Profiling Kinase Activities for Precision Oncology in Diffuse Gastric Cancer.

Gastric cancer (GC) remains a leading cause of cancer-related mortality globally. This is due to the fact that majority of the cases of GC are diagnosed at an advanced stage when the treatment options are limited and prognosis is poor. The diffuse subtype of gastric cancer (DGC) under Lauren's classification is more aggressive and usually occurs in younger patients than the intestinal subtype. The concept of personalized medicine is leading to the identification of multiple biomarkers in a large variety of cancers using different combinations of omics technologies. Proteomic changes including post-translational modifications are crucial in oncogenesis. We analyzed the phosphoproteome of DGC by using paired fresh frozen tumor and adjacent normal tissue from five patients diagnosed with DGC. We found proteins involved in the epithelial-to-mesenchymal transition (EMT), c-MYC pathway, and semaphorin pathways to be differentially phosphorylated in DGC tissues. We identified three kinases, namely, bromodomain adjacent to the zinc finger domain 1B (BAZ1B), WNK lysine-deficient protein kinase 1 (WNK1), and myosin light-chain kinase (MLCK) to be hyperphosphorylated, and one kinase, AP2-associated protein kinase 1 (AAK1), to be hypophosphorylated. LMNA hyperphosphorylation at serine 392 (S392) was demonstrated in DGC using immunohistochemistry. Importantly, we have detected heparin-binding growth factor (HDGF), heat shock protein 90 (HSP90), and FTH1 as potential therapeutic targets in DGC, as drugs targeting these proteins are currently under investigation in clinical trials. Although these new findings need to be replicated in larger study samples, they advance our understanding of signaling alterations in DGC, which could lead to potentially novel actionable targets in GC.

In Silico Analysis of Ion Channels and Their Correlation with Epithelial to Mesenchymal Transition in Breast Cancer.

Uncontrolled growth of breast cells due to altered gene expression is a key feature of breast cancer. Alterations in the expression of ion channels lead to variations in cellular activities, thus contributing to attributes of cancer hallmarks. Changes in the expression levels of ion channels were observed as a consequence of EMT. Additionally, ion channels were reported in the activation of EMT and maintenance of a mesenchymal phenotype. Here, to identify altered ion channels in breast cancer patients, differential gene expression and weighted gene co-expression network analyses were performed using transcriptomic data. Protein-protein interactions network analysis was carried out to determine the ion channels interacting with hub EMT-related genes in breast cancer. Thirty-two ion channels were found interacting with twenty-six hub EMT-related genes. The identified ion channels were further correlated with EMT scores, indicating mesenchymal phenotype. Further, the pathway map was generated to represent a snapshot of deregulated cellular processes by altered ion channels and EMT-related genes. Kaplan-Meier five-year survival analysis and Cox regressions indicated the expression of CACNA1B, ANO6, TRPV3, VDAC1 and VDAC2 to be potentially associated with poor survival. Deregulated ion channels correlate with EMT-related genes and have a crucial role in breast cancer-associated tumorigenesis. Most likely, they are potential candidates for the determination of prognosis in patients with breast cancer.

Metallothionein immunohistochemistry has high sensitivity and specificity for detection of Wilson disease.

Diagnosis of Wilson disease (WD) can be difficult because of its protean clinical presentations, but early diagnosis is important because effective treatment is available and can prevent disease progression. Similarly, diagnosis of WD on liver biopsy specimens is difficult due to the wide range of histologic appearances. A stain that could help identify WD patients would be of great value. The goal of this study was to use mass spectrometry-based proteomics to identify potential proteins that are differentially expressed in WD compared to controls, and could serve as potential immunohistochemical markers for screening. Several proteins were differentially expressed in WD and immunohistochemical stains for two (metallothionein (MT) and cytochrome C oxidase copper chaperone (COX17)) were tested and compared to other methods of diagnosis in WD including copper staining and quantitative copper assays. We found diffuse metallothionein immunoreactivity in all liver specimens from patients with WD (n = 20); the intensity of the staining was moderate to strong. This staining pattern was distinct from that seen in specimens from the control groups (none of which showed strong, diffuse staining), which included diseases that may be in the clinical or histologic differential of WD (steatohepatitis (n = 51), chronic viral hepatitis (n = 40), autoimmune hepatitis (n = 50), chronic biliary tract disease (n = 42), and normal liver (n = 20)). COX17 immunostain showed no significant difference in expression between the WD and control groups. MT had higher sensitivity than rhodanine for diagnosis of WD. While the quantitative liver copper assays also had high sensitivity, they require more tissue, have a higher cost, longer turnaround time, and are less widely available than an immunohistochemical stain. We conclude that MT IHC is a sensitive immunohistochemical stain for the diagnosis of WD that could be widely deployed as a screening tool for liver biopsies in which WD is in the clinical or histologic differential diagnosis.

