Midkine and chronic kidney disease-associated multisystem organ dysfunctions.

Chronic kidney disease (CKD) is a progressive multisystem condition with yet undefined mechanistic drivers and multiple implicated soluble factors. If identified, these factors could be targeted for therapeutic intervention for a disease that currently lacks specific treatment. There is increasing preclinical evidence that the heparin/endothelial glycocalyx-binding molecule midkine (MK) has a pathological role in multiple CKD-related, organ-specific disease processes, including CKD progression, hypertension, vascular and cardiac disease, bone disease and CKD-related cancers. Concurrent with this are studies documenting increases in circulating and urine MK proportional to glomerular filtration rate (GFR) loss in CKD patients and evidence that administering soluble MK reverses the protective effects of MK deficiency in experimental kidney disease. This review summarizes the growing body of evidence supporting MK's potential role in driving CKD-related multisystem disease, including MK's relationship with the endothelial glycocalyx, the deranged MK levels and glycocalyx profile in CKD patients and a proposed model of MK organ interplay in CKD disease processes and highlights the importance of ongoing research into MK's potential as a therapeutic target.

The CD151-midkine pathway regulates the immune microenvironment in inflammatory breast cancer.

The immune microenvironment in inflammatory breast cancer (IBC) is poorly characterised, and molecular and cellular pathways that control accumulation of various immune cells in IBC tissues remain largely unknown. Here, we discovered a novel pathway linking the expression of the tetraspanin protein CD151 in tumour cells with increased accumulation of macrophages in cancerous tissues. It is notable that elevated expression of CD151 and a higher number of tumour-infiltrating macrophages correlated with better patient responses to chemotherapy. Accordingly, CD151-expressing IBC xenografts were characterised by the increased infiltration of macrophages. In vitro migration experiments demonstrated that CD151 stimulates the chemoattractive potential of IBC cells for monocytes via mechanisms involving midkine (a heparin-binding growth factor), integrin α6ß1, and production of extracellular vesicles (EVs). Profiling of chemokines secreted by IBC cells demonstrated that CD151 increases production of midkine. Purified midkine specifically stimulated migration of monocytes, but not other immune cells. Further experiments demonstrated that the chemoattractive potential of IBC-derived EVs is blocked by anti-midkine antibodies. These results demonstrate for the first time that changes in the expression of a tetraspanin protein by tumour cells can affect the formation of the immune microenvironment by modulating recruitment of effector cells to cancerous tissues. Therefore, a CD151-midkine pathway can be considered as a novel target for controlled changes of the immune landscape in IBC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Stratification of asthma phenotypes by airway proteomic signatures.

BACKGROUND: Stratification by eosinophil and neutrophil counts increases our understanding of asthma and helps target therapy, but there is room for improvement in our accuracy in prediction of treatment responses and a need for better understanding of the underlying mechanisms. OBJECTIVE: We sought to identify molecular subphenotypes of asthma defined by proteomic signatures for improved stratification. METHODS: Unbiased label-free quantitative mass spectrometry and topological data analysis were used to analyze the proteomes of sputum supernatants from 246 participants (206 asthmatic patients) as a novel means of asthma stratification. Microarray analysis of sputum cells provided transcriptomics data additionally to inform on underlying mechanisms. RESULTS: Analysis of the sputum proteome resulted in 10 clusters (ie, proteotypes) based on similarity in proteomic features, representing discrete molecular subphenotypes of asthma. Overlaying granulocyte counts onto the 10 clusters as metadata further defined 3 of these as highly eosinophilic, 3 as highly neutrophilic, and 2 as highly atopic with relatively low granulocytic inflammation. For each of these 3 phenotypes, logistic regression analysis identified candidate protein biomarkers, and matched transcriptomic data pointed to differentially activated underlying mechanisms. CONCLUSION: This study provides further stratification of asthma currently classified based on quantification of granulocytic inflammation and provided additional insight into their underlying mechanisms, which could become targets for novel therapies.

