Ion Efflux Research Articles

Phenylurea-type Cytokinin ameliorates the performance of young pea plants under salt stress

Cytokinins are known to enhance stress tolerance in plants. The present study aimed to assess the possible protective effect of exogenous phenylurea-type cytokinin (4PU-30) on alleviating salt (NaCl) stress. Young pea (Pisum sativum L.) plants were sprayed with cytokinin 4PU-30 and were subsequently subjected to NaCl treatment. The effect of 4PU-30 on cell membrane stability was assessed based on electrolyte leakage from leaves of control and NaCl stressed plants. A previously established model system employing the kinetics of ion leakage served to evaluate the effect of the 4PU-30 application on plants response to salinity. Salt treatment caused a moderate decrease in leaf water content. Accumulation of proline, malondialdehyde (MDA), and hydrogen peroxide (H2O2) in the leaves of NaCl treated plants indicated the development of oxidative stress, which was significantly alleviated by pretreatment with phenylurea-type cytokinin 4PU-30. Foliar application of 4PU-30 reduced the damaging effect of NaCl, as evidenced by decreased electrolyte leakage. Distinct roles of cell walls and plasmalemma in the processes of ion efflux due to salt stress are discussed.

A mini-review on ion fluxes that regulate NLRP3 inflammasome activation.

The activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome can be induced by a wide spectrum of activators. This is unlikely achieved by the binding of different activators directly to the NLRP3 protein itself, as the activators found so far show different forms of chemical structures. Previous studies have shown that these activators can induce potassium ion (K+) and chloride ion (Cl-) efflux, calcium (Ca2+) and other ion mobilization, mitochondrial dysfunction, and lysosomal disruption, all of which are believed to cause NLRP3 inflammasome activation; how these events are induced by the activators and how they coordinate with each other in inducing the NLRP3 inflammasome activation are not fully understood. Increasing evidence suggests that the coordinated change of intracellular ion concentrations may be a common mechanism for the NLRP3 activation by different activators. In this mini-review, we present a brief summary of the current knowledge about how different ionic flows (including K+, sodium ion, Ca2+, magnesium ion, manganese ion, zinc ion, iron ion, and Cl-) are involved in regulating the NLRP3 inflammasome activation in macrophages.

(R)-Roscovitine and CFTR modulators enhance killing of multi-drug resistant Burkholderia cenocepacia by cystic fibrosis macrophages

Cystic fibrosis (CF) is characterized by chronic bacterial infections and heightened inflammation. Widespread ineffective antibiotic use has led to increased isolation of drug resistant bacterial strains from respiratory samples. (R)-roscovitine (Seliciclib) is a unique drug that has many benefits in CF studies. We sought to determine roscovitine’s impact on macrophage function and killing of multi-drug resistant bacteria. Human blood monocytes were isolated from CF (F508del/F508del) and non-CF persons and derived into macrophages (MDMs). MDMs were infected with CF clinical isolates of B. cenocepacia and P. aeruginosa. MDMs were treated with (R)-roscovitine or its main hepatic metabolite (M3). Macrophage responses to infection and subsequent treatment were determined. (R)-roscovitine and M3 significantly increased killing of B. cenocepacia and P. aeruginosa in CF MDMs in a dose-dependent manner. (R)-roscovitine-mediated effects were partially dependent on CFTR and the TRPC6 channel. (R)-roscovitine-mediated killing of B. cenocepacia was enhanced by combination with the CFTR modulator tezacaftor/ivacaftor and/or the alternative CFTR modulator cysteamine. (R)-roscovitine also increased MDM CFTR function compared to tezacaftor/ivacaftor treatment alone. (R)-roscovitine increases CF macrophage-mediated killing of antibiotic-resistant bacteria. (R)-roscovitine also enhances other macrophage functions including CFTR-mediated ion efflux. Effects of (R)-roscovitine are greatest when combined with CFTR modulators or cysteamine, justifying further clinical testing of (R)-roscovitine or optimized derivatives.

The Roles of ZnT1 and ZnT4 in Glucose-Stimulated Zinc Secretion in Prostate Epithelial Cells.

