Hypotonic Conditions Research Articles

Bacterial-host adhesion dominated by collagen subtypes remodelled by osmotic pressure.

Environmental osmolarity plays a crucial role in regulating the functions and behaviors of both host cells and pathogens. However, it remains unclear whether and how environmental osmotic stimuli modulate bacterial‒host interfacial adhesion. Using single-cell force spectroscopy, we revealed that the interfacial adhesion force depended nonlinearly on the osmotic prestimulation of host cells but not bacteria. Quantitatively, the adhesion force increased dramatically from 25.98 nN under isotonic conditions to 112.45 or 93.10 nN after the host cells were treated with the hypotonic or hypertonic solution. There was a strong correlation between the adhesion force and the number of host cells harboring adherent/internalized bacteria. We further revealed that enhanced overexpression levels of collagen XV and II were responsible for the increases in interfacial adhesion under hypotonic and hypertonic conditions, respectively. This work provides new opportunities for developing host-directed antibacterial strategies related to interfacial adhesion from a mechanobiological perspective.

Antineoplastic with DNA fragmentation assay and anti-oxidant, anti-inflammatory with gene expression activity of Lactobacillus plantarum isolated from local Egyptian milk products

Many lactic acid bacteria (LAB), known for their human health benefits, are derived from milk and utilized in biotherapeutic applications or for producing valuable nutraceuticals. However, the specific role of milk-associated LAB in biotherapeutics remains underexplored. To address this, eight milk product samples were randomly selected from the Egyptian market, diluted, and then cultured anaerobically on MRS agar. Subsequently, 16 suspected LAB isolates were recovered and underwent rapid preliminary identification. Among these isolates, the Lactobacillus plantarum strain with accession number (OQ547261.1) was identified due to its strong antioxidant activity depending on the DPPH assay, L. plantarum displayed notable antioxidant activities of 71.8% and 93.8% at concentrations of 125–1000 µg/mL, respectively. While ascorbic acid showed lower concentrations of 7.81, 3.9, and 1.95 µg/mL which showed activities of 45.1%, 34.2%, and 27.2%, respectively. The anti-inflammatory efficacy of L. plantarum was evaluated based on its capability to prevent hemolysis induced by hypotonic conditions. At a concentration of 1000 µg/mL, L. plantarum could reduce hemolysis by 97.7%, nearly matching the 99.5% inhibition rate achieved by the standard drug, indomethacin, at an identical concentration. Moreover, L. plantarum exhibited high hemolytic activity at 100 µg/mL (14.3%), which decreased to 1.4% at 1000 µg/mL. The abundance of phenolic acids and flavonoids was determined by high-performance liquid chromatography (HPLC) in L. plantarum. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that L. plantarum increased gene expression of the inflammatory marker TLR2 by 133%, and cellular oxidation markers SOD1 and SOD2 by 65% and 74.2%, respectively, while suppressing CRP expression by 33.3%. These results underscore L. plantarum’s exceptional anti-inflammatory and antioxidant activities. Furthermore, L. plantarum induces cancer cell death through necrotic nuclear DNA fragmentation. These findings suggest that L. plantarum is not only suitable for nutraceutical production but also holds potential as a probiotic strain. Future research should focus on enhancing the capacity of this strain across various industries and fostering innovation in multiple fields.

Role of Piezo2 in Schwann Cell Volume Regulation and Its Impact on Neurotrophic Release Regulation.

Tactile perception relies on mechanoreceptors and nerve fibers, including c-fibers, Aβ-fibers and Aδ-fibers. Schwann cells (SCs) play a crucial role in supporting nerve fibers, with non-myelinating SCs enwrapping c-fibers and myelinating SCs ensheathing Aβ and Aδ fibers. Recent research has unveiled new functions for cutaneous sensory SCs, highlighting the involvement of nociceptive SCs in pain perception and Meissner corpuscle SCs in tactile sensation. Furthermore, Piezo2, previously associated with Merkel cell tactile sensitivity, has been identified in SCs. The goal of this study was to investigate the channels implicated in SC mechanosensitivity and the release process of neurotrophic factor secretion. Immortalized IFRS1 SCs and human primary SCs generated two distinct subtypes of SCs: undifferentiated and differentiated SCs. Quantitative PCR was employed to evaluate the expression of differentiation markers and mechanosensitive channels, including TRP channels (TRPV4, TRPM7 and TRPA1) and Piezo channels (Piezo1 and Piezo2). To validate the functionality of specific mechanosensitive channels, Ca2+ imaging and electronic cell sizing experiments were conducted under hypotonic conditions, and inhibitors and siRNAs were used. Protein expression was assessed by Western blotting and immunostaining. Additionally, secretome analysis was performed to evaluate the release of neurotrophic factors in response to hypotonic stimulation, with BDNF, a representative trophic factor, quantified using ELISA. Induction of differentiation increased Piezo2 mRNA expression levels both in IFRS1 and in human primary SCs. Both cell types were responsive to hypotonic solutions, with differentiated SCs displaying a more pronounced response. Gd3+ and FM1-43 effectively inhibited hypotonicity-induced Ca2+ transients in differentiated SCs, implicating Piezo2 channels. Conversely, inhibitors of Piezo1 and TRPM7 (Dooku1 and NS8593, respectively) had no discernible impact. Moreover, Piezo2 in differentiated SCs appeared to participate in regulatory volume decreases (RVD) after cell swelling induced by hypotonic stimulation. A Piezo2 deficiency correlated with reduced RVD and prolonged cell swelling, leading to heightened release of the neurotrophic factor BDNF by upregulating the function of endogenously expressed Ca2+-permeable TRPV4. Our study unveils the mechanosensitivity of SCs and implicates Piezo2 channels in the release of neurotrophic factors from SCs. These results suggest that Piezo2 may contribute to RVD, thereby maintaining cellular homeostasis, and may also serve as a negative regulator of neurotrophic factor release. These findings underscore the need for further investigation into the role of Piezo2 in SC function and neurotrophic regulation.

