<b>Background and Objective:</b> Horseshoe crabs are widely used in both traditional and modern pharmaceutical applications. Most of the previous studies on horseshoe crabs focused on their blood which contains hemolymph and amoebocyte lysate. This study aimed to determine the potential antibacterial and antifouling properties of different extracts from the carapace and the book gills of <i>Carcinoscorpius rotundicauda</i>. <b>Materials and Methods:</b> The crude extracts were subjected to the bioactivity tests using the disc-diffusion and the inhibition of biofilm-formation measurement assays, for both the antibacterial and antifouling activities respectively. <b>Results:</b> The results obtained indicated that the carapace extracts had stronger antibacterial and antifouling effects compared to the book gills extracts. Extracts obtained from the male displayed more activity compared to the extracts from the female with a few exceptions. Methanol and acetone carapace crude extracts showed the best overall performance. A sterol compound was isolated from the carapace acetone extracts of the male of <i>C. rotundicauda</i>. However, the compound did not display strong activity compared to the crude extract. The compound might be contributing to the observed activity with other components through a synergistic effect. <b>Conclusion:</b> The presence of antibacterial and antifouling activities in the carapace and book gills extracts could be added to the complexity of the defence mechanisms of horseshoe crabs. The results of this study, therefore, may contribute to the knowledge of the defence mechanisms of <i>C. rotundicauda</i>. Further research is needed to determine the bioactivities of other parts of the animal and to explore their potential applications.
In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
Constitutive secretion is predominantly measured by collecting the media from cells and performing plate-based assays. This approach is particularly sensitive to changes in cell number, and a significant amount of effort has to be spent to overcome this. We have developed a panel of quantitative flow cytometry-based assays and reporter cell lines that can be used to measure constitutive secretion. These assays are insensitive to changes in cell number making them very robust and well suited to functional genomic and chemical screens. Here, we outline the key steps involved in generating and using these assays for studying constitutive secretion.
Introduction: Cervical cancer is a leading cause of death in women. Current cancer treatment comes with side effects. Clinacanthus nutans has been known traditionally to treat cancer. This study was aimed to characterize C. nutans standardized fraction (SF1) and to investigate its anticancer mechanism against SiHa cells.
Materials and Methods: SF1 was produced by optimized methodology for bioassay-guided fractionation. Fourier transform infrared (FTIR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) were carried out to characterize the SF1. SF1 was screened for cytotoxicity activity toward HeLa, SiHa, and normal cells (NIH) cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The anticancer mechanism of SF1 was evaluated toward SiHa cells, which showed highest cytotoxicity toward SF1 treatment. The mechanism includes cell cycle progression and protein expression, which was detected using specific antibody-conjugated fluorescent dye, p53-FITC, by flow cytometry.
Results: Major constituents of SF1 were alkaloids with amines as functional group. SF1 showed highest cytotoxic activity against SiHa (half-maximal inhibitory concentration [IC50] < 10 µg/mL) compared to HeLa cells. Cytoselectivity of SF1 was observed with no IC50 detected on normal NIH cells. On flow cytometry analysis, SF1 was able to induce apoptosis on SiHa cells by arresting cell cycle at G1/S and upregulation of p53 protein.
Conclusion: SF1 showed anticancer activity by inducing apoptosis through arrested G1/S cell cycle checkpoint-mediated mitochondrial pathway.
Multiple dinoflagellate species from the genus Karlodinium have been well known to form massive and toxic blooms that consequently cause fish kills in many coastal waters around the world. Karlodinium australe is a mixotrophic and potentially ichthyotoxic species associated with fish kills. Here, we investigated phagotrophy of K. australe (isolate KaJb05) established from a bloom event in the West Johor Strait, Malaysia, using several prey species (phytoplankton, zooplankton, and larval fish). The results showed that K. australe ingested relatively small prey cells of co-occurring microalgae by direct engulfment, while it fed on larger prey cells of microalgae by tube feeding. The results of animal exposure bioassays using rotifer (Brachionus plicatilis), brine shrimp (Artemia salina), and larval fish (Oryzias melastigma) demonstrated that phagotrophy (in terms of the trophic mode of the dinoflagellate), or micropredation (in terms of the mechanism of lethal effects on prey), played a more important role than the toxicity did in causing the lethal effects of K. australe on these aquatic animals under low cell densities of K. australe, while the mortalities of animals observed in the exposure to cell lysates of K. australe were solely caused by the toxicity. A comparison of the lethal effects between K. australe and K. veneficum revealed that the lethal effect of K. australe on rotifers was much stronger than that of K. veneficum at all cell densities applied in the experiments and the more "aggressive" micropredation of K. australe is suggested to explain the difference in lethal effect between K. austale and K. veneficum. Our results may explain why K. australe exhibited fish killings during moderate blooms at cell densities < 2.34 × 106 cells L-1, whereas K. veneficum was observed to cause massive fish kills only if the cell density was above 107 cells L-1. We believe these findings provide new insights into the ecological consequences of phagotrophy exhibited in some mixotrophic and harmful algae such as species of Karlodinium and of HAB events in general.
