Infection of Alfalfa Cotyledons by an Incompatible but Not a Compatible Species of Colletotrichum Induces Formation of Paramural Bodies and Secretion of EVs.

Hemibiotrophic fungi in the genus Colletotrichum employ a biotrophic phase to invade host epidermal cells followed by a necrotrophic phase to spread through neighboring mesophyll and epidermal cells. We used serial block face-scanning electron microscopy (SBF-SEM) to compare subcellular changes that occur in Medicago sativa (alfalfa) cotyledons during infection by Colletotrichum destructivum (compatible on M. sativa) and C. higginsianum (incompatible on M. sativa). Three-dimensional reconstruction of serial images revealed that alfalfa epidermal cells infected with C. destructivum undergo massive cytological changes during the first 60 h following inoculation to accommodate extensive intracellular hyphal growth. Conversely, inoculation with the incompatible species C. higginsianum resulted in no successful penetration events and frequent formation of papilla-like structures and cytoplasmic aggregates beneath attempted fungal penetration sites. Further analysis of the incompatible interaction using focused ion beam-scanning electron microscopy (FIB-SEM) revealed the formation of large multivesicular body-like structures that appeared spherical and were not visible in compatible interactions. These structures often fused with the host plasma membrane, giving rise to paramural bodies that appeared to be releasing extracellular vesicles (EVs). Isolation of EVs from the apoplastic space of alfalfa leaves at 60 h postinoculation showed significantly more vesicles secreted from alfalfa infected with incompatible fungus compared with compatible fungus, which in turn was more than produced by noninfected plants. Thus, the increased frequency of paramural bodies during incompatible interactions correlated with an increase in EV quantity in apoplastic wash fluids. Together, these results suggest that EVs and paramural bodies contribute to immunity during pathogen attack in alfalfa. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Three-Dimensional Ultrastructure of Arabidopsis Cotyledons Infected with Colletotrichum higginsianum.

We used serial block-face scanning electron microscopy (SBF-SEM) to study the host-pathogen interface between Arabidopsis cotyledons and the hemibiotrophic fungus Colletotrichum higginsianum. By combining high-pressure freezing and freeze-substitution with SBF-SEM, followed by segmentation and reconstruction of the imaging volume using the freely accessible software IMOD, we created 3D models of the series of cytological events that occur during the Colletotrichum-Arabidopsis susceptible interaction. We found that the host cell membranes underwent massive expansion to accommodate the rapidly growing intracellular hypha. As the fungal infection proceeded from the biotrophic to the necrotrophic stage, the host cell membranes went through increasing levels of disintegration culminating in host cell death. Intriguingly, we documented autophagosomes in proximity to biotrophic hyphae using transmission electron microscopy (TEM) and a concurrent increase in autophagic flux between early to mid/late biotrophic phase of the infection process. Occasionally, we observed osmiophilic bodies in the vicinity of biotrophic hyphae using TEM only and near necrotrophic hyphae under both TEM and SBF-SEM. Overall, we established a method for obtaining serial SBF-SEM images, each with a lateral (x-y) pixel resolution of 10 nm and an axial (z) resolution of 40 nm, that can be reconstructed into interactive 3D models using the IMOD. Application of this method to the Colletotrichum-Arabidopsis pathosystem allowed us to more fully understand the spatial arrangement and morphological architecture of the fungal hyphae after they penetrate epidermal cells of Arabidopsis cotyledons and the cytological changes the host cell undergoes as the infection progresses toward necrotrophy. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

In Situ Structure Determination of Bacterial Surface Nanomachines Using Cryo-Electron Tomography.

Bacterial surface nanomachines are often refractory to structural determination in their intact form due to their extensive association with the cell envelope preventing them from being properly purified for traditional structural biology methods. Cryo-electron tomography (cryo-ET) is an emerging branch of cryo-electron microscopy that can visualize supramolecular complexes directly inside frozen-hydrated cells in 3D at nanometer resolution, therefore posing a unique capability to study the intact structures of bacterial surface nanomachines in situ and reveal their molecular association with other cellular components. Furthermore, the resolution of cryo-ET is continually improving alongside methodological advancement. Here, using the type IV pilus machine in Myxococcus xanthus as an example, we describe a step-by-step workflow for in situ structure determination including sample preparation and screening, microscope and camera tuning, tilt series acquisition, data processing and tomogram reconstruction, subtomogram averaging, and structural analysis.

