μM Concentration Research Articles

Multiresponsive Nanoscale Self-Assembly of Azurin-Elastin-like Polypeptide Fusion Protein for Enhanced Prostate Cancer Therapy.

A fusion protein composed of a bacterial protein, azurin, having antineoplastic properties and a thermally responsive structural cationic elastin-like protein (ELP), is designed, cloned, expressed, and purified. A simple method of inverse transition cycle (ITC) is employed to purify the fusion protein azurin-ELP diblock copolymer (d-bc). The molecular weight of the azurin-ELP fusion protein is ∼32 kDa. Further, its self-assembly properties are investigated. Interestingly, the engineered azurin-ELP d-bc in response to increasing temperature shows a dual-step phase separation into biofunctional nanostructures. Around the physiological temperature, azurin-ELP d-bc forms stable coacervates, which is dependent on the concentration and time of incubation. These coacervates are formed below the lower critical solubility temperature (LCST) of the ELP block at physiological temperature. Above LCST, i.e., 50-55°C, micelles of size ranging from 25 to 30 nm are formed. The cytotoxicity of azurin-ELP d-bc depends on the size of the coacervates formed and their cellular uptake at physiological temperature. Further, MTT assay of azurin-ELP d-bc in the cross-linked micelles prepared ex situ shows > six times higher killing of LNCaP cells than the unimeric form of azurin-ELP at 5 μM concentration. The flow cytometric results of these micelles at 20 μM concentration show ∼97% LNCaP cells in the apoptotic phase. Thus, azurin-ELP cross-linked micelles have enhanced potential for anticancer therapy due to their higher avidity.

Non‐enzymatic electrochemical biosensor based on MgO@rGO‐MoS2 nanohybrid for phenolic compounds detection

In the present work, efforts have been made to fabricate a non‐enzymatic electrochemical biosensor based on MgO nanoparticles modified graphene oxide‐molybdenum disulfide (MgO@rGO‐MoS2) nanohybrid for ultra‐sensitive detection of water‐polluting dihydroxy benzene compound. The one‐pot hydrothermal method has been used to synthesize MgO@rGO‐MoS2 nanohybrid followed by electrophoretic deposition of the nanohybrid onto ITO‐coated glass substrate. The excellent conducting and catalytic ability of MoS2 and MgO facilitates the superior electroanalytical ability of fabricated electrodes, as revealed by the electrochemical studies, whereas the non‐enzymatic approach boosts the stability and sensitivity of the transducing matrix. The fabricated biosensor exhibits linear response in the 0.1 to 200.0 μM concentration range for hydroquinone (Hqn) with a detection limit of 0.086 μM and 0.04 μA μM−1 cm−2 sensitivity. The validation of the biosensor with tap and running river water shows an outstanding recovery rate.

Endoplasmic reticulum stress and pro-inflammatory responses induced by phthalate metabolites monoethylhexyl phthalate and monobutyl phthalate in 1.1B4 pancreatic beta cells

In recent years, phthalates and their metabolites have been associated with metabolic diseases such as diabetes mellitus. To investigate the effects of phthalate metabolites exposure on insulin production and release, 1.1B4 pancreatic beta cells were treated with different concentrations (0.001–1000 µM) of monoethylhexyl phthalate (MEHP) and monobutyl phthalate (MBP). For such purpose, the 1.1B4 cells were evaluated for their viability, apoptosis rate, lysosomal membrane permeabilization (LMP), mitochondrial membrane potential (ΔΨm), oxidative stress, ER stress status, in addition to their secretory functions. MEHP, not MBP, exhibited a notable reduction in metabolic viability, particularly at higher concentrations (500 and 1000 µM) following 24-hour exposure. Similarly, both MEHP and MBP induced decreased metabolic viability at high concentrations after 48- and 72-hour exposure. Notably, neither MEHP nor MBP demonstrated a significant impact on apoptosis rates after 24-hour exposure, and MBP induced mild necrosis at 1000 µM concentration. Cell proliferation rates, indicated by PCNA expression, decreased with 10 and 1000 µM MEHP and 0.1 and 10 µM MBP exposures. LMP analysis revealed an increase in 1000 µM MBP group. Exposure to 0.001 µM of both MEHP and MBP significantly reduced cellular glutathione (GSH) levels. No significant change in intracellular reactive oxygen species (ROS) levels and ΔΨm was observed, but MBP-exposed cells exhibited elevated levels of lipid peroxidation. Functional assessments of pancreatic beta cells unveiled reduced insulin secretion at low glucose concentrations following exposure to both MEHP and MBP, with concurrent alterations in the expression levels of key proteins associated with beta cell function, including GLUT1, GCK, PDX1, and MafA. Moreover, MEHP and MBP exposures were associated with alterations in ER stress-related pathways, including JNK, GADD153, and NF-κB expression, as well as PPARα and PPARγ levels. In conclusion, this study provides comprehensive insights into the diverse impacts of MEHP and MBP on 1.1B4 pancreatic beta cells, emphasizing their potential role in modulating cell survival, metabolic function, and stress response pathways.

