Molecular histopathology of matrix proteins through autofluorescence super-resolution microscopy.

Extracellular matrix diseases like fibrosis are elusive to diagnose early on, to avoid complete loss of organ function or even cancer progression, making early diagnosis crucial. Imaging the matrix densities of proteins like collagen in fixed tissue sections with suitable stains and labels is a standard for diagnosis and staging. However, fine changes in matrix density are difficult to realize by conventional histological staining and microscopy as the matrix fibrils are finer than the resolving capacity of these microscopes. The dyes further blur the outline of the matrix and add a background that bottlenecks high-precision early diagnosis of matrix diseases. Here we demonstrate the multiple signal classification method-MUSICAL-otherwise a computational super-resolution microscopy technique to precisely estimate matrix density in fixed tissue sections using fibril autofluorescence with image stacks acquired on a conventional epifluorescence microscope. We validated the diagnostic and staging performance of the method in extracted collagen fibrils, mouse skin during repair, and pre-cancers in human oral mucosa. The method enables early high-precision label-free diagnosis of matrix-associated fibrotic diseases without needing additional infrastructure or rigorous clinical training.

Utilization of DNA and 2D metal oxide interaction for an optical biosensor.

The efficient monitoring and early detection of viruses may provide essential information about diseases. In this work, we have highlighted the interaction between DNA and a two-dimensional (2D) metal oxide for developing biosensors for further detection of viral infections. Spectroscopic measurements have been used to probe the efficient interactions between single-stranded DNA (ssDNA) and the 2D metal oxide and make them ideal candidates for detecting viral infections. We have also used fully atomistic molecular dynamics (MD) simulation to give a microscopic understanding of the experimentally observed ssDNA-metal oxide interaction. The adsorption of ssDNA on the inorganic surface was found to be driven by favourable enthalpy change, and 5'-guanine was identified as the interacting nucleotide base. Additionally, the in silico assessment of the conformational changes of the ssDNA chain during the adsorption process was also performed in a quantitative manner. Finally, we comment on the practical implications of these developments for sensing that could help design advanced systems for preventing virus-related pandemics.

FTIR microspectroscopy and multivariate analysis facilitate identification of dynamic changes in epithelial-to-mesenchymal transition induced by TGF-ß in prostate cancer cells.

The standard diagnosis of prostate cancer is accomplished by the identification of cytomorphological deviations in biopsied tissues while immunohistochemistry is used to resolve the equivocal cases. Accumulating evidence favors the concept that epithelial-to-mesenchymal transition (EMT) is a stochastic process composed of multiple intermediate states instead of a single binary switch. Despite its significant role in promoting cancer aggressiveness, the current tissue-based risk stratification tools do not include any of the EMT phenotypes as a metric. As a proof-of-concept, the present study analyzes the temporal progression of EMT in transforming growth factor-beta (TGF-ß) treated PC3 cells encompassing multifarious characteristics such as morphology, migration and invasion, gene expression, biochemical fingerprint, and metabolic activity. Our multimodal approach reinstates EMT plasticity in TGF-ß treated PC3 cells. Further, it highlights that mesenchymal transition is accompanied by discernible changes in cellular morphometry and molecular signatures particularly in the range of 1800-1600 cm-1 and 3100-2800 cm-1 of Fourier-transformed infrared (FTIR) spectra signifying Amide III and lipid, respectively. Investigation of attenuated total reflectance (ATR)-FTIR spectra of extracted lipids from PC3 cell populations undergoing EMT identifies changes in stretching vibration at FTIR peaks at 2852, 2870, 2920, 2931, 2954, and 3010 cm-1 characteristics of fatty acids and cholesterol. Chemometric analysis of these spectra indicates that the level of unsaturation and acyl chain length of fatty acid coregister with differential epithelial/mesenchymal states of TGF-ß treated PC3 cells. Observed changes in lipids also correlate with cellular nicotinamide adenine dinucleotide hydrogen (NADH) and flavin adenine dinucleotide dihydrogen (FADH2) levels and mitochondrial oxygen consumption rate. In summary, our study establishes that morphological and phenotypic traits of epithelial/mesenchymal variants of PC3 cells concur with their respective biochemical and metabolic properties. It also underscores that spectroscopic histopathology has a definitive potential to refine the diagnosis of prostate cancer reckoning its molecular and biochemical heterogeneities.

