Traditional herbal medicine: Therapeutic potential in acute gouty arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Acute gouty arthritis (AGA) is characterized by a rapid inflammatory reaction caused by the build-up of monosodium urate (MSU) crystals in the tissues surrounding the joints. This condition often associated with hyperuricemia (HUA), is distinguished by its symptoms of intense pain, active inflammation, and swelling of the joints. Traditional approaches in AGA management often fall short of desired outcomes in clinical settings. However, recent ethnopharmacological investigations have been focusing on the potential of Traditional Herbal Medicine (THM) in various forms, exploring their therapeutic impact and targets in AGA treatment. AIM OF THE REVIEW: This review briefly summarizes the current potential pharmacological mechanisms of THMs - including active ingredients, extracts, and prescriptions -in the treatment of AGA, and discusses the relevant potential mechanisms and molecular targets in depth. The objective of this study is to offer extensive information and a reference point for the exploration of targeted AGA treatment using THMs. MATERIALS AND METHODS: This review obtained scientific publications focused on in vitro and in vivo studies of anti-AGA THMs conducted between 2013 and 2023. The literature was collected from various journals and electronic databases, including PubMed, Elsevier, ScienceDirect, Web of Science, and Google Scholar. The retrieval and analysis of relevant articles were guided by keywords such as "acute gouty arthritis and Chinese herbal medicine," "acute gouty arthritis herbal prescription," "acute gouty arthritis and immune cells," "acute gouty arthritis and inflammation," "acute gouty arthritis and NOD-like receptor thermoprotein domain associated protein 3 (NLRP3)," "acute gouty arthritis and miRNA," and "acute gouty arthritis and oxidative stress." RESULTS: We found that AGA has a large number of therapeutic targets, highlighting the effectiveness the potential of THMs in AGA treatment through in vitro and in vivo studies. THMs and their active ingredients can mitigate AGA symptoms through a variety of therapeutic targets, such as influencing macrophage polarization, neutrophils, T cells, natural killer (NK) cells, and addressing factors like inflammation, NLRP3 inflammasome, signaling pathways, oxidative stress, and miRNA multi-target interactions. The anti-AGA properties of THMs, including their active components and prescriptions, were systematically summarized and categorized based on their respective therapeutic targets. CONCLUSION: phenolic, flavonoid, terpenoid and alkaloid compounds in THMs are considered the key ingredients to improve AGA. THMs and their active ingredients achieve enhanced efficacy through interactions with multiple targets, of which NLRP3 is a main therapeutic target. Nonetheless, given the intricate composition of traditional Chinese medicine (TCM), additional research is required to unravel the underlying mechanisms and molecular targets through which THMs alleviate AGA.

Radiomics features based on dual-area CT predict the expression levels of fatty acid binding protein 4 and outcome in hepatocellular carcinoma.

RATIONALE AND OBJECTIVES: To evaluate the predictive value of tumor and peritumor radiomics in the fatty acid binding protein 4 (FABP4) expression levels and overall survival in patients with hepatocellular carcinoma. MATERIALS AND METHODS: The genomic data of HCC patients were obtained from The Cancer Genome Atlas. The Dual-area CT images of corresponding patients were downloaded from The Cancer Imaging Archive, for radiomics feature extraction, model construction and prognosis analysis. Simultaneously, using patients from Sichuan Provincial People's Hospital, the prognostic value of the radiomics model in HCC patients was validated. RESULTS: In the TCIA database, the area under the curve (AUC) values of the volumes of interest (VOI)whole model in the training set and internal validation set were 0.812 and 0.754, respectively, and the AUC value of VOIwhole+periphery in the training set and internal validation set were 0.866 and 0.779, respectively. In the VOIwhole and the VOIwhole+periphery model of the independent cohort, there were significant differences in OS between the high and low rad-score groups (P = 0.009, P = 0.021, respectively). Significant positive correlations can be observed between FABP4 expression and correlations with rad-score of VOIwhole model (r = 0.691) and VOIwhole+periphery model (r = 0.732) in the independent cohort. CONCLUSION: Radiomics models of tumor and peritumor Dual-area CT images could predict stably the expression levels of FABP4 and may be helping in personalized treatment strategies.

Identification of immunological characteristics and cuproptosis-related molecular clusters in primary Sjögren's syndrome.

