures?

The effects of dietary zinc supplement on intracellular zinc concentration in bovine immune cells

Abstract Zinc (Zn) is one of the most abundant metals in our body and is critical for innate and adaptive immune functions. In humans and mice, Zn deficiency may lead to immunodeficiency, reduced reproductive health, and increased disease susceptibility. However, trace mineral requirements and evidence of Zn dysregulation are poorly defined in cattle. This study aimed to evaluate intracellular Zn concentrations in immune cells from young growing steers with different Zn dietary supplements. Eighteen Angus crossbred steers were stratified by body weight and randomly assigned to two dietary treatments groups: 1) control, no supplemental Zn, or 2) high Zn; 150 ppm of Zn delivered in premixes in total mixed rations. To evaluate the intracellular Zn concentration of immune cells, whole blood cells were loaded with FluoZinTM-3, a fluorescent cell-permeant Zn indicator, then stimulated with Zn pyrithione (100 μM) or TPEN [N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine] (50 μM) to measure maximum and minimum mean fluorescent intensity. Cells were stained with surface antibodies to identify immune cell populations and data were collected by flow cytometry. The intracellular Zn concentrations of circulating CD8 T cells and γδ T cells, but not other populations, differed significantly between treatment groups. These results indicate that intracellular Zn concentration can be influenced by dietary Zn intake, and that certain immune populations are differentially affected by Zn status. This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2020-06540 from the USDA National Institute of Food and Agriculture.

Synthesis of A Tetrahydrocannabinol (THC) Analog For Analyzing Cellular Proliferation in Concert With Epigallocatechin Gallate (EGCG) on A Bone‐like Cancer Cell Line (UMR 106‐01 BSP)

Cannabinoids have been extensively studied in the field of cancer research. Tetrahydrocannabinol (THC) has shown promising results in influencing cellular proliferation when in association with other cannabinoids. This traditional entourage effect solely focuses on the study of THC with other cannabinoids. However, not many studies have been done to explore the synergistic effect of THC analogs when used with non‐cannabinoid compounds. THC in its isolate form for experimentation is very strictly regulated. Therefore, this study was conducted in the pursuit of synthesizing and experimenting with analogs of THC to observe a potential entourage effect with epigallocatechin gallate (EGCG), a compound known for its efficacy to reduce proliferation at higher concentrations in UMR cells. It was hypothesized that active analogs of THC can be synthesized and used in concert with EGCG to potentiate decreased proliferation in the bone‐like cancer cell line UMR 106‐01 BSP (UMR cells). Briefly, a Knoevenagel condensation and a Diels‐Alder reaction using 1,3‐cyclohexanediol dissolved in methanol (MeOH) and citronellal with ethylenediamine diacetic acid (EDDA) at a temperature of 60℃ was used to synthesize a novel THC analog, 3,10,10‐Trimethyl‐1,2,3,4,4a,6,7,8,10,10a‐decahydro‐9‐oxa‐5‐phenanthrenone (TDP). UMR cells were routinely passaged, counted, plated in six‐well culture plates at 480,00 cells/mL, then treated with 10‐fold dilutions of TDP. The plates were incubated for 72 hours in a humidified incubator at 37 degrees Celsius with 5% carbon dioxide infusion. At the end of the experiment, the cells were routinely washed with HANKS buffered saline solution (HBSS), then routinely counted using the Luna Automated Cell Counter. In another experiment, designated cells were co‐treated with TDP+EGCG, following the protocol above. F test ANOVA was used to compare variances and all values in the results were expressed as means ± SD. The results from the attempted cannabinoid analog synthesis yielded a novel active THC analog, TDP. Serial dilutions treatment of the UMR cells with TDP alone showed its ability to decrease cell count in a concentration dependent manner. However, when coupled with higher concentrations of EGCG, the co‐treatment increased cell count rather than potentiating the effect of decreasing cellular proliferation. The F Test ANOVA showed robust statistical significance (p values <0.05) with regard to TDP’s effect of decreasing cell proliferation in UMR cells in a concentration‐dependent manner. Overall, the outcomes of this study suggest that active forms of THC analogs can be synthesized and tested in concert with other non‐cannabinoid compounds like EGCG. This study opens the door to explore the entourage effect of TDP with other non‐cannabinoid compounds that may provide another tool in the therapeutic treatment of bone cancer cells.