You get what you test for: The killing effect of phage lysins is highly dependent on buffer tonicity and ionic strength.

The phage lysin field has done nothing but grow in the last decades. As a result, many different research groups around the world are contributing to the field, often with certain methodological differences that pose a challenge to the interpretation and comparison of results. In this work, we present the case study of three Acinetobacter baumannii-targeting phage lysins (wild-type endolysin LysMK34 plus engineered lysins eLysMK34 and 1D10) plus one lysin with broad activity against Gram-positive bacteria (PlySs2) to provide exemplary evidence on the risks of generalization when using one of the most common lysin evaluation assays: the killing assay with resting cells. To that end, we performed killing assays with the aforementioned lysins using hypo-, iso- and hypertonic buffers plus human serum either as the reaction or the dilution medium in a systematic manner. Our findings stress the perils of creating hypotonic conditions or a hypotonic shock during a killing assay, suggesting that hypotonic buffers should be avoided as a test environment or as diluents before plating to avoid overestimation of the killing effect in the assayed conditions. As a conclusion, we suggest that the nature of both the incubation and the dilution buffers should be always clearly identified when reporting killing activity data, and that for experimental consistency the same incubation buffer should be used as a diluent for posterior serial dilution and plating unless explicitly required by the experimental design. In addition, the most appropriate buffer mimicking the final application must be chosen to obtain relevant results.

Assessing the Impact of Fencing on Postural Parameters: Observational Study Findings on Elite Athletes.

The aim of the study was to evaluate whether the static stabilometric parameters among elite fencers, were affected by prolonged, asymmetric training regimen. A sample of 26 elite fencers of both genders, aged 19.15 ± 2.24 years, practising one of the three disciplines foil, épée, or sabre, was recruited for the study. Anthropometric measurements including thigh and calf circumferences and postural assessment based on the weight distribution on a stabilimeter platform were performed. Postural tone, as indicated by measures such as sway length and sway area ratio was calculated.. No notable anthropometric asymmetries were detected within the examined group The weight distribution patterns on the support quadrants in static stabilometric measurements did not suggest clinically significant issues. There were no significant differences among subgroups based on gender and lower limb dominance for both anthropometric and stabilimeter variables. However, 30.8% of participants showed anomalies in postural tone (hypertonic and hypotonic condition). Five out of eight athletes found with abnormal postural tone were foil fencers, suggesting a potential discipline-specific effect. Individual adjustments were found in foil fencers. These findings provide insights into the potential effects of fencing training on postural parameters among elite athletes.

Role of Protein Phosphatase 1 (PP1) Upon KCCs Regulation by WNK3 Kinase During Cell Volume Regulation and Ion Transport

