Sequential trypsin and ProAlanase digestions unearth immunological protein biomarkers shrouded by skeletal collagen.

This study investigates the efficacy of proteomic analysis of human remains to identify active infections in the past through the detection of pathogens and the host response to infection. We advance leprosy as a case study due to the sequestering of sufferers in leprosaria and the suggestive skeletal lesions that can result from the disease. Here we present a sequential enzyme extraction protocol, using trypsin followed by ProAlanase, to reduce the abundance of collagen peptides and in so doing increase the detection of non-collagenous proteins. Through our study of five individuals from an 11th to 18th century leprosarium, as well as four from a contemporaneous non-leprosy associated cemetery in Barcelona, we show that samples from 2 out of 5 leprosarium individuals extracted with the sequential digestion methodology contain numerous host immune proteins associated with modern leprosy. In contrast, individuals from the non-leprosy associated cemetery and all samples extracted with a trypsin-only protocol did not. Through this study, we advance a palaeoproteomic methodology to gain insights into the health of archaeological individuals and take a step toward a proteomics-based method to study immune responses in past populations.

Probing the salivary proteome for prognostic biomarkers in response to non-surgical periodontal therapy.

AIM: This prospective study investigated the salivary proteome before and after periodontal therapy. MATERIALS AND METHODS: Ten systemically healthy, non-smoking, stage III, grade C periodontitis patients underwent non-surgical periodontal treatment. Full-mouth periodontal parameters were measured, and saliva (n = 30) collected pre- (T0), and one (T1) and six (T6) months post-treatment. The proteome was investigated by label-free quantitative proteomics. Protein expression changes were modelled over time, with significant protein regulation considered at false discovery rate <0.05. RESULTS: Treatment significantly reduced bleeding scores, percentages of sites with pocket depth ≥5 mm, plaque and gingival indexes. One thousand seven hundred and thirteen proteins were identified and 838 proteins (human = 757, bacterial = 81) quantified (≥2 peptides). At T1, 80 (T1 vs. T0: 60↑:20↓), and at T6, 118 human proteins (T6 vs. T0: 67↑:51↓) were regulated. The salivary proteome at T6 versus T1 remained stable. Highest protein activity post- versus pre-treatment was observed for cellular movement and inflammatory response. The small proline-rich protein 3 (T1 vs. T0: 5.4-fold↑) and lymphocyte-specific protein 1 (T6 vs. T0: 4.6-fold↓) were the top regulated human proteins. Proteins from Neisseria mucosa and Treponema socranskii (T1 vs. T0: 8.0-fold↓, 4.9-fold↓) were down-regulated. CONCLUSIONS: Periodontal treatment reduced clinical disease parameters and these changes were reflected in the salivary proteome. This underscores the potential of utilizing saliva biomarkers as prognostic tools for monitoring treatment outcomes.

Functional proteomics of colon cancer Consensus Molecular Subtypes.

BACKGROUND: The Colorectal Cancer Subtyping Consortium established four Consensus Molecular Subtypes (CMS) in colorectal cancer: CMS1 (microsatellite-instability [MSI], Immune), CMS2 (Canonical, epithelial), CMS3 (Metabolic), and CMS4 (Mesenchymal). However, only MSI tumour patients have seen a change in their disease management in clinical practice. This study aims to characterise the proteome of colon cancer CMS and broaden CMS's clinical utility. METHODS: One-hundred fifty-eight paraffin samples from stage II-III colon cancer patients treated with adjuvant chemotherapy were analysed through DIA-based mass-spectrometry proteomics. RESULTS: CMS1 exhibited overexpression of immune-related proteins, specifically related to neutrophils, phagocytosis, antimicrobial response, and a glycolytic profile. These findings suggested potential therapeutic strategies involving immunotherapy and glycolytic inhibitors. CMS3 showed overexpression of metabolic proteins. CMS2 displayed a heterogeneous protein profile. Notably, two proteomics subtypes within CMS2, with different protein characteristics and prognoses, were identified. CMS4 emerged as the most distinct group, featuring overexpression of proteins related to angiogenesis, extracellular matrix, focal adhesion, and complement activation. CMS4 showed a high metastatic profile and suggested possible chemoresistance that may explain its worse prognosis. CONCLUSIONS: DIA proteomics revealed new features for each colon cancer CMS subtype. These findings provide valuable insights into potential therapeutic targets for colorectal cancer subtypes in the future.

