Two-Dimensional Gel Electrophoresis for Protein Separation of Plant Samples.

This chapter provides a description of the procedure for two-dimensional electrophoresis that can be performed for any given gel size and isoelectric focusing range. This will enable the operator to recognize critical steps and gain sufficient information to generate 2D images suitable for computer-assisted analysis of 2D-gel, as well as mass spectrometry analysis for protein identification and characterization.

Carbon content and other soil properties of near-surface peats before and after peatland restoration.

Peatland restoration usually aims at restarting the peatlands' function to store carbon within peat. The soil properties of the near-surface peat can give a first understanding of this process. Therefore, we sampled pH value, total organic carbon content (TOC), total nitrogen content (TN), C/N ratio as well as dry bulk density (BD), and describe the structure of near-surface peats in six restored fens in North-East Germany before (2002-2004) and after (2019-2021) restoration. Before restoration, the study sites showed peat degradation to various extents in their near-surface peats. pH values remained relatively stable over time. Comparing the degraded peat horizons, TOC increased significantly in four study sites, ranging from 35.7% to 47.8% in 2002-2004 and from 42.5% to 54.0% in 2019-2021. TN varied from 1.5% to 3.5% in 2002-2004 and from 1.8% to 3.2% in 2019-2021, but changes were only significant in one site, showing a slight decrease. In three sites, the increase in C/N ratio was significant, indicating lower nutrient availability. BD ranged from 0.08 to 0.48 g/cm3 in 2002-2004 and from 0.10 to 0.16 g/cm3 in 2019-2021, decreasing significantly in four sites. The structure of the degraded peat horizons changed after restoration to a more homogenous, sludge mass with larger re-aggregates. In three sites, new peat moss peat layers above the degraded soil horizon were present in 2019-2021, with a mean thickness of 6.8 to 36.1 cm. The structure was comparable to typical, slightly decomposed peat moss peat. Our findings suggest that within about 17 years after fen restoration, and thereby a water table rise close to surface, TOC of the near-surface peats increased to values that are typical for undisturbed peatlands. This indicates that restoration can lead to the re-establishment of peatlands as potential carbon sinks, with TOC within the near-surface peat as one key factor in this process. Further, we assume that the decrease in nutrient availability, decrease of BD, and new, undisturbed peat layers can favor the establishment of mire-specific biodiversity and support ecosystem services similar to near-natural mires.

Direct Observation of Particle-To-Particle Variability in Ambient Aerosol pH Using a Novel Analytical Approach Based on Surface-Enhanced Raman Spectroscopy.

The pH of atmospheric aerosols is a key characteristic that profoundly influences their impacts on climate change, human health, and ecosystems. Despite widely performed aerosol pH research, determining the pH levels of individual atmospheric aerosol particles has been a challenge. This study presents a novel analytical technique that utilizes surface-enhanced Raman spectroscopy to assess the pH of individual ambient PM2.5-10 aerosol particles in conjunction with examining their hygroscopic behavior, morphology, and elemental compositions. The results revealed a substantial pH variation among simultaneously collected aerosol particles, ranging from 3.3 to 5.7. This variability is likely related to each particle's unique reaction and aging states. The extensive particle-to-particle pH variability suggests that atmospheric aerosols present at the same time and location can exhibit diverse reactivities, reaction pathways, phase equilibria, and phase separation properties. This pioneering study paves the way for in-depth investigations into particle-to-particle variability, size dependency, and detailed spatial and temporal variations of aerosol pH, thus deepening our understanding of atmospheric chemistry and its environmental implications.

Effects of pH-shifting and ultrasound on the structural and emulsifying properties of peanut globulin fractions.

