Phase Contrast Light Microscopy Research Articles

Integrative redescription of the Xerobiotus euxinus Pilato, Kiosya, Lisi, Inshina & Biserov, 2011 (Tardigrada: Eutardigrada: Macrobiotidae) population from Ukraine

Using integrative taxonomy, we redescribe an eutardigrade Xerobiotus euxinus Pilato et al. 2011 based on material from the type locality and additional population from the Black Sea Biosphere Reserve in the south of Ukraine. Integrative redescription is based on morphological and morphometric data of animals and eggs from both phase contrast light microscopy (PCM), as well as, from scanning electron microscopy (SEM), and four DNA markers, three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI). Originally, the species was described solely based on adults’ morphology. The uncorrected genetic distances indicated that X. euxinus is the most similar to X. gretae with a p-distance value of COI fragment. Morphologically, X. euxinus differs from all Xerobiotus taxa mainly by the shape of claws on legs IV, but also by some other morphological and morphometrical characters of eggs and animals.

Epithelial-mesenchymal Transition (EMT) and the Effect of Atorvastatin on it in ARPE-19 cells

Proliferative vitreoretinopathy (PVR) develops after an unsuccessful or complicated recovery from rhegmatogenous retinal detachment (RRD) surgery. Intraocular scar formation with the contribution of epithelial-mesenchymal transition (EMT) in RPE cells is prominent in the pathology of PVR. In the present study, the EMT process was experimentally induced in human retinal pigment epithelium (RPE; ARPE-19) cells, and the effect of atorvastatin on the process was studied. The mRNA and protein levels of mesenchymal markers actin alpha 2 (ACTA2) / alpha-smooth muscle actin (α-SMA) and fibronectin (FN), and epithelial markers occludin (OCLN) and zonula occludens-1 (ZO-1) were measured using quantitative real-time PCR (qRT-PCR) and western blot methods, respectively. In addition, α-SMA and FN were visualized using immunofluorescence staining. Cells were photographed under a phase contrast light microscope. Changes in the functionality of cells following the EMT process were studied using the IncuCyte scratch wound cell migration assay and the collagen cell invasion assay with confocal microscopy. The induction of EMT in ARPE-19 cells increased the expression of mesenchymal markers ACTA2/α-SMA and fibronectin and reduced the expression of epithelial marker OCLN both at mRNA and protein levels. The mRNA levels of ZO-1 were lower after EMT, as well. Increased levels of α-SMA and FN were confirmed by immunofluorescence staining. Atorvastatin further increased the mRNA levels of mesenchymal markers ACTA2 and FN as well as the protein levels of α-SMA and reduced the mRNA levels of epithelial markers OCLN and ZO-1 under the EMT process. EMT promoted wound closure and cell invasion into the 3D collagen matrix when compared to untreated control cells. These data present cellular changes upon the induction of the EMT process in ARPE-19 cells and the propensity of atorvastatin to complement the effect. More studies are needed to confirm the exact influence of the EMT process and atorvastatin treatment on the PVR development after RRD surgery.

The role of intestinal microbiota in the development of dysfunction of the biliary system in children

Objective: to study the state of the intestinal and biliary microbiota in children with intestinal and gallbladder dysfunction and to determine the features of the component composition of the biliary microbiota. Methods: We examined 20 children with irritable bowel syndrome (IBS). The presence of small intestinal bacterial overgrowth (SIBO) syndrome was determined (using a hydrogen breath test with lactulose), microbial markers of the intestinal and biliary microbiota (using gas chromatography-mass spectrometry). We also performed microscopy of bile (phase-contrast light microscopy) obtained through duodenal intubation. Results: In all children with IBS, SIBO was verified in the small intestine, with insufficiency of the ileocecal valve in half of them, associated with hypomotor dyskinesia of the gallbladder. Microscopy of the bile revealed a large number of crystals and leukocytes. A study of microbial markers of intestinal and biliary microbiota in all children with IBS and SIBO revealed excessive bacterial associative growth of gram-negative opportunistic bacteria in combination with viruses of the Herpes family. An increase in the level of lipopolysaccharides (2 times higher than normal) and a deficiency of microbial markers of the main groups of microorganisms (anaerobic bacteria) responsible for metabolization processes in the enterohepatic circulation (plasmalogen less than 50 μg/ml) were recorded. Excessive growth of 28 species of opportunistic bacteria was detected in bile in diagnostically significant concentrations associated with hypomotor dysfunction of the gallbladder and lithogenicity of bile. Conclusion: A violation of the biliary microbiota was established with an increase in the proportion of gram-negative opportunistic bacteria, the spectrum of which was close to that of the intestine and their association with intestinal and biliary dyskinesia