Proteomic Signatures of Diffuse and Intestinal Subtypes of Gastric Cancer.

Gastric cancer is a leading cause of death from cancer globally. Gastric cancer is classified into intestinal, diffuse and indeterminate subtypes based on histology according to the Laurén classification. The intestinal and diffuse subtypes, although different in histology, demographics and outcomes, are still treated in the same fashion. This study was designed to discover proteomic signatures of diffuse and intestinal subtypes. Mass spectrometry-based proteomics using tandem mass tags (TMT)-based multiplexed analysis was used to identify proteins in tumor tissues from patients with diffuse or intestinal gastric cancer with adjacent normal tissue control. A total of 7448 or 4846 proteins were identified from intestinal or diffuse subtype, respectively. This quantitative mass spectrometric analysis defined a proteomic signature of differential expression across the two subtypes, which included gremlin1 (GREM1), bcl-2-associated athanogene 2 (BAG2), olfactomedin 4 (OLFM4), thyroid hormone receptor interacting protein 6 (TRIP6) and melanoma-associated antigen 9 (MAGE-A9) proteins. Although GREM1, BAG2, OLFM4, TRIP6 and MAGE-A9 have all been previously implicated in tumor progression and metastasis, they have not been linked to intestinal or diffuse subtypes of gastric cancer. Using immunohistochemical labelling of a tissue microarray comprising of 124 cases of gastric cancer, we validated the proteomic signature obtained by mass spectrometry in the discovery cohort. Our findings should help investigate the pathogenesis of these gastric cancer subtypes and potentially lead to strategies for early diagnosis and treatment.

Olive leaf-derived PPAR agonist complex induces collagen IV synthesis in human skin models.

INTRODUCTION: Peroxisome proliferator-activated receptor (PPAR) agonists are known to modulate the synthesis of dermal lipids and proteins including collagens. Olive (Olea europaea) leaves have been reported to contain PPAR-binding ligands. Collagen IV, a major dermal-epidermal junction (DEJ) protein, degrades with both age and disease. Here, we report the formulation of a novel multi-ligand complex, Linefade, and its effects on collagen IV synthesis. METHODS: Linefade prepared from the leaves of Olea europaea contains 2% w/w plant extract solids dissolved in a mixture of glyceryl monoricinoleate and dimethyl isosorbide. In silico docking was performed with PPAR-α (PDB ID: 2P54). Linefade was evaluated for PPAR-α-dependent transcription in a luciferase reporter assay system. Cell viability and collagen IV levels in human dermal fibroblast cultures were measured using the MTT method and ELISA assay, respectively. Transcriptome analysis was conducted on a full-thickness reconstituted human skin (EpiDermFT) model. Ex vivo cell viability and collagen IV immunostaining were performed on human skin explants. RESULTS: In silico docking model of the major constituents (oleanolic acid and glyceryl monoricinoleate) produced a co-binding affinity of -6.7 Kcal/mole. Linefade significantly increased PPAR-α transcriptional activity in CHO cells and collagen IV synthesis in adult human dermal fibroblasts. Transcriptome analysis revealed that 1% Linefade modulated the expression of 280 genes with some related to epidermal differentiation, DEJ, PPAR, Nrf2 and retinoid pathways. An ex vivo human explant study showed that 1% Linefade, delivered via a triglycerides excipient, increased collagen IV levels along the dermal-epidermal junction by 52%. CONCLUSION: In silico modelling and in vitro and ex vivo analyses confirmed Linefade-mediated activation of PPAR-α and stimulation of collagen IV synthesis.