Large-Scale Label-Free Quantitative Mapping of the Sputum Proteome.

Analysis of induced sputum supernatant is a minimally invasive approach to study the epithelial lining fluid and, thereby, provide insight into normal lung biology and the pathobiology of lung diseases. We present here a novel proteomics approach to sputum analysis developed within the U-BIOPRED (unbiased biomarkers predictive of respiratory disease outcomes) international project. We present practical and analytical techniques to optimize the detection of robust biomarkers in proteomic studies. The normal sputum proteome was derived using data-independent HDMSE applied to 40 healthy nonsmoking participants, which provides an essential baseline from which to compare modulation of protein expression in respiratory diseases. The "core" sputum proteome (proteins detected in ≥40% of participants) was composed of 284 proteins, and the extended proteome (proteins detected in ≥3 participants) contained 1666 proteins. Quality control procedures were developed to optimize the accuracy and consistency of measurement of sputum proteins and analyze the distribution of sputum proteins in the healthy population. The analysis showed that quantitation of proteins by HDMSE is influenced by several factors, with some proteins being measured in all participants' samples and with low measurement variance between samples from the same patient. The measurement of some proteins is highly variable between repeat analyses, susceptible to sample processing effects, or difficult to accurately quantify by mass spectrometry. Other proteins show high interindividual variance. We also highlight that the sputum proteome of healthy individuals is related to sputum neutrophil levels, but not gender or allergic sensitization. We illustrate the importance of design and interpretation of disease biomarker studies considering such protein population and technical measurement variance.

Urine and serum midkine levels in an Australian chronic kidney disease clinic population: an observational study.

BACKGROUND AND OBJECTIVES: The cytokine midkine (MK) is pathologically implicated in progressive chronic kidney disease (CKD) and its systemic consequences and has potential as both a biomarker and therapeutic target. To date, there are no published data on MK levels in patients with different stages of CKD. This study aims to quantify MK levels in patients with CKD and to identify any correlation with CKD stage, cause, progression, comorbid disease or prescribed medication. METHODS: In this observational, single-centre study, demographic data were collected, and serum and urine assayed from 197 patients with CKD and 19 healthy volunteers in an outpatient setting. RESULTS: The median serum and urine MK level in volunteers was 754 pg/mL (IQR: 554-1025) and 239 pg/mL (IQR: 154-568), respectively. Compared with serum MK in stage 1 CKD (660 pg/mL, IQR: 417-893), serum MK increased in stage 3 (1878 pg/mL, IQR: 1188-2756; p<0.001), 4 (2768 pg/mL, IQR: 2065-4735; p<0.001) and 5 (4816 pg/mL, IQ: 37477807; p<0.001). Urine MK levels increased from stage 1 CKD (343 pg/mL, IQR: 147-437) to stage 3 (1007 pg/mL, IQR: 465-2766; p=0.07), 4 (2961 pg/mL, IQR: 1368-5686; p=0.005) and 5 (6722 pg/mL, IQR: 3796-10 060; p=0.001). Fractional MK excretion (FeMK) increased from stage 1 CKD (0.159, IQR: 0.145-0.299) to stage 3 (1.024, IQR: 0.451-1.886, p=0.047), 4 (3.39, IQR: 2.10-5.82, p=0.004) and 5 (11.95, IQR: 5.36-24.41, p<0.001). When adjusted for estimated glomerular filtration rate, neither serum nor urine MK correlated with primary CKD diagnosis or CKD progression (small sample). There was a positive correlation between protein:creatinine ratio and FeMK (p=0.003). Angiotensin blockade (adjusted for proteinuria) was associated with lower urine MK (p=0.018) and FeMK (p=0.025). CONCLUSION: MK levels sequentially rise with CKD stage beyond stage 2, and our data support existing animal evidence for an MK/renin angiotensin-system/proteinuria relationship. To what extent this is related to renal clearance versus pathology, or the consequences of chronically elevated MK levels requires further exploration.