Revised description of the blueberry bud mite, Acalitus vaccinii (Acari: Trombidiformes: Eriophyidae), and a key to all Eriophyoidea on Vaccinium

In 2014 the blueberry bud mite, Acalitus vaccinii (Acari: Trombidformes: Eriophyidae), was detected in the Mpumalanga province of South Africa for the first time causing significant damage to cultivated blueberries. This was the first detection of this pest outside of North America, to which it is native. However, its taxonomic description at that time lacked critical detail and omitted characters and life stages important for easy and accurate identification. Using an integrative taxonomic approach, we combined phase contrast light microscopy with low-temperature SEM and DNA barcoding data to revise the description of A. vaccinii using South African specimens. Additional characters not included in previous descriptions but reported here are the h1 (accessory) setae, leg I & II u’ (mesal) setae, and leg II bv (femoral) setae. Detailed descriptions and measurements of all life stages are included, along with a discussion of morphological variation and biology. Two DNA sequences of common barcode regions (nuclear and mitochondrial) are provided to further aid in identification. In addition, a key to all known species of eriophyoid mites present on Vaccinium is provided.

Effect of Silica in the Manufacture of Autologous Serum Eye Drops on Corneal Stromal Cells.

The aim of this study was to evaluate the effect of serum clot activator, silica (SiO 2 ), which may be used for making autologous serum eye drops, on human corneal fibroblasts. Cultured human corneal fibroblasts were exposed to 10%, 20%, and 30% silica for 1, 6, and 24 hours; methyl thiazolyl tetrazolium-based colorimetric assay was performed to determine the survival rate of fibroblasts and lactate dehydrogenase leakage assay to assess the cytotoxicity. The apoptotic response was evaluated by flow cytometric analysis and fluorescence staining with Annexin V and propidium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The survival rate of human corneal fibroblasts and cytotoxicity showed both dose-dependent and time-dependent responses. The fluorescent micrograph and flow cytometry showed that as the exposure time increased, more cells underwent apoptosis or necrosis after treatment with 30% silica. When observed with light and electron microscopy, the number of corneal fibroblasts decreased and they were more detached from the dish. In addition, damaged corneal fibroblasts showed degenerative changes after exposure to 30% silica. Silica showed dose-dependent and time-dependent toxicity in human corneal fibroblasts. It is safer to keep the blood in tubes without a clot activator when manufacturing autologous serum eye drops to prevent possible corneal cytotoxicity.

Interactions of Neuronally Induced Stem Cells from Apical Papilla Spheres, Stems Cells from Apical Papilla, and Human Umbilical Vascular Endothelial Cells on Vasculogenesis and Neurogenesis