INTRODUCTION: Les agonistes du récepteur activé par les proliférateurs de peroxysomes (PPAR) sont connus pour moduler la synthèse des lipides cutanés et des protéines du derme, y compris des collagènes. Il a été signalé que les feuilles d'olivier (Olea europaea) contiennent des ligands de liaison aux PPAR. Le collagène IV, une protéine majeure de la jonction dermo-épidermique (DEJ), se dégrade avec l'âge et la maladie. Nous rapportons ici la formulation d'un nouveau complexe multi ligand, Linefade, et ses effets sur la synthèse du collagène IV. MÉTHODES: Le complexe Linefade préparé à partir des feuilles d'Olea europaea contient 2 % p/p de solides d'extraits végétaux dissous dans un mélange de monoricinoléate de glycéryle et d'isosorbide de diméthyle. Un docking in silico a été réalisé avec PPAR-α (PDB ID : 2P54). Linefade a été évalué pour la transcription dépendante du PPAR-α dans un système de test rapporteur à la luciférase. La viabilité cellulaire et les niveaux de collagène IV dans les cultures de fibroblastes dermiques humains ont été respectivement mesurés en utilisant la méthode MTT et le test ELISA. L'analyse du transcriptome a été réalisée sur un modèle de peau humaine reconstitué sur toute son épaisseur (EpiDermFT). La viabilité cellulaire ex vivo et l'immunomarquage du collagène IV ont été réalisés sur des explants de peau humaine. RÉSULTATS: Le modèle de docking in silico des principaux constituants (acide oléanolique et monoricinoléate de glycéryle) a produit une affinité de liaison conjointe de -6,7 Kcal/mole. Linefade a augmenté de manière significative l'activité transcriptionnelle du PPAR-α dans les cellules CHO et la synthèse du collagène IV dans les fibroblastes dermiques humains chez les personnes adultes. L'analyse du transcriptome a révélé que 1% de Linefade modulait l'expression de 280 gènes dont certains étaient liés à la différenciation épidermique, à la DEJ, au PPAR, à la voie Nrf2 et aux voies rétinoïdes. Une étude ex vivo sur des explants humains a montré que 1% de Linefade, délivré via un excipient de triglycérides, augmentait de 52% les niveaux de collagène IV le long de la jonction dermo-épidermique. CONCLUSION: La modélisation in silico et les analyses in vitro et ex vivo ont confirmé l'activation du PPAR-- α et la stimulation de la synthèse du collagène IV par Linefade.

Acute Kidney Injury in Severe COVID-19 Has Similarities to Sepsis-Associated Kidney Injury: A Multi-Omics Study.

OBJECTIVE: To compare coronavirus disease 2019 (COVID-19) acute kidney injury (AKI) to sepsis-AKI (S-AKI). The morphology and transcriptomic and proteomic characteristics of autopsy kidneys were analyzed. PATIENTS AND METHODS: Individuals 18 years of age and older who died from COVID-19 and had an autopsy performed at Mayo Clinic between April 2020 to October 2020 were included. Morphological evaluation of the kidneys of 17 individuals with COVID-19 was performed. In a subset of seven COVID-19 cases with postmortem interval of less than or equal to 20 hours, ultrastructural and molecular characteristics (targeted transcriptome and proteomics analyses of tubulointerstitium) were evaluated. Molecular characteristics were compared with archived cases of S-AKI and nonsepsis causes of AKI. RESULTS: The spectrum of COVID-19 renal pathology included macrophage-dominant microvascular inflammation (glomerulitis and peritubular capillaritis), vascular dysfunction (peritubular capillary congestion and endothelial injury), and tubular injury with ultrastructural evidence of mitochondrial damage. Investigation of the spatial architecture using a novel imaging mass cytometry revealed enrichment of CD3+CD4+ T cells in close proximity to antigen-presenting cells, and macrophage-enriched glomerular and interstitial infiltrates, suggesting an innate and adaptive immune tissue response. Coronavirus disease 2019 AKI and S-AKI, as compared to nonseptic AKI, had an enrichment of transcriptional pathways involved in inflammation (apoptosis, autophagy, major histocompatibility complex class I and II, and type 1 T helper cell differentiation). Proteomic pathway analysis showed that COVID-19 AKI and to a lesser extent S-AKI were enriched in necroptosis and sirtuin-signaling pathways, both involved in regulatory response to inflammation. Upregulation of the ceramide-signaling pathway and downregulation of oxidative phosphorylation in COVID-19 AKI were noted. CONCLUSION: This data highlights the similarities between S-AKI and COVID-19 AKI and suggests that mitochondrial dysfunction may play a pivotal role in COVID-19 AKI. This data may allow the development of novel diagnostic and therapeutic targets.