We have previously demonstrated by MRI that high glucose stimulates efflux of zinc ions from the prostate. To our knowledge, this phenomena had not been reported previously and the mechanism remains unknown. Here, we report some initial observations that provide new insights into zinc processing during glucose-stimulated zinc secretion (GSZS) in the immortalized human prostate epithelial cell line, PNT1A. Additionally, we identified the subtypes of zinc-containing cells in human benign prostatic hyperplasia (BPH) tissue to further identify which cell types are likely responsible for zinc release in vivo. An intracellular fluorescence marker, FluoZin-1-AM, was used to assess the different roles of ZnT1 and ZnT4 in zinc homeostasis in wild type (WT) and mRNA knockdown PNT1A cell lines. Additionally, Bafilomycin A1 (Baf) was used to disrupt lysosomes and assess the role of lysosomal storage during GSZS. ZIMIR, an extracellular zinc-responsive fluorescent marker, was used to assess dynamic zinc efflux of WT and ZnT1 mRNA knockdown cells exposed to high glucose. Electron microscopy was used to assess intracellular zinc storage in response to high glucose and evaluate how Bafilomycin A1 affects zinc trafficking. BPH cells were harvested from transurtheral prostatectomy tissue and stained with fluorescent zinc granule indicator (ZIGIR), an intracellular zinc-responsive fluorescent marker, before being sorted for cell types using flow cytometry. Fluorescent studies demonstrate that ZnT1 is the major zinc efflux transporter in prostate epithelial cells and that loss of ZnT1 via mRNA knockdown combined with lysosomal storage disruption results in a nearly 4-fold increase in cytosolic zinc. Knockdown of ZnT1 dramatically reduces zinc efflux during GSZS. Electron microscopy (EM) reveals that glucose stimulation significantly increases lysosomal storage of zinc; disruption of lysosomes via Baf or ZnT4 mRNA knockdown increases multi-vesicular body (MVB) formation and cytosolic zinc levels. In human BPH tissue, only the luminal epithelial cells contained significant amounts of zinc storage granules. Exposure of prostate epithelial cells to high glucose alters zinc homeostasis by inducing efflux of zinc ions via ZnT1 channels and increasing lysosomal storage via ZnT4. Given that prostate cancer cells undergo profound metabolic changes that result in reduced levels of total zinc, understanding the complex interplay between glucose exposure and zinc homeostasis in the prostate may provide new insights into the development of prostate carcinogenesis.

Bacterial Spore mRNA - What's Up With That?

Bacteria belonging to the orders Bacillales and Clostridiales form spores in response to nutrient starvation. From a simplified morphological perspective, the spore can be considered as comprising a central protoplast or core, that is, enveloped sequentially by an inner membrane (IM), a peptidoglycan cortex, an outer membrane, and a proteinaceous coat. All of these structures are characterized by unique morphological and/or structural features, which collectively confer metabolic dormancy and properties of environmental resistance to the quiescent spore. These properties are maintained until the spore is stimulated to germinate, outgrow and form a new vegetative cell. Spore germination comprises a series of partially overlapping biochemical and biophysical events – efflux of ions from the core, rehydration and IM reorganization, disassembly of cortex and coat – all of which appear to take place in the absence of de novo ATP and protein synthesis. If the latter points are correct, why then do spores of all species examined to date contain a diverse range of mRNA molecules deposited within the spore core? Are some of these molecules “functional,” serving as translationally active units that are required for efficient spore germination and outgrowth, or are they just remnants from sporulation whose sole purpose is to provide a reservoir of ribonucleotides for the newly outgrowing cell? What is the fate of these molecules during spore senescence, and indeed, are conditions within the spore core likely to provide any opportunity for changes in the transcriptional profile of the spore during dormancy? This review encompasses a historical perspective of spore ribonucleotide biology, from the earliest biochemical led analyses – some of which in hindsight have proved to be remarkably prescient – through the transcriptomic era at the turn of this century, to the latest next generation sequencing derived insights. We provide an overview of the key literature to facilitate reasoned responses to the aforementioned questions, and many others, prior to concluding by identifying the major outstanding issues in this crucial area of spore biology.

