Free Buffer Research Articles

Undulating Free Energy Landscapes Buffer Redox Chains from Environmental Fluctuations.

Roller-coaster or undulating free energy landscapes, with alternating high and low potential cofactors, occur frequently in biological redox chains. Yet, there is little understanding of the possible advantages created by these landscapes. We examined the tetraheme subunit associated with Blastochloris viridis reaction centers, comparing the dynamics of the native protein and of hypothetical (in silico) mutants. We computed the variation in the total number of electrons in wild type (WT) and mutant tetrahemes connected to an electron reservoir in the presence of a time-varying potential, as a model for a fluctuating redox environment. We found that roller-coaster free energy landscapes buffer the redox cofactor populations from these fluctuations. The WT roller-coaster landscape slows forward and backward electron transfer in the face of fluctuations, and may offer the advantage of sustaining the reduction of essential cofactors, such as the chlorophyll special pair in photosynthesis, even though an undulating landscape introduces thermodynamically uphill steps in multistep redox chains.

Purinergic agonists increase [Ca2+]i in rat conjunctival goblet cells through ryanodine receptor type 3.

ATP and benzoylbenzoyl-ATP (BzATP) increase free cytosolic Ca2+ concentration ([Ca2+]i) in conjunctival goblet cells (CGCs) resulting in mucin secretion. The purpose of this study was to investigate the source of the Ca2+i mobilized by ATP and BzATP. First-passage cultured rat CGCs were incubated with Fura-2/AM, and [Ca2+]i was measured under several conditions with ATP and BzATP stimulation. The following conditions were used: 1) preincubation with the Ca2+ chelator EGTA, 2) preincubation with the SERCA inhibitor thapsigargin (10-6 M), which depletes ER Ca2+ stores, 3) preincubation with phospholipase C (PLC) or protein kinase A (PKA) inhibitor, or 4) preincubation with the voltage-gated calcium channel antagonist nifedipine (10-5 M) and the ryanodine receptor (RyR) antagonist dantrolene (10-5 M). Immunofluorescence microscopy (IF) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to investigate RyR presence in rat and human CGCs. ATP-stimulated peak [Ca2+]i was significantly lower after chelating Ca2+i with 2 mM EGTA in Ca2+-free buffer. The peak [Ca2+]i increase in CGCs preincubated with thapsigargin, the PKA inhibitor H89, nifedipine, and dantrolene, but not the PLC inhibitor, was reduced for ATP at 10-5 M and BzATP at 10-4 M. Incubating CGCs with dantrolene alone decreased [Ca2+]i and induced CGC cell death at a high concentration. RyR3 was detected in rat and human CGCs with IF and RT-qPCR. We conclude that ATP- and BzATP-induced Ca2+i increases originate from the ER and that RyR3 may be an essential regulator of CGC [Ca2+]i. This study contributes to the understanding of diseases arising from defective Ca2+ signaling in nonexcitable cells.NEW & NOTEWORTHY ATP and benzoylbenzoyl-ATP (BzATP) induce mucin secretion through an increase in free cytosolic calcium concentration ([Ca2+]i) in conjunctival goblet cells (CGCs). The mechanisms through which ATP and BzATP increase [Ca2+]i in CGCs are unclear. Ryanodine receptors (RyRs) are fundamental in [Ca2+]i regulation in excitable cells. Herein, we find that ATP and BzATP increase [Ca2+]i through the activation of protein kinase A, voltage-gated calcium channels, and RyRs, and that RyRs are crucial for nonexcitable CGCs' Ca2+i homeostasis.

Time-dependent effects of tumor necrosis factor α on Ca2+-dependent secretion in murine small intestinal organoids.

