Ultrastructural Morphology Research Articles

Galangin Triggers Eryptosis and Hemolysis Through Ca2+ Nucleation and Metabolic Collapse Mediated by PKC/CK1α/COX/p38/Rac1 Signaling Axis.

Anticancer drugs cause anemia in patients through eryptosis and hemolysis. We thus studied the in vitro toxicity of galangin (GAL) in red blood cells (RBCs). RBCs were exposed to 50-500 μM of GAL and analyzed for markers of eryptosis and hemolysis. Ca2+ nucleation, phosphatidylserine (PS) externalization, oxidative stress, and cell size were detected via fluorescence-activated cell sorting using Fluo4/AM, annexin-V-FITC, 2',7'-dichlorodihydrofluorescein diacetate, and forward scatter (FSC), respectively. Acetylcholinesterase (AChE) activity was measured via Ellman's assay and ultrastructural morphology was examined via scanning electron microscopy. Membrane rupture and extracellular hemoglobin, aspartate transaminase (AST), and lactate dehydrogenase (LDH) were assessed via colorimetric methods. Distinct experiments were carried out to identify protective agents and signaling pathways using small-molecule inhibitors. GAL triggered sucrose-sensitive hemolysis with AST and LDH leakage, increased annexin-V-FITC and Fluo4 fluorescence, and decreased FSC and AChE activity which was associated with the formation of granulated echinocytes. Ca2+ omission and energy replenishment with glucose, adenine, and guanosine blunted PS externalization and preserved cellular volume. Moreover, caffeine, Trolox, heparin, and uric acid had similar ameliorative effects. Hemolysis was abrogated via caffeine, Trolox, heparin, mannitol, lactate, melatonin, and PEG 8000. Notably, co-treatment of cells with GAL and staurosporin, D4476, or acetylsalicylic acid prevented PS externalization whereas only the presence of SB203580 and NSC23766 rescued the cells from GAL-induced hemolysis. Ca2+ nucleation and metabolic collapse mediated by PKC/CK1α/COX/p38/Rac1 drive GAL-induced eryptosis and hemolysis. These novel findings carry ramifications for the clinical prospects of GAL in anticancer therapy.

Diversity of Chlorophyceae in Some Wetlands of Nkwen in the City of Bamenda (North-West Region, Cameroon)

Chlorophyceae are one of the classes of green algae, distinguished mainly on the basis of ultra-structural morphology and at the basis of trophic chains. Human activities lead to imbalance of aquatic ecosystems and disappearance of primary producers. The aim of the study was to determine the diversity and distribution of Chlorophyceae in some wetlands of Bamenda. Data collection and samples were taken from June 2023 to May 2024 in all the study sites. The sampling of Chlorophyceae was done using plankton net for phytoplankton, and scrabbing for periphyton. Diversity indices showed significant variations in the different study sites. The species richness amounted to 3 orders divided in 11 families, 13 genera and 22 species. The most dominant family was Chlamydomonadaceae with 5 species; followed by Selenastraceae with 4 species. Chlorophyceae are not more diversified in Nkwen Rivers. They are greatly adaptive and characterized organisms, and may survive in different environmental conditions.

Impact of oxidized low-density lipoprotein on rat liver sinusoidal endothelial cell morphology and function

Atherogenesis is associated with elevated plasma levels of oxidized low-density lipoproteins (oxLDL). In vivo, oxLDL causes liver endothelial swelling, and disrupts liver sinusoidal endothelial cell (LSECs) fenestrations. We mapped the nanoscale kinetics of these changes in vitro in isolated rat LSECs challenged with oxLDL and monitored viability with endocytosis and cytotoxicity assays. OxLDL disrupted LSEC ultrastructure – increasing oxLDL concentrations and oxidation levels caused sieve plate loss, fenestration fusion, and gap formation. Importantly, these effects were not uniform across all LSECs. LSECs retained the ability to endocytose ligands irrespective of the presence of oxLDL. However, increasing oxidation levels and concentrations of oxLDL inhibited LSEC mediated degradation of endocytosed ligands. Viability was unaffected by any oxLDL challenge. In conclusion, oxLDL disrupts LSEC ultrastructural morphology in vitro but LSECs remain viable and mostly maintain the scavenging function during oxLDL challenge.

