Cellular Inclusions Research Articles

Histo-anatomical features of garden lupin (lupinus polyphyllus lindl.)

Lupinus polyphyllus Lindl. (garden lupin, large-leaved lupin) is a perennial herbaceous legume, used for ornamental purposes, for improving soil quality, as forage or in human food products. However, little is known about anatomical features this species’Lupin vegetative organs (roots, petioles, leaf blades) were collected, cross sectioned and analyzed by bright-field microscopy. Lupin roots showed primary and incipient secondary structures, with a cortex featuring large-sized cells with rich cellular content and granular solid inclusions. Leaf petioles had rare peltate hairs and large sub-stomatal lacunes, a large medullary lacune and rich parenchyma. Leaf blades had typical bifacial structure, with three-layered palisade tissue and less proeminent midrib, with a collateral bundle, protected by small caps of collenchyma. Bangladesh J. Bot. 53(3): 423-428, 2024 (September)

Experimental and Computational Analyses of Sustainable Approaches in Railways

Railway transportation is widely recognized as an environment-friendly and sustainable means for conveying freight and passengers over long distances. This article investigates the effectiveness of utilizing scrap tire rubber granules and geosynthetics to enhance track performance in response to the growing demands for railway transport and the consequent escalation of train-induced loading. A multi-faceted methodology, incorporating experimental, numerical, and analytical techniques, is employed to examine the efficacy of these sustainable approaches. Results from three-dimensional (3D) finite element (FE) analyses conducted on slab tracks for high-speed railways reveal that the addition of a resilient layer, comprising scrap tire rubber granules, reduces vertical stress within the track substructure. Laboratory investigations on an innovative composite material consisting of soil, scrap rubber granules, and polyurethane demonstrate its potential to enhance track performance. Findings from two-dimensional (2D) FE analyses conducted on pile-supported railway embankments highlight an enhanced transfer of load to the pile head following the installation of a geogrid layer at the embankment base. Finally, the results from the analytical approach indicate a reduction in track settlement and a decrease in the track geometry degradation rate on reinforcing the ballast layer with 3D cellular geoinclusion. The novelty of this study lies in the comprehensive assessment of the innovative composite material under drained and cyclic loading conditions, the investigation of the influence of train loading on geosynthetic tension and the load transfer mechanism in railway embankments, and the development of an innovative computational methodology capable of assessing the effectiveness of 3D cellular inclusions in improving the ballasted railway track performance. The findings from this article underscore the effectiveness of these sustainable approaches in mitigating the challenges posed by increased loads on railway tracks, providing valuable insights for the ongoing efforts to optimize railway transportation infrastructure.

An animal study on the effectiveness of platelet-rich plasma as a direct pulp capping agent

Direct pulp capping (DPC) is a conservative approach for preserving tooth vitality without requiring more invasive procedures by enhancing pulp healing and mineralized tissue barrier formation. We investigated the effectiveness of Platelet Rich Plasma (PRP) vs. Mineral Trioxide Aggregate (MTA) as a DPC agent. Forty-two teeth from three mongrel dogs were divided into two equal groups. After three months, the animals were sacrificed to evaluate teeth radiographically using cone-beam computerized tomography, histopathologically, and real-time PCR for dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein (MEPE), and nestin (NES) mRNA expression. Radiographically, hard tissue formation was evident in both groups without significant differences (p = 0.440). Histopathologic findings confirmed the dentin bridge formation in both groups; however, such mineralized tissues were homogenous without cellular inclusions in the PRP group, while was osteodentin type in the MTA group. There was no significant difference in dentin bridge thickness between the PRP-capped and MTA-capped teeth (p = 0.732). The PRP group had significantly higher DSPP, MEPE, and NES mRNA gene expression than the MTA group (p < 0.05). In conclusion, PRP enables mineralized tissue formation following DPC similar to MTA, and could generate better cellular dentinogenic responses and restore dentin with homogenous architecture than MTA, making PRP a promising alternative DPC agent.