Intestinal organoids are stem cell-derived, 3D "mini-guts" with similar functions as the native intestinal epithelium such as electrolyte transport or establishment of an epithelial barrier. During intestinal inflammation, epithelial functions are dysregulated by proinflammatory cytokines like tumor necrosis factor α (TNFα) and other messengers from the immune system resulting in a loss of electrolytes and water due to an impaired epithelial barrier and higher net secretion. A murine small intestinal organoid model was established to study (long-term) effects of TNFα on the intestinal epithelium in vitro using live imaging, immunohistochemical staining and qPCR. TNFα induced apoptosis in intestinal organoids as indicated by an increased number of cells with immunoreactivity for cleaved caspase 3. Furthermore, TNFα exposure led to swelling of the organoids which was inhibited by bumetanide and was concomitant with an upregulation of the bumetanide-sensitive Na+-K+-2Cl- symporter 1 (NKCC1) as shown by qPCR. Fura-2 imaging experiments revealed time-dependent changes in Ca2+ signaling consisting of a rise in the basal cytosolic Ca2+ concentration at day 1 and an increase of the carbachol-induced Ca2+ response after 3days TNFα exposure. This was prevented by preincubation with La3+, an inhibitor of non-selective cation channels, or by using a Ca2+-free buffer indicating an enhancement of the Ca2+ influx from the extracellular side by the cytokine. No significant changes in cDNA levels of epithelial barrier proteins could be observed in the presence of TNFα. Intestinal organoids are a useful tool to study the mechanism underlying the TNFα-induced secretion on enterocytes such as the regulation of NKCC1 expression or the modulation of cellular Ca2+ signaling.

Study on the health risk of cyanuric acid in swimming pool water and its prevention and control measures.

Cyanuric acid is a widely used fine chemical intermediate that acts as a free chlorine buffer in swimming pool water, wherein it is often used as a stabilizer to maintain the germicidal efficacy of chlorinated disinfectants. However, it has also been associated with health risks. Herein, we introduced the sources and functions of cyanuric acid in swimming pool water, focusing on potential health risks associated with excessive concentration of the component and the current control standards worldwide. Also, the prevention and control measures were summarized in terms of physical chemistry, biodegradation, and ultraviolet radiation to provide a basis for the development of public health policies for swimming pool management.

Knowledge, attitude and practices of farmers and experts about the effects of pesticide residues on agricultural product users and ecosystems: A case of Fogera District, Ethiopia

Pesticides are chemicals used to control different types of pests. Though pesticides played a role in improving the quantity and quality of production, they have been threatening ecosystems and posed effects on humans in different parts of the world. Unfortunately, there were no studies made about the effects of pesticide residues on ecosystems and consumers in the Fogera District of Amhara Region, Ethiopia. Hence, the main objective of this study was to understand the knowledge, attitude, and practices of respondents about the effects of pesticide residues on ecosystems and consumers. A cross-sectional survey complemented by focus group discussions and field observations was used to gather the required data for the study. The close-ended data were analyzed using descriptive statistics, logistic regressions, and independent t-test, and data from open-ended questions were grouped and summarized based on their similarities. The findings of the study confirmed that there was significant knowledge, attitude, and practices difference between farmers and consumers about the effects of pesticide residues on ecosystems and humans. Farmers used highly toxic pesticides to control pests and improve the glossiness of vegetables and khat. Though they didn’t use the sprayed vegetables for their home consumption, some of the farmers deliberately supplied pesticide-sprayed vegetables without worrying about the negative effects of the pesticides on the consumers. There were also fishing practices from rivers after intoxicating the fish using the pesticide sprayed feed. This, in turn, might poison individuals who consume the fish. In general, pesticide application practices and consumption of pesticide-sprayed foodstuffs and surface water might pose serious health risks to ecosystems and humans. To minimize the negative effects of pesticides, rigorous awareness-raising on the effects and management of pesticides, enforcement of laws, delineation of the pesticide free buffer zone for waters, the establishment of a clear pesticide supply chain to the end users, ecosystem assessment and food safety monitoring schemes are highly required.