Promotion of anagen, increased hair density and reduction of hair fall in a clinical setting following identification of FGF5-inhibiting compounds via a novel 2-stage process.

BACKGROUND: There are very few effective, scientifically validated treatments with known mechanisms of action for treatment of hair loss in both men and women. Fibroblast growth factor 5 (FGF5) is an important factor in the irreversible transition from anagen to catagen, and inhibition of FGF5 prolongs anagen phase and reduces hair loss. OBJECTIVE: We aimed to screen botanically derived molecules for FGF5 inhibitory activity in vitro and assess efficacy in a clinical setting. METHODS: We screened for FGF5 inhibitory efficacy via a novel 2-step in vitro pipeline consisting of an engineered FGF5 responsive cell line, followed by an activated dermal papillae (DP) cell method. Efficacy in a clinical setting was assessed in a randomized, single-blind, placebo-controlled trial against early- to mid-stage pattern hair loss in men and women. RESULTS: We observed FGF5 inhibitory activity for a number of compounds from the monoterpenoid family, many showing greater inhibitory efficacy than our previously reported crude plant extracts. Evaluation of a lead candidate in a clinical study over 112 days showed a significant improvement in anagen:telogen (AT) ratio (p = 0.002), reduced hair fall (p = 0.007) and improved visual grading (p = 0.004). Scientifically matched photography on a subgroup of randomly chosen participants highlighted significant improvement in hair density, with increases evident in all tested participants compared to baseline. CONCLUSION: Isolates from the monoterpenoid family displayed efficacy in FGF5 inhibition in vitro. A topical formulation containing a leading isolate significantly improved AT ratio, reduced hair fall and increased apparent hair density in the tested population of men and women.

Application of 'omics technologies to biomarker discovery in inflammatory lung diseases.

Inflammatory lung diseases are highly complex in respect of pathogenesis and relationships between inflammation, clinical disease and response to treatment. Sophisticated large-scale analytical methods to quantify gene expression (transcriptomics), proteins (proteomics), lipids (lipidomics) and metabolites (metabolomics) in the lungs, blood and urine are now available to identify biomarkers that define disease in terms of combined clinical, physiological and patho-biological abnormalities. The aspiration is that these approaches will improve diagnosis, i.e. define pathological phenotypes, and facilitate the monitoring of disease and therapy, and also, unravel underlying molecular pathways. Biomarker studies can either select predefined biomarker(s) measured by specific methods or apply an "unbiased" approach involving detection platforms that are indiscriminate in focus. This article reviews the technologies presently available to study biomarkers of lung disease within the 'omics field. The contributions of the individual 'omics analytical platforms to the field of respiratory diseases are summarised, with the goal of providing background on their respective abilities to contribute to systems medicine-based studies of lung disease.

Activation of thermogenesis in brown adipose tissue and dysregulated lipid metabolism associated with cancer cachexia in mice.

Cancer cachexia/anorexia is a complex syndrome that involves profound metabolic imbalances and is directly implicated as a cause of death in at least 20% to 30% of all cancers. Brown adipose tissue (BAT) plays a key role in thermogenesis and energy balance and potentially contributes to the physiologic perturbations associated with cachexia. In this study, we investigated the impact of cachexia-inducing colorectal tumor on BAT in mice. We found that brown adipocytes were smaller and exhibited profound delipidation in cachectic tumor-bearing mice. Diurnal expression profiling of key regulators of lipid accumulation and fatty acid ß-oxidation and their corresponding target genes revealed dramatic molecular changes indicative of active BAT. Increased Ucp1, Pbe, and Cpt1α expression at specific points coincided with higher BAT temperatures during the dark cycle, suggestive of a temporal stimulation of thermogenesis in cachexia. These changes persisted when cachectic mice were acclimatized to 28°C confirming inappropriate stimulation of BAT despite thermoneutrality. Evidence of inflammatory signaling also was observed in the BAT as an energetically wasteful and maladaptive response to anorexia during the development of cachexia.