Histopathological Features of Shell Teeth: A Case Report

Shell Teeth are a type of radiographic appearance of teeth seen in Dentinogenesis Imperfecta type II (DGI II). Radiographically, they are characterised by an enlarged pulp chamber enclosed by a thin layer of dentin. The clinical appearance of these teeth ranges from yellow to bluish-grey in colour. The histological features of shell teeth have not been updated in the literature, unlike clinical and radiographic appearance. These features are not available in the literature compared to other dental anomalies. Here, the authors present a case of an 18-year-old female who came with the complaint of poor aesthetic dentition. The dentition aligned with the clinical and radiographic features of shell teeth. Histopathological features of the present case included an irregular arrangement of the dentinal matrix and a few cellular inclusions. The final diagnosis was given as DGI type II. This developmental disorder requires prevention and early intervention of its dental implications, such as dental caries and attrition. The treatment usually requires a wellplanned multidisciplinary protocol involving endodontic and prosthodontic approaches. Histopathological features of shell teeth are required to aid in the identification of the precise pathogenesis and genetic mechanism behind this structural disorder of dentin. It also helps in the modification of certain treatment procedures to accommodate the structural abnormality. The case report aimed to bridge the literature gap on the histopathological features of shell teeth.

Almyronema epifaneia gen. & sp. nov. (Cyanobacteria, Nodosilineaceae) isolated from an Indian mangrove forest

ABSTRACT Three simple filamentous cyanobacteria (strains S1, S6 and S10) were isolated from the upper surface of intertidal soil of an Indian mangrove forest, the Sundarbans. The three cyanobacterial strains formed a new cluster within the phylogenetic tree calculated based on 16S rRNA gene sequences, which was distinctive from the clade of the ecologically and morphologically most similar genus, Euryhalinema. Moreover, the sequence similarity of 16S rRNA gene was less than 94.4% compared with the reference strain of Euryhalinema, which confirmed that the new strains belonged to a novel genus. The folded secondary structure of D1-D1’, Box-B and V3 helices of the internal transcribed spacer (ITS) region were different from those of Euryhalinema and other members of family Nodosilineaceae. P-distance analysis of the ITS region of the test strains showed 22.2% dissimilarity with the reference genus Euryhalinema, which exceeded the cut off (7%) and strongly supported that it is a novel species within a novel genus. Differential morphological and ultrastructural characters, such as the presence of various cellular inclusions (prominent large cyanophycin granule, glycogen α granules, lipid β granules and mucilaginous sheath), conical-shaped apical cell as well as cell width, supported the description of a new taxon. Therefore, based on morphological, ultrastructural and molecular analysis we propose that cyanobacterial strains S1, S6 and S10 belong to a new monophyletic genus and species Almyronema epifaneia gen. & sp. nov., in the recently established family Nodosilineaceae.

Rosmarinic Acid Inhibits Rift Valley Fever Virus: In Vitro , Computational and Analytical Studies

Aim: The antiviral potentials of rosmarinic acid (RA) against Rift Valley fever (RVF) virus were investigated. Methods: Antiviral activity was investigated by evaluating the reduction in the viral infectivity titer. Computational and LC–MS studies were performed for investigating the mechanism of action. This is via testing the interaction between RA and its major metabolite with the key infectivity proteins and determination of RA cellular permeability. Results: A superior reduction in RVF infectivity titer (45.5%) was observed when RA was applied post-infection compared to 17.7% reduction following its application before infection in addition to time-dependent inactivation kinetics. Recorded data showed in-silico inhibitory potential of RA and its metabolite against RVFV cap-binding protein and glycoprotein C, which are integral for viral transcription. LC–MS revealed cellular inclusion of RA, supporting its intracellular viral interaction. Conclusion: These antiviral potentials might suggest a promising foundation for future anti-RVF drug development.

Synaptic expression of TAR-DNA-binding protein 43 in the mouse spinal cord determined using super-resolution microscopy.