No Benefit of 3% Hypertonic Saline Following Experimental Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a stroke subtype with a high mortality rate (~ 40%). After ICH, the mass effect of the hematoma and edema contribute to raised intracranial pressure (ICP) and poor outcome. Endogenous compensatory mechanisms that blunt ICP elevations include redirection of venous blood and cerebrospinal fluid, along with brain tissue compliance (e.g., decreased cell volume, increased cell density); however, these limited reserves can be exhausted after severe stroke, resulting in decompensated ICP that requires careful clinical management. Management strategies can include administration of hypertonic saline (HTS), an osmotic agent that putatively attenuates edema, and thereby ICP elevations. Evidence regarding the efficacy of HTS treatment following ICH remains limited. In this study, adult male rats were given a collagenase-induced striatal ICH and a bolus of either 3% HTS or 0.9% saline vehicle at 2- and 14-hours post-stroke onset. Neurological deficits, edema, ipsilateral cell volume and density (in areas S1 and CA1), and contralateral CA1 ultrastructural morphology were assessed 24 h post-ICH. Animals had large bleeds (median 108.2 µL), extensive edema (median 83.9% brain water content in ipsilateral striatum), and evident behavioural deficits (median 5.4 neurological deficit scale score). However, HTS did not affect edema (p ≥ 0.4797), behaviour (p = 0.6479), cell volume (p ≥ 0.1079), or cell density (p ≥ 0.0983). Qualitative ultrastructural assessment of contralateral area CA1 suggested that HTS administration was associated with paradoxical cellular swelling in ICH animals. Overall, there was no benefit with administering 3% HTS after ICH.

The Effect of Metallic Nanoparticles Supplementation in Semen Extender on Post-thaw Quality and Fertilizing Ability of Egyptian Buffalo (Bubalus bubalis) Spermatozoa.

Nanomaterials offer several promising prospects in the field of farm animal reproduction, encompassing a broad range of applications such as transgenesis and the precise delivery of substances to sperm cells, antimicrobial, antioxidants properties as well as their potent role in improving cryopreservation methods. The aim of the present study is to explore the effect of supplementing the semen extender with 10µg/mL nano gold (Au-NPs10), 10µg/mL nano silver (Ag-NPs10), 1µg/mL nano selenium (Se-NPs1), and 100µg/mL nano zinc oxide (ZnO-NPs100) on sperm characteristics and kinematics parameters, acrosome integrity, oxidative biomarkers, morphological and apoptosis-like changes of frozen-thawed buffalo bull sperm, and, ultimately, their fertilizing capacity. The results revealed that all aforementioned nano materials significantly improved viability, progressive motility, membrane integrity, acrosome integrity, and kinematic parameters as well as apoptosis-like changes of post-thawed buffalo bull sperm compared to the control (p < 0.05). No discernible effects were observed on sperm ultrastructure morphology measures as a response to the addition of these metallic nanoparticles to the extender. The values of caspase 3 significantly decreased by 64.22, 45.99, 75.59, and 49.39% in Au-NPs10, Ag-NPs10, Se-NPs1, and ZnO-NPs100treated groups, respectively, compared to the control. The addition of 100µg ZnO-NPs to the extender significantly decreased the total count of bacteria, fungi, and yeast compared to the control (p < 0.05). The AuNPs10 and SeNPs1 treated groups showed lower content of malondialdehyde, hydrogen peroxide, and nitric oxide concentrations and higher values of total antioxidant capacity of post-thawed extended semen (p < 0.05). Pregnancy rates increased by 17.5, 20, and 30% in buffalo cows inseminated with sperm treated with ZnO-NPs100, Se-NPs1, and Au-NPs10, respectively, compared to the control group. The present results indicate that the freezing extender supplemented with metallic nanoparticles can be an effective strategy to enhance the cryotolerance and fertility potential of buffalo bull sperm.

Decursin ameliorates neurotoxicity induced by glutamate through restraining ferroptosis by up-regulating FTH1 in SH-SY5Y neuroblastoma cells

Alzheimer’s disease (AD) is the most common form of neurodegeneration which currently has no effective treatment. Ferroptosis is a new style of programmed cell death and is widely implicated in the pathogenesis and progression of AD. Decursin has been shown widely neuroprotective effects but poorly understood about the underlying mechanisms between decursin and ferroptosis in AD. Here, the protective effect of decursin and the underlying mechanism under glutamate treatment in SH-SY5Y cells was investigated. SH-SY5Y cells were cultured with glutamate in the presence or absence of decursin. The safe concentrations of decursin on SH-SY5Y cells were measured via CCK-8. Furthermore, LDH content, antioxidant enzyme activities including GPx, CAT and SOD, MDA contents, GSH levels, ROS formation, MMP, mitochondria ultrastructure morphology change, and intracellular Fe2+ levels were measured to investigate the influence of decursin and Fer-1 on ferroptosis in glutamate-treated SH-SY5Y cells. Moreover, the expressions of ferroptosis-related proteins were determined by Western blot. As a result, glutamate-induced cell survival was markedly elevated and morphological change was improved by decursin administrated in SH-SY5Y cells. Furthermore, decursin could reversed the decreased antioxidant enzyme activities, GSH levels, GPX4n and FTH1 expression, as well as the increased iron levels, LDH, MDA, ROS formation, and MMP, which showed similar effects to Fer-1, the specific ferroptosis inhibitor. Therefore, the inhibitory effect of decursin on ferroptosis probably was partially governed by FTH1 expression to regulate the cellular iron homeostasis. Additionally, decursin facilitated the translocation of Nrf2 from the cytoplasm to the nucleus. Taken together, our data for the first time suggest that decursin could ameliorate neurotoxicity induced by glutamate by attenuating ferroptosis via alleviating cellular iron levels by up-regulating FTH1 expression which is attributing to its promotion of Nrf2 translocation into the nucleus in SH-SY5Y neuroblastoma cells. Hence, decursin might be a novel and promising therapeutic option for AD. In addition, our study also provided some new clues to potential target for the intervention and therapy of AD.

