Diseased Human Tissues Research Articles

Safety and practicality of an excisional lymph node study driving HIV cure research in South Africa.

Studying diseased human tissues offers better insights into the intricate interactions between pathogens and the human host. In conditions such as HIV and cancers, where diseases primarily manifest in tissues, peripheral blood studies are limited in providing a thorough understanding of disease processes and localized immune responses. We describe a study designed to obtain excisional lymph nodes from volunteers for HIV reservoir studies. Since study commencement in 2015, 181 lymph node excisions have been performed, resulting in collection of 138 lymph node tissues. Lymph nodes were surgically excised from study volunteers using a minimally invasive procedure, performed in a minor theater under local anesthesia. The surgery takes less than 30 minutes to complete, minimizing risk and stress on the volunteer. The small incision made during the procedure typically heals within a week. The associated discomfort is generally manageable, and participants are often able to resume their regular activities within a day. Only 5.5% of the study participants experienced minor adverse events, such as swelling and prolonged wound healing, recovering within 2 weeks with no serious adverse events reported. Our study demonstrates that when done with outmost care, obtaining excised lymph nodes for research is relatively safe and practical.

Expression of miRNAs (146a and 155) in human peri-implant tissue affected by peri-implantitis: a case control study

BackgroundIn literature, the levels of miRNA-146a and miRNA-155 are increased in periodontitis. Limited data are available regarding the expression of miRNA-146a and miR-NA-155 in diseased human peri-implant tissue. Therefore, the objective of this study was to explore the expression of miRNA-146a and miRNA-155 in human gingival peri-implant tissue affected by peri-implantitis.MethodsAfter recording the clinical parameters, human peri-implant pocket tissues were harvested from sites diagnosed with peri-implantitis (n = 15 cases) in addition to healthy peri-implant sulcus tissues (n = 15 controls). The levels of miRNA-146a and miRNA-155 were assessed using real-time qPCR.ResultsCases exhibited a significantly higher mean expression of miRNA-155 (5.2-fold increase) and miRNA-146a (2.8-fold increase) than controls. MiRNA-155 and miRNA-146a demonstrated an appropriate sensitivity (87.5% and 87.5%, respectively) and specificity (73.3% and 66.7%, respectively) in discriminating cases from controls. A moderate correlation (r = 0.544, p = 0.029) was found between miRNA-155 and miRNA-146a levels in the case group.ConclusionsThe expressions of miRNA-146a and miR-NA-155 are different between healthy and peri-implantitis affected tissues. Both miRNAs might potentially able to discriminate healthy from peri-implantitis affected tissues.

Binary fabrication of decellularized lung extracellular matrix hybridgels for in vitro chronic obstructive pulmonary disease modeling

Limited treatments and a lack of appropriate animal models have spurred the study of scaffolds to mimic lung disease in vitro. Decellularized human lung and its application in extracellular matrix (ECM) hydrogels has advanced the development of these lung ECM models. Controlling the biochemical and mechanical properties of decellularized ECM hydrogels continues to be of interest due to inherent discrepancies of hydrogels when compared to their source tissue. To optimize the physiologic relevance of ECM hydrogel lung models without sacrificing the native composition we engineered a binary fabrication system to produce a Hybridgel composed of an ECM hydrogel reinforced with an ECM cryogel. Further, we compared the effect of ECM-altering disease on the properties of the gels using elastin poor Chronic Obstructive Pulmonary Disease (COPD) vs non-diseased (ND) human lung source tissue. Nanoindentation confirmed the significant loss of elasticity in hydrogels compared to that of ND human lung and further demonstrated the recovery of elastic moduli in ECM cryogels and Hybridgels. These findings were supported by similar observations in diseased tissue and gels. Successful cell encapsulation, distribution, cytotoxicity, and infiltration were observed and characterized via confocal microscopy. Cells were uniformly distributed throughout the Hybridgel and capable of survival for 7 days. Cell-laden ECM hybridgels were found to have elasticity similar to that of ND human lung. Compositional investigation into diseased and ND gels indicated the conservation of disease-specific elastin to collagen ratios. In brief, we have engineered a composited ECM hybridgel for the 3D study of cell-matrix interactions of varying lung disease states that optimizes the application of decellularized lung ECM materials to more closely mimic the human lung while conserving the compositional bioactivity of the native ECM. Statement of significanceThe lack of an appropriate disease model for the study of chronic lung diseases continues to severely inhibit the advancement of treatments and preventions of these otherwise fatal illnesses due to the inability to recapture the biocomplexity of pathologic cell-ECM interactions. Engineering biomaterials that utilize decellularized lungs offers an opportunity to deconstruct, understand, and rebuild models that highlight and investigate how disease specific characteristics of the extracellular environment are involved in driving disease progression. We have advanced this space by designing a binary fabrication system for a ECM Hybridgel that retains properties from its source material required to observe native matrix interactions. This design simulates a 3D lung environment that is both mechanically elastic and compositionally relevant when derived from non-diseased tissue and pathologically diminished both mechanically and compositionally when derived from COPD tissue. Here we describe the ECM hybridgel as a model for the study of cell-ECM interactions involved in COPD.

