ABSTRACT
Microbial genomes produced by standard single-cell amplification methods are largely incomplete. Here, we show that primary template-directed amplification (PTA), a novel single-cell amplification technique, generated nearly complete genomes from three bacterial isolate species. Furthermore, taxonomically diverse genomes recovered from aquatic and soil microbiomes using PTA had a median completeness of 81%, whereas genomes from standard multiple displacement amplification-based approaches were usually <30% complete. PTA-derived genomes also included more associated viruses and biosynthetic gene clusters.
ABSTRACT
Woodsmoke (WS) exposure is associated with significant health-related sequelae. Different populations can potentially exhibit varying susceptibility, based on endocrine phenotypes, to WS and investigating neurological impacts following inhaled WS is a growing area of research. In this study, a whole-body inhalation chamber was used to expose both male and female C57BL/6 mice (n = 8 per group) to either control filtered air (FA) or acute WS (0.861 ± 0.210 mg/m3) for 4 h/d for 2 days. Neuroinflammatory and lipid-based biological markers were then assessed. In a second set of studies, female mice were divided into two groups: one group was ovariectomized (OVX) to simulate an ovarian hormone-deficient state (surgical menopause), and the other underwent Sham surgery as controls, to mechanistically assess the impact of ovarian hormone presence on neuroinflammation following FA and acute WS exposure to simulate an acute wildfire episode. There was a statistically significant impact of sex (P ≤ 0.05) and statistically significant interactions between sex and treatment in IL-1ß, CXCL-1, TGF-ß, and IL-6 brain relative gene expression. Hippocampal and cortex genes also exhibited significant changes in acute WS-exposed Sham and OVX mice, particularly in TGF-ß (hippocampus) and CCL-2 and CXCL-1 (cortex). Cortex GFAP optical density (OD) showed a notable elevation in male mice exposed to acute WS, compared to the control FA. Sham and OVX females demonstrated differential GFAP expression, depending on brain region. Overall, targeted lipidomics in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) serum and brain lipids demonstrated more significant changes between control FA and acute WS exposure in female mice, compared to males. In summary, male and female mice show distinct neuroinflammatory markers in response to acute WS exposure. Furthermore, ovarian hormone deficiency may impact the neuroinflammatory response following an acute WS event.
Subject(s)
Mice, Inbred C57BL , Neuroinflammatory Diseases , Animals , Female , Male , Mice , Neuroinflammatory Diseases/metabolism , Neuroinflammatory Diseases/etiology , Sex Factors , Ovariectomy/adverse effects , Brain/metabolism , Ovary/metabolismABSTRACT
Selective staining of extracellular vesicles (EVs) is a major challenge for diagnostic and therapeutic applications. Herein, the EV labeling properties of a new class of tetranuclear polypyridylruthenium(II) complexes, Rubb7-TNL and Rubb7-TL, as phosphorescent stains are described. These new stains have many advantages over standard stains to detect and characterize EVs, including: high specificity for EV staining versus cell staining; high phosphorescence yields; photostability; and a lack of leaching from EVs until incorporation with target cells. As an example of their utility, large EVs released from control (basal) or lipopolysaccharide (LPS)-stimulated THP-1 monocytic leukemia cells were studied as a model of immune system EVs released during bacterial infection. Key findings from EV staining combined with flow cytometry were as follows: (i) LPS-stimulated THP-1 cells generated significantly larger and more numerous large EVs, as compared with those from unstimulated cells; (ii) EVs retained native EV physical properties after staining; and (iii) the new stains selectively differentiated intact large EVs from artificial liposomes, which are models of cell membrane fragments or other lipid-containing debris, as well as distinguished two distinct subpopulations of monocytic EVs within the same experiment, as a result of biochemical differences between unstimulated and LPS-stimulated monocytes. Comparatively, the staining patterns of A549 epithelial lung carcinoma-derived EVs closely resembled those of THP-1 cell line-derived EVs, which highlighted similarities in their selective staining despite their distinct cellular origins. This is consistent with the hypothesis that these new phosphorescent stains target RNA within the EVs.