Round Robin based Arbitration Mechanism for Signaling Approach based Router Architecture

In Network-on-Chip the effectiveness of the network resource allocation is demonstrated by the flow control mechanism. There are two types of flow control mechanisms: buffered and bufferless. Compared to buffered flow control methods, buffer less flow control mechanisms are easier to use, need less power, and take up less space. When there are congestion and resource conflicts, it experiences higher packet loss and packet misrouting inside the network. A good buffered control mechanism useful as it overcomes the limitations of buffer less mechanism. There are numerous buffered and bufferless flow control methods available. In this paper, signaling-based Virtual Output Queue Router Arbiter Mechanism is used to explore credit-based flow control. This mechanism worked on new concept that is “stress value”. This information is generated in the form of credit whenever any input buffer has free space. Then, using this credit data, the node's stress value is determined. Free buffer space takes precedence over stress value if it is bigger. The stress value will increase if there is less available buffer space. To handle the congestion problem, the signaling block then sends this stress value to a neighboring router. To help the arbitrator make a more accurate decision, the crediting system constantly operates in tandem with arbitration.

A fixed 20:1 combination of cafedrine/theodrenaline increases cytosolic Ca2+ concentration in human tracheal epithelial cells via ryanodine receptor-mediated Ca2+ release

Mucociliary clearance is a pivotal physiological mechanism that protects the lung by cleaning the airways from pollution and colonization, thereby preventing infection. Ciliary function is influenced by various signal transduction cascades, and Ca2+ represents a key second messenger. A fixed 20:1 combination of cafedrine and theodrenaline has been widely used to treat perioperative hypotension and emergency hypotensive states since the 1960s; however, its effect on the intracellular Ca2+ concentration ([Ca2+]i) of respiratory epithelium remains unknown. Therefore, human tracheal epithelial cells were exposed to the clinically applied 20:1 mixture of cafedrine/theodrenaline and the individual substances separately. [Ca2+]i was assessed by FURA-2 340/380 fluorescence ratio. Pharmacological inhibitors were applied to elucidate relevant signal transduction cascades, and reverse transcription polymerase chain reaction (RT-PCR) was performed on murine tracheal epithelium to analyze ryanodine receptor (RyR) subtype expression. All three pharmacological preparations instantaneously induced a steep increase in [Ca2+]i that quickly returned to its baseline value despite the persistence of each substance. Peak [Ca2+]i following the administration of 20:1 cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone increased in a dose-dependent manner, with median effective concentrations of 0.35 mM (7.32 mM cafedrine and 0.35 mM theodrenaline), 3.14 mM, and 3.45 mM, respectively. When extracellular Ca2+ influx was inhibited using a Ca2+-free buffer solution, the peak [Ca2+]i following the administration of cafedrine alone and theodrenaline alone were reduced but not abolished. No alteration in [Ca2+]i compared with baseline [Ca2+]i was observed during β-adrenergic receptor inhibition. Depletion of caffeine-sensitive stores and inhibition of RyR, but not IP3 receptors, completely abolished any increase in [Ca2+]i. However, [Ca2+]i still increased following the depletion of mitochondrial Ca2+ stores using 2,4-dinitrophenol. RT-PCR revealed RyR-2 and RyR-3 expression on murine tracheal epithelium. Although our experiments showed that cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone release Ca2+ from intracellular stores through mechanisms that are exclusively triggered by β-adrenergic receptor stimulation, which most probably lead to RyR activation, clinical plasma concentrations are considerably lower than those used in our experiments to elicit an increase in [Ca2+]i; therefore, further studies are needed to evaluate the ability of cafedrine/theodrenaline to alter mucociliary clearance in clinical practice.

Oxidized Phosphatidylcholines Trigger TRPA1 and Ryanodine Receptor-dependent Airway Smooth Muscle Contraction.