Evaluating the radioprotective effect of Cimetidine, IMOD, and hybrid radioprotectors agents: An in-vitro study.

INTRODUCTION: There are various radioprotective agents with different mechanisms that help to decrease ionizing radiation side effects. The radioprotective effect of Cimetidine and IMOD was assessed individually and compared with the hybrid radioprotectors agents (HRPAs-IMOD and Cimetidine) on human lymphocyte cells. METHODS: Twenty healthy volunteers (ten men and ten women) participated in the present study. About 75 mL peripheral blood lymphocytes from each individual were collected, and they were divided into 36 groups. Briefly, the blood samples were treated with different concentrations of Cimetidine (12.6 and 25.2 µg/mL) and IMOD (0.04, 0.08, and 0.12 mg/mL), and also a combination of these agents, namely hybrid radioprotectors agents (HRPAs). Besides, the irradiated groups were exposed to 2 and 4 Gy of Co-60 gamma irradiation. The amount of cellular damage was assessed using the micronucleus assay. The repeated measurements and paired T-test statistical analysis were used to compare the micronucleus frequencies in different groups. RESULTS: The micronucleus frequencies were significantly reduced (p < 0.05) in irradiated groups when the non-toxic concentrations of Cimetidine, IMOD, and HRPAs have been used. The reduction in micronucleus frequency was obtained 5-29% for Cimetidine and 40-51% for IMOD in peripheral blood lymphocytes irradiated with 2 Gy. This reduction in 4 Gy irradiation was 8-17% for Cimetidine and 27-37% for IMOD. The HRPAs resulted in a higher radioprotective effect, in a way that they cause up to 58% and 43% micronucleus frequency reduction in 2 and 4 Gy, respectively. CONCLUSION: In conclusion, the HRPAs showed the highest level of radioprotective. In addition, IMOD was remarkably higher radioprotective than Cimetidine, which may be related to its greater non-toxic concentrations.

A Newly Discovered Forest of the Whip Coral Viminella flagellum (Anthozoa, Alcyonacea) in the Mediterranean Sea: A Non-Invasive Method to Assess Its Population Structure.

Coral forests are vulnerable marine ecosystems formed by arborescent corals (e.g., Anthozoa of the orders Alcyonacea and Antipatharia). The population structure of the habitat-forming corals can inform on the status of the habitat, representing an essential aspect to monitor. Most Mediterranean corals live in the mesophotic and aphotic zones, and their population structures can be assessed by analyzing images collected by underwater vehicles. This is still not possible in whip-like corals, whose colony lengths and flexibilities impede the taking of direct length measurements from images. This study reports on the occurrence of a monospecific forest, of the whip coral Viminella flagellum in the Aeolian Archipelago (Southern Tyrrhenian Sea; 149 m depth), and the assessment of its population structure through an ad-hoc, non-invasive method to estimate a colony height based on its width. The forest of V. flagellum showed a mean density of 19.4 ± 0.2 colonies m-2 (up to 44.8 colonies m-2) and no signs of anthropogenic impacts. The population was dominated by young colonies, with the presence of large adults and active recruitment. The new model proved to be effective for non-invasive monitoring of this near threatened species, representing a needed step towards appropriate conservation actions.

Cryo-Electron Tomography and Automatic Segmentation of Cultured Hippocampal Neurons.

Cryo-electron tomography is fast becoming a preferred method for studying intracellular environments at the molecular scale. Increases in data collection throughput means that large numbers of tomograms can be generated at rates too fast for humans to easily explore quantitatively. Currently, there is a large effort to make data collection and segmentation tools more automated. Here, we describe a workflow for preparing cultured neurons on electron microscopy grids, batch tomographic data collection, reconstruction and automatic segmentation using freely and commercially available software.

Experimental steering of electron microscopy studies using prior X-ray computed tomography.