Cytokinin-mediated shoot proliferation and its correlation with phytoremediation effects in Cd-hyperaccumulator ecotype of Sedum alfredii

The phytohormones cytokinins (CKs) are known to regulate apical/auxiliary meristems, control shoot growth and are associated with nutrient uptake and high biomass production. In this study, different cytokinins were tested on Sedum alfredii (S.alfredii) for shoot proliferation and growth performance as well as their correlation with phytoextraction efficiency. Among the tested cytokinins, Zeatin (ZTN) treatments produced the highest number of shoots (5–6 per explant) with 5 and 10 μM ZTN concentrations which are shown as zeatin (ZTN) > kinetin (KTN) > benzylaminopurine (BA) > thidiazuron (TDZ). Maximum biomass production was produced on these media. The maximum biomass (0.14 g) was found in 10 μM ZTN concentration with a 1-fold difference (mean value: 0.02 g) from CK (0.12 g). However, the lowest biomass (0.11 g) was found with 4 μM TDZ, with a 1-fold difference (mean value: 0.02 g) from CK (0.13 g) which suppressed shoot growth. The leaf area and leaf chlorophyll index were significantly increased in all cytokinins except TDZ, and the relation was ZTN > KTN > BA>CK > TDZ. Cadmium accumulation was significantly higher in treatments containing cytokinins as compared to cytokinin-free media. Zeatin at 10 μM concentration was the most effective for high biomass production and correlated with higher cadmium uptake efficiency. The results suggest that cytokinins particularly ZTN, play a crucial role in enhancing both biomass production and cadmium, uptake efficiency in S. alfredii. Therefore, in large-scale phytoremediation initiatives conducted in field conditions, cytokinins can be utilized as growth regulators to enhance biomass production and cadmium extraction efficiency in S.alfredii.

In Vivo Wound Healing in Drosophila melanogaster and Mouse Models: Synergistic Effect of Bovine Serum Albumin and Graphene Quantum Dots.

Bovine serum albumin (BSA)-based biomaterials have garnered significant attention for their remarkable potential in wound healing, primarily due to their effective biological actions in addressing the skin inflammation phase and mitigating hypoalbuminemia. Motivated by these attributes, a nanocomposite hydrogel is developed by blending BSA with poly(vinyl alcohol) (PVA), complemented by the incorporation of graphene quantum dot (GQD). The FTIR study establishes a hydrogen-bonding interaction between the -NH2 groups of BSA and the -OH group of PVA. Microscopic investigations establish that the dispersion of GQDs with an average size of 22.5 nm results in smoothening of the surface of the nanocomposite. The nanocomposite hydrogel reveals excellent swelling attributes of about 920% in a period of 6 h due to its optimum cross-linking condition. Furthermore, the hydrogel exhibits a water vapor transmission rate of 8.45 mg cm-2 h-1, akin to the transmission rate of wounded skin. The PVA/BSA@GQD nanocomposite's antibacterial efficacy is evaluated against Morganella morganii bacteria, showing 99% killing, while its cytotoxicity assay against HeLa cells exhibited a minimum cell viability of 76% at a 20 μM concentration, which is ideal for a wound dressing material. In vivo wound healing investigations are conducted on Drosophila, showcasing a 100% wound surface closure within 4 h. This outcome is further substantiated through in vivo studies involving mice, where complete re-epithelialization is achieved within a span of 13 days. The combined results establish the PVA/BSA@GQD nanocomposite as a potential wound dressing material.