Proteomics of extremophiles.

Functional genomic approaches, such as proteomics, greatly enhance the value of genome sequences by providing a global level assessment of which genes are expressed, when genes are expressed and at what cellular levels gene products are synthesized. With over 1000 complete genome sequences of different microorganisms available, and DNA sequencing for environmental samples (metagenomes) producing vast amounts of gene sequence data, there is a real opportunity and a clear need to generate associated functional genomic data to learn about the source microorganisms. In contrast to the technological advances that have led to the accelerated rate and ease at which DNA sequence data can be generated, mass spectrometry based proteomics remains a technically sophisticated and exacting science. In recognition of the need to make proteomics more accessible to a growing number of environmental microbiologists so that the 'functional genomics gap' may be bridged, this review strives to demystify proteomic technologies and describe ways in which they have been applied, and more importantly, can be applied to study the physiology and ecology of extremophiles.

Chaperonins from an Antarctic archaeon are predominantly monomeric: crystal structure of an open state monomer.

Archaea are abundant in permanently cold environments. The Antarctic methanogen, Methanococcoides burtonii, has proven an excellent model for studying molecular mechanisms of cold adaptation. Methanococcoides burtonii contains three group II chaperonins that diverged prior to its closest orthologues from mesophilic Methanosarcina spp. The relative abundance of the three chaperonins shows little dependence on organism growth temperature, except at the highest temperatures, where the most thermally stable chaperonin increases in abundance. In vitro and in vivo, the M. burtonii chaperonins are predominantly monomeric, with only 23-33% oligomeric, thereby differing from other archaea where an oligomeric ring form is dominant. The crystal structure of an N-terminally truncated chaperonin reveals a monomeric protein with a fully open nucleotide binding site. When compared with closed state group II chaperonin structures, a large-scale ≈ 30° rotation between the equatorial and intermediate domains is observed resulting in an open nucleotide binding site. This is analogous to the transition observed between open and closed states of group I chaperonins but contrasts with recent archaeal group II chaperonin open state ring structures. The predominance of monomeric form and the ability to adopt a fully open nucleotide site appear to be unique features of the M. burtonii group II chaperonins.

Defining the response of a microorganism to temperatures that span its complete growth temperature range (-2°C to 28°C) using multiplex quantitative proteomics.

The growth of all microorganisms is limited to a specific temperature range. However, it has not previously been determined to what extent global protein profiles change in response to temperatures that incrementally span the complete growth temperature range of a microorganism. As a result it has remained unclear to what extent cellular processes (inferred from protein abundance profiles) are affected by growth temperature and which, in particular, constrain growth at upper and lower temperature limits. To evaluate this, 8-plex iTRAQ proteomics was performed on the Antarctic microorganism, Methanococcoides burtonii. Methanococcoides burtonii was chosen due to its importance as a model psychrophilic (cold-adapted) member of the Archaea, and the fact that proteomic methods, including subcellular fractionation procedures, have been well developed. Differential abundance patterns were obtained for cells grown at seven different growth temperatures (-2°C, 1°C, 4°C, 10°C, 16°C, 23°C, 28°C) and a principal component analysis (PCA) was performed to identify trends in protein abundances. The multiplex analysis enabled three largely distinct physiological states to be described: cold stress (-2°C), cold adaptation (1°C, 4°C, 10°C and 16°C), and heat stress (23°C and 28°C). A particular feature of the thermal extremes was the synthesis of heat- and cold-specific stress proteins, reflecting the important, yet distinct ways in which temperature-induced stress manifests in the cell. This is the first quantitative proteomic investigation to simultaneously assess the response of a microorganism to numerous growth temperatures, including the upper and lower growth temperatures limits, and has revealed a new level of understanding about cellular adaptive responses.