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) can provide unrivalled high-resolution images of specific features and volumes of interest. However, the regions interrogated are typically very small, and sample preparation is both time-consuming and destructive. Here we consider how prior X-ray micro-computed tomography (microCT) presents an opportunity to increase the efficiency of electron microscopy in biology. We demonstrate how it can be used to; select the most promising samples and target site-specific locations; provide a wider context of the location being interrogated (multiscale correlative imaging); guide sample preparation and 3D imaging schemes; as well as quantify the effects of destructive sample preparation and staining procedures. We present a workflow utilising open source software in which microCT can be used either broadly, or precisely, to experimentally steer and inform subsequent electron microscopy studies. As automated sample registration procedures are developed to enable correlative microscopy, experimental steering by prior CT could be beneficially routinely incorporated into many experimental workflows.

Protective Effects of IMOD and Cimetidine against Radiation-induced Cellular Damage.

Radiation damage is to a large extent caused by overproduction of reactive oxygen species (ROS). Radioprotectors are agents or substances that reduce the effects of radiation in healthy normal tissues while maintaining the sensitivity to radiation damage in tumor cells. Radioprotectors are agents or substances that reduce the effects of radiation in healthy normal tissues while maintaining the sensitivity to radiation damage in tumor cells Cimetidine was found more effective when used in vivo; this effect might be due to the augmentation of the presence of Sulphur atom in the compound which is ýimportant for their scavenging activity. Recently, a new herbal-based medicine with immunomodulatory capacities, Setarud (IMOD), was introduced as an additional therapy in various inflammatory diseases and HIV infection. IMOD is a mixture of herbal extracts enriched with selenium. Selenium confers protection by inducing or activating cellular free-radical scavenging systems and by enhancing peroxide breakdown. This article suggests that nontoxic amount of IMOD and cimetidine have radioprotective properties and could reduce cytotoxic effects of radiation.

Effects of IMOD™ on angiogenesis, miR-503 and CDC25 expression levels in heart tissue of diabetic male rats.

OBJECTIVE: Diabetes is associated with vascular complications and impaired angiogenesis. Since angiogenesis plays a crucial role in vascular homeostasis in ischemic heart diseases, in this study, the effect of IMOD™ on miR-503 and CDC25 expression level which are altered in impaired angiogenesis were investigated in heart tissue of diabetic rats. MATERIALS AND METHODS: Forty male Wistar rats (200-250 g) were randomly classified into 4 groups: control (C), IMOD™ (I), diabetes (D), and diabetes+IMOD™ (D+I). For induction of experimental diabetes in animals, a single dose of streptozotocin (STZ; 60mg/kg) was injected intraperitoneally. After 8 weeks of treatment with IMOD™ (20 mg/kg/day), heart tissue samples were removed and used for measurement of miR-503 and CDC25 expression level as well as histological studies. RESULTS: Results of this study showed that diabetes decreased heart tissue angiogenesis which was associated with increased miR-503 and reduced CDC25 expression levels (p<0.05) and IMOD™ could reduce the expression of miR-503 and increase the expression of CDC25 (p<0.05). Moreover, IMOD™ extensively induced angiogenesis in the heart tissue of diabetic group. However, IMOD™ had no significant effect on expressions of miR-503 and CDC25, or angiogenesis in healthy rats. CONCLUSION: This study showed that IMOD™ is able to increase angiogenesis in the heart tissue of diabetic rats. The angiogenic effect of IMOD™ is associated with reduction of miR-503 expression and increased expression of CDC25.

The effects of setarud on the immunological status of HIV-positive patients: Efficacy of a novel multi-herbal drug.

OBJECTIVE: This study examines the effect of the addition of IMOD, a novel multi-herbal drug to the highly active anti-retroviral therapy (HAART) regimen, on the immunological status of HIV-positive patients. MATERIALS AND METHODS: A randomized two-parallel-group (HAART group versus HAART+IMOD group), pretest-posttest design was used.Sixty patients with indications for treatment with the HAART regimen participated. One week before and 2 days after the treatments, immunological parameters including total lymphocyte count (TLC) and CD4 cell count were assessed.The intervention group received the HAART regimen plus IMOD every day for 3 months. The control group received only the HAART regimen every day for 3 months. RESULTS: In the intervention group, a significant difference was observed in CD4between before and after drug therapy (CD4 was increased). However, in the control group, the difference in CD4 was not significant before and after drug therapy. The difference in TLC was not significantly different between the two groups before and after therapy. Nevertheless, TLC was higher in the intervention group. CONCLUSION: IMOD (as a herbal drug) has been successfully added to the HAART regimen to improve the immunological status of HIV-positive patients.