Mild excitotoxic effect on mitochondrial membrane potential, Ca2+ homeostasis and energy production in a mouse model of Alzheimer’s disease

AbstractBackgroundNeurons are bound to maintain a tight control over their cellular homeostasis in order to preserve their many functions, and specifically to precisely regulate ion fluxes for the generation of action potential and neurotransmission. In particular, the energetic balance has to be finely adjusted since its alteration may hamper cellular functionality with consequent cell suffering and death. Mitochondria play a central role in the homeostatic regulation within the cells due to the many vital tasks they perform in terms of energy production, participation in the regulation of Ca2+ concentration, modulation of intracellular signaling that allow the cell to respond to external solicitationMethodPrimary hippocampal neuronal cultures of 14‐17 DIV from WT and AD mouse model were challenged with μM concentration of glutamate to induce a mild excitotoxic effect; mitochondrial membrane potential, mitochondrial an cytosolic Ca2+ transients and cytosolic ATP levels were evaluated with fluorescent probes. Cells were treated also with inhibitors of the permeability transition pore to evaluate its effect on cell functionalityResultWe observed that in a model of Familial Alzheimer’s disease, hippocampal neurons are sensitive to a modest excitotoxic signal that produces a transient cytosolic and mitochondrial matrix Ca2+ with consequent partial loss of mitochondrial membrane potential and delayed recovery of mitochondrial matrix [Ca2+] which might contribute in the activation of the mitochondria permeability transition pore and consequent impairment of mitochondrial and cellular functions. This is accompanied by a reduced cytosolic ATP content that can result in an energy deficit for the cell. A brief treatment with inhibitors of the permeability transition pore is able to reduce these effects and might have protective roles against exogenous insults to the cells and help mitigate the neuronal loss characteristic of Alzheimer’s disease.ConclusionAD hippocampal neurons are more sensitive to a mild excitotoxic insult that affect mitochondrial functionality and in particular the activation of the permeability transition pore (PTP). Treatment with μM concentrations of two different inhibitors of the PTP prevent mitochondrial impairment.

Jasmonates and salicylic acid as enigmatic orchestrators of capitula senescence in Cosmos sulphureus Cav.

The fine-tuning of the intricate network of plant growth hormones empowers the balanced responses of plants to environmental and developmental signals. Salicylic acid and jasmonates are emerging as advanced hormones that provide plants with resistance to environmental stresses. Senescence is characterized by coordinated and systematic crosstalk between phytohormones that remodels the biochemical and physiological mechanisms in plants, resulting in cell death. The present investigation examines the role of jasmonates (methyl jasmonate and jasmonic acid) and salicylic acid (SA) in regulating the petal senescence of detached stalks of Cosmos sulphureus. Based on our results, it was revealed that SA and jasmonic acid (JA) at 40μM and methyl jasmonate (MJ) at 0.75μM concentration delayed the senescence of detached flowers of C. sulphureus considerably. These growth regulators improved the membrane stability, reinforced the antioxidant enzyme activities and averted the upsurge of hydrogen peroxide (H2O2) content in the petals. Additionally, SA and jasmonates preserved higher content oftotal phenols, reducing sugars and soluble proteins in the petals, besides impeding the bacterial growth in testing solutions which corroborated with themaximum solution uptake. The elevated soluble protein content was found to be associated with low specific protease activity (SPA) and α-amino acid content in the petal tissues. Our study concluded that SA and jasmonates delayed flower senescence by averting oxidative stress and maintaining the nutritional status of the petals.

Reduction of cryopreservation-induced structural, functional and molecular damages in ram sperm by hydrated C60 fullerene.

This study aimed to investigate the morphological, functional and molecular changes in frozen-thawed ram sperm using an extender containing different concentrations of hydrated carbon 60 fullerene (C60 HyFn), a nanotechnological product. Semen taken from each of the seven Akkaraman rams were pooled. Semen collection was done twice a week and it continued for 3 weeks. Each pooled semen sample was divided into six equal groups and diluted with tris + egg yolk extender including 0 (control), 200, 400, 800 nM, 1 and 5 μM concentrations of C60 HyFn at 37°C. They were then frozen in liquid nitrogen vapour at -140°C, stored in liquid nitrogen container (-196°C) and thawed at 37°C for 25 s before analysis. In comparison with control, C60 HyFn addition prior to freezing procedure provided significant increases in total and progressive motility rates, glutathione peroxidase, catalase activities and percentage of highly active mitochondria, and significant decreases in dead and abnormal sperm rates, lipid peroxidation, caspase-3 and DNA fragmentation levels in frozen-thawed ram semen. When compared to control, C60 HyFn supplementation significantly down-regulated the expression levels of miR-200a and KCNJ11, and significantly up-regulated the expression levels of miR-3958-3p (at the concentrations of 200, 400, 800 nM and 1 μM), CatSper1 (at the concentrations of 200, 400 nM and 5 μM), CatSper2 (at the concentrations of 1 and 5 μM), CatSper3 (at the concentrations of 200, 400 nM, 1 and 5 μM), CatSper4 (at all concentrations), ANO1 (at the concentrations of 800 nM, 1 and 5 μM) and TRPV5 (at the concentrations of 200, 400 and 800 nM). The addition of C60 HyFn had no effect on global DNA methylation rates. As a result, C60 HyFn supplementation to ram semen extenders may be beneficial in reducing some of the functional, structural and molecular damages in sperm induced by the freeze-thawing procedure.

