Mass spectrometry imaging protocol for spatial mapping of lipids, N-glycans and peptides in murine lung tissue.

RATIONALE: The application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to murine lungs is challenging due to the spongy nature of the tissue. Lungs consist of interconnected air sacs (alveoli) lined by a single layer of flattened epithelial cells, which requires inflation to maintain its natural structure. Therefore, a protocol that is compatible with both lung instillation and high spatial resolution is essential to enable multi-omic studies on murine lung disease models using MALDI-MSI. METHODS AND RESULTS: To maintain the structural integrity of the tissue, murine lungs were inflated with 8% (w/v) gelatin for lipid MSI of fresh frozen tissues or 4% (v/v) paraformaldehyde neutral buffer for N-glycan and peptide MSI of FFPE tissues. Tissues were sectioned and prepared for enzymatic digestion and/or matrix deposition. Glycerol-free PNGase F was applied for N-glycan MSI, while Trypsin Gold was applied for peptide MSI using the iMatrixSpray and ImagePrep Station, respectively. For lipid, N-glycan and peptide MSI, α-cyano-4-hydroxycinnamic acid matrix was deposited using the iMatrixSpray. MS data were acquired with 20 µm spatial resolution using a timsTOF fleX MS instrument followed by MS fragmentation of lipids, N-glycans and peptides. For lipid MSI, trapped ion mobility spectrometry was used to separate isomeric/isobaric lipid species. SCiLS™ Lab was used to visualize all MSI data. For analyte identification, MetaboScape®, GlycoMod and Mascot were used to annotate MS fragmentation spectra of lipids, N-glycans and tryptic peptides, respectively. CONCLUSIONS: Our protocol provides instructions on sample preparation for high spatial resolution MALDI-MSI, MS/MS data acquisition and lipid, N-glycan and peptide annotation and identification from murine lungs. This protocol will allow non-biased analyses of diseased lungs from preclinical murine models and provide further insight into disease models.

Protein Profiling in Human Papillomavirus-Associated Cervical Carcinogenesis: Cornulin as a Biomarker for Disease Progression.

Nearly 90% of cervical cancers are linked to human papillomavirus (HPV). Uncovering the protein signatures in each histological phase of cervical oncogenesis provides a path to biomarker discovery. The proteomes extracted from formalin-fixed paraffin-embedded tissues of the normal cervix, HPV16/18-associated squamous intraepithelial lesion (SIL), and squamous cell carcinoma (SCC) were compared using liquid chromatography-mass spectrometry (LC-MS). A total of 3597 proteins were identified, with 589, 550, and 1570 proteins unique to the normal cervix, SIL, and SCC groups, respectively, while 332 proteins overlapped between the three groups. In the transition from normal cervix to SIL, all 39 differentially expressed proteins were downregulated, while all 51 proteins discovered were upregulated in SIL to SCC. The binding process was the top molecular function, while chromatin silencing in the SIL vs. normal group, and nucleosome assembly in SCC vs. SIL groups was the top biological process. The PI3 kinase pathway appears crucial in initiating neoplastic transformation, while viral carcinogenesis and necroptosis are important for cell proliferation, migration, and metastasis in cervical cancer development. Annexin A2 and cornulin were selected for validation based on LC-MS results. The former was downregulated in the SIL vs. normal cervix and upregulated in the progression from SIL to SCC. In contrast, cornulin exhibited the highest expression in the normal cervix and lowest in SCC. Although other proteins, such as histones, collagen, and vimentin, were differentially expressed, their ubiquitous expression in most cells precluded further analysis. Immunohistochemical analysis of tissue microarrays found no significant difference in Annexin A2 expression between the groups. Conversely, cornulin exhibited the strongest expression in the normal cervix and lowest in SCC, supporting its role as a tumor suppressor and potential biomarker for disease progression.

Altered N-linked glycosylation in endometrial cancer.