Amyotrophic Lateral Sclerosis (ALS) is characterised by a loss of motor neurons in the brain and spinal cord that is preceded by early-stage changes in synapses that may be associated with TAR-DNA-Binding Protein 43 (TDP-43) pathology. Cellular inclusions of hyperphosphorylated TDP-43 (pTDP-43) are a key hallmark of neurodegenerative diseases such ALS. However, there has been little characterisation of the synaptic expression of TDP-43 inside subpopulations of spinal cord synapses. This study utilises a range of high-resolution and super-resolution microscopy techniques with immunolabelling, as well as an aptamer-based TDP-43 labelling strategy visualised with single-molecule localisation microscopy, to characterise and quantify the presence of pTDP-43 in populations of excitatory synapses near where motor neurons reside in the lateral ventral horn of the mouse lumbar spinal cord. We observe that TDP-43 is expressed in approximately half of spinal cord synapses as nanoscale clusters. Synaptic TDP-43 clusters are found most abundantly at synapses associated with VGLUT1-positive presynaptic terminals, compared to VGLUT2-associated synapses. Our nanoscopy techniques showed no difference in the subsynaptic expression of pTDP-43 in the ALS mouse model, SOD1G93a, compared to healthy controls, despite prominent structural deficits in VGLUT1-associated synapses in SOD1G93a mice. This research characterises the basic synaptic expression of TDP-43 with nanoscale precision and provides a framework with which to investigate the potential relationship between TDP-43 pathology and synaptic pathology in neurodegenerative diseases.

Ethanol Exposure to Ethanol-Oxidizing HEPG2 Cells Induces Intracellular Protein Aggregation.

Aggresomes are collections of intracellular protein aggregates. In liver cells of patients with alcoholic hepatitis, aggresomes appear histologically as cellular inclusions known as Mallory-Denk (M-D) bodies. The proteasome is a multicatalytic intracellular protease that catalyzes the degradation of both normal (native) and abnormal (misfolded and/or damaged) proteins. The enzyme minimizes intracellular protein aggregate formation by rapidly degrading abnormal proteins before they form aggregates. When proteasome activity is blocked, either by specific inhibitors or by intracellular oxidants (e.g., peroxynitrite, acetaldehyde), aggresome formation is enhanced. Here, we sought to verify whether inhibition of proteasome activity by ethanol exposure enhances protein aggregate formation in VL-17A cells, which are recombinant, ethanol-oxidizing HepG2 cells that express both alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1). We exposed ethanol-non-oxidizing HepG2 cells (ADH-/CYP2E1-) or ethanol-oxidizing VL-17A (ADH+/CYP2E1+) to varying levels of ethanol for 24 h or 72 h. After these treatments, we stained cells for aggresomes (detected microscopically) and quantified their numbers and sizes. We also conducted flow cytometric analyses to confirm our microscopic findings. Additionally, aggresome content in liver cells of patients with alcohol-induced hepatitis was quantified. After we exposed VL-17A cells to increasing doses of ethanol for 24 h or 72 h, 20S proteasome activity declined in response to rising ethanol concentrations. After 24 h of ethanol exposure, aggresome numbers in VL-17A cells were 1.8-fold higher than their untreated controls at all ethanol concentrations employed. After 72 h of ethanol exposure, mean aggresome numbers were 2.5-fold higher than unexposed control cells. The mean aggregate size in all ethanol-exposed VL-17A cells was significantly higher than in unexposed control cells but was unaffected by the duration of ethanol exposure. Co-exposure of cells to EtOH and rapamycin, the latter an autophagy activator, completely prevented EtOH-induced aggresome formation. In the livers of patients with alcohol-induced hepatitis (AH), the staining intensity of aggresomes was 2.2-fold higher than in the livers of patients without alcohol use disorder (AUD). We conclude that ethanol-induced proteasome inhibition in ethanol-metabolizing VL-17A hepatoma cells causes accumulation of protein aggregates. Notably, autophagy activation removes such aggregates. The significance of these findings is discussed.

Homotypic fibrillization of TMEM106B across diverse neurodegenerative diseases.