TCF1-LEF1 co-expression identifies a multipotent progenitor cell (TH2-MPP) across human allergic diseases.

Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (TH2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed TH2-multipotent progenitors (TH2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (Treg) cells and follicular helper T (TFH) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between TH2-MPP, TH2 effectors, Treg cells and TFH cells. TH2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify TH2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells.

Abstract 2930: Recovery of limited viable cells from low-quality precious samples for scRNA-seq

Abstract Biomarker discovery is a critical milestone in pursuing personalized and targeted cancer treatments, yet it faces formidable challenges hindering success rates. Limited access to high-quality samples restricts in-depth biological investigation, with scarce and compromised viable cells acting as barriers to progress. Levitation Technology™ addresses these challenges by significantly improving sample quality and preserving maximum viable cells for single-cell RNA sequencing (scRNA-seq). This innovative approach overcomes limitations in sample quality and availability and heralds a new era of possibilities, propelling significant advancements in biomarker discovery for personalized cancer treatment. To assess the performance of Levitation Technology using the LeviCell® 1.0 system, 105 cryopreserved samples derived from diseased human tissues were processed by Discovery Life Sciences™. This diverse sample set covered 20 disease indications, including 17 types of cancer. A staggering 70% of the samples (n=74) exhibited cell viability ranging between 14-69%, underscoring the need for enhanced viability for effective scRNA-seq analysis. Notably, 45 of these samples presented fewer than 500,000 cells, a scenario marked by low quality and limited viable cells. Traditional methods fail to recover viable cells from such samples without considerable or complete viable cell loss. After processing the samples with the LeviCell 1.0 system, all samples had improved quality, with an impressive 90% of the most difficult samples (n=40) presenting a viability increase to 70-94%, meeting the minimum quality criteria for scRNA-seq. A notable success story was a particular breast cancer sample with only 31,000 cells and 14% viability. Processing with the LeviCell 1.0 system yielded 13,300 viable cells and a substantial 4.5-fold increase in viability to 64%. Such enhancements in sample quality translate into significant improvements in scRNA-seq data quality, enabling more single-cell data extraction from each invaluable sample. Levitation Technology represents a paradigm shift in sample processing, rescuing even the most challenging samples. This groundbreaking technology maximizes the retention of viable cells while efficiently eliminating dead cells and debris, factors that have traditionally compromised scRNA-seq data quality. By revolutionizing this aspect of sample preparation, Levitation Technology opens up unparalleled opportunities to explore deeper into disease biology, providing a leap forward in biomarker discovery. Citation Format: Camila Egidio, Dinesh Bhosale, Lanxin Shen, Susana Jett, Amir Malek, Teddy de Groot, Suzanne Weaver, Gang Sun, Kevin Travers, Geoff Facer. Recovery of limited viable cells from low-quality precious samples for scRNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2930.