Carvacrol alters the membrane phospholipids in erythromycin-resistant Streptococcus pyogenes

BackgroundPhospholipids are significant constituents of bacterial membranes. Bacteria can alter their cytoplasmic membrane fatty acid composition in response to changing environmental stress conditions. Carvacrol, a mono-terpenoid phenol, has excellent antibacterial properties against Streptococcus pyogenes. PurposeThis study examined modifications of the membrane phospholipid composition in the presence of sublethal carvacrol concentrations using erythromycin-resistant S. pyogenes. MethodsThe cell membrane isolates (CMIs) were prepared from S. pyogenes strain Spy 1558, erm. The ultrastructural morphology was studied using transmission electron microscopy (TEM). Carvacrol-treated CMIs were analyzed for fluidity changes using fluorescence-anisotropy. Changes in membrane lipid composition were assessed using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS/MS). ResultsFluidity of the bacterial membrane increased time-dependently when exposed to carvacrol. The CMI isolated from cells treated with carvacrol showed a change in cardiolipins (CL), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) percentages compared to the untreated control. ConclusionCarvacrol induces modulation of specific membrane phospholipid and fatty acid composition of S. pyogenes. Further investigations are necessary to understand how fatty acid and phospholipid biosynthesis can be influenced by carvacrol.

Toxicity of 6-gingerol and Cymbopogon citratus against Pediculus humanus capitis De Geer (Phthiraptera: Pediculidae): Mortality, detoxifying enzymes, and morphological ultrastructure alterations in lice

Pediculus humanus capitis (head louse), which causes pediculosis capitis, remains a global health concern. Plant products are efficient alternative pediculicides for treating the human ectoparasite P. h. capitis which is resistant to permethrin. The study evaluates the toxicity and mechanisms of 6-gingerol and Cymbopogon citratus leaf extract on P. h. capitis. Pediculus humanus capitis adult stages were exposed to three different dosages of 6-gingerol and C. citratus crude leaf extract on filter sheets for 5, 10, and 30 min, respectively. The biochemical approach was used to assess the activity of detoxifying enzymes including acetylcholinesterase (AChE), glutathione S-transferase (GST), and oxidase. Scanning electron microscope (SEM) was used to investigate the ultrastructure of the morphological body of lice. After 30 min, 6-gingerol and C. citratus leaf extract killed P. h. capitis completely. Bioassay periods significantly affected lice mortality (P < 0.05). The LC50 values for 6-gingerol and C. citratus extract were 1.79 μg/cm2 and 25.0 μg/cm2, respectively. 6-Gingerol and C. citratus leaf extract significantly lower AChE and GST activity (P < 0.05). Cymbopogon citratus also caused morphological ultrastructure changes in P. h. capitis, including an irregularly formed head, thorax, abdominal respiratory spiracles, and belly. 6-Gingerol and C. citratus leaf extracts could be used as an alternate pediculicide to decrease P. h. capitis populations.

Isolation, characterization, and immunomodulatory effects of extracellular vesicles isolated from fish pathogenic Aeromonas hydrophila