Different Roles of p62 (SQSTM1) Isoforms in Keratin-Related Protein Aggregation.

p62/Sequestosome-1 (p62) is a multifunctional adaptor protein and is also a constant component of disease-associated protein aggregates, including Mallory-Denk bodies (MDBs), in steatohepatitis and hepatocellular carcinoma. We investigated the interaction of the two human p62 isoforms, p62-H1 (full-length isoform) and p62-H2 (partly devoid of PB1 domain), with keratins 8 and 18, the major components of MDBs. In human liver, p62-H2 is expressed two-fold higher compared to p62-H1 at the mRNA level and is present in slightly but not significantly higher concentrations at the protein level. Co-transfection studies in CHO-K1 cells, PLC/PRF/5 cells as well as p62- total-knockout and wild-type mouse fibroblasts revealed marked differences in the cytoplasmic distribution and aggregation behavior of the two p62 isoforms. Transfection-induced overexpression of p62-H2 generated large cytoplasmic aggregates in PLC/PRF/5 and CHO-K1 cells that mostly co-localized with transfected keratins resembling MDBs or (transfection without keratins) intracytoplasmic hyaline bodies. In fibroblasts, however, transfected p62-H2 was predominantly diffusely distributed in the cytoplasm. Aggregation of p62-H2 and p62ΔSH2 as well as the interaction with K8 (but not with K18) involves acquisition of cross-ß-sheet conformation as revealed by staining with luminescent conjugated oligothiophenes. These results indicate the importance of considering p62 isoforms in protein aggregation disease.

Comprehensive Evaluation of PAXgene Fixation on Oral Cancer Tissues Using Routine Histology, Immunohistochemistry, and FTIR Microspectroscopy.

The choice of tissue fixation is critical for preserving the morphology and biochemical information of tissues. Fragile oral tissues with lower tensile strength are challenging to process for histological applications as they are prone to processing damage, such as tissue tear, wrinkling, and tissue fall-off from slides. This leads to loss of morphological information and unnecessary delay in experimentation. In this study, we have characterized the new PAXgene tissue fixation system on oral buccal mucosal tissue of cancerous and normal pathology for routine histological and immunohistochemical applications. We aimed to minimize the processing damage of tissues and improve the quality of histological experiments. We also examined the preservation of biomolecules by PAXgene fixation using FTIR microspectroscopy. Our results demonstrate that the PAXgene-fixed tissues showed significantly less tissue fall-off from slides. Hematoxylin and Eosin staining showed comparable morphology between formalin-fixed and PAXgene-fixed tissues. Good quality and slightly superior immunostaining for cancer-associated proteins p53 and CK5/6 were observed in PAXgene-fixed tissues without antigen retrieval than formalin-fixed tissues. Further, FTIR measurements revealed superior preservation of glycogen, fatty acids, and amide III protein secondary structures in PAXgene-fixed tissues. Overall, we present the first comprehensive evaluation of the PAXgene tissue fixation system in oral tissues. This study concludes that the PAXgene tissue fixation system can be applied to oral tissues to perform diagnostic molecular pathology experiments without compromising the quality of the morphology or biochemistry of biomolecules.

Road Map to Understanding SARS-CoV-2 Clinico-Immunopathology and COVID-19 Disease Severity.

SARS-CoV-2, a novel coronavirus, was first identified in Wuhan, China in December 2019. The rapid spread of the virus worldwide prompted the World Health Organization (WHO) to declare COVID-19 a pandemic in March 2020. COVID-19 discontinuing's a global health crisis. Approximately 80% of the patients infected with SARS-CoV-2 display undetectable to mild inflammation confined in the upper respiratory tract. In remaining patients, the disease turns into a severe form affecting almost all major organs predominantly due to an imbalance of innate and adaptive arms of host immunity. The purpose of the present review is to narrate the virus's invasion through the system and the host's reaction. A thorough discussion on disease severity is also presented regarding the behavior of the host's immune system, which gives rise to the cytokine storm particularly in elderly patients and those with comorbidities. A multifaceted yet concise description of molecular aspects of disease progression and its repercussion on biochemical and immunological features in infected patients is tabulated. The summary of pathological, clinical, immunological, and molecular accounts discussed in this review is of theranostic importance to clinicians for early diagnosis of COVID-19 and its management.