This work investigated and compared the structural and emulsifying properties of peanut globulin fractions (conarachin and arachin) after ultrasonication (US) and pH2.5-shifting treatments, singly and in combination. Results showed that pH2.5-shifting was more effective in degrading peanut protein subunits and unfolding their structures than US treatment. Conarachin tended to aggregate during US and pH2.5-shifting treatments possibly due to higher free sulfhydryl content, while high molecular weight arachin tended to disaggregate during these treatments. pH2.5-shifting or US+pH2.5-shifting treatments significantly increased the surface hydrophobicity of conarachin (from 72 to 314) and arachin (from 336 to 888), which may be responsible for the enhancement of protein emulsifying activity. All treatments significantly improved the physical stability of arachin-stabilized emulsions with higher absolute potentials but lowered that of conarachin-stabilized emulsions. However, pH2.5-shifting or US+pH2.5-shifting treatments could improve the stability of conarachin-stabilized emulsions in the presence of salts.

A pH-responsive nanoplatform with dual-modality imaging for enhanced cancer phototherapy and diagnosis of lung metastasis.

To address the limitations of traditional photothermal therapy (PTT)/ photodynamic therapy (PDT) and real-time cancer metastasis detection, a pH-responsive nanoplatform (NP) with dual-modality imaging capability was rationally designed. Herein, 1 H,1 H-undecafluorohexylamine (PFC), served as both an oxygen carrier and a 19F magnetic resonance imaging (MRI) probe, and photosensitizer indocyanine green (ICG) were grafted onto the pH-responsive peptide hexahistidine (H6) to form H6-PFC-ICG (HPI). Subsequently, the heat shock protein 90 inhibitor, gambogic acid (GA), was incorporated into hyaluronic acid (HA) modified HPI (HHPI), yielding the ultimate HHPI@GA NPs. Upon self-assembly, HHPI@GA NPs passively accumulated in tumor tissues, facilitating oxygen release and HA-mediated cell uptake. Once phagocytosed by lysosomes, protonation of H6 was triggered due to the low pH, resulting in the release of GA. With near-infrared laser irradiation, GA-mediated decreased HSP90 expression and PFC-mediated increased ROS generation amplified the PTT/PDT effect of HHPI@GA, leading to excellent in vitro and in vivo anticancer efficacies. Additionally, the fluorescence and 19F MRI dual-imaging capabilities of HHPI@GA NPs enabled effective real-time primary cancer and lung metastasis monitoring. This work offers a novel approach for enhanced cancer phototherapy, as well as precise cancer diagnosis.

Extraction and analysis of high-quality chloroplast DNA with reduced nuclear DNA for medicinal plants.

BACKGROUND: Obtaining high-quality chloroplast genome sequences requires chloroplast DNA (cpDNA) samples that meet the sequencing requirements. The quality of extracted cpDNA directly impacts the efficiency and accuracy of sequencing analysis. Currently, there are no reported methods for extracting cpDNA from Erigeron breviscapus. Therefore, we developed a suitable method for extracting cpDNA from E. breviscapus and further verified its applicability to other medicinal plants. RESULTS: We conducted a comparative analysis of chloroplast isolation and cpDNA extraction using modified high-salt low-pH method, the high-salt method, and the NaOH low-salt method, respectively. Subsequently, the number of cpDNA copies relative to the nuclear DNA (nDNA ) was quantified via qPCR. As anticipated, chloroplasts isolated from E. breviscapus using the modified high-salt low-pH method exhibited intact structures with minimal cell debris. Moreover, the concentration, purity, and quality of E. breviscapus cpDNA extracted through this method surpassed those obtained from the other two methods. Furthermore, qPCR analysis confirmed that the modified high-salt low-pH method effectively minimized nDNA contamination in the extracted cpDNA. We then applied the developed modified high-salt low-pH method to other medicinal plant species, including Mentha haplocalyx, Taraxacum mongolicum, and Portulaca oleracea. The resultant effect on chloroplast isolation and cpDNA extraction further validated the generalizability and efficacy of this method across different plant species. CONCLUSIONS: The modified high-salt low-pH method represents a reliable approach for obtaining high-quality cpDNA from E. breviscapus. Its universal applicability establishes a solid foundation for chloroplast genome sequencing and analysis of this species. Moreover, it serves as a benchmark for developing similar methods to extract chloroplast genomes from other medicinal plants.

Soft Materials with Time-Programmed Changes in Physical Properties through Lyotropic Phase Transitions Induced by pH-Changing Reactions.