It is well established that cell surface glycans play a vital role in biological processes and their altered form can lead to carcinogenesis. Mass spectrometry-based techniques have become prominent for analysing N-linked glycans, for example using matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Additionally, MALDI MS can be used to spatially map N-linked glycans directly from cancer tissue using a technique termed MALDI MS imaging (MALDI MSI). This powerful technique combines mass spectrometry and histology to visualise the spatial distribution of N-linked glycans on a single tissue section. Here, we performed N-glycan MALDI MSI on six endometrial cancer (EC) formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMA) consisting of eight EC patients with lymph node metastasis (LNM) and twenty without LNM. By doing so, several putative N-linked glycan compositions were detected that could significantly distinguish normal from cancerous endometrium. Furthermore, a complex core-fucosylated N-linked glycan was detected that could discriminate a primary tumour with and without LNM. Structural identification of these putative N-linked glycans was performed using porous graphitized carbon liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS). Overall, we observed higher abundance of oligomannose glycans in tumour compared to normal regions with AUC ranging from 0.85-0.99, and lower abundance of complex N-linked glycans with AUC ranges from 0.03-0.28. A comparison of N-linked glycans between primary tumours with and without LNM indicated a reduced abundance of a complex core-fucosylated N-glycan (Hex)2(HexNAc)2(Deoxyhexose)1+(Man)3(GlcNAc)2, in primary tumour with associated lymph node metastasis. In summary, N-linked glycan MALDI MSI can be used to differentiate cancerous endometrium from normal, and endometrial cancer with LNM from endometrial cancer without.

Uncovering Tumor-Stroma Inter-relationships Using MALDI Mass Spectrometry Imaging.

Tumorigenesis involves a complex interplay between genetically modified cancer cells and their adjacent normal tissue, the stroma. We used an established breast cancer mouse model to investigate this inter-relationship. Conditional activation of Rho-associated protein kinase (ROCK) in a model of mammary tumorigenesis enhances tumor growth and progression by educating the stroma and enhancing the production and remodeling of the extracellular matrix. We used peptide matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to quantify the proteomic changes occurring within tumors and their stroma in their regular spatial context. Peptides were ranked according to their ability to discriminate between the two groups, using a receiver operating characteristic tool. Peptides were identified by liquid chromatography tandem mass spectrometry, and protein expression was validated by quantitative immunofluorescence using an independent set of tumor samples. We have identified and validated four key proteins upregulated in ROCK-activated mammary tumors relative to those expressing kinase-dead ROCK, namely, collagen I, α-SMA, Rab14, and tubulin-ß4. Rab14 and tubulin-ß4 are expressed within tumor cells, whereas collagen I is localized within the stroma. α-SMA is predominantly localized within the stroma but is also expressed at higher levels in the epithelia of ROCK-activated tumors. High expression of COL1A, the gene encoding the pro-α 1 chain of collagen, correlates with cancer progression in two human breast cancer genomic data sets, and high expression of COL1A and ACTA2 (the gene encoding α-SMA) are associated with a low survival probability (COLIA, p = 0.00013; ACTA2, p = 0.0076) in estrogen receptor-negative breast cancer patients. To investigate whether ROCK-activated tumor cells cause stromal cancer-associated fibroblasts (CAFs) to upregulate expression of collagen I and α-SMA, we treated CAFs with medium conditioned by primary mammary tumor cells in which ROCK had been activated. This led to abundant production of both proteins in CAFs, clearly highlighting the inter-relationship between tumor cells and CAFs and identifying CAFs as the potential source of high levels of collagen 1 and α-SMA and associated enhancement of tissue stiffness. Our research emphasizes the capacity of MALDI-MSI to quantitatively assess tumor-stroma inter-relationships and to identify potential prognostic factors for cancer progression in human patients, using sophisticated mouse cancer models.

The Emerging Role of Cytoskeletal Proteins as Reliable Biomarkers.