A Reverse-Osmosis Model of Apoptotic Shrinkage.

The standard theory of apoptotic volume decrease (AVD) posits activation of potassium and/or chloride channels, causing an efflux of ions and osmotic loss of water. However, in view of the multitude of possible channels that are known to support apoptosis, a model based on specific signaling to a channel presents certain problems. We propose another mechanism of apoptotic dehydration based on cytoskeletal compression. As is well known, cytoskeleton is not strong enough to expel a substantial amount of water against an osmotic gradient. It is possible, however, that an increase in intracellular pressure may cause an initial small efflux of water, and that will create a small concentration gradient of ions, favoring their exit. If the channels are open, some ions will exit the cell, relieving the osmotic gradient; in this way, the process will be able to continue. Calculations confirm the possibility of such a mechanism. An increase in membrane permeability for water or ions may also result in dehydration if accompanied even by a constant cytoskeletal pressure. We review the molecular processes that may lead to apoptotic dehydration in the context of this model.

Physiological Processes Modulated by the Chloride-Sensitive WNK-SPAK/OSR1 Kinase Signaling Pathway and the Cation-Coupled Chloride Cotransporters.

The role of Cl– as an intracellular signaling ion has been increasingly recognized in recent years. One of the currently best described roles of Cl– in signaling is the modulation of the With-No-Lysine (K) (WNK) – STE20-Proline Alanine rich Kinase (SPAK)/Oxidative Stress Responsive Kinase 1 (OSR1) – Cation-Coupled Cl– Cotransporters (CCCs) cascade. Binding of a Cl– anion to the active site of WNK kinases directly modulates their activity, promoting their inhibition. WNK activation due to Cl– release from the binding site leads to phosphorylation and activation of SPAK/OSR1, which in turn phosphorylate the CCCs. Phosphorylation by WNKs-SPAK/OSR1 of the Na+-driven CCCs (mediating ions influx) promote their activation, whereas that of the K+-driven CCCs (mediating ions efflux) promote their inhibition. This results in net Cl– influx and feedback inhibition of WNK kinases. A wide variety of alterations to this pathway have been recognized as the cause of several human diseases, with manifestations in different systems. The understanding of WNK kinases as Cl– sensitive proteins has allowed us to better understand the mechanistic details of regulatory processes involved in diverse physiological phenomena that are reviewed here. These include cell volume regulation, potassium sensing and intracellular signaling in the renal distal convoluted tubule, and regulation of the neuronal response to the neurotransmitter GABA.

Mechanism of action of secondary metabolites from marine-derived Streptoymces on bacterial isolates by membrane permeability

This study was designed to further investigate the potential mechanism of action of our previously characterized and identified marine-derived Streptomyces extracts (ESC003 and ESC012) on selected bacterial isolates from our culture bank. Time-kill kinetics, protein and lipid leakages assay, cell membrane permeability, phosphate and potassium ions efflux assay, extracellular adenosine triphosphate (ATP) concentration and membrane potential (MP) were all carried out using the marine-derived Streptomyces extract to determine and understand the probable mode of action at which the extract inhibit or kill bacterial cell. The MIC of ESC003 and ESC012 ranged from 0.16 to 6.25 mg/mL while the MBC ranged from 1.25 to >10 mg/mL. On the time-kill kinetics, a reduction in mean viable cell amount was detected at respective time studied. For the impermeability of the bacterial isolates, the relative electric conductivity increased with increase in concentration and time interval of exposure. As regards the protein leakage, lipid leakage, 260 nm absorbing materials leakage, phosphate and potassium ions efflux; considerable amount of these products were leaked with increase in concentration and time of exposure to the bacterial isolates. The extracellular ATP concentration from respective bacterial isolates increased appreciably with increased concentration of exposure with a simultaneous decrease in membrane potential. Findings from this study revealed that the Streptomyces extracts revealed a significant breakthrough against susceptible bacterial isolates via the permeability of the bacterial cell membrane, and thus resulted in the outflow of ATP, electrolytes, DNA materials, and proteins. These changes lead to disruption, and eventually cell death, which were proportional to a concurrent decrease in the viability of the bacterial cell.