Tyrosine Phosphoproteomics of Patient-Derived Xenografts Reveals Ephrin Type-B Receptor 4 Tyrosine Kinase as a Therapeutic Target in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma is a recalcitrant tumor with minimal response to conventional chemotherapeutic approaches. Oncogenic signaling by activated tyrosine kinases has been implicated in cancers resulting in activation of diverse effector signaling pathways. Thus, the discovery of aberrantly activated tyrosine kinases is of great interest in developing novel therapeutic strategies in the treatment and management of pancreatic cancer. Patient-derived tumor xenografts (PDXs) in mice serve as potentially valuable preclinical models as they maintain the histological and molecular heterogeneity of the original human tumor. Here, we employed high-resolution mass spectrometry combined with immunoaffinity purification using anti-phosphotyrosine antibodies to profile tyrosine phosphoproteome across 13 pancreatic ductal adenocarcinoma PDX models. This analysis resulted in the identification of 1199 tyrosine-phosphorylated sites mapping to 704 proteins. The mass spectrometric analysis revealed widespread and heterogeneous activation of both receptor and non-receptor tyrosine kinases. Preclinical studies confirmed ephrin type-B receptor 4 (EphB4) as a potential therapeutic target based on the efficacy of human serum albumin-conjugated soluble EphB4 in mice bearing orthotopic xenografts. Immunohistochemistry-based validation using tissue microarrays from 346 patients with PDAC showed significant expression of EphB4 in >70% of patients. In summary, we present a comprehensive landscape of tyrosine phosphoproteome with EphB4 as a promising therapeutic target in pancreatic ductal adenocarcinoma.

A mass spectrometry-based targeted assay for detection of SARS-CoV-2 antigen from clinical specimens.

BACKGROUND: The COVID-19 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has overwhelmed health systems worldwide and highlighted limitations of diagnostic testing. Several types of diagnostic tests including RT-PCR-based assays and antigen detection by lateral flow assays, each with their own strengths and weaknesses, have been developed and deployed in a short time. METHODS: Here, we describe an immunoaffinity purification approach followed a by high resolution mass spectrometry-based targeted qualitative assay capable of detecting SARS-CoV-2 viral antigen from nasopharyngeal swab samples. Based on our discovery experiments using purified virus, recombinant viral protein and nasopharyngeal swab samples from COVID-19 positive patients, nucleocapsid protein was selected as a target antigen. We then developed an automated antibody capture-based workflow coupled to targeted high-field asymmetric waveform ion mobility spectrometry (FAIMS) - parallel reaction monitoring (PRM) assay on an Orbitrap Exploris 480 mass spectrometer. An ensemble machine learning-based model for determining COVID-19 positive samples was developed using fragment ion intensities from the PRM data. FINDINGS: The optimized targeted assay, which was used to analyze 88 positive and 88 negative nasopharyngeal swab samples for validation, resulted in 98% (95% CI = 0.922-0.997) (86/88) sensitivity and 100% (95% CI = 0.958-1.000) (88/88) specificity using RT-PCR-based molecular testing as the reference method. INTERPRETATION: Our results demonstrate that direct detection of infectious agents from clinical samples by tandem mass spectrometry-based assays have potential to be deployed as diagnostic assays in clinical laboratories, which has hitherto been limited to analysis of pure microbial cultures. FUNDING: This study was supported by DBT/Wellcome Trust India Alliance Margdarshi Fellowship grant IA/M/15/1/502023 awarded to AP and the generosity of Eric and Wendy Schmidt.

