Comprehensive Embryological Analyses of Common Buckwheat (Fagopyrym esculentum Moench).

Knowledge of detailed reproductive biology of cultivated species is important as requirements for fruit and seed production allow the development of effective management strategies and a sustainable use. Embryological processes of common buckwheat (Fagopyrum esculentum Moench) are difficult to interpret due to the influence of genetic determinants, i.e., dimorphic heterostyly resulting in the production of long- and short-styled flowers, and environmental predisposition, i.e., sensitivity of ovules to thermal stress. Furthermore, the situation is complicated by overproduction of flowers and depletion of resources as the plant ages. Herein we provide protocols that allow to visualize both basic and more specific embryological features and also disturbances in sexual reproduction of common buckwheat resulting from external and internal factors. All stages of plant material fixation, preparation, staining, and observation are described and explained in detail. Technical tips and pictures of properly prepared microscopic sections are also provided.

Two-Dimensional Gel Electrophoresis in Studies of Flower and Leaf Proteome of Common Buckwheat.

Two-dimensional gel electrophoresis (2-DE) is a proteomic tool used for the separation of protein mixtures according to protein isoelectric point and molecular mass. Although gel-free quantitative and qualitative proteomic study techniques are now available, 2-DE remains a useful analytical tool. The presented protocol was performed to analyze the flower and leaf proteome of common buckwheat using 24 cm immobilized pH gradient strips (pH 4-7) and visualization of proteins on gels via colloidal Coomassie G-250 staining.

3, 5-Dihydroxy 4', 7-dimethoxyflavone-DNA interaction study for nucleic acid detection and differential cell staining.

The present study is focused on application of a natural compound, 3, 5-dihydroxy 4', 7-dimethoxyflavone (DHDM) from a medicinal plant Alpinia nigra for nucleic acid detection and differential cell staining. DHDM was found to interact with nucleic acid and forms complex, which was investigated for various applications. It was successfully utilized to visualize plasmid, genomic, and ds-linear DNA in agarose gel electrophoresis without affecting the DNA mobility in the gel. Fluorescence of DHDM increased several fold upon binding to dsDNA. Photostability of the compound was assessed and showed photobleaching effect that decreased gradually over time. Application of the compound was further extended to differential cell staining. When observed in fluorescence microscope, DHDM stained the dead cells and differentiated them from live cells in the case of bacterial, yeast, and mammalian cells. Higher concentration of the compound was found to be less cytotoxic to cancerous cells. Nucleic acid staining dyes like Ethidium bromide (EtBr), Propidium iodide (PI), etc. are carcinogens and environmental pollutants and therefore DHDM a natural compound, is a major benefit and thus can serve as an alternative to the current dyes.

In-vitro Antioxidant Activity and Flow Cytometric Analysis of Simarouba glauca DC Bark Extract Induced Apoptosis in Triple Negative Breast Cancer Cells.

OBJECTIVE: Ethnomedicinally Simarouba glauca DC is an important plant containing major class of phenols and terpenoids as bioactive compounds. The present study focuses on the evaluation of the anticancer effects of S. glauca bark UAE-EA (Ultrasonicator Assisted Extraction - Ethyl Acetate) fraction (SG-Fraction) against MDA-MB-231 triple negative breast cancer cell lines. METHODS: UAE-EA technique was used for the extraction of phytochemicals from S. glauca bark. Fractionation method was carried out to obtain Ethyl acetate fraction and PPS, TPC, and DPPH assays were performed to characterize the extract. MTT assay was then applied to analyse the viability of cells and MMP assay to confirm the initiation of drug induced apoptosis. Apoptotic morphology and quantification were assessed by DAPI and Annexin V/propidium iodide (PI) staining. RESULTS: UAE yielded 53g of crude extract in methanol. 16g Ethyl acetate fraction was obtained from fractionation. Phytoconstituents such as alkaloids, phenols, flavonoids, and triterpenoids were detected. The TPC was 278.65 mg GAE/100ml. The SG-Fraction showed maximum 66.38% RSA at 200 µg/ml and IC50 value was 101.72 µg/ml. MMP confirmed the induction of apoptosis. DAPI showed the reduction of nuclei with bright chromatin condensation, blebbing, nuclear fragmentation and apoptotic bodies. Annexin-V FITC/PI study showed 59.48% apoptosis induction. This fraction showed a similar trend of antioxidant effect as compared to ascorbic acid but, prominently lower cell viability than Camptothecin (P<0.005). In line with higher TPC in the SG-fraction, free radical scavenging activity was increased (r = 0.098**, p=0.002) and cell viability was reduced significantly (r = -0.097*** p<0.01). CONCLUSION: These results indicate that UAE-EA fraction of S. glauca bark inhibits the growth of MDA-MB-231 cells and can be considered for further neo-adjuvant chemotherapy drug research.