We present the development of time-programmable functional soft materials. The materials undergo reversible phase transitions between lyotropic phases with different topologies and symmetries, which in turn have very different physical properties: viscosity, diffusion, and optical transparency. Here, this behavior is achieved by combining pH-responsive lyotropic phases made from the lipid monoolein doped with 10% oleic acid, with chemical reactions that have well-defined controllable kinetics: autocatalytic urea-urease and methyl formate hydrolysis, which increase and decrease pH, respectively. In this case, we use small-angle X-ray scattering (SAXS) and optical imaging to show temporally controlled transitions between the cloudy hexagonal phase, which is a two-dimensional (2D) array of cylindrical inverse micelles, and the transparent, highly viscous three-dimensional (3D) bicontinuous cubic phases. By combining these into a single reaction mixture where the pH increases and then decreases again, we can induce a sequential transformation cycle from hexagonal to cubic and back to hexagonal over several hours. The sample therefore changes from cloudy to transparent and back again as a proof-of-concept demonstration for a wider range of soft materials with time-programmable changes in physical properties.

Microbiome-derived acidity protects against microbial invasion in Drosophila.

Microbial invasions underlie host-microbe interactions resulting in pathogenesis and probiotic colonization. In this study, we explore the effects of the microbiome on microbial invasion in Drosophila melanogaster. We demonstrate that gut microbes Lactiplantibacillus plantarum and Acetobacter tropicalis improve survival and lead to a reduction in microbial burden during infection. Using a microbial interaction assay, we report that L. plantarum inhibits the growth of invasive bacteria, while A. tropicalis reduces this inhibition. We further show that inhibition by L. plantarum is linked to its ability to acidify its environment via lactic acid production by lactate dehydrogenase, while A. tropicalis diminishes the inhibition by quenching acids. We propose that acid from the microbiome is a gatekeeper to microbial invasions, as only microbes capable of tolerating acidic environments can colonize the host. The methods and findings described herein will add to the growing breadth of tools to study microbe-microbe interactions in broad contexts.

Multiparameter Sensing of Oxygen and pH at Biological Interfaces via Hyperspectral Imaging of Luminescent Sensor Nanoparticles.

Chemical dynamics in biological samples are seldom stand-alone processes but represent the outcome of complicated cascades of interlinked reaction chains. In order to understand these processes and how they correlate, it is important to monitor several parameters simultaneously at high spatial and temporal resolution. Hyperspectral imaging is a promising tool for this, as it provides broad-range spectral information in each pixel, enabling the use of multiple luminescent indicator dyes, while simultaneously providing information on sample structures and optical properties. In this study, we first characterized pH- and O2-sensitive indicator dyes incorporated in different polymer matrices as optical sensor nanoparticles to provide a library for (hyperspectral) chemical imaging. We then demonstrate the successful combination of a pH-sensitive indicator dye (HPTS(DHA)3), an O2-sensitive indicator dye (PtTPTBPF), and two reference dyes (perylene and TFPP), incorporated in polymer nanoparticles for multiparameter chemical imaging of complex natural samples such as green algal biofilms (Chlorella sorokiniana) and seagrass leaves (Zostera marina) with high background fluorescence. We discuss the system-specific challenges and limitations of our approach and further optimization possibilities. Our study illustrates how multiparameter chemical imaging with hyperspectral read-out can now be applied on natural samples, enabling the alignment of several chemical parameters to sample structures.

Ionic liquids adsorption and interfacial tension reduction for synthetic resinous and asphaltenic oils: salinity and pH effects.

