Novel combinatorial autophagy inhibition therapy for triple negative breast cancers.

BACKGROUND: Triple negative breast cancer (TNBC) has the worst prognosis among breast cancer subtypes. It is characterized by lack of estrogen, progesterone and human epidermal growth factor 2 receptors, and thus, have limited therapeutic options. Autophagy has been found to be correlated with poor prognosis and aggressive behaviour in TNBC. This study aimed to target autophagy in TNBC via a novel approach to inhibit TNBC progression. METHODS: Immunoblotting and confocal microscopy were carried out to examine the effect of tumor microenvironmental stressors on autophagy. Cellular proliferation and migration assays were used to test the effect of different autophagy inhibitors and standard chemotherapy alone or in combination. In vivo xenograft mouse model was utilized to assess the effect of autophagy inhibitors alone or in combination with Paclitaxel. High resolution mass spectrometry based proteomic analysis was performed to explore the mechanisms behind chemoresistance in TNBC. Lastly, immunohistochemistry was done to assess the correlation between autophagy related proteins and clinical characteristics in TNBC tissue specimens. RESULTS: Metabolic stressors were found to induce autophagy in TNBC cell lines. Autophagy initiation inhibitors, SAR405 and MRT68921, showed marked synergy in their anti-proliferative activity in both chemosensitive and chemoresistant TNBC cell models. Paradoxically, positive expression of autophagosome marker LC3 was shown to be associated with better overall survival of TNBC patients. CONCLUSION: In this study, a novel combination between different autophagy inhibitors was identified which inhibited tumor cell proliferation in both chemosensitive and chemoresistant TNBC cells and could result in development of a novel treatment modality against TNBC.

Novel serum protein biomarker panel for early diagnosis of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Late presentation of disease at the time of diagnosis is one of the major reasons for dismal prognostic outcomes for PDAC patients. Currently, there is a lack of clinical biomarkers, which can be used to diagnose PDAC patients at an early resectable stage. This study performed proteomic mass spectrometry to identify novel blood-based biomarkers for early diagnosis of PDAC. Serum specimens from 88 PDAC patients and 88 healthy controls (60 discovery cohort and 28 validation cohort) were analyzed using data independent acquisition high resolution mass spectrometry to identify candidate biomarker proteins. A total of 249 proteins were identified and quantified by the mass spectrometric analysis. Six proteins were markedly (>1.5 fold) and significantly (p < .05; q < 0.1) increased in PDAC patients compared to healthy controls in discovery cohort. Notably, four of these six proteins were significantly upregulated in an independent validation cohort. The top three upregulated proteins (i.e., Polymeric Immunoglobulin Receptor [PIGR], von Willebrand Factor [vWF], and Fibrinogen) were validated using enzyme linked immunosorbent assay, which led to selection of PIGR and vWF as a diagnostic biomarker panel for PDAC. The panel showed high ability to diagnose early stage (stage I and II) PDAC patients (area under the curve [AUC]: 0.8926), which was further improved after the addition of clinically used prognostic biomarker (Ca 19-9) to the panel (AUC: 0.9798). In conclusion, a novel serum protein biomarker panel for early diagnosis of PDAC was identified.

Proteome profiling of enriched membrane-associated proteins unraveled a novel sophorose and cello-oligosaccharide transporter in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized. RESULTS: To address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose. CONCLUSION: This study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.