Effect of Moringa olifera leaves on growth and gut microbiota of Nile tilapia (Oreochromis niloticus) / Efeito das folhas da Moringa olifera no crescimento e na microbiota intestinal da tilápia do Nilo (Oreochromis niloticus)

Abstract The study was conducted to evaluate the effect of Moringa olifera on the growth and gut health of Tilapia (Oreochromis niloticus). The feed having 30% crude protein was prepared as an experimental diet with 4%, 8% and 10% M. olifera leaf supplementation, respectively. The control diet was devoid of M. olifera leaves. The 10 weeks feeding trial was carried out on 60 fish in aquaria. Fish was fed @ 3% of body weight twice a day. Diet with the high level of inclusion of M. olifera leaves significantly increased the growth rate, Survival Rate (SR), Specific Growth Rate (SGR) and Feed Conversion Efficiency (FCE) in all treatment groups compared to the control group. Similarly, Feed Conversion Ratio (FCR) gradually decreased and found highly-significant. To check the gut health of the Tilapia, random samples were selected and dissected. Nutrient agar was used as culture media to check the growth of bacteria. Pour Plate Method was used for viable colonies count by colony counter. Through staining method, the different bacteria such as Escherichia coli, Salmonella, Shigella and Pseudomonas aeruginosa were identify abundantly in the intestine of control diet fish but less number present in treatment diets groups. These results showed that M. olifera leaves up to 10% of dietary protein can be used for Nile tilapia for significant growth and healthy gut microbiota of fish.

Resumo O estudo foi conduzido para avaliar o efeito da Moringa olifera no crescimento e saúde intestinal da tilápia (Oreochromis niloticus). A ração com 30% de proteína bruta foi preparada como dieta experimental com 4%, 8% e 10% de suplementação de folhas de M. olifera, respectivamente. A dieta controle foi desprovida de folhas de M. olifera. O ensaio de alimentação de 10 semanas foi realizado em 60 peixes em aquários. O peixe pesava 3% do peso corporal duas vezes ao dia. A dieta com alto nível de inclusão de folhas de M. olifera aumentou significativamente a taxa de crescimento, taxa de sobrevivência (SR), taxa de crescimento de sobrevivência (SGR) e eficiência de conversão alimentar (FCE) em todos os grupos de tratamento em comparação com o grupo de controle. Da mesma forma, a taxa de conversão de alimentação (FCR) diminuiu gradualmente e foi considerada altamente significativa. Para verificar a saúde intestinal da tilápia, amostras aleatórias foram selecionadas e dissecadas. O ágar nutriente foi usado como meio de cultura para verificar o crescimento das bactérias. O método da placa de Verter foi usado para a contagem de colônias viáveis ​​por contador de colônias. Através do método de coloração, diferentes como Escherichia coli, Salmonella, Shigella e Pseudomonas aeruginosa foram identificados abundantemente no intestino de peixes da dieta controle, mas em menor número nos grupos de dieta de tratamento. Esses resultados mostraram que M. olifera deixa até 10% da proteína dietética e pode ser usado para tilápia do Nilo para um crescimento significativo e microbiota intestinal saudável de peixes.

Pharmacologically active herbal remedies against atherosclerosis, characterization and DoE based marker quantification by densitometry, and cell based assays on THP-1 cell lines.