Developmental stage-specific proteome analysis of the legume pod borer Maruca vitrata provides insights on relevant proteins.

The spotted pod borer, Maruca vitrata (Lepidoptera: Crambidae) is a destructive insect pest that inflicts significant productivity losses on important leguminous crops. Unravelling insect proteomes is vital to comprehend their fundamental molecular mechanisms. This research delved into the proteome profiles of four distinct stages -three larval and pupa of M. vitrata, utilizing LC-MS/MS label-free quantification-based methods. Employing comprehensive proteome analysis with fractionated datasets, we mapped 75 % of 3459 Drosophila protein orthologues out of which 2695 were identified across all developmental stages while, 137 and 94 were exclusive to larval and pupal stages respectively. Cluster analysis of 2248 protein orthologues derived from MaxQuant quantitative dataset depicted six clusters based on expression pattern similarity across stages. Consequently, gene ontology and protein-protein interaction network analyses using STRING database identified cluster 1 (58 proteins) and cluster 6 (25 proteins) associated with insect immune system and lipid metabolism. Furthermore, qRT-PCR-based expression analyses of ten selected proteins-coding genes authenticated the proteome data. Subsequently, functional validation of these chosen genes through gene silencing reduced their transcript abundance accompanied by a marked increase in mortality among dsRNA-injected larvae. Overall, this is a pioneering study to effectively develop a proteome atlas of M. vitrata as a potential resource for crop protection programs.

Changes in Medicago truncatula seed proteome along the rehydration-dehydration cycle highlight new players in the genotoxic stress response.

Introduction: Several molecular aspects underlying the seed response to priming and the resulting vigor profile are still poorly understood. Mechanisms involved in genome maintenance deserve attention since the balance between stimulation of germination and DNA damage accumulation versus active repair is a key determinant for designing successful seed priming protocols. Methods: Changes in the Medicago truncatula seed proteome were investigated in this study, using discovery mass spectrometry and label-free quantification, along the rehydration-dehydration cycle of a standard vigorization treatment (hydropriming plus dry-back), and during post-priming imbibition. Resuts and discussion: From 2056 to 2190 proteins were detected in each pairwise comparison, among which six were differentially accumulated and 36 were detected only in one condition. The following proteins were selected for further investigation: MtDRP2B (DYNAMIN-RELATED PROTEIN), MtTRXm4 (THIOREDOXIN m4), and MtASPG1 (ASPARTIC PROTEASE IN GUARD CELL 1) showing changes in seeds under dehydration stress; MtITPA (INOSINE TRIPHOSPHATE PYROPHOSPHORYLASE), MtABA2 (ABSCISIC ACID DEFICIENT 2), MtRS2Z32 (SERINE/ARGININE-RICH SPLICING FACTOR RS2Z32), and MtAQR (RNA HELICASE AQUARIUS) that were differentially regulated during post-priming imbibition. Changes in the corresponding transcript levels were assessed by qRT-PCR. In animal cells, ITPA hydrolyses 2'-deoxyinosine triphosphate and other inosine nucleotides, preventing genotoxic damage. A proof of concept was performed by imbibing primed and control M. truncatula seeds in presence/absence of 20 mM 2'-deoxyinosine (dI). Results from comet assay highlighted the ability of primed seeds to cope with dI-induced genotoxic damage. The seed repair response was assessed by monitoring the expression profiles of MtAAG (ALKYL-ADENINE DNA GLYCOSILASE) and MtEndoV (ENDONUCLEASE V) genes that participate in the repair of the mismatched I:T pair in BER (base excision repair) and AER (alternative excision repair) pathways, respectively.

Proteomic profiling of canine fibrosarcoma and adjacent peritumoral tissue.