Inhibition of autophagy initiation: A novel strategy for oral squamous cell carcinomas.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is one of the most common forms of oral cancer and is known to have poor prognostic outcomes. Autophagy is known to be associated with aggressive tumor biology of OSCC. Hence, this study aimed to develop a novel therapeutic strategy against OSCC by targeting the autophagic pathway. METHODS: Immunoblotting, and confocal microscopy were used to examine the effect of tumor microenvironmental stressors on the autophagy activity. Cellular proliferation and migration assays were performed to assess the anti-cancer activity of standard chemotherapy and autophagy initiation inhibitors, either alone or in combination. High resolution mass-spectrometry based proteomic analysis was utilized to understand the mechanisms behind chemoresistance in OSCC models. Finally, immunohistochemistry was performed to determine associations between autophagy markers and clinicopathological characteristics. RESULTS: Tumor microenvironmental stressors were shown to induce autophagy activity in OSCC cell lines. Novel combinations of chemotherapy and autophagy inhibitors as well as different classes of autophagy inhibitors were identified. Combination of MRT68921 and SAR405 demonstrated marked synergy in their anti-proliferative activity and also showed synergy with chemotherapy in chemoresistant OSCC cell models. Autophagy was identified as one of the key pathways involved in mediating chemoresistance in OSCC. Furthermore, TGM2 was identified as a key upstream regulator of chemoresistance in OSCC models. Finally, positive staining for autophagosome marker LC3 was shown to be associated with low histological grade OSCC. CONCLUSION: In conclusion, this study identified a combination of novel autophagy inhibitors which can potently inhibit proliferation of both chemosensitive as well as chemoresistant OSCC cells and could be developed as a novel therapy against advanced OSCC tumors.

Quantification of short-chain fatty acids in human stool samples by LC-MS/MS following derivatization with aniline analogues.

The gut microbiome produces a range of short chain fatty acids (SCFA) crucially linked with diet and nutrition, metabolism, gastrointestinal health and homeostasis. SCFA are primarily measured using gas or liquid chromatography-mass spectrometry (LC/MS) after undergoing chemical derivatization. Here we assess the merits of a derivatization protocol using aniline and two aniline analogues (3-phenoxyaniline and 4-(benzyloxy)aniline) for the targeted LC-MS/MS quantification of nine SCFA (acetic, propionic, butyric, valeric, caproic acid, isobutyric, isovaleric, 2-methylbutyric, and 2-ethylbutyric acid). Evaluation of product ion spectra and optimization of MS detection conditions, provided superior detection sensitivity for 3-phenoxyaniline and 4-(benzyloxy)aniline compared to aniline. We developed a facile SCFA derivatization method using 3-phenoxyaniline under mild reaction conditions which allows for the simultaneous quantification of these SCFA in human stool samples in under eleven minutes using multiple reaction monitoring LC-MS/MS. The method was successfully validated and demonstrates intra- and inter-day accuracy (88.5-103% and 86.0-109%) and precision (CV of 0.55-7.00% and 0.33-9.55%) with recoveries (80.1-87.2% for LLOQ, 88.5-93.0% for ULOQ) and carry-over of (2.68-17.9%). Selectivity, stability and matrix effects were also assessed and satisfied validation criteria. Method applicability was demonstrated by analysing SCFA profiles in DNA-stabilized human stool samples from newly diagnosed colorectal cancer patients prior to surgery. The development of this improved method and its compatibility to measure SCFAs from DNA-stabilized stool will facilitate studies investigating the gut microbiome in health and disease.

Proteome Analysis of Whole Blood Collected by Volumetric Absorptive Microsampling.

Proteomic biomarker discovery and analysis from human biofluids using liquid chromatography-mass spectrometry (LC-MS) is an area of intense biomedical research. There is a growing interest to analyze microsampled patient blood specimens as this is potentially more patient-friendly enabling at-home and bedside self-collection of small blood volumes. However, there are limited studies applying LC-MS proteomic analysis of whole blood as it is dominated by red blood cell proteins such as hemoglobin which suppresses the detection of other less abundant proteins. Volumetric absorptive microsampling (VAMS) devices overcome this issue in part by providing a trapping matrix which allows depletion of abundant blood cell proteins through washing, prior to proteolysis and LC-MS. This approach allows the analysis of proteins from erythrocytes, leukocytes, and plasma and leads to deeper proteomic coverage compared to conventional plasma proteomics, increasing the prospects to discover novel biomarker proteins.