Gene expression levels and inhibitory effect of 7-[(7-methoxy-4,5-dihydro-1H-benzo[g]indazol-3-yl)carbonyl]-2-phenyl-5,6,7,8-tetrahydropyrazolo[1,5-a]pyrido[4,3-d]Pyrimidin-9(1H)-one (MCL) against AP2-I and BDP1 in malaria experimental models

Resistance to antimalarial drugs leads a global recurrence of malaria, posing a significant challenge to malaria control. This study aimed to assess gene expression and resistance profiles in clinical isolates, and validate the inhibitory effect of MCL compound against PbAP2-I and PbBDP1 genes in vivo. Malaria prevalence rate throughout the clinical study period stood at 12.24%. Blood samples were obtained from 182 malaria outpatients and 30 P. berghei-infected mice treated with varying MCL concentrations. DNA and RNA were extracted. PfAP2-I, PfBDP1, PfMDR1, and PfK13 genes were amplified using Real-time qPCR and sequenced for mutation profiling. Relative gene expression of AP2-I and BDP1 genes in P. falciparum and P. berghei-treated mice was carried out by Reverse-transcription PCR. The data were analyzed using ANOVA at P<0.05 indicating a significant difference. Relative gene expression results from clinical isolates showed PfAP2-I and PfBDP1 are highly expressed in the trophozoite and schizont stages. The clinical isolates show conserved nucleotide sequences for PfAP2-I, PfBDP1, and PfK13 genes compared to the 3D7 strain. However, Asn86Tyr mutation implicated in antimalarial drug resistance and Leu1312Iso mutation not previously reported to be associated with antimalarial drug resistance were observed for the PfMDR1 gene. MCL inhibited the expression of PbAP2-I (by 4.52 and 8.08 folds) and PbBDP1 (by 37.92 and 79.07 folds) at 40µM and 50µM concentrations after a 7-day, single oral daily dose. Our findings validate MCL's inhibitory activity against AP2-I and BDP1 genes, hence representing potential new targets of antimalarial therapy for controlling malaria parasite invasion.

Novel Pi3kδ Inhibitor Roginolisib Synergizes with the Bcl-2 Inhibitor Venetoclax in Hematological Malignancies