Impacts of 25 years of groundwater extraction on subsidence in the Mekong delta, Vietnam.

Many major river deltas in the world are subsiding and consequently become increasingly vulnerable to flooding and storm surges, salinization and permanent inundation. For the Mekong Delta, annual subsidence rates up to several centimetres have been reported. Excessive groundwater extraction is suggested as the main driver. As groundwater levels drop, subsidence is induced through aquifer compaction. Over the past 25 years, groundwater exploitation has increased dramatically, transforming the delta from an almost undisturbed hydrogeological state to a situation with increasing aquifer depletion. Yet the exact contribution of groundwater exploitation to subsidence in the Mekong delta has remained unknown. In this study we deployed a delta-wide modelling approach, comprising a 3D hydrogeological model with an integrated subsidence module. This provides a quantitative spatially-explicit assessment of groundwater extraction-induced subsidence for the entire Mekong delta since the start of widespread overexploitation of the groundwater reserves. We find that subsidence related to groundwater extraction has gradually increased in the past decades with highest sinking rates at present. During the past 25 years, the delta sank on average ∼18 cm as a consequence of groundwater withdrawal. Current average subsidence rates due to groundwater extraction in our best estimate model amount to 1.1 cm yr-1, with areas subsiding over 2.5 cm yr-1, outpacing global sea level rise almost by an order of magnitude. Given the increasing trends in groundwater demand in the delta, the current rates are likely to increase in the near future.

In vitro protection of human lymphocytes from toxic effects of chlorpyrifos by selenium-enriched medicines.

OBJECTIVES: Chlorpyrifos (CP) is a broad-spectrum organophosphorus pesticide used extensively in agricultural and domestic pest control, accounting for 50% of the global insecticidal use. In the present study, protective effects of two selenium-enriched strong antioxidative medicines IMOD and Angipars were examined in human lymphocytes treated with CP in vitro. MATERIALS AND METHODS: Isolated lymphocytes were exposed to 12 µg/ml CP either alone or in combination with effective doses (ED50) of IMOD (0.2 µg/ml) and Angipars (1 µg/ml). After 3 days incubation, the viability and oxidative stress markers including cellular lipid peroxidation (LPO), myeloperoxidase (MPO), total thiol molecules (TTM), and total antioxidant power (TAP) were evaluated. Also, the levels of tumor necrosis factor-α (TNF-α), as inflammatory index along with acetylcholinesterase (AChE) activity and cell apoptosis were assessed by flow cytometry. RESULTS: Results indicated that effective doses of IMOD and Angipars reduced CP-exposed lymphocyte mortality rate along with oxidative stress. Both agents restored CP-induced elevation of TNF-α and protected the lymphocytes from CP-induced apoptosis and necrosis. CONCLUSION: Overall, results confirm that IMOD and Angipars reduce the toxic effects associated with CP through free radical scavenging and protection from apoptosis and necrosis.

Herbal medicine IMOD suppresses LPS-induced production of proinflammatory cytokines in human dendritic cells.

Traditional medicines that stimulate or modulate the immune system can be used as innovative approaches to treat immunological diseases. The herbal medicine IMOD has been shown to strongly modulate immune responses in several animal studies as well as in clinical trials. However, little is known about the mechanisms of IMOD to modulate immunity. Here we have investigated whether IMOD modulates the immunological function of human dendritic cells (DCs). IMOD alone did not induce DC maturation nor production of cytokines. Notably, IMOD decreased the production of pro-inflammatory cytokines IL-6, IL-12 p70, and TNFα by LPS-activated DCs at both mRNA and protein levels in a dose dependent manner. In contrast, treatment with IMOD did not affect LPS induced-production of the anti-inflammatory cytokine IL-10. Furthermore, IMOD inhibited T cell activation/proliferation by LPS-treated DCs and skewed T-cells responses toward the T helper type 2 polarization. These data strongly indicate that IMOD has a potent immunomodulatory ability that affects TLR signaling and thereby modulates DC function. Insight into the immunomodulatory effect of herbal medicine IMOD may provide innovative strategies to affect the immune system and to help combat various diseases.

