Biological and Molecular Effects of Trypanosoma cruzi Residence in a LAMP-Deficient Intracellular Environment

Trypanosoma cruzi invades non-professional phagocytic cells by subverting their membrane repair process, which is dependent on membrane injury and cell signaling, intracellular calcium increase, and lysosome recruitment. Cells lacking lysosome-associated membrane proteins 1 and 2 (LAMP1 and LAMP2) are less permissive to parasite invasion but more prone to parasite intracellular multiplication. Several passages through a different intracellular environment can significantly change T. cruzi’s gene expression profile. Here, we evaluated whether one single passage through LAMP-deficient (KO) or wild-type (WT) fibroblasts, thus different intracellular environments, could influence T. cruzi Y strain trypomastigotes’ ability to invade L6 myoblasts and WT fibroblasts host cells. Parasites released from LAMP2 KO cells (TcY-L2−/−) showed higher invasion, calcium signaling, and membrane injury rates, for the assays in L6 myoblasts, when compared to those released from WT (TcY-WT) or LAMP1/2 KO cells (TcY-L1/2−/−). On the other hand, TcY-L1/2−/− showed higher invasion, calcium signaling, and cell membrane injury rates, for the assays in WT fibroblasts, compared to TcY-WT and TcY-L1/2−/−. Albeit TcY-WT presented an intermediary invasion and calcium signaling rates, compared to the others, in WT fibroblasts, they induced lower levels of injury, which reinforces that signals mediated by surface membrane protein interactions also have a significant contribution to trigger host cell calcium signals. These results clearly show that parasites released from WT or LAMP KO cells are distinct from each other. Additionally, these parasites’ ability to invade the cell may be distinct depending on which cell type they interact with. Since these alterations most likely would reflect differences among parasite surface molecules, we also evaluated their proteome. We identified few protein complexes, membrane, and secreted proteins regulated in our dataset. Among those are some members of MASP, mucins, trans-sialidases, and gp63 proteins family, which are known to play an important role during parasite infection and could correlate to TcY-WT, TcY-L1/2−/−, and TcY-L2−/− biological behavior.

Exogenous Putrescine Alleviates Drought Stress by Altering Reactive Oxygen Species Scavenging and Biosynthesis of Polyamines in the Seedlings of Cabernet Sauvignon

Climate change imposes intensive dry conditions in most grape-growing regions. Drought stress is one of the most devastating abiotic factors threatening grape growth, yield, and fruit quality. In this study, the alleviation effect of exogenous putrescine (Put) was evaluated using the seedlings of Cabernet Sauvignon (Vitis vinifera L.) subjected to drought stress. The phenotype, photosynthesis index, membrane injury index (MII), and antioxidant system, as well as the dynamic changes of endogenous polyamines (PAs) of grape seedlings, were monitored. Results showed that drought stress increased the MII, lipid peroxidation, and the contents of reactive oxygen species (ROS) (H2O2 and O2–), while it decreased the antioxidant enzyme activity and the net photosynthesis rate (Pn). However, the application of Put alleviated the effects of drought stress by altering ROS scavenging, enhancing the antioxidant system, and increasing the net Pn. Put distinctly increased the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as the contents of ascorbic acid (AsA) and glutathione (GSH). Meanwhile, exogenous Put also promoted the metabolism of endogenous PAs by upregulating their synthetic genes. Our results confirmed that the exogenous application of Put can enhance the antioxidant capacity as well as alter the PA pool, which provides better drought tolerance for Cabernet Sauvignon seedlings.

Priming Effect of Exogenous ABA on Heat Stress Tolerance in Rice Seedlings is Associated with the Upregulation of Antioxidative Defense Capability and Heat Shock-Related Genes