BHLHE40/41 regulate microglia and peripheral macrophage responses associated with Alzheimer’s disease and other disorders of lipid-rich tissues

Genetic and experimental evidence suggests that Alzheimer’s disease (AD) risk alleles and genes may influence disease susceptibility by altering the transcriptional and cellular responses of macrophages, including microglia, to damage of lipid-rich tissues like the brain. Recently, sc/nRNA sequencing studies identified similar transcriptional activation states in subpopulations of macrophages in aging and degenerating brains and in other diseased lipid-rich tissues. We collectively refer to these subpopulations of microglia and peripheral macrophages as DLAMs. Using macrophage sc/nRNA-seq data from healthy and diseased human and mouse lipid-rich tissues, we reconstructed gene regulatory networks and identified 11 strong candidate transcriptional regulators of the DLAM response across species. Loss or reduction of two of these transcription factors, BHLHE40/41, in iPSC-derived microglia and human THP-1 macrophages as well as loss of Bhlhe40/41 in mouse microglia, resulted in increased expression of DLAM genes involved in cholesterol clearance and lysosomal processing, increased cholesterol efflux and storage, and increased lysosomal mass and degradative capacity. These findings provide targets for therapeutic modulation of macrophage/microglial function in AD and other disorders affecting lipid-rich tissues.

Bioactive and chemically defined hydrogels with tunable stiffness guide cerebral organoid formation and modulate multi-omics plasticity in cerebral organoids.

Organoids are an emerging technology with great potential in human disease modelling, drug development, diagnosis, tissue engineering, and regenerative medicine. Organoids as 3D-tissue culture systems have gained special attention in the past decades due to their ability to faithfully recapitulate the complexity of organ-specific tissues. Despite considerable successes in culturing physiologically relevant organoids, their real-life applications are currently limited by challenges such as scarcity of an appropriate biomimetic matrix. Peptide amphiphiles (PAs) due to their well-defined chemistry, tunable bioactivity, and extracellular matrix (ECM)-like nanofibrous architecture represent an attractive material scaffold for organoids development. Using cerebral organoids (COs) as exemplar, we demonstrate the possibility to create bio-instructive hydrogels with tunable stiffness ranging from 0.69kPa to 2.24kPa to culture and induce COs growth. We used orthogonal chemistry involving oxidative coupling and supramolecular interactions to create two-component hydrogels integrating the bio-instructive activity and ECM-like nanofibrous architecture of a laminin-mimetic PAs (IKVAV-PA) and tunable crosslinking density of hyaluronic acid functionalized with tyramine (HA-Try). Multi-omics technology including transcriptomics, proteomics, and metabolomics reveals the induction and growth of COs in soft HA-Tyr hydrogels containing PA-IKVAV such that the COs display morphology and biomolecular signatures similar to those grown in Matrigel scaffolds. Our materials hold great promise as a safe synthetic ECM for COs induction and growth. Our approach represents a well-defined alternative to animal-derived matrices for the culture of COs and might expand the applicability of organoids in basic and clinical research. STATEMENT OF SIGNIFICANCE: Synthetic bio-instructive materials which display tissue-specific functionality and nanoscale architecture of the native extracellular matrix are attractive matrices for organoids development. These synthetic matrices are chemically defined and animal-free compared to current gold standard matrices such as Matrigel. Here, we developed hydrogel matrices with tunable stiffness, which incorporate laminin-mimetic peptide amphiphiles to grow and expand cerebral organoids. Using multi-omics tools, the present study provides exciting data on the effects of neuro-inductive cues on the biomolecular profiles of brain organoids.

Latest Patents for Endoscopic Cleaning, Disinfection Devices, and Lavage Fluid Collection & Filtration Devices

Background: With ageing, the population, and the increasing number of people with compromised immunity, environmental problems are becoming increasingly severe. Infants and older people are greatly affected by respiratory airway blockages, such as phlegm accumulation leading to poor breathing or foreign bodies entering the body, thereby, increasingly complicating respiratory infectious diseases, and raising the demand for endoscopes. The endoscope can observe the pathological conditions and morphological changes of human diseased tissues and carry out corresponding ‘dredging". Endoscope lavage is an essential medical means to improve dyspnea and extract foreign bodies, so it has been widely used in medicine. Objective: To review the research status of the endoscope by combining the latest developments and technologies of endoscope cleaning and disinfection systems, lavage collection, and filtration systems in recent years (from 2015 to now) to achieve the purpose of continuous improvement and optimization of the endoscope Methods: By analyzing the patent features of the endoscope in the cleaning and disinfecting system and the irrigation fluid collection and filtration system, it shows that there is a greater risk of hospital cross-infection when the endoscope uses to make a diagnosis of a disease before it is applied to the human body and under direct vision, or when biopsy of a lesion is performed to make a pathological diagnosis, which explains that it is necessary to clean and disinfect the medical endoscope and collect the irrigation fluid beforehand. At the same time, to reduce patients' pain by performing only one irrigation procedure, it is crucial to filter the irrigation fluid to achieve a more accurate examination of the irrigation fluid biopsy. Results: By comparing the main problems existing in the endoscope, to prospect the development trend of the endoscope. Conclusion: By improving and optimizing the cleaning and disinfection system, the irrigation fluid collection, and the filtration system of the endoscope, the loopholes in the current technology can be solved and made more practical.