BACKGROUND: Primary Sjögren's syndrome (pSS) is a chronic systemic autoimmune disease characterized by lymphocyte infiltration of the exocrine glands. The typical clinical symptoms of pSS include dryness of the mouth (xerostomia) and eyes (xerophthalmia), fatigue, and joint pain. Cuproptosis is a recently identified mode of programmed cell death that leads to the progression of multiple diseases, and the precise etiology and pathophysiology of pSS remain unknown. Consequently, the aim of our study was to explore cuproptosis-related molecular clusters and identify key genes in pSS. METHOD: Gene expression profiles of the peripheral blood in the GSE84844 dataset were downloaded to identify the expression characteristics of cuproptosis regulators and immune cell infiltration. Subsequently, further exploration was conducted on the clusters involving cuproptosis-related genes (CRGs) and the corresponding immune cell infiltration, and the WGCNA algorithm was applied to explore the cluster-specific differentially expressed genes. Finally, the best machine prediction model was selected for candidate hub cuproptosis-associated genes and the accuracy of predictive efficiency was verified by the salivary gland in an external dataset (GSE143153) and enzyme-linked immunosorbent assay. RESULT: Through a comparison of patients with pSS and controls, 7 CRGs and 4 types of immune cells were identified. Immune cell infiltration revealed significant immune heterogeneity in three cuproptosis-related molecular clusters in pSS. The random forest machine model showed the best discriminatory performance (area under the receiver operating characteristic curve (AUC) = 1.000) and built a predictive model based on 5 genes, which demonstrated satisfactory performance (AUC = 0.70) in the GSE143153 dataset. Based on serum samples, EED (AUC = 0.557), CBL (AUC = 0.635), and NFU1 (AUC = 0.655) showed lower expression levels in patients with pSS (p = 0.037, p = 0.000, p = 0.000, respectively). CONCLUSION: In this study, we systematically analyzed the association between pSS and cuproptosis, established a predictive model that screened for high-risk genes linked to the advancement of pSS, and explored the pathogenic mechanisms and novel therapeutic strategies for pSS, targeting EED, CBL and NFU1.

CircNOLC1 Promotes Colorectal Cancer Liver Metastasis by Interacting with AZGP1 and Sponging miR-212-5p to Regulate Reprogramming of the Oxidative Pentose Phosphate Pathway.

Liver metastasis is a common cause of death in progressive colorectal cancer patients, but the molecular mechanisms remain unclear. Here, it is reported that a conserved and oxidative pentose phosphate pathway-associated circular RNA, circNOLC1, plays a crucial role in colorectal cancer liver metastasis. It is found that circNOLC1 silencing reduces the oxidative pentose phosphate pathway-related intermediate metabolites and elevates NADP+ /NADPH ratio and intracellular ROS levels, thereby attenuating colorectal cancer cell proliferation, migration, and liver metastasis. circNOLC1 interacting with AZGP1 to activate mTOR/SREBP1 signaling, or sponging miR-212-5p to upregulate c-Met expression, both of which can further induce G6PD to activate oxidative pentose phosphate pathway in colorectal cancer liver metastasis. Moreover, circNOLC1 is regulated by the transcription factor YY1 and specifically stabilized HuR induces its parental gene mRNA expression. The associations between circNOLC1 and these signaling molecules are validated in primary CRC and corresponding liver metastasis tissues. These findings reveal that circNOLC1 interacting with AZGP1 and circNOLC1/miR-212-5p/c-Met axis plays a key role in oxidative pentose phosphate pathway-mediated colorectal cancer liver metastasis, which may provide a novel target for precision medicine of colorectal cancer.