The effects of sulfate salts under low and high salinity conditions and pH of 3.5-11 on interfacial tension (IFT) reduction and IL adsorption using resinous (RSO) and asphaltenic (8 wt/wt%) synthetic oils are investigated. The measurements showed the increasing effect of pH on the IFT of RSO/DW from 23.5 to 27.3 mN/m (pH = 3.5 → 7) in the first place and a reducing effect (0.4 mN/m) if pH = 7 → 11. Using a high concentration of 50,000 ppm for MgSO4, and Na2SO4 revealed an extensive IFT reduction for a pH value of 11 with the value of 0.20 mN/m for Na2SO4. The measured IFT values showed the significant impact of IL (500 ppm) on the IFT (minimum value of 0.01 mN/m for RSO/50,000 Na2SO4 + 500 ppm 1-decyl-3-methyl imidazolium triflate ([C10mim][TfO])) for pH = 11. The IL adsorption measurements showed the role of in-situ surfactant production (saponification process) on the 1-decyl-3-methyl imidazolium chloride ([C10mim][Cl]) and [C10mim][TfO] adsorption reduction from 3.67 to 2.33 and 4.21 to 3.34 mg IL/g rock, respectively. The performed core flooding experiments using the optimum chemical formulation showed the possibility of tertiary oil recovery with maximum oil recovery of 28.8% based on original oil in place in the presence of 500 ppm.

Deciphering the importance of culture pH on CD22 CAR T-cells characteristics.

BACKGROUND: Chimeric antigen receptor (CAR) T-cells have demonstrated significant efficacy in targeting hematological malignancies, and their use continues to expand. Despite substantial efforts spent on the optimization of protocols for CAR T-cell manufacturing, critical parameters of cell culture such as pH or oxygenation are rarely actively monitored during cGMP CAR T-cell generation. A comprehensive understanding of the role that these factors play in manufacturing may help in optimizing patient-specific CAR T-cell therapy with maximum benefits and minimal toxicity. METHODS: This retrospective study examined cell culture supernatants from the manufacture of CAR T-cells for 20 patients with B-cell malignancies enrolled in a phase 1/2 clinical trial of anti-CD22 CAR T-cells. MetaFLEX was used to measure supernatant pH, oxygenation, and metabolites, and a Bio-Plex assay was used to assess protein levels. Correlations were assessed between the pH of cell culture media throughout manufacturing and cell proliferation as well as clinical outcomes. Next-generation sequencing was conducted to examine gene expression profiles of the final CAR T-cell products. RESULTS: A pH level at the lower range of normal at the beginning of the manufacturing process significantly correlated with measures of T-cell expansion and metabolism. Stable or rising pH during the manufacturing process was associated with clinical response, whereas a drop in pH was associated with non-response. CONCLUSIONS: pH has potential to serve as an informative factor in predicting CAR T-cell quality and clinical outcomes. Thus, its active monitoring during manufacturing may ensure a more effective CAR T-cell product.

Selective Bacterial Growth Inactivation by pH-Sensitive Sulfanilamide Functionalized Carbon Dots.

Carbon dots (CDs) were synthesized hydrothermally by mixing citric acid (CA) and an antifolic agent, sulfanilamide (SNM), employed for pH sensing and bacterial growth inactivation. Sulfanilamide is a prodrug; aromatic hetero cyclization of the amine moiety along with other chemical modifications produces an active pharmacological compound (chloromycetin and miconazole), mostly administered for the treatment of various microbial infections. On the other hand, the efficacy of the sulfanilamide molecule as a drug for antimicrobial activity was very low. We anticipated that the binding of the sulfanilamide molecule on the carbon dot (CD) surface may form antibacterial CDs. Citric acid was hybridized with sulfanilamide during the hydrothermal preparation of the CDs. The molecular fragments of bioactivated sulfanilamide molecule play a crucial role in bacterial growth inactivation for Gram-positive and Gram-negative bacteria. The functional groups of citric acid and sulfanilamide were conserved during the CD formation, facilitating the zwitterionic behavior of CDs associated with its photophysical activity. At low concentrations of CDs, the antibacterial activity was apparent for Gram-positive bacteria only. This Gram-positive bacteria selectivity was also rationalized by zeta potential measurement.

Fine tuning of the net charge alternation of polyzwitterion surfaced lipid nanoparticles to enhance cellular uptake and membrane fusion potential.

