Effects of yeast culture supplementation on milk yield, rumen fermentation, metabolism, and bacterial composition in dairy goats.

The effects of yeast culture (YC) on dairy goat milk yield and potential effects of rumen microbial population changes on rumen fermentation are poorly understood. This study aimed to evaluate the effects of YC on milk yield and rumen fermentation in dairy goats and explore the potential microbial mechanisms. Forty Laoshan dairy goats with a weight of 51.23 ± 2.23 kg and daily milk yield of 1.41 ± 0.26 kg were randomly divided into 4 groups: control (no YC), YC1 (10 g/day per goat), YC2 (25 g/day per goat), and YC3 (40 g/day per goat). The pre-feeding period was 15 days, and the official period was 60 days. Laoshan dairy goats were milked twice daily, and the individual milk yield was recorded. On the last day of the official period, rumen fluid was collected to measure rumen fermentation, perform quantitative polymerase chain reaction (PCR), and detect metabolites. Compared to the control group, the YC group had greater milk yield; higher acetic acid, butyric acid, and total volatile fatty acid contents; and lower ammonia-N (NH3-N) content in the rumen (p < 0.05). YC increased the abundance of Clostridia_UCG-014 and Paraprevotella (p < 0.05). Differential metabolites L-leucine and aspartic acid were screened. This study revealed the microbial mechanisms linking the relative abundance of Paraprevotella and Clostridia_UCG-014 to L-leucine and aspartic acid utilization. These results describe the potential benefits of supplementing 10 g/day per goat YC in the diets of Laoshan dairy goats for improving the rumen environment and milk yield.

Effects of Ginger Straw Silage with Enzymes on Growth Performance, Digestion and Metabolism, Meat Quality and Rumen Microflora Diversity of Laiwu Black Goat.

Laiwu black goats comprise an excellent local germplasm resource; however, a shortage of feed resources has led to the application of unconventional feed. Ginger straw feed has good physiological effects, but research on this feed source for ruminant animals is lacking. The aim of this study was to determine the effects of enzymatic silage ginger straw on Laiwu black goat performance. The experiment used an independent sample t-test analysis method; 24 healthy Laiwu black goats with a body weight of 20.05 ± 1.15 kg and age of 5.67 ± 0.25 months were randomly divided into two groups with three replicates (bars) per group and four goats per replicate. The experimental diet was composed of mixed concentrate, silage, and garlic peel at a 2:7:1 ratio. The silage used in the two groups was whole corn silage (CON group) and 60% whole corn silage plus 40% enzymatic silage ginger straw (SG group), and the other components were identical. Daily feed intake/daily gain (F/G) was significantly higher in the SG group than in the CON group (p < 0.05), but there were no significant differences in dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) digestibility between the groups. The shear force, cooking loss, centrifugal loss, and pressure loss of the longissimus dorsi muscle group were significantly lower in the SG than in the CON group (p < 0.05). Compared with those in the CON group, the serum and liver total antioxidant capacity was significantly increased in the SG group, and in the liver, the O2·-, malondialdehyde, and OH· contents were significantly decreased. Collectively, the rumen fluid microbial diversity was changed in the SG group. It was concluded that enzymatic silage ginger straw usage instead of 40% whole silage corn as feed for Laiwu black goats can significantly improve the muscle quality, antioxidant capacity, and intestinal flora, with no adverse effects on production performance. In conclusion, our study provides a basis for ginger straw processing and storage and its rational application in the Laiwu black goat diet.

Density Functional Theory Calculation May Confirm Arsenic-Thiol Adhesion as the Primary Mechanism of Arsenical Toxicity.

Previously, it was believed that methylation was the body's primary method to detoxify inorganic arsenic. However, recent research has shown that the metabolized intermediate known as MMAIII is more toxic than arsenite and arsenate, contradicting a previous understanding. Another important question arises: is arsenical toxicity truly caused by arsenic binding to proteins through arsenic thiol adhesion? Based on the toxicity order of the experiment, with MMAIII being the most toxic, followed by arsenite, arsenate, DMAV, and MMAV, density functional theory (DFT) calculations can provide a straightforward assessment of this issue. Our practice captures all the transition states associated with a specific imaginary-frequency vibration mode, including proton transfer and simultaneous departure of leaving group. We have obtained the energy barriers for five arsenicals reacting with thiol, alcohol, and amine separately. In addition to energetic favorability, the following are the energy barriers for arsenic's reaction with thiol ranked from low to high: MMAIII (25.4 kcal/mol), arsenite (27.7 kcal/mol), arsenate (32.8 kcal/mol), DMAV (36.2 kcal/mol), and MMAV (38.3 kcal/mol). Results show that the toxicity of arsenicals is mainly caused by their reaction with thiol rather than with alcohol or amine, as supported by the trend of decreasing toxicity and increasing energy barriers. Thus, this DFT calculation may confirm the paradigm that arsenic-thiol adhesion is the primary cause of arsenic toxicity in the body.