Mass Spectrometric Analysis of Urine from COVID-19 Patients for Detection of SARS-CoV-2 Viral Antigen and to Study Host Response.

SARS-CoV-2 infection has become a major public health burden and affects many organs including lungs, kidneys, the liver, and the brain. Although the virus is readily detected and diagnosed using nasopharyngeal swabs by reverse transcriptase polymerase chain reaction (RT-PCR), detection of its presence in body fluids is fraught with difficulties. A number of published studies have failed to detect viral RNA by RT-PCR methods in urine. Although microbial identification in clinical microbiology using mass spectrometry is undertaken after culture, here we undertook a mass spectrometry-based approach that employed an enrichment step to capture and detect SARS-CoV-2 nucleocapsid protein directly from urine of COVID-19 patients without any culture. We detected SARS-CoV-2 nucleocapsid protein-derived peptides from 13 out of 39 urine samples. Further, a subset of COVID-19 positive and COVID-19 negative urine samples validated by mass spectrometry were used for the quantitative proteomics analysis. Proteins with increased abundance in urine of SARS-CoV-2 positive individuals were enriched in the acute phase response, regulation of complement system, and immune response. Notably, a number of renal proteins such as podocin (NPHS2), an amino acid transporter (SLC36A2), and sodium/glucose cotransporter 5 (SLC5A10), which are intimately involved in normal kidney function, were decreased in the urine of COVID-19 patients. Overall, the detection of viral antigens in urine using mass spectrometry and alterations of the urinary proteome could provide insights into understanding the pathogenesis of COVID-19.

In silico strategies for probing novel DPP-IV inhibitors as anti-diabetic agents.

Identification of new DPP-IV inhibitors by integrating validated in silico approach is being presented herein. Novel hits were identified by combining pharmacophore and structure based virtual screening of ZINC and Knowledge Base in house database followed by ADME profiling, consensus docking studies. Six potential hits were identified and analysed for their synthetic accessibility score, novelty analysis and pan assay interference compounds filtration. Out of six, three hits viz., ZINC25060187, ZINC53746227 and KB-10 were analysed for stability studies using Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) and Molecular Dynamics (MD) simulation. The simulation studies of the identified hits revealed that these hits have good selectivity and stability in DPP-IV binding pocket. Important interactions with amino acids viz., Tyr547, Glu205 and Glu206 similar to co-crystallized ligand were also observed. One of the hits viz., KB-10 was synthesized and evaluated for its biological potential. The compound KB-10 showed good DPP-IV inhibition in both in vitro and in vivo studies with IC50: 22.69 µM. This study supports the fact that these techniques hold potential for efficient screening of compounds with unknown affinity for DPP-IV that could serve as candidates for therapeutic development.Communicated by Ramaswamy H. Sarma.

Spectrum of metastatic neoplasms of the brain: A clinicopathological study in a tertiary care cancer centre.

BACKGROUND: While brain metastases (BM) are the most common causes of neurologic disorders in patients with known systemic malignancies, they can often be the initial manifestations of an undetected primary elsewhere. BM are major causes of morbidity and mortality in cancer patients. AIMS: We describe a mixed population (data from both retrospective and prospective collection) having a BM from a solid tumor. We report the percentage distribution of the most frequent types of BM, confirming the data published in the literature. This paper may play a role in presenting the Southeast Asian reality compared with the Western countries. SETTING: A tertiary-care cancer centre. MATERIALS AND METHODS: Data for 4 years were retrieved from the records of the Department of Pathology of our institute. Hematolymphoid and meningeal tumors were excluded. Hematoxylin and eosin (H and E) stained slides were reviewed, and in cases with an unknown primary, immunohistochemistry (IHC) was advised. The panel of markers was chosen based on the histomorphology on H and E sections. IHC was done in cases with an unknown primary where paraffin blocks were available. RESULTS: Lung cancer was found to be the most common primary malignancy (n = 30; 48.4%) followed by breast cancer (n = 13; 21%), colorectal cancer (n = 6; 9.6%), and skin cancer (melanoma) [n = 3; 4.8%]. CONCLUSION: The incidence of BM from lung and breast cancer was similar to that seen in the Western studies. However, BM from colorectal cancer and melanoma show a higher and lower incidence, respectively, in comparison with the Western literature.