Effects of some natural immunomodulatory compounds in combination with thalidomide on survival rate and tumor size in fibrosarcoma-bearing mice.

PURPOSE: Despite significant advances have been achieved in cancer therapy, response to conventional treatments like surgery, radiotherapy and chemotherapy varies among individuals. Immunotherapy is known to be an effective strategy for patients who are resistant to the currently available interventions. METHODS: Ninety-six male Balb/c mice (aged 6-8 weeks) were selected and divided into twelve groups of eight. Approximately, 1×10(6)of WEHI-164 cells were injected to each mouse for tumor genesis. Five immunotherapy treatments were considered in this study, including Heat Shock Proteins (HSP), Bacillus Calmette-Guérin (BCG), Bifidobacterium, Immuno-Modulator Drug (IMOD) and Thalidomide. After tumor formation, the groups were treated with one or more of these therapies. Tumor size and survival rate was regularly monitored. RESULTS: Depending on the treatment group, tumor sizes were different. In some groups, combined treatments demonstrated more inhibitory effects on tumor growth rate. The mice in group (IMOD+ Thalidomide) had the lowest survival rate but group (BCG+ HSP+ Thalidomide) survived until the end of the experiment. CONCLUSION: The (HSP+ BCG+ Thalidomide) group exhibited satisfactory outcomes and two third of the mice in this group went into complete remission. Some combination therapies in test groups had significant impacts on survival and tumor growth rate.

Evaluation of a Novel Herbal Immunomodulator Drug (IMOD) in Treatment of Experimental Canine Visceral leishmaniasis.

Toxicity and drug resistance against pentavalent antimonials, medications of choice in treatment of leishmaniasis for more than 5 decades, have become important subjects globally. This study was a randomized, open labeled trial that was designed to determine efficacy and safety of IMOD as a novel herbal immunomodulator drug for treatment of canine visceral leishmaniasis (CVL). Twenty healthy mongrel dogs were infected with Iranian strain of L. Infantum amastigotes and randomly divided to 5 groups with four animals for each included on: I: negative control (non-infected) II: Glucantime® III: Glucantime® plus IMOD (immune-chemotherapy) IV: IMOD and V: positive control (non-treated). Physical examination, hematological, biochemical, serological, parasitological, pathological and imaging evaluations were performed pre-/post- interventions every month for 3 months. Comparing with control groups (I&V), immune-chemotherapy group (Glucantime® plus IMOD) showed significantly higher efficacy in resolving the clinical signs and hematobiochemistry factors. Based on our results, using IMOD in combination with meglumine antimoniate (Glucantime®) has significantly improved CVL than the latter drug alone. So, it seems this new herbal medicine is useful as adjuvant therapy for canine visceral leishmaniasis.

The in vitro anti-viral potential of Setarud (IMOD™), a commercial herbal medicine with protective activity against acquired immune deficiency syndrome in clinical trials.

OBJECTIVES: Setarud (IMOD™) is a herbal medicine with beneficial effect for patients suffering Human immunodeficiency virus (HIV) infection and has been approved for IV (intra venues) injection. The beneficial effect of IMOD administration for acquired immune deficiency syndrome (AIDS) patient has been proved in previous clinical trials. Here the in vitro inhibitory effect of IMOD against HIV-1, Herpes simplex virus (HSV) and murine leukemia viruses (MLV) was evaluated. MATERIALS AND METHODS: HIV single cycle replication and HSV plaque reduction assays were used to evaluate the anti-viral effect. The level of HIV replication was monitored by p24 capture Enzyme-linked immunosorbent assay (ELISA). The single round infection [with green fluorescent protein (GFP) reporter MLV and HIV], virucidal and time-of-additions (HSV) assays were utilized to determine the mode of anti-viral activity. The toxicity of IMOD for cells was monitored by XTT (sodium 3_-[1 (phenylaminocarbonyl)- 3,4-tetrazolium]-bis (4-methoxy-6-nitro)benzene sulfonic acid) cell proliferation assay kit. RESULTS: IMOD inhibited 50% of HIV-1 and HSV replication (IC(50)) at 6.5 × 10(-4) and 4.3 × 10(-3)V/V concentrations, respectively. The IC(50) value against HIV-1 and MLV infection were 6 × 10(-4)V/V and 4.9 × 10(-4)V/V. Virucidal assay showed that IMOD reduces the potency of HIV and HSV particles to 41 and 54% of control, respectively. Time-of-addition study revealed that IMOD inhibits the replication of HSV at a stage after penetration of virions to the target cells. CONCLUSIONS: Data from this study indicate that IMOD has significant anti-viral activity against HIV, HSV and MLV. Setarud could be subjected to further investigation after isolation of the constituents and determination of the toxic components.

