Biophysical Alterations Research Articles

Diabetes and Cognitive Decline: An Innovative Approach to Analyzing the Biophysical and Vibrational Properties of the Hippocampus.

Diabetes Mellitus (DM) is a disease characterized by high blood glucose levels, known as hyperglycemia. Diabetes represents a risk factor for the development of neurodegenerative diseases, such as Alzheimer's Disease (AD), one of the most prevalent neurodegenerative diseases worldwide, which leads to progressive mental, behavioral, and functional decline, affecting many brain structures, especially the hippocampus. Here, we aim to characterize the ultrastructural, nanomechanical, and vibrational changes in hyperglycemic hippocampal tissue using atomic force microscopy (AFM) and Raman spectroscopy. DM was induced in rats by streptozotocin injection (type 1) or dietary intervention (type 2). Cryosections of the hippocampus were prepared and analyzed on an MM8 AFM (Bruker) in Peak Force Quantitative Nanomechanics mode, performing 25 μm2 scans in 9 regions of 3 samples from each group. Ultrastructural and nanomechanical data such as surface roughness, area, volume, Young's modulus, and adhesion were evaluated. The hippocampal samples were also analyzed on a T64000 Spectrometer (Horiba), using a laser λ = 632.8 nm, and for each sample, four spectra were obtained in different regions. AFM analyses show changes on the ultrastructural scale since diabetic animals had hippocampal tissue with greater roughness and volume. Meanwhile, diabetic tissues had decreased adhesion and Young's modulus compared to control tissues. These were corroboratedby Raman data that shows changes in the molecular composition of diabetic tissues. The individual spectra show that the most significant changes are in the amide, cholesterol, and lipid bands. Overall, the data presented here show that hyperglycemia induces biophysical alterations in the hippocampal tissue of diabetic rats, providing novel biophysical and vibrational cues on the relationship between hyperglycemia and dementia.

A note on the silent decline of the Caspian environment

The Caspian Sea, the world's largest enclosed water body, experiences significant transformations in its physico-chemical properties and a decline in bioresources due to extensive anthropogenic activities. These activities include the discharge of diverse pollutants and bio-physical alterations such as over-fishing, hunting, and physical alterations to rivers. While acute manifestations such as a fall in the Caspian water levels and wetland desiccation are more overt, the pervasive impact of human activities contributes to a likely irreversible decline in environmental quality that we aim to spotlight in this discussion in order to facilitate its restoration.

Vestibular afferent neurons develop normally in the absence of quantal/glutamatergic input.

The vestibular nerve is comprised of neuron sub-groups with diverse functions related to their intrinsic biophysical properties. This diversity is partly due to differences in the types and numbers of low-voltage-gated potassium channels found in the neurons' membranes. Expression for some low-voltage gated ion channels like KCNQ4 is upregulated during early post-natal development; suggesting that ion channel composition and neuronal diversity may be shaped by hair cell activity. This idea is consistent with recent work showing that glutamatergic input from hair cells is necessary for the normal diversification auditory neurons. To test if biophysical diversity is similarly dependent on glutamatergic input in vestibular neurons, we examined the maturation of the vestibular epithelium and ganglion neurons in Vglut3-ko mice whose hair cell synapses lack glutamate. Despite lacking glutamatergic input, the knockout mice showed no notable balance deficits and crossed challenging balance beams with little difficulty. Immunolabeling of the Vglut3-ko vestibular epithelia showed normal development as indicated by an identifiable striolar zone with calyceal terminals labeled by molecular marker calretinin, and normal expression of KCNQ4 by the end of the second post-natal week. We found similar numbers of Type I and Type II hair cells in the knockout and wildtype animals, regardless of epithelial zone. Thus, the presumably quiescent Type II hair cells are not cleared from the epithelium. Patch-clamp recordings showed that biophysical diversity of vestibular ganglion neurons in the Vglut3-ko mice is comparable to that found in wildtype controls, with a similar range firing patterns at both immature and juvenile ages. However, our results suggest a subtle biophysical alteration to the largest ganglion cells (putative somata of central zone afferents); those in the knockout had smaller net conductance and were more excitable than those in the wild type. Thus, unlike in the auditory nerve, glutamatergic signaling is unnecessary for producing biophysical diversity in vestibular ganglion neurons. And yet, because the input signals from vestibular hair cells are complex and not solely reliant on quantal release of glutamate, whether diversity of vestibular ganglion neurons is simply hardwired or regulated by a more complex set of input signals remains to be determined.

Resting cell formation in the marine diatom Thalassiosira pseudonana.