Identification of immunological characteristics and cuproptosis-related molecular clusters in Rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by progressive articular damage, functional loss, and comorbidities. The relationship between cuproptosis, a form of programmed cell death, and RA remains unknown. Therefore, this study aimed to explore cuproptosis-related molecular clusters in RA. METHODS: Gene expression profiles of GSE93272 were downloaded from the Gene Expression Omnibus to identify the expression profiles of cuproptosis regulators and the immune infiltration characteristics of RA. The molecular clusters of cuproptosis-related genes and the related immune cell infiltration were explored. Cluster-specific differentially expressed genes were identified using the weighted gene co-expression network analysis. Further, an external dataset (GSE15573) was used, and an enzyme-linked immunosorbent assay was performed to validate the predictive efficiency. RESULTS: Thirteen cuproptosis-related genes and activated immune responses were identified between patients with RA and controls. Immune infiltration revealed significant immunological heterogeneity in the two cuproptosis-related molecular clusters in RA. Functional enrichment indicated that Cluster1 and Cluster2 were predominantly enriched in the toll-like receptor signalling pathway and regulation of autophagy, respectively. Further, the performance of FAM96A and CGRRF1 genes in the external validation dataset was observed to be relatively satisfactory (area under the receiver operating characteristic curve = 0.687 and 0.674, respectively). Based on our serum samples, FAM96A and CGRRF1 both exhibited higher expression levels in patients with RA (p = 0.001; p = 0.000). CONCLUSIONS: Our study systematically illustrated the involvement of cuproptosis in the progression of RA, and explored the pathogenic mechanisms and novel therapeutic strategies for RA, targeting FAM96A and CGRRF1.

Identification of a Stress-Sensitive Anorexigenic Neurocircuit From Medial Prefrontal Cortex to Lateral Hypothalamus.

BACKGROUND: A greater understanding of how the brain controls appetite is fundamental to developing new approaches for treating diseases characterized by dysfunctional feeding behavior, such as obesity and anorexia nervosa. METHODS: By modeling neural network dynamics related to homeostatic state and body mass index, we identified a novel pathway projecting from the medial prefrontal cortex (mPFC) to the lateral hypothalamus (LH) in humans (n = 53). We then assessed the physiological role and dissected the function of this mPFC-LH circuit in mice. RESULTS: In vivo recordings of population calcium activity revealed that this glutamatergic mPFC-LH pathway is activated in response to acute stressors and inhibited during food consumption, suggesting a role in stress-related control over food intake. Consistent with this role, inhibition of this circuit increased feeding and sucrose seeking during mild stressors, but not under nonstressful conditions. Finally, chemogenetic or optogenetic activation of the mPFC-LH pathway is sufficient to suppress food intake and sucrose seeking in mice. CONCLUSIONS: These studies identify a glutamatergic mPFC-LH circuit as a novel stress-sensitive anorexigenic neural pathway involved in the cortical control of food intake.

Monolithic 3D phase profile formation in glass for spatial and temporal control of optical waves.

Optical manufacturing technologies play a central role in modern science and engineering. Progress on both subtractive and additive fabrications is transforming the implementation of optical technologies. Despite the recent advances, modern fabrication still faces challenges in the accuracy, dimension, durability, intensity, and wavelength range. Here we present a direct monolithic 3D phase profile formation in glass and demonstrate its versatile applications for high-accuracy spatial and temporal control of optical waves in the extreme wavelength and intensity domains, direct fabrication of microlenses, and in situ aberration correction for refractive components. These advances and flexibilities will provide a new dimension for high-performance optical design and manufacture and enable novel applications in a broad range of disciplines.

Proteomic Analysis of Human Follicular Fluid Reveals the Pharmacological Mechanisms of the Chinese Patent Drug Kunling Pill for Improving Diminished Ovarian Reserve.

Objective: To explore the pharmacological mechanism of a Chinese patent drug (Kunling Pill (KLP)) on improving diminished ovarian reserve based on proteomic analysis. Methods: A total of 18 patients divided into three groups (the normal ovary reserve (NOR), diminished ovary reserve (DOR), and KLP groups) undergoing assisted reproductive technology by standard ovarian stimulation protocols were recruited to collect follicular fluid. Data-independent acquisition mass spectrometry was used to identify differentially expressed proteins by nano-LC-MS/MS. Bioinformatic analysis was conducted to predict the functions and pathways of the identified proteins. Clinical, hormonal, and biochemical parameters were also analyzed in the three groups. Results: A total of 144 differentially expressed proteins were screened out, including 56 proteins that were downregulated and 88 proteins that were upregulated in the DOR group compared with the NOR group, while 27 proteins were shared in the KLP-treated group. Among them, 10 proteins were upregulated and 17 proteins were downregulated in the KLP-treated group compared with the DOR group. The most enriched biological processes accounted for 28 GO terms, including cellular process, biological regulation, metabolic process, and regulation of biological process. Significant pathways were associated with fatty acid elongation, fatty acid degradation, fatty acid metabolism, nicotinate and nicotinamide metabolism, and valine, leucine, and isoleucine degradation. Conclusion: Our study provides the proteome profiles of human follicular fluid from DOR patients treated by KLP. Functional analyses of proteome datasets revealed that core proteins (SAA1, MIF, and PRDX5) and related pathways (fatty acid metabolism, nicotinate and nicotinamide metabolism, and tyrosine and purine metabolism) are possible pharmacological mechanisms through which KLP improves DOR. Therefore, these findings may help better understand the complex mechanisms through which DOR is treated by the Chinese patent drug KLP.