Antibiotic Resistance of Bacillus cereus in Plant Foods and Edible Wild Mushrooms in a Province.

Bacillus cereus is a common pathogen causing foodborne diseases, secreting and producing a large number of toxins that can cause a variety of diseases and pose many threats to human health. In this study, 73 strains of Bacillus cereus were isolated and identified from six types of foods from seven different cities in a province, and the antibiotic-resistant phenotype was detected by using the Bauer-Kirby method. Results showed that the 73 isolates were completely sensitive to gentamicin and 100% resistant to chloramphenicol, in addition to which all strains showed varying degrees of resistance to 13 other common antibiotics, and a large number of strains resistant to multiple antibiotics were found. A bioinformatic analysis of the expression of resistance genes in Bacillus cereus showed three classes of antibiotic-resistant genes, which were three of the six classes of antibiotics identified according to the resistance phenotype. The presence of other classes of antibiotic-resistant genes was identified from genome-wide information. Antibiotic-resistant phenotypes were analyzed for correlations with genotype, and remarkable differences were found among the phenotypes. The spread of antibiotic-resistant strains is a serious public health problem that requires the long-term monitoring of antimicrobial resistance in Bacillus cereus, and the present study provides important information for monitoring antibiotic resistance in bacteria from different types of food.

Contamination of Plant Foods with Bacillus cereus in a Province and Analysis of Its Traceability.

Bacillus cereus is an important zoonotic foodborne conditional pathogen. It is found in vegetables, dairy products, rice, and other foods, thereby greatly endangering human health. Investigations on B. cereus contamination in China primarily focus on raw milk, dairy products, meat, and others, and limited research has been conducted on plant-based foodstuffs. The rapid development of sequencing technology and the application of bioinformatics-related techniques means that analysis based on whole-genome sequencing has become an important tool for the molecular-epidemiology investigation of B. cereus. In this study, we investigated the contamination of B. cereus in six types of commercially available plant foods from eight regions of a province. The molecular epidemiology of the isolated B. cereus was analyzed by whole-genome sequencing. We aimed to provide fundamental data for the surveillance and epidemiology analysis of B. cereus in food products in China. The rapid traceability system of B. cereus established in this study can provide a basis for rapid molecular epidemiology analysis of B. cereus, as well as for the prevention and surveillance of B. cereus. Moreover, it can also be expanded to monitoring and rapid tracing of more foodborne pathogens.

Deep Learning-Based Automatic Duckweed Counting Using StarDist and Its Application on Measuring Growth Inhibition Potential of Rare Earth Elements as Contaminants of Emerging Concerns.

In recent years, there have been efforts to utilize surface water as a power source, material, and food. However, these efforts are impeded due to the vast amounts of contaminants and emerging contaminants introduced by anthropogenic activities. Herbicides such as Glyphosate and Glufosinate are commonly known to contaminate surface water through agricultural industries. In contrast, some emerging contaminants, such as rare earth elements, have started to enter the surface water from the production and waste of electronic products. Duckweeds are angiosperms from the Lemnaceae family and have been used for toxicity tests in aquatic environments, mainly those from the genus Lemna, and have been approved by OECD. In this study, we used duckweed from the genus Wolffia, which is smaller and considered a good indicator of metal pollutants in the aquatic environment. The growth rate of duckweed is the most common endpoint in observing pollutant toxicity. In order to observe and mark the fronds automatically, we used StarDist, a machine learning-based tool. StarDist is available as a plugin in ImageJ, simplifying and assisting the counting process. Python also helps arrange, manage, and calculate the inhibition percentage after duckweeds are exposed to contaminants. The toxicity test results showed Dysprosium to be the most toxic, with an IC50 value of 14.6 ppm, and Samarium as the least toxic, with an IC50 value of 279.4 ppm. In summary, we can provide a workflow for automatic frond counting using StarDist integrated with ImageJ and Python to simplify the detection, counting, data management, and calculation process.