Development of small molecule inhibitors of natural killer group 2D receptor (NKG2D)

Natural killer group 2D (NKG2D) is a homodimeric activating immunoreceptor whose function is to detect and eliminate compromised cells upon binding to the NKG2D ligands (NKG2DL) major histocompatibility complex (MHC) molecules class I-related chain A (MICA) and B (MICB) and UL16 binding proteins (ULBP1-6). While typically present at low levels in healthy cells and tissue, NKG2DL expression can be induced by viral infection, cellular stress or transformation. Aberrant activity along the NKG2D/NKG2DL axis has been associated with autoimmune diseases due to the increased expression of NKG2D ligands in human disease tissue, making NKG2D inhibitors an attractive target for immunomodulation. Herein we describe the discovery and optimization of small molecule PPI (protein–protein interaction) inhibitors of NKG2D/NKG2DL. Rapid SAR was guided by structure-based drug design and accomplished by iterative singleton and parallel medicinal chemistry synthesis. These efforts resulted in the identification of several potent analogs (14, 21, 30, 45) with functional activity and improved LLE.

Understanding painful versus non-painful dental pain in female and male patients: A transcriptomic analysis of human biopsies.

Dental pain from apical periodontitis is an infection induced-orofacial pain condition that presents with diversity in pain phenotypes among patients. While 60% of patients with a full-blown disease present with the hallmark symptom of mechanical allodynia, nearly 40% of patients experience no pain. Furthermore, a sexual dichotomy exists, with females exhibiting lower mechanical thresholds under basal and diseased states. Finally, the prevalence of post-treatment pain refractory to commonly used analgesics ranges from 7-19% (∼2 million patients), which warrants a thorough investigation of the cellular changes occurring in different patient cohorts. We, therefore, conducted a transcriptomic assessment of periapical biopsies (peripheral diseased tissue) from patients with persistent apical periodontitis. Surgical biopsies from symptomatic male (SM), asymptomatic male (AM), symptomatic female (SF), and asymptomatic female (AF) patients were collected and processed for bulk RNA sequencing. Using strict selection criteria, our study found several unique differentially regulated genes (DEGs) between symptomatic and asymptomatic patients, as well as novel candidate genes between sexes within the same pain group. Specifically, we found the role of cells of the innate and adaptive immune system in mediating nociception in symptomatic patients and the role of genes involved in tissue homeostasis in potentially inhibiting nociception in asymptomatic patients. Furthermore, sex-related differences appear to be tightly regulated by macrophage activity, its secretome, and/or migration. Collectively, we present, for the first time, a comprehensive assessment of peripherally diseased human tissue after a microbial insult and shed important insights into the regulation of the trigeminal system in female and male patients.

Infection and inflammation stimulate expansion of a CD74+ Paneth cell subset to regulate disease progression.

Paneth cells (PCs), a specialized secretory cell type in the small intestine, are increasingly recognized as having an essential role in host responses to microbiome and environmental stresses. Whether and how commensal and pathogenic microbes modify PC composition to modulate inflammation remain unclear. Using newly developed PC-reporter mice under conventional and gnotobiotic conditions, we determined PC transcriptomic heterogeneity in response to commensal and invasive microbes at single cell level. Infection expands the pool of CD74+ PCs, whose number correlates with auto or allogeneic inflammatory disease progressions in mice. Similar correlation was found in human inflammatory disease tissues. Infection-stimulated cytokines increase production of reactive oxygen species (ROS) and expression of a PC-specific mucosal pentraxin (Mptx2) in activated PCs. A PC-specific ablation of MyD88 reduced CD74+ PC population, thus ameliorating pathogen-induced systemic disease. A similar phenotype was also observed in mice lacking Mptx2. Thus, infection stimulates expansion of a PC subset that influences disease progression.