A chemically modified alpha-amylase with a molten-globule state has entropically driven enhanced thermal stability.

The thermostability properties of TAA were investigated by chemically modifying carboxyl groups on the surface of the enzyme with AMEs. The TAA(MOD) exhibited a 200% improvement in starch-hydrolyzing productivity at 60 degrees C. By studying the kinetic, thermodynamic and biophysical properties, we found that TAA(MOD) had formed a thermostable, MG state, in which the unfolding of the tertiary structure preceded that of the secondary structure by at least 20 degrees C. The X-ray crystal structure of TAA(MOD) revealed no new permanent interactions (electrostatic or other) resulting from the modification. By deriving thermodynamic activation parameters of TAA(MOD), we rationalised that thermostabilisation have been caused by a decrease in the entropy of the transition state, rather than being enthalpically driven. Far-UV CD shows that the origin of decreased entropy may have arisen from a higher helical content of TAA(MOD). This study provides new insight into the intriguing properties of an MG state resulting from the chemical modification of TAA.

Analyzing the hydrophobic proteome of the antarctic archaeon Methanococcoides burtonii using differential solubility fractionation.

Proteomic studies have proven useful for studying the Antarctic archaeon Methanococcoides burtonii; however, little has been learned about the hydrophobic and membrane proteins, despite knowledge of their biological importance. In this study, new methods were developed to analyze and maximize the coverage of the hydrophobic proteome. Central to the analysis was a differential solubility fractionation (DSF) procedure using n-octyl-beta-D-glucopyranoside. The study achieved a significant increase (330) in the total number of known expressed proteins. From 612 identified, 185 were predicted to contain transmembrane domains or be associated with the membrane and 190 to be hydrophobic. The DSF procedure increased the efficacy of identifying membrane proteins by up to 169% and was economical, requiring far fewer runs (12% of machine time) to analyze the proteome compared to procedures without DSF. The analysis of peptide spectral counts enabled the assessment of growth temperature specific proteins. This semiquantitative analysis was particularly useful for identifying low abundance proteins unable to be quantified using labeling strategies. The proteogenomic analysis of the newly identified proteins revealed many cellular processes not previously associated with adaptation of the cell. This DSF-based approach is likely to benefit proteomic analyses of hydrophobic proteins for a broad range of biological systems.

Global proteomic analysis of the insoluble, soluble, and supernatant fractions of the psychrophilic archaeon Methanococcoides burtonii. Part I: the effect of growth temperature.

The response of the cold-adapted (psychrophilic) methanogenic archaeon Methanococcoides burtonii to growth temperature was investigated using differential proteomics (postincorporation isobaric labeling) and tandem liquid chromatography-mass spectrometry (LC/LC-MS/MS). This is the first proteomic study of M. burtonii to include techniques that specifically enrich for both surface and membrane proteins and to assess the effects of growth temperature (4 vs 23 degrees C) and carbon source (trimethylamine vs methanol) on cellular protein levels. Numerous surface layer proteins were more abundant at 4 degrees C, indicating an extensive remodeling of the cell envelope in response to low temperature. Many of these surface proteins contain domains associated with cell adhesion. Within the cell, small proteins each composed of a single TRAM domain were recovered as important cold adaptation proteins and might serve as RNA chaperones, in an analogous manner to Csp proteins (absent from M. burtonii). Other proteins that had higher abundances at 4 degrees C can be similarly tied to relieving or resolving the adverse affects of cold growth temperature on translational capacity and correct protein folding. The proteome of M. burtonii grown at 23 degrees C was dominated by oxidative stress proteins, as well as a large number of integral membrane proteins of unknown function. This is the first truly global proteomic study of a psychrophilic archaeon and greatly expands knowledge of the cellular mechanisms underpinning cold adaptation in the Archaea.