IntroductionStem cell–based dental pulp regeneration has been extensively studied, mainly focusing on exploiting dental stem cells’ osteogenic and angiogenic potentials. Dental stem cells’ neurogenic role is often overlooked. Stem cells from apical papilla (SCAPs), originating from the neural crest and capable of sphere formation, display potent neurogenic capacity. This study aimed to investigate the interactions of neuronally induced stem cells from apical papilla (iSCAP) spheres, SCAPs, and human umbilical vascular endothelial cells (HUVECs) on vasculogenesis and neurogenesis. MethodsSCAPs were isolated and characterized using flow cytometry and multilineage differentiation assays. SCAP monolayer culture and spheres were neuronally induced by a small molecule neural induction medium, and the neural gene expression and neurite formation at days 0, 3, and 7 were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and using phase-contrast light and fluorescence microscopy. Direct coculture or pulp-on-chip was used to investigate iSCAP sphere interaction with SCAPs and HUVECs. RT-qPCR, fluorescence microscopy, and immunostaining with β-tubulin III, alpha-smooth muscle actin, and CD31 were used to study neural gene expression, neurite formation, and neurovascular cell interactions. ResultsNeural induction medium with small molecules rapidly induced SCAP differentiation toward neural-like cells. Gene expression of Nestin, β-tubulin III, microtubule-associated protein 2, neuron-specific enolase, and NeuN was higher in iSCAP spheres than in iSCAPs. iSCAP spheres formed more and longer neurites compared with iSCAPs. iSCAP sphere, HUVEC, and SCAP direct coculture significantly enhanced vessel formation along with up-regulated VEGF (P < .001) and multiple neural markers, such as Nestin (P < .01), microtubule-associated protein 2 (P < .001), S100 (P < .001), and NG2 (P < .001). iSCAP spheres, SCAPs, and HUVECs cultured in a pulp-on-chip system promoted endothelial and neural cell migration toward each other and alpha-smooth muscle actin–positive and CD31-positive cells assembling for the vascular constitution. ConclusionsiSCAP-formed spheres interact with SCAPs and HUVECs, promoting vasculogenesis and neurogenesis.

In-silico and in-vitro study reveals ziprasidone as a potential aromatase inhibitor against breast carcinoma

Aromatase enzyme plays a fundamental role in the development of estrogen receptors, and due to this functionality, the enzyme has gained significant attention as a therapeutic for reproductive disorders and cancer diseases. The currently employed aromatase inhibitors have severe side effects whereas our novel aromatase inhibitor is more selective and less toxic, therefore has greater potential to be developed as a drug. The research framework of this study is to identify a potent inhibitor for the aromatase target by profiling molecular descriptors of the ligand and to find a functional pocket in the target by docking and MD simulations. For assessing cellular and metabolic activities as indicators of cell viability and cytotoxicity, in-vitro studies were performed by using the colorimetric MTT assay. Aromatase activities were determined by a fluorometric method. Cell morphology was assessed by phase-contrast light microscopy. Flow cytometry and Annexin V-FITC/PI staining assay determined cell cycle distribution and apoptosis. This study reports that CHEMBL708 (Ziprasidone) is the most promising compound that showed excellent aromatase inhibitory activity. By using better drug design methods and experimental studies, our study identified a novel compound that could be effective as a high-potential drug candidate against aromatase enzyme. We conclude that the compound ziprasidone effectively blocks the cell cycle at the G1-S phase and induces cancer cell death. Further, in-vivo studies are vital for developing ziprasidone as an anticancer agent. Lastly, our research outcomes based on the results of the in-silico experiments may pave the way for identifying effective drug candidates for therapeutic use in breast cancer.

Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae)

In a moss sample collected in Ribeiro Frio, Madeira, Paramacrobiotus gadabouti sp. nov. was found and described using the integrative taxonomy approach. The new species is described based on morphological and morphometric data from both phase-contrast light microscopy (PCM), as well as scanning electron microscopy (SEM). Moreover, four DNA markers, three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) markers, were used to elucidate the phylogenetic position of the new species within the family Macrobiotidae. The new species has a microplacoid that placed it within Parmacrobiotus richtersi group and exhibit richtersi-type eggs having processes terminated with cap-like structures. Paramacrobiotus gadabouti sp. nov. is most similar to Pam. alekseevi, Pam. filipi and Pam. garynahi, but differs from them mainly in details of egg morphology and morphometrics. Unlike other species from this group, which were confirmed as bisexual and showed limited distribution, Paramacrobiotus gadabouti sp. nov. is yet another parthenogenetic species with a wide distribution, demonstrating that at least some tardigrades confirm to the hypothesis of 'everything is everywhere'.