Systematic transcriptional analysis of human cell lines for gene expression landscape and tumor representation

Cell lines are valuable resources as model for human biology and translational medicine. It is thus important to explore the concordance between the expression in various cell lines vis-à-vis human native and disease tissues. In this study, we investigate the expression of all human protein-coding genes in more than 1,000 human cell lines representing 27 cancer types by a genome-wide transcriptomics analysis. The cell line gene expression is compared with the corresponding profiles in various tissues, organs, single-cell types and cancers. Here, we present the expression for each cell line and give guidance for the most appropriate cell line for a given experimental study. In addition, we explore the cancer-related pathway and cytokine activity of the cell lines to aid human biology studies and drug development projects. All data are presented in an open access cell line section of the Human Protein Atlas to facilitate the exploration of all human protein-coding genes across these cell lines.

Human Alzheimer’s disease reactive astrocytes exhibit a loss of homeostastic gene expression

Astrocytes are one of the brain’s major cell types and are responsible for maintaining neuronal homeostasis via regulating the extracellular environment, providing metabolic support, and modulating synaptic activity. In neurodegenerative diseases, such as Alzheimer’s disease, astrocytes can take on a hypertrophic appearance. These reactive astrocytes are canonically associated with increases in cytoskeletal proteins, such as glial fibrillary acidic protein and vimentin. However, the molecular alterations that characterize astrocytes in human disease tissues have not been extensively studied with single cell resolution. Using single nucleus RNA sequencing data from normal, pathologic aging, and Alzheimer’s disease brains, we identified the transcriptomic changes associated with reactive astrocytes. Deep learning-based clustering algorithms denoised expression data for 17,012 genes and clustered 15,529 astrocyte nuclei, identifying protoplasmic, gray matter and fibrous, white matter astrocyte clusters. RNA trajectory analyses revealed a spectrum of reactivity within protoplasmic astrocytes characterized by a modest increase of reactive genes and a marked decrease in homeostatic genes. Amyloid but not tau pathology correlated with astrocyte reactivity. To identify reactivity-associated genes, linear regressions of gene expression versus reactivity were used to identify the top 52 upregulated and 144 downregulated genes. Gene Ontology analysis revealed that upregulated genes were associated with cellular growth, responses to metal ions, inflammation, and proteostasis. Downregulated genes were involved in cellular interactions, neuronal development, ERBB signaling, and synapse regulation. Transcription factors were significantly enriched among the downregulated genes. Using co-immunofluorescence staining of Alzheimer’s disease brain tissues, we confirmed pathologic downregulation of ERBB4 and transcription factor NFIA in reactive astrocytes. Our findings reveal that protoplasmic, gray matter astrocytes in Alzheimer’s disease exist within a spectrum of reactivity that is marked by a strong loss of normal function.

3D genomics identify alternate mechanisms of homologous recombination deficiency in uterine sarcoma.