Evaluation of Priming Efficiency of Forskolin in Tissue-Specific Human Mesenchymal Stem Cells into Dopaminergic Neurons: An In Vitro Comparative Study

Background: Human mesenchymal stem cells (hMSC) can be derived from various tissue sources and differentiated into dopaminergic (DAergic) neurons using various types of inducers. There are several strategies that have been reported to generate functional dopaminergic neuronal cells from hMSCs in the most efficient manner possible. However, this area is still under extensive research. In this study, we aim to compare hMSCs derived from bone marrow (BM), adipose tissue (AD) and dental pulp (DP) to generate functional dopaminergic neurons, using FGF2 and forskolin. Post-differentiation, multiple factors were used to characterize the cells at morphological, morphometric, ultra-structural, mRNA and protein levels for various markers (Nestin, NF, MAP2, Tuj1, TH, DAT, PitX3, Ngn2, Kv4.2, SCN5A). Cells’ functionality was studied by calcium ion imaging, along with the amount of dopamine secreted by the cells in the culture medium. Results: Data analysis revealed that forskolin has comparable effect on BM- and AD-derived MSC (28.43% and 29.46% DAergic neurons, respectively), whereas DP-MSC (42.78 ± 1.248% DAergic neurons) show better outcome in terms of efficient generation of DAergic neuronal cells, expression of neuronal associated markers, dopamine release and calcium ion efflux. Ultra-structural studies by SEM and TEM also revealed a substantial change in both cellular morphology and composition of cellular organelles. It was observed that AD-MSCs showed the best neuronal features, at morphological, gene, and protein levels upon induction with the above-mentioned induction cocktail. Conclusion: It may be concluded that a combination of FGF2 and forskolin yields functionally active dopaminergic neuronal cells in vitro, with highest percentage of the same from AD-MSCs, as compared to that in BM-MSCs and DP-MSCs. The outcomes and comparative evaluation provide a substantial platform for further studies on molecular pathways involved in the process of DAergic neurogenesis in individual cases.

CrossTalk proposal: Apical NKCC1 of choroid plexus epithelial cells works in the net inward flux mode under basal conditions, maintaining intracellular Cl- and cell volume.

CrossTalk proposal: Apical NKCC1 of choroid plexus epithelial cells works in the net inward flux mode under basal conditions, maintaining intracellular Cl- and cell volume.

BAFF-driven NLRP3 inflammasome activation in B cells

BAFF supports B-cell survival and homeostasis by activating the NF-κB pathway. While NF-κB is also involved in the priming signal of NLRP3 inflammasome, the role of BAFF in NLRP3 inflammasome regulation is unknown. Here we report BAFF engagement to BAFF receptor elicited both priming and activating signals for NLRP3 inflammasomes in primary B cells and B lymphoma cell lines. This induction of NLRP3 inflammasomes by BAFF led to increased NLRP3 and IL-1β expression, caspase-1 activation, IL-1β secretion, and pyroptosis. Mechanistically, BAFF activated NLRP3 inflammasomes by promoting the association of cIAP-TRAF2 with components of NLRP3 inflammasomes, and by inducing Src activity-dependent ROS production and potassium ion efflux. B-cell receptor (BCR) stimulation on the Lyn signaling pathway inhibited BAFF-induced Src activities and attenuated BAFF-induced NLRP3 inflammasome activation. These findings reveal an additional function of BAFF in B-cell homeostasis that is associated with BCR activities.

Plasma Membrane Pores Drive Inflammatory Cell Death.