Background: The K-Cl cotransporters (KCCs) are SLC12A cation/chloride cotransporters (CCCs) that mediate epithelial transport, maintain cellular volume, and regulate GABA neurotransmission. These mechanisms are complex, and contain multiple sensors, transducers, and effectors, often operating in parallel pathways. It has been shown that KCCs activity is affected by direct phosphorylation and dephosphorylation. In particular dephosphorylation of important regulatory sites on KCCs is necessary to become fully activated, whereas phosphorylation by SPAK/OSR1 kinases will result in KCCs inhibition. Protein phosphatases (PP) inhibitors reduce KCCs function and so, it is generally accepted the presence of phosphatases on the KCCs signaling pathway. Several studies have proposed protein phosphatase 1 (PP1) as the major PP responsible for KCCs dephosphorylation. On the other hand, WNK3 kinases have been shown to directly regulate KCCs activity bypassing their osmotic regulation. That is wild type (WT) WNK3 phosphorylates KCCs under hypotonic conditions where they are normally active, whereas kinase dead WNK3 (DA) activates KCCs function under isotonic conditions where they are normally inhibited. On the present study we analyze the role of protein phosphatase 1 (PP1) upon KCCs regulation by WNK3 kinase during cell volume regulation and ion transport. Methods: HEK 293 cells and Xenopus laevis oocytes were either transfected or microinjected with KCC3 or KCC4 and one of the following PP1 plasmids: WT, H248K (inactive) or T320A (constitutive active), and/or WT WNK3 /DA WNK3. KCCs dephosphorylation in response to cell swelling, hypotonic conditions, was evaluated by Western blot analysis of KCCs regulatory phosphorylation site (pThr1048 in KCC3a). Phosphatase/kinase interaction and the possible formation of a complex was analyzed by transfecting cells or microinjecting oocytes with WNK3 PP1 putative binding sites mutants (WNK3 PP1a y PP1b). Phosphorylation of SPAK/OSR1 activating regulatory site Ser373 was analyzed in parallel under the same conditions. Results: WT or constitutive active mutant PP1 T320A induced KCCs Thr1048 dephosphorylation in HEK cells and X. laevis oocytes even under isotonic conditions, where KCCs are normally phosphorylated. WT WNK3 prevented KCCs T1048 dephosphorylation under hypotonic conditions, ablating their response to cell swelling. Nevertheless, when co-expressed with PP1 T320A, KCCs became dephosphorylated in both osmotic conditions eliminating WT WNK3 inhibitory effect. PP1 inactivating mutation H248K prevented KCCs Thr1048 dephosphorylation in response to hypotonic conditions. However, when co-expressed with DA WNK3, this KCCs regulatory site, became dephosphorylated under hypotonic conditions. SPAK Ser373 regulation was unaffected in any off the studied conditions. Conclusions: Our results show a direct role for PP1 on KCCs function. PP1WT and PP1T320A regulate KCCs activity by its dephosphorylation. In presence of WT WNK3, PP1 activity predominates over WNK3 surpassing the inhibitory phosphorylation of the regulatory Thr residue. KCCs are not dephosphorylated by PP1H248K under hypotonic conditions. Coexpression of kinase dead WNK3 overcomes this effect, supporting the hypothesis of a phosphatase/kinase complex regulating KCCs cell volume regulatory response. AM CF-2023-I-532, INCar 22-1345 PdlH Papiit IN228123. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Multifaceted Chemico-Pharmacological Insights into Cynometra ramiflora L.: Unveiling its GC-MS, Cytotoxic, Thrombolytic, Anti-Inflammatory, Antioxidant, Anti-Diarrheal, Hypoglycemic, and Analgesic Potentials

Objective: This study aimed to comprehensively evaluate the chemico-pharmacological properties of methanolic extractives derived from Cyanometra ramiflora leaves, encompassing a spectrum of activities including cytotoxic, thrombolytic, membrane-stabilizing, antioxidant, antidiarrheal, hypoglycemic, and analgesic effects. Methods: Phytomolecules were identified using Gas Chromatography-Mass Spectrometry (GC-MS), while antioxidant activity was assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and total phenolic content tests. Cytotoxicity was determined through the brine shrimp bioassay, while thrombolytic activity was evaluated via clot lysis assays, and membrane-stabilizing effects were investigated for potential anti-inflammatory properties. In-vivo experiments using mice were conducted solely with the methanolic extract to assess central and peripheral analgesic, hypoglycemic, and antidiarrheal properties. Results: The presence of 47 compounds in the methanolic extract, with tetratriacontyl pentafluoropropionate (9.25%), pentatriacontane (8.75%), and 2-methylhexacosane (5.9%) being predominant. The dichloromethane-soluble fraction exhibited the highest cytotoxicity, with a 50% lethal concentration (LC50) of 11.05 µg/mL. Both petroleum-ether and methanol-soluble fractions demonstrated more than 50% lysis in thrombolytic activity assays. Under hypotonic and heat-induced conditions, the ethyl acetate and dichloromethane-soluble fractions of C. ramiflora significantly reduced erythrocyte hemolysis. Notably, the methanol-soluble fraction exhibited significant antioxidant potential with 50% inhibitory concentration (IC50) of 17.71 μg/mL compared to the standard EDTA (IC50 = 75.94 μg/mL). The hypoglycemic investigation revealed a 63.73% reduction at 90 min with a 600 mg/kg dose, while mild antidiarrheal activity was observed with a 37.5% reduction in diarrhea at 4 h intervals. Significant analgesic effects were demonstrated in both central and peripheral tests, with a 68.53% reduction in writhing during the peripheral test and an average tail immersion time of 5.38 s at 90 min. Conclusion: C. ramiflora leaves show promise as natural sources for various pharmacological activities, including cytotoxicity, thrombosis, inflammation, antioxidant, hypoglycemic, and analgesic effects. However, further comprehensive research is recommended to elucidate the precise mechanisms of action and analyze the phytochemical composition of C. ramiflora plants.