Misfolding and aggregation of disease-specific proteins, resulting in the formation of filamentous cellular inclusions, is a hallmark of neurodegenerative disease with characteristic filament structures, or conformers, defining each proteinopathy. Here we show that a previously unsolved amyloid fibril composed of a 135 amino acid C-terminal fragment of TMEM106B is a common finding in distinct human neurodegenerative diseases, including cases characterized by abnormal aggregation of TDP-43, tau, or a-synuclein protein. A combination of cryoelectron microscopy and mass spectrometry was used to solve the structures of TMEM106B fibrils at a resolution of 2.7 A ̊ from postmortem human brain tissue afflicted with frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP, n = 8), progressive supranuclear palsy (PSP, n = 2), or dementia with Lewy bodies (DLB, n = 1). The commonality of abundant amyloid fibrils composed of TMEM106B, a lysosomal/endosomal protein, to a broad range of debilitating human disorders indicates a shared fibrillization pathway that may initiate or accelerate neurodegeneration.

The Applications and Challenges of the Development of In Vitro Tumor Microenvironment Chips.

The tumor microenvironment (TME) plays a critical, yet mechanistically elusive role in tumor development and progression, as well as drug resistance. To better understand the pathophysiology of the complex TME, a reductionist approach has been employed to create in vitro microfluidic models called "tumor chips". Herein, we review the fabrication processes, applications, and limitations of the tumor chips currently under development for use in cancer research. Tumor chips afford capabilities for real-time observation, precise control of microenvironment factors (e.g. stromal and cellular components), and application of physiologically relevant fluid shear stresses and perturbations. Applications for tumor chips include drug screening and toxicity testing, assessment of drug delivery modalities, and studies of transport and interactions of immune cells and circulating tumor cells with primary tumor sites. The utility of tumor chips is currently limited by the ability to recapitulate the nuances of tumor physiology, including extracellular matrix composition and stiffness, heterogeneity of cellular components, hypoxic gradients, and inclusion of blood cells and the coagulome in the blood microenvironment. Overcoming these challenges and improving the physiological relevance of in vitro tumor models could provide powerful testing platforms in cancer research and decrease the need for animal and clinical studies.

Accumulation of TMEM106B C-terminal fragments in neurodegenerative disease and aging.

Several studies using cryogenic electron microscopy (cryo-EM) techniques recently reported the isolation and characterization of novel protein filaments, composed of a C-terminal fragment (CTF) of the endolysosomal transmembrane protein 106B (TMEM106B), from human post-mortem brain tissue with various neurodegenerative conditions and normal aging. Genetic variation in TMEM106B is known to influence the risk and presentation of several neurodegenerative diseases, especially frontotemporal dementia (FTD) caused by mutations in the progranulin gene (GRN). To further elucidate the significance of TMEM106B CTF, we performed immunohistochemistry with antibodies directed against epitopes within the filament-forming C-terminal region of TMEM106B. Accumulation of TMEM106B C-terminal immunoreactive (TMEM-ir) material was a common finding in all the conditions evaluated, including frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP), Alzheimer's disease, tauopathies, synucleinopathies and neurologically normal aging. TMEM-ir material was present in a wide range of brain cell types and in a broad neuroanatomical distribution; however, there was no co-localization of TMEM-ir material with other neurodegenerative proteins in cellular inclusions. In most conditions, the presence and abundance of TMEM-ir aggregates correlated strongly with patient age and showed only a weak correlation with the TMEM106B haplotype or the primary pathological diagnosis. However, all patients with FTD caused by GRN mutations were found to have high levels of TMEM-ir material, including several who were relatively young (< 60years). These findings suggest that the accumulation of TMEM106B CTF is a common age-related phenomenon, which may reflect lysosomal dysfunction. Although its significance in most neurodegenerative conditions remains uncertain, the consistent finding of extensive TMEM-ir material in cases of FTLD-TDP with GRN mutations further supports a pathomechanistic role of TMEM106B and lysosomal dysfunction in this specific disease population.

Exploring the Oxidative Stress Regulation of Mice with Hyperglycemia by Lactiplantibacillus plantarum SCS4.