Bacterial extracellular vesicles (BEVs) are natural nanocarriers that have shown great potential for biomedical applications such as biomarkers, cancer therapy, immunomodulators, vaccines, wound healing, tissue engineering, and drug carriers. In the present study, BEVs were isolated from the gram-negative bacterium, Aeromonas hydrophila using the ultracentrifugation method and denoted as AhEVs. Using transmission electron microscopy imaging, we confirmed the ultrastructure and spherical shape morphology of AhEVs. Nanoparticle-tracking analysis results showed a mean particle size of 105.5 ± 2.0 nm for AhEVs. Moreover, the particle concentration of AhEVs was 2.34 ± 0.12 × 1011 particles/mL of bacterial supernatant. AhEV-treated fathead minnow (FHM) cells did not show cytotoxicity effects up to 50 μg/mL with no significant decrease in cells. Moreover, no mortality was observed in larval zebrafish up to 50 μg/mL which indicates that the AhEVs are biocompatible at this concentration. Furthermore, fluorescent-labeled AhEVs were internalized into FHM cells. Results of qRT-PCR analysis in FHM cells revealed that cellular pro-inflammatory cytokines such as nuclear factor (NF)-κB, interferon (Ifn), Irf7, interleukin (Il) 8, and Il11 were upregulated while downregulating the expression of anti-inflammatory Il10 in a concentration-dependent manner. AhEV-treated adult zebrafish (5 μg/fish) induced toll-like receptor (tlr) 2 and tlr4; tumor necrosis factor-alpha (tnfα); heat shock protein (hsp) 70; and il10, il6, and il1β in kidney. Protein expression of NF-κB p65 and Tnfα presented amplified levels in the spleen of AhEVs-treated zebrafish. Based on the collective findings, we conclude that AhEVs exhibited morphological and physicochemical characteristics to known EVs of gram (−)ve bacteria. At biocompatible concentrations, the immunomodulatory activity of AhEVs was demonstrated by inducing different immune response genes in FHM cells and zebrafish. Hence, we suggest that AhEVs could be a novel vaccine candidate in fish medicine due to their ability to elicit strong immune responses.

P-357 Exposure to bleomycin in vitro reduces biomarkers of human male gamete competence

Abstract Study question Does male human sperm exposure to bleomycin in vitro affect their aptitude to breed? Summary answer Bleomycin, an antitumor antibiotic, adversely impacts human male gamete morphology and functionality, thus potentially compromising male fertility. What is known already Bleomycin is a chemotherapeutic agent used to treat a number of tumors that affect males of reproductive age. Hitherto, its effect on human male reproductive capacity alone has not been previously shown. The little information available on this subject relates to in vivo animal studies and supports the feasibility of this study. Study design, size, duration Between November 2022 and June 2023, individuals of reproductive age were enrolled in this study and had their clinical semen analyzed. The remaining ejaculate was supplied by 45 participants with sperm concentration above the World Health Organization (WHO) threshold for scientific purposes. Sperm were isolated from seminal plasma in the ejaculate of each individual, diluted to a concentration of 16 X 106 sz/mL (raw). Participants/materials, setting, methods In vitro incubation was performed in sperm preparation medium (SPM; control) or bleomycin (100 µg/mL; exposed) augmented for two hours at 37oC and 5% CO2. The results of exposed group were compared to raw and control. To evaluate the competence of human sperm, markers such as acrosome and DNA integrity, mitochondrial membrane potential, sperm motility, vitality, and oxidative stress were evaluated. Ultrastructural data from transmission electron microscopy was also obtained and correlated with functional experiments. Main results and the role of chance The participants were all of reproductive age (median age of 36 years old). The raw sperm parameters fulfilled the normozoospermia threshold set by the WHO, 2021. Prior to the current trial, none of the participants had been diagnosed with cancer or had received chemotherapy, radiation therapy, or both. Bleomycin exposure for two hours in vitro significantly (P &amp;lt; 0.001) reduced sperm motility, vitality, and chromatin condensation as compared to raw and control sperm. Moreover, bleomycin significantly (P &amp;lt; 0.001) increased the percentage of sperm with DNA fragmentation (sDNAfrag) and low mitochondrial membrane potential (MMP). Broad strands were significantly (P &amp;lt; 0.001) attained by bleomycin-induced breaks in the sperm DNA (Head staining pattern), which were succeeded by histone-rich areas (PAR-post-acrossomic region staining pattern). Additionally, bleomycin significantly (P &amp;lt; 0.05) delayed the acrossomic response, but had no significantly influence on the generation of extracellular and intracellular reactive oxygen species or in its reconstitution. Modifications in ultrastructural morphology caused by bleomycin corroborate the results obtained by functional tests. Limitations, reasons for caution Bleomycin impact on male fertility competence in vivo remains crucial. Increased sample sizes, including prepubertal subjects, are needed to pinpoint the precise bleomycin’s mechanism to cause oxidative stress, apoptosis, and sDNAfrag. Long-term implications on fertility and reproductive health, besides methods to lessen or offset bleomycin’s effects, should also be pursued. Wider implications of the findings Bleomycin adversely affects male gamete competence in humans. Along with its accompanying lung toxicity, healthcare practitioners should actively monitor and further investigate bleomycin’s gonadotoxicity in males. It is advised that all cancer patients undergoing chemotherapy treatments that contain bleomycin be provided with a fertility preservation plan in the meantime. Trial registration number not applicable