Integrative description of Macrobiotus kosmali sp. nov. (hufelandi group) from the Island of Madeira (Portugal)

Using integrative taxonomy, we describe Macrobiotus kosmali sp. nov. from Ribeira Brava, Madeira (Portugal). Based on morphological and morphometric data from both phase contrast light microscopy (PCM), as well as, from scanning electron microscopy (SEM), description of the new species is provided. Additionally, four DNA markers, three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) were used to calculate the p-distances between Macrobiotus kosmali sp. nov. and other species of the genus Macrobiotus. The uncorrected genetic distances indicated that the new species is the most similar to its four congeners, namely Mac. cf. recens and morphologically, new species is most similar to Mac. dariae, Mac. glebkai, Mac. noemiae, Mac. recens, Mac. rybaki and Mac. scoticus, but differs from them mainly in details of its egg chorion and egg processes. http://urn:lsid:zoobank.org:pub:B371F633-B35C-4578-8C75-9FA2B5E80588

Bending Elasticity of Phospholipid Bilayers Containing an Amphipathic Peptide with Low Mammalian Cytotoxicity

&#x0D; &#x0D; &#x0D; Peptide mimetics imitate natural peptides’ structure but they could be specifically designed to be more selective concerning their toxicity to mammalian cells. In most cases this specificity is due to their ability to form α-helix in amphipathic environment. In addition, the specific activity depends on the ability of final structure to penetrate cell membrane. Being responsible for the cell integrity and compartmentalization, biomembranes also play a major role in cellular processes, in which the membrane deformations are important. In the present study we probe peptide-membrane interactions for a shortened amino acid sequence KLAKLAK-NH2 of an antimicrobial peptide with apoptotic effect. The bending rigidity of model lipid bilayers is measured by flicker spectroscopy of quasispherical unilamellar vesicles monitored and analyzed in phase contrast light microscopy. At high peptide concentrations ∼ 30 μmol/L and peptide-to-lipid total molar ratios ∼ 0.90 bilayer stacking formation is observed. A reduction of the bending constant is reported at peptide-to-lipid total molar ratio ∼ 0.80. The membrane softening indicates peripheral peptide orientation at the lipid bilayer, which is considered a prerequisite for channel formation. Based on KLAKLAK-NH2 effect on the membrane bending elasticity we provide an evaluation of the peptide partition coefficient characterizing its affinity to POPC bilayers. The acquired results might be helpful in efforts to further tailor the pharmacokinetic properties of antimicrobial peptides in combination with strengthened stability towards enzymatic degradation.&#x0D; &#x0D; &#x0D;

Development and evaluation of a bovine lung-on-chip (bLOC) to study bovine respiratory diseases

PurposeCurrent air-liquid interface (ALI) models of bovine proximal airways have their limitations. They do not simulate blood flow necessary to mimic systemic drug administration, and repeated sampling requires multiple, independent cultures. A bovine lung-on-chip (bLOC) would overcome these limitations, providing a convenient and cost-effective model for pharmacokinetic or pathogenicity studies.MethodsBovine pulmonary arterial endothelial cells seeded into the endothelial channel of an Emulate Lung-Chip were interfaced with bovine bronchial epithelial cells in the epithelial channel. Cells were cultured at ALI for up to 21 days. Differentiation was assessed by mucin quantification, phase-contrast light microscopy and immunofluorescence of cell-specific markers in fixed cultures. Barrier integrity was determined by FITC-labelled dextran 3–5 kDa permeability. To evaluate the model, endothelial-epithelial transport of the antibiotic drug, danofloxacin, was followed using liquid chromatography-mass spectrometry, with the aim of replicating data previously determined in vivo.ResultsbLOC cultures secreted quantifiable mucins, whilst cilia formation was evident in the epithelial channel. Barrier integrity of the model was demonstrated by resistance to FITC-Dextran 3–5 kDa permeation. Bronchial epithelial and endothelial cell-specific markers were observed. Close to plasma, representative PK data for danofloxacin was observed in the endothelial channel; however, danofloxacin in the epithelial channel was mostly below the limit of quantification.ConclusionA co-culture model of the bovine proximal airway was successfully generated, with potential to replace in vivo experimentation. With further optimisation and characterisation, the bLOC may be suitable to perform drug pharmacokinetic studies for bovine respiratory disease (BRD), and other applications.