Fibrosarcoma (FSA) are rare soft tissue tumors that display aggressive local behavior and invasive growth leading to high rates of tumor recurrence. While the low incidence in humans hampers detailed understanding of the disease, FSA are frequent in dogs and present potential models for the human condition. However, a lack of in-depth molecular characterization of FSA and unaffected peritumoral tissue (PTT) in both species impedes the translational potential of dogs. To address this shortcoming, we characterized canine FSA and matched skeletal muscle, adipose and connective tissue using laser-capture microdissection (LCM) and LC-MS/MS in 30 formalin-fixed paraffin embedded (FFPE) specimens. Principal component analysis of 3'530 different proteins detected across all samples clearly separates the four tissues, with several targets strongly differentiating tumor from all three PTTs. 25 proteins were exclusively found in tumor tissue in ≥80% of cases. Among these, CD68 (a macrophage marker), Optineurin (OPTN), Nuclear receptor coactivator 5 (NCOA5), RAP1GDS1 (Rap1 GTPase-GDP dissociation stimulator 1) and Stromal cell derived factor 2 like 1 (SDF2L1) were present in ≥90% of FSA. Protein expression across all FSA was highly homogeneous and characterized by MYC and TP53 signaling, hyperactive EIF2 and immune-related changes as well as strongly decreased oxidative phosphorylation and oxidative lipid metabolism. Finally, we demonstrate significant molecular homology between canine FSA and human soft-tissue sarcomas, emphasizing the relevance of studying canine FSA as a model for human FSA. In conclusion, we provide the first detailed overview of proteomic changes in FSA and surrounding PTT with relevance for the human disease.

Dairying enabled Early Bronze Age Yamnaya steppe expansions.

During the Early Bronze Age, populations of the western Eurasian steppe expanded across an immense area of northern Eurasia. Combined archaeological and genetic evidence supports widespread Early Bronze Age population movements out of the Pontic-Caspian steppe that resulted in gene flow across vast distances, linking populations of Yamnaya pastoralists in Scandinavia with pastoral populations (known as the Afanasievo) far to the east in the Altai Mountains1,2 and Mongolia3. Although some models hold that this expansion was the outcome of a newly mobile pastoral economy characterized by horse traction, bulk wagon transport4-6 and regular dietary dependence on meat and milk5, hard evidence for these economic features has not been found. Here we draw on proteomic analysis of dental calculus from individuals from the western Eurasian steppe to demonstrate a major transition in dairying at the start of the Bronze Age. The rapid onset of ubiquitous dairying at a point in time when steppe populations are known to have begun dispersing offers critical insight into a key catalyst of steppe mobility. The identification of horse milk proteins also indicates horse domestication by the Early Bronze Age, which provides support for its role in steppe dispersals. Our results point to a potential epicentre for horse domestication in the Pontic-Caspian steppe by the third millennium BC, and offer strong support for the notion that the novel exploitation of secondary animal products was a key driver of the expansions of Eurasian steppe pastoralists by the Early Bronze Age.

The protein landscape of chronic lymphocytic leukemia.

Many functional consequences of mutations on tumor phenotypes in chronic lymphocytic leukemia (CLL) are unknown. This may be in part due to a scarcity of information on the proteome of CLL. We profiled the proteome of 117 CLL patient samples with data-independent acquisition mass spectrometry and integrated the results with genomic, transcriptomic, ex vivo drug response, and clinical outcome data. We found trisomy 12, IGHV mutational status, mutated SF3B1, trisomy 19, del(17)(p13), del(11)(q22.3), mutated DDX3X and MED12 to influence protein expression (false discovery rate [FDR] = 5%). Trisomy 12 and IGHV status were the major determinants of protein expression variation in CLL as shown by principal-component analysis (1055 and 542 differentially expressed proteins, FDR = 5%). Gene set enrichment analyses of CLL with trisomy 12 implicated B-cell receptor (BCR)/phosphatidylinositol 3-kinase (PI3K)/AKT signaling as a tumor driver. These findings were supported by analyses of protein abundance buffering and protein complex formation, which identified limited protein abundance buffering and an upregulated protein complex involved in BCR, AKT, MAPK, and PI3K signaling in trisomy 12 CLL. A survey of proteins associated with trisomy 12/IGHV-independent drug response linked STAT2 protein expression with response to kinase inhibitors, including Bruton tyrosine kinase and mitogen-activated protein kinase kinase (MEK) inhibitors. STAT2 was upregulated in unmutated IGHV CLL and trisomy 12 CLL and required for chemokine/cytokine signaling (interferon response). This study highlights the importance of protein abundance data as a nonredundant layer of information in tumor biology and provides a protein expression reference map for CLL.