LncRNA SNHG6 promotes glycolysis reprogramming in hepatocellular carcinoma by stabilizing the BOP1 protein.

Metabolic reprogramming is an important feature in tumor progression. Long noncoding RNA's (lncRNA) small nucleolar RNA host gene 6 (SNHG6) acts as a proto-oncogene in hepatocellular carcinoma (HCC) but its role in glycolysis is mostly unknown. The role of SNHG6 and Block of proliferation 1 (BOP1) on glycolysis is assessed by glucose uptake, lactate production, oxygen consumptive rate (OCR) and extracellular acidification rate (ECAR) and glycolytic enzyme levels. The regulatory effect of SNHG6 on BOP1 protein was confirmed by Western blotting, MS2 pull-down, RNA pull-down, and RIP assay. SNHG6 and BOP1 levels were increased in HCC tissues and cells. SNHG6 and BOP1 were prognostic factors in HCC patients and significantly correlated to TP53 mutant and tumor grade. SNHG6 promoted proliferation, inhibited apoptosis, enhanced glucose uptake and lactate production, decreased OCR, and increased ECAR in HCC cell lines. SNHG6 could bind the BOP1 protein and enhance its stability. BOP1 overexpression rescued the change of proliferation, apoptosis, and glycolysis in HCCLM3 and SMMC-7721 cells. Our data indicate that SNHG6 accelerates proliferation and glycolysis and inhibits the apoptosis of HCC cell lines by binding the BOP1 protein and enhancing its stability. Both SNHG6 and BOP1 are promising prognostic and therapeutic markers in HCC.

High resolution ultrasonic neural modulation observed via in vivo two-photon calcium imaging.

Neural modulation plays a major role in delineating the circuit mechanisms and serves as the cornerstone of neural interface technologies. Among the various modulation mechanisms, ultrasound enables noninvasive label-free deep access to mammalian brain tissue. To date, most if not all ultrasonic neural modulation implementations are based on ∼1 MHz carrier frequency. The long acoustic wavelength results in a spatially coarse modulation zone, often spanning over multiple function regions. The modulation of one function region is inevitably linked with the modulation of its neighboring regions. Moreover, the lack of in vivo cellular resolution cell-type-specific recording capabilities in most studies prevents the revealing of the genuine cellular response to ultrasound. To significantly increase the spatial resolution, we explored the application of high-frequency ultrasound. To investigate the neuronal response at cellular resolutions, we developed a dual-modality system combining in vivo two-photon calcium imaging and focused ultrasound modulation. The studies show that the ∼30 MHz ultrasound can suppress the neuronal activity in awake mice at 100-µm scale spatial resolutions, paving the way for high-resolution ultrasonic neural modulation. The dual-modality in vivo system validated through this study will serve as a general platform for studying the dynamics of various cell types in response to ultrasound.

Clear optically matched panoramic access channel technique (COMPACT) for large-volume deep brain imaging.

To understand neural circuit mechanisms underlying behavior, it is crucial to observe the dynamics of neuronal structure and function in different regions of the brain. Since current noninvasive imaging technologies allow cellular-resolution imaging of neurons only within ~1 mm below the cortical surface, the majority of mouse brain tissue remains inaccessible. While miniature optical imaging probes allow access to deep brain regions, cellular-resolution imaging is typically restricted to a small tissue volume. To increase the tissue access volume, we developed a clear optically matched panoramic access channel technique (COMPACT). With probe dimensions comparable to those of common gradient-index lenses, COMPACT enables a two to three orders of magnitude greater tissue access volume. We demonstrated the capabilities of COMPACT by multiregional calcium imaging in mice during sleep. We believe that large-volume in vivo imaging with COMPACT will be valuable to a variety of deep tissue imaging applications.