Asthma pathobiology includes oxidative stress that modifies cell membranes and extracellular phospholipids. Oxidized phosphatidylcholines (OxPCs) in lung lavage from allergen-challenged human participants correlate with airway hyperresponsiveness and induce bronchial narrowing in murine thin-cut lung slices. OxPCs activate many signaling pathways, but mechanisms for these responses are unclear. We hypothesize that OxPCs stimulate intracellular free Ca2+ flux to trigger airway smooth muscle contraction. Intracellular Ca2+ flux was assessed in Fura-2-loaded, cultured human airway smooth muscle cells. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) induced an approximately threefold increase in 20 kD myosin light chain phosphorylation. This correlated with a rapid peak in intracellular cytoplasmic Ca2+ concentration ([Ca2+]i) (143 nM) and a sustained plateau that included slow oscillations in [Ca2+]i. Sustained [Ca2+]i elevation was ablated in Ca2+-free buffer and by TRPA1 inhibition. Conversely, OxPAPC-induced peak [Ca2+]i was unaffected in Ca2+-free buffer, by TRPA1 inhibition, or by inositol 1,4,5-triphosphate receptor inhibition. Peak [Ca2+]i was ablated by pharmacologic inhibition of ryanodine receptor (RyR) Ca2+ release from the sarcoplasmic reticulum. Inhibiting the upstream RyR activator cyclic adenosine diphosphate ribose with 8-bromo-cyclic adenosine diphosphate ribose was sufficient to abolish OxPAPC-induced cytoplasmic Ca2+ flux. OxPAPC induced ∼15% bronchial narrowing in thin-cut lung slices that could be prevented by pharmacologic inhibition of either TRPA1 or RyR, which similarly inhibited OxPC-induced myosin light chain phosphorylation in cultured human airway smooth muscle cells. In summary, OxPC mediates airway narrowing by triggering TRPA1 and RyR-mediated mobilization of intracellular and extracellular Ca2+ in airway smooth muscle. These data suggest that OxPC in the airways of allergen-challenged subjects and subjects with asthma may contribute to airway hyperresponsiveness.

Reliable Data Delivery Using Fuzzy Reinforcement Learning in Wireless Sensor Networks

Wireless sensor networks (WSNs) has been envisioned as a potential paradigm in sensing technologies. Achieving energy efficiency in a wireless sensor network is challenging since sensor nodes have confined energy. Due to the multi-hop communication, sensor nodes spend much energy re-transmitting dropped packets. Packet loss may be minimized by finding efficient routing paths. In this research, a routing using fuzzy logic and reinforcement learning procedure is designed for WSNs to determine energy-efficient paths; to achieve reliable data delivery. Using the node’s characteristics, the reward is determined via fuzzy logic. For this paper, we employ reinforcement learning to improve the rewards, computed by considering the quality of the link, available free buffer of node, and residual energy. Further, simulation efforts have been made to illustrate the proposed mechanism’s efficacy in energy consumption, delivery delay of the packets, number of transmissions, and lifespan.

A free base porphyrin as an effective modifier of the cathode interlayer for organic solar cells

Herein, we demonstrate the co-optimization of power conversion efficiency (PCE) and air stability of the organic solar cells (OSCs) by free porphyrin-based buffer of zinc oxide (ZnO) cathode. By combining first-principle calculations and experimental measurements, we investigated the adsorption behaviors and working mechanism of free base porphyrin (TPP) and zinc porphyrin (ZnTPP) on ZnO cathode. The N atoms of TPP are capable of forming Zn—N coordination with the Zn atom of ZnO substrate, thereby generating interfacial dipoles and reducing the work function of ITO/ZnO cathode, which is beneficial for the charge transfer in the OSCs. More importantly, the large adsorption energy between ZnO and TPP, as well as the good compatibility between TPP and the organic layer, enables TPP to act as a “molecular glue” between the inorganic interface and the organic active layers, which improves not only the PCE but also the stability of the devices under high humidity air environment. In contrast, the already coordinated porphyrin ZnTPP has no such positive effects on the device performance when used as ZnO buffer. The results are of great significance for the study of the working mechanism of interlayer buffers in OSCs, and are also of guidance for designing the buffer molecules.