Identification of disease-promoting stromal components by comparative proteomic and transcriptomic profiling of canine mammary tumors using laser-capture microdissected FFPE tissue.

Cancer-associated stroma (CAS) profoundly influences progression of tumors including mammary carcinoma (mCA). Canine simple mCA represent relevant models of human mCA, notably also with respect to CAS. While transcriptomic changes in CAS of mCA are well described, it remains unclear to what extent these translate to the protein level. Therefore, we sought to gain insight into the proteomic changes in CAS and compare them with transcriptomic changes in the same tissue. To this end, we analyzed CAS and matched normal stroma using laser-capture microdissection (LCM) and LC-MS/MS in a cohort of 14 formalin-fixed paraffin embedded (FFPE) canine mCAs that we had previously characterized using LCM-RNAseq. Our results reveal clear differences in protein abundance between CAS and normal stroma, which are characterized by changes in the extracellular matrix, the cytoskeleton, and cytokines such as TNF. The proteomics- and RNAseq-based analyses of LCM-FFPE show a substantial degree of correlation, especially for the most deregulated targets and a comparable activation of pathways. Finally, we validate transcriptomic upregulation of LTBP2, IGFBP2, COL6A5, POSTN, FN1, COL4A1, COL12A1, PLOD2, COL4A2, and IGFBP7 in CAS on the protein level and demonstrate their adverse prognostic value for human breast cancer. Given the relevance of canine mCA as a model for the human disease, our analysis substantiates these targets as disease-promoting stromal components with implications for breast cancer in both species.

Network integration and modelling of dynamic drug responses at multi-omics levels.

Uncovering cellular responses from heterogeneous genomic data is crucial for molecular medicine in particular for drug safety. This can be realized by integrating the molecular activities in networks of interacting proteins. As proof-of-concept we challenge network modeling with time-resolved proteome, transcriptome and methylome measurements in iPSC-derived human 3D cardiac microtissues to elucidate adverse mechanisms of anthracycline cardiotoxicity measured with four different drugs (doxorubicin, epirubicin, idarubicin and daunorubicin). Dynamic molecular analysis at in vivo drug exposure levels reveal a network of 175 disease-associated proteins and identify common modules of anthracycline cardiotoxicity in vitro, related to mitochondrial and sarcomere function as well as remodeling of extracellular matrix. These in vitro-identified modules are transferable and are evaluated with biopsies of cardiomyopathy patients. This to our knowledge most comprehensive study on anthracycline cardiotoxicity demonstrates a reproducible workflow for molecular medicine and serves as a template for detecting adverse drug responses from complex omics data.

Unraveling the proteomic changes involved in the resistance response of Cajanus platycarpus to herbivory by Helicoverpa armigera.

The pigeonpea wild relative Cajanus platycarpus is resistant to Helicoverpa armigera, one of the major pests responsible for yield losses in Cajanus cajan. Deciphering the molecular mechanism underlying host plant resistance is pertinent to identify proteins that aid in the mitigation of the insect pest. The present study adopted comparative proteomics as a tool to interpret the resistance mechanism(s) in C. platycarpus vis-à-vis C. cajan during continued herbivory (up to 96 h). Over-representation analysis of the differentially expressed proteins implicated a multi-dimensional resistance response accomplished by both physical and chemical barriers in C. platycarpus. While the chemical basis for resistance was depicted by the upregulation of proteins playing a rate limiting role in the phenylpropanoid pathway, the physical basis was provided by the regulation of proteins involved in microtubule assembly and synthesis of lignins. Upregulation of proteins in the polyamine pathway indicated the role of metabolite conjugates to be negatively affecting herbivore growth. Reallocation of resources and diversion of metabolic flux to support the production of secondary metabolites could be the probable approach in the wild relative against herbivory. Our study provided deeper insights into the pod borer resistance mechanism in C. platycarpus for utility in crop improvement. KEY POINTS: • Pod borer resistance in Cajanus platycarpus is multi-dimensional. • Pod borer resistance has been arbitrated to cell wall rigidity and secondary metabolites. • Phenylpropanoid pathway derivatives apparently shaped the plant chemical defense against pod borer.