SS-31 treatment ameliorates cardiac mitochondrial morphology and defective mitophagy in a murine model of Barth syndrome

Barth syndrome (BTHS) is a lethal rare genetic disorder, which results in cardiac dysfunction, severe skeletal muscle weakness, immune issues and growth delay. Mutations in the TAFAZZIN gene, which is responsible for the remodeling of the phospholipid cardiolipin (CL), lead to abnormalities in mitochondrial membrane, including alteration of mature CL acyl composition and the presence of monolysocardiolipin (MLCL). The dramatic increase in the MLCL/CL ratio is the hallmark of patients with BTHS, which is associated with mitochondrial bioenergetics dysfunction and altered membrane ultrastructure. There are currently no specific therapies for BTHS. Here, we showed that cardiac mitochondria isolated from TAFAZZIN knockdown (TazKD) mice presented abnormal ultrastructural membrane morphology, accumulation of vacuoles, pro-fission conditions and defective mitophagy. Interestingly, we found that in vivo treatment of TazKD mice with a CL-targeted small peptide (named SS-31) was able to restore mitochondrial morphology in tafazzin-deficient heart by affecting specific proteins involved in dynamic process and mitophagy. This agrees with our previous data showing an improvement in mitochondrial respiratory efficiency associated with increased supercomplex organization in TazKD mice under the same pharmacological treatment. Taken together our findings confirm the beneficial effect of SS-31 in the amelioration of tafazzin-deficient dysfunctional mitochondria in a BTHS animal model.

Comparison of the transcriptome, lipidome, and c-di-GMP production between BCGΔBCG1419c and BCG, with Mincle- and Myd88-dependent induction of proinflammatory cytokines in murine macrophages

We have previously reported the transcriptomic and lipidomic profile of the first-generation, hygromycin-resistant (HygR) version of the BCGΔBCG1419c vaccine candidate, under biofilm conditions. We recently constructed and characterized the efficacy, safety, whole genome sequence, and proteomic profile of a second-generation version of BCGΔBCG1419c, a strain lacking the BCG1419c gene and devoid of antibiotic markers. Here, we compared the antibiotic-less BCGΔBCG1419c with BCG. We assessed their colonial and ultrastructural morphology, biofilm, c-di-GMP production in vitro, as well as their transcriptomic and lipidomic profiles, including their capacity to activate macrophages via Mincle and Myd88. Our results show that BCGΔBCG1419c colonial and ultrastructural morphology, c-di-GMP, and biofilm production differed from parental BCG, whereas we found no significant changes in its lipidomic profile either in biofilm or planktonic growth conditions. Transcriptomic profiling suggests changes in BCGΔBCG1419c cell wall and showed reduced transcription of some members of the DosR, MtrA, and ArgR regulons. Finally, induction of TNF-α, IL-6 or G-CSF by bone-marrow derived macrophages infected with either BCGΔBCG1419c or BCG required Mincle and Myd88. Our results confirm that some differences already found to occur in HygR BCGΔBCG1419c compared with BCG are maintained in the antibiotic-less version of this vaccine candidate except changes in production of PDIM. Comparison with previous characterizations conducted by OMICs show that some differences observed in BCGΔBCG1419c compared with BCG are maintained whereas others are dependent on the growth condition employed to culture them.

#2928 Fabry disease may imbalance integrin α3 related to endothelial dysfunction

Abstract Background and Aims Fabry Disease (FD) is a rare disease characterized by a mutation in the GLA gene and consequently by the functional anomaly of the α-Galactosidase A (α-Gal) enzyme. The lack of this functional enzyme leads to lysosomal accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different cell types, causing several symptoms throughout the body, which aggravate during the patient's longevity. We already demonstrated that exposure of endothelial cells to chloroquine, to mimic FD effects, leads to lysosomal accumulation in human endothelial cells. In the present study, we analyzed the endothelial changes in ultrastructural morphology and cell adhesion under evaluation of Integrin α3 expression, an important integrin that stabilizes cell adhesion and is involved in several signaling pathways in endothelial cells. Method Human endothelial cells (EA.hy926, ATCC CRL-2922) were exposed to chloroquine diphosphate (0.5 µg/mL) for 72 hours. The ultrastructural morphology was analyzed through scanning electron microscopy (SEM) and the Integrin α3 expression was analyzed through Western Blot and RT-qPCR. Results Through SEM (Fig. 1C-F) it was observed that after the exposure to chloroquine diphosphate, there was a decrease in cell expansion and adhesion and an increase in vesicular content. Curiously, the analysis of the Integrin α3 gene (ITGA3) and protein expression (Fig. 1A-B) demonstrated a significant increase (P &amp;lt; 0.05) in cells that were exposed to chloroquine diphosphate compared to non-exposed cells (control cells). Conclusion The in vitro condition of the FD in human endothelial cells demonstrated an increase in Integrin α3; however, there was a decrease in cell adhesion and expansion. The evaluation of the Integrin α3 expression could be important for monitoring the endothelial dysfunction in FD, as it is intrinsically involved in cell adhesion, remodeling, and fibrosis. These important changes can lead to renal impairment, proteinuria, and decreased glomerular filtration rate.