The Cytoplasmic Domain of the SARS-CoV-2 Envelope Protein Assembles into a ß-Sheet Bundle in Lipid Bilayers.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) protein forms a pentameric ion channel in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of the infected cell. The cytoplasmic domain of E interacts with host proteins to cause virus pathogenicity and may also mediate virus assembly and budding. To understand the structural basis of these functions, here we investigate the conformation and dynamics of an E protein construct (residues 8-65) that encompasses the transmembrane domain and the majority of the cytoplasmic domain using solid-state NMR. 13C and 15N chemical shifts indicate that the cytoplasmic domain adopts a ß-sheet-rich conformation that contains three ß-strands separated by turns. The five subunits associate into an umbrella-shaped bundle that is attached to the transmembrane helices by a disordered loop. Water-edited NMR spectra indicate that the third ß-strand at the C terminus of the protein is well hydrated, indicating that it is at the surface of the ß-bundle. The structure of the cytoplasmic domain cannot be uniquely determined from the inter-residue correlations obtained here due to ambiguities in distinguishing intermolecular and intramolecular contacts for a compact pentameric assembly of this small domain. Instead, we present four structural topologies that are consistent with the measured inter-residue contacts. These data indicate that the cytoplasmic domain of the SARS-CoV-2 E protein has a strong propensity to adopt ß-sheet conformations when the protein is present at high concentrations in lipid bilayers. The equilibrium between the ß-strand conformation and the previously reported α-helical conformation may underlie the multiple functions of E in the host cell and in the virion.

A Validated Assay to Quantify Osimertinib and Its Metabolites, AZ5104 and AZ7550, from Microsampled Dried Blood Spots and Plasma.

BACKGROUND: Osimertinib is an oral small-molecule tyrosine kinase receptor inhibitor used to treat non-small cell lung cancer (NSCLC) with a sensitizing epidermal growth factor receptor mutation. Patients may experience drug toxicity and require dose deescalation. The study aimed to quantitate osimertinib and its 2 active metabolites, AZ5104 and AZ7550, in microsampled dried blood spots (DBS) collected from patients with NSCLC using a hemaPEN device and compare them with plasma drug levels. METHODS: A 6-min ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed and validated using plasma and DBS. The accuracy, selectivity, matrix effect, recovery, and stability were assessed using bioanalytical validation criteria. The hematocrit effect was investigated in DBS. Drug levels were measured in 15 patients with NSCLC, and the Bland-Altman method was used to compare measurements between plasma and DBS. RESULTS: The validated assay determined accurate and precise quantities, respectively, for osimertinib in both plasma (93.2%-99.3%; 0.2%-2.3%) and DBS (96.7%-99.6%; 0.5%-10.3%) over a concentration of 1-729 ng/mL. The osimertinib metabolites, AZ5104 and AZ7550, were similarly validated in accordance with bioanalytical guidelines. For 30%-60% patient hematocrit, no hematocrit bias was observed with DBS for all analytes. The Bland-Altman method showed high concordance between plasma and DBS analyte levels. Stability experiments revealed that osimertinib and its metabolites were poorly stable in plasma at room temperature, whereas all analytes were stable in DBS for 10 days at room temperature. CONCLUSIONS: The measurement of osimertinib, AZ5104, and AZ7550 from hemaPEN microsampled DBS is a convenient and reliable approach for therapeutic drug monitoring that produces measurements consistent with plasma drug levels.

pH- and Calcium-Dependent Aromatic Network in the SARS-CoV-2 Envelope Protein.

The envelope (E) protein of the SARS-CoV-2 virus is a membrane-bound viroporin that conducts cations across the endoplasmic reticulum Golgi intermediate compartment (ERGIC) membrane of the host cell to cause virus pathogenicity. The structure of the closed state of the E transmembrane (TM) domain, ETM, was recently determined using solid-state NMR spectroscopy. However, how the channel pore opens to mediate cation transport is unclear. Here, we use 13C and 19F solid-state NMR spectroscopy to investigate the conformation and dynamics of ETM at acidic pH and in the presence of calcium ions, which mimic the ERGIC and lysosomal environment experienced by the E protein in the cell. Acidic pH and calcium ions increased the conformational disorder of the N- and C-terminal residues and also increased the water accessibility of the protein, indicating that the pore lumen has become more spacious. ETM contains three regularly spaced phenylalanine (Phe) residues in the center of the peptide. 19F NMR spectra of para-fluorinated Phe20 and Phe26 indicate that both residues exhibit two sidechain conformations, which coexist within each channel. These two Phe conformations differ in their water accessibility, lipid contact, and dynamics. Channel opening by acidic pH and Ca2+ increases the population of the dynamic lipid-facing conformation. These results suggest an intricate aromatic network that regulates the opening of the ETM channel pore. This aromatic network may be a target for E inhibitors against SARS-CoV-2 and related coronaviruses.