Cytoskeletal proteins are essential building blocks of cells. More than 100 cytoskeletal and cytoskeleton-associated proteins are known and for some, their function and regulation are understood in great detail. Apart from cell shape and support, they facilitate many processes such as intracellular signaling and transport, and cancer related processes such as proliferation, migration, and invasion. During the last decade, comparative proteomic studies have identified cytoskeletal proteins as in vitro markers for tumor progression and metastasis. Here, these results are summarized and a number of unrelated studies are highlighted, identifying the same cytoskeletal proteins as potential biomarkers. These findings might indicate that the abundance of these potential markers of tumor progression is associated with the biological outcome and are independent of the cancer origin. This correlates well with recently published results from the Cancer Genome Atlas, indicating that cancers show remarkable similarities in their analyzed molecular information, independent of their organ of origin. It is postulated that the quantification of cytoskeletal proteins in healthy tissues, tumors, in adjacent tissues, and in stroma, is a great source of molecular information, which might not only be used to classify tumors, but more importantly to predict patients' outcome or even best treatment choices.

Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) for Monitoring of Drug Response in Primary Cancer Spheroids.

Malignant ascites is a fluid, which builds up in the abdomen and contains cancer cells in the form of single cells or multicellular clusters called spheroids. Malignant ascites has been observed in patients suffering from ovarian, cervical, gastric, colorectal, pancreatic, endometrial, or primary liver cancer. The spheroids are believed to play a major role in chemo resistance and metastasis of the cancer. To ease the discomfort of patients, malignant ascites (MA) is often drained from the abdomen using a procedure called paracentesis. MA retrieved via this minimal invasive procedure is a great source for cancer spheroids, which can be used for testing chemotherapeutic drugs and drug combinations. Herein, the existing workflow is adapted to make concurrent monitoring of drug accumulation, drug response, and drug metabolites feasible using primary spheroids or spheroids grown without a scaffolding matrix. To achieve this, those spheroids are embedded in matrigel, before fixing them with formalin. This makes it possible to process, store, and ship samples at room temperature. This new approach might be used to choose the best targeted therapy for each patient and thereby facilitate personalized medicine.

MALDI Mass Spectrometry Imaging of Early- and Late-Stage Serous Ovarian Cancer Tissue Reveals Stage-Specific N-Glycans.

Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N-glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor-specific N-glycan alterations in ovarian cancer development and progression. matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N-glycan distribution on formalin-fixed paraffin-embedded ovarian cancer tissue sections from early- and late-stage patients. Tumor-specific N-glycans are identified and structurally characterized by porous graphitized carbon-liquid chromatography-electrospray ionization-tandem mass spectrometry (PGC-LC-ESI-MS/MS), and then assigned to high-resolution images obtained from MALDI-MSI. Spatial distribution of 14 N-glycans is obtained by MALDI-MSI and 42 N-glycans (including structural and compositional isomers) identified and structurally characterized by LC-MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N-glycan families are localized to the tumor regions of late-stage ovarian cancer patients relative to early-stage patients. Potential N-glycan diagnostic markers that emerge include the oligomannose structure, (Hex)6 + (Man)3 (GlcNAc)2 , and the complex neutral structure, (Hex)2 (HexNAc)2 (Deoxyhexose)1 + (Man)3 (GlcNAc)2 . The distribution of these markers is evaluated using a tissue microarray of early- and late-stage patients.

Egg White as a Quality Control in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI).

The strength of MALDI-MSI is to analyze and visualize spatial intensities of molecular features from an intact tissue. The distribution of the intensities can then be visualized within a single tissue section or compared in between sections, acquired consecutively. This method can be reliably used to reveal physiological structures and has the potential to identify molecular details, which correlate with biological outcomes. MALDI-MSI implementation in clinical laboratories requires the ability to ensure method quality and validation to meet diagnostic expectations. To be able to get consistent qualitative and quantitative results, standardized sample preparation and data acquisition are of highest priority. We have previously shown that the deposition of internal standards onto the tissue section during sample preparation can be used to improve the mass accuracy of monitored m/z features across the sample. Here, we present the use of external and internal controls for the quality check of sample preparation and data acquisition, which is particularly relevant when either many spectra are acquired during a single MALDI-MSI experiment or data from independent experiments are processed together. To monitor detector performance and sample preparation, we use egg white as an external control for peptide and N-glycan MALDI-MSI throughout the experiment. We have also identified endogenous peptides from cytoskeletal proteins, which can be reliably monitored in gynecological tissue samples. Lastly, we summarize our standard quality control workflow designed to produce reliable and comparable MALDI-MSI data from single sections and tissue microarrays (TMAs).