Alterations of Myocardial Mitochondrial Morphology and Function in a Canine Model of Premature Ventricular Contractions-Induced Cardiomyopathy.

Changes in myocardial mitochondrial morphology and function in premature ventricular contractions (PVCs)-induced cardiomyopathy (PVCCM) remain poorly studied. Here, we investigated the effects of PVCs with different coupling intervals (CIs) on myocardial mitochondrial remodelling in a canine model of PVCCM. Twenty-one beagles underwent pacemaker implantation and were randomised into the sham (n = 7), short-coupled PVCs (SCP, n = 7), and long-coupled PVCs (LCP, n = 7) groups. Right ventricular (RV) apical bigeminy was produced for 12-week to induce PVCCM in the SCP (CI, 250 ms) and LCP (CI, 350 ms) groups. Echocardiography was performed at baseline and biweekly thereafter to evaluate cardiac function. Masson's trichrome staining measured ventricular interstitial fibrosis. The ultrastructural morphology of the myocardial mitochondria was analysed using transmission electron microscopy. Mitochondrial Ca2+ concentration, reactive oxygen species (ROS) levels, adenosine triphosphate (ATP) content, membrane potential, and electron transport chain (ETC) complex activity were measured to assess myocardial mitochondrial function. Twelve-week-PVCs led to left ventricular (LV) enlargement with systolic dysfunction, disrupted mitochondrial morphology, increased mitochondrial Ca2+ concentration and ROS levels, decreased mitochondrial ATP content and membrane potential, and impaired ETC complex activity in both the SCP and LCP groups (all p < 0.01 vs the sham group). Ventricular fibrosis was observed only in canines with LCP. Worse cardiac function and more pronounced abnormalities in mitochondrial morphology and function were observed in the LCP group than to the SCP group (all p < 0.05). We demonstrated myocardial mitochondrial abnormalities in dogs with PVCCM, characterised by abnormal mitochondrial morphology, mitochondrial Ca2+ overload, oxidative stress, and impaired mitochondrial energy metabolism. Compared to SCP, long-term LCP exposure resulted in more severe mitochondrial remodelling and cardiac dysfunction in dogs.

The protective effect of Lonicera japonica Thunb. against lipopolysaccharide-induced acute lung injury in mice: Modulation of inflammation, oxidative stress, and ferroptosis

Ethnopharmacological relevanceVarious components of Lonicera japonica Thunb. (LJT) exhibit pharmacological activities, including anti-inflammatory and antioxidant effects. Nevertheless, the relationship between LJT and ferroptosis remains largely unexplored. Aim of the studyThe purpose of this research was to look into the role of LJT in regulating LPS-induced ferroptosis in ALI and to compare the effects of different parts of LJT. Materials and methodsWe established a mice ALI model by treating with LPS. Administered mice with different doses of Lonicerae Japonicae Flos (LJF), Lonicera Japonica Leaves (LJL) and Lonicerae Caulis (LRC) extracts, respectively. The levels of IL-6, IL-1β, TNF-α, IL-4, IL-10, and PGE2 in bronchoalveolar lavage fluid (BALF) were measured using enzyme-linked immunosorbent assay. Furthermore, the concentrations of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), reactive oxygen species (ROS), and total ferrous ions (Fe2+) in lung tissues were evaluated. Hematoxylin and eosin staining was conducted to examine the morphological structure of lung tissues. Transmission electron microscopy was used to investigate the ultrastructural morphology of mitochondria. Furthermore, the effects of LJT were evaluated via immunohistochemical staining, western blotting, and quantitative real-time polymerase chain reaction analyses. Finally, employing molecular docking and molecular dynamics research techniques, we aimed to identify crucial components in LJT that might inhibit ferroptosis by targeting nuclear factor erythroid 2-related factor 2 (Nrf2) and glutathione peroxidase 4 (GPX4). ResultsWe observed that pretreatment with LJT significantly mitigated LPS-induced lung injury and suppressed ferroptosis. This was supported by reduced accumulation of pro-inflammatory cytokines, ROS, MDA, and Fe2+, along with increased levels of anti-inflammatory cytokines, SOD, GSH, Nrf2, and GPX4 in the lung tissues of ALI mice. Luteolin-7-O-rutinoside, apigenin-7-O-rutinoside, and amentoflavone in LJT exhibit excellent docking effects with key targets of ferroptosis, Nrf2 and GPX4. ConclusionsPretreatment with LJT may alleviate LPS-induced ALI, possibly by suppressing ferroptosis. Our initial results indicate that LJT activates the Nrf2/GPX4 axis, providing protection against ferroptosis in ALI. This finding offers a promising therapeutic candidate for ALI treatment.