Proteomic Analysis of Whole Blood Using Volumetric Absorptive Microsampling for Precision Medicine Biomarker Studies.

Microsampling of patient blood promises several benefits over conventional phlebotomy practices to facilitate precision medicine studies. These include at-home patient blood collection, supporting telehealth monitoring, minimal postcollection processing, and compatibility with nonrefrigerated transport and storage. However, for proteomic biomarker studies, mass spectrometry of whole blood has generally been avoided in favor of using plasma or serum obtained from venepuncture. We evaluated the use of a volumetric absorptive microsampling (VAMS) device as a sample preparation matrix to enable LC-MS proteomic analyses of dried whole blood. We demonstrated the detection and robust quantitation of up to 1600 proteins from single-shot shotgun-LC-MS analysis of dried whole blood, greatly enhancing proteome depth compared with conventional single-shot LC-MS analyses of undepleted plasma. Some proteins not previously reported in blood were detected using this approach. Various washing reagents were used to demonstrate that proteins can be preferentially removed from VAMS devices prior to downstream analyses. We provide a demonstration that archival frozen blood cell pellets housed under long-term storage (exceeding 5 years) are compatible with VAMS to enable quantitation of potential biomarker proteins from biobank repositories. These demonstrations are important steps in establishing viable analysis workflows to underpin large-scale precision medicine studies. Data are available via ProteomeXchange with the identifier PXD028605.

Patterns of gene expression in pollen of cotton (Gossypium hirsutum) indicate downregulation as a feature of thermotolerance.

Reproductive performance in plants is impaired as maximum temperatures consistently approach 40°C. However, the timing of heatwaves critically affects their impact. We studied the molecular responses during pollen maturation in cotton to investigate the vulnerability to high temperature. Tetrads (TEs), uninucleate and binucleate microspores, and mature pollen were subjected to SWATH-MS and RNA-seq analyses after exposure to 38/28°C (day/night) for 5 days. The results indicated that molecular signatures were downregulated progressively in response to heat during pollen development. This was even more evident in leaves, where three-quarters of differentially changed proteins decreased in abundance during heat. Functional analysis showed that translation of genes increased in TEs after exposure to heat; however, the reverse pattern was observed in mature pollen and leaves. For example, proteins involved in transport were highly abundant in TEs whereas in later stages of pollen formation and leaves, heat suppressed synthesis of proteins involved in cell-to-cell communication. Moreover, a large number of heat shock proteins were identified in heat-affected TEs, but these proteins were less abundant in mature pollen and leaves. We speculate that the sensitivity of TE cells to heat is related to high rates of translation targeted to pathways that might not be essential for thermotolerance. Molecular signatures during stages of pollen development after heatwaves could provide markers for future genetic improvement.

Lipid-Dependent Titration of Glutamic Acid at a Bilayer Membrane Interface.

The ionization properties of protein side chains in lipid-bilayer membranes will differ from the canonical values of side chains exposed to an aqueous solution. While the propensities of positively charged side chains of His, Lys, and Arg to release a proton in lipid membranes have been rather well characterized, the propensity for a negatively charged Glu side chain to receive a proton and achieve the neutral state in a bilayer membrane has been less well characterized. Indeed, the ionization of the glutamic acid side chain has been predicted to depend on its depth of burial in a lipid membrane but has been difficult to verify experimentally. To address the issue, we incorporated an interfacial Glu residue at position 4 of a distinct 23-residue transmembrane helix and used 2H NMR to examine the helix properties as a function of pH. We observe that the helix tilt and azimuthal rotation vary little with pH, but the extent of helix unraveling near residues 3 and 4 changes as the Glu residue E4 titrates. Remarkably, the 2H quadrupolar splitting for the side chain of alanine A3 responds to pH with an apparent pK a of 4.8 in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 6.3 in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), but is unchanged up to pH 8.0 in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the presence of residue E4. With bilayers composed of alkali-stable ether-linked lipids, the side chain of A3 responds to pH with an apparent pK a of 11.0 in the ether analogue of DOPC. These results suggest that the depth dependence of Glu ionization in lipid-bilayer membranes may be steeper than previously predicted or envisioned.