Resting cells represent a survival strategy employed by diatoms to endure prolonged periods of unfavourable conditions. In the oceans, many diatoms sink at the end of their blooming season and therefore need to endure cold and dark conditions in the deeper layers of the water column. How they survive these conditions is largely unknown. We conducted an integrative analysis encompassing methods from histology, physiology, biochemistry, and genetics to reveal the biological mechanism of resting-cell formation in the model diatom Thalassiosira pseudonana. Resting-cell formation was triggered by a decrease in light and temperature with subsequent catabolism of storage compounds. Resting cells were characterised by an acidic and viscous cytoplasm and altered morphology of the chloroplast ultrastructure. The formation of resting cells in T. pseudonana is an energy demanding process required for a biophysical alteration of the cytosol and chloroplasts to endure the unfavourable conditions of the deeper ocean as photosynthetic organisms. However, most resting cells (> 90%) germinate upon return to favorable growth conditions.

Evaluation of biophysical alterations in the epithelial and endothelial layer of patients with Bullous Keratopathy

The cornea is a fundamental ocular tissue for the sense of sight. Thanks to it, the refraction of two-thirds of light manages to participate in the visual process and protect against mechanical damage. Because it is transparent, avascular, and innervated, the cornea comprises five main layers: Epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Each layer plays a key role in the functionality and maintenance of ocular tissue, providing unique ultrastructural and biomechanical properties. Bullous Keratopathy (BK) is an endothelial dysfunction that leads to corneal edema, loss of visual acuity, epithelial blisters, and severe pain, among other symptoms. The corneal layers are subject to changes in their biophysical properties promoted by Keratopathy. In this context, the Atomic Force Microscopy (AFM) technique in air was used to investigate the anterior epithelial surface and the posterior endothelial surface, healthy and with BK, using a triangular silicone tip with a nominal spring constant of 0.4 N/m. Six human corneas (n = 6) samples were used for each analyzed group. Roughness data, calculated by third-order polynomial adjustment, adhesion, and Young's modulus, were obtained to serve as a comparison and identification of morphological and biomechanical changes possibly associated with the pathology, such as craters and in the epithelial layer and exposure of a fibrotic layer due to loss of the endothelial cell wall. Endothelial cell membrane area and volume data were calculated, obtaining a relevant comparison between the control and patient. Such results may provide new data on the physical properties of the ocular tissue to understand the physiology of the cornea when it has pathology.

Marginated aberrant red blood cells induce pathologic vascular stress fluctuations in a computational model of hematologic disorders.

Red blood cell (RBC) disorders such as sickle cell disease affect billions worldwide. While much attention focuses on altered properties of aberrant RBCs and corresponding hemodynamic changes, RBC disorders are also associated with vascular dysfunction, whose origin remains unclear and which provoke severe consequences including stroke. Little research has explored whether biophysical alterations of RBCs affect vascular function. We use a detailed computational model of blood that enables characterization of cell distributions and vascular stresses in blood disorders and compare simulation results with experimental observations. Aberrant RBCs, with their smaller size and higher stiffness, concentrate near vessel walls (marginate) because of contrasts in physical properties relative to normal cells. In a curved channel exemplifying the geometric complexity of the microcirculation, these cells distribute heterogeneously, indicating the importance of geometry. Marginated cells generate large transient stress fluctuations on vessel walls, indicating a mechanism for the observed vascular inflammation.

Inhibitory Effects of LRP1-Based Immunotherapy on Cardiac Extracellular Matrix Biophysical Alterations Induced by Hypercholesterolemia.

The accumulation of lipids in cardiomyocytes contributes to cardiac dysfunction. The specific blockage of cardiomyocyte cholesteryl ester (CE) loading by antibodies (Abs) against the P3 sequence (Gly1127-Cys1140) of the LRP1 receptor improves cardiac insulin sensitivity. The impact of anti-P3 Abs on high-fat diet (HFD)-induced cardiac extracellular matrix (ECM) biophysical alterations was analyzed. Both IrP (without Abs) and P3-immunized rabbits (with Abs) were randomized into groups fed either HFD or a standard chow diet. Cardiac lipids, proteins, and carbohydrates were characterized by Fourier transform infrared spectroscopy in the attenuated total reflectance mode. The hydric organization and physical structure were determined by differential scanning calorimetry. HFD increased the levels of esterified lipids, collagen, and α-helical structures and upregulated fibrosis, bound water, and ECM plasticization in the heart. The inhibitory effect of anti-P3 Abs on cardiac CE accumulation was sufficient to reduce the collagen-filled extracellular space, the level of fibrosis, and the amount of bound water but did not counteract ECM plasticization in the heart of hypercholesterolemic rabbits.