Subject(s)
Extracellular Vesicles , Flow Cytometry , Monocytes , Humans , Extracellular Vesicles/metabolism , Flow Cytometry/methods , Monocytes/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Nucleic Acids/metabolism , Staining and Labeling/methods , THP-1 Cells , Coordination Complexes/chemistry , Coordination Complexes/pharmacology , Lipopolysaccharides/pharmacology , Cell Line, Tumor , A549 CellsABSTRACT
This review focusses on the significance of fluorescent, phosphorescent labelling and tracking of extracellular vesicles (EVs) for unravelling their biology, pathophysiology, and potential diagnostic and therapeutic uses. Various labeling strategies, such as lipid membrane, surface protein, luminal, nucleic acid, radionuclide, quantum dot labels, and metal complex-based stains, are evaluated for visualizing and characterizing EVs. Direct labelling with fluorescent lipophilic dyes is simple but generally lacks specificity, while surface protein labelling offers selectivity but may affect EV-cell interactions. Luminal and nucleic acid labelling strategies have their own advantages and challenges. Each labelling approach has strengths and weaknesses, which require a suitable probe and technique based on research goals, but new tetranuclear polypyridylruthenium(II) complexes as phosphorescent probes have strong phosphorescence, selective staining, and stability. Future research should prioritize the design of novel fluorescent probes and labelling platforms that can significantly enhance the efficiency, accuracy, and specificity of EV labeling, while preserving their composition and functionality. It is crucial to reduce false positive signals and explore the potential of multimodal imaging techniques to gain comprehensive insights into EVs.
Subject(s)
Extracellular Vesicles , Fluorescent Dyes , Extracellular Vesicles/chemistry , Extracellular Vesicles/metabolism , Humans , Fluorescent Dyes/chemistry , Radioactive Tracers , Magnetic Resonance Imaging/methods , Animals , Contrast Media/chemistry , Contrast Media/metabolismABSTRACT
BACKGROUND: Rural regions of the western United States have experienced a noticeable surge in both the frequency and severity of acute wildfire events, which brings significant challenges to both public safety and environmental conservation efforts, with impacts felt globally. Identifying factors contributing to immune dysfunction, including endocrinological phenotypes, is essential to understanding how hormones may influence toxicological susceptibility. METHODS: This exploratory study utilized male and female C57BL/6 mice as in vivo models to investigate distinct responses to acute woodsmoke (WS) exposure with a focus on sex-based differences. In a second set of investigations, two groups were established within the female mouse cohort. In one group, mice experienced ovariectomy (OVX) to simulate an ovarian hormone-deficient state similar to surgical menopause, while the other group received Sham surgery as controls, to investigate the mechanistic role of ovarian hormone presence in driving immune dysregulation following acute WS exposure. Each experimental cohort followed a consecutive 2-day protocol with daily 4-h exposure intervals under two conditions: control HEPA-filtered air (FA) and acute WS to simulate an acute wildfire episode. RESULTS: Metals analysis of WS particulate matter (PM) revealed significantly increased levels of 63Cu, 182W, 208Pb, and 238U, compared to filtered air (FA) controls, providing insights into the specific metal components most impacted by the changing dynamics of wildfire occurrences in the region. Male and female mice exhibited diverse patterns in lung mRNA cytokine expression following WS exposure, with males showing downregulation and females displaying upregulation, notably for IL-1ß, TNF-α, CXCL-1, CCL-5, TGF-ß, and IL-6. After acute WS exposure, there were notable differences in the responses of macrophages, neutrophils, and bronchoalveolar lavage (BAL) cytokines IL-10, IL-6, IL-1ß, and TNF-α. Significant diverse alterations were observed in BAL cytokines, specifically IL-1ß, IL-10, IL-6, and TNF-α, as well as in the populations of immune cells, such as macrophages and polymorphonuclear leukocytes, in both Sham and OVX mice, following acute WS exposure. These findings elucidated the profound influence of hormonal changes on inflammatory outcomes, delineating substantial sex-related differences in immune activation and revealing altered immune responses in OVX mice due to ovarian hormone deficiency. In addition, the flow cytometry analysis highlighted the complex interaction between OVX surgery, acute WS exposure, and their collective impact on immune cell populations within the hematopoietic bone marrow niche. CONCLUSIONS: In summary, both male and female mice, alongside females subjected to OVX and those who had sham surgery, exhibit significant variations in the expression of proinflammatory cytokines, chemokines, lung mRNA gene expression, and related functional networks linked to signaling pathways. These differences potentially act as mediators of sex-specific and hormonal influences in the systemic inflammatory response to acute WS exposure during a wildfire event. Understanding the regulatory roles of genes expressed differentially under environmental stressors holds considerable implications, aiding in identifying sex-specific therapeutic targets for addressing acute lung inflammation and injury.
