Comprehensive Proteomics Analysis of Polyhydroxyalkanoate (PHA) Biology in Pseudomonas putida KT2440: The Outer Membrane Lipoprotein OprL is a Newly Identified Phasin.

Pseudomonas putida KT2440 is an important bioplastic-producing industrial microorganism capable of synthesizing the polymeric carbon-rich storage material, polyhydroxyalkanoate (PHA). PHA is sequestered in discrete PHA granules, or carbonosomes, and accumulates under conditions of stress, for example, low levels of available nitrogen. The pha locus responsible for PHA metabolism encodes both anabolic and catabolic enzymes, a transcription factor, and carbonosome-localized proteins termed phasins. The functions of phasins are incompletely understood but genetic disruption of their function causes PHA-related phenotypes. To improve our understanding of these proteins, we investigated the PHA pathways of P.putida KT2440 using three types of experiments. First, we profiled cells grown in nitrogen-limited and nitrogen-excess media using global expression proteomics, identifying sets of proteins found to coordinately increase or decrease within clustered pathways. Next, we analyzed the protein composition of isolated carbonosomes, identifying two new putative components. We carried out physical interaction screens focused on PHA-related proteins, generating a protein-protein network comprising 434 connected proteins. Finally, we confirmed that the outer membrane protein OprL (the Pal component of the Pal-Tol system) localizes to the carbonosome and shows a PHA-related phenotype and therefore is a novel phasin. The combined datasets represent a valuable overview of the protein components of the PHA system in P.putida highlighting the complex nature of regulatory interactions responsive to nutrient stress.

Back to the future: Comparing yeast as an outmoded artificial tracer for simulating microbial transport in karst aquifer systems to more modern approaches.

Bacterial contamination of karst groundwater is a major concern for public health. Artificial tracing studies are crucial for establishing links between locations where pollutants can rapidly reach the aquifer systems and subsequent receptors, as well as for enhanced understanding of pollutant transport. However, widely used solute artificial tracers do not always move through the subsurface in the same manner as particles and microorganisms, hence may not be ideal proxies for predicting movement of bacterial contaminants. This study evaluates whether a historically used microbial tracer (yeast) which is readily available, inexpensive, and environmentally friendly, but usually overlooked in modern karst hydrogeological studies due to challenges associated with its detection and quantification in the past, can reemerge as a valuable tracer using the latest technology for its detection. Two field-based studies on separate karst systems were carried out during low-flow conditions using a portable particle counter along with flow cytometry measurements to monitor the recovery of the yeast at the springs. Soluble fluorescent dyes were also injected simultaneously with the yeast for comparison of transport dynamics. On one tracer test, through a karst conduit of much higher velocities, the injected yeast and fluorescent dye arrived at the same time at the spring, in comparison to the tracer test on a conduit system with lower groundwater velocities in which the yeast particles were detected before the dye at the sampling site. Both a portable particle counter and flow cytometry successfully detected yeast during both tests, thereby demonstrating the applicability of this tracer with contemporary instrumentation. Even though no significant advantages of flow cytometry over the portable counter system can be reported on the basis of the presented results, this study has shown that flow cytometry can be successfully used to detect and quantify introduced microbial tracers in karst environments with extremely high precision.

The use of sterol profiles, supported with other faecal source tracking methods, to apportion septic tanks contamination in rural catchments.

Identifying the origin of faecal pollution in water is needed for effective water management decisions to protect both human health and aquatic ecosystems. Traditionally used indicators of faecal contamination, such as E. coli, only indicate pollution from warm-blooded animals and not the specific source of contamination; hence, more source specific tracers are required. The study has focussed on separating the two main sources of contaminants within rural catchments in Ireland, agriculture and on-site wastewater treatment systems (predominantly septic tanks). While human-specific effluent tracers may assist in identifying potential pathways from individual septic tanks to surface waters, it is difficult to quantify the cumulative impact of such systems at a catchment scale. This study has investigated faecal sterols as a method to quantify such an impact on four small catchments in areas of low subsoil permeability with high densities of septic tanks. The results demonstrate the usefulness of faecal sterols which provide a quantitative evaluation of the respective impact between agricultural pasture inputs and on-site effluent showing differences between the four catchments. The study also highlights the need to derive more specific local reference sterol profile databases for specific countries or regions, using local source material of animal faeces and effluent. Two intensive sampling campaigns on the four catchments then used faecal sterols in parallel to fluorescent whitening compounds (FWCs), caffeine, artificial sweeteners and selected pharmaceuticals to gain further insights and confirmation about contamination hotspots as well as providing comparison between the different parameters. The combination of sterols, FWCs, caffeine, acesulfame and cyclamate has proven suitable to provide an estimate of the extent of human contamination in these rural catchments and has yielded additional information about potential pollution pathways and proximity of contamination. Overall, this methodology can help to facilitate a targeted and effective water management in such catchments.