Atherosclerosis is a complex condition that develops at varying rates in multiple configurations and blood vessels. The primary cause of morbidity and mortality worldwide, particularly in the industrialized nations, continues to be atherosclerosis. Ayurveda, Siddha, and Unani systems of medicine, among other traditional medical systems, utilize polyherbal compositions. The treatment of atherosclerosis has been improved with a novel multibotanical combination. In this study, we sought to formulate, characterize, and standardize a polyherbal formulation based on design of experiments (DoE), densitometric studies and to predict for antioxidant activity using molecular docking analysis based on LC- MS identified phytomarkers. In addition we have assessed its cell viability by MTT assay along with Ao/EtBr staining technique and intracellular ROS assay using THP-1 cell lines. Reported findings showed that the HPTLC based quantified components of selected multiherbals has the ability to treat for atherosclerosis. This document could be used to quickly authenticate the formulation as the method optimized was based on CCD design which shows desirability of 0.962 and 0.839. Cell based assays scientifically proves that the formulation was not toxic based on MTT assay along with AO/EtBr staining technique and has excellent antioxidant activities based on intracellular ROS assay using THP-1 cell lines. The observed findings would be crucial for future clinical aspects since the bioactive molecules contained in the extracts may have anticipated effects with other compounds and show a superior therapeutic potential. As a result, this study offers standardized and potentially therapeutic information about effective polyherbal formulation for atherosclerosis.

Chryseobacterium herbae Isolated from the Rhizospheric Soil of Pyrola calliantha H. Andres in Segrila Mountain on the Tibetan Plateau.

A non-motile, Gram-staining-negative, orange-pigmented bacterium called herbae pc1-10T was discovered in Tibet in the soil around Pyrola calliantha H. Andres' roots. The isolate thrived in the temperature range of 10-30 °C (optimal, 25 °C), pH range of 5.0-9.0 (optimum, pH = 6.0), and the NaCl concentration range of 0-1.8% (optimal, 0%). The DNA G+C content of the novel strain was 37.94 mol%. It showed the function of dissolving organophosphorus, acquiring iron from the environment by siderophore and producing indole acetic acid. Moreover, the genome of strain herbae pc1-10T harbors two antibiotic resistance genes (IND-4 and AdeF) encoding a ß-lactamase, and the membrane fusion protein of the multidrug efflux complex AdeFGH; antibiotic-resistance-related proteins were detected using the Shotgun proteomics technology. The OrthoANIu values between strains Chryseobacterium herbae pc1-10T; Chryseobacterium oleae CT348T; Chryseobacterium kwangjuense KJ1R5T; and Chryseobacterium vrystaatense R-23566T were 90.94%, 82.96%, and 85.19%, respectively. The in silico DDH values between strains herbae pc1-10T; C. oleae CT348T; C. kwangjuense KJ1R5T; and C. vrystaatense R-23566T were 41.7%, 26.6%, and 29.7%, respectively. Chryseobacterium oleae, Chryseobacterium vrystaatense, and Chryseobacterium kwangjuense, which had 16S rRNA gene sequence similarity scores of 97.80%, 97.52%, and 96.75%, respectively, were its closest phylogenetic relatives. Chryseobacterium herbae sp. nov. is proposed as the designation for the strain herbae pc1-10T (=GDMCC 1.3255 = JCM 35711), which represented a type species based on genotypic and morphological characteristics. This study provides deep knowledge of a Chryseobacterium herbae characteristic description and urges the need for further genomic studies on microorganisms living in alpine ecosystems, especially around medicinal plants.

Cell Biological Analyses of Anther Morphogenesis and Pollen Viability in Arabidopsis and Rice.

Major advances have been made in our understanding of anther developmental processes in flowering plants through a combination of genetic studies, cell biological technologies, biochemical analyses, microarray and high-throughput sequencing-based approaches. In this chapter, we summarize widely used protocols for pollen viability staining, investigation of anther morphogenesis by scanning electron microscopy (SEM), light microscopy of semi-thin sections, ultrathin section-based transmission electron microscopy (TEM), TUNEL (terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate (dUTP) nick end labeling) assay for tapetum programmed cell death, and laser microdissection procedures to obtain specific cells or cell layers for transcriptome analysis.

Melanin-dependent tissue interactions induced by a 755-nm picosecond-domain laser: complementary visualization by optical imaging and histology.