Abstract 1191: Targeting the tumor extracellular matrix to enhance therapeutic interventions in glioblastoma

Abstract Brain cancers remain some of the most challenging tumors to treat. The goal of our research is to use a 3D in vitro model to acquire in-depth understanding on the unique environment of brain tumors in order to find new biomarkers of disease and develop more efficient therapeutic approaches. Brain extracellular matrix (ECM) plays a key role in glioblastoma invasion and therapeutic resistance. In particular, the aberrant biosynthesis of the main component of brain ECM, hyaluronic acid (HA), has been associated to pathological conditions. In this study, we investigate the influence of tumor extracellular microenvironment in glioblastoma progression. We focus here on the tumor-associated HA biosynthesis in response to biophysical alterations, such as hypoxia and stiffness, and after radio and chemotherapeutic interventions. The ability to manipulate tumor ECM can improve therapeutic outcomes and restrict glioblastoma growth and infiltration. We have established engineered brain tumor biomaterials based on functionalized gelatin hydrogels decorated with covalently bound HA. This biomaterial approach can monitor the response of patient-derived xenograft cell populations with different molecular signatures (1, 2) in combination with microfluidic devices. Here, we characterized tumor cell response to targeted inhibitors and ionizing radiation. We quantified HA molecular weight (MW) distribution of secreted HA, and the tumor cell response to inhibition of HA synthase 2 (HAS2) and hyaluronidase (Hyal). Analysis of HA biosynthesis suggests that GBM cells compensate for a lack of matrix-bound HA by producing soluble HA to stimulate invasion. Moreover, we revealed that different signaling pathways are altered in the tumor cells depending on the microenvironment as they respond to an EGFR and CD44 inhibitor. While the decrease in invasion and proliferation in gelatin-only matrices comes from the negative regulation of ERK pathway, HA plays a favorable role in the inhibition of EGFR, through STAT3 deactivation (3). The MW and abundance of HA are mediated by the activity of HA synthases and hyaluronidases. These enzymes vary in expression, rate, and MW of the HA produced. We have found a heterogeneous profile of MW distributions secreted by different tumor types when exposed to different targeted inhibitors. We have also shown an increase in HA secretion after exposure to radiation and inhibition of hyaluronidase, also associated to significant changes in cell proliferation. The oxidative stress induced by the hypoxic environment also induces the production of hyaluronic acid and significantly affects glycolysis and mitochondrial oxidative phosphorylation, that are further altered by the molecular weight of the HA present in the extracellular space. (1) Pedron S. Adv. Healthcare Mater. 2017; 6:1700529; (2) Pedron S. MRS Commun. 2017; 7:442-449; (3) Pedron S. Biomaterials 2019; 219:119371. Citation Format: Edward R. Neves, Joseph Mueller, Achal Anand, Kimberly A. Selting, Brendan A. Harley, Sara Pedron-Haba. Targeting the tumor extracellular matrix to enhance therapeutic interventions in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1191.

Age-related matrix stiffening epigenetically regulates α-Klotho expression and compromises chondrocyte integrity

Extracellular matrix stiffening is a quintessential feature of cartilage aging, a leading cause of knee osteoarthritis. Yet, the downstream molecular and cellular consequences of age-related biophysical alterations are poorly understood. Here, we show that epigenetic regulation of α-Klotho represents a novel mechanosensitive mechanism by which the aged extracellular matrix influences chondrocyte physiology. Using mass spectrometry proteomics followed by a series of genetic and pharmacological manipulations, we discovered that increased matrix stiffness drove Klotho promoter methylation, downregulated Klotho gene expression, and accelerated chondrocyte senescence in vitro. In contrast, exposing aged chondrocytes to a soft matrix restored a more youthful phenotype in vitro and enhanced cartilage integrity in vivo. Our findings demonstrate that age-related alterations in extracellular matrix biophysical properties initiate pathogenic mechanotransductive signaling that promotes Klotho promoter methylation and compromises cellular health. These findings are likely to have broad implications even beyond cartilage for the field of aging research.

A Cantú syndrome mutation produces dual effects on KATP channels by disrupting ankyrin B regulation.