The Effects of Ibuprofen, Naproxen and Diclofenac on cell Apoptosis, Cell Proliferation and Histology Changes in Human Cholangiocarcinoma Cell Lines.

Objective:To examine the effects of ibuprofen, naproxen and diclofenac, non-steroidal anti-inflammatory drugs (NSAIDs) on cell proliferation activity of the human CCA cell lines. Methods:KKU-M139 and KKU-213B cell lines were used in this study. The cell viability was assessed by the MTT assay. Lipid synthesis determined by Oil red O staining and colorimetric assay. An inverted phase-contrast light microscope was used to investigate the histological change of the cells. Caspases 3/7 activity and AnnexinV/PI were used to assess apoptosis by multiple microplate reader. Results:The results showed that ibuprofen, naproxen and diclofenac suppressed the viability of the KKU-M139 and KKU-213B cells in a dose-dependent manner, as measured especially diclofenac. However, these three NSAIDs slightly decreased lipid synthesis determined by Oil red O staining and colorimetric assay. The histological change observations showed the shrinking cell and become star-shaped in high dose treated groups. Interestingly, these NSAIDs exhibited in both of KKU-M139 and KKU-213B cell lines, the diclofenac-treated cells had the most injury cells. The cells exhibited cell injury features. In addition, the detection of caspase 3/7 and AnnexinV/PI in this investigation revealed early cell apoptotic characteristics. Conclusion:These finding suggest that ibuprofen, naproxen and diclofenac suppress cell viability. The results reveal that ibuprofen, naproxen and diclofenac, which induce the histological change and apoptosis. This study indicates that these NSAIDs may be used as an anti-proliferation agent for the treatment of CCA in the future.

Ibuprofen and diclofenac differentially affect cell viability, apoptosis and morphology changes of human cholangiocarcinoma cell lines

ObjectivesCholangiocarcinoma is a malignant biliary epithelial duct neoplasm caused by chronic inflammation after liver fluke infection. It is a major public health concern in the Greater Mekong sub-region in northeast Thailand. Herein, the effects of the non-steroidal anti-inflammatory drugs (NSAIDs) ibuprofen and diclofenac on the cell proliferation activity of the human cholangiocarcinoma cell lines KKU-M139 and KKU-213B were studied. MethodsCell viability was assessed with MTT assays. Inverted phase-contrast light microscopy, scanning electron microscopy and transmission electron microscopy were used to investigate the cells’ morphological alterations. Caspase 3/7 and Annexin V/PI were detected with a multimode microplate reader. ResultsIbuprofen and diclofenac decreased viability in both cell lines, and ibuprofen-treated cells exhibited reversible cell injury. In both KKU-M139 and KKU-213B cell lines, the diclofenac-treated cells had the greatest injury. The cells exhibited features of irreversible cell injury. In addition, caspase 3/7 and Annexin V/PI detection revealed early cell apoptotic characteristics. ConclusionThese findings suggest that NSAIDs may potentially suppress cell viability. Ibuprofen and diclofenac both induced morphological changes and apoptosis.