Fractional picosecond-domain lasers (PSL) induce optical breakdown, which correlates histologically to vacuolization in the epidermis and dermis. In this ex vivo porcine study, we sought to establish a framework for the investigation of laser-tissue interactions and their dependence on melanin density. Light- (melanin index: 24.5 [0-100]), medium- (58.7), and dark-pigmented (> 98) porcine skin samples were exposed to a 755-nm fractional PSL and examined with dermoscopy, line-field confocal optical coherence tomography (LC-OCT), conventional OCT, and subsequently biopsied for digitally stained ex vivo confocal microscopy (EVCM) and histology, using hematoxylin and eosin (HE) and Warthin-Starry (WS) melanin staining. Dermoscopy showed focal whitening in medium- and dark-pigmented skin. Similarly, LC-OCT and OCT visualized melanin-dependent differences in PSL-induced tissue alterations. Vacuoles were located superficially in the epidermis in dark-pigmented skin but at or below the dermal-epidermal junction in medium-pigmented skin; in light-pigmented skin, no vacuoles were observed. Histology confirmed the presence of vacuoles surrounded by areas void of WS staining and disrupted stratum corneum in darker skin. The combined use of optical imaging for multiplanar visualization and histological techniques for examination of all skin layers may mitigate the effect of common artifacts and attain a nuanced understanding of melanin-dependent laser-tissue interactions.

A novel method for age identification of mountain-cultivated ginseng.

Panax ginseng, a slow-growing perennial herb, is the most praised and popular traditional medicinal herb. Mountain-cultivated ginseng (MCG) and cultivated ginseng (CG) both belong to Panax ginseng C. A. Meyer. The market price and medical effects of this popular health product are closely related to its age. It is widely acknowledged that CG is typically harvested after 4-6 years of growth, but MCG is often collected after 10 years. Until now, the age identification of MCG or mountain wild ginseng (MWG) has remained a major challenge. In this study, we established a novel and rapid method for staining xylem vessels with phloroglucinol and identifying the "annual growth rings" of ginseng by utilizing a stereoscope, which serves as a reliable indicator of the age of MCG. Statistical analysis of the ring radius and the ring density of MCG aged from 1 to 20 years shows that the secondary xylem of MCG increases rapidly in the first 3 years but then gradually slows down from 4 to 10 years, and minor fluctuation is observed in the next 10 years. Meanwhile, the space between the growth rings (ring density) becomes increasingly small with age. This straightforward staining approach can reveal the age of MCG with remarkable clarity and can distinguish MCG from CG. RESEARCH HIGHLIGHTS: A novel rapid staining method for Panax ginseng was established. The age of mountain-cultivated ginseng (MCG) can be identified by microscopic techniques. MCG and cultivated ginseng (CG) can be discriminated by microstructure characteristics.

Cellular Localization of Orexin 1 Receptor in Human Hypothalamus and Morphological Analysis of Neurons Expressing the Receptor.

The orexin system is related to food behavior, energy balance, wakefulness and the reward system. It consists of the neuropeptides orexin A and B, and their receptors, orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R). OX1R has selective affinity for orexin A, and is implicated in multiple functions, such as reward, emotions, and autonomic regulation. This study provides information about the OX1R distribution in human hypothalamus. The human hypothalamus, despite its small size, demonstrates a remarkable complexity in terms of cell populations and cellular morphology. Numerous studies have focused on various neurotransmitters and neuropeptides in the hypothalamus, both in animals and humans, however, there is limited experimental data on the morphological characteristics of neurons. The immunohistochemical analysis of the human hypothalamus revealed that OX1R is mainly found in the lateral hypothalamic area, the lateral preoptic nucleus, the supraoptic nucleus, the dorsomedial nucleus, the ventromedial nucleus, and the paraventricular nucleus. The rest of the hypothalamic nuclei do not express the receptor, except for a very low number of neurons in the mammillary bodies. After identifying the nuclei and neuronal groups that were immunopositive for OX1R, a morphological and morphometric analysis of those neurons was conducted using the Golgi method. The analysis revealed that the neurons in the lateral hypothalamic area were uniform in terms of their morphological characteristics, often forming small groups of three to four neurons. A high proportion of neurons in this area (over 80%) expressed the OX1R, with particularly high expression in the lateral tuberal nucleus (over 95% of neurons). These results were analyzed, and shown to represent, at the cellular level, the distribution of OX1R, and we discuss the regulatory role of orexin A in the intra-hypothalamic areas, such as its special role in the plasticity of neurons, as well as in neuronal networks of the human hypothalamus.

Cowanin induces apoptosis in breast cancer cells via Bcl-2 signaling pathway.