Trends in Serum Cytokine Expression in Pediatric Skeletal Dysplasia.

The skeletal dysplasias are a heterogeneous group of genetic conditions caused by abnormalities of growth, development, and maintenance of bone and cartilage. Little is known about the roles that cytokines play in the inflammatory and non-inflammatory pathophysiology of skeletal dysplasia. We sought to test our hypothesis that cytokines would be differentially expressed in children with skeletal dysplasia as compared to typically growing controls. Cytokine levels were analyzed using the Cytokine Human Magnetic 25-Plex Panel (Invitrogen, Waltham, MA, USA); 136 growing individuals with skeletal dysplasia and compared to a cohort of 275 healthy pediatric control subjects. We focused on the expression of 12 cytokines across nine dysplasia cohorts. The most common skeletal dysplasia diagnoses were: achondroplasia (58), osteogenesis imperfecta (19), type II collagenopathies (11), multiple epiphyseal dysplasia (MED: 9), diastrophic dysplasia (8), metatropic dysplasia (8), and microcephalic osteodysplastic primordial dwarfism type II (MOPDII: 8). Of the 108 specific observations made, 45 (41.7%) demonstrated statistically significant differences of expression between controls and individuals with skeletal dysplasia. Four of the 12 analyzed cytokines demonstrated elevated expression above control levels in all of the dysplasia cohorts (interleukin 12 [IL-12], IL-13, interferon γ-induced protein 10 kDa [IP-10], regulated on activation, normal T cell expressed and secreted [RANTES]) and two demonstrated expression below control levels across all dysplasia cohorts (monocyte chemoattractant protein 1 [MCP-1], macrophage inflammatory protein-1ß [MIP-1ß]). The highest levels of overexpression were seen in MOPDII, with expression levels of IP-10 being increased 3.8-fold (p < 0.0001). The lowest statistically significant levels of expressions were in type II collagenopathies, with expression levels of MCP-1 being expressed 0.43-fold lower (p < 0.005). With this data, we hope to lay the groundwork for future directions in dysplasia research that will enhance our understanding of these complex signaling pathways. Looking forward, validating these early trends in cytokine expression, and associating the observed variations with trends in the progression of dysplasia may offer new candidates for clinical biomarkers or even new therapeutics. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Identification of potential non-invasive biomarkers in diastrophic dysplasia.

Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by pathogenic variants in the SLC26A2 gene encoding for a cell membrane sulfate/chloride antiporter crucial for sulfate uptake and glycosaminoglycan (GAG) sulfation. Research on a DTD animal model has suggested possible pharmacological treatment approaches. In view of future clinical trials, the identification of non-invasive biomarkers is crucial to assess the efficacy of treatments. Urinary GAG composition has been analyzed in several metabolic disorders including mucopolysaccharidoses. Moreover, the N-terminal fragment of collagen X, known as collagen X marker (CXM), is considered a real-time marker of endochondral ossification and growth velocity and was studied in individuals with achondroplasia and osteogenesis imperfecta. In this work, urinary GAG sulfation and blood CXM levels were investigated as potential biomarkers for individuals affected by DTD. Chondroitin sulfate disaccharide analysis was performed on GAGs isolated from urine by HPLC after GAG digestion with chondroitinase ABC and ACII, while CXM was assessed in dried blood spots. Results from DTD patients were compared with an age-matched control population. Undersulfation of urinary GAGs was observed in DTD patients with some relationship to the clinical severity and underlying SLC26A2 variants. Lower than normal CXM levels were observed in most patients, even if the marker did not show a clear pattern in our small patient cohort because CXM values are highly dependent on age, gender and growth velocity. In summary, both non-invasive biomarkers are promising assays targeting various aspects of the disorder including overall metabolism of sulfated GAGs and endochondral ossification.