In silico identification of potential drug compound against Peroxisome proliferator-activated receptor-gamma by virtual screening and toxicity studies for the treatment of Diabetic Nephropathy.

Diabetic Nephropathy is a serious complication of diabetes mellitus. Current therapeutic strategies of Diabetic Nephropathy are based on control of modifiable risks like hypertension, glucose levels, and dyslipidemia. Peroxisome proliferator activated receptor-gamma (PPAR-γ) is implicated in several metabolic syndromes including Diabetic Nephropathy, besides obesity, insulin insensitivity, dislipidemia, inflammation, and hypertension. In the present study, virtual screening of 617 compounds from two different public databases was done against PPAR-γ with an objective to find a possible lead compound. Two softwares, PyRx and iGEMDOCK, were used to achieve the docking accuracy in order to avoid loss of candidate compounds. Rosiglitazone (used to treat Diabetic Nephropathy) was taken as the standard compound. A total of 30 compounds with good binding affinity with PPAR-γ were selected for further filtering, on the basis of absorption, distribution, metabolism, excretion, and toxicity (ADMET). The interaction profiling of these 30 compounds, showed a minimum of one and maximum of three interactions with reference to rosiglitazone (SER-289, HIS-449, HIS-323, TYR-473). The fulfilling of ADMET analysis criteria of 30 compounds led to the selection of four compounds (ZINC ID 00181552, 00276456, 00298314, 00448009). Molecular dynamics simulation of these lead compounds in complex with PPAR-γ revealed that three of the four compounds formed a stable complex in the ligand-binding pocket of PPAR-γ during 20-ns simulation. Hence, these three (ZINC ID 00181552, 00276456, 00298314) of the four compounds are potential candidates for experimental validation of biological activity against PPAR-γ in future drug discovery studies.

Development and evaluation of animal models for sex steroid deficient dry eye.

PURPOSE: The present study was conducted to develop animal models to mimic postmenopausal/androgen deficiency dry eye and to evaluate the expression of sex steroid receptors (NR3A1, NR3A2 and NR3C4) in the ocular tissues of the developed models. METHODS: The study was conducted in healthy Wistar rats of either sex weighing 180-250g. Bilateral ovariectomy was performed in female rats and oral finasteride (dose of 1.16mg/kg/day) challenge was given to both male and female rats. Along with time tear film stability was assessed by using cotton thread method and tear breakup time (TBUT). Dew point calculation was done using August-Roche-Magnus approximation during the tear assessments to correlate environmental factors affecting the tear function tests. At the end, animals were sacrificed and ocular tissues (lacrimal gland and cornea) were subjected for the quantification of the expression of NR3A1 (ER-α), NR3A2 (ER-ß) and androgen (NR3C4) receptors. RESULTS: The impact of ovariectomy caused a significant tear film deficiency from the 20th day onwards in all female rats. The ten day finasteride administration also showed a significant tear film deficiency in both male and female rats. However, subjecting 60days post ovariectomy rats to finasteride challenge did not show any further decrease in tear flow. Gene expression analysis also revealed a significant downregulation of sex steroid receptors in ocular tissues after ovariectomy and finasteride challenge. DISCUSSION AND CONCLUSION: From this study, it has been concluded that ovariectomized and finasteride treated antiandrogenic models produced a significant tear deficiency in the rats which can be explored for pharmacological screening of topical agents and understanding the disease process in postmenopausal and androgen deficiency dry eye disorders.