Gut microbiome contributes to impairment of immunity in pulmonary tuberculosis patients by alteration of butyrate and propionate producers.

Tuberculosis (TB) is primarily associated with decline in immune health status. As gut microbiome (GM) is implicated in the regulation of host immunity and metabolism, here we investigate GM alteration in TB patients by 16S rRNA gene and whole-genome shotgun sequencing. The study group constituted of patients with pulmonary TB and their healthy household contacts as controls (HCs). Significant alteration of microbial taxonomic and functional capacity was observed in patients with active TB as compared to the HCs. We observed that Prevotella and Bifidobacterium abundance were associated with HCs, whereas butyrate and propionate-producing bacteria like Faecalibacterium, Roseburia, Eubacterium and Phascolarctobacterium were significantly enriched in TB patients. Functional analysis showed reduced biosynthesis of vitamins and amino acids in favour of enriched metabolism of butyrate and propionate in TB subjects. The TB subjects were also investigated during the course of treatment, to analyse the variation of GM. Although perturbation in microbial composition was still evident after a month's administration of anti-TB drugs, significant changes were observed in metagenome gene pool that pointed towards recovery in functional capacity. Therefore, the findings from this pilot study suggest that microbial dysbiosis may contribute to pathophysiology of TB by enhancing the anti-inflammatory milieu in the host.

Annexin A2 and alpha actinin 4 expression correlates with metastatic potential of primary endometrial cancer.

The prediction of lymph node metastasis using clinic-pathological data and molecular information from endometrial cancers lacks accuracy and is therefore currently not routinely used in patient management. Consequently, although only a small percentage of patients with endometrial cancers suffer from metastasis, the majority undergo radical surgery including removal of pelvic lymph nodes. Upon analysis of publically available data and published research, we compiled a list of 60 proteins having the potential to display differential abundance between primary endometrial cancers with versus those without lymph node metastasis. Using data dependent acquisition LC-ESI-MS/MS we were able to detect 23 of these proteins in endometrial cancers, and using data independent LC-ESI-MS/MS the differential abundance of five of those proteins was observed. The localization of the differentially expressed proteins, was visualized using peptide MALDI MSI in whole tissue sections as well as tissue microarrays of 43 patients. The proteins identified were further validated by immunohistochemistry. Our data indicate that annexin A2 protein level is upregulated, whereas annexin A1 and α actinin 4 expression are downregulated in tumours with lymph node metastasis compared to those without lymphatic spread. Moreover, our analysis confirmed the potential of these markers, to be included in a statistical model for prediction of lymph node metastasis. The predictive model using highly ranked m/z values identified by MALDI MSI showed significantly higher predictive accuracy than the model using immunohistochemistry data. In summary, using publicly available data and complementary proteomics approaches, we were able to improve the prediction model for lymph node metastasis in EC.

Classification of MALDI-MS imaging data of tissue microarrays using canonical correlation analysis-based variable selection.

Applying MALDI-MS imaging to tissue microarrays (TMAs) provides access to proteomics data from large cohorts of patients in a cost- and time-efficient way, and opens the potential for applying this technology in clinical diagnosis. The complexity of these TMA data-high-dimensional low sample size-provides challenges for the statistical analysis, as classical methods typically require a nonsingular covariance matrix that cannot be satisfied if the dimension is greater than the sample size. We use TMAs to collect data from endometrial primary carcinomas from 43 patients. Each patient has a lymph node metastasis (LNM) status of positive or negative, which we predict on the basis of the MALDI-MS imaging TMA data. We propose a variable selection approach based on canonical correlation analysis that explicitly uses the LNM information. We apply LDA to the selected variables only. Our method misclassifies 2.3-20.9% of patients by leave-one-out cross-validation and strongly outperforms LDA after reduction of the original data with principle component analysis.