Bio‐Engineering Yeast Cells Biolenses by Reshaping Intracellular Vacuoles

AbstractAn intriguing field of research has recently emerged in which biological cells can be demonstrated to behave as photonics devices, such as optical microlenses. This research highlights yeast cells as promising candidates for engineering biolenses with customizable focal properties. Typically serving as positive biolenses due to their quasi‐spherical shape and positive refractive index (RI) contrast, yeast cells can achieve a negative lensing effect by manipulating their intracellular content, without altering their overall morphology. In fact, it shows that exposure to hypotonic conditions induces the formation of a large, roundish vacuole with lower RI values with respect to the surrounding cytoplasm, thus altering the cell's optical properties. This behavior is investigated by using 3D RI tomograms obtained through a holo‐tomographic phase imaging flow cytometry system, demonstrating that the anisotropic nature of yeast cells results in focal properties dependent on their specific orientations in respect to the incident light. Furthermore, Zemax OpticStudio is utilized to perform comprehensive assessments for optical design analysis of the obtained cell biolenses. The methods described here and the related results represent a pioneering investigation of biological cells’ metamorphoses via intracellular content manipulation for biolens applications.

Microballoons: Osmotically-inflated elastomer shells for ultrafast release of encapsulants and mechanical energy

HypothesisMicrocapsules with osmotically-inflated elastic shells exhibit an ultrafast release of encapsulants while mechanically stimulating the microenvironments, akin to popping balloons. ExperimentsTo prepare elastic shells with uniform thickness and size, monodisperse water-in-oil-in-water (W/O/W) double-emulsion drops are produced in a capillary microfluidic device. The polydimethylsiloxane (PDMS)-containing oil phase is thermally cured to create the elastic shell. The elastic shells are inflated by pumping water into the lumen in hypotonic conditions. The inflated microcapsules produced undergo mechanical compression, and their release properties are studied. FindingsBy controlling the osmotic pressure difference, Microballoons are inflated into a diameter of 200 μm – 316 μm and shell thickness of 7.8 μm – 0.7 µm, respectively. The inflated shell pops due to mechanical failure when subjected to mechanical stress above a certain threshold, resembling a balloon. During popping, the stretched shell rapidly retracts to the original uninflated state, resulting in an ultrafast release of encapsulants from the lumen within a millisecond. This process converts elastic potential energy stored in the shell into mechanical energy with substantial power. The microballoons mechanically stimulate the local environment, leading to the direct and rapid release of encapsulants. This has the potential to improve absorption efficiency.

Efficient flocculation pretreatment of coal gasification wastewater by halophilic bacterium Halovibrio variabilis TG-5.

The isolated halophilic bacterial strain Halovibrio variabilis TG-5 showed a good performance in the pretreatment of coal gasification wastewater. With the optimum culture conditions of pH = 7, a temperature of 46°C, and a salinity of 15%, the chemical oxygen demand and volatile phenol content of pretreated wastewater were decreased to 1721mg/L and 94mg/L, respectively. The removal rates of chemical oxygen demand and volatile phenol were over 90% and 70%, respectively. At the optimum salinity conditions of 15%, the total yield of intracellular compatible solutes and the extracellular transient released yield under hypotonic conditions were increased to 6.88g/L and 3.45g/L, respectively. The essential compatible solutes such as L-lysine, L-valine, and betaine were important in flocculation mechanism in wastewater pretreatment. This study provided a new method for pretreating coal gasification wastewater by halophilic microorganisms, and revealed the crucial roles of compatible solutes in the flocculation process.

Management of Functional Status of a Floppy Child with Ayurveda and Panchakarma: A Case Report

ABSTRACT The floppy (hypotonic) condition often presents with abnormally lower muscle tone, resulting in the limpness of the body. Antigravity muscle movements are difficult to perform. This case had a brain lesion due to hypoxic–ischemic encephalopathy inside the cerebrum. Hypotonic cases in Ayurveda are close to Vata Vayadhi (~Diseases due to Vata dosha alleviation) with Avarana (~occlusion) due to Shiromarmaghata (~damage to the brain). This state is with symptoms such as Chestanasha (~loss of movement), Gatra vikshep sanga (~restricted body movements), and Sarveshu Skhalitam Cha Gatau Bhrusham (~fall during the walk) close to hypotonic status. In this article, a 2 and a ½-year-old male child with diagnosed hypotonic status and delayed milestones attended Kaumarbhritya outpatient department. He was not able to balance or stand with support and would lie down while sitting. He was managed according to Ayurveda guidelines for 180 days. The case was provided oral Ayurveda medications, Panchakarma therapy, and some basic exercises. The case was assessed after 60 days for the outcome of the treatment. After 180 days, a multifaceted improvement is seen. The patient started standing and walking without support, and his power grade of muscle and joints also improved. His advanced daily living activities that scored 10, before treatment improved to 70, after treatment (AT). With all-around improvement, his Gross Motor Functional Classification System score also improved from 3 to 1, AT. Magnetic resonance imaging changes were seen after a period of treatment, showing gliotic changes AT reflecting healing and neuroplastic changes. Conclusively, early intervention with Ayurveda can change the functional status in hypotonic conditions.