Subject(s)
Inhalation Exposure , Mice, Inbred C57BL , Animals , Female , Male , Inhalation Exposure/adverse effects , Wildfires , Particulate Matter/toxicity , Sex Factors , Cytokines/metabolism , Cytokines/immunology , Lung/immunology , Lung/drug effects , Lung/metabolism , Smoke/adverse effects , Air Pollutants/toxicity , Bronchoalveolar Lavage Fluid/immunology , Bronchoalveolar Lavage Fluid/chemistry , Ovariectomy , Mice , Ovary/immunology , Ovary/drug effects , Ovary/metabolismABSTRACT
Rhenium(I) tricarbonyl complexes are widely studied for their cell imaging properties and anti-cancer and anti-microbial activities, but the complexes with S-donor ligands remain relatively unexplored. A series of six fac-[Re(NN)(CO)3(SR)] complexes, where (NN) is 2,2'-bipyridyl (bipy) or 1,10-phenanthroline (phen), and RSH is a series of thiocarboxylic acid methyl esters, have been synthesized and characterized. Cellular uptake and anti-proliferative activities of these complexes in human breast cancer cell lines (MDA-MB-231 and MCF-7) were generally lower than those of the previously described fac-[Re(NN)(CO)3(OH2)]+ complexes; however, one of the complexes, fac-[Re(CO)3(phen)(SC(Ph)CH2C(O)OMe)] (3b), was active (IC50 â¼ 10 µM at 72 h treatment) in thiol-depleted MDA-MB-231 cells. Moreover, unlike fac-[Re(CO)3(phen)(OH2)]+, this complex did not lose activity in the presence of extracellular glutathione. Taken together these properties show promise for further development of 3b and its analogues as potential anti-cancer drugs for co-treatment with thiol-depleting agents. Conversely, the stable and non-toxic complex, fac-[Re(bipy)(CO)3(SC(Me)C(O)OMe)] (1a), predominantly localized in the lysosomes of MDA-MB-231 cells, as shown by live cell confocal microscopy (λex = 405 nm, λem = 470-570 nm). It is strongly localized in a subset of lysosomes (25 µM Re, 4 h treatment), as shown by co-localization with a Lysotracker dye. Longer treatment times with 1a (25 µM Re for 48 h) resulted in partial migration of the probe into the mitochondria, as shown by co-localization with a Mitotracker dye. These properties make complex 1a an attractive target for further development as an organelle probe for multimodal imaging, including phosphorescence, carbonyl tag for vibrational spectroscopy, and Re tag for X-ray fluorescence microscopy.
Subject(s)
Antineoplastic Agents , Cell Proliferation , Coordination Complexes , Rhenium , Sulfur , Humans , Rhenium/chemistry , Rhenium/pharmacology , Cell Proliferation/drug effects , Coordination Complexes/chemistry , Coordination Complexes/pharmacology , Coordination Complexes/chemical synthesis , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Ligands , Sulfur/chemistry , Sulfur/pharmacology , Cell Line, Tumor , Drug Screening Assays, Antitumor , Molecular StructureABSTRACT
Microbial genomes produced by single-cell amplification are largely incomplete. Here, we show that primary template amplification (PTA), a novel single-cell amplification technique, generated nearly complete genomes from three bacterial isolate species. Furthermore, taxonomically diverse genomes recovered from aquatic and soil microbiomes using PTA had a median completeness of 81%, whereas genomes from standard amplification approaches were usually <30% complete. PTA-derived genomes also included more associated viruses and biosynthetic gene clusters.