Bile Duct Ligation Changes the Inhibitory Control of Vasopressin Neurons in Male Rats

Dilutional hyponatremia typically is caused by increased Arginine Vasopressin (AVP) secretion. This increases the risk of complications and mortality in patients with liver cirrhosis. Liver cirrhosis in male rats increases AVP release but the underlying mechanisms are still under investigation. In other models of chronic AVP release, changes in chloride homeostasis alters the inhibitory control of AVP neurons in the supraoptic nucleus (SON) causing GABA mediated excitation instead of inhibition. In the bile duct ligation (BDL) model of cirrhosis, changes in either intracellular chloride concentration or cell firing activity have not been directly tested. We hypothesize that liver cirrhosis increases intracellular chloride concentration leading to a decrease in GABA‐mediated inhibition of SON neurons from male rats.To test this hypothesis, we injected an adeno‐associated virus with a vasopressin promoter and a ratiometric chloride indicator, AAV2‐0VP1‐ClopHensorN into the SON of adult male Sprague‐Dawley rats. The rats either received BDL surgery, which was used to model liver cirrhosis, or a sham ligation surgery. All BDL rats had significantly higher liver weight to body weight ratio (n = 10‐20, p < 0.05), lower plasma osmolality (n = 10‐20, p < 0.001) and lower hematocrit (n = 10‐20, p < 0.05) as compared to sham rats. All rats were anesthetized with isoflurane (2‐3%), their brains were collected, and slices containing the SON were prepared. Loose‐cell patch recordings were made at 31±1oC using standard artificial cerebrospinal fluid. Labeled AVP cells and unlabeled SON cells were tested with focal application of muscimol (100µM), a GABAA receptor agonist.Cells from BDL rats showed impaired GABAA‐mediated inhibition in response to muscimol, as compared to those from sham rats. All putative AVP SON neurons from sham rats (4/4) showed decreased activity following muscimol while half of the putative AVP SON neurons (7/14) from BDL rats were excited (p < 0.001). Similar results were observed in experiments were the injections missed the SON. In these experiments, all SON neurons from sham rats were inhibited by muscimol (26/26) while thirty‐five percent of SON neurons from BDL rats (16/45) were excited by muscimol (p = 0.001). ClopHensorN based chloride imaging showed that muscimol application was associated with decreased intracellular chloride concentrations in putative AVP cells from BDL rats as compared to sham rats (n = 31‐32, p = 0.001). The results show that liver cirrhosis impairs the normal inhibitory response of SON neurons to GABAA receptor activation which could contribute to increased cell activity and dilutional hyponatremia in male rats.

Antitumor Activity of Tubulin-Binding Agent MPC-6827 on Different Types of Cancer Cell Lines.

In this study, the antitumor effects of tubulin-binding agent MPC-6827 on HeLa, MCF-7 and A549 cell lines originated from cervix carcinoma, metastatic breast adenocarcinoma and adenocarcinomic human alveolar basal epithelial cells respectively were determined. Cell index, BrdU labelling index, mitotic index and apoptotic index were evaluated in experiments. In cell index experiment 2 nM, 4 nM, 6 nM, 8 nM, 10 nM MPC-6827 applied to three cell lines. These parameters showed that 4 nM was the optimum concentration for HeLa and A549 cells, while 2 nM was the optimum concentration for MCF-7 cells. The use of optimum concentrations for each cell line has shown that while there was a significant decrease in mitotic index, BrdU labelling index, there was a significant increase in apoptotic index.

Investigation of the Effects of the Endogenous Cannabinoid Anandamide on Luminal A Breast Cancer Cell Line MCF-7

The present study was carried out to investigate anti-tumoral effects of Anandamide (AEA) in luminal A breast cancer cell line MCF-7. Cell viability was measured by MTT assay and cell index was measured by xCelligence DP analyzer system. The Feulgen method was used to determine the mitotic index parameter, and the 3H-Thymidine method was used to determine the labeling index parameter. The apoptotic index parameter was determined using a fluorescent dye DAPI. The results of this study showed that 25 µM Anandamide concentration was the optimum concentration for MCF-7 cells. While this concentration decreased the proportion of cells in the mitotic phase and synthesis phase, it increased the proportion of apoptotic cells.