e17624 Background: Uterine sarcomas are rare tumors with diverse morphologic appearances, genetic underpinnings, and clinical behavior. Molecular characterization of these tumors by next generation sequencing (NGS) has improved disease classification and prognostication, but its impact on therapeutic selection remains limited. Analysis of 3D genomics datasets has recently been shown to detect large complex structural variants (SV) and chromatin architecture in normal and diseased human tissues. We applied genome-wide chromosome conformation capture (Hi-C) techniques in a variety of uterine sarcomas to identify novel therapeutic targets. Methods: Formalin-fixed paraffin-embedded tissue from 48 primary or recurrent uterine sarcomas were subjected to Hi-C to identify chromosomal rearrangements. The cohort included leiomyosarcoma (LMS, n = 7), adenosarcoma (AS, n = 7), endometrial stromal sarcoma (n = 7; high grade (HGESS), n = 4; low grade (LGESS), n = 3), sarcoma not otherwise specified (n = 17; high grade (HGNOS), n = 10, low grade (LGNOS), n = 7), undifferentiated uterine sarcoma (UUS, n = 6) and heterologous sarcoma (n = 2). Tumors were previously subjected to targeted DNA (n = 10) and/or RNA (n = 26) NGS with no therapeutic targets identified and only BCOR internal tandem duplication detected in a HGESS. Results: Hi-C detected SV in 73% of uterine sarcomas (n = 35/48), including those previously negative by targeted RNA NGS (n = 25/26). Homologous recombination repair (HRR) gene alterations were detected by Hi-C in 42% of tumors (n = 20/48), including AS (n = 5/7), HGESS (n = 2/4), LGESS (n = 1/3), LMS (n = 2/7), UUS (n = 2/6), heterologous sarcoma (n = 1/2), HGNOS (n = 3/10) and LGNOS (n = 1/7). SV included gene (gene to gene, n = 3; gene to non-coding DNA, n = 8) and proximity (n = 13) fusions disrupting HRR genes ( ATM, BRIP1, CDK12, CHEK1, CHEK2, NBN, PARP3, RAD51B, RAD51C, RAD51D) with enrichment for RAD51B fusions (50%, n = 10/20). Among all tumors with HRR gene alterations detected by Hi-C, only one uterine sarcoma harbored a missense ATM mutation by targeted DNA NGS (n = 1/5). Conclusions: Molecular characterization of uterine sarcomas by Hi-C may uncover HRR gene alterations not currently detectable by targeted DNA and RNA NGS. Homologous recombination deficiency (HRD) is not limited to LMS and may frequently result from SV, particularly between HRR gene to non-coding DNA in a variety of uterine sarcomas. These alternate mechanisms for HRD may serve as a potential therapeutic biomarker for HRD-targeted therapy in uterine sarcoma.

An approach for in-situ detection of gold colloid aggregates amyloid formations within the hippocampus of the Cohen’s Alzheimer’s disease rat model by surface enhanced raman scattering methods

BackgroundAmyloid beta (Aβ) peptides, such as Aβ1–40 or Aβ1–42 are regarded as hallmark neuropathological biomarkers associated with Alzheimer’s disease (AD). The formation of an aggregates by Aβ1–40 or Aβ1–42-coated gold nano-particles are hypothesized to contain conformation of Aβ oligomers, which could exist only at an initial stage of fibrillogenesis. New MethodThe attempt of in-situ detection of externally initiated gold colloid (ca. 80 nm diameter) aggregates in the middle section of the hippocampus of the Long Evans Cohen’s Alzheimer’s disease rat model was conducted through the Surface Enhanced Raman Scattering (SERS) method. ResultsThe SERS spectral features contained modes associated with β-sheet interactions and a significant number of modes that were previously reported in SERS shifts for Alzheimer diseased rodent and human brain tissues; thereby, strongly implying a containment of amyloid fibrils. The spectral patterns were further examined and compared with those collected from in-vitro gold colloid aggregates which were formed from Aβ1–40 - or Aβ1–42 -coated 80 nm gold colloid under pH ∼4, pH ∼7, and pH ∼10, and the best matched datasets were found with that of the aggregates of Aβ1–42 -coated 80 nm gold colloid at ∼pH 4.0. The morphology and physical size of this specific gold colloid aggregate was clearly different from those found in-vitro. Comparison with Existing Method(s)The amyloid fibril with a β-sheet conformation identified in previously reported in AD mouse/human brain tissues was involved in a formation of the gold colloid aggregates. However, to our surprise, best explanation for the observed SERS spectral features was possible with those in vitro Aβ1–42 -coated 80 nm gold colloid under pH ∼4. ConclusionsA formation of gold colloid aggregates was confirmed in the AD rat hippocampal brain section with unique physical morphology compared to those observed in in-vitro Aβ1–42 or Aβ1–40 mediated gold colloid aggregates. It was concluded that a β-sheet conformation identified in previously reported in AD mouse/human brain tissues was in volved in a formation of the gold colloid aggregates.