Metabolomics of Gastric Cancer.

Gastric cancer is the fourth most common malignancy worldwide and the third leading cause of cancer deaths. Recent metabolomics research has advanced our understanding of the relationship between metabolic reprogramming and gastric cancer progression and led to the discovery of metabolic targets for potential clinical applications and therapeutic interventions. As a powerful tool for metabolite and flux measurement, metabolomics not only allows a comprehensive analysis of metabolites and related metabolic pathways but also can investigate the interactions between gastric cancer cells and tumour microenvironment as well as between the cancer cells and gastric microbiome. In this chapter, we aim to summarize the recent advances in gastric cancer metabolism and discuss the applications of metabolomics for target discovery in gastric cancer.

Long-range remote focusing by image-plane aberration correction.

Laser scanning plays an important role in a broad range of applications. Toward 3D aberration-free scanning, a remote focusing technique has been developed for high-speed imaging applications. However, the implementation of remote focusing often suffers from a limited axial scan range as a result of unknown aberration. Through simple analysis, we show that the sample-to-image path length conservation is crucially important to the remote focusing performance. To enhance the axial scan range, we propose and demonstrate an image-plane aberration correction method. Using a static correction, we can effectively improve the focus quality over a large defocusing range. Experimentally, we achieved ∼three times greater defocusing range than that of conventional methods. This technique can broadly benefit the implementations of high-speed large-volume 3D imaging.

Classification of Schizophrenia by Functional Connectivity Strength Using Functional Near Infrared Spectroscopy.

Functional near-infrared spectroscopy (fNIRS) has been widely employed in the objective diagnosis of patients with schizophrenia during a verbal fluency task (VFT). Most of the available methods depended on the time-domain features extracted from the data of single or multiple channels. The present study proposed an alternative method based on the functional connectivity strength (FCS) derived from an individual channel. The data measured 100 patients with schizophrenia and 100 healthy controls, who were used to train the classifiers and to evaluate their performance. Different classifiers were evaluated, and support machine vector achieved the best performance. In order to reduce the dimensional complexity of the feature domain, principal component analysis (PCA) was applied. The classification results by using an individual channel, a combination of several channels, and 52 ensemble channels with and without the dimensional reduced technique were compared. It provided a new approach to identify schizophrenia, improving the objective diagnosis of this mental disorder. FCS from three channels on the medial prefrontal and left ventrolateral prefrontal cortices rendered accuracy as high as 84.67%, sensitivity at 92.00%, and specificity at 70%. The neurophysiological significance of the change at these regions was consistence with the major syndromes of schizophrenia.

Enhanced Immune Responses Against Japanese Encephalitis Virus Infection Using Japanese Encephalitis Live-Attenuated Virus Adjuvanted with Montanide GEL 01 ST in Mice.

Japanese encephalitis virus (JEV) is one of the major causes of acute encephalitis in human and animal. To prevent JEV infection, an effective live-attenuated vaccine is needed. In the article, JEV attenuated strain, SCYA201201 of GI genotype, which was mixed with 10% concentrate GEL 01 ST adjuvant (Montanide™ GEL 01 ST), was selected for a vaccine candidate and its immunogenicity was evaluated in mice. Our results showed that JEV mixed with GEL 01 ST elicited production of both IgG1 and IgG2a antibodies, and enhanced virus-specific crossprotective intergenotypic response in mice. Proliferation of splenocytes was observed in all immunized groups and a relatively higher proliferation activity was detected in JEV mixed with GEL 01 ST group (p < 0.05). In the JEV + 10% GEL 01 ST vaccinated group, the proportion of CD4+ T cells in spleen was significantly higher than that of control group (p < 0.05), and the yields of interleukin (IL)-2, IL-4, and interferon-γ in the splenocyte supernatant were also significantly higher than that of control group (p < 0.05). Moreover, complete protection was provided after JEV challenge in mice in JEV mixed with GEL 01 ST group, and early immunity was detected in those mice immunized with JEV mixed with GEL 01 ST. These findings confirm that GEL 01 ST can enhance JEV live-attenuated immunogenicity, and JEV +10% GEL 01 ST used as vaccine candidates provide protection against JEV infection in a mouse model, which could be used as potential vaccine candidates in pig.