Gastrointestinal nematode infections can cause great losses in revenue due to decrease livestock production and animal death. The use of anthelmintic to control gastrointestinal nematode put a selection pressure on nematode populations which led to emergence of anthelmintic resistance. Because of that, this study was carried out to investigate the efficacy of aqueous and methanol extract of Cassia siamea against the motility of C. elegans Bristol N2 and C. elegans DA1316. Caenorhabditis elegans Bristol N2 is a susceptible strain and C. elegans DA1316 is an ivermectin resistant strain. In vitro bioassay of various concentrations of (0.2, 0.6, 0.8, 1.0 and 2.0 mg mL-1) aqueous and methanol extracts of C. siamea was conducted against the motility of L4 larvae of C. elegans Bristol N2 and C. elegans DA1316. The L4 larvae were treated with 0.02 μg mL-1 of ivermectin served as positive control while those in M9 solution served as negative control. The activity of the extracts was observed after 24 h and 48 h. A significant difference was recorded in the extract performance compared to control at (P < 0.001) after 48 h against the motility of the larvae of both strains. The methanol extracts inhibited the motility of C. elegans Bristol N2 by 86.7% as well as DA1316 up to 84.9% at 2.0 mg mL-1 after 48 h. The methanol extract was more efficient than aqueous extract (P < 0.05) against the motility of both strains of C. elegans. Cassia siamea may be used as a natural source of lead compounds for the development of alternative anthelmintic against parasitic nematodes as well ivermectin resistant strains of nematodes.
The aim of this study was to develop an in vitro assay for use in place of in vivo assays of snake venom lethality and antivenom neutralizing potency. A novel in vitro assay has been developed based on the binding of post-synaptically acting α-neurotoxins to nicotinic acetylcholine receptor (nAChR), and the ability of antivenoms to prevent this binding. The assay gave high correlation in previous studies with the in vivo murine lethality tests (Median Lethal Dose, LD50), and the neutralization of lethality assays (Median Effective Dose, ED50) by antisera against Naja kaouthia, Naja naja and Bungarus candidus venoms. Here we show that, for the neurotoxic venoms of 20 elapid snake species from eight genera and four continents, the in vitro median inhibitory concentrations (IC50s) for α-neurotoxin binding to purified nAChR correlated well with the in vivo LD50s of the venoms (R2 = 0.8526, p < 0.001). Furthermore, using this assay, the in vitro ED50s of a horse pan-specific antiserum against these venoms correlated significantly with the corresponding in vivo murine ED50s, with R2 = 0.6896 (p < 0.01). In the case of four elapid venoms devoid or having a very low concentration of α-neurotoxins, no inhibition of nAChR binding was observed. Within the philosophy of 3Rs (Replacement, Reduction and Refinement) in animal testing, the in vitro α-neurotoxin-nAChR binding assay can effectively substitute the mouse lethality test for toxicity and antivenom potency evaluation for neurotoxic venoms in which α-neurotoxins predominate. This will greatly reduce the number of mice used in toxicological research and antivenom production laboratories. The simpler, faster, cheaper and less variable in vitro assay should also expedite the development of pan-specific antivenoms against various medically important snakes in many parts of the world.
The present study focused on investigating the storage stability of oil-in-water (O/W) emulsions with high oil volume fractions prepared with palm olein-based diacylglycerol oil (POL-DAG)/soybean oil (SBO) blends at 25 °C. The incorporation of different ratios of oil blends significantly influenced (p < 0.05) the texture, color, droplet size distribution, and rheological parameters of the emulsions. Only emulsions incorporated with 10% to 20% POL-DAG in oil phase exhibited pseudoplastic behavior that fitted the Power Law model well. Furthermore, the O/W emulsions prepared with POL-DAG/SBO blends exhibited elastic properties, with G' higher than G". During storage, the emulsion was found to be less solid-like with the increase in tan δ values. All emulsions produced with POL-DAG/SBO blends also showed thixotropic behavior. Optical microscopy revealed that the POL-DAG incorporation above 40% caused aggregated droplets to coalesce and flocculate and, thus, larger droplet sizes were observed. The current results demonstrated that the 20% POL-DAG substituted emulsion was more stable than the control emulsion. The valuable insights gained from this study would be able to generate a lot more possible applications using POL-DAG, which could further sustain the competitiveness of the palm oil industry.