Instantaneous extracellular solution exchange for concurrent evaluation of membrane permeability of single cells.

The osmotic stress imposed on microorganisms by hypotonic conditions is perceived to regulate water and solute flux via cell membranes, which are crucial for survival. Some cells that fail to perceive osmotic stress die because this results in the rupture of the cell membrane. The flux through the membrane is characterized by the membrane permeability, which is measured using a stopped-flow apparatus in response to a millisecond-order osmolarity change. However, the obtained data are an ensemble average of each cell response. Additionally, the measurement of permeability, considering cellular viability, contributes to a more accurate evaluation of osmoadaptation. Here, we present a novel on-chip instantaneous extracellular solution exchange method using an air-liquid interface. The presented method provides a concurrent evaluation at the single-cell level in response to a millisecond-order osmotic shock, considering cellular viability by solution exchange. This method utilizes a liquid bridge with a locally formed droplet on the surface of a micropillar fabricated inside a microchannel. We evaluated a solution exchange time of 3.6 ms and applied this method to Synechocystis PCC 6803 under two different osmolarity conditions. The live/dead ratio of 1 M to 0.5 M osmotic down shock condition was 78.8/21.2% while that of 1 M to 0.25 M osmotic down shock condition was 40.0/60.0%. We evaluated the water permeability of two groups: cells that were still live before and after osmotic shock (hereafter named cell type 1), and cells that were live before but were dead 10 minutes after osmotic shock (hereafter named cell type 2). The results indicated that the water permeability of cell type 2 was higher than that of cell type 1. The results obtained using the presented methods confirmed that the effect of osmotic stress can be accurately evaluated using single-cell analysis.

Mechanisms of aquaporin-4 vesicular trafficking in mammalian cells.

The aquaporin-4 (AQP4) water channel is abundantly expressed in the glial cells of the central nervous system and facilitates brain swelling following diverse insults, such as traumatic injury or stroke. Lack of specific and therapeutic AQP4 inhibitors highlights the need to explore alternative routes to control the water permeability of glial cell membranes. The cell surface abundance of AQP4 in mammalian cells fluctuates rapidly in response to changes in oxygen levels and tonicity, suggesting a role for vesicular trafficking in its translocation to and from the cell surface. However, the molecular mechanisms of AQP4 trafficking are not fully elucidated. In this work, early and recycling endosomes were investigated as likely candidates of rapid AQP4 translocation together with changes in cytoskeletal dynamics. In transiently transfected HEK293 cells a significant amount of AQP-eGFP colocalised with mCherry-Rab5-positive early endosomes and mCherry-Rab11-positive recycling endosomes. When exposed to hypotonic conditions, AQP4-eGFP rapidly translocated from intracellular vesicles to the cell surface. Co-expression of dominant negative forms of the mCherry-Rab5 and -Rab11 with AQP4-eGFP prevented hypotonicity-induced AQP4-eGFP trafficking and led to concentration at the cell surface or intracellular vesicles respectively. Use of endocytosis inhibiting drugs indicated that AQP4 internalisation was dynamin-dependent. Cytoskeleton dynamics-modifying drugs also affected AQP4 translocation to and from the cell surface. AQP4 trafficking mechanisms were validated in primary human astrocytes, which express high levels of endogenous AQP4. The results highlight the role of early and recycling endosomes and cytoskeletal dynamics in AQP4 translocation in response to hypotonic and hypoxic stress and suggest continuous cycling of AQP4 between intracellular vesicles and the cell surface under physiological conditions.

Assessment of Expression Levels and Localization Patterns of Phospholipase C zeta in Different Grades of HOST in Human Sperm.

Phospholipase C zeta (PLC-ζ) deficiency in sperm can underlie oocyte activation failure after intracytoplasmic sperm injection (ICSI). The aim of this study was to determine PLC-ζ expression and location in individual spermatozoa in each host score so that a hypo-osmotic swelling test (HOST) may be used to help routine sperm selection for ICSI. In this experimental study, fresh semen samples were randomly obtained from 30 men who were referred to the Andrology Unit of the Infertility Center. Samples were processed by density gradient centrifugation (DGC) and exposed to hypotonic conditions. Seven different tail patterns, classified from 'a' to 'g' can be detected according to World Health Organization (WHO) criteria. Then, the PLC-ζ protein localization pattern was assessed by quantitative Immunofluorescence in individual sperm Host grades. Moreover, the sperm content of PLC-ζ protein was evaluated by flow cytometry correlated with semen analysis parameters. In the present study, quantitive immunofluorescence analysis indicated that sperm from different host grades exhibited seven localization patterns of PLC-ζ of acrosomal (A); equatorial (EQ), and postacrosomal (PA) patterns. A+EQ=acrosomal and equatorial, A+PA=acrosomal and post-acrosomal, EQ+PA=equatorial and post-crosomal, and A+EQ+PA. The sperm from HOST grade 'd' exhibited significantly higher PLC-ζ (A+PA) and (A+EQ+PA) staining compared to sperm from other grades (P=0.006). The sperm from grade 'd' exhibited higher PLC-ζ (EQ+PA) compared with other grades (P=0.001). However, grade 'd' was not significantly different from 'c' (P=0.087). Analysis of the combined results confirmed that there was a clear reduction in PLC-ζ immunofluorescence in Host grades 'a', 'f' and 'g' sperms. Our data suggest that HOST may represent a useful diagnostic tool for the selection of sperms exhibiting a higher level of PLC-ζ expression.