Spontaneous hypocarbia without mechanical ventilation in preterm infants with cystic periventricular leukomalacia.

AIM: Hypocarbia induced by mechanical ventilation has been considered a main cause of cystic periventricular leukomalacia (cPVL). However, hypocarbia may occur spontaneously in response to intracellular metabolic acidosis. We aimed to assess whether hypocarbia is more common during mechanical respiratory support than during spontaneous ventilation in infants with cPVL. METHOD: In this single-centre, retrospective chart analysis, we compared partial pressure of carbon dioxide (pCO2 ) during the first 96 hours of life in infants with cPVL during endotracheal mechanical ventilation, non-invasive respiratory support, or without respiratory support. RESULTS: Cystic periventricular leukomalacia was diagnosed in 23 infants born between 2006 and 2017. Gestational age was 24 weeks in two infants and ranged between 28 and 32 weeks in 21 infants. In these 21 infants, pCO2 less than 35 mm Hg during the first 96 ours of life accounted for 9/60 (15%) measurements during endotracheal mechanical ventilation, 16/116 (14%) during non-invasive respiratory support and 14/42 (33%) in infants without respiratory support (P = .014). CONCLUSION: In our series of infants with cPVL, hypocarbia was more common without respiratory support than during endotracheal mechanical ventilation and non-invasive respiratory support. This would suggest that hypocarbia is a symptom rather than a cause of cPVL in these infants.

Characterization of circulating plasma proteins in dairy cows with cytological endometritis.

Early diagnosis of endometritis in dairy cattle is currently requires invasive techniques and specialist expertise. The goal of this study is to utilize a gel-free mass-spectrometry based proteomics approach to compare the plasma proteome of dairy cattle with cytological endometritis to those without. Blood samples were collected from cows (N = 112) seven days postpartum (DPP). Plasma samples from a cohort of 20 animals with cytological endometritis (n = 10) and without (n = 10) as classified 21 DPP were selected for proteomic analysis. Differential abundances of proteins between the two animal groups were determined using both fold change (≥1.5 fold change) and statistical significance threshold (p < .05). A total of 181 non-redundant proteins were quantified, and 25 proteins were found with differential abundance. These include 4 binding protein alpha and mannose binding lectin 2 involved in immune responses. Differentially abundant proteins between the animals were then processed using PANTHER for gene ontology. Gene ontology included associations with innate immune processes, acute phase responses and immune regulation. A potential marker for disease identified here is the "uncharacterized protein G5E513," a protein previously defined by RNA-transcripts. These proteins may form the basis for endometritis prognosis, the development of which is proceeded by systemic changes in immune function. SIGNIFICANCE: Endometritis is a costly reproductive disease of lactating dairy cows that warrants timely diagnosis. We utilized a gel-free mass-spectrometry based proteomics approach to compare the plasma proteome of dairy cattle with cytological endometritis to those without, for the characterization of changes in the proteomic profile associated with uterine disease postpartum. Furthermore, we compared the plasma proteome of healthy and affected cows in the same physiological status of production to better understand the relationship between changes in expression of circulating proteins and to unravel essential biological mechanisms involved in bovine cytological endometritis.

Generation and Characterization of Anti-VGLUT Nanobodies Acting as Inhibitors of Transport.

The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transporters in excitatory neurotransmission underpins their importance as pharmacological targets. Although several compounds inhibit VGLUTs, highly specific inhibitors were so far unavailable, thus limiting applications to in vitro experiments. Besides their potential in pharmacology, specific inhibitors would also be beneficial for the elucidation of transport mechanisms. To overcome this shortage, we generated nanobodies (Nbs) by immunization of a llama with purified rat VGLUT1 and subsequent selection of binders from a phage display library. All identified Nbs recognize cytosolic epitopes, and two of the binders greatly reduced the rate of uptake of glutamate by reconstituted liposomes and subcellular fractions enriched with synaptic vesicles. These Nbs can be expressed as functional green fluorescent protein fusion proteins in the cytosol of HEK cells for intracellular applications as immunocytochemical and biochemical agents. The selected binders thus provide valuable tools for cell biology and neuroscience.