Red palm weevil (RPW), Rhynchophorus ferrugineus, is a polyphagous insect that caused economic damage in various palm species, particularly coconut plantation in Malaysia. Therefore, entomopathogenic fungus Metarhizium anisopliae was being introduced in attempts to control biologically the RPW. The entomopathogenicity of an indigenous (Met-Gra4) and foreign (Met-TH) strains of M. anisopliae isolated from the soil of Malaysia and Thailand, were tested against RPW adults in laboratory bioassays at 50, 70, 90% relative humidity (RH). Bioassays indicate no significance differences in efficacy between both the conidia of M. anisopliae strains against RPW adults. Met-Gra4 showed the highest efficacy at 90% RH (LT50 = 6.17 days). However, LT50 only slightly differed from Met-TH (6.33 days; 90% RH). Scanning el ectron microscopy for the treated RPWs showed that Met-Gra4 (90% RH) was densely sporulated within the abdomen, while Met-TH can be found mainly across cuticular surface of RPW.
Toxicity Identification Evaluation (TIE) is a useful method for the classification and identification of toxicants in a composite environment water sample. However, its extension to a larger sample size has been restrained owing to the limited throughput of toxicity bioassays. Here we reported the development of a high-throughput method of TIE Phase I. This newly developed method was assisted by the fluorescence-based cellular oxidation (CO) biosensor fabricated with roGFP2-expressing bacterial cells in 96-well microplate format. The assessment of four river water samples from Langat river basin by this new method demonstrated that the contaminant composition of the four samples can be classified into two distinct groups. The entire toxicity assay consisted of 2338 tests was completed within 12 h with a fluorescence microplate reader. Concurrently, the sample volume for each assay was reduced to 50 μL, which is 600 to 4700 times lesser to compare with conventional bioassays. These imply that the throughput of the CO biosensor-assisted TIE Phase I is now feasible for constructing a large-scale toxicity monitoring system, which would cover a whole watershed scale.
Stereospermum fimbriatum or locally known as "Chicha" is traditionally used for itchy skin, earache, stomachache and postpartum treatments. This study was designed to evaluate the antimicrobial potential of S. fimbriatum's stem bark against 11 pathogens and isolate its bioactive compound. Successive soxhlet extraction was conducted using n-hexane, dichloromethane (DCM) and methanol. Disc diffusion, minimum inhibitory and bactericidal concentration (MIC & MBC) assays were done to examine the antimicrobial activity. Bioassay-guided isolation was conducted on S. fimbriatum's extract. The DCM extract of stem bark (DS) was the most potent extract followed by n-hexane extract of the stem bark (NS). A novel compound was isolated and coded as C1 which demonstrated potent antibacterial effects with the MIC values as low as 3.13 µg/mL to 6.25 µg/mL, against S. epidermidis, MRSA and S. aureus. Thus, S. fimbriatum could be a potential source of antimicrobial agents for the treatment of skin infections, specifically, MRSA.
A high-performance thin-layer chromatography (HPTLC) method combined with effect-directed-analysis (EDA) was developed to screen the antioxidant, neuroprotective and antidiabetic effects in essential oils derived from lavender flower, lemon myrtle, oregano, peppermint, sage, and rosemary leaves (Lamiaceae family). HPTLC hyphenated with microchemical (DPPH•, p-anisaldehyde, and ferric chloride) derivatizations, was used to evaluate antioxidant activity, presence of phytosterols and terpenoids, and polyphenolic content, while the combination with biochemical (α-amylase and acetylcholine esterase (AChE) enzymatic) derivatizations was used to asses α-amylase and AChE inhibitory activities. The superior antioxidant activity of oregano leaf extract is attributed to the presence of high levels of aromatic compounds, like polyphenolic acids. The strongest α-amylase inhibition was observed in lemon myrtle and rosemary plus extracts due to the presence of monoterpenes. Rosemary and sage extracts exhibit the highest AChE inhibition activity, with 1 μL essential oils being more potent than the recommended daily dose of donepezil. This superior neuroprotection was attributed to the presences of di- and triterpenes that displayed strong AChE inhibition and antioxidant potential in DPPH• free radical assay. Antioxidant activity was related to phenolic content (R = 0.49), while α-amylase inhibitory activity was positively related to antioxidant activity (R = 0.20) and terpenoid/sterol content (R = 0.31). AChE inhibitory activity was correlated (R = 0.80) to the combined effect of phenolics and terpenoids. Thus, the superior AChE inhibitory and neuroprotection potential of rosemary and sage essential oils could be attributed to joint effects of main phenolic and terpene constituents. The hyphenated HPTLC method provided rapid bioanalytical profiling of highly complex essential oil samples.