Calmodulin Regulates Ciliary Beats in the Human Nasal Mucosa Through Adenylate/Guanylate Cyclases and Protein Kinases A/G

Background: The ciliary beat of the airway epithelium, including the sinonasal epithelium, has a significant role in frontline defense and is thought to be controlled by the level of intracellular Ca²⁺. Involvement of calmodulin and adenylate/guanylate cyclases in the regulation of ciliary beats has been reported, and here we investigated the interrelation between these components of the ciliary beat regulatory pathway. Methods: The inferior turbinates were collected from 29 patients with chronic hypertrophic rhinitis/rhinosinusitis during endoscopic sinonasal surgery. The turbinate mucosa was cut into thin strips, and mucociliary movement was observed under a phase-contrast light microscope equipped with a high-speed digital video camera. Results: The ciliary beat frequency (CBF) was significantly increased by stimulation with 100 μM CALP3 (calmodulin agonist), which was completely suppressed by adding 100 µM SQ22536 (adenylate cyclase inhibitor) and 10 µM ODQ (guanylate cyclase inhibitor) together and by adding 1 µM KT5720 (protein kinase A inhibitor) and 1 µM KT5823 (protein kinase G inhibitor) together. The CBF was significantly increased by stimulation with 10 µM forskolin (adenylate cyclase activator) and 10 µM BAY41-2272 (guanylate cyclase activator) and by stimulation with 100 µM 8-bromo-cAMP (cAMP analog) and 100 µM 8-bromo-cGMP (cGMP analog), which was not changed by adding 1 µM calmidazolium (calmodulin antagonist). Conclusions: These results confirmed that the regulatory pathway of ciliary beats in the human nasal mucosa involves calmodulin, adenylate/guanylate cyclases, and protein kinases A/G and indicate that adenylate/guanylate cyclases and protein kinases A/G act downstream of calmodulin, but not vice versa, and that these cyclases relay calmodulin signaling.

Comprehensive hygienic assessment of the dust factor in the manufacturing heat-insulating building products from man-made mineral fibers

Introduction. Air pollution with aerosols of mineral fibers in the manufacturing heat-insulating building products is the primary harmful production factor that can increase the risks of developing several diseases of various organs and systems. It determines the relevance of conducting comprehensive studies of the dust factor in such industries, assessing the relationship between mass measurements and fibre counts, the development of targeted preventive measures, considering the features of the technological process. The study aims to conduct comprehensive hygienic studies of the dust factor in the manufacturing heat-insulating building products from basalt fiber and determine the morphological features of dust at different stages of the technological process, the presence of a relationship between mass measurements and fibre counts. Materials and methods. The studies were carried out based on manufacturing heat-insulating building products from basalt fiber. To assess the dust factor, the authors carried out the analysis of mass measurements of dust and fibre counts using phase-contrast light microscopy. The authors used linear regression analysis to study the dependence of the mass measurements and fibre counts. Results. The mass measurements of dust in the working area don't exceed hygienic standards and ranges from 0.51 mg/m3 to 1.19 mg/m3, and the fibre counts are from 0.45 f/cm3 to 1.23 f/cm3. The morphological analysis of the dust samples' preparations collected on filters from the air of the working area made it possible to reveal the characteristic features of the composition of the dust at various stages of the technological process. The study confirmed a positive correlation of weak strength between fibre counts and the mass measurements. The resulting dependence model is statistically significant, but the coefficient of determination of this model (R2) is 0.043. Fibre counts in the first phase of the production cycle are significantly higher than in the second. Simultaneously, the mass measurements do not have significant differences in different phases of the production cycle. Conclusions. There is a positive correlation of weak strength between fibre counts and mass measurements, but the resulting linear regression model can explain only 4.3% of the variation of fibre counts based on the mass measurements, which does not allow using this model for converting of the results of measurements of several particles. Microscopy of dust preparations makes it possible to determine the morphological picture characteristic of various stages of the technological process. Fibre counts depend on the phase of the production cycle. The data obtained can be used to design air purification systems and operating modes at various stages of the technological process.