The aim of this study was to evaluate the effects of Lactiplantibacillus plantarum SCS4 (L. plantarum SCS4) on oxidative stress in streptozocin-induced hyperglycemic mice. After establishment of the hyperglycemic model, control group mice were gavaged daily with phosphate-buffered saline, while different experimental groups (AG, BG, and CG) mice were gavaged with L. plantarum SCS4 suspension, cellular inclusion suspension, and inactivated inclusion suspension for 10weeks, respectively. Compared with the model group (MG) group, the results showed that fasting blood glucose levels in BG and CG groups decreased, and postprandial 2-h blood glucose levels in BG groups decreased, whereas glucose tolerance improved. Meanwhile, ROS and MDA levels in serum of AG mice were decreased significantly (P < 0.05). Compared with the MG group, serum levels of GPx, HO-1, and NQO1 were increased in the BG group, whereas serum levels of CAT, HO-1, and GSH were increased in the CG group. Our results indicate that L. plantarum SCS4 can alleviate oxidative stress induced by hyperglycemia, and there may be synergistic effects among the different treatments.

Performance improvement of ballasted railway tracks using three-dimensional cellular geoinclusions

Three-dimensional (3D) cellular inclusions such as geocells and scrap rubber tyres improve the engineering properties of the infill materials by providing all-around confinement. Although the 3D geoinclusions possess immense potential in the railway industry, their application is still limited due to a lack of adequate techniques to evaluate the magnitude of improvement provided by these artificial inclusions. This article presents an innovative computational approach to evaluate the effectiveness of 3D cellular geoinclusions in improving the performance of ballasted railway tracks. The proposed method is an integrated approach that combines the additional confinement model with the geotechnical rheological model for a railway track. The methodology is applied to an open track-bridge transition, and the results revealed that the geoinclusions substantially reduce the differential settlement. However, the magnitude of improvement depends on the opening size, placement location within the track and material used to manufacture the cellular inclusions. Moreover, the magnitude of settlement reduction also depends on the axle load and subgrade soil properties. The proposed methodology can assist the railway engineers in assessing the efficacy of 3D inclusions in improving the performance of railway tracks and help select the most appropriate material, size, and location of reinforcement for deriving maximum benefits.

The Use of Computed Tomography in the Diagnosis of Jejunal Diverticulosis Complicated by Diverticulitis and Intestinal Bleeding

The jejunal diverticulosis is rare and accounts for approximately 0.5–1 % of the general population. Diagnosis of the pathology is difficult even in the presence of symptomatic complications (perforation, abscess, peritonitis, sepsis, intestinal obstruction and bleeding), therefore, it is often overlooked or delayed, which requires high awareness and vigilance of physicians. Emergency surgical interventions are performed due to the lack of clear clinical symptoms and reliable radiological data in an average of 25 % of patients with complications of diverticulosis. In the presented case, multislice computed tomography (MSCT) with intravenous contrasting revealed multiple diverticula of the jejunum with reactively altered walls, infiltration of the adjacent cellular tissue and small gas inclusions, indicating diverticulum microperforation. Esophagogastroduodenoscopy (EGD) was performed which confirmed the jejunum bleeding, and was followed by laparotomy which confirmed multiple jejunal diverticula with microperforation and bleeding. Thus, timely MSCT in combination with EGD allowed to avoid complications of jejunal diverticulitis.

Revisiting biocrystallization: purine crystalline inclusions are widespread in eukaryotes.

Despite the widespread occurrence of intracellular crystalline inclusions in unicellular eukaryotes, scant attention has been paid to their composition, functions, and evolutionary origins. Using Raman microscopy, we examined >200 species from all major eukaryotic supergroups. We detected cellular crystalline inclusions in 77% species out of which 80% is composed of purines, such as anhydrous guanine (62%), guanine monohydrate (2%), uric acid (12%) and xanthine (4%). Our findings shifts the paradigm assuming predominance of calcite and oxalates. Purine crystals emerge in microorganisms in all habitats, e.g., in freshwater algae, endosymbionts of reef-building corals, deadly parasites, anaerobes in termite guts, or slime molds. Hence, purine biocrystallization is a general and ancestral eukaryotic process likely present in the last eukaryotic common ancestor (LECA) and here we propose two proteins omnipresent in eukaryotes that are likely in charge of their metabolism: hypoxanthine-guanine phosphoribosyl transferase and equilibrative nucleoside transporter. Purine crystalline inclusions are multifunctional structures representing high-capacity and rapid-turnover reserves of nitrogen and optically active elements, e.g., used in light sensing. Thus, we anticipate our work to be a starting point for further studies spanning from cell biology to global ecology, with potential applications in biotechnologies, bio-optics, or in human medicine.