Structure and function of the EA1 surface layer of Bacillus anthracis

The Gram-positive spore-forming bacterium Bacillus anthracis is the causative agent of anthrax, a deadly disease mostly affecting wildlife and livestock, as well as representing a bioterrorism threat. Its cell surface is covered by the mutually exclusive S-layers Sap and EA1, found in early and late growth phases, respectively. Here we report the nanobody-based structural characterization of EA1 and its native lattice contacts. The EA1 assembly domain consists of 6 immunoglobulin-like domains, where three calcium-binding sites structure interdomain contacts that allow monomers to adopt their assembly-competent conformation. Nanobody-induced depolymerization of EA1 S-layers results in surface defects, membrane blebbing and cell lysis under hypotonic conditions, indicating that S-layers provide additional mechanical stability to the cell wall. Taken together, we report a complete model of the EA1 S-layer and present a set of nanobodies that may have therapeutic potential against Bacillus anthracis.

Glycosaminoglycans modulate compressive stiffness and circumferential residual stress in the porcine thoracic aorta

The mechanical properties of the aorta are influenced by the extracellular matrix, a network mainly comprised of fibers and glycosaminoglycans (GAG). In this work, we demonstrate that GAG contribute to the opening angle (a marker of circumferential residual stresses) in intact and glycated aortic tissue. Enzymatic GAG depletion was associated with a decrease in the opening angle, by approximately 25% (p = 0.009) in the ascending (AS) region, 32% (p = 0.003) in the aortic arch (AR), and 42% (p = 0.001) in the lower descending thoracic (LDT) region. A similar effect of GAG depletion on aortic ring opening angle was also observed in previously glycated tissues. Using indentation testing, we found that the radial compressive stiffness significantly increased in the AS region following GAG depletion, compared to fresh (p = 0.006) and control samples (p = 0.021), and that the compressive properties are heterogeneous along the aortic tree. A small loss of water content was also detected after GAG depletion, which was most prominent under hypotonic conditions. Finally, the AS region was also associated with a significant loss of compressive deformation (circumferential stretch that is < 1) in the inner layer of the aorta following GAG depletion, suggesting that GAG interact with ECM fibers in their effect on aortic mechanics. The importance of this work lies in its identification of the role of GAG in modulating the mechanical properties of the aorta, namely the circumferential residual stresses and the radial compressive stiffness, as well as contributing to the swelling state and the level of circumferential prestretch in the tissue. Statement of significanceThe mechanical properties of the aorta are influenced by the composition and organization of its extracellular matrix (ECM) and are highly relevant to medical conditions affecting the structural integrity of the aorta. The extent of contribution of glycosaminoglycans (GAG), a relatively minor ECM component, to the mechanical properties of the aorta, remains poorly characterized. This works shows that GAG contribute on average 30% to the opening angle (an indicator of circumferential residual stresses) of porcine aortas, and that GAG-depletion is associated with an increased radial compressive stiffness of the aorta. GAG-depletion was also associated with a loss of water content and compressive deformation in the inner layers of the aortic wall providing insight into potential mechanisms for their biomechanical role.

Optimization of the Green Chemistry-like Extraction of Phenolic Compounds from Grape (Vitis labrusca L.) and Blackberry (Rubus fruticosus L.) Seeds with Concomitant Biological and Antioxidant Activity Assessments.

The objective of this work was to determine the phenolic composition, chemical and cellular antioxidant activity, cytotoxicity in human cells, and peroxidative inhibition of the defatted fraction of grape (Vitis labrusca) and blackberry (Rubus fruticosus) seeds. Soxhlet extraction (Sox) was used to extract the fat and obtain the degreased material. A statistical optimization study was developed to maximize the extraction of bioactive compounds and antioxidant activity from defatted grape and blackberry seeds. Simultaneous optimization was applied with a combination of 35.9 min of extraction and a solid-to-solvent ratio of 1 g of defatted grape seed to 61.28 mL of an extracting solvent (60% ethanol) and 62.1 min of extraction and a solid-to-solvent ratio of 1 g of defatted blackberry seed to 64.1 mL of an extracting solvent (60% ethanol). In the cell viability assay, HepG2 cancer cells seemed more sensitive to grape and blackberry extracts, while Ea.hy926 hybrid cells showed more resistance to their effects. In general, the extracts presented low/no cytotoxicity, exhibited a protective effect against H2O2-induced ROS production, and demonstrated antioxidant activity and a protective effect on the erythrocytes when subjected to hypotonic and isotonic conditions not presenting hemolytic behavior (5.0 to 10.0 μg GAE/mL). Thus, the results provided a broad assessment of the bioactivity of the extracts obtained using a simple and low-cost process developed by employing non-toxic solvents and with the potential to be used in technological applications.