Lymph node metastasis of primary endometrial cancers: Associated proteins revealed by MALDI imaging.

Metastasis is a crucial step of malignant progression and is the primary cause of death from endometrial cancer. However, clinicians presently face the challenge that conventional surgical-pathological variables, such as tumour size, depth of myometrial invasion, histological grade, lymphovascular space invasion or radiological imaging are unable to predict with accuracy if the primary tumour has metastasized. In the current retrospective study, we have used primary tumour samples of endometrial cancer patients diagnosed with (n = 16) and without (n = 27) lymph node metastasis to identify potential discriminators. Using peptide matrix assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI), we have identified m/z values which can classify 88% of all tumours correctly. The top discriminative m/z values were identified using a combination of in situ sequencing and LC-MS/MS from digested tumour samples. Two of the proteins identified, plectin and α-Actin-2, were used for validation studies using LC-MS/MS data independent analysis (DIA) and immunohistochemistry. In summary, MALDI-MSI has the potential to identify discriminators of metastasis using primary tumour samples.

MALDI Mass Spectrometry Imaging Reveals Decreased CK5 Levels in Vulvar Squamous Cell Carcinomas Compared to the Precursor Lesion Differentiated Vulvar Intraepithelial Neoplasia.

Vulvar cancer is the fourth most common gynecological cancer worldwide. However, limited studies have been completed on the molecular characterization of vulvar squamous cell carcinoma resulting in a poor understanding of the disease initiation and progression. Analysis and early detection of the precursor lesion of HPV-independent vulvar squamous cell carcinoma (VSCC), differentiated vulvar intraepithelial neoplasia (dVIN), is of great importance given dVIN lesions have a high level of malignant potential. Here we present an examination of adjacent normal vulvar epithelium, dVIN, and VSCC from six patients by peptide Matrix-assisted laser desorption/ionization Mass Spectrometry Imaging (MALDI-MSI). The results reveal the differential expression of multiple peptides from the protein cytokeratin 5 (CK5) across the three vulvar tissue types. The difference observed in the relative abundance of CK5 by MALDI-MSI between the healthy epithelium, dVIN, and VSCC was further analyzed by immunohistochemistry (IHC) in tissue from eight VSCC patients. A decrease in CK5 immunostaining was observed in the VSCC compared to the healthy epithelium and dVIN. These results provide an insight into the molecular fingerprint of the vulvar intraepithelial neoplasia that appears to be more closely related to the healthy epithelium than the VSCC.

Phthiriasis palpebrarum masquerading as blepharitis.

Pthiriasis palpebrarum, a rare condition caused by Phthirus pubis, can mimic blepharitis and manifest as intense itching, inflammation and eyelid redness. We describe a case of Pthiriasis palpebrarum in a young girl with right upper eyelid discomfort. A slit lamp examination revealed eggs on her eyelashes and adult lice on the eyelids' surface. Dermatology consultation confirmed the diagnosis and successful treatment followed. Family evaluation uncovered genital Phthiriasis pubis in her parents, who received appropriate treatment. This report emphasises the importance of accurate diagnosis and management of this masquerading condition by careful slit lamp and microscopic evaluation. It also highlights the significance of comprehensive family history and examination.

A systematic review and in silico analysis of studies investigating the ischaemic penumbra proteome in animal models of experimental stroke.