Is Your Henderson–Hasselbalch Calculation of Buffer pH Correct?

A survey of 21 free online buffer calculators and apps found only 2 that correctly calculated pH under conditions where [H+] and [OH–] cannot be ignored in the ratio term of the Henderson–Hasselbalch equation. Traditional guidelines for when use of formal concentrations is acceptable were found to be flawed. For acidic (pKa ≤ 7) buffers, the use of formal concentrations is valid when FA–/Ka > 20. For alkaline (pKa ≥ 7) buffers, the approximation is valid when FBH+/Kb > 20. Robust equations for predicting pH of acidic and alkaline buffers are presented, which could be used with programmable calculators and should be used for online buffer calculators and apps.

Application of High Kinetic Energy Buffer Control Technology after Free Recoil

Aiming at the problem of high recoil kinetic energy caused by launching long-range guided projectiles, the main effect and Pareto of barrel diameter, projectile mass, initial velocity and charge on recoil kinetic energy are studied based on Latin hypercube test design under certain bore pressure conditions, which provides reference for the evaluation and control of high recoil kinetic energy brought by new ammunition. At the same time, in order to solve the difficulty of free recoil high kinetic energy buffer braking, four mathematical models and technical schemes of recoil buffer braking are introduced and designed, and the buffer characteristics are compared and analyzed. Based on the analysis of reducing the buffer force and the length of recoil motion, a free recoil test bench scheme for the combination of blocking cables and recoil buffer devices is also proposed. The recoil braking force is planned based on NCGA intelligent optimization algorithm. The results show that the combined buffer braking principle is feasible and the buffer effect is good.

DynaQ: Enabling Protocol-Independent Service Queue Isolation in Cloud Data Centers

Switches in cloud data centers support multiple service queues per port to provide differentiated network performance among different traffic classes. To isolate service queues, recent solutions leverage the power of Explicit Congestion Notification (ECN). However, this causes a fundamental dependency on ECN-based transport protocols, making it hard to use generic transport protocols. To this end, we design DynaQ, a protocol-independent multi-queue management solution that enables service queue isolation with generic transport protocols. The key idea of DynaQ is to adjust the packet dropping threshold of service queues dynamically. Specifically, DynaQ allows a service queue to occupy free buffer space but prevents the queue from hurting other active queues. Our solution requires only a few additional clock cycles to implement on hardware. To evaluate DynaQ comprehensively, we conduct a series of testbed experiments and large-scale simulations. Our evaluation results show that, compared to alternative schemes, DynaQ is the only solution that achieves work-conserving weighted fair sharing and low latency without protocol dependency.

On the discontinuous precipitation during recrystallization in a multicomponent alloy

Discontinuous precipitation (DP) reactions are among the most important solid-state phenomena that occur during the migration of grain boundaries (GBs) in many engineering alloys. Migration involves solute redistribution across the moving GB and the solute diffusion assumed to occur only along it. In this work, we show the atomic scale compositional analysis across a migrating GB during recrystallization in a multicomponent alloy that reveals evidence of bulk/lattice diffusion in a precipitate free buffer zone ahead of the RF, and solute segregations at local interfaces that aid to determine the rate-limiting solutes for DP reaction kinetics.

Achieving Per-Flow Fairness and High Utilization With Limited Priority Queues in Data Center

Modern data centers often host multiple applications with diverse network demands. To provide fair bandwidth allocation to several thousand traversing flows, Approximate Fair Queueing (AFQ) utilizes multiple priority queues in switch to approximate ideal fair queueing. However, due to limited number of queues in programmable switches, AFQ easily experiences high packet loss and low link utilization. In this paper, we propose Elastic Fair Queueing (EFQ), which leverages limited priority queues to flexibly achieve both high network utilization and fair bandwidth allocation. EFQ dynamically assigns the free buffer space in priority queues for each packet to obtain high utilization without sacrificing flow-level fairness. The results of simulation experiments and real implementations show that EFQ reduces the average flow completion time by up to 82% over the state-of-the-art fair bandwidth allocation mechanisms.