Roginolisib (IOA-244) is a first in class allosteric modulator and non-ATP competitive, selective PI3Kδ inhibitor currently in a Phase 1 clinical study in lymphoma and solid tumors. Initial structural and biochemical studies identified unique chemical structure, excellent selectivity, and excellent PK properties for roginolisib as compared to earlier PI3Kδ inhibitors, while the clinical trial has demonstrated a favorable safety profile. Consistent with prior PI3Kδ inhibitors, roginolisib inhibits the in vitro growth of lymphoma cells. In contrast to other PI3Kδ inhibitors, roginolisib activity is correlated with the expression levels of PIK3CD, suggesting specific and on target cancer cell-intrinsic effects (PMID: 37066023). To identify rational combination strategies, initial pharmacological screening of 474 compounds in combination with roginolisib was carried out using a MTT assay in the lymphoma cell lines HH (cutaneous T cell lymphoma, CTCL) and SP-53 (mantle cell lymphoma, MCL). Initial screening identified that roginolisib synergized with the bcl-2 inhibitor venetoclax in the HH cell line with a combination index value (CI) of 0.003 by the Chou-Talalay method. To further validate this finding, the combination of roginolisib with venetoclax or with another bcl-2 inhibitor S55746 was tested in a broad range of lymphoma cell lines including GRANTA519, JVM2, SP49 (MCL); FARAGE, TMD8 (diffuse large B cell lymphoma); MEC1 (chronic lymphocytic leukemia); MJ (cutaneous T cell lymphoma); and YT (NK lymphoma). The combination showed synergism in all the cell lines (CI 0.81 - 0.05) except SP49 (CI- 1.3). Similarly, the combination of roginolisib with S55746 showed synergism in most of the cell lines (CI- 0.46 - 0.089), additive effect in SP49 (CI- 1) and antagonistic effect in GRANTA519 (CI- 1.3) and FARAGE (CI- 2.17). To assess the tumor-intrinsic effect of roginolisib in patient derived CLL cells, PBMCs isolated from CLL patients responding to prior BTK inhibitor (i) (n=4) or progressed on BTKi (n=6) were tested to check the effect of roginolisib on BCR signalling. Cells were treated with increasing doses of roginolisib from 0.625 μM to 5 μM in the presence of anti-IgM. Other BCRi such as idelalisib, duvelisib and acalabrutinib were used at 5 μM concentrations as positive controls. Roginolisib showed a dose dependent suppression of the activity of BCR downstream kinases such as AKT and ERK. The suppression of phosphorylation of AKT and ERK were comparable with other BCRi tested. Additionally, the apoptosis analysis by annexin/7AAD showed comparable activity of roginolisib with idelalisib, duvelisib or acalabrutinib at 5 μM concentration. To investigate the possible synergy of roginolisib with venetoclax in CLL patient samples who are BTKi responders (n=6) or BTKi progressors (n=8), apoptosis analysis was carried out in patient PBMCs. Viability of CLL cells following 48h of ex vivo treatment with increasing concentrations of roginolisib (0.625 to 5 μM) and venetoclax (1 to 8 nM) alone or in combination was tested. Both the BTKi responders and progressors showed strong synergy with the combination, with slightly better cell killing activity in BTKi progressors ( p=0.007) than responders ( p=0.019). To understand the mechanism by which roginolisib increases the sensitivity to venetoclax, changes in the expression levels of various Bcl-2 family proteins that have been implicated in venetoclax resistance such as Mcl-1, Bcl-xL, Bcl-2, A1 and BIM were measured by immunoblotting. Changes in expression were evaluated in PBMCs from both the BTKi responders (n=8) and progressors (n=7) after 24h treatment with 0.625 and 5 μM concentrations of roginolisib. A significant dose dependent downregulation of Mcl-1 was observed compared to DMSO treated cells in both BTKi responders ( p=0.0033) and progressors ( p= 0.0025). In addition, a significant increase in proapoptotic BIM EL was also observed at both the 0.625 μM ( p=0.0054) and 5 μM ( p=0.0194) concentrations compared to DMSO. In summary, the novel PI3Kδ selective inhibitor roginolisib shows tumor cell killing on various hematologic malignancies including CLL cells from patients with disease progression on BTK inhibitors. In addition, roginolisib synergized with venetoclax in lymphoma cell lines and CLL patient samples. Our data support extending this combination strategy to clinical trials in hematological malignancies.

A fluorescence turn “on-off” imaging probe for sequential detection of Al3+ and L-Cysteine in HeLa cells

At this “Aluminum Age”, exposure to aluminum (metallic or ionic form) is inevitable and inestimable. The presence of aluminum in biological systems is evident but more often aluminum toxicity is less understood. Therefore, the presence of biologically reactive aluminum needs to be identified and quantified. Alongside metals, L-cysteine, an essential amino acid, plays a pivotal role in the homeostasis of cellular oxidative and reductive stress. However, excess (<7g) could be lethal and can lead to death. Thus, in-situ selective detection of aluminum and L-cysteine is of larger interest. Here we report a fluorogenic probe (R) for the sequential selective detection and quantification of Al3+ and L-cysteine in a semi-aqueous medium (3:7; water: DMSO). The probe (R) was synthesized by a one-step acid-mediated condensation reaction between pyridine-3,4-diamine and 2-hydroxy-1-napthaldehyde. The synthesized probe was characterized using 1H and 13C NMR, and HR-Mass spectroscopic techniques. The probe (R) is non-emissive in nature, but on recognition of Al3+, the probe R showed “turn-on” emission (bright yellow colour) showing two emission maxima (522 nm and 547 nm), and no naked eye observable color change. Other competing cations do not show any noticeable fluorescence outcome. The R + Al3+ ensemble can specifically detect L-cysteine among all the essential amino acids by showing a fluorescence “turn-off” response. The sensing mechanism of Al3+ is obeying the chelation-enhanced fluorescence (CHEF) effect. The binding constant of R + Al3+ is 0.3 × 104 M−1. The limit of detection (LoD) for Al3+ and L-cysteine are 2.02 × 10-7 M and 0.5 × 10-5 M respectively. The probe (R) can show maximum efficiency within the pH range (7.0–10.0). The probe is found non-toxic (>80 % cell viability with 15 µM concentration) and employed for the in-vitro fluorescence imaging in the HeLa cell.