SDPR expression in human trabecular meshwork and its potential role in racial disparities of glaucoma

In order to identify how differential gene expression in the trabecular meshwork (TM) contributes to racial disparities of caveolar protein expression, TM dysfunction and development of primary open angle glaucoma (POAG), RNA sequencing was performed to compare TM tissue obtained from White and Black POAG surgical (trabeculectomy) specimens. Healthy donor TM tissue from White and Black donors was analyzed by PCR, qPCR, immunohistochemistry staining, and Western blot to evaluate SDPR (serum deprivation protein response; Cavin 2) and CAV1/CAV2 (Caveolin 1/Caveolin 2). Standard transmission electron microscopy (TEM) and immunogold labeled studies were performed. RNA sequencing demonstrated reduced SDPR expression in TM from Black vs White POAG patients’ surgical specimens, with no significant expression differences in other caveolae-associated genes, confirmed by qPCR analysis. No racial differences in SDPR gene expression were noted in healthy donor tissue by PCR analysis, but there was greater expression as compared to specimens from patients with glaucoma. Analysis of SDPR protein expression confirmed specific expression in the TM regions, but not in adjacent tissues. TEM studies of TM specimens from healthy donors did not demonstrate any racial differences in caveolar morphology, but a significant reduction of caveolae with normal morphology and immuno-gold staining of SDPR were noted in glaucomatous TM as compared to TM from healthy donors. Linkage of SDPR expression levels in TM, POAG development, and caveolar ultrastructural morphology may provide the basis for a novel pathway of exploration of the pathologic mechanisms of glaucoma. Differential gene expression of SDPR in TM from Black vs White subjects with glaucoma may further our understanding of the important public health implications of the racial disparities of this blinding disease.

Ultrastructural insights into cellular organization, energy storage and ribosomal dynamics of an ammonia-oxidizing archaeon from oligotrophic oceans.

Nitrososphaeria, formerly known as Thaumarchaeota, constitute a diverse and widespread group of ammonia-oxidizing archaea (AOA) inhabiting ubiquitously in marine and terrestrial environments, playing a pivotal role in global nitrogen cycling. Despite their importance in Earth's ecosystems, the cellular organization of AOA remains largely unexplored, leading to a significant unanswered question of how the machinery of these organisms underpins metabolic functions. In this study, we combined spherical-chromatic-aberration-corrected cryo-electron tomography (cryo-ET), scanning transmission electron microscopy (STEM), and energy dispersive X-ray spectroscopy (EDS) to unveil the cellular organization and elemental composition of Nitrosopumilus maritimus SCM1, a representative member of marine Nitrososphaeria. Our tomograms show the native ultrastructural morphology of SCM1 and one to several dense storage granules in the cytoplasm. STEM-EDS analysis identifies two types of storage granules: one type is possibly composed of polyphosphate and the other polyhydroxyalkanoate. With precise measurements using cryo-ET, we observed low quantity and density of ribosomes in SCM1 cells, which are in alignment with the documented slow growth of AOA in laboratory cultures. Collectively, these findings provide visual evidence supporting the resilience of AOA in the vast oligotrophic marine environment.

The Effect of Cell Culture Passage on the Efficacy of Mesenchymal Stromal Cells as a Cell Therapy Treatment.