Therapeutic Benefits of Pomegranate Flower Extract: A Novel Effect That Reduces Oxidative Stress and Significantly Improves Diastolic Relaxation in Hyperglycemic In Vitro in Rats.

The pomegranate flower is an ancient herb in traditional Chinese medicine with multiple properties. Recent studies have shown that pomegranate flower extract is beneficial, especially for hyperglycemia. In this experiment, we investigated the diastolic effect of pomegranate flower polyphenol (PFP) extract on the isolated thoracic aorta of rats in both the absence and presence of high glucose levels. Isotonic contractile forces were recorded from aortic rings (about 3 mm in length) from rats using the BL-420F Biological Function Test System. Tissues were precontracted with 60 mM KCl to obtain maximum tension under 1.0 g load for 1 hour before the balance was achieved, and the fluid was changed every 15 minutes. PFP (700 mg/L–900 mg/L) showed a concentration-dependent relaxant effect on the aortic rings; vasodilation in the endothelium-intact was significantly higher than that in the de-endothelialized segments (P < 0.01). The endothelium-dependent vasorelaxant effect of PFP was partially attenuated by K+ channel blockers, tetraethylammonium (TEA), glibenclamide (Glib), and BaCl2, as well as L-NAME (eNOS inhibitor) on the denuded endothelium artery ring. Concentration-dependent inhibition of PFP on releasing intracellular Ca2+ in the Ca2+-free solution and vasoconstriction of CaCl2 in Ca2+-free buffer plus K+ (60 mM) was observed. In addition, PFP (0.1–10 mg/L) showed significant inhibition of acetylcholine-induced endothelial-dependent relaxation in the aorta of rats in the presence of high glucose (44 mmol/L). Nevertheless, the vasodilating effect of PFP was inhibited by atropine and L-NAME. The results indicated that PFP-induced vasodilation was most likely related to the antioxidant effects through enhanced NO synthesis, as well as the blocking of K+ channels and inhibition of extracellular Ca2+ entry. In conclusion, these observations showed that PFP ameliorates vasodilation in hyperglycemic rats. Hence, our results suggest that PFP supplementation may be beneficial for hypertensive patients with diabetes.

VANET Clustering-Based Routing Protocol for Vehicular Ad-Hoc Network Using Two Metaheuristic Algorithms

Vehicular ad hoc networks (VANET) are a special model of mobile ad-hoc networks (MANETs)and have many applications in safe driving, and entertainment by exchanging information between vehicles (e.g. road conditions, weather, congestion, etc.). the problem is that these Nodes(vehicles) in VANETs have very high mobility and due to this dynamic nature and the instability of wireless links between vehicles, providing an efficient and reliable routing method is a serious challenge for VANETs. Therefore, in order to meet this challenge and maintain the necessary information exchange between vehicles without loss, deletion, or access late, it is necessary to solve this problem by (designing an efficient and secure routing protocol for the exchange of important data in VANET). In this paper two algorithms were used together (Harris Hawks optimization algorithm (HHO) and Artificial Bee Colony algorithm (ABC)), taking into account the best parameters such as (the amount of free buffer size, distance between nodes, speed of nodes, node degree, and the quality of link connection) to improve the results compared to the rest of the articles and HIR. the simulation results indicated that the methodology improves other algorithms with Relevance in terms of parameters of packet delivery rate, throughput, and end-to-end delay.