Lipid nanoparticles (LNPs) coated with functional and biocompatible polymers have been widely used as carriers to deliver oligonucleotide and messenger RNA therapeutics to treat diseases. Poly(ethylene glycol) (PEG) is a representative material used for the surface coating, but the PEG surface-coated LNPs often have reduced cellular uptake efficiency and pharmacological activity. Here, we demonstrate the effect of pH-responsive ethylenediamine-based polycarboxybetaines with different molecular weights as an alternative structural component to PEG for the coating of LNPs. We found that appropriate tuning of the molecular weight around polycarboxybetaine-modified LNP, which incorporated small interfering RNA, could enhance the cellular uptake and membrane fusion potential in cancerous pH condition, thereby facilitating the gene silencing effect. This study demonstrates the importance of the design and molecular length of polymers on the LNP surface to provide effective drug delivery to cancer cells.

The study presents the unique characteristics of small interfering RNA (siRNA)-loaded lipid nanoparticles (LNPs) with different lengths of PGlu(DET-Car), revealing the length of PGlu(DET-Car) critically affects the formation of a stable LNP, the cellular uptake, membrane fusion, and gene silencing abilities.

Effects of soil pH on the growth, soil nutrient composition, and rhizosphere microbiome of Ageratina adenophora.

Ageratina adenophora is an invasive weed species found in many countries. Methods to control the spread of this weed have been largely unsuccessful. Soil pH is the most important soil factor affecting the availability of nutrients for plant and impacting its growth. Understanding the mechanisms of the influence of soil pH on the growth of A. adenophora may help to develop effective control measures. In this study, we artificially changed the soil pH in pot experiments for A. adenophora. We studied the effects of acidic (pH 5.5), weakly acidic (pH 6.5), neutral (pH 7.2), and alkaline (pH 9.0) soils on the growth, availability of soil nutrients, activity of antioxidant enzymes, levels of redox markers in the leaves, and the structure and diversity of the rhizosphere microbiome. Soil with a pH 7.2 had a higher (47.8%) below-ground height versus soils of pH 5.5 at day 10; plant had a higher (11.3%) above-ground height in pH 7.2 soils than pH 9.0 soils at day 90; no differences in the fresh and dry weights of its above- and belowground parts, plant heights, and root lengths were observed in plants growing in acid, alkaline, or neutral pH soil were observed at day 180. Correspondingly, the antioxidant enzymes SOD (superoxide dismutase), POD (peroxidase), CAT (catalase) and redox markers GSH (glutathione) and MDA (malondialdehyde) were measured in the leaves. Significant differences existed in the activities of CAT and the levels of GSH between those growing in acidic and alkaline soils and those in neutral pH soil at day 90; however, only lower (36.8%) CAT activities in those grown at pH 5.5 than those grown at pH 7.2 were found at day 180. Similarly, significant differences in available P (16.89 vs 3.04 mg Kg-1) and total K (3.67 vs 0.96 mg Kg-1), total P (0.37 vs 0.25 g Kg-1) and total N (0.45 vs 1.09 g Kg-1) concentrations were found between the rhizosphere soils of A. adenophora grown at pH 9.0 and 7.2 at day 90; no such differences were seen at day 180. High throughput analyses of the 16S rRNA and ITS fragments showed that the rhizosphere microbiome diversity and composition under different soil pH conditions changed over 180 days. The rhizosphere microbiomes differed in diversity, phylum, and generic composition and population interactions under acid and alkaline conditions versus those grown in neutral soils. Soil pH had a greater impact on the diversity and composition of the prokaryotic rhizosphere communities than those of the fungal communities. A. adenophora responded successfully to pH stress by changing the diversity and composition of the rhizosphere microbiome to maintain a balanced nutrient supply to support its normal growth. The unusual pH tolerance of A. adenophora may be one crucial reason for its successful invasion. Our results suggest that attempts use soil pH to control its invasion by changing the soil pH (for example, using lime) will fail.

Metabolomics and proteomics insights into subacute ruminal acidosis etiology and inhibition of proliferation of yak rumen epithelial cells in vitro.