A New Duplex Recombinase Polymerase Amplification (D-RPA) Method for the Simultaneous and Rapid Detection of Shigella and Bacillus cereus in Food.

Shigella and Bacillus cereus are two common foodborne pathogens that cause intestinal diseases and seriously affect human life and health. Traditional microbiological culture methods are time-consuming and laborious, and polymerase chain reaction (PCR)-based methods rely on expensive thermal cyclers and lengthy reaction times. In this study, on the basis of the specific gene ipaH7 of Shigella and the virulence gene nheABC of B. cereus, a duplex detection system was established for the first time by using the recombinase polymerase amplification technique (D-RPA). After optimization, D-RPA could be effectively amplified at 42 °C for 25 min with excellent specificity, and the detection limits of D-RPA for Shigella and B. cereus in artificially contaminated samples were 2.7 × 101 and 5.2 × 102 CFU/mL, respectively. This study provides a certain research basis for multiple detection with RPA, an isothermal amplification technology. Furthermore, it lays a good foundation for high-throughput rapid detection of foodborne pathogens.

A Novel Cu2+ Quantitative Detection Nucleic Acid Biosensors Based on DNAzyme and "Blocker" Beacon.

In this paper, a "turn-off" biosensor for detecting copper (II) ions based on Cu2+-dependent DNAzyme and a "blocker" beacon were developed. Upon the copper ion being added, the Cu2+-dependent DNAzyme substrate strand was irreversibly cleaved, thereby blocking the occurrence of the ligation reaction and PCR, which inhibited the G-rich sequence from forming the G-quadruplex structure, efficiently reducing the detection signal. This method had the characteristics of strong specificity and high sensitivity compared with the existing method due to the application of ligation-dependent probe signal recognition and amplification procedures. Under the optimized conditions, this method proved to be highly sensitive. The signal decreased as the concentration of copper ions increased, exhibiting a linear calibration from 0.03125 µM to 0.5 µM and a limit of detection of 18.25 nM. Subsequently, the selectivity of this biosensor was verified to be excellent by testing different relevant metal ions. Furthermore, this detection system of copper (II) ions was successfully applied to monitor Cu2+ contained in actual water samples, which demonstrated the feasibility of the biosensor.

Effects of nonantibiotic growth promoter combinations on growth performance, nutrient utilization, digestive enzymes, intestinal morphology, and cecal microflora of broilers.

Given the ban on antibiotic growth promoters, the effects of nonantibiotic alternative growth promoter combinations (NAGPCs) on the growth performance, nutrient utilization, digestive enzyme activity, intestinal morphology, and cecal microflora of broilers were evaluated. All birds were fed pellets of two basal diets-starter (0-21 d) and grower (22-42 d)-with either enramycin (ENR) or NAGPC supplemented. 1) control + ENR; 2) control diet (CON, basal diet); 3) control + mannose oligosaccharide (MOS) + mannanase (MAN) + sodium butyrate (SB) (MMS); 4) control + MOS + MAN + Bacillus subtilis (BS) (MMB); 5) control + MOS + fruit oligosaccharide (FOS) + SB (MFS); 6) control + FOS + BS (MFB); 7) control + MOS + FOS + MAN (MFM); 8) control + MOS + BS + phytase (PT) (MBP). ENR, MOS, FOS, SB, MAN, PT, and BS were added at 100, 2,000, 9,000, 1,500, 300, 37, and 500 mg/kg, respectively. The experiment used a completely random block design with six replicates per group: 2400 Ross 308 broilers in the starter phase and 768 in the grower phase. All NAGPCs significantly improved body weight gain (P < 0.01), utilization of dry matter, organic matter, and crude protein (P < 0.05), villus height and villus height/crypt depth in the jejunum and ileum (P < 0.01), and decreased the feed conversion ratio (P < 0.01) at d 21 and 42. MMS, MMB, MFB, and MFM duodenum trypsin, lipase, and amylase activities increased significantly (P < 0.05) at d 21 and 42. On d 21 and 42, MMS, MMB, and MBP increased the abundance of Firmicutes and Bacteroides whereas MMB, MFB, and MBP decreased the abundance of Proteobacteria, compared to ENR and CON. Overall, the NAGPCs were found to have some beneficial effects and may be used as effective antibiotic replacements in broilers.