OBJECTIVES: This study aimed to investigate the effect of cowanin the mechanism of cowanin toward cell death and BCL-2 protein (antiapoptotic) expression of T47D breast cancer. METHODS: The cell death was evaluated by double staining, namely acridine orange and propidium iodide, and then observed under a fluorescence microscope. Meanwhile, the BCL-2 protein expression was determined by western blotting with measurement of protein area and protein density. RESULTS: The result found T47D breast cancer cells were viable, apoptosis, and necrosis after treatment with cowanin. The average viable cells, apoptosis, and necrosis percentages were 54.13 %, 45.43 %, and 0.44 %, respectively. Statistical analysis showed cowanin could significantly induce death in T47D breast cancer cells by apoptosis (p<0.05). It was also revealed that cowanin and positive control (doxorubicin) treatment had a significantly decreased protein area and protein density (p<0.05). CONCLUSIONS: It can be concluded that cowanin can induce death in T47D breast cancer cells by apoptosis and affect the expression of Bcl-2 protein in T47D breast cancer cells.

Effects of fatty acid-ethanol amine (FA-EA) derivatives on lipid accumulation and inflammation.

This study aimed to investigate the effect of fatty acid-ethanol amine (FA-EA) derivatives (L1-L10) on the mitigation of intracellular lipid accumulation and downregulation of pro-inflammatory cytokines in vitro. First, the series of FA-EA derivatives were synthesized and characterized. Then, their cytotoxic, intracellular lipid accumulation and inhibition of pro-inflammatory cytokines were evaluated. The oil red O staining experiment showed that the tested compounds L4, L6, L8, L9, and L10 could reduce intracellular lipid accumulation induced by palmitic acid (PA). Moreover, ω-3/ω-6 PUFA-EA derivatives showed inhibitory effect on the production of pro-inflammatory cytokines in lipopolysaccharide (LPS) -stimulated RAW 264.7 cells. ω-3/ω-6 PUFA-EA derivatives at a concentrations of 10 µM could significantly decrease mRNA levels of IL-6, IL-1ß, and TNF-α, inhibit NO production, and alleviate the protein expression of IL-1ß in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. These data suggest that ω-3 PUFA-EA derivatives can be beneficial for further pharmaceutical development to treat chronic low-grade inflammation diseases such as obesity.

Increased Brain-Derived Neurotrophic Factor and Hippocampal Dendritic Spine Density Are Associated with the Rapid Antidepressant-like Effect of Iron-citalopram and Iron-Imipramine Combinations in Mice.

Iron supplementation previously demonstrated antidepressant-like effects in post-partum rats. The present study evaluates the possible synergistic antidepressant effect of sub-therapeutic dose of iron co-administered with citalopram or imipramine in female Institute of Cancer Research mice. Depression-like symptoms were induced in the forced swim (FST), tail suspension (TST), and open space swim (OSST) tests while open field test (OFT) was used to assess locomotor activity. Mice (n = 8) received iron (0.8-7.2 mg/kg), citalopram (3-30 mg/kg), imipramine (3-30 mg/kg), desferrioxamine (50 mg/kg) or saline in the single treatment phase of each model and subsequently a sub-therapeutic dose of iron co-administered with citalopram or imipramine. Assessment of serum brain derived neurotrophic factor (BDNF) and dendritic spine density was done using ELISA and Golgi staining techniques respectively. Iron, citalopram and imipramine, unlike desferrioxamine, reduced immobility score in the TST, FST and OSST without affecting locomotor activity, suggesting antidepressant-like effect. Sub-therapeutic dose of iron in combination with citalopram or imipramine further enhanced the antidepressant-like effect, producing a more rapid effect when compared to the iron, citalopram or imipramine alone. Iron, citalopram and imipramine or their combinations increased serum BDNF concentration, hippocampal neuronal count and dendritic spine densities. Our study provides experimental evidence that iron has antidepressant-like effect and sub-therapeutic dose of iron combined with citalopram or imipramine produces more rapid antidepressant-like effect. We further show that iron alone or its combination with citalopram or imipramine attenuates the neuronal loss associated with depressive conditions, increases dendritic spines density and BDNF levels. These finding suggest iron-induced neuronal plasticity in the mice brain.