Congenital hypotonia: systematic approach for prenatal detection.

Congenital hypotonic conditions are rare and heterogeneous, and some are severely debilitating or lethal. Contrary to its prominent postnatal manifestation, the prenatal presentation of hypotonia is frequently subtle, inhibiting prenatal detection. We aimed to characterize the prenatal sonographic manifestation of congenital hypotonia throughout pregnancy, evaluate the yield of diagnostic tests and propose diagnostic models to increase its prenatal detection. This was a retrospective observational study of singleton pregnancies with congenital hypotonia, diagnosed either prenatally or immediately after birth, at a single tertiary center between the years 2012 and 2020. Prenatally, hypotonia was diagnosed if a fetus showed sonographic or clinical signs suggestive of hypotonia and had a confirmed underlying genetic condition, or in the absence of a known genetic abnormality if the fetus exhibited multiple prominent signs suggestive of hypotonia. Postnatally, it was diagnosed in neonates displaying reduced muscle tone leading to reduced spontaneous movement, reduced swallowing or feeding difficulty. We reviewed the medical records of pregnant patients carrying fetuses subsequently diagnosed with congenital hypotonia and assessed the yield of ultrasound scans, fetal magnetic resonance imaging, computed tomography and genetic tests. The detection rate of sonographic signs suggesting fetal hypotonia was calculated. The prevalence of non-specific signs, including polyhydramnios, persistent breech presentation, intrauterine growth restriction and maternal perception of reduced fetal movement, were compared between the study group and the local liveborn singleton population. Potential detection rates of different theoretical semiotic diagnostic models, differing in the threshold for referral for a targeted scan, were assessed based on the cohort's data. The study group comprised 26 cases of congenital hypotonia, of which 10 (38.5%) were diagnosed prenatally, and the controls included 95 105 singleton live births, giving a prevalence of congenital hypotonia of 1:3658. Nuchal translucency thickness and the early anomaly scan at 13-17 weeks were normal in all 22 and 23 cases, respectively, in which this was performed. The mid-trimester scan performed at 19-25 weeks was abnormal in four of 24 (16.7%) cases. The overall prenatal detection rate of congenital hypotonic conditions in our cohort was 38.5%. Only cases which underwent a targeted scan were detected and, among the 16 cases which underwent this scan, the prenatal detection rate was 62.5% compared with 0% in pregnancies that did not undergo this scan (P = 0.003). An abnormal genetic diagnosis was obtained in 21 (80.8%) cases using the following modalities: chromosomal microarray analysis (CMA) in two (9.5%), whole-exome sequencing (WES) in 14 (66.7%) and methylation analysis in five (23.8%). CMA was abnormal in 8% (2/25) of the cases and WES detected a causative genetic mutation in 87.5% (14/16) of the cases in which these were performed. Comparison of non-specific signs in the study group with those in the local singleton population showed that hypotonic fetuses had significantly more polyhydramnios (64.0% vs 3.0%, P < 0.0001), persistent breech presentation (58.3% vs 4.2%, P < 0.0001), intrauterine growth restriction (30.8% vs 3.0%, P < 0.0001) and maternal perception of reduced fetal movement (32.0% vs 4.7%, P < 0.0001). Prenatally, the most commonly detected signs supporting a diagnosis of hypotonia were structural anomaly (62.5%, 10/16), reduced fetal movement (46.7%, 7/15), joint contractures (46.7%, 7/15) and undescended testes ≥ 30 weeks (42.9%, 3/7 males). Proposed diagnostic strategies that involved performing a targeted scan for a single non-specific ultrasound sign or two such signs, and then carrying out a comprehensive genetic evaluation for any additional sign, offered theoretical detection rates in our cohort of 88.5% and 57.7%, respectively. Congenital hypotonic conditions are rare and infrequently detected prenatally. Sonographic signs are visible from the late second trimester. A targeted scan increases prenatal detection significantly. Comprehensive genetic testing, especially WES, is the cornerstone of diagnosis in congenital hypotonia. Theoretical diagnostic models which may increase prenatal detection are provided. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

P74: Plasma Osmolarity as a Determinant of Platelet Morphology and Function in Heart Failure