Effect of Low Concentration of Nitroxides on SH-SY5Y Cells Transfected with the Tau Protein

Nitroxides, stable synthetic free radicals, are promising antioxidants, showing many beneficial effects both at the cellular level and in animal studies. However, the cells are usually treated with high millimolar concentrations of nitroxides which are not relevant to the concentrations that could be attained in vivo. This paper aimed to examine the effects of low (≤10 μM) concentrations of three nitroxides, 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO), 4-hydroxy-TEMPO (TEMPOL) and 4-amino-TEMPO (TEMPAMINE), in pure chemical systems and on SH-SY5Y cells transfected with the human tau protein (TAU cells), a model of chronic cellular oxidative stress, and transfected with the empty plasmid (EP cells). All nitroxides were active in antioxidant-activity tests except for the 2,2′-azinobis-(3-ethylbenzthiazolin-6-sulfonate) radical (ABTS•) decolorization assay and reduced Fe3+, inhibited autoxidation of adrenalin and pyrogallol and oxidation of dihydrorhodamine123 by 3-morpholino-sydnonimine SIN-1. TEMPO protected against fluorescein bleaching from hypochlorite, but TEMPAMINE enhanced the bleaching. Nitroxides showed no cytotoxicity and were reduced by the cells to non-paramagnetic derivatives. They decreased the level of reactive oxygen species, depleted glutathione, and increased mitochondrial-membrane potential in both types of cells, and increased lipid peroxidation in TAU cells. These results demonstrate that even at low micromolar concentrations nitroxides can affect the cellular redox equilibrium and other biochemical parameters.

Engraulisin: A novel marine derived cell penetrating peptide with activity against drug resistant bacteria

Cell penetrating peptides (CPP) with their intrinsic ability to penetrate plasma membranes facilitate intracellular uptake of various macromolecules. Although a substantial number of CPPs have been reported over the last three decades, the number is still inadequate when compared to the theoretically feasible peptides with similar physicochemical composition.Marine organisms, due to their hostile environment, are an immense source of several high-valued therapeutically relevant peptides. Various marine derived antibacterial, antimycotic and anticancer peptides have demonstrated improved activity in comparison to peptides of terrestrial origin. While a significant number of marine bioactive peptides exist, cell penetrating peptides from marine organisms remain unravelled.In this study, we report Engraulisin from Engraulis japonicus, a computationally derived novel cell penetrating peptide of marine origin. Engraulisin manifest successful uptake in mammalian cells at 5 μM concentration with negligible cytotoxicity observed through MTT assay. Analysis of its cellular uptake mechanism revealed significant inhibition at 4 °C suggesting endocytosis as the major route of cellular entry. Interestingly, the novel peptide also demonstrated selective antimicrobial activity against Methicillin-resistant Staphylococcus aureus (MRSA). Additionally, molecular dynamics simulation with POPC and POPG bilayer system unveiled significance of positively charged residues in forming a stable membrane interaction. Engraulisin represents a novel marine-derived cell penetrating peptide which can be explored for cellular delivery of pharmaceutically relevant molecules.

Synergistic effects of punicic acid and alpha lipoic acid ameliorate inflammatory and metabolic genes expression in C2C12 myoblast cells under oxidative stress condition.

Inflammation is a reaction of the immune system to infection and injury; in fact, it positioned at the center of metabolic disorders, particularly obesity, type 2 diabetes, and cardiovascular diseases. Thus play a major role not only in their development, but also exerts as a crucial linking factor among those diseases. In this regard, one of the strategies for tackling this problem is application of antioxidants to treat such diseases. The present study was performed to evaluate the synergistic effects of punicic acid (PUA) and alpha-lipoic acid (ALA) as antioxidants and radical scavenging reagents on the expression of some inflammatory and metabolism-related genes under oxidative stress in the muscle cells. The experimental treatments consisted of a range of 20, 40, 80, 160, and 320 µM of PUA, and 5, 25, 50, 100, and 200 µM of ALA with a 200 µM concentration of H2 O2 as an oxidative stress inducer. Accordingly, fatty acid treatments were applied for 24 h, and H2 O2 was treated for 1 h. Our results indicated that the simultaneous treatment of PUA and ALA at optimal concentrations (80 and 50 µM, respectively) decreased the expression of inflammation genes and increased the expression of regulatory genes (Pparγ, Pgc-1α) related to metabolism (p < .05). Unexpectedly, H2 O2 treatment increased the Fndc5 expression (p < .05). Maximal upregulation of Pparγ, Pgc-1α were obtained when fatty acids combination (PUA and ALA) were used in the culture of H2 O2 treated cells (p < .05). Therefore, our findings suggest that the simultaneous use of PUA and ALA fatty acids could reduce oxidative stress, and the expression of inflammatory genes, thereby improving the cell metabolism.

Revisiting the hydrolysis of ampicillin catalyzed by Temoneira-1 β-lactamase, and the effect of Ni(II), Cd(II) and Hg(II).