A stilbene derivative as dual-channel fluorescent probe for mitochondrial G-quadruplex DNA in living cells

G-quadruplex DNA has attracted the widespread attention as a novel target of anticancer strategy. Herein, two novel stilbene derivatives 2a and 2b were designed and synthesized under mild reaction conditions, and their interactions with G-quadruplex DNA, cytotoxicity, and distribution in living cells were investigated in detail. Both compounds display a low cytotoxicity and the higher affinity to G-quadruplex DNA than to the other secondary structures, including duplex, single-stranded and i-motif DNA, moreover, the affinity of 2b with m-allyl pyridine salt group to G-quadruplex DNA is about 10-fold stronger than that of 2a with p-allyl pyridine salt group. The interactions of the compounds with the promoter G-quadruplexes are enthalpy-driven by an ITC assay. 2a and 2b not only stabilize the G-quadruplex structure but also induce the G-rich sequences (bcl-2, HRCC and KSS) to fold into the mixed-type G-quadruplex in Na+/K+ free Tris-HCl buffer at pH 7.0, and 2b presents the higher stabilization to G-quadruplex than 2a by a FRET-melting assay. 2b presents a dual-emission at 508 and 600 nm and gives a turn-on and stronger and more sensitive fluorescence response over 2a to the promoter (bcl-2, c-kit 2 and c-myc) and mitochondrial (HRCC and KSS) G-quadruplex DNA at both emission wavelengths, moreover, the peak at 508 nm is blue-shifted to 466 nm after binding to DNA. The blue and red dual-channel CLSM images indicate that 2b is mainly distributed in the mitochondrion of living HepG2 cells. The results show that 2b is a potential dual-channel fluorescent probe for mitochondrial G-quadruplex DNA in living cells.

Crescent-Shaped Carbazole Derivatives as Light-Up Fluorescence Probes for G-Quadruplex DNA and Live Cell Imaging.

In recent years, studies on the organic small molecule fluorescent probes targeting G-quadruplexes have attracted a wide attention, because G-quadruplexes play an important role in various biological functions. Herein, we reported three crescent-shaped carbazole derivatives (4a-4c) and studied their interactions with the single-stranded, duplex, G-quadruplex and i-motif DNA. Both 4b and 4c have an above 100 nm Stokes shift and low fluorescence intensity, moreover, they exhibit the stronger affinity to G-quadruplex than to the other DNA structures. 4b with a cyanovinyl pyridine salt group displays a specific light-up fluorescence response to G-quadruplexes. FRET and CD results suggest that both 4b and 4c are able to improve the stability of G-quadruplexes and maintain their topology, moreover, they induce G-rich sequences (bcl-2, HTG, and KSS) to fold into G-quadruplexes in Na+ /K+ free buffer. In addition, CLSM images suggest that 4b and 4c are mainly distributed in the mitochondrion of living HepG2 cells, and a weak fluorescence signal is also observed in the nucleus for 4c. Given that the two compounds have the larger association constants to G-quadruplexes over to duplex and single-stranded DNA, we speculate that the fluorescence signals in cells may mainly be attributed to the compound/G-quadruplex DNA complexes.

ANALYSIS OF SPERMATOZOA QUALITY USING PERCOLL DENSITY GRADIENT CENTRIFUGATION THROUGH THE ADMINISTRATION OF PHOSPHOLIPID + EGTA

The Percoll density gradient centrifugation (PDGC) method is frequently used in the sexing of spermatozoa. However, this method causes damage to the spermatozoa membranes, resulting in a decreased quality of spermatozoa. We analysed the impacts of phospholipid PC (phosphatidylcholine) and EGTA (ethylene glycol bis (β-aminoethyl ether) N,N,N',N'-tetraacetic acid) Ca2+ free buffer on the quality of bovine spermatozoa after the PDGC process, using semen from Friesian Holstein (FH) bulls aged 5 - 8 years. The following variables were observed: spermatozoa motility, spermatozoa viability, spermatozoa membrane integrity, spermatozoa that have not experienced capacitation, spermatozoa that have experienced capacitation and spermatozoa that have undergone acrosomal reaction. The results showed that the administration of phospholipid PC + EGTA Ca2+ free buffer to spermatozoa, followed by the PDGC process, could maintain or further improve the values of all variables. In the PDGC process, phospholipid PC 10% + EGTA Ca2+ free buffer at 1 mM was most suitable.