Global proteomic analysis of the insoluble, soluble, and supernatant fractions of the psychrophilic archaeon Methanococcoides burtonii. Part II: the effect of different methylated growth substrates.

Methanococcoides burtonii is a cold-adapted methanogenic archaeon from Ace Lake in Antarctica. Methanol and methylamines are the only substrates it can use for carbon and energy. We carried out quantitative proteomics using iTRAQ of M. burtonii cells grown on different substrates (methanol in defined media or trimethylamine in complex media), using techniques that enriched for secreted and membrane proteins in addition to cytoplasmic proteins. By integrating proteomic data with the complete, manually annotated genome sequence of M. burtonii, we were able to gain new insight into methylotrophic metabolism and the effects of methanol on the cell. Metabolic processing of methanol and methylamines is initiated by methyltransferases specific for each substrate, with multiple paralogs for each of the methyltransferases (similar to other members of the Methanosarcinaceae). In M. burtonii, most methyltransferases appear to have distinct roles in the metabolism of methylated substrates, although two methylamine methyltransferases appear to be nonfunctional. One set of methyltransferases for trimethylamine catabolism appears to be membrane associated, potentially providing a mechanism to directly couple trimethylamine uptake to demethylation. Important roles were highlighted for citrate synthase, glutamine synthetase, acetyl-CoA decarbonylase/synthase, and pyruvate synthase in carbon and nitrogen metabolism during growth on methanol. M. burtonii had only a marginal response to the provision of exogenous amino acids (from yeast extract), indicating that it is predisposed to the endogenous synthesis of amino acids. Growth on methanol appeared to cause oxidative stress in the cell, possibly through the formation of reactive nonoxygen species and formaldehyde, and the oxidative inactivation of corrinoid proteins, with the cell responding by elevating the synthesis of universal stress (Usp) proteins, several nucleic acid binding proteins, and a serpin. In addition, changes in levels of cell envelope proteins were linked to counteracting the disruptive solvent effects of methanol on cell membranes. This is the first global proteomic study to examine the effects of different carbon sources on the growth of an obligately methylotrophic methanogen.

The genome sequence of the psychrophilic archaeon, Methanococcoides burtonii: the role of genome evolution in cold adaptation.

Psychrophilic archaea are abundant and perform critical roles throughout the Earth's expansive cold biosphere. Here we report the first complete genome sequence for a psychrophilic methanogenic archaeon, Methanococcoides burtonii. The genome sequence was manually annotated including the use of a five-tiered evidence rating (ER) system that ranked annotations from ER1 (gene product experimentally characterized from the parent organism) to ER5 (hypothetical gene product) to provide a rapid means of assessing the certainty of gene function predictions. The genome is characterized by a higher level of aberrant sequence composition (51%) than any other archaeon. In comparison to hyper/thermophilic archaea, which are subject to selection of synonymous codon usage, M. burtonii has evolved cold adaptation through a genomic capacity to accommodate highly skewed amino-acid content, while retaining codon usage in common with its mesophilic Methanosarcina cousins. Polysaccharide biosynthesis genes comprise at least 3.3% of protein coding genes in the genome, and Cell wall, membrane, envelope biogenesis COG genes are overrepresented. Likewise, signal transduction (COG category T) genes are overrepresented and M. burtonii has a high 'IQ' (a measure of adaptive potential) compared to many methanogens. Numerous genes in these two overrepresented COG categories appear to have been acquired from epsilon- and delta-Proteobacteria, as do specific genes involved in central metabolism such as a novel B form of aconitase. Transposases also distinguish M. burtonii from other archaea, and their genomic characteristics indicate they have an important role in evolving the M. burtonii genome. Our study reveals a capacity for this model psychrophile to evolve through genome plasticity (including nucleotide skew, horizontal gene transfer and transposase activity) that enables adaptation to the cold, and to the biological and physical changes that have occurred over the last several thousand years as it adapted from a marine to an Antarctic lake environment.