β-Lactamases grant resistance to bacteria against β-lactam antibiotics. The active center of TEM-1 β-lactamase accommodates a Ser-Xaa-Xaa-Lys motif. TEM-1 β-lactamase is not a metalloenzyme but it possesses several putative metal ion binding sites. The sites composed of His residue pairs chelate borderline transition metal ions such as Ni(II). In addition, there are many sulfur-containing donor groups that can coordinate soft metal ions such as Hg(II). Cd(II) may bind to both types of the above listed donor groups. No significant change was observed in the circular dichroism spectra of TEM-1 β-lactamase on increasing the metal ion content of the samples, with the exception of Hg(II) inducing a small change in the secondary structure of the protein. A weak nonspecific binding of Hg(II) was proven by mass spectrometry and 119m Hg perturbed angular correlation spectroscopy. The hydrolytic process of ampicillin catalyzed by TEM-1 β-lactamase was described by the kinetic analysis of the set of full catalytic progress curves, where the slow, yet observable conversion of the primary reaction product into a second one, identified as ampilloic acid by mass spectrometry, needed also to be considered in the applied model. Ni(II) and Cd(II) slightly promoted the catalytic activity of the enzyme while Hg(II) exerted a noticeable inhibitory effect. Hg(II) and Ni(II), applied at 10 μM concentration, inhibited the growth of E. coli BL21(DE3) in M9 minimal medium in the absence of ampicillin, but addition of the antibiotic could neutralize this toxic effect by complexing the metal ions.

Interactive effect of air pollutant and meteorological factors on seasonal influenza transmission, Shanghai, China

BackgroundPrevious studies have provided evidence of seasonal influenza transmission linked to various meteorological and environmental factors. However, the joint effect from both air pollutant exposure and meteorological factors that modify the seasonal influenza incidence is not well discussed. MethodsData on weekly laboratory-confirmed influenza positive case numbers were obtained from Pudong New Area, Shanghai, China between January 1, 2011 and December 31, 2019. Weekly average weather data (absolute humidity (AH), minimum temperature (Min Temp) and particulate matter <2.5 μm (PM2.5) concentration levels) at the same period were collected from Copernicus Atmosphere Monitoring Services (CAMS).A General additive model (GAM) and distributed lag nonlinear model (DLNM) with Quasi-Poisson likelihood were used to evaluate the independent and interaction effect of weather factors and PM2.5 on seasonal influenza transmission along with lag effects. ResultsOur research showed that both AH and Min Temp had U-shape interactive relationships with PM2.5 at high PM2.5 concentration level. Interaction between PM2.5 and AH had positive relative risk (RR) effect at lag of 5 weeks when AH = 5 g/m3and 15 g/m3, as well as the same lag period when Min Temp = 5 °C and 20 °C. ConclusionOur study found that an interactive effect between air pollutant and weather factors exists and significantly affects influenza transmission. For future studies, lag interactive effects among air pollutant and temperature or humidity should be considered in the evaluation of the environment-influenza association, as such interactions might change the disease response to short-term effects.

Macrolide and Ketolide Antibiotics Inhibit the Cytotoxic Effect of Trastuzumab Emtansine in HER2-Positive Breast Cancer Cells: Implication of a Potential Drug-ADC Interaction in Cancer Chemotherapy.

Macrolides are widely used for the long-term treatment of infections and chronic inflammatory diseases. The pharmacokinetic features of macrolides include extensive tissue distribution because of favorable membrane permeability and accumulation within lysosomes. Trastuzumab emtansine (T-DM1), a HER2-targeting antibody-drug conjugate (ADC), is catabolized in the lysosomes, where Lys-SMCC-DM1, a potent cytotoxic agent, is processed by proteinase degradation and subsequently released from the lysosomes to the cytoplasm through the lysosomal membrane transporter SLC46A3, resulting in an antitumor effect. We recently demonstrated that erythromycin and clarithromycin inhibit SLC46A3 and attenuate the cytotoxicity of T-DM1; however, the effect of other macrolides and ketolides has not been determined. In this study, we evaluated the effect of macrolide and ketolide antibiotics on T-DM1 cytotoxicity in a human breast cancer cell line, KPL-4. Macrolides used in the clinic, such as roxithromycin, azithromycin, and josamycin, as well as solithromycin, a ketolide under clinical development, significantly attenuated T-DM1 cytotoxicity in addition to erythromycin and clarithromycin. Of these, azithromycin was the most potent inhibitor of T-DM1 efficacy. These antibiotics significantly inhibited the transport function of SLC46A3 in a concentration-dependent manner. Moreover, these compounds extensively accumulated in the lysosomes at the levels estimated to be 0.41-13.6 mM when cells were incubated with them at a 2 μM concentration. The immunofluorescence staining of trastuzumab revealed that azithromycin and solithromycin inhibit the degradation of T-DM1 in the lysosomes. These results suggest that the attenuation of T-DM1 cytotoxicity by macrolide and ketolide antibiotics involves their lysosomal accumulation and results in their greater lysosomal concentrations to inhibit the SLC46A3 function and T-DM1 degradation. This suggests a potential drug-ADC interaction during cancer chemotherapy.