Ischaemic stroke results in the formation of a cerebral infarction bordered by an ischaemic penumbra. Characterising the proteins within the ischaemic penumbra may identify neuro-protective targets and novel circulating markers to improve patient care. This review assessed data from studies using proteomic platforms to compare ischaemic penumbra tissues to controls following experimental stroke in animal models. Proteins reported to differ significantly between penumbra and control tissues were analysed in silico to identify protein-protein interactions and over-represented pathways. Sixteen studies using rat (n = 12), mouse (n = 2) or primate (n = 2) models were included. Heterogeneity in the design of the studies and definition of the penumbra were observed. Analyses showed high abundance of p53 in the penumbra within 24 hours of permanent ischaemic stroke and was implicated in driving apoptosis, cell cycle progression, and ATM- MAPK- and p53- signalling. Between 1 and 7 days after stroke there were changes in the abundance of proteins involved in the complement and coagulation pathways. Favourable recovery 1 month after stroke was associated with an increase in the abundance of proteins involved in wound healing. Poor recovery was associated with increases in prostaglandin signalling. Findings suggest that p53 may be a target for novel therapeutics for ischaemic stroke.

From biomolecules to biogeochemistry: Exploring the interaction of an indigenous bacterium with gold.

Specialised microbial communities colonise the surface of gold particles in soils/sediments, and catalyse gold dissolution and re-precipitation, thereby contributing to the environmental mobility and toxicity of this 'inert' precious metal. We assessed the proteomic and physiological response of Serratia proteamaculans, the first metabolically active bacterium enriched and isolated directly from natural gold particles, when exposed to toxic levels of soluble Au3+ (10 µM). The results were compared to a metal-free blank, and to cultures exposed to similarly toxic levels of soluble Cu2+ (0.1 mM); Cu was chosen for comparison because it is closely associated with Au in nature due to similar geochemical properties. A total of 273 proteins were detected from the cells that experienced the oxidative effects of soluble Au, of which 139 (51%) were upregulated with either sole expression (31%) or had synthesis levels greater than the Au-free control (20%). The majority (54%) of upregulated proteins were functionally different from up-regulated proteins in the bacteria-copper treatment. These proteins were related to broad functions involving metabolism and biogenesis, followed by cellular process and signalling, indicating significant specificity for Au. This proteomic study revealed that the bacterium upregulates the synthesis of various proteins related to oxidative stress response (e.g., Monothiol-Glutaredoxin, Thiol Peroxidase, etc.) and cellular damage repair, which leads to the formation of metallic gold nanoparticles less toxic than ionic gold. Therefore, indigenous bacteria may mediate the toxicity of Au through two different yet simultaneous processes: i) repairing cellular components by replenishing damaged proteins and ii) neutralising reactive oxygen species (ROS) by up-regulating the synthesis of antioxidants. By connecting the fields of molecular bacteriology and environmental biogeochemistry, this study is the first step towards the development of biotechnologies based on indigenous bacteria applied to gold bio-recovery and bioremediation of contaminated environments.

Intracameral Drug Delivery: A Review of Agents, Indications, and Outcomes.

An intracameral (IC) injection directly delivers the drug into the anterior chamber of the eye. This targeted drug delivery technique overcomes the ocular barriers and offers a high therapeutic concentration of medication at the desired site and consequently better clinical outcomes. IC drug delivery is a safe and effective modality with many advantages over topical delivery. These include excellent bioavailability, reduced systemic risk, and minimal ocular toxicity. Agents delivered via IC injection have shown promising results against infection, inflammation, ocular hypertension, and neovascularization. Current literature shows that IC antibiotics, including cefuroxime, vancomycin, and moxifloxacin, are routinely used for prophylaxis of endophthalmitis. Other drugs available for IC use are steroids, anesthetics, mydriatics, miotics, antivascular endothelial growth factor, antiglaucoma, and alkylating agents. Introduction of sustained-release devices containing dexamethasone or Bimatoprost in anterior chamber via IC route has the potential in treating ocular inflammation and raised intraocular pressure. The complications such as hemorrhagic occlusive retinal vasculitis and toxic anterior segment syndrome have been documented with IC prophylaxis but are rare. In this review, we provide an overview of available IC drugs, their pharmacokinetics, the spectrum of activity, dosage and preparation, prophylactic and therapeutic usage, clinical efficacy, and safety profiles.