BACKGROUND: Untargeted metabolomics and proteomics were employed to investigate the intracellular response of yak rumen epithelial cells (YRECs) to conditions mimicking subacute rumen acidosis (SARA) etiology, including exposure to short-chain fatty acids (SCFA), low pH5.5 (Acid), and lipopolysaccharide (LPS) exposure for 24 h. RESULTS: These treatments significantly altered the cellular morphology of YRECs. Metabolomic analysis identified significant perturbations with SCFA, Acid and LPS treatment affecting 259, 245 and 196 metabolites (VIP > 1, P < 0.05, and fold change (FC) ≥ 1.5 or FC ≤ 0.667). Proteomic analysis revealed that treatment with SCFA, Acid, and LPS resulted in differential expression of 1251, 1396, and 242 proteins, respectively (FC ≥ 1.2 or ≤ 0.83, P < 0.05, FDR < 1%). Treatment with SCFA induced elevated levels of metabolites involved in purine metabolism, glutathione metabolism, and arginine biosynthesis, and dysregulated proteins associated with actin cytoskeleton organization and ribosome pathways. Furthermore, SCFA reduced the number, morphology, and functionality of mitochondria, leading to oxidative damage and inhibition of cell survival. Gene expression analysis revealed a decrease the genes expression of the cytoskeleton and cell cycle, while the genes expression associated with inflammation and autophagy increased (P < 0.05). Acid exposure altered metabolites related to purine metabolism, and affected proteins associated with complement and coagulation cascades and RNA degradation. Acid also leads to mitochondrial dysfunction, alterations in mitochondrial integrity, and reduced ATP generation. It also causes actin filaments to change from filamentous to punctate, affecting cellular cytoskeletal function, and increases inflammation-related molecules, indicating the promotion of inflammatory responses and cellular damage (P < 0.05). LPS treatment induced differential expression of proteins involved in the TNF signaling pathway and cytokine-cytokine receptor interaction, accompanied by alterations in metabolites associated with arachidonic acid metabolism and MAPK signaling (P < 0.05). The inflammatory response and activation of signaling pathways induced by LPS treatment were also confirmed through protein interaction network analysis. The integrated analysis reveals co-enrichment of proteins and metabolites in cellular signaling and metabolic pathways. CONCLUSIONS: In summary, this study contributes to a comprehensive understanding of the detrimental effects of SARA-associated factors on YRECs, elucidating their molecular mechanisms and providing potential therapeutic targets for mitigating SARA.

Evaluation of Diuretic Activity of Aqueous and Hydro Methanolic Crude Extracts and Solvent Fraction of the Hydromethanolic Flower Extract of Erica Arborea L. (Ericaceae) in Swiss Albino Mice.

Purpose: To evaluate the diuretic effects of aqueous (AQ) and hydromethanolic crude extract (HM) the as well as the solvent fractions of the HM extract from Erica arborea flowers in mice. Methods: Mice were administered AQ and HM crude extracts, along with solvent fractions of HM extracts of E. arborea flowers, including HXF (n-hexane fraction), EAF (ethyl acetate fraction), and AQF (aqueous fraction), at doses ranging from 100 to 400 mg/kg orally. The effects of these extracts and solvent fractions on urine and salt excretion over 5 hours were compared to the effects of the solvent used for reconstitution and a standard drug (furosemide 10 mg/kg), as well as to each other. Results: The HM crude extract at a lower dose (100 mg/kg) significantly increased urine volume and salt excretion starting from the 3rd h compared to the AQ crude extract. Similar effects were observed for EAF. Notably, the HM extract and its EAF at 400 mg/kg showed comparable urine and salt excretion profiles to the standard drug. Conclusion: This study demonstrated that HM extract and EAF promote better diuresis, likely due to their saluretic properties. Furthermore, it confirms the diuretic activity of Erica arborea flowers.

On the Importance of Acidity in Cancer Cells and Therapy.

Cancer cells are associated with high glycolytic activity, which results in acidification of the tumor microenvironment. The occurrence of this stressful condition fosters tumor aggressiveness, with the outcome of invasiveness and metastasis that are linked to a poor clinical prognosis. Acidosis can be both the cause or consequence of alterations in the functions and expressions of transporters involved in intracellular acidity regulation. This review aims to explore the origin of acidity in cancer cells and the various mechanisms existing in tumors to resist, survive, or thrive in the acidic environment. It highlights the difficulties in measuring the intracellular pH evolution that impedes our understanding of the many regulatory and feedback mechanisms. It finally presents the consequences of acidity on tumor development as well as the friend or foe role of acidity in therapy.