Herbal plants are traditionally utilized to treat various illnesses. They contain phytochemicals that can be extracted using conventional methods such as maceration, soxhlet, and boiling, as well as non-conventional methods including ultrasonic, microwave, and others. Carica papaya leaves have been used for the treatment of dengue, fungal, and bacterial infections as well as an ingredient in anti-aging products. Phytochemicals analysis detected the presence of kaempferol, myricetin, carpaine, pseudocarpaine, dehydrocarpaine I and II, ferulic acid, caffeic acid, chlorogenic acid, β-carotene, lycopene, and anthraquinones glycoside. Conventional preparation by boiling and simple maceration is practical, simple, and safe; however, only polar phytochemicals are extracted. The present study aims to investigate the effects of three different non-conventional extraction techniques (ultrasonic-assisted extraction, reflux, and agitation) on C. papaya phytochemical constituents, the antioxidant capacity, and wound-healing activities. Among the three techniques, the reflux technique produced the highest extraction yield (17.86%) with the presence of saponins, flavonoids, coumarins, alkaloids, and phenolic metabolites. The reflux technique also produced the highest 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging with an IC50 value of 0.236 mg/mL followed by ultrasonic-assisted extraction (UAE) (IC50: 0.377 mg/mL) and agitation (IC50: 0.404 mg/mL). At tested concentrations (3.125 µg/mL to 500 µg/mL), all extracts do not exhibit a cytotoxicity effect on the human skin fibroblast, HSF1184. Interestingly, reflux and UAE were active fibroblast proliferators that support 85% (12.5 µg/mL) and 41% (6.25 µg/mL) better cell growth, respectively. Additionally, during the early 24 h of the scratch assay, the migration rate at 12.5 µg/mL was faster for all extracts with 51.8% (reflux), 49.3% (agitation), and 42.5% (UAE) as compared to control (21.87%). At 48 h, proliferated cells covered 78.7% of the scratch area for reflux extract, 63.1% for UAE, 61% for agitation, and 42.6% for control. Additionally, the collagen synthesis was enhanced for 31.6% and 65% after 24 and 48 h of treatment for reflux. An HPLC-MS/MS-QTOF (quadruple time-of-flight) analysis of reflux identified nine phytochemicals, including carpaine, kaempferol 3-(2G-glucosylrutinoside), kaempferol 3-(2″-rhamnosylgalactoside), 7-rhamnoside, kaempferol 3-rhamnosyl-(1->2)-galactoside-7-rhamnoside, luteolin 7-galactosyl-(1->6)-galactoside, orientin 7-O-rhamnoside, 11-hydroperoxy-12,13-epoxy-9-octadecenoic acid, palmitic amide, and 2-hexaprenyl-6-methoxyphenol. The results suggested that reflux was the best technique as compared to ultrasonic and agitation.
The continuous and sole dependence on imidazolinone (IMI) herbicides for weedy rice control has led to the evolution of herbicide resistance in weedy rice populations across various countries growing IMI herbicide-resistant rice (IMI-rice), including Malaysia. A comprehensive study was conducted to elucidate occurrence, level, and mechanisms endowing resistance to IMI herbicides in putative resistant (R) weedy rice populations collected from three local Malaysian IMI-rice fields. Seed bioassay and whole-plant dose-response experiments were conducted using commercial IMI herbicides. Based on the resistance index (RI) quantification in both experiments, the cross-resistance pattern of R and susceptible (S) weedy rice populations and control rice varieties (IMI-rice variety MR220CL2 and non-IMI-rice variety MR219) to imazapic and imazapyr was determined. A molecular investigation was carried out by comparing the acetohydroxyacid synthase (AHAS) gene sequences of the R and S populations and the MR220CL2 and MR219 varieties. The AHAS gene sequences of R weedy rice were identical to those of MR220CL2, exhibiting a Ser-653-Asn substitution, which was absent in MR219 and S plants. In vitro assays were conducted using analytical grade IMI herbicides of imazapic (99.3%) and imazapyr (99.6%) at seven different concentrations. The results demonstrated that the AHAS enzyme extracted from the R populations and MR220CL2 was less sensitive to IMI herbicides than that from S and MR219, further supporting that IMI herbicide resistance was conferred by target-site mutation. In conclusion, IMI resistance in the selected populations of Malaysian weedy rice could be attributed to a Ser-653-Asn mutation that reduced the sensitivity of the target site to IMI herbicides. To our knowledge, this study is the first to show the resistance mechanism in weedy rice from Malaysian rice fields.