SPINT2 is hypermethylated in both IDH1 mutated and wild-type glioblastomas, and exerts tumor suppression via reduction of c-Met activation.

PURPOSE: Both IDH1-mutated and wild-type gliomas abundantly display aberrant CpG island hypermethylation. However, the potential role of hypermethylation in promoting gliomas, especially the most aggressive form, glioblastoma (GBM), remains poorly understood. METHODS: We analyzed RRBS-generated methylation profiles for 11 IDH1WT gliomas (including 7 GBMs), 24 IDH1MUT gliomas (including 6 GBMs), and 5 normal brain samples and employed TCGA GBM methylation profiles as a validation set. Upon classification of differentially methylated CpG islands by IDH1 status, we used integrated analysis of methylation and gene expression to identify SPINT2 as a top cancer related gene. To explore functional consequences of SPINT2 methylation in GBM, we validated SPINT2 methylation status using targeted bisulfite sequencing in a large cohort of GBM samples. We assessed DNA methylation-mediated SPINT2 gene regulation using 5-aza-2'-deoxycytidine treatment, DNMT1 knockdown and luciferase reporter assays. We conducted functional analyses of SPINT2 in GBM cell lines in vitro and in vivo. RESULTS: We identified SPINT2 as a candidate tumor-suppressor gene within a group of CpG islands (designated GT-CMG) that are hypermethylated in both IDH1MUT and IDH1WT gliomas but not in normal brain. We established that SPINT2 downregulation results from promoter hypermethylation, and that restoration of SPINT2 expression reduces c-Met activation and tumorigenic properties of GBM cells. CONCLUSIONS: We defined a previously under-recognized group of coordinately methylated CpG islands common to both IDH1WT and IDH1MUT gliomas (GT-CMG). Within GT-CMG, we identified SPINT2 as a top cancer-related candidate and demonstrated that SPINT2 suppressed GBM via down-regulation of c-Met activation.

Procurement and Storage of Surgical Biospecimens.

A biobank is an important nexus between clinical and research aspects of pathology. The collection and storage of high quality surgical samples is essential for diagnosis post-surgery, and can also be used to create vaccines, identify therapeutic targets or establish eligibility of cancer patients in a clinical trial. Therefore, personnel handling surgical tissues should follow standard operating procedures (SOP) to maximize efficiency and preserve tissue quality. This chapter is intended to familiarize novice biobank personnel with the issues associated with different steps of surgical tissue collection including patient consent, sample collection, tissue storage, quality control, and distribution.

An Introduction to the Performance of Immunohistochemistry.

Immunohistochemistry (IHC) is a powerful technique that exploits the specific binding between an antibody and antigen to detect and localize specific antigens in cells and tissue, most commonly detected and examined with the light microscope. A standard tool in many fields in the research setting, IHC has become an essential ancillary technique in clinical diagnostics in anatomic pathology (Lin F, Chen Z. Arch Pathol Lab Med 138:1564-1577, 2014) with the advent of antigen retrieval methods allowing it to be performed conveniently on formalin fixed paraffin embedded (FFPE) tissue (Taylor CR, Shi S-R, Barr NJ. Techniques of immunohistochemistry: principles, pitfalls, and standardization. In: Dabbs DJ (ed) Diagnostic immunohistochemistry: theranostic and genomic applications, 3rd edn. Saunders, Philadelphia, 2010; Shi SR, Key ME, Kalra KL. J Histochem Cytochem 39:741-748, 1991) and automated methods for high volume processing with reproducibility (Prichard J, Hicks D, Hammond E. Automated immunohistochemistry overview. In: Fan L, Jeffrey P (eds) Handbook of practical immunohistochemistry: frequently asked questions, 2nd edn. Springer, New York, 2015). IHC is frequently utilized to assist in the classification of neoplasms, determination of a metastatic tumor's site of origin and detection of tiny foci of tumor cells inconspicuous on routine hematoxylin and eosin (H&E) staining. Furthermore, it is increasingly being used to provide predictive and prognostic information, such as in testing for HER2 amplification in breast cancer (Wolff AC, Hammond MEH, Hicks DG et al. Arch Pathol Lab Med 138:241-256, 2014) in addition to serving as surrogate markers for molecular alterations in neoplasms, including IDH1 and ATRX mutations in brain tumors (Appin CL, Brat DJ. Mol Aspects Med. 45:87-96, 2015). In this chapter we describe the basic methods of immunohistochemical staining which has become an essential tool in the daily practice of anatomic pathology worldwide.