Purpose: Heart failure (HF) is associated with increased sodium and water retention, though disproportionately, leading to hypervolemia and reduced plasma osmolality (<275 mOsm/kg). HF is also associated with thrombosis/thromboembolic adverse advents, either in the native heart, or in mechanical circulatory support (MCS) devices implanted to restore hemodynamics. Platelets, critical cells in thrombosis, are mechanosensitive, responding to altered shear flow associated with HF or MCS devices. Excess plasma water and hypo-osmolarity can induce a gradient via which cells become swollen and stiff as water is forced into cells. It is recognized in cell biology that volume regulation and swelling can impact cell mechanics and function. However, it is not well understood how these processes are regulated or affect platelet function. Here, we investigate the affect of altered tonicity on platelet morphology/geometry and corresponding affect on platelet activation and aggregation. Methods: Human gel-filtered platelets (GFP; 20,000/µL) or platelet rich plasma (PRP; 100,000/µL) were obtained from fresh ACD-A anticoagulated whole blood. Modified Tyrode’s buffer osmolality was measured (Advanced Instruments; Osmo1), mixed with platelets and adjusted to a final concentration of either 250mOsm/kg (“Hypotonic”), 285mOsm/kg (“Isotonic”), or 325mOsm/kg (“Hypertonic”). Scanning ion conductance microscopy (SICM; Park Systems NX12) was utilized to quantify platelet morphology and volume. Platelet activation was measured as thrombin generation rate utilizing a previously-described modified prothrombinase assay. Platelet aggregation was quantified from agonist-mediated light-transmission aggregometry (BIO/DATA Corp; PAP-8E). All experiments were performed in duplicate with N ≥ 3 donors. Results: Overall, platelet geometry and function were altered in an osmolarity-dependent fashion. Hypotonic conditions led to an average 2.1µm3 (17.7%) increase in platelet volume, compared to isotonic conditions (Fig 1A). Despite this large increase in volume, there was a non-significant increase in thrombin generation rate in hypotonic conditions; however, a hypertonic environment led to a significant increase (0.14 min-1 average increase; Fig 1B). Collagen-mediated aggregation had a negative correlation to osmolarity, with significantly higher aggregation in hypotonic conditions (average 11.7% increase; Fig 1C). Conclusions: Hypotonicity leads to increased platelet volume and increased aggregability to collagen; however, platelet thrombin generation remains unaffected. The results of our study provide insight into operative mechanisms of thrombogenicity and altered platelet function under heart failure hypo-osmotic conditions. Further, these studies are hypothesis-generating as to the potential role of water, ion, and membrane mechanisms, providing the foundation for defining the impact of platelet volume regulation on shear-mediated platelet dysfunction in future studies.

Human KCC2a Protein Expression and Phosphorylation is Altered by Elimination of the Second Cryo-EM Identified Chloride Binding Site

Background: Potassium-chloride cotransporters (KCCs) are responsible for the symport of Cl- with K+ in an electroneutral manner. They play important roles in numerous crucial physiological processes, from cell volume regulation to transepithelial solute transport, intracellular ion homeostasis and regulation of GABA neurotransmission. Under isotonicity, except for KCC2, KCCs remain inhibited due to phosphorylation of residues located in cytoplasmic domains, whereas in response to hypotonic cell swelling, dephosphorylation of these residues stimulates transport activity. After several decades of studies, new breakthroughs with the cryo-EM structural determination of KCCs have deepened our understanding of their molecular basis and transport function, including architecture, ion binding sites, and coupled ion transport. In the present study, we mutated and analyzed the two chloride-binding sites in human KCC2a recently reported by cryo-EM to determine whether the phosphorylation status is modified upon their removal. Methods: Xenopus laevis oocytes and HEK-293 cells were either microinjected or transfected with human KCC2a wild type (WT) or chloride-binding site mutants, G136/V137/I138A (S1-Cl), and G436/I437/M438/Y592A (S2-Cl). KCC2a phosphorylation status in response to isotonic and hypotonic conditions was evaluated by immunoblot analysis of regulatory phosphorylation sites pThr926 and pThr1030. In parallel, phosphorylation status of SPAK/OSR1 activating regulatory site pSer373/pSer325 was analyzed under the same conditions. Results: Elimination of either chloride-binding site induced changes in total expression of KCC2a. Removal of S1-Cl slightly increased total KCC2a expression, whereas lack of S2-Cl showed a significant reduction of total protein in Xenopus laevis oocytes and HEK-293 cells. Phosphorylation of regulatory Thr1030 in S2-Cl mutant is significatively decreased on both biological models, even under isotonic inhibitory (phosphorylated) conditions. pThr1030 on S1-Cl mutant behaves similarly to WT KCC2a. Phosphorylation of regulatory SPAK kinase is not affected on both mutants. Conclusions: Our results suggest that while both sites affect KCC2a total expression, only the S2-Cl appears to be affecting KCC2a activating response. It appears S2-Cl mutation dysregulates SPAK signaling pathway, since the kinase osmotic response remains unchanged on presence of KCC2a chloride binding mutants. AM INCar 22-1345 and PdlH UNAM Papiit IN222320 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.