Could Phosphorous MR Spectroscopy Help Predict the Severity of Vasospasm? A Pilot Study.

One of the main causes of the dismal prognosis in patients who survive the initial bleeding after aneurysmal subarachnoidal hemorrhage is the delayed cerebral ischaemia caused by vasospasm. Studies suggest that cerebral magnesium and pH may potentially play a role in the pathophysiology of this adverse event. Using phosphorous magnetic resonance spectrocopy (31P-MRS), we calculated the cerebral magnesium (Mg) and pH levels in 13 patients who suffered from aSAH. The values between the group that developed clinically significant vasospasm (n = 7) and the group that did not (n = 6) were compared. The results of this study show significantly lower cerebral Mg levels (p = 0.019) and higher pH levels (p < 0.001) in the cumulative group (all brain voxels together) in patients who developed clinically significant vasospasm. Further clinical studies on a larger group of carefully selected patients are needed in order to predict clinically significant vasospasm.

Tunable Oxidized-Chitin Hydrogels with Customizable Mechanical Properties by Metal or Hydrogen Ion Exposure.

This study focuses on the optimization of chitin oxidation in C6 to carboxylic acid and its use to obtain a hydrogel with tunable resistance. After the optimization, water-soluble crystalline ß-chitin fibrils (ß-chitOx) with a degree of functionalization of 10% were obtained. Diverse reaction conditions were also tested for α-chitin, which showed a lower reactivity and a slower reaction kinetic. After that, a set of hydrogels was synthesized from ß-chitOx 1 wt.% at pH 9, inducing the gelation by sonication. These hydrogels were exposed to different environments, such as different amounts of Ca2+, Na+ or Mg2+ solutions, buffered environments such as pH 9, PBS, pH 5, and pH 1, and pure water. These hydrogels were characterized using rheology, XRPD, SEM, and FT-IR. The notable feature of these hydrogels is their ability to be strengthened through cation chelation, being metal cations or hydrogen ions, with a five- to tenfold increase in their storage modulus (G'). The ions were theorized to alter the hydrogen-bonding network of the polymer and intercalate in chitin's crystal structure along the a-axis. On the other hand, the hydrogel dissolved at pH 9 and pure water. These bio-based tunable hydrogels represent an intriguing material suitable for biomedical applications.

Host-Pathogen Interactions and Correlated Factors That Are Affected in Replicative-Aged Cryptococcus neoformans.

Cryptococcus neoformans is a facultative intracellular fungal pathogen. Ten-generation-old (10GEN) C. neoformans cells are more resistant to phagocytosis and killing by macrophages than younger daughter cells. However, mechanisms that mediate this resistance and intracellular parasitism are poorly understood. Here, we identified important factors for the intracellular survival of 10GEN C. neoformans, such as urease activity, capsule synthesis, and DNA content using flow cytometry and fluorescent microscopy techniques. The real-time visualization of time-lapse imaging was applied to determine the phagosomal acidity, membrane permeability, and vomocytosis (non-lytic exocytosis) rate in J774 macrophages that phagocytosed C. neoformans of different generational ages. Our results showed that old C. neoformans exhibited higher urease activity and enhanced Golgi activity. In addition, old C. neoformans were more likely to be arrested in the G2 phase, resulting in the occasional formation of aberrant trimera-like cells. To finish, the advanced generational age of the yeast cells slightly reduced vomocytosis events within host cells, which might be associated with increased phagolysosome pH and membrane permeability. Altogether, our results suggest that old C. neoformans prevail within acidic phagolysosomes and can manipulate the phagosome pH. These strategies may be used by old C. neoformans to resist phagosomal killing and drive cryptococcosis pathogenesis. The comprehension of these essential host-pathogen interactions could further shed light on mechanisms that bring new insights for novel antifungal therapeutic design.