Background/Objective: Mesenchymal Stromal Cells (MSCs) have been considered a promising treatment for several diseases, such as cardiac injuries. Many studies have analyzed their functional properties; however, few studies have characterized MSCs through successive culture passages. The main objective of this work was to analyze the phenotype and functionality of MSCs isolated from two different sources in five culture passages to determine if the culture passage might influence the efficacy of MSCs as a cell therapy treatment. Methods: Bone Marrow (BM)-MSCs were harvested from the femur of Wistar rats (n = 17) and Adipose Tissue(AT)-MSCs were isolated from inguinal fat (n = 17). MSCs were cultured for five culture passages, and the immunophenotype was analyzed by flow cytometry, the functionality was characterized by adipogenic, osteogenic, and chondrogenic differentiation assays, and cytokine secretion capacity was determined through the quantification of the Vascular Endothelial Growth-Factor, Fibroblast Growth-Factor2, and Transforming Growth-Factorβ1 in the cell supernatant. The ultrastructure of MSCs was analyzed by transmission electron microscopy. Results: BM-MSCs exhibited typical phenotypes in culture passages two, four, and five, and their differentiation capacity showed an irregular profile throughout the five culture passages analyzed. AT-MSCs showed a normal phenotype and differentiation capacity in all the culture passages. BM- and AT-MSCs did not modify their secretion ability or ultrastructural morphology. Conclusions: Throughout the culture passages, BM-MSCs, but not AT-MSCs, exhibited changes in their functional and phenotypic characteristic that might affect their efficacy as a cell therapy treatment. Therefore, the culture passage selected should be considered for the application of MSCs as a cell therapy treatment.

Updating the morphological phylogenetics of Nopinae (Araneae: Caponiidae): novel terminals and characters, with two new species

A re-analysis of the morphological phylogeny of the Nopinae is made, based on an update in the description of Aamunops Galán-Sánchez &amp; Álvarez-Padilla, 2022 and the addition of the recently described genera Nopsma Sánchez-Ruiz, Brescovit &amp; Bonaldo, 2020 and Roddemberryus Sánchez-Ruiz &amp; Bonaldo, 2023. Two new species, Aamunops hoof sp. nov. (male) and Aamunops yiselae sp. nov. (male and female), are also described, which allows a better understanding of the genus morphology and resulted in an emended diagnosis. The description of Aamunops has been updated to include several characteristics of the ultrastructural morphology, legs, chelicerae, palps and female genitalia. The inclusion of these new characters of Aamunops along with those of Nopsma and Roddemberryus in the previous data matrix resulted in a new, completely different hypothesis of the relationships of the nopine genera: Nopsma is part of a group formed by Cubanops and Nyetnops, while Aamunops and Roddemberryus are grouped with representatives of Tarsonops. The four-eyed Nopsides ceralbonus Chamberlin, 1924 was recovered as the most basal species of Nopinae. The relationships among genera of Nopinae and the phylogenetic position of three species, whose taxonomic position is doubtful (Cubanops luquillo Sánchez-Ruiz, Brescovit &amp; Alayón, 2015, Orthonops confuso Galán-Sánchez &amp; Álvarez-Padilla, 2022 and Tarsonops irataylori Bond &amp; Taylor, 2013), is also discussed.

The effect and mechanism of volatile oil emulsion from leaves of Clausena lansium (Lour.) Skeels on Staphylococcus aureus in vitro.

This study aimed to develop a suitable dosage form of volatile oil from wampee leaves and to explore its antibacterial mechanism in vitro. The chemical composition of the volatile oil from wampee leaves was determined by gas chromatography-mass spectrometry (GC-MS). Different microemulsion ratios were tested and their stabilities were investigated to determine the optimal ratio. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the wampee leaves volatile oil emulsion (WVOE) against Salmonella typhimurium (S. typhimurium) and Staphylococcus aureus (S. aureus) were determined using double-dilution and plate-counting methods, respectively. Morphological changes in these two bacteria were observed using scanning electron microscopy. Death, ultrastructural morphology, and biofilm formation were also assessed for S. aureus. Finally, we established an S. aureus-infected Lewis lung carcinoma (LLC) cell model to evaluate the protective effects of the volatile oil emulsion and the associated mechanisms. The volatile oil extracted from wampee leaves contained 37 compounds, of which 96.49% were aromatic hydrocarbons, terpenoids, and their oxygen-containing derivatives. The emulsion was most stable at 1:1 in the oil phase and 1:9 in the water phase. WVOE had poor antibacterial activity against S. typhimurium, but the MIC and MBC against S. aureus were 312.5 and 2,500 μg/mL, respectively. S. aureus survival rates were 84.6%, 14.5%, and 12.8% in the 1/2, 1, and 4 × MIC groups, respectively, compared with 97.2% in the control group. S. typhimurium survival was not affected by WVOE treatment. WVOE administration induced cavity formation and abnormal binary fission, and significantly inhibited biofilm formation in S. aureus cells. The WVOE notably reduced the number of S. aureus and inhibited TLR4, NLRP3, NF-κB, IL-6, IL-18, and TNF-α gene expression in S. aureus-infected LLC cells. The WVOE had a significant inhibitory effect on S. aureus and altered its cell membrane permeability. Moreover, it alleviated inflammation by inhibiting the NF-κB-NLRP3 pathway in S. aureus-infected LLC cells.