Topological Arrangement of Cardiac Fibroblasts Regulates Cellular Plasticity.

RATIONALE: Cardiac fibroblasts do not form a syncytium but reside in the interstitium between myocytes. This topological relationship between fibroblasts and myocytes is maintained throughout postnatal life until an acute myocardial injury occurs, when fibroblasts are recruited to, proliferate and aggregate in the region of myocyte necrosis. The accumulation or aggregation of fibroblasts in the area of injury thus represents a unique event in the life cycle of the fibroblast, but little is known about how changes in the topological arrangement of fibroblasts after cardiac injury affect fibroblast function. OBJECTIVE: The objective of the study was to investigate how changes in topological states of cardiac fibroblasts (such as after cardiac injury) affect cellular phenotype. METHODS AND RESULTS: Using 2 and 3-dimensional (2D versus 3D) culture conditions, we show that simple aggregation of cardiac fibroblasts is sufficient by itself to induce genome-wide changes in gene expression and chromatin remodeling. Remarkably, gene expression changes are reversible after the transition from a 3D back to 2D state demonstrating a topological regulation of cellular plasticity. Genes induced by fibroblast aggregation are strongly associated and predictive of adverse cardiac outcomes and remodeling in mouse models of cardiac hypertrophy and failure. Using solvent-based tissue clearing techniques to create optically transparent cardiac scar tissue, we show that fibroblasts in the region of dense scar tissue express markers that are induced by fibroblasts in the 3D conformation. Finally, using live cell interferometry, a quantitative phase microscopy technique to detect absolute changes in single cell biomass, we demonstrate that conditioned medium collected from fibroblasts in 3D conformation compared with that from a 2D state significantly increases cardiomyocyte cell hypertrophy. CONCLUSIONS: Taken together, these findings demonstrate that simple topological changes in cardiac fibroblast organization are sufficient to induce chromatin remodeling and global changes in gene expression with potential functional consequences for the healing heart.

Early experience with formalin-fixed paraffin-embedded (FFPE) based commercial clinical genomic profiling of gliomas-robust and informative with caveats.

BACKGROUND: Commercial targeted genomic profiling with next generation sequencing using formalin-fixed paraffin embedded (FFPE) tissue has recently entered into clinical use for diagnosis and for the guiding of therapy. However, there is limited independent data regarding the accuracy or robustness of commercial genomic profiling in gliomas. METHODS: As part of patient care, FFPE samples of gliomas from 71 patients were submitted for targeted genomic profiling to one commonly used commercial vendor, Foundation Medicine. Genomic alterations were determined for the following grades or groups of gliomas; Grade I/II, Grade III, primary glioblastomas (GBMs), recurrent primary GBMs, and secondary GBMs. In addition, FFPE samples from the same patients were independently assessed with conventional methods such as immunohistochemistry (IHC), Quantitative real-time PCR (qRT-PCR), or Fluorescence in situ hybridization (FISH) for three genetic alterations: IDH1 mutations, EGFR amplification, and EGFRvIII expression. RESULTS: A total of 100 altered genes were detected by the aforementioned targeted genomic profiling assay. The number of different genomic alterations was significantly different between the five groups of gliomas and consistent with the literature. CDKN2A/B, TP53, and TERT were the most common genomic alterations seen in primary GBMs, whereas IDH1, TP53, and PIK3CA were the most common in secondary GBMs. Targeted genomic profiling demonstrated 92.3%-100% concordance with conventional methods. The targeted genomic profiling report provided an average of 5.5 drugs, and listed an average of 8.4 clinical trials for the 71 glioma patients studied but only a third of the trials were appropriate for glioma patients. CONCLUSIONS: In this limited comparison study, this commercial next generation sequencing based-targeted genomic profiling showed a high concordance rate with conventional methods for the 3 genetic alterations and identified mutations expected for the type of glioma. While it may not be feasible to exhaustively independently validate a commercial genomic profiling assay, examination of a few markers provides some reassurance of its robustness. While potential targeted drugs are recommended based on genetic alterations, to date most targeted therapies have failed in glioblasomas so the usefulness of such recommendations will increase with development of novel and efficacious drugs.