Malignant potentiality assessment of oral submucous fibrosis through semi-quantitative approach.

BACKGROUND: In the context of early diagnosis and prevention of oral cancer, precise assessment of malignant potentiality of the oral potentially malignant disorders, particularly oral submucous fibrosis (OSF) is crucial. Till date, the assessment of malignant potentiality suffers from predictive ambiguity due to the lack of precision in the gold standard techniques. This can be addressed by integrating heuristic domain knowledge with quantitative analysis. AIM: The aim of this study is to propose an index for enhancing accuracy in malignant potentiality evaluation. MATERIALS AND METHODS: The present study analyzes important histomorphometric attributes (epithelial thickness, basal cell nuclear size, nuclear-to-cytoplasmic area ratio of basal cells, chromaticity of basal cell nucleus, thickness of basement membrane, ratio of vasculature in juxta-epithelial connective tissue [i.e., area covered by blood vessels/total area], collagen density in the lamina propria) of oral mucosa in dysplastic and nondysplastic OSF in association with relevant oncopathological appreciations (weightage of different features as suggested by oral pathologists) toward proposing a "Malignant Potentiality Index" (MPI). RESULTS: Analysis of variance and notch box plot analysis depict statistically significant differences (P < 0.0001) in the histopathological features among different study groups (normal oral mucosa, OSF without dysplasia, OSF with dysplasia). Histopathological observation of one OSF patient with calculated MPI is shown. CONCLUSION: This newly proposed diagnostic cum prognostic decision-making parameter, the "MPI" may bring a value addition to the conventional diagnostic gold standard.

Growth, serum biochemical, and histopathological responses of broilers administered with silver nanoparticles as a drinking water disinfectant.

The supplementation of 50 ppm dosed silver nanoparticles (AgNPs) as a disinfectant in broilers drinking water was investigated to examine their growth performance, blood serum biochemistry, and organ histology in the case group, compared to the control. The growth performance parameters, such as water intake, feed intake, and body weight were recorded 6 times, each in an interval of 7 days, over a period of 42 days. At the end of each 42 days, the blood and major organs of the 1 case boiler out of 75 and 1 control broiler out of 75 were collected in random. The procedure was repeated 3 sets one after another, each consisting 42 day intervening period. The liver enzyme, lipid profile, glucose level, organ histology, and concentration of AgNPs in liver, spleen, heart, and small intestine were determined. The obtained results show that the growth performance of the case broilers was significantly higher than the control section (p < 0.05). However, in all the three sets the changes in lipid profile, liver enzyme, and glucose level of the case broilers were not statistically significantly different compared to the control (p > 0.05). The histology of liver, kidney, heart, spleen, and small intestine of broilers has not shown any damages to the cells as compared to the control samples. In the case samples, the highest concentration of AgNPs was observed in the small intestine (5.44 µg/g) followed by liver (4.32 µg/g), kidney (3.94 µg/g), heart (3.82 µg/g), and spleen (3.49 µg/g). The present study concludes that the administering 50 ppm AgNPs of average 15 nm size in the poultry drinking water was found safe for consumption as well as for growth enhancing, due to better bird comfort.

The PPARα Agonist Fenofibrate Prevents Formation of Protein Aggregates (Mallory-Denk bodies) in a Murine Model of Steatohepatitis-like Hepatotoxicity.

Chronic intoxication of mice with the porphyrinogenic compound 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) leads to morphological and metabolic changes closely resembling steatohepatitis, a severe form of metabolic liver disease in humans. Since human steatohepatitis (both the alcoholic and non-alcoholic type) is characterized by reduced expression of PPARα and disturbed lipid metabolism we investigated the role of this ligand-activated receptor in the development of DDC-induced liver injury. Acute DDC-intoxication was accompanied by early significant downregulation of Pparα mRNA expression along with PPARα-controlled stress-response and lipid metabolism genes that persisted in the chronic stage. Administration of the specific PPARα agonist fenofibrate together with DDC prevented the downregulation of PPARα-associated genes and also improved the stress response of Nrf2-dependent redox-regulating genes. Moreover, oxidative stress and inflammation were strongly reduced by DDC/fenofibrate co-treatment. In addition, fenofibrate prevented the disruption of hepatocyte intermediate filament cytoskeleton and the formation of Mallory-Denk bodies at late stages of DDC intoxication. Our findings show that, like in human steatohepatitis, PPARα is downregulated in the DDC model of steatohepatitis-like hepatocellular damage. Its downregulation and the pathomorphologic features of steatohepatitis are prevented by co-administration of fenofibrate.