Transmission-Mode MALDI Mass Spectrometry Imaging of Single Cells: Optimizing Sample Preparation Protocols.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) makes it possible to simultaneously visualize the spatial distribution of dozens to hundreds of different biomolecules (e.g., phospho- and glycolipids) in tissue sections and in cell cultures. The implementation of novel desorption and (post-)ionization techniques has recently pushed the pixel size of this imaging technique to the low micrometer scale and below and thus to a cellular and potentially sub-cellular level. However, to fully exploit this potential for cell biology and biomedicine, sample preparation becomes highly demanding. Here, we investigated the effect of several key parameters on the quality of the sample preparation and achievable spatial resolution, that include the washing, drying, chemical fixation, and matrix coating steps. The incubation of cells with formalin for about 5 min in combination with isotonic washing and mild drying produced a robust protocol that largely preserved not only cell morphologies, but also the molecular integrities of amine group-containing cell membrane phospholipids (phosphatidylethanolamines and -serines). A disadvantage of the chemical fixation is an increased permeabilization of cell membranes, resulting in leakage of cytosolic compounds. We demonstrate the pros and cons of the protocols with four model cell lines, cultured directly on indium tin oxide (ITO)-coated glass slides. Transmission (t-)mode MALDI-2-MSI enabled on a Q Exactive plus Orbitrap mass spectrometer was used to analyze the cultures at a pixel size of 2 μm. Phase contrast light microscopy and scanning electron microscopy were used as complementary methods. The protocols described could prove to be an important contribution to the advancement of single-cell MALDI imaging, especially for the characterization of cell-to-cell heterogeneities at a molecular level.

Hemocyte profile, phagocytosis, and antibacterial activity in response to immune challenge of the date fruit stalk borer, Oryctes elegans

The date fruit stalk borer, Oryctes elegans Prell, is a destructive pest on date palms in Saudi Arabia. We evaluated the immune response of the third instars (last instars), by the intrahemocoelic injection of lipopolysaccharide (LPS) (a pathogen-associated molecular peptide, PAMP), which significantly increased the total hemocyte count. Phase-contrast light microscopy revealed the presence of five hemocyte types: prohemocytes, granulocytes, plasmatocytes, oenocytoids, and spherulocytes. Transmission electron microscopy demonstrated that, among these hemocytes, only the granulocytes and plasmatocytes phagocytosed latex beads. Injection with LPS also significantly decreased the number of oenocytoids. The antibacterial activity of plasma proteins of larvae injected with LPS, measured using the agar well diffusion method, against Gram-positive and Gram-negative bacteria varied based on the bacterial strain, the total concentration of plasma protein, and time post-injection with LPS. The results of the current study may be useful in the biological control of O. elegans. Furthermore, new compounds with antibacterial activity that might be useful for the development of innovative drugs of natural origin can be identified in O. elegans.

CircPDE4B inhibits retinal pathological angiogenesis via promoting degradation of HIF-1α though targeting miR-181c.

Retinopathy of prematurity is a major cause of childhood blindness worldwide. Hence, exploring the proper treatment methods is a must in tacking this disease. qRT-PCR and western blot were used to detect the expression of genes and proteins, respectively. The proliferation of human retinal vascular endothelial cells (HRECs) was ensured by MTT assay. The luciferase activity was measured through luciferase assay. The inverted phase-contrast light microscope was used to observe the formation of a vascular tube. In the present study, our data demonstrated that circPDE4B was downregulated, while hypoxia-inducible factor-1α (HIF-1α) and VEGFA were upregulated in the retinopathy of prematurity model in vitro and in vivo. CircPDE4B increasing remarkably inhibited the expression of HIF-1α and VEGFA in hypoxia-induced HRECs and subsequent repressed cell proliferation and pathological angiogenesis. We further found that miR-181c suppressed the expression of von Hippel-Lindau (VHL), while circPDE4B could promote VHL expression via binding to miR-181c. Finally, our results revealed that circPDE4B inhibited the expression of VEGFA and pathological angiogenesis via facilitating VHL-mediated ubiquitin degradation of HIF-1α. In conclusion, circPDE4B suppressed the expression of VEGFA and pathological angiogenesis via promoting VHL-mediated ubiquitin degradation of HIF-1α through binding to miR-181c. Our study indicated that circPDE4B might be an effective therapeutic target of retinopathy of prematurity.

