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BACKGROUND AND AIMS IgA nephropathy (IgAN) is characterized by the mesangial deposition of IgA-containing immune complexes (IgA-IC). There is, however, no clear association between extent of mesangial IgA-IC deposition and disease severity in IgAN, suggesting that the composition of the IgA-IC, rather than the actual amount, may be critical in determining pathogenic consequences. The NEFIGAN trial (NCT01738035) assessed the safety and efficacy of a novel targeted-release formulation of budesonide (Nefecon®), designed to deliver the drug to the gut-associated lymphoid tissue (GALT)-rich distal ileum in patients with IgAN. The trial comprised a 6-month run-in, a 9-month treatment and a 3-month follow-up phase. A total of 48 patients received Nefecon® 16 mg/day, 51 patients received Nefecon® 8 mg/day and 50 patients received placebo. The key result of the study was that Nefecon® 16 mg/day, added to optimized renin–angiotensin system blockade, reduced proteinuria and stabilized estimated glomerular filtration rate in patients with IgAN. These findings have now been replicated in the NefIgArd study, which released data in 2021. Here, we report the composition of IgA-IC in the serum of patients treated with Nefecon® 16 mg/day or placebo in the NEFIGAN trial, assessed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). This study was supported by a grant from Calliditas Therapeutics AB. METHOD Using Jacalin Agarose and elution with 0.1 M d-galactose, IgA-ICs were affinity-purified from serum of patients who received placebo (n=18) or Nefecon® 16 mg/day (n=18) at the start of treatment (SOT) and end of treatment (EOT). All protein samples were reduced and alkylated prior to digestion with trypsin, dried using a Savant SpeedVac (Thermo Fisher Scientific, UK) and dissolved in 40 µL of 0.1% formic acid. Each sample, spiked with iRT standard peptides (Biognosys, Switzerland), was loaded onto an Evotip (Evosep Biosystems, Denmark) column. Mass spectrometry analyses were performed on the Evosep One (Evosep) platform, coupled to the TIMS TOF Pro (Bruker, US). For protein identification and quantification, raw data were processed with FragPipe (V16), based on at least two peptides per protein. The protein intensities thus generated were Log_2-transformed and internally normalized using IGHA1 protein as standard. For each patient, the normalized protein amount at SOT was subtracted from the amount at EOT. A probabilistic dropout model was applied to the data to estimate fold changes between treatment and placebo groups at EOT. The proteins were ranked using t-statistic, and a gene set enrichment analysis was performed to determine the gene sets significantly affected by Nefecon® 16 mg/day versus placebo. RESULTS Gene ontology analysis revealed that treatment with Nefecon® 16 mg/day led to a significant protein enrichment in IgA-IC associated with pathways driving biological processes involved in the negative regulation of gene expression and transcription, gene silencing and epigenetics. In parallel, treatment with Nefecon® was also associated with an enrichment of proteins associated with extracellular vesicle and exosome formation. CONCLUSION For the first time, we show that it is possible to pharmacologically modify the composition of circulating IgA-IC in IgAN. Treatment with Nefecon® 16 mg/day for 9 months selectively altered the protein composition of IgA-IC in a way that is likely to have significant consequences on the downstream mesangial response to IgA-IC accumulation; this may, in part, explain the antiproteinuric effect of this approach and the longer-term kidney function protection that has been reported in both the NEFIGAN and NefIgArd clinical trials. Studies are ongoing to precisely delineate the impact of these compositional changes on the pathogenicity of IgA-IC in IgAN.

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.

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., Neoplasia, 23 (4), ISSN:1522-8002, ISSN:1476-5586

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 Mountains(1,2) and Mongolia(3). Although some models hold that this expansion was the outcome of a newly mobile pastoral economy characterized by horse traction, bulk wagon transport(4-6) and regular dietary dependence on meat and milk(5), 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., Nature, 598 (7882), ISSN:0028-0836, ISSN:1476-4687

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.

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., Using a network propagation approach with integrated multi-omic data, Selevsek et al. develop a reproducible workflow for identifying drug toxicity effects in cellular systems. This is demonstrated with the analysis of anthracycline cardiotoxicity in cardiac microtissues under the effect of multiple drugs.

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, Journal of Proteomics, 205, ISSN:1874-3919, ISSN:1876-7737

This paper analyses CO2 emissions reduction costs based on project data from the Climate Cent Foundation (CCF), a climate policy instrument in Switzerland. Four conclusions are drawn. First, for the projects investigated, the CCF on average pays €63/ton. Due to the Kyoto Protocol, the CCF buys reductions only until 2012. This cut-off increases reported per ton reduction costs, as the additional lifetime project costs are set in relation to reductions only until 2012, rather than to reductions realised over the whole lifetime. Lifetime reduction costs are €45/t. Second, correlation between CCF's payments and lifetime reduction costs per ton is low. Projects with low per ton reduction costs should thus be identified based on lifetime per ton reduction costs. Third, the wide range of project costs per ton observed casts doubts on the widely used identification of the merit order of reduction measures based on average per ton costs for technology types. Finally, the CCF covers only a fraction of additional re...

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.