Necroptosis and pyroptosis are two forms of regulated cell death. They are executed by the proteins mixed-lineage kinase domain-like (MLKL) and gasdermin D (GSDMD), respectively. Once activated by numerous pathways, these proteins form membrane pores that allow the influx and efflux of various ions, proteins, and water, ultimately resulting in the death of the cell. These modalities of cell death are considered highly inflammatory because of the release of inflammatory cytokines and damage-associated molecular patterns, and are thereby not only deleterious for the dying cell itself, but also its environment or the entire organism. The relevance for these processes has been observed in various physiological and pathophysiological conditions, ranging from viral and bacterial infections over autoimmune and chronic inflammatory diseases to ischemic organ damage. In recent years, initial in vitro experiments have shed light on a range of connections between necroptosis and pyroptosis. Initial in vivo studies also indicate that, in many disease models, these two forms of cell death cannot be considered individually, as they demonstrate a complex interaction. In this article, we provide an overview of the currently known structure, pathways of activation, and functions of MLKL and GSDMD. With emerging evidence for an interconnection between necroptosis and pyroptosis in not only in vitro, but also in vivo models of disease, we highlight in particular the clinical relevance of the crosslinks between these two forms of inflammatory cell death and their implications for novel therapeutic strategies in a variety of diseases.

Mitochondrial dysfunction caused by outer membrane vesicles from Gram-negative bacteria activates intrinsic apoptosis and inflammation.

Sensing of microbes activates the innate immune system, depending on functional mitochondria. However, pathogenic bacteria inhibit mitochondrial activity by delivering toxins via outer membrane vesicles (OMVs). How macrophages respond to pathogenic microbes that target mitochondria remains unclear. Here, we show that macrophages exposed to OMVs from Neisseria gonorrhoeae, uropathogenic Escherichia coli and Pseudomonas aeruginosa induce mitochondrial apoptosis and NLRP3 inflammasome activation. OMVs and toxins that cause mitochondrial dysfunction trigger inhibition of host protein synthesis, which depletes the unstable BCL-2 family member MCL-1 and induces BAK-dependent mitochondrial apoptosis. In parallel with caspase-11-mediated pyroptosis, mitochondrial apoptosis and potassium ion efflux activate the NLRP3 inflammasome after OMV exposure in vitro. Importantly, in the in vivo setting, the activation and release of interleukin-1β in response to N. gonorrhoeae OMVs is regulated by mitochondrial apoptosis. Our data highlight how innate immune cells sense infections by monitoring mitochondrial health.

Antimicrobial, Aflatoxin B1 Inhibitory and Lipid Oxidation Suppressing Potential of Anethole-Based Chitosan Nanoemulsion as Novel Preservative for Protection of Stored Maize

Aflatoxins (AFs) are the most frequent contaminants of maize and maize-based products, and its consumption can cause severe adverse effects to humans and animals. The efficacy of essential oils (EOs) and their bioactive compounds as potential antifungal agents has been well documented against food-borne fungi. This study evaluates the preservative potency of anethole-based chitosan nanoemulsion (Ant-eCsNe) to control deterioration of stored maize samples from fungal infestation, aflatoxin B1 (AFB1) contamination and lipid oxidation. Release study indicated a relatively good sustainable release profile for the encapsulated anethole after 10 days. The Ant-eCsNe showed improved efficacy against A. flavus (AF-LHP-VS8) and other common food-borne moulds and inhibited growth and AFB1 biosynthesis at 0.8 and 0.4 μL/mL, respectively. Ant-eCsNe caused concentration-dependent inhibition of ergosterol content and increased efflux of cellular ions (Ca+2, Mg+2 and K+) and 260 and 280 nm absorbing materials, suggesting damage of fungal plasma membrane. Inhibition of methylglyoxal in fungal cells treated with Ant-eCsNe signifies its novel antiaflatoxigenic mechanism of action. Ant-eCsNe exhibited strong in vitro DPPH• and ABTS+• scavenging activity with IC50 value 89.36 and 45.05 μL/mL, respectively, and inhibited lipid oxidation in stored maize samples. Further, Ant-eCsNe exhibited reasonably strong efficacy in preserving maize samples from fungal and AFB1 contamination during in vivo investigations and did not change the sensory attributes as well. Overall results revealed that Ant-eCsNe holds good potential to be applied as food preservative to reduce fungal and aflatoxin contamination causing deterioration of stored maize.