Use of a Recombinant Biomarker Protein DDA Library Increases DIA Coverage of Low Abundance Plasma Proteins.

Credible detection and quantification of low abundance proteins from human blood plasma is a major challenge in precision medicine biomarker discovery when using mass spectrometry (MS). In this proof-of-concept study, we employed a mixture of selected recombinant proteins in DDA libraries to subsequently identify (not quantify) cancer-associated low abundance plasma proteins using SWATH/DIA. The exemplar DDA recombinant protein spectral library (rPSL) was derived from tryptic digestion of 36 recombinant human proteins that had been previously implicated as possible cancer biomarkers from both our own and other studies. The rPSL was then used to identify proteins from nondepleted colorectal cancer (CRC) EDTA plasmas by SWATH-MS. Most (32/36) of the proteins used in the rPSL were reliably identified from CRC plasma samples, including 8 proteins (i.e., BTC, CXCL10, IL1B, IL6, ITGB6, TGFα, TNF, TP53) not previously detected using high-stringency protein inference MS according to PeptideAtlas. The rPSL SWATH-MS protocol was compared to DDA-MS using MARS-depleted and postdigestion peptide fractionated plasmas (here referred to as a human plasma DDA library). Of the 32 proteins identified using rPSL SWATH, only 12 could be identified using DDA-MS. The 20 additional proteins exclusively identified using the rPSL SWATH approach were almost exclusively lower abundance (i.e., <10 ng/mL) proteins. To mitigate justified FDR concerns, and to replicate a more typical library creation approach, the DDA rPSL library was merged with a human plasma DDA library and SWATH identification repeated using such a merged library. The majority (33/36) of the low abundance plasma proteins added from the rPSL were still able to be identified using such a merged library when high-stringency HPP Guidelines v3.0 protein inference criteria were applied to our data set. The MS data set has been deposited to ProteomeXchange Consortium via the PRIDE partner repository (PXD022361).

Structure and drug binding of the SARS-CoV-2 envelope protein transmembrane domain in lipid bilayers.

An essential protein of the SARS-CoV-2 virus, the envelope protein E, forms a homopentameric cation channel that is important for virus pathogenicity. Here we report a 2.1-Å structure and the drug-binding site of E's transmembrane domain (ETM), determined using solid-state NMR spectroscopy. In lipid bilayers that mimic the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membrane, ETM forms a five-helix bundle surrounding a narrow pore. The protein deviates from the ideal α-helical geometry due to three phenylalanine residues, which stack within each helix and between helices. Together with valine and leucine interdigitation, these cause a dehydrated pore compared with the viroporins of influenza viruses and HIV. Hexamethylene amiloride binds the polar amino-terminal lumen, whereas acidic pH affects the carboxy-terminal conformation. Thus, the N- and C-terminal halves of this bipartite channel may interact with other viral and host proteins semi-independently. The structure sets the stage for designing E inhibitors as antiviral drugs.