Wrinkled MXene-modified screen-printed electrodes for highly efficient sarcosine detection

MXenes are gaining immense attention owing to their robust conductivity, presence of favorable surface-functional groups and excellent hydrophilicity. Herein, a simple solution-based approach was adopted to functionalize few-layer thick MXene-Ti3C2Tx with chitosan (Chi) molecules, creating a stable electro-active screen-printed electrode (SPE) platform for the detection of sarcosine (SAR), a new biomarker for prostate cancer. In comparison to pristine MXene, the chitosan-functionalized Ti3C2Tx (MX-Chi/SPE) offered favorable surface functionalization for easy immobilization of sarcosine oxidase (SOx) enzymes, which led to the improved enzymatic oxidation of SAR. The detection mechanism relied upon the efficient reduction of enzymatically produced H2O2 over MX-Chi/SPE, enabling selective detection of SAR both in low (0.1–0.3 µM) and high (1.5–3.5 µM) concentration ranges, with an estimated limit of detection (LOD) values of 0.012 µM and 0.14 µM (S/N = 3), respectively. Due to its robust enzymatic configuration, the biosensor enabled selective detection of SAR even in the presence of interfering species like ascorbic acid, uric acid, and glucose. Furthermore, the biosensor demonstrated excellent signal repeatability and stability and effectively recovered spiked SAR levels (98–106%) from artificial urine samples. This approach demonstrated the successful integration of MXenes as redox-active materials in clinical biosensors with a simple electrode preparation procedure, which has the potential to be extended to other MXene compositions.

Repurposing Synthetic Congeners of a Natural Product Aurone Unveils a Lead Antitumor Agent Inhibiting Folded P-Loop Conformation of MET Receptor Tyrosine Kinase.

A library of 24 congeners of the natural product sulfuretin were evaluated against nine panels representing nine cancer diseases. While sulfuretin elicited very weak activities at 10 µM concentration, congener 1t was identified as a potential compound triggering growth inhibition of diverse cell lines. Mechanistic studies in HCT116 colon cancer cells revealed that congener 1t dose-dependently increased levels of cleaved-caspases 8 and 9 and cleaved-PARP, while it concentration-dependently decreased levels of CDK4, CDK6, Cdc25A, and Cyclin D and E resulting in induction of cell cycle arrest and apoptosis in colon cancer HCT116 cells. Mechanistic study also presented MET receptor tyrosine kinase as the molecular target mediating the anticancer activity of compound 1t in HCT116 cells. In silico study predicted folded p-loop conformation as the form of MET receptor tyrosine kinase responsible for binding of compound 1t. Together, the current study presents compound 1t as an interesting anticancer lead for further development.

Pyrogallol-based dipodal optical probe as new smart analytical tool for sustainable detection of cobalt in biosystem

The present research focuses on the micro-level detection of cobalt ions in biological and environmental samples using a new probe. The probe is a multifunctional symmetrical dipodal molecule with two pyrogallol binding units attached to the malonate scaffold through a propylene spacer. It was synthesized and characterized by 1H NMR, 13C NMR, IR, electronic spectroscopy, and mass spectrometry. The molecule's binding, thermodynamic, and photophysical properties are also described. The designed probe demonstrates an excellent sensing ability for Co(II) based on the ESIPT “OFF-ON” fluorescence mechanism. The experiments explore the high selectivity of the ligand for cobalt sensing over a wide range of metal ions of biological and environmental importance. The fluorescence intensity shows a linear response to Co(II) in 5–100 μM concentration with a detection limit of 8.75 x 10−5 and a 2.65-fold enhancement in the intensity. These results establish its potential application as a fluorescence sensor. The probe is also employed as a colorimetric sensor for the qualitative determination of cobalt ions in DMSO solution. The interesting behavior of the probe motivated us further to study its coordination properties with divalent cobalt in solution. The pre-organized assembly with an appropriate cavity size favors the ligand for an efficient Co(II) encapsulation by coordinating through imine-Ns and aromatic ring-Os donors, giving high formation constants.