Color stability of bulk-fill compared to conventional resin-based composites: A scoping review.

OBJECTIVES: To conduct a scoping review on color stability of bulk-fill compared to conventional resin-based composites exposed to colored beverages. MATERIALS AND METHODS: In vitro studies, investigating bulk-fill resin-based composites color stability submitted to artificial staining by colored beverages were included. Studies investigating color change induced by smoke and whitening treatments as well as papers not written in English language were excluded. A systematic search, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) was performed on four databases (Embase, Medline, Scopus, Web of Science) for articles published until October 01st 2022. The study selection was then performed by two authors who screened the abstracts independently and followed the JBI approach. RESULTS: Of 717 screened abstracts, 59 were selected for full-text analysis. Finally, 19 studies were included in this review. They investigated 19 different bulk-fill composites and different artificial-staining-by-liquids-protocols including coffee, red wine, tea, coke, and others. Seven papers reported higher color change in the investigated bulk fill materials than in conventional resin-based composites, while nine studies reported the opposite. SonicFill showed the highest color stability for most of the papers. CONCLUSIONS: The available evidence suggests that bulk-fill materials show variability in color stability. This behavior can be attributed to the heterogeneity of composition and staining procedures in the selected studies. CLINICAL SIGNIFICANCE: Clinicians should keep in mind that bulk-fill materials may change color when exposed to colorant drinks or food.

Transcutaneous Electrical Stimulation for the Prevention of Dry Eye Disease after Photorefractive Keratectomy: Randomized Controlled Trial.

Objective: To evaluate the efficacy and safety of transcutaneous electrical stimulation (TES) for the prevention of dry eye after photorefractive keratectomy (PRK). Design: Prospective, single-center, single-blinded, parallel group, placebo-controlled, randomized clinical trial. Participants: Between February 2020 and October 2020, patients at the Samsung Medical Center scheduled to undergo PRK to correct myopia were screened and enrolled. Methods: The participants in the TES group were instructed to use the electrical stimulation device (Nu Eyne 01, Nu Eyne Co) at the periocular region after the operation, whereas those in the control group were to use the sham device. Dry eye symptoms were evaluated preoperatively and postoperatively at weeks 1, 4, and 12 using the Ocular Surface Disease Index (OSDI) questionnaire, the 5-Item Dry Eye Questionnaire (DEQ-5), and the Standard Patient Evaluation for Eye Dryness II (SPEED II) questionnaire. Dry eye signs were assessed using tear break-up time (TBUT), total corneal fluorescein staining (tCFS), and total conjunctival staining score according to the National Eye Institute/Industry scale. The pain intensity was evaluated using a visual analog scale. Main Outcome Measures: Primary outcomes were OSDI and TBUT. Results: Twenty-four patients were enrolled and completed follow-up until the end of the study (12 patients in the TES group, 12 patients in the control group). Refractive outcomes and visual acuity were not different between the groups. No serious adverse event was reported with regard to device use. No significant difference in OSDI and SPEED II questionnaires and the DEQ-5 was observed between the groups in the 12th week after surgery. The TBUT scores 12 weeks after the surgery were 9.28 ± 6.90 seconds in the TES group and 5.98 ± 2.55 seconds in the control group with significant difference (P = 0.042). The tCFS and total conjunctival staining score were significantly lower in the TES group than in the control group at postoperative 4 weeks. Pain intensity at the first week was significantly lower in the TES group than in the control group by 65% (P = 0.011). Conclusion: The application of TES is safe and effective in improving dry eye disease after PRK. Financial Disclosures: The author(s) have no proprietary or commercial interest in any materials discussed in this article.

A simple and low-cost method to visualize musculature and other aspects of anatomy by confocal microscopy.