Comparison of the psychoactive activity of four primary Areca nut alkaloids in zebrafish by behavioral approach and molecular docking.

Areca palm nut (Areca catechu) has been listed as one of the most addictive substances, along with tobacco, alcohol, and caffeine. It belongs to the family Arecaceae and is widely used in Asia. Areca nut contains seven psychoactive alkaloids; however, the effects of these alkaloids on behaviors are rarely to be addressed in zebrafish. Therefore, this study aims to compare the psychoactive and potential adverse effects of four primary alkaloids (arecoline, arecaidine, guvacine, and guvacoline) isolated from areca nut on zebrafish. We found that four alkaloids induced hyperactivity-like behaviors in zebrafish larvae. Cooperating the results with the previous study, molecular docking scores suggested these alkaloids might bind to multiple muscarinic acetylcholine receptors (mAChRs), and various best binding modes were shown. According to the adult zebrafish behavioral test, arecoline was found to slightly increase the locomotor activity and caused tightening shoaling formations of adult zebrafish. Meanwhile, zebrafish exposed to arecaidine have reduced aggressiveness and conspecific social interaction. Similar to arecaidine, guvacoline treatment also caused abnormalities in zebrafish social behaviors. Furthermore, the fish displayed abnormal exploratory behaviors after being exposed to guvacoline. Interestingly, altered fear response behaviors were only displayed by guvacine-treated fish besides their lower locomotor activity. Based on the results of molecular docking, we hypothesize that the behavior alterations might be a consequence of the interaction between alkaloids and multiple mAChRs in the nervous system. In summary, our study found that each alkaloid specifically affects adult zebrafish behaviors.

Lanthanides Toxicity in Zebrafish Embryos Are Correlated to Their Atomic Number.

Rare earth elements (REEs) are critical metallic materials with a broad application in industry and biomedicine. The exponential increase in REEs utilization might elevate the toxicity to aquatic animals if they are released into the water due to uncareful handling. The specific objective of our study is to explore comprehensively the critical factor of a model Lanthanide complex electronic structures for the acute toxicity of REEs based on utilizing zebrafish as a model animal. Based on the 96 h LC50 test, we found that the majority of light REEs display lower LC50 values (4.19-25.17 ppm) than heavy REEs (10.30-41.83 ppm); indicating that they are atomic number dependent. Later, linear regression analyses further show that the average carbon charge on the aromatic ring (aromatic Cavg charge) can be the most significant electronic structural factor responsible for the Lanthanides' toxicity in zebrafish embryos. Our results confirm a very strong correlation of LC50 to Lanthanide's atomic numbers (r = 0.72), Milliken charge (r = 0.70), and aromatic Cavg charge (r = -0.85). This most significant correlation suggests a possible toxicity mechanism that the Lanthanide cation's capability to stably bind to the aromatic ring on the residue of targeted proteins via a covalent chelating bond. Instead, the increasing ionic bond character can reduce REEs' toxicity. In addition, Lanthanide toxicity was also evaluated by observing the disruption of photo motor response (PMR) activity in zebrafish embryos. Our study provides the first in vivo evidence to demonstrate the correlation between an atomic number of Lanthanide ions and the Lanthanide toxicity to zebrafish embryos.

Discovery of 7, 4'-dimethoxy-3-hydroxyflavone as a protease-activated receptor 4 antagonist with antithrombotic activity and less bleeding tendency in mice.