132 Colorimetric Assessment of Household Settled Dust Captured on Silicon Nanomembranes

Abstract Humans spend 70-90% of their time indoors, however, there is a significant lack of knowledge regarding human exposure to microplastic particles and fibers (MPs) within the indoor environment. Fibers make up more than 90% of household settled dust worldwide and have been found in indoor air. Studies have identified MPs larger than 50 micrometers in indoor dust, but these are unlikely to be respirable and little information is available regarding smaller airborne particles. It is evident that humans are inhaling these particles as they have been found in excised diseased and healthy human lung tissues. We have developed methods to identify plastic particles in settled household dust larger than one micrometer in diameter by distinguishing plastic from cellulosic, proteinaceous, and inorganic materials using two different stains, Nile Red for plastics, and Trypan Blue for cellulosic materials. Proteinaceous and inorganic materials remain unstained. Household settled dust is collected onto silicon nitride nanomembranes where in situ analysis of particles can be characterized. The innovative use of nanomembranes allow for particle capture and multiple analyses to take place on the same substrate. Particle analysis via colorimetric staining and imaging is followed by polymer identification via Raman spectroscopy and subsequently characterized via scanning electron microscopy for size and surface morphology. Using this innovative approach, microplastic particles larger than one micrometer in diameter have been identified in all settled dust samples.

Overexpression of MicroRNA-155 aggravates pulpitis by targeting kinesin superfamily Proteins-5C based on illumina high-throughput sequencing.

To investigate the regulatory role of miR-155 and Kinesin Superfamily Proteins-5C (KIF-5C) in the progression of pulpitis based on bioinformatic analysis. Normal pulp tissues and pulpitis pulp tissues were collected and subjected to high-throughput sequencing and the differentially expressed miRNAs were determined. An in vitro and in vivo pulpitis model was established. HE, IHC staining and histological evaluation were used to verify the inflammatory state of human and mouse pulp tissues. The mRNA expression of IL-1β and TGF-β1 were determined by RT-qPCR and protein expression of IL-1α, IL-4, IL-8, IL-13, IFN-γ, IL-6, IL-10 and MCP-1 were determined by protein chip. The target genes of miR-155 were predicted by miRanda database and verified by Dual-luciferase reporter assay, RT-qPCR and western blotting. MiR-155 lentivirus were used to upregulate or downregulate miR-155 and the siRNA of KIF-5C was used to downregulate KIF-5C. The expression of miR-155 or KIF-5C was determined by RT-qPCR. All statistics were analysed using GraphPad prism 8.2. The high-throughput sequencing results showed that 6 miRNAs (miR-155, miR-21, miR-142, miR-223, miR-486, miR-675) were significantly upregulated in diseased human pulp tissues, and miR-155 was significantly elevated among the six miRNAs. RT-qPCR results demonstrated that miR-155 expression was upregulated in human pulpitic tissue, mice pulpitic tissue and LPS-HDPCs. IL-1β was increased while TGF-β1 was decreased in lenti-miR-155 transfected LPS-HDPCs. Analysis of protein chip results indicated that lenti-miR-155 transfected LPS-HDPCs produced higher levels of IL-8, IL-6, MCP-1. The opposite results were obtained when miR-155 was inhibited. Through miRanda database screen and Dual-luciferase reporter assay, the target gene (KIF-5C) of miR-155 was identified. In lenti-miR-155 transfected LPS-HDPCs, the expression of KIF-5C was downregulated. However, when shRNA-miR-155 was transfected to LPS-HDPCs, the opposite result was obtained. Silent RNA was used to knock down KIF-5C, the results showed that when both KIF-5C and miR-155 were knocked down simultaneously, the downregulated expression of inflammatory factors observed in LPS-HDPCs following miR-155 knockdown was rescued. MiR-155 plays an important role in promoting pulpitis through targeting KIF-5C and may serve as a potential therapeutic target.

Raman microspectroscopy identifies fibrotic tissues in collagen-related disorders via deconvoluted collagen type I spectra.