Confocal microscopy study of musculature and other anatomical structures in whole-mount preparations of arthropods and some other cuticle-bearing animals often presents a significant difficulty because the cuticle poses a barrier to fluorescent dyes and their pigmented tissues can cause attenuation of fluorescent signal. This paper describes a simple and inexpensive procedure based on the use of clove oil as a tissue-clearing, staining, and mounting medium that helps overcome the problem of slow dye penetration and tissue opaqueness and allows muscles and several other organ systems to be visualized by confocal or epifluorescent microscopy. This clove oil-induced fluorescence (COIF) method relies on the ability of clove oil to accumulate in muscles and some other tissues and become steadily fluorescent if irradiated at 488 nm. For this method, animals were fixed in 70% ethanol or 4% formaldehyde, then dehydrated and mounted in clove oil. Heavily pigmented animals were additionally bleached in hydrogen peroxide prior to the dehydration step. The COIF method showed excellent results in all major groups of arthropods and some mollusks and annelids revealing the three-dimensional arrangement of muscles, gonads, glands, cellular nuclei and some parts of the nervous and digestive systems. In the other animal groups tested, clove oil stained all tissues making it difficult to observe the anatomical details. The COIF method is advantageous in some respects over other methods such as phalloidin staining because of its tissue penetration and clearing abilities, low cost of the reagents, resistance to photodamage and the possibility of staining museum specimens.

Identification of microorganisms from fermented biowaste and the potential for wastewater treatment.

Food loss or waste is a far-reaching problem and has indeed become a worrying issue that is growing at an alarming rate. Fruits and vegetables are lost or wasted at the highest rate among the composition of food waste. Furthermore, the world is progressing toward sustainable development; hence, an efficient approach to valorise fruit and vegetable waste (FVW) is necessary. A simple phenotypic characterisation of microbiota isolated from the fermented FVW was conducted, and its effectiveness toward wastewater treatment was investigated. Presumptive identification suggested that yeast is dominant in this study, accounting for 85% of total isolates. At the genus level, the enriched medium's microbial community consists of Saccharomyces, Bacillus and Candida. Ammonium in the wastewater can enhance certain bacteria to grow, such as lactic acid bacteria, resulting in decreased NH4+ concentration at the end of the treatment to 0.5 mg/L. In addition, the fermented biowaste could reduce PO43- by 90% after the duration of treatment. Overall, FVW is a valuable microbial resource, and the microbial population enables a reduction in organic matter such as NH4+ and PO43-. This study helps explore the function and improve the effectiveness of utilising biowaste by understanding the microorganisms responsible for producing eco-enzyme.

Live Fluorescence Visualization of Cellulose and Pectin in Plant Cell Walls.

The plant cell wall comprises various types of macromolecules whose abundance and spatial distribution change dynamically and are crucial for plant architecture. High-resolution live cell imaging of plant cell wall components is, therefore, a powerful tool for plant cell biology and plant developmental biology. To acquire suitable data, the experimental setup for staining and imaging of non-fixed samples must be straightforward and avoid creating stress-induced artifacts. We present a detailed sample preparation and live image acquisition protocol for fluorescence visualization of cell wall components using commercially available probes and stains.

Effects of black tea tooth staining previously to 35% hydrogen peroxide bleaching

Aim: To determine if the artificial staining with black tea (BT) influences the enamel microhardness before in-office bleaching and if BT staining is necessary to evaluate the efficacy of bleaching with 35% hydrogen peroxide Methods: Enamel/dentin blocks were randomized into groups according to the staining protocol (n=5/group): (CO) control ­ maintained in artificial saliva solution (AS); (BT4) immersed in black tea solution for 4 h; (BT24) immersed in black tea solution for 24 h. After the staining protocols, all specimens were kept in AS for one week, followed by bleaching (three sessions of HP application for 40 min). Knoop surface microhardness (kgF/mm2) was determined at baseline (T0), after staining (T1), after 7 days of storage in AS (T2), and after bleaching (T3). The color (∆E00) and coordinate changes (∆L, ∆a, ∆b) were measured using a digital spectrophotometer at T0 and T3. Data were submitted to one-way (∆E00, ∆L, ∆a, ∆b) or two-way ANOVA repeated measures (kgF/mm2) and Tukey's test (a=5%). Results: The staining protocols (BT4 and BT24) promoted significantly lower microhardness (T1 and T2, p<0.05) than CO, whereas CO was the only group to maintain microhardness values over time. Bleaching promoted perceptible ∆E00 without a significant difference among the groups regardless of the staining protocol (p=0.122). CO and BT4 showed no differences in terms of ∆L and ∆a (p>0.05), but BT4 displayed a higher ∆b than CO. Conclusion:The artificial staining with BT negatively affected the enamel surface microhardness and was not essential to evaluate the efficacy of 35% hydrogen peroxide bleaching