ABSTRACT
A new photoluminescent polypyridylruthenium(II) stain for extracellular vesicles (EVs) released from lipopolysaccharide-stimulated THP-1 monocytes enabled important new insights into how the bacteria-induced immune system affects the blood-brain barrier (BBB). These included previously unknown aspects of EV interactions with BBB microvascular endothelial cells and the extracellular matrix relevant to human brain diseases.
Subject(s)
Endothelial Cells , Extracellular Vesicles , Humans , Endothelium , Brain , Blood-Brain BarrierABSTRACT
BACKGROUND AND AIM: Some individuals in Indonesia consume intact goat gallbladder to prevent and treat malaria. The acute and subacute toxicity tests of goat bile (GB) have shown mild diarrhea in mice. Therefore, this study aimed to evaluate the suppressive effect of GB on parasitemia, splenomegaly, hepatomegaly, and blood biochemistry to assess liver and kidney function in BALB/c mice infected with Plasmodium berghei ANKA. MATERIALS AND METHODS: Fifty healthy mice were infected with P. berghei ANKA and divided into five groups. Mice in three groups were administered 0.5 mL of 25%, 50%, or 100% of GB by gavage. Animals in Group 4 were administered 187.2 mg/kg BW of dihydroartemisinin-piperaquine phosphate as a positive control (POS Group). Mice in fifth group were administered sterile water as negative (NEG) controls. Further, 30 uninfected mice were divided into groups 6-8 and administered GB as were mice in the first three groups. Group 9 included 10 uninfected and untreated animals as healthy controls. Treatments were administered in a 4-day suppressive test followed by daily observation of Giemsa-stained blood smears. On day 7, mice were sacrificed to measure the length and weight of spleens and livers, plasma levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine. RESULTS: GB suppressed parasitemia but did not affect the size and weight of spleens or livers or plasma levels of AST and ALT compared to uninfected GB-treated and healthy control animals. Conversely, plasma levels of BUN and creatinine were suppressed and remained in the normal range in all groups of mice. CONCLUSION: GB suppresses parasitemia with no significant impact on hepatic enzymes in GB-treated infected mice. Liver dysfunction in GB-treated infected mice was due to P. berghei rather than GB treatment.
ABSTRACT
AIM: The aim of this study was to investigate the toxicity of goat bile in BALB/c mice since some Indonesian people consume raw goat gallbladder to treat malaria and increase stamina. MATERIALS AND METHODS: Acute toxicity test was done in six groups of BALB/c mice using 100%, 50%, 25%, 12.5%, and 6.75% of goat bile and negative control. The death of mice was observed within 14 days. In the subacute toxicity test, the body weight and hematology parameters on day 0 and day 4 post-treatment were evaluated. The mice were closely observed for 28 days before plasma collection for the blood biochemistry evaluation. RESULTS: Mild diarrhea was observed in acute and subacute toxicity tests. No death of mice was observed in acute test. Goat bile did not inhibit the increase of the body weight of mice. A slight reduction in hemoglobin and hematocrit levels in mice treated with 25% and 50% goat bile, however, remained normal in mice treated with 100% goat bile. The red and white blood cell count were not affected. Liver and kidney functions were not affected by goat bile treatment as revealed by the plasma level of aspartate aminotransferase and alanine aminotransferase, blood urea nitrogen, and creatinine, which remained in the normal range. CONCLUSION: Goat bile treatment in BALB/c mice caused mild toxicity in mice. Hydrophobic bile acids may cause the toxicity of goat bile in mice; therefore, it is recommended that goat bile consumption not to be taken oftenly to avoid its harmful effect.