There is growing evidence of the importance of protease-activated receptor 4 (PAR4), one of thrombin receptors, as a therapeutic target in thrombotic cardiovascular diseases. In the present study, we utilized ligand-based virtual screening, bioassay, and structure-activity relationship study to discover PAR4 antagonists with new chemical scaffolds from natural origin, and examined their application as antiplatelet agents. By using these approaches, we have identified a flavonoid, 7, 4'-dimethoxy-3-hydroxyflavone, that exhibits anti-PAR4 activity. 7, 4'-Dimethoxy-3-hydroxyflavone inhibited PAR4-mediated human platelet aggregation, GPIIb/IIIa activation, and P-selectin secretion. Also, it inhibited PAR4 downstream signaling pathways, including Ca2+/protein kinase C, Akt, and MAP kinases ERK and p38, in human platelets, and suppressed PAR4-mediated ß-arrestin recruitment in CHO-K1 cells exogenously expressed human PAR4. In a microfluidic system, 7, 4'-dimethoxy-3-hydroxyflavone reduced thrombus formation on collagen-coated chambers at an arterial shear rate in recalcified whole blood. Furthermore, mice treated with 7, 4'-dimethoxy-3-hydroxyflavone were significantly protected from FeCl3-induced carotid arterial occlusions, without significantly affecting tail bleeding time. In conclusion, 7, 4'-dimethoxy-3-hydroxyflavone represents a new class of nature-based PAR4 antagonist, it shows effective in vivo antithrombotic properties with less bleeding tendency, and could be a potential candidate for developing new antiplatelet agents.

Genetically Modified Soybean Detection Using a Biosensor Electrode with a Self-Assembled Monolayer of Gold Nanoparticles.

In this study, we proposed a genosensor that can qualitatively and quantitatively detect genetically modified soybeans using a simple electrode with evenly distributed single layer gold nanoparticles. The DNA sensing electrode is made by sputtering a gold film on the substrate, and then sequentially depositing 1,6-hexanedithiol and gold nanoparticles with sulfur groups on the substrate. Then, the complementary to the CaMV 35S promoter (P35S) was used as the capture probe. The target DNA directly extracted from the genetically modified soybeans rather than the synthesized DNA segments was used to construct the detection standard curve. The experimental results showed that our genosensor could directly detect genetically modified genes extracted from soybeans. We obtained two percentage calibration curves. The calibration curve corresponding to the lower percentage range (1-6%) exhibits a sensitivity of 2.36 Ω/% with R2 = 0.9983, while the calibration curve corresponding to the higher percentage range (6-40%) possesses a sensitivity of 0.1 Ω/% with R2 = 0.9928. The limit of detection would be 1%. The recovery rates for the 4% and 5.7% GMS DNA were measured to be 104.1% and 102.49% with RSD at 6.24% and 2.54%. The gold nanoparticle sensing electrode developed in this research is suitable for qualitative and quantitative detection of genetically modified soybeans and can be further applied to the detection of other genetically modified crops in the future.

Evaluation of Effects of Ractopamine on Cardiovascular, Respiratory, and Locomotory Physiology in Animal Model Zebrafish Larvae.

Ractopamine (RAC) is a beta-adrenoceptor agonist that is used to promote lean and increased food conversion efficiency in livestock. This compound has been considered to be causing behavioral and physiological alterations in livestock like pig. Few studies have addressed the potential non-target effect of RAC in aquatic animals. In this study, we aimed to explore the potential physiological response after acute RAC exposure in zebrafish by evaluating multiple endpoints like locomotor activity, oxygen consumption, and cardiovascular performance. Zebrafish larvae were subjected to waterborne RAC exposure at 0.1, 1, 2, 4, or 8 ppm for 24 h, and the corresponding cardiovascular, respiratory, and locomotion activities were monitored and quantified. In addition, we also performed in silico molecular docking for RAC with 10 zebrafish endogenous ß-adrenergic receptors to elucidate the potential acting mechanism of RAC. Results show RAC administration can significantly boost locomotor activity, cardiac performance, oxygen consumption, and blood flow rate, but without affecting the cardiac rhythm regularity in zebrafish embryos. Based on structure-based flexible molecular docking, RAC display similar binding affinity to all ten subtypes of endogenous ß-adrenergic receptors, from adra1aa to adra2db, which are equivalent to the human one. This result suggests RAC might act as high potency and broad spectrum ß-adrenergic receptors agonist on boosting the locomotor activity, cardiac performance, and oxygen consumption in zebrafish. To validate our results, we co-incubated a well-known ß-blocker of propranolol (PROP) with RAC. PROP exposure tends to minimize the locomotor hyperactivity, high oxygen consumption, and cardiac rate in zebrafish larvae. In silico structure-based molecular simulation and binding affinity tests show PROP has an overall lower binding affinity than RAC. Taken together, our studies provide solid in vivo evidence to support that RAC plays crucial roles on modulating cardiovascular, respiratory, and locomotory physiology in zebrafish for the first time. In addition, the versatile functions of RAC as ß-agonist possibly mediated via receptor competition with PROP as ß-antagonist.