Label-Free Quantification Mass Spectrometry Identifies Protein Markers of Chemotherapy Response in High-Grade Serous Ovarian Cancer.

Eighty percent of ovarian cancer patients initially respond to chemotherapy, but the majority eventually experience a relapse and die from the disease with acquired chemoresistance. In addition, 20% of patients do not respond to treatment at all, as their disease is intrinsically chemotherapy resistant. Data-independent acquisition nano-flow liquid chromatography-mass spectrometry (DIA LC-MS) identified the three protein markers: gelsolin (GSN), calmodulin (CALM1), and thioredoxin (TXN), to be elevated in high-grade serous ovarian cancer (HGSOC) tissues from patients that responded to chemotherapy compared to those who did not; the differential expression of the three protein markers was confirmed by immunohistochemistry. Analysis of the online GENT2 database showed that mRNA levels of GSN, CALM1, and TXN were decreased in HGSOC compared to fallopian tube epithelium. Elevated levels of GSN and TXN mRNA expression correlated with increased overall and progression-free survival, respectively, in a Kaplan-Meier analysis of a large online repository of HGSOC patient data. Importantly, differential expression of the three protein markers was further confirmed when comparing parental OVCAR-5 cells to carboplatin-resistant OVCAR-5 cells using DIA LC-MS analysis. Our findings suggest that GSN, CALM1, and TXN may be useful biomarkers for predicting chemotherapy response and understanding the mechanisms of chemotherapy resistance. Proteomic data are available via ProteomeXchange with identifier PXD033785.

A Protocol for the Acquisition of Comprehensive Proteomics Data from Single Cases Using Formalin-Fixed Paraffin Embedded Sections.

The molecular analysis of small or rare patient tissue samples is challenging and often limited by available technologies and resources, such as reliable antibodies against a protein of interest. Although targeted approaches provide some insight, here, we describe the workflow of two complementary mass spectrometry approaches, which provide a more comprehensive and non-biased analysis of the molecular features of the tissue of interest. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) generates spatial intensity maps of molecular features, which can be easily correlated with histology. Additionally, liquid chromatography tandem mass spectrometry (LC-MS/MS) can identify and quantify proteins of interest from a consecutive section of the same tissue. Here, we present data from concurrent precancerous lesions from the endometrium and fallopian tube of a single patient. Using this complementary approach, we monitored the abundance of hundreds of proteins within the precancerous and neighboring healthy regions. The method described here represents a useful tool to maximize the number of molecular data acquired from small sample sizes or even from a single case. Our initial data are indicative of a migratory phenotype in these lesions and warrant further research into their malignant capabilities.

Sustained Activity of Stimuli-Responsive Curcumin and Acemannan Based Hydrogel Patches in Wound Healing.

Natural plant extract, namely acemannan (Ac) and curcumin (Cur), coencapsulated pluronic micelles, showing thermoresponsive properties, were designed for efficient and safe in vivo wound healing applications. Ac and Cur, widely used antimicrobials, find limited applications because of their low stability, short biological half-life, poor solubility, and low bioavailability. Herein, we report the extraction of Ac from aloe vera and coencapsulation of it with Cur in pluronic micelles to take advantage of the combined effects of both components. Both Ac and Cur preserved their bioactive functionality upon encapsulation. Single photon emission computed tomography imaging confirmed that NPAcC2 hydrogel masked the whole wound by forming a layer. Cur and Ac synergistically resulted in rapid wound closure on the seventh day, and full-grown hair was observed on the 10th day. Individually they both take more than 20 days for wound closure. The increase in the concentration of curcumin increases the healing properties of the material. For days 1, 6, and 10 of the wound dressing experiment, the percentages of wound closure of the mice were the highest for NPAcC2 (i.e., 100%) compared to the untreated control (25%) while maintaining the integrity of the skin. These natural product-based hydrogels have limited side effects vs those caused by commercial drugs in wound healing.