Heat stress is a major restrictive factor that suppresses rice production. In this study, we investigated the potential priming effect of exogenous abscisic acid (ABA) on heat tolerance in rice seedlings. Seedlings were pretreated with 10 μM ABA by root drenching for 24 h and then subjected to heat stress conditions of 40 °C day/35 °C night. ABA pretreatment significantly decreased leaf withering by 2.5– 28.5% and chlorophyll loss by 12.8–35.1% induced by heat stress in rice seedlings. ABA application also mitigated cell injury, as shown by lower malondialdehyde (MDA) content, membrane injury and expression of cell death-related genes OsKOD1 , OsCP1 and OsNAC4 , while expression of OsBI1 , a cell death-suppressor gene, was upregulated by ABA pretreatment. Moreover, ABA pretreatment improved antioxidant defense capacity, as shown by an obvious upregulation of ROS-scavenging genes and a decrease in ROS content (O 2 – and H 2 O 2 ) and downregulation of the OsRbohs gene. The application of fluridone, an ABA biosynthesis inhibitor, increased membrane injury and the accumulation of ROS under heat stress. Exogenous potent antioxidants (proanthocyanidins, PC) significantly alleviated leaf withering by decreasing ROS overaccumulation and membrane injury induced by heat stress. In addition, ABA pretreatment significantly superinduced the expression of ABA-responsive genes SalT and OsWsi18 , the ABA biosynthesis genes OsNCED3 and OsNCED4 , and the heat shock-related genes OsHSP23.7 , OsHSP17.7 , OsHSF7 and OsHsfA2a . Taken together, these results suggest that exogenous ABA has a potential priming effect for enhancing heat stress tolerance of rice seedlings mainly by improving antioxidant defense capacity and heat shock-related genes.

Fundamentals of Membrane Lipid Replacement: A Natural Medicine Approach to Repairing Cellular Membranes and Reducing Fatigue, Pain, and Other Symptoms While Restoring Function in Chronic Illnesses and Aging

Membrane Lipid Replacement (MLR) uses natural membrane lipid supplements to safely replace damaged, oxidized lipids in membranes in order to restore membrane function, decrease symptoms and improve health. Oral MLR supplements contain mixtures of cell membrane glycerolphospholipids, fatty acids, and other lipids, and can be used to replace and remove damaged cellular and intracellular membrane lipids. Membrane injury, caused mainly by oxidative damage, occurs in essentially all chronic and acute medical conditions, including cancer and degenerative diseases, and in normal processes, such as aging and development. After ingestion, the protected MLR glycerolphospholipids and other lipids are dispersed, absorbed, and internalized in the small intestines, where they can be partitioned into circulating lipoproteins, globules, liposomes, micelles, membranes, and other carriers and transported in the lymphatics and blood circulation to tissues and cellular sites where they are taken in by cells and partitioned into various cellular membranes. Once inside cells, the glycerolphospholipids and other lipids are transferred to various intracellular membranes by lipid carriers, globules, liposomes, chylomicrons, or by direct membrane–membrane interactions. The entire process appears to be driven by ‘bulk flow’ or mass action principles, where surplus concentrations of replacement lipids can stimulate the natural exchange and removal of damaged membrane lipids while the replacement lipids undergo further enzymatic alterations. Clinical studies have demonstrated the advantages of MLR in restoring membrane and organelle function and reducing fatigue, pain, and other symptoms in chronic illness and aging patients.

Effective Management of Cucumber Powdery Mildew with Essential Oils

This research evaluated the efficacy of essential oils in the management of cucumber powdery mildew. Essential oils of lemongrass, lemon, thyme, peppermint, abundance blend, purification blend, and thieves blend were tested in vitro and under greenhouse conditions in two separate experiments. The effects of essential oils were tested against powdery mildew disease at concentrations of 1.0–2.5 mL/L, and the consequent impact of the oils on plant growth was evaluated. Powdery mildew fungus, Podosphaera xanthii, was identified using sequencing of the ITS region. The essential oils significantly reduced disease incidence up to 77.3% compared with the positive control (p < 0.5). Moreover, the essential oils increased the plant length (up to 187 cm), leaf area (up to 27.5 cm2), fresh weight (up to 123 g), dry weight (up to 22.5 g), number of flowers (16.3), and metabolite content compared with the positive control (p < 0.5). Cell membrane injury decreased significantly in the oil-treated pants (p < 0.5), indicating the protective effect of essential oils. This study recommends the application of essential oils in an appropriate dose (2.5 mL/L) to protect cucumber plants against powdery mildew. Overdose of the oils (more than 2.5 mL/L) should be avoided due to adverse effects.