Fibrosis is a consequence of the pathological remodeling of extracellular matrix (ECM) structures in the connective tissue of an organ. It is often caused by chronic inflammation, which over time, progressively leads to an excess deposition of collagen type I (COL I) that replaces healthy tissue structures, in many cases leaving a stiff scar. Increasing fibrosis can lead to organ failure and death; therefore, developing methods that potentially allow real-time monitoring of early onset or progression of fibrosis are highly valuable. In this study, the ECM structures of diseased and healthy human tissue from multiple organs were investigated for the presence of fibrosis using routine histology and marker-independent Raman microspectroscopy and Raman imaging. Spectral deconvolution of COL I Raman spectra allowed the discrimination of fibrotic and non-fibrotic COL I fibers. Statistically significant differences were identified in the amide I region of the spectral subpeak at 1608cm-1, which was deemed to be representative for structural changes in COL I fibers in all examined fibrotic tissues. Raman spectroscopy-based methods in combination with this newly discovered spectroscopic biomarker potentially offer a diagnostic approach to non-invasively track and monitor the progression of fibrosis. STATEMENT OF SIGNIFICANCE: Current diagnosis of fibrosis still relies on histopathological examination with invasive biopsy procedures. Although, several non-invasive imaging techniques such as positron emission tomography, single-photon emission computed tomography and second harmonic generation are gradually employed in preclinical or clinical studies, these techniques are limited in spatial resolution and the morphological interpretation highly relies on individual experience and knowledge. In this study, we propose a non-destructive technique, Raman microspectroscopy, to discriminate fibrotic changes of collagen type I based on a molecular biomarker. The changes of the secondary structure of collagen type I can be identified by spectral deconvolution, which potentially can provide an automatic diagnosis for fibrotic tissues in the clinical applicaion.

Cardiac RGS Proteins in Human Heart Failure and Atrial Fibrillation: Focus on RGS4.

The regulator of G protein signaling (RGS) proteins are crucial for the termination of G protein signals elicited by G protein-coupled receptors (GPCRs). This superfamily of cell membrane receptors, by far the largest and most versatile in mammals, including humans, play pivotal roles in the regulation of cardiac function and homeostasis. Perturbations in both the activation and termination of their G protein-mediated signaling underlie numerous heart pathologies, including heart failure (HF) and atrial fibrillation (AFib). Therefore, RGS proteins play important roles in the pathophysiology of these two devasting cardiac diseases, and several of them could be targeted therapeutically. Although close to 40 human RGS proteins have been identified, each RGS protein seems to interact only with a specific set of G protein subunits and GPCR types/subtypes in any given tissue or cell type. Numerous in vitro and in vivo studies in animal models, and also in diseased human heart tissue obtained from transplantations or tissue banks, have provided substantial evidence of the roles various cardiomyocyte RGS proteins play in cardiac normal homeostasis as well as pathophysiology. One RGS protein in particular, RGS4, has been reported in what are now decades-old studies to be selectively upregulated in human HF. It has also been implicated in protection against AFib via knockout mice studies. This review summarizes the current understanding of the functional roles of cardiac RGS proteins and their implications for the treatment of HF and AFib, with a specific focus on RGS4 for the aforementioned reasons but also because it can be targeted successfully with small organic molecule inhibitors.

Proteome architecture of human-induced pluripotent stem cell-derived three-dimensional organoids as a tool for early diagnosis of neuronal disorders.

Induced pluripotent stem cells (iPSCs) derived three-dimensional (3D) model for rare neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) is emerging as a novel alternative to human diseased tissue to explore the disease etiology and potential drug discovery. In the interest of the same, we have generated a TDP-43-mutated human iPSCs (hiPSCs) derived 3D organoid model of ALS disease. The high-resolution mass spectrometry (MS)-based proteomic approach is used to explore the differential mechanism under disease conditions and the suitability of a 3D model to study the disease. The hiPSCs cell line was procured from a commercial source, grown, and characterized following standard protocols. The mutation in hiPSCs was accomplished using CRISPR/Cas-9 technology and predesigned gRNA. The two groups of organoids were produced by normal and mutated hiPSCs and subjected to the whole proteomic profiling by high-resolution MS in two biological replicates with three technical replicas of each. The proteomic analysis of normal and mutated organoids revealed the proteins associated with pathways of neurodegenerative disorders, proteasomes, autophagy, and hypoxia-inducible factor-1 signaling. Differential proteomic analysis revealed that the mutation in TDP-43 gene caused proteomic deregulation, which impaired protein quality mechanisms. Furthermore, this impairment may contribute to the generation of stress conditions that may ultimately lead to the development of ALS pathology. The developed 3D model represents the majority of candidate proteins and associated biological mechanisms altered in ALS disease. The study also offers novel protein targets that may uncloud the precise disease pathological mechanism and be considered for future diagnostic and therapeutic purposes for various neurodegenerative disorders.