Efficacy of Setarud (IMOD™), a novel electromagnetically-treated multi-herbal compound, in mouse immunogenic type-1 diabetes.

INTRODUCTION: The aim of this study was to evaluate the effects and mechanisms of Setarud (IMOD™) as a multi-herbal medicinal formula on a mouse model of type 1 diabetes. METERIAL AND METHODS: Autoimmune diabetes was induced by multiple low-dose intraperitoneal injection of 40 mg/kg of streptozotocin (STZ) for five consecutive days. IMOD™ was administered at an effective dose of 20 mg/kg/day for 21 days. After 21 days of treatment, the pancreases of the animals were separated and homogenized. In the pancreas tissue, the level of lipid peroxidation as thiobarbituric acid reactive substances (TBARS), total antioxidant power as ferric reducing ability of pancreas (FRAP), myeloperoxidase (MPO), and the concentrations of interleukin-1 (IL-1ß) and tumour necrosis factor-α (TNF-α) were evaluated. Glucose changes were tested in the blood. Microscopic changes in the pancreas were followed by histological examinations. RESULTS: No significant difference was found between IMOD™ and diabetic control groups in blood glucose pattern. STZ-exposed mice showed a significant increase in pancreatic TBARS, MPO, IL-1ß, and TNF-α levels, along with a significant decrease in FRAP value. Co-administration of IMOD™ significantly improved all the mentioned parameters disrupted by STZ administration except for blood glucose and histological changes. CONCLUSION: IMOD™ could ameliorate oxidative and immunological distresses of type-1 immunogenic diabetes but could not normalize blood glucose. Further studies are recommended to clarify the effects of IMOD™ on immunological factors to address whether this new agent could be applied in diabetes prevention or therapy.

Examination of Setarud (IMOD™) in the management of patients with severe sepsis.

BACKGROUND AND THE PURPOSE OF THE STUDY: Analysis of current immunomodulating strategies indicates that monovalent approaches are unlikely to restore immunostasis or achieve complete therapy of sepsis. Setarud (IMOD) as a mixture of urtica, carotenoids, urea, and selenium has been recently patented for its potential in reduction of Tumor Necrosis Factor alpha (TNF-α) and Interferon-γ and Interleukin-2 levels. The aim of this study was to examine efficacy of IMOD in the management of patients with severe sepsis. METHODS: Twenty patients with severe sepsis and acute physiology and chronic health evaluation (APACHE) score of more than 20 were randomized to receive standard treatment of severe sepsis (control group) or standard treatment plus IMOD (IMOD group). The group treated with IMOD for 14 days was according to the pilot study and regarding the stability of patient's conditions in the ICU. Of course patients in both groups received standard treatment and all were monitored for 28 days. Blood samples were analyzed for interleukins (IL-1, IL-2, IL-6), plasminogen activator inhibitor (PAI-1), TNF-α, total thiol molecules (TTM), nitric oxide (NO), total antioxidant power (TAP), and lipid peroxidation (LPO). Daily APACHE, Sequential Organ Failure Assessment (SOFA), and Simplified Acute Physiology Score (SAPS) were calculated. RESULTS AND MAJOR CONCLUSION: Comparing with controls, IMOD was significantly effective in improving SAPS, SOFA, and APACHE scores, and reduction of mortality rate. Among tested inflammatory biomarkers, IMOD significantly improved TTM and TNF-α values. It is concluded that IMOD might be added as a safe adjutant to standard treatment of severe sepsis.