Internal quality control of red cell concentrates in a regional blood center.

Objective: To assess the quality of the whole blood-derived red cell concentrates by measuring hematocrit. Study Design: Cross Sectional study. Setting: Regional Transfusion Center in Rawalpindi. Period: February to December 2019. Material & Methods: A total of 390 whole blood-derived red cell concentrates were included using a random sampling technique. These units were evaluated for hematocrit which is a hematological parameter using Sysmex Hematology Analyzer XP 100. The measurement of hematocrit was expressed as the mean range and standard deviation (mean ± SD) using descriptive statistics. Results: A total of 390 whole blood-derived red cell concentrates were subjected to quality analysis by measuring the hematocrit. The mean range of hematocrit was found to be 68.4 ± 4.8 g/dL. The hematocrit of 98.7% units was in compliance with standard criteria according to the American Association of blood banks which suggested it to be less than 80%. Conclusion: The results of this study showed an optimum level of conformity of the quality of whole blood-derived Red Cell Concentrates with International Standards.

LPIN1 Induces Gefitinib Resistance in EGFR Inhibitor-Resistant Non-Small Cell Lung Cancer Cells.

Simple SummaryHere, we attempted to identify targets that could be used to overcome resistance toward epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC). To accomplish this, we chose LPIN1 among the candidate targets that were identified from a previously performed genome-wide RNAi screening assay and validated it as a key factor regulating gefitinib resistance in EGFR-mutant NSCLC cells. We confirmed that LPIN1 depletion increased gefitinib sensitivity in drug-resistant H1650 NSCLC cells, as well as patient-derived YL05 lung cancer cells. Moreover, we found that LPIN1 expression was induced following gefitinib treatment, and activities of protein kinase C delta and nuclear factor kappa B, and lipid droplet formation were induced in an LPIN1-dependent manner. Additionally, we validated that targeting LPIN1 synergistically retarded tumor growth in an in vivo mouse xenograft model.Drug resistance limits the efficacy of targeted therapies, including tyrosine kinase inhibitors (TKIs); however, a substantial portion of the drug resistance mechanisms remains unexplained. In this study, we identified LPIN1 as a key factor that regulates gefitinib resistance in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) cells. Unlike TKI-sensitive HCC827 cells, gefitinib treatment induced LPIN1 expression and increased diacylglycerol concentration in TKI-resistant H1650 cells, followed by the activation of protein kinase C delta and nuclear factor kappa B (NF-κB) in an LPIN1-dependent manner, resulting in cancer cell survival. Additionally, LPIN1 increased the production of lipid droplets, which play an important role in TKI drug resistance. All results were recapitulated in a patient-derived EGFR-mutant NSCLC cell line. In in vivo tumorigenesis assay, we identified that both shRNA-mediated depletion and pharmaceutical inhibition of LPIN1 clearly reduced tumor growth and confirmed that gefitinib treatment induced LPIN1 expression and LPIN1-dependent NF-κB activation (an increase in p-IκBα level) in tumor tissues. These results suggest an effective strategy of co-treating TKIs and LPIN1 inhibitors to prevent TKI resistance in NSCLC patients.

Valosin-containing protein regulates the stability of fused in sarcoma granules in cells by changing ATP concentrations.

Fused in sarcoma (FUS), a DNA/RNA-binding protein, undergoes liquid-liquid phase separation to form granules in cells. Aberrant FUS granulation is associated with neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We found that FUS granules contain a multifunctional AAA ATPase, valosin-containing protein (VCP), which is known as a key regulator of protein degradation. FUS granule stability depends on ATP concentrations in cells. VCP ATPase changes the FUS granule stability time-dependently by consuming ATP to reduce its concentrations in the granules: VCPs in de novo FUS granules stabilize the granules, while long-lasting VCP colocalization destabilizes the granules. The proteolysis-promoting function of VCP may subsequently dissolve the unstabilized granules. We propose that VCP colocalized to the FUS granules acts as a timer to limit the residence time of the granules in cells.