Stochastic phenotype switching leads to intratumor heterogeneity in human liver cancer.

Intratumor heterogeneity is increasingly recognized as a major factor impacting diagnosis and personalized treatment of cancer. We characterized stochastic phenotype switching as a mechanism contributing to intratumor heterogeneity and malignant potential of liver cancer. Clonal analysis of primary tumor cell cultures of a human sarcomatoid cholangiocarcinoma identified different types of self-propagating subclones characterized by stable (keratin-7-positive or keratin-7-negative) phenotypes and an unstable phenotype consisting of mixtures of keratin-7-positive and keratin-7-negative cells, which lack stem cell features but may reversibly switch their phenotypes. Transcriptome sequencing and immunohistochemical studies with the markers Zeb1 and CD146/MCAM demonstrated that switching between phenotypes is linked to changes in gene expression related but not identical to epithelial-mesenchymal transition. Stochastic phenotype switching occurred during mitosis and did not correlate with changes in DNA methylation. Xenotransplantation assays with different cellular subclones demonstrated increased tumorigenicity of cells showing phenotype switching, resulting in tumors morphologically resembling the invasive component of primary tumor and metastasis. CONCLUSION: Our data demonstrate that stochastic phenotype switching contributes to intratumor heterogeneity and that cells with a switching phenotype have increased malignant potential. (Hepatology 2017).

p62/Sequestosome-1 Is Indispensable for Maturation and Stabilization of Mallory-Denk Bodies.

Mallory-Denk bodies (MDBs) are hepatocytic protein aggregates found in steatohepatitis and several other chronic liver diseases as well as hepatocellular carcinoma. MDBs are mainly composed of phosphorylated keratins and stress protein p62/Sequestosome-1 (p62), which is a common component of cytoplasmic aggregates in a variety of protein aggregation diseases. In contrast to the well-established role of keratins, the role of p62 in MDB pathogenesis is still elusive. We have generated total and hepatocyte-specific p62 knockout mice, fed them with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to induce MDBs and allowed the mice to recover from DDC intoxication on a standard diet to investigate the role of p62 in MDB formation and elimination. In the absence of p62, smaller, granular and less distinct MDBs appeared, which failed to mature to larger and compact inclusions. Moreover, p62 deficiency impaired the binding of other proteins such as NBR1 and Hsp25 to MDBs and altered the cellular defense mechanism by downregulation of Nrf2 target genes. Upon recovery from DDC intoxication on a standard diet, there was an enhanced reduction of p62-deficient MDBs, which was accompanied by a pronounced decrease in ubiquitinated proteins. Our data provide strong evidence that keratin aggregation is the initial step in MDB formation in steatohepatitis-related mouse models. Interaction of p62 with keratin aggregates then leads to maturation i.e., enlargement and stabilization of the MDBs as well as recruitment of other MDB-associated proteins.

Sequestosome 1/p62 Protein Is Associated with Autophagic Removal of Excess Hepatic Endoplasmic Reticulum in Mice.

Xenobiotics exposure increases endoplasmic reticulum (ER) proliferation and cytochrome P-450 (CYP) induction to sustain metabolic requirements. Whether autophagy is essential for the removal of excess ER and CYP and whether an autophagy receptor is involved in this process in mammals remains elusive. In this study, we show that autophagy is induced in mouse livers after withdrawal of the hepatic mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP). Although isolated autophagosomes, autolysosomes, and lysosomes from mouse livers after withdrawal of TCPOBOP contained ER proteins, those in control mouse livers did not. Liver-specific Atg5 knockout mice had higher basal hepatic ER content that was further increased and sustained after withdrawal of TCPOBOP compared with wild-type mice. In addition to regulating ER degradation, our results also suggest that autophagy plays a role in regulating the homeostasis of hepatic CYP because blocking autophagy led to increased CYP2B10 accumulation either at the basal level or following TCPOBOP withdrawal. Furthermore, we found that the autophagy receptor protein sequestosome 1 (SQSTM1)/p62 is associated with the ER. After withdrawal of TCPOBOP, p62 knockout mice had increased ER content in the liver compared with wild-type mice. These results suggest that p62 may act as an autophagy receptor for the autophagic removal of excess ER in the mouse liver. Taken together, our results indicate that autophagy is important for the removal of excess ER and hepatic CYP enzymes in mouse livers, a process associated with the autophagy receptor protein p62.