Structure and Drug Binding of the SARS-CoV-2 Envelope Protein in Phospholipid Bilayers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic. Successful development of vaccines and antivirals against SARS-CoV-2 requires a comprehensive understanding of the essential proteins of the virus. The envelope (E) protein of SARS-CoV-2 assembles into a cation-selective channel that mediates virus budding, release, and host inflammation response. E blockage reduces virus pathogenicity while E deletion attenuates the virus. Here we report the 2.4 Å structure and drug-binding site of E's transmembrane (TM) domain, determined using solid-state nuclear magnetic resonance (NMR) spectroscopy. In lipid bilayers that mimic the endoplasmic reticulum Golgi intermediate compartment (ERGIC) membrane, ETM forms a five-helix bundle surrounding a narrow central pore. The middle of the TM segment is distorted from the ideal a-helical geometry due to three regularly spaced phenylalanine residues, which stack within each helix and between neighboring helices. These aromatic interactions, together with interhelical Val and Leu interdigitation, cause a dehydrated pore compared to the viroporins of influenza and HIV viruses. Hexamethylene amiloride and amantadine bind shallowly to polar residues at the N-terminal lumen, while acidic pH affects the C-terminal conformation. These results indicate that SARS-CoV-2 E forms a structurally robust but bipartite channel whose N- and C-terminal halves can interact with drugs, ions and other viral and host proteins semi-independently. This structure establishes the atomic basis for designing E inhibitors as antiviral drugs against SARS-CoV-2.

Flanking aromatic residue competition influences transmembrane peptide helix dynamics.

To address biophysical principles and lipid interactions that underlie the properties of membrane proteins, modifications that vary the neighbors of tryptophan residues in the highly dynamic transmembrane helix of GW4,20 ALP23 (acetyl-GGAW4 A(LA)6 LAW20 AGA-amide) were examined using deuterium NMR spectroscopy. It was found that L5,19 GW4,20 ALP23, a sequence isomer of the low to moderately dynamic GW5,19 ALP23, remains highly dynamic. By contrast, a removal of W4 to produce F4,5 GW20 ALP23 restores a low level of dynamic averaging, similar to that of the F4,5 GW19 ALP23 helix. Interestingly, a high level of dynamic averaging requires the presence of both tryptophan residues W4 and W20, on opposite faces of the helix, and does not depend on whether residue 5 is Leu or Ala. Aspects of helix unwinding and potential oligomerization are discussed with respect to helix dynamic averaging and the locations of particular residues at a phosphocholine membrane interface.

Evaluating bioanalytical capabilities of paper spray ionization for abiraterone drug quantification in patient plasma.

Paper spray ionization (PSI) is a direct, fast, and low-cost ambient ionization technique which may have clinical utility for qualitative and quantitative analysis of therapeutic drugs and metabolites from patient specimens. We developed and validated a PSI-mass spectrometry (PSI-MS/MS) method according to the US-FDA guidelines for bioanalytical studies to measure the prostate cancer drug abiraterone directly from patient plasma. The established linearity range was 3.1-156.8 ng/mL with a precision (%CV) and an accuracy (%) range of 0.5-10.7 and 93.5-103.2, respectively. The mean internal standard normalized matrix factor for abiraterone was just below 1 with highest %CV of 10.2 at the low-level quality control. In benchmarking the performance of this assay against a published LC-MS/MS assay, we showed they were mostly equivalent, with the exception of accuracy with clinical samples. We found the quantitative values observed for abiraterone measured directly from patient plasma using PSI-MS/MS showed positive bias. Upon investigation, we concluded the increased values were due to summed quantitation of isomeric abiraterone conjugates and metabolites which are separable by LC-MS/MS, but not with the current PSI-MS/MS configuration. Despite demonstrating the utility of PSI-MS/MS for rapid bioanalysis, this study also highlighted a limitation encountered with the direct analysis of abiraterone in clinical samples.

Retinal proteomics of experimental glaucoma model reveal intraocular pressure-induced mediators of neurodegenerative changes.