High‐Resolution NMR H/D Exchange of Human Superoxide Dismutase Inclusion Bodies Reveals Significant Native Features Despite Structural Heterogeneity

AbstractProtein aggregation is central to aging, disease and biotechnology. While there has been recent progress in defining structural features of cellular protein aggregates, many aspects remain unclear due to heterogeneity of aggregates presenting obstacles to characterization. Here we report high‐resolution analysis of cellular inclusion bodies (IBs) of immature human superoxide dismutase (SOD1) mutants using NMR quenched amide hydrogen/deuterium exchange (qHDX), FTIR and Congo red binding. The extent of aggregation is correlated with mutant global stability and, notably, the free energy of native dimer dissociation, indicating contributions of native‐like monomer associations to IB formation. This is further manifested by a common pattern of extensive protection against H/D exchange throughout nine mutant SOD1s despite their diverse characteristics. These results reveal multiple aggregation‐prone regions in SOD1 and illuminate how aggregation may occur via an ensemble of pathways.

High-Resolution NMR H/D Exchange of Human Superoxide Dismutase Inclusion Bodies Reveals Significant Native Features Despite Structural Heterogeneity.

Protein aggregation is central to aging, disease and biotechnology. While there has been recent progress in defining structural features of cellular protein aggregates, many aspects remain unclear due to heterogeneity of aggregates presenting obstacles to characterization. Here we report high-resolution analysis of cellular inclusion bodies (IBs) of immature human superoxide dismutase (SOD1) mutants using NMR quenched amide hydrogen/deuterium exchange (qHDX), FTIR and Congo red binding. The extent of aggregation is correlated with mutant global stability and, notably, the free energy of native dimer dissociation, indicating contributions of native-like monomer associations to IB formation. This is further manifested by a common pattern of extensive protection against H/D exchange throughout nine mutant SOD1s despite their diverse characteristics. These results reveal multiple aggregation-prone regions in SOD1 and illuminate how aggregation may occur via an ensemble of pathways.

Homotypic fibrillization of TMEM106B across diverse neurodegenerative diseases

Misfolding and aggregation of disease-specific proteins, resulting in the formation of filamentous cellular inclusions, is a hallmark of neurodegenerative disease with characteristic filament structures, or conformers, defining each proteinopathy. Here we show that a previously unsolved amyloid fibril composed of a 135 amino acid C-terminal fragment of TMEM106B is a common finding in distinct human neurodegenerative diseases, including cases characterized by abnormal aggregation of TDP-43, tau, or α-synuclein protein. A combination of cryoelectron microscopy and mass spectrometry was used to solve the structures of TMEM106B fibrils at a resolution of 2.7Å from postmortem human brain tissue afflicted with frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP, n= 8), progressive supranuclear palsy (PSP, n= 2), or dementia with Lewy bodies (DLB, n= 1). The commonality of abundant amyloid fibrils composed of TMEM106B, a lysosomal/endosomal protein, to a broad range of debilitating human disorders indicates a shared fibrillization pathway that may initiate or accelerate neurodegeneration.

Identifying and characterizing effectors of low complexity protein domain assembly

TDP-43 is an RNA and DNA processing protein with a highly conserved, C-terminal low-complexity domain. In vitro, the TDP-43 low complexity domain can form liquid-liquid phase separations which readily mature into amyloid fibrils. Fibrils containing C-terminal fragments of TDP-43 have been identified within cellular inclusions of post-mortem neuronal tissue in a considerable range of neurodegenerative diseases, including ALS, FTD, and Alzheimer’s. Nearly all ALS-associated mutations are found in the low complexity domain, further highlighting its significance to disease.

The Medical Applications of Transmission Electron Microscope: Subject Review

Transmission electron microscope TEM is a tool used to visualization intracellular components of certain samples ranging from very little dimension one micrometer up to one nanometer, Therefore, TEM can reveal a tiny detail that can’t investigate through the light microscope. The application of TEM in the medical field may help the researchers to analyze the morphological structure of samples obtained from small organisms like bacteria and viruses, as well as study the samples of cellular inclusions on basis of three-dimensional images.