Pharmaceutical Assessment Suggests Locomotion Hyperactivity in Zebrafish Triggered by Arecoline Might Be Associated with Multiple Muscarinic Acetylcholine Receptors Activation.

Arecoline is one of the nicotinic acid-based alkaloids, which is found in the betel nut. In addition to its function as a muscarinic agonist, arecoline exhibits several adverse effects, such as inducing growth retardation and causing developmental defects in animal embryos, including zebrafish, chicken, and mice. In this study, we aimed to study the potential adverse effects of waterborne arecoline exposure on zebrafish larvae locomotor activity and investigate the possible mechanism of the arecoline effects in zebrafish behavior. The zebrafish behavior analysis, together with molecular docking and the antagonist co-exposure experiment using muscarinic acetylcholine receptor antagonists were conducted. Zebrafish larvae aged 96 h post-fertilization (hpf) were exposed to different concentrations (0.001, 0.01, 0.1, and 1 ppm) of arecoline for 30 min and 24 h, respectively, to find out the effect of arecoline in different time exposures. Locomotor activities were measured and quantified at 120 hpf. The results showed that arecoline caused zebrafish larvae locomotor hyperactivities, even at a very low concentration. For the mechanistic study, we conducted a structure-based molecular docking simulation and antagonist co-exposure experiment to explore the potential interactions between arecoline and eight subtypes, namely, M1a, M2a, M2b, M3a, M3b, M4a, M5a, and M5b, of zebrafish endogenous muscarinic acetylcholine receptors (mAChRs). Arecoline was predicted to show a strong binding affinity to most of the subtypes. We also discovered that the locomotion hyperactivity phenotypes triggered by arecoline could be rescued by co-incubating it with M1 to M4 mAChR antagonists. Taken together, by a pharmacological approach, we demonstrated that arecoline functions as a highly potent hyperactivity-stimulating compound in zebrafish that is mediated by multiple muscarinic acetylcholine receptors.

Dual-acting antibacterial porous chitosan film embedded with a photosensitizer.

This study proposes to develop a dual-acting antibacterial film of porous chitosan (Cs) embedded with small molecular compound, which possesses photosensitive characteristics with bactericidal efficacy, to promote the accelerated recovery of infectious wounds. The Cs/small molecular compound (Cs-cpd.2) dressing was prepared using the freeze-drying method. Characterization of the synthesized Cs-cpd.2 indicated that it has high porosity and moisture absorption effect, hence enhancing the absorption of wound exudate. Experimental results showed that Cs-cpd.2 dressing has good bactericidal and bacteriostatic effects on Staphylococcus aureus under visible-light irradiation and has antibacterial effect in the dark. It was also found that the small molecular compound does not have cytotoxicity at a dose of 0-5 µM. Furthermore, Cs-cpd.2 that contained small molecular compound with a concentration of 0.3-1 µM has positive effect on both the cell viability rate and cell proliferation rate of human fibroblast CG1639. Cs-cpd.2 can significantly promote cell proliferation when the small molecular compound and the basic fibroblast growth factor bFGF were added together. Therefore, the proposed Cs-cpd.2 dressing is feasible for photodynamic therapy (PDT) and clinical wound dressing applications.

Identifying new liver X receptor alpha modulators and distinguishing between agonists and antagonists by crystal ligand pocket screening.

Background: Modulators of LXRα are of high pharmacological interest as LXRα regulates fatty acid metabolism, inflammatory processes and cancer. We aim to identify new LXRα modulators and to recognize a distinguishable feature of agonists. Results&methodology: The ligand self-dock and largest-cavity-size searching purposely located two appropriate ligand-binding sites to reach the two aims. One is identifying the new modulators from Maybridge library. 20 new compounds are confirmed by the in vitro reporter gene assay. The other is denoting an agonist by at least one best docking pose having one hydrogen bond to LXRα Helix12 His421. Conclusion: Based on the quality x-ray binding pocket, we can identify new LXRα modulators and distinguish between agonists and antagonists by molecular docking.

Green extraction of healthy and additive free mitochondria with a conventional centrifuge.