Current evidence suggests that exposure to chronically induced intraocular pressure (IOP) leads to neurodegenerative changes in the inner retina. This study aimed to determine retinal proteomic alterations in a rat model of glaucoma and compared findings with human retinal proteomics changes in glaucoma reported previously. We developed an experimental glaucoma rat model by subjecting the rats to increased IOP (9.3 ± 0.1 vs 20.8 ± 1.6 mm Hg) by weekly microbead injections into the eye (8 weeks). The retinal tissues were harvested from control and glaucomatous eyes and protein expression changes analysed using a multiplexed quantitative proteomics approach (TMT-MS3). Immunofluorescence was performed for selected protein markers for data validation. Our study identified 4304 proteins in the rat retinas. Out of these, 139 proteins were downregulated (≤0.83) while the expression of 109 proteins was upregulated (≥1.2-fold change) under glaucoma conditions (P ≤ .05). Computational analysis revealed reduced expression of proteins associated with glutathione metabolism, mitochondrial dysfunction/oxidative phosphorylation, cytoskeleton, and actin filament organisation, along with increased expression of proteins in coagulation cascade, apoptosis, oxidative stress, and RNA processing. Further functional network analysis highlighted the differential modulation of nuclear receptor signalling, cellular survival, protein synthesis, transport, and cellular assembly pathways. Alterations in crystallin family, glutathione metabolism, and mitochondrial dysfunction associated proteins shared similarities between the animal model of glaucoma and the human disease condition. In contrast, the activation of the classical complement pathway and upregulation of cholesterol transport proteins were exclusive to human glaucoma. These findings provide insights into the neurodegenerative mechanisms that are specifically affected in the retina in response to chronically elevated IOP.

Two of a kind: transmissible Schwann cell cancers in the endangered Tasmanian devil (Sarcophilus harrisii).

Devil facial tumour disease (DFTD) comprises two genetically distinct transmissible cancers (DFT1 and DFT2) endangering the survival of the Tasmanian devil (Sarcophilus harrisii) in the wild. DFT1 first arose from a cell of the Schwann cell lineage; however, the tissue-of-origin of the recently discovered DFT2 cancer is unknown. In this study, we compared the transcriptome and proteome of DFT2 tumours to DFT1 and normal Tasmanian devil tissues to determine the tissue-of-origin of the DFT2 cancer. Our findings demonstrate that DFT2 expresses a range of Schwann cell markers and exhibits expression patterns consistent with a similar origin to the DFT1 cancer. Furthermore, DFT2 cells express genes associated with the repair response to peripheral nerve damage. These findings suggest that devils may be predisposed to transmissible cancers of Schwann cell origin. The combined effect of factors such as frequent nerve damage from biting, Schwann cell plasticity and low genetic diversity may allow these cancers to develop on rare occasions. The emergence of two independent transmissible cancers from the same tissue in the Tasmanian devil presents an unprecedented opportunity to gain insight into cancer development, evolution and immune evasion in mammalian species.

Radiation-Stimulated Translocation of CD166 and CRYAB to the Endothelial Surface Provides Potential Vascular Targets on Irradiated Brain Arteriovenous Malformations.

Vascular targeting with pro-thrombotic antibody-conjugates is a promising biological treatment for brain arteriovenous malformations (bAVMs). However, targeted drug delivery relies on the identification of unique or overexpressed markers on the surface of a target cell. In the absence of inherent biological markers, stereotactic radiosurgery may be used to prime induction of site-specific and targetable molecular changes on the endothelial surface. To investigate lumen-accessible, endothelial targets induced by radiation, we combined Gamma knife surgery in an AVM animal model with in vivo biotin-labeling and comparative proteomics. Two proteins, αB-crystallin (CRYAB)-a small heat shock protein that normally acts as an intracellular chaperone to misfolded proteins-and activated leukocyte cell adhesion molecule CD166, were further validated for endothelial surface expression after irradiation. Immunostaining of endothelial cells in vitro and rat AVM tissue ex vivo confirmed de novo induction of CRYAB following irradiation (20 Gy). Western analysis demonstrated that CRYAB accumulated intracellularly as a 20 kDa monomer, but, at the cell surface, a novel 65 kDa protein was observed, suggesting radiation stimulates translocation of an atypical CRYAB isoform. In contrast, CD166 had relatively high expression in non-irradiated cells, localized predominantly to the lateral surfaces. Radiation increased CD166 surface exposure by inducing translocation from intercellular junctions to the apical surface without significantly altering total protein levels. These findings reinforce the dynamic molecular changes induced by radiation exposure, particularly at the cell surface, and support further investigation of radiation as a priming mechanism and these molecules as putative targets for focused drug delivery in irradiated tissue.