The Efficiency of Different Priming Agents for Improving Germination and Early Seedling Growth of Local Tunisian Barley under Salinity Stress

The current work aimed to investigate the effect of seed priming with different agents (CaCl2, KCl, and KNO3) on germination and seedling establishment in seeds of the barley species of both Hordeum vulgare (L. Manel) and Hordeum maritimum germinated with three salt concentrations (0, 100, and 200 mM NaCl). The results showed that under unprimed conditions, salt stress significantly reduced the final germination rate, the mean daily germination, and the seedling length and dry weight. It led to a decrease in the essential nutrient content (iron, calcium, magnesium, and potassium) against an increase in sodium level in both of the barley species. Moreover, this environmental constraint provoked a membrane injury caused by a considerable increase in electrolyte leakage and the malondialdehyde content (MDA). Data analysis proved that seed priming with CaCl2, KCl, and KNO3 was an effective method for alleviating barley seed germination caused by salt stress to varying degrees. Different priming treatments clearly stimulated germination parameters and the essential nutrient concentration, in addition to increasing the seedling growth rate. The application of seed priming reduced the accumulation of sodium ions and mitigated the oxidative stress of seeds caused by salt. This mitigation was traduced by the maintenance of low levels of MDA and electrolyte leakage. We conclude that the priming agents can be classed into three ranges based on their efficacy on the different parameters analyzed; CaCl2 was placed in the first range, followed closely by KNO3, while the least effective was KCl, which placed in the third range.

Short-term transcriptomic response to plasma membrane injury

Plasma membrane repair mechanisms are activated within seconds post-injury to promote rapid membrane resealing in eukaryotic cells and prevent cell death. However, less is known about the regeneration phase that follows and how cells respond to injury in the short-term. Here, we provide a genome-wide study into the mRNA expression profile of MCF-7 breast cancer cells exposed to injury by digitonin, a mild non-ionic detergent that permeabilizes the plasma membrane. We focused on the early transcriptional signature and found a time-dependent increase in the number of differentially expressed (> twofold, P < 0.05) genes (34, 114 and 236 genes at 20-, 40- and 60-min post-injury, respectively). Pathway analysis highlighted a robust and gradual three-part transcriptional response: (1) prompt activation of immediate-early response genes, (2) activation of specific MAPK cascades and (3) induction of inflammatory and immune pathways. Therefore, plasma membrane injury triggers a rapid and strong stress and immunogenic response. Our meta-analysis suggests that this is a conserved transcriptome response to plasma membrane injury across different cell and injury types. Taken together, our study shows that injury has profound effects on the transcriptome of wounded cells in the regeneration phase (subsequent to membrane resealing), which is likely to influence cellular status and has been previously overlooked.

Competency of Clove and Cinnamon Essential Oil Fumigation against Toxigenic and Atoxigenic Aspergillus flavus Isolates

Aspergillus flavus is a frequent contaminant of maize grain. We isolated this fungus, determined the colony morphology and species (by internal transcribed spacer sequencing) and measured the aflatoxin content. The selected A. flavus fungi were placed into two groups, toxigenic and atoxigenic; both appeared similar morphologically, except that the atoxigenic group lacked sclerotia. An essential oil fumigation test with clove and cinnamon oils as antifungal products was performed on fungal conidial discs and fungal colonies in Petri plates. Cinnamon oil at 2.5 to 5.0 μL/plate markedly inhibited the mycelial growth from conidial discs of both strains, whereas clove oil showed less activity. The oils had different effects on fungal mycelia. The higher clove fumigation doses of 10.0 to 20.0 μL/plate controlled fungal growth, while cinnamon oil caused less inhibition. Compared with atoxigenic groups, toxigenic A. flavus responded stably. Within abnormal A. flavus hyphae, the essential oils degenerated the hyphal morphology, resulting in exfoliated flakes and shrinkage, which were related to fungal membrane injury and collapse of vacuoles and phialide. The treatments, especially those with cinnamon oil, increased the electroconductivity, which suggested a weak mycelium membrane structure. Moreover, the treatments with essential oils reduced the ergosterol content in mycelia and the aflatoxin accumulation in the culture broth. The fumigations with clove and cinnamon oils inhibited the development of both conidia and colonies of A. flavus in dose-dependent manners.

Monocular-Vision-Based Retinal Membrane Peeling with a Handheld Robot

Retinal membrane peeling requires delicate manipulation. The presence of the surgeon's physiological tremor, the high variability and often low quality of the ophthalmic image, and excessive forces make the tasks more challenging. Preventing unintended movement caused by tremor and unintentional forces can reduce membrane injury. With the use of an actively stabilized handheld robot, we employ a monocular camera-based surface reconstruction method to estimate the retinal plane and we propose the use of a virtual fixture with application of hard and soft stops and motion scaling to improve control of the tool tip during delaminating in a laboratory simulation of retinal membrane peeling. A hard stop just below the membrane surface helps to limit downward force exerted on the surface. Motion scaling also improves the user's control of contact force when delaminating. We demonstrate a reduction of maximum force and maximum surface-penetration distance from the estimated retinal plane using the proposed technique.