In this research, we propose a novel centrifugal device for the massive extraction of healthy mitochondria with a centrifuge used in general laboratories within 30 minutes. The device mainly consists of two key components. One component is a microfluidic device, which is fabricated by photolithography, nickel electroforming, and polydimethylsiloxane casting, for the efficient disruption of the cell membrane. The other component is a stainless steel container, which is manufactured by computer numerical control machining, for the storage of the cell suspension. After assembly, the appropriate number of cells is pushed through the microfluidic device for cell membrane disruption by centrifugal force generated by a general laboratory centrifuge. The solution which contains cell debris and mitochondria are collected to purify the crude mitochondria via differential centrifugation. Compared with the quantity and efficiency of mitochondria isolated from the same number of cells using a conventional kit, device-extracted mitochondria show a more complete mitochondrial electron transport chain complex and a similar number of mitochondria verified by Western blot analysis of mitochondrial complexes I-V and mitochondrial outer membrane protein Tom20, respectively, as well as a normal mitochondrial structure revealed by transmission electron microscopy. Moreover, the mitochondrial membrane potential of device-extracted mitochondria stained with tetramethylrhodamine ethyl ester is higher than that of kit-extracted mitochondria. Furthermore, the coculture of device-extracted mitochondria with fibroblasts revealed that fibroblasts could uptake foreign mitochondria through endocytosis without drug treatment. These results show that the proposed microfluidic device preserves mitochondrial protein structure, membrane integrity, and membrane potential within 30 minutes of extraction and is a useful tool for therapeutic mitochondrial transplantation and regenerative medicine.

Cardiac Rhythm and Molecular Docking Studies of Ion Channel Ligands with Cardiotoxicity in Zebrafish.

Safety is one of the most important and critical issues in drug development. Many drugs were abandoned in clinical trials and retracted from the market because of unknown side effects. Cardiotoxicity is one of the most common reasons for drug retraction due to its potential side effects, i.e., inducing either tachycardia, bradycardia or arrhythmia. The zebrafish model could be used to screen drug libraries with potential cardiotoxicity in a high-throughput manner. In addition, the fundamental principles of replacement, reduction, and refinement of laboratory animal usage, 3R, could be achieved by using zebrafish as an alternative to animal models. In this study, we used a simple ImageJ-based method to evaluate and screen 70 ion channel ligands and successfully identify six compounds with strong cardiotoxicity in vivo. Next, we conducted an in silico-based molecular docking simulation to elucidate five identified compounds that might interact with domain III or domain IV of the Danio rerio L-type calcium channel (LTCC), a known pharmaceutically important target for arrhythmia. In conclusion, in this study, we provide a web lab and dry lab combinatorial approach to perform in vivo cardiotoxicity drug screening and in silico mechanistic studies.

Selective Inhibition of PAR4 (Protease-Activated Receptor 4)-Mediated Platelet Activation by a Synthetic Nonanticoagulant Heparin Analog.

Objective- PAR4 (protease-activated receptor 4), one of the thrombin receptors in human platelets, has emerged as a promising target for the treatment of arterial thrombotic disease. Previous studies implied that thrombin exosite II, known as a binding site for heparin, may be involved in thrombin-induced PAR4 activation. In the present study, a heparin octasaccharide analog containing the thrombin exosite II-binding domain of heparin was chemically synthesized and investigated for anti-PAR4 effect. Approach and Results- PAR4-mediated platelet aggregation was examined using either thrombin in the presence of a PAR1 antagonist or γ-thrombin, which selectively activates PAR4. SCH-28 specifically inhibits PAR4-mediated platelet aggregation, as well as the signaling events downstream of PAR4 in response to thrombin. Moreover, SCH-28 prevents thrombin-induced ß-arrestin recruitment to PAR4 but not PAR1 in Chinese Hamster Ovary-K1 cells using a commercial enzymatic complementation assay. Compared with heparin, SCH-28 is more potent in inhibiting PAR4-mediated platelet aggregation but has no significant anticoagulant activity. In an in vitro thrombosis model, SCH-28 reduces thrombus formation under whole blood arterial flow conditions. Conclusions- SCH-28, a synthetic small-molecular and nonanticoagulant heparin analog, inhibits thrombin-induced PAR4 activation by interfering with thrombin exosite II, a mechanism of action distinct from other PAR4 inhibitors that target the receptor. The characteristics of SCH-28 provide a new strategy for targeting PAR4 with the potential for the treatment of arterial thrombosis.