The influence of dissolved oxygen around rice roots on salt tolerance during pre-tillering and tillering phases

Rice is a strategic crop that has a high tolerance to hypoxia compared to other species. Our purpose was to investigate the salinity-acclimation mechanisms in rice plants triggered by hypoxia in the nutrient solution. So, the development of radial O2-loss barriers and physiological and biochemical responses against salinity were evaluated in rice plants (cv. SCSBRS 113) grown under saline stress and subjected to different levels of dissolved oxygen around of root in the pre-tillering and tillering phases. Our results reported an interaction between hypoxia and salinity treatments. Salinity acted as a much harsher pressure than severe hypoxia, particularly in the pre-tillering stage. However, severe hypoxia was able to aggravate the physiological stress imposed by salinity in the tillering stage, and combination with salinity contributed to a reduction of Ca2+ and K+ content. Besides, the radial O2-loss barrier did not reduce the influx or efflux of water and ions. It also was unrelated to a root shortening and decreased relative water content in leaves. The hypoxia attenuation did not contribute much to the better growth and development of rice plants, and the radial O2-loss barriers did not confer salinity tolerance by hindering Na+ and Cl− uptake.

Are Aquaporins Expressed in Stomatal Complexes Promising Targets to Enhance Stomatal Dynamics?

The opening and closure of stomata depend on the turgor pressure adjustment by the influx or efflux of ions and water in guard cells. In this process, aquaporins may play important roles by facilitating the transport of water and other small molecules. In this perspective, we consider the potential roles of aquaporins in the membrane diffusion of different molecules (H2O, CO2, and H2O2), processes dependent on abscisic acid and CO2 signaling in guard cells. While the limited data already available emphasizes the roles of aquaporins in stomatal movement, we propose additional approaches to elucidate the specific roles of single or several aquaporin isoforms in the stomata and evaluate the perspectives aquaporins might offer to improve stomatal dynamics.

The S. mutans mntE gene encodes a manganese efflux transporter.

Streptococcus mutans is a colonizer of the human dentition, and under conditions of dysbiosis is the primary causative agent of dental caries. The pathogenic potential of S. mutans depends, in part, on its ability to regulate the transport of metal ions across the plasma membrane to maintain intracellular metal ion homeostasis. Research in our laboratory has focused on the Mn2+ -specific SloC lipoprotein importer and its regulator encoded by the S. mutans sloR gene. Herein, we used a bioinformatics approach to identify a gene on the S. mutans UA159 chromosome, SMU_1176, as a metal ion efflux transporter that contributes to S. mutans manganese ion homeostasis. Metal ion sensitivity assays performed with the wild-type S. mutans UA159 strain and an isogenic SMU_1176 insertion-deletion mutant, called GMS3000, revealed significantly heightened sensitivity of GMS3000 to MnSO4 challenge. 54 Mn uptake experiments support the accumulation of 54 Mn in GMS3000 cell pellets when compared to 54 Mn concentrations in UA159 or in a complemented strain of GMS3000, called GMS3001. Inductively coupled plasma mass spectrometry (ICP-MS) studies were performed in parallel to quantify intracellular manganese concentrations in these strains, the results of which corroborate the 54 Mn uptake studies, and support the SMU_1176 gene product as a Mn2+ efflux protein. Expression profiling experiments revealed de-repression of SMU_1176 gene transcription in the SloR-deficient GMS584 strain of S. mutans, especially under high manganese conditions. In conclusion, the S. mutans SMU_1176 gene, which we renamed mntE, is a manganese efflux transporter that contributes to essential metal ion homeostasis as part of the SloR regulon.