Many researchers have focused chitosan as a source of potential bioactive material during the past few decades. However, chitosan has several drawbacks to be utilised in biological applications, including poor solubility under physiological conditions. Therefore, a new interest has recently emerged on partially hydrolysed chitosan, chitosan oligosaccharides (COS). In this study, degradation of chitosan was performed by Cellulase from Trichoderma reesei® 1.5L and Response Surface Methodology (RSM) were employed to optimize the hydrolysis temperature, pH, enzyme concentration and substrate concentration. Optimization of cellulase T. reesei® using central composite design (CCD) was to obtain optimum parameters and all the factors showed significant effects (p˂0.05). The maximum response, Celluclast® activity (1.268 U) was obtained by assaying the process at 49.79oC, pH 4.5, 3% (v/w) of enzyme concentration and 25% (w/v) concentration of chitosan for 24 hours.
T4 genotype Acanthamoeba are opportunistic pathogens that cause two types of infections, including vision-threatening Acanthamoeba keratitis (AK) and a fatal brain infection known as granulomatous amoebic encephalitis (GAE). Due to the existence of ineffective treatments against Acanthamoeba, it has become a potential threat to all contact lens users and immunocompromised patients. Metal nanoparticles have been proven to have various antimicrobial properties against bacteria, fungi, and parasites. Previously, different types of cobalt nanoparticles showed some promise as anti-acanthamoebic agents. In this study, the objectives were to synthesize and characterize the size, morphology, and crystalline structure of cobalt phosphate nanoparticles, as well as to determine the effects of different sizes of cobalt metal-based nanoparticles against A. castellanii. Cobalt phosphate octahydrate (CHP), Co3(PO4)2•8H2O, was synthesized by ultrasonication using a horn sonicator, then three different sizes of cobalt phosphates Co3(PO4)2 were produced through calcination of Co3(PO4)2•8H2O at 200 °C, 400 °C and 600 °C (CP2, CP4, CP6). These three types of cobalt phosphate nanoparticles were characterized using a field emission scanning electron microscope (FESEM), energy dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD) analysis. Next, the synthesized nanoparticles were subjected to biological assays to investigate their amoebicidal, amoebistatic, anti-encystation, and anti-excystation effects against A. castellanii, as well as cell cytotoxicity. The overall results showed that 1.30 ± 0.70 µm of CHP microflakes demonstrated the best anti-acanthemoebic effects at 100 µg/mL, followed by 612.50 ± 165.94 nm large CP6 nanograins. However, amongst the three tested cobalt phosphates, Co3(PO4)2, the smaller nanoparticles had stronger antiamoebic effects against A. castellanii. During cell cytotoxicity analysis, CHP exhibited only 15% cytotoxicity against HeLa cells, whereas CP6 caused 46% (the highest) cell cytotoxicity at the highest concentration, respectively. Moreover, the composition and morphology of nanoparticles is suggested to be important in determining their anti-acathamoebic effects. However, the molecular mechanisms of cobalt phosphate nanoparticles are still unidentified. Nevertheless, the results suggested that cobalt phosphate nanoparticles hold potential for development of nanodrugs against Acanthamoeba.