Keratin 23 is a stress-inducible marker of mouse and human ductular reaction in liver disease.

BACKGROUND & AIMS: Keratins (K) constitute the epithelial intermediate filaments. Among them, K7/K19 are widely used markers of the regenerative liver response termed ductular reaction (DR) that consists of activated biliary epithelial cells (BECs) and hepatic progenitor cells (HPCs) and correlates with liver disease severity. In the present study we aimed to characterize K23 in the liver. METHODS: We analyzed the expression and localization of K23 in the digestive system under basal conditions as well as in various human and mouse liver diseases/stress models. Cell culture studies were used to study factors regulating K23 expression. RESULTS: In untreated mice, K23 was restricted to biliary epithelia. It was (together with K7/K19) markedly upregulated in three different DR/cholestatic injury models, i.e., multidrug resistance protein 2 (Mdr2) knockouts, animals treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine or subjected to bile duct ligation. K23 levels correlated with the DR marker Fn14 and immunofluorescence staining showed a distinct co-localization with K7/K19. In chronic human liver disease, K23 expression increased in patients with a more prominent inflammation/fibrosis. A dramatic upregulation (>200times) was observed in patients with acute liver failure (ALF) and end-stage primary biliary cholangitis (PBC). Patients with alcoholic liver cirrhosis displayed increased K23 serum levels. In primary hepatocytes as well as hepatobiliary cell lines, treatment with TNF-related weak inducer of apoptosis (TWEAK), and the type I acute phase inducer interleukin (IL)-1ß but not the type II inducer IL-6 elevated K23 expression. CONCLUSIONS: K23 represents a specific, stress-inducible DR marker, whose levels correlate with liver disease severity. K23 may represent a useful non-invasive DR marker. LAY SUMMARY: Ductular reaction represents a basic response to liver injury and correlates with liver disease severity. Our study identifies K23 as a novel ductular reaction marker in mice and humans.

Epigenetic silencing of apoptosis-inducing gene expression can be efficiently overcome by combined SAHA and TRAIL treatment in uterine sarcoma cells.

The lack of knowledge about molecular pathology of uterine sarcomas with a representation of 3-7% of all malignant uterine tumors prevents the establishment of effective therapy protocols. Here, we explored advanced therapeutic options to the previously discovered antitumorigenic effects of the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) by combined treatment with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L). In addition, we investigated the uterine sarcoma cell lines, MES-SA and ESS-1, regarding the underlying molecular mechanisms of SAHA and TRAIL-induced apoptosis and their resistance towards TRAIL. Compared to single SAHA or TRAIL treatment, the combination of SAHA with TRAIL led to complete cell death of both tumor cell lines after 24 to 48 hours. In contrast to single SAHA treatment, apoptosis occured faster and was more pronounced in ESS-1 cells than in MES-SA cells. Induction of SAHA- and TRAIL-induced apoptosis was accompanied by upregulation of the intrinsic apoptotic pathway via reduction of mitochondrial membrane potential, caspase-3, -6, and -7 activation, and PARP cleavage, but was also found to be partially caspase-independent. Apoptosis resistance was caused by reduced expression of caspase-8 and DR 4/TRAIL-R1 in ESS-1 and MES-SA cells, respectively, due to epigenetic silencing by DNA hypermethylation of gene promoter sequences. Treatment with the demethylating agent 5-Aza-2'-deoxycytidine or gene transfer therefore restored gene expression and increased the sensitivity of both cell lines against TRAIL-induced apoptosis. Our data provide evidence that deregulation of epigenetic silencing by histone acetylation and DNA hypermethylation might play a fundamental role in the origin of uterine sarcomas. Therefore, tumor growth might be efficiently overcome by a cytotoxic combinatorial treatment of HDAC inhibitors with TRAIL.