23 Na-MRI as a Noninvasive Biomarker for Cancer Diagnosis and Prognosis.

The influx of sodium (Na+) ions into a resting cell is regulated by Na+ channels and by Na+/H+ and Na+/Ca2+ exchangers, whereas Na+ ion efflux is mediated by the activity of Na+/K+‐ATPase to maintain a high transmembrane Na+ ion gradient. Dysfunction of this system leads to changes in the intracellular sodium concentration that promotes cancer metastasis by mediating invasion and migration. In addition, the accumulation of extracellular Na+ ions in cancer due to inflammation contributes to tumor immunogenicity. Thus, alterations in the Na+ ion concentration may potentially be used as a biomarker for malignant tumor diagnosis and prognosis. However, current limitations in detection technology and a complex tumor microenvironment present significant challenges for the in vivo assessment of Na+ concentration in tumor. 23Na‐magnetic resonance imaging (23Na‐MRI) offers a unique opportunity to study the effects of Na+ ion concentration changes in cancer. Although challenged by a low signal‐to‐noise ratio, the development of ultrahigh magnetic field scanners and specialized sodium acquisition sequences has significantly advanced 23Na‐MRI. 23Na‐MRI provides biochemical information that reflects cell viability, structural integrity, and energy metabolism, and has been shown to reveal rapid treatment response at the molecular level before morphological changes occur. Here we review the basis of 23Na‐MRI technology and discuss its potential as a direct noninvasive in vivo diagnostic and prognostic biomarker for cancer therapy, particularly in cancer immunotherapy. We propose that 23Na‐MRI is a promising method with a wide range of applications in the tumor immuno‐microenvironment research field and in cancer immunotherapy monitoring.Level of Evidence2Technical EfficacyStage 2

Dielectric spectroscopy of in-vitro human blood of diabetic and non-diabetic patients through low level laser therapy

The research work investigates the dielectric properties (dielectric constant, dielectric loss and conductivity) of both diabetic and non-diabetic patients’ blood in order to enhance of low level laser therapy (LLLT). Knowing the dielectric properties of blood, profile dose threshold exposure duration can improve LLLT for diabetic mellitus disease. This was achieved, using impedance analyser 4294A, frequency range 40 kHz - 30 MHz. Measurements were taken before and after blood irradiated with a portable diode-pumped solid state laser of wavelength 532 nm at power of 60 mW in standard cuvettes. Control diabetic patient’s bloods were high in dielectric parameter compared with the control non-diabetic patient’s blood. After exposure for 5 and 10 minute’s duration, the value of dielectric loss for the blood exposed for 5 minutes duration appreciated significantly. This is attributed to the energy acquired by haemoglobin and oxygen activation that increased cell membrane resistance, protecting the K+ions efflux and thus prevent perilous balance of Ca+2 ions. Laser power of 60 mW for 5 minutes exposure proved effective in LLL diabetic mellitus therapy.

Microsecond Simulation of the Proteoglycan-like Region of Carbonic Anhydrase IX and Design of Chemical Inhibitors Targeting pH Homeostasis in Cancer Cells

Carbonic anhydrase IX (CAIX) is a membrane-bound enzyme associated with tumor hypoxia and found to be over expressed in various tumor conditions. Targeting CAIX catalytic activity is proven to be efficient modality in modulating pH homeostasis in cancer cells. Proteoglycan-like (PG) region is unique to CAIX and is proposed to serve as an antenna enhancing the export of protons in conjunction with facilitated efflux of lactate ions via monocarboxylate transporters. Moreover, the PG region is also reported to contribute to the assembly and maturation of focal adhesion links during cellular attachment and dispersion on solid supports. Thus, drug targeting of this region shall efficiently modulate pH homeostasis and cell adhesion in cancer cells. As the PG region is intrinsically disordered, the complete crystal structure is not elucidated. Hence, in this study, we intend to sample the conformational landscape of the PG region at microsecond scale simulation in order to sample the most probable conformations that shall be utilized for structure-based drug design. In addition, the sampled conformations were subjected to high-throughput virtual screening against NCI and Maybridge datasets to identify potential hits based on consensus scoring and validation by molecular dynamics simulation. Further, the identified hits were experimentally validated for efficacy by in vitro and direct enzymatic assays. The results reveal 5-(2-aminoethyl)-1,2,3-benzenetriol to be the most promising hit as it showed significant CAIX inhibition at all levels of in silico and experimental validation.