Rice fungal pathogens, responsible for severe rice yield loss and biotoxin contamination, cause increasing concerns on environmental safety and public health. In the paddy environment, we observed that the asymptomatic rice phyllosphere microenvironment was dominated by an indigenous fungus, Aspergillus cvjetkovicii, which positively correlated with alleviated incidence of Magnaporthe oryzae, one of the most aggressive plant pathogens. Through the comparative metabolic profiling for the rice phyllosphere microenvironment, two metabolites were assigned as exclusively enriched metabolic markers in the asymptomatic phyllosphere and increased remarkably in a population-dependent manner with A. cvjetkovicii. These two metabolites evidenced to be produced by A. cvjetkovicii in either a phyllosphere microenvironment or artificial media were purified and identified as 2(3H)-benzofuranone and azulene, respectively, by gas chromatography coupled to triple quadrupole mass spectrometry and nuclear magnetic resonance analyses. Combining with bioassay analysis in vivo and in vitro, we found that 2(3H)-benzofuranone and azulene exerted dissimilar actions at the stage of infection-related development of M. oryzae. A. cvjetkovicii produced 2(3H)-benzofuranone at the early stage to suppress MoPer1 gene expression, leading to inhibited mycelial growth, while azulene produced lately was involved in blocking of appressorium formation by downregulation of MgRac1. More profoundly, the microenvironmental interplay dominated by A. cvjetkovicii significantly blocked M. oryzae epidemics in the paddy environment from 54.7 to 68.5% (p < 0.05). Our study first demonstrated implication of the microenvironmental interplay dominated by indigenous and beneficial fungus to ecological balance and safety of the paddy environment.
Human skin pigmentation is a result of constitutive and facultative pigmentation. Facultative pigmentation is frequently stimulated by UV radiation, pharmacologic drugs, and hormones whereby leads to the development of abnormal skin hyperpigmentation. To date, many state-of-art depigmenting compounds have been studied using in vitro model to treat hyperpigmentation problems for cosmetic dermatological applications; little attention has been made to compare the effectiveness of these depigmenting compounds and their mode of actions. In this present article, new and recent depigmenting compounds, their melanogenic pathway targets, and modes of action are reviewed. This article compares the effectiveness of these new depigmenting compounds to modulate several melanogenesis-regulatory enzymes and proteins such as tyrosinase (TYR), TYR-related protein-1 (TRP1), TYR-related protein-2 (TRP2), microphthalmia-associated transcription factor (MITF), extracellular signal-regulated kinase (ERK) and N-terminal kinases (JNK) and mitogen-activated protein kinase p38 (p38 MAPK). Other evidences from in vitro assays such as inhibition on melanosomal transfer, proteasomes, nitric oxide, and inflammation-induced melanogenesis are also highlighted. This article also reviews analytical techniques in different assays performed using in vitro model as well as their advantages and limitations. This article also provides an insight on recent finding and re-examination of some protocols as well as their effectiveness and reliability in the evaluation of depigmenting compounds. Evidence and support from related patents are also incorporated in this present article to give an overview on current patented technology, latest trends, and intellectual values of some depigmenting compounds and protocols, which are rarely highlighted in the literatures.
This study highlighted the performance of the developed integrated loop-mediated isothermal amplification (LAMP) coupled with a colorimetric DNA-based magnetogenosensor. The biosensor operates through a DNA hybridization system in which a specific designed probe captures the target LAMP amplicons. We demonstrated the magnetogenosensor assay by detecting pathogenic Leptospira, which causes leptospirosis. The color change of the assay from brown to blue indicated a positive result, whereas a negative result was indicated by the assay maintaining its brown color. The DNA biosensor was able to detect DNA at a concentration as low as 200 fg/μl, which is equivalent to 80 genomes/reaction. The specificity of the biosensor assay was 100% when it was evaluated with 172 bacterial strains. An integrated LAMP and probe-specific magnetogenosensor was successfully developed, promising simple and rapid visual detection in clinical diagnostics and service as a point-of-care device.
Reversible valves on centrifugal microfluidic platforms facilitate the automation of bioanalytical assays, especially of those requiring a series of steps (such as incubation) in a single reaction chamber. In this study, we present fixed elastic reversible (FER) valves and tunable elastic reversible (TER) valves that are easy to fabricate, implement and control. In the FER valve the compression of an elastic barrier/patch against a microchamber's outlet prevents the release of liquid. The valve sealing pressure was determined by adjusting the engraving depth of the valve-seat at which the elastic patch was located, this allows to set the sealing pressure during disc fabrication. In the TER valve, the patch compression value and sealing pressure is controlled by the penetration depth of a plastic screw into the valve-seat. The ER valves prevent liquid flow until the centrifugal force overcomes their sealing pressure. Moreover, at a constant spin speed, turning the screw of a TER valve reduces its sealing pressure and opens the valve. Therefore, the TER valve allows for controlling of the liquid transfer volume at various spin speeds. The FER and TER valves' behavior is mathematically described and equations for the prediction of their operation under centrifugal forces are provided. As a point-of-care (POC) application of ER valves, we have developed a microfluidic disc with a series of TER valves and peptide microarrays for automated multiplexed detection of five different proteins from a single serum sample.