ATP-sensitive potassium (KATP) channels composed of Kir6.x and sulfonylurea receptor (SURs) subunits couple cellular metabolism to electrical activity. Cantú syndrome (CS) is a rare disease caused by mutations in the genes encoding Kir6.1 (KCNJ8) and SUR2A (ABCC9) that produce KATP channel hyperactivity due to a reduced channel block by physiological ATP concentrations. We functionally characterized the p.S1054Y SUR2A mutation identified in two CS carriers, who exhibited a mild phenotype although the mutation was predicted as highly pathogenic. We recorded macroscopic and single-channel currents in CHO and HEK-293 cells and measured the membrane expression of the channel subunits by biotinylation assays in HEK-293 cells. The mutation increased basal whole-cell current density and at the single-channel level, it augmented opening frequency, slope conductance, and open probability (Po), and promoted the appearance of multiple conductance levels. p.S1054Y also reduced Kir6.2 and SUR2A expression specifically at the membrane. Overexpression of ankyrin B (AnkB) prevented these gain- and loss-of-function effects, as well as the p.S1054Y-induced reduction of ATP inhibition of currents measured in inside-out macropatches. Yeast two-hybrid assays suggested that SUR2A WT and AnkB interact, while p.S1054Y interaction with AnkB is decreased. The p.E322K Kir6.2 mutation, which prevents AnkB binding to Kir6.2, produced similar biophysical alterations than p.S1054Y. Our results are the first demonstration of a CS mutation whose functional consequences involve the disruption of AnkB effects on KATP channels providing a novel mechanism by which CS mutations can reduce ATP block. Furthermore, they may help explain the mild phenotype associated with this mutation.

Pathologic mechanobiological interactions between red blood cells and endothelial cells directly induce vasculopathy in iron deficiency anemia

SummaryThe correlation between cardiovascular disease and iron deficiency anemia (IDA) is well documented but poorly understood. Using a multi-disciplinary approach, we explore the hypothesis that the biophysical alterations of red blood cells (RBCs) in IDA, such as variable degrees of microcytosis and decreased deformability may directly induce endothelial dysfunction via mechanobiological mechanisms. Using a combination of atomic force microscopy and microfluidics, we observed that subpopulations of IDA RBCs (idRBCs) are significantly stiffer and smaller than both healthy RBCs and the remaining idRBC population. Furthermore, computational simulations demonstrated that the smaller and stiffer idRBC subpopulations marginate toward the vessel wall causing aberrant shear stresses. This leads to increased vascular inflammation as confirmed with perfusion of idRBCs into our “endothelialized” microfluidic systems. Overall, our multifaceted approach demonstrates that the altered biophysical properties of idRBCs directly lead to vasculopathy, suggesting that the IDA and cardiovascular disease association extends beyond correlation and into causation.

Relationship between lymphedema after breast cancer treatment and biophysical characteristics of the affected tissue.

The treatment of breast cancer is often complicated by lymphedema of the upper limbs. Standard lymphedema evaluation methodologies are not able to measure tissue fibrosis. The ultrasound aspects related to tissue microstructures of lymphedema are neglected in clinical evaluations. The objective of this study was to identify and measure the degree of impairment, topography, and biophysical alterations of subcutaneous lymphedema tissue secondary to the treatment of breast cancer by ultrasonography. Forty-two women at a mean age of 58 (±9.7) years, with unilateral lymphedema due to breast cancer treatment, were evaluated. The upper limbs were divided into affected (affected by lymphedema) and control (contralateral limb). Each limb was subdivided into seven areas, defined by perimetry, evaluated in pairs. The biophysical characteristics thickness, entropy, and echogenicity were evaluated by ultrasonography. The results showed a significant difference in the echogenicity and thickness variables between the affected and unaffected upper limb, in all the extent of the upper limb, while entropy showed no significant difference. The findings indicate that the data presented were consistent both in identifying and measuring the degree of impairment and biophysical changes in the subcutaneous tissue of lymphedema secondary to the treatment of breast cancer.

Balanites aegyptiaca (Heglig Dates) Reduces Oxidative Stress and Biophysical Alterations of Erythrocyte Membranes in Streptozotocin-Induced Diabetic Rats

Balanites aegyptiaca (Heglig Dates) Reduces Oxidative Stress and Biophysical Alterations of Erythrocyte Membranes in Streptozotocin-Induced Diabetic Rats

Alcohol exacerbated biochemical and biophysical alterations in liver mitochondrial membrane of diabetic male wistar rats – A possible amelioration by green tea

Alcohol exacerbated biochemical and biophysical alterations in liver mitochondrial membrane of diabetic male wistar rats – A possible amelioration by green tea

New Insights into Hemolytic Anemias: Ultrastructural and Nanomechanical Investigation of Red Blood Cells Showed Early Morphological Changes.