Cockle Shell-Derived Aragonite CaCO3 Nanoparticles for Co-Delivery of Doxorubicin and Thymoquinone Eliminates Cancer Stem Cells

Cancer stem cells CSCs (tumour-initiating cells) are responsible for cancer metastasis and recurrence associated with resistance to conventional chemotherapy. This study generated MBA MD231 3D cancer stem cells enriched spheroids in serum-free conditions and evaluated the influence of combined doxorubicin/thymoquinone-loaded cockle-shell-derived aragonite calcium carbonate nanoparticles. Single loaded drugs and free drugs were also evaluated. WST assay, sphere forming assay, ALDH activity analysis, Surface marker of CD44 and CD24 expression, apoptosis with Annexin V-PI kit, cell cycle analysis, morphological changes using a phase contrast light microscope, scanning electron microscopy, invasion assay and migration assay were carried out; The combination therapy showed enhanced apoptosis, reduction in ALDH activity and expression of CD44 and CD24 surface maker, reduction in cellular migration and invasion, inhibition of 3D sphere formation when compared to the free drugs and the single drug-loaded nanoparticle. Scanning electron microscopy showed poor spheroid formation, cell membrane blebbing, presence of cell shrinkage, distortion in the spheroid architecture; and the results from this study showed that combined drug-loaded cockle-shell-derived aragonite calcium carbonate nanoparticles can efficiently destroy the breast CSCs compared to single drug-loaded nanoparticle and a simple mixture of doxorubicin and thymoquinone.

New records of tardigrade species of the Macrobiotus hufelandi group (Tardigrada: Eutardigrada: Macrobiotidae) from Ukraine

Species of the Macrobiotus hufelandi group are among the most well known and commonly found tardigrades. They are reported in almost every study of Tardigrada diversity in terrestrial habitats. In spite of this, zoogeographical data on this group remain ambiguous and insufficient. Since most species of the M. hufelandi group have been described in the last three decades, many older records need to be confirmed or revised. In this study we aimed to investigate, which species may be present in Ukraine, since most Ukrainian records of this group are outdated. We studied samples of mosses and lichens collected from different regions of Ukraine (mainly in the East and South of the country) in 2007–2019. Tardigrades were extracted from samples and mounted on permanent slides in Faure's medium. After primary identification of genera and species groups we focused on 13 samples containing local populations of the M. hufelandi group. Details of animal and egg morphology were studied under high magnifications of phase contrast light microscopy. Species were identified based on morphology of the oral cavity armature, egg shell morphology, animal cuticle granulation, as well as some morphometric characters of claws and bucco-pharyngeal apparatus. This research revealed the presence of at least six different species in Ukraine: M. glebkai Biserov, 1990; M. hufelandi C.A.S. Schultze, 1834; M. macrocalix Bertolani &amp; Rebecchi, 1993; M. polonicus Pilato, Kaczmarek, Michalczyk &amp; Lisi, 2003; M. sottilei Pilato, Kiosya, Lisi &amp; Sabella, 2012; M. vladimiri Bertolani, Biserov, Rebecchi &amp; Cesari, 2011. Three of them: M. macrocalix, M. sottilei, and M. vladimiri are reported in this study from Ukraine for the first time. Adding three more species recorded in earlier studies: M. diversus Biserov, 1990; M. persimilis Binda &amp; Pilato, 1972, and M. sapiens Binda &amp; Pilato, 1984 – gives the total count of at least 9 species of the group in Ukraine. However, according to the recent studies on species complexes the real species diversity is very likely to be higher. Nevertheless, more sampling and obtaining molecular data from different Ukrainian populations of the M. hufelandi group from Ukraine are required.