Several diseases are characterized by changes in the mechanical properties of erythrocytes. Hemolytic anemias are an example of these diseases. Among the hemolytic anemias, Sickle Cell Disease and Thalassemia are the most common, characterized by alterations in the structure of their hemoglobin. Sickle cell disease has a pathological origin in synthesizing abnormal hemoglobin, HbS. In contrast, thalassemia results in extinction or decreased synthesis of α and β hemoglobin chains. This work presents a detailed study of biophysical and ultrastructural early erythrocytes membrane alterations at the nanoscale using Atomic Force Microscopy (AFM). Cells from individuals with sickle cell anemia and thalassemia mutations were studied. The analysis methodology in the AFM was given by blood smear and exposure of the inner membrane for ghost analysis. A robust statistic was used with 65,536 force curves for each map, ten cells of each type, with three individuals for each sample group. The results showed significant differences in cell rigidity, adhesion, volume, and roughness at early morphological alterations, bringing new perspectives for understanding pathogenesis. The sickle cell trait (HbAS) results stand out. Significant alterations were observed in the membrane properties, bringing new perspectives for the knowledge of this mutation. This work presents ultrastructural and biomechanical signatures of sickle cell anemia and thalassemia genotypes, which may help determine a more accurate biophysical description and clinical prognosis for these diseases.

The unusual cell wall of the Lyme disease spirochaete Borrelia burgdorferi is shaped by a tick sugar

Peptidoglycan—a mesh sac of glycans that are linked by peptides—is the main component of bacterial cell walls. Peptidoglycan provides structural strength, protects cells from osmotic pressure and contributes to shape. All bacterial glycans are repeating disaccharides of N-acetylglucosamine (GlcNAc) β-(1–4)-linked to N-acetylmuramic acid (MurNAc). Borrelia burgdorferi, the tick-borne Lyme disease pathogen, produces glycan chains in which MurNAc is occasionally replaced with an unknown sugar. Nuclear magnetic resonance, liquid chromatography–mass spectroscopy and genetic analyses show that B. burgdorferi produces glycans that contain GlcNAc–GlcNAc. This unusual disaccharide is chitobiose, a component of its chitinous tick vector. Mutant bacteria that are auxotrophic for chitobiose have altered morphology, reduced motility and cell envelope defects that probably result from producing peptidoglycan that is stiffer than that in wild-type bacteria. We propose that the peptidoglycan of B. burgdorferi probably evolved by adaptation to obligate parasitization of a tick vector, resulting in a biophysical cell-wall alteration to withstand the atypical torque associated with twisting motility.

Morphological and mechanical changes induced by quercetin in human T24 bladder cancer cells

Quercetin is a flavonoid found in a great variety of foods such as vegetables and fruits. This compound has been shown to inhibit the proliferation of various types of cancer cells, as well as the growth of tumors in animal models. In the present study, we analyze morphological and mechanical changes produced by quercetin in T24 bladder cancer cells. Decreased cell viability and cell number were observed following quercetin treatment at 40 μM and 60 μM, respectively, as observed by the MTT assay and trypan blue exclusion test, supporting the hypothesis of quercetin anticancer effect. These assays also allowed us to determine the 40, 60, and 80 μM quercetin concentrations for the following analyses, Lactate Dehydrogenase assay (LDH); Nuclear Morphometric Analysis (NMA); and atomic force microscopy (AFM). The LDH assay showed no cytotoxic effect of quercetin on T24 cancer cells. The AFM showed morphological changes following quercetin treatment, namely decreased cell body, cytoplasmic retraction, and membrane condensation. Following quercetin treatment, the NMA evidenced an increased percentage of nuclei characteristic to the apoptotic and senescence processes. Cells also presented biophysical alterations consistent with cell death by apoptosis, as increased roughness and aggregation of membrane proteins, in a dose-dependent manner. Cellular elasticity, obtained through force curves, showed increased stiffness after quercetin treatment. Data presented herein demonstrate, for the first time, in a quantitative and qualitative form, the morphological and mechanical alterations induced by quercetin on bladder cancer cells.

Physiological and biophysical alterations in maize plants caused by Colletotrichum graminicola infection verified by OJIP study

Physiological and biophysical alterations in maize plants caused by Colletotrichum graminicola infection verified by OJIP study

Biophysical and biochemical alterations in extravillous trophoblast cells infected by Zika virus

Biophysical and biochemical alterations in extravillous trophoblast cells infected by Zika virus

Withdrawal notice to “Alcohol exacerbated biochemical and biophysical alterations in liver mitochondrial membrane of diabetic male wistar rats – A possible amelioration by green tea” [Phytochem. Lett. 44C (2021) 227–238

Withdrawal notice to “Alcohol exacerbated biochemical and biophysical alterations in liver mitochondrial membrane of diabetic male wistar rats – A possible amelioration by green tea” [Phytochem. Lett. 44C (2021) 227–238