Macrophage Galactose Lectin Contributes to the Regulation of FVIII (Factor VIII) Clearance in Mice-Brief Report.

BACKGROUND: Although most plasma FVIII (Factor VIII) circulates in complex with VWF (von Willebrand factor), a minority (3%-5%) circulates as free-FVIII, which is rapidly cleared. Consequently, 20% of total FVIII may be cleared as free-FVIII. Critically, the mechanisms of free-FVIII clearance remain poorly understood. However, recent studies have implicated the MGL (macrophage galactose lectin) in modulating VWF clearance. METHODS: Since VWF and FVIII share similar glycosylation, we investigated the role of MGL in FVIII clearance. FVIII binding to MGL was assessed in immunosorbent and cell-based assays. In vivo, FVIII clearance was assessed in MGL1-/- and VWF-/-/FVIII-/- mice. RESULTS: In vitro-binding studies identified MGL as a novel macrophage receptor that binds free-FVIII in a glycan-dependent manner. MGL1-/- and MGL1-/- mice who received an anti-MGL1/2 blocking antibody both showed significantly increased endogenous FVIII activity compared with wild-type mice (P=0.036 and P<0.0001, respectively). MGL inhibition also prolonged the half-life of infused FVIII in FVIII-/- mice. To assess whether MGL plays a role in the clearance of free FVIII in a VWF-independent manner, in vivo clearance experiments were repeated in dual VWF-/-/FVIII-/- mice. Importantly, the rapid clearance of free FVIII in VWF-/-/FVIII-/- mice was significantly (P=0.012) prolonged in the presence of anti-MGL1/2 antibodies. Finally, endogenous plasma FVIII levels in VWF-/- mice were significantly increased following MGL inhibition (P=0.016). CONCLUSIONS: Cumulatively, these findings demonstrate that MGL plays an important role in regulating macrophage-mediated clearance of both VWF-bound FVIII and free-FVIII in vivo. We propose that this novel FVIII clearance pathway may be of particular clinical importance in patients with type 2N or type 3 Von Willebrand disease.

Multiple fluorescence approaches to identify rapid changes in microbial indicators at karst springs.

Karst springs are globally important for drinking water supply but are often also exceptionally vulnerable to contamination. Such springs usually exhibit strong variation in microbial water quality in sharp response to rainfall events, thus, posing a health hazard to consumers of water supplied from these sources. The rapid detection of such changes is extremely important as well as being able to establish a link to the sources of such pollution, so that appropriate measures can be taken both in terms of immediate protection of human health and the management of karst aquifers. In this study, a fluorescence-based multi-parameter approach was trialed in order to evaluate which methods can be used to monitor rainfall-induced rapid changes in microbial water quality at karst springs, as well as determine whether such changes can be linked to sources of human effluent contamination. The results from three monitoring periods at two karst springs revealed marked responses to rainfall events for all of the microbial parameters measured. Total cell count (TCC) measurements using flow cytometry (FCM) showed very strong positive correlations with the more conventionally monitored faecal indicator bacteria (FIB) and total coliforms (TC), indicating that such a fluorescence-based and cultivation-independent technique can be very useful to indicate rapid changes in microbial water quality at karst springs. Furthermore, very strong positive correlations were also found between tryptophan-like fluorescence (TLF) measurements and concentrations of all monitored microbial parameters, again demonstrating that such a fluorescence-based approach can also be useful for detecting rapid changes in concentrations of traditional faecal indicators. Interestingly, it was found that fluorescent whitening compounds (FWCs) signals do not necessarily follow temporal variations of microbial indicators. However, the frequency of detection of positive FWCs signals may still reveal useful information about the overall magnitude of human wastewater effluent impacts on karst aquifer systems.

The Importance of Timing, Fundamental Attitudes, and Appropriate Interventions as Key Aspects of Chaplain Suicide Prevention: A European Expert Panel of Mental Health Chaplains.

An expert panel consisting of six mental health chaplains from several European countries was convened to ascertain the specific contribution of mental health chaplains to suicide prevention, and to generate good practices for suicide prevention. Three themes emerged: (1) the importance of timing; (2) fundamental attitudes of the chaplain towards the patient, and (3) appropriate interventions.

Application of visible light active photocatalysis for water contaminants: A review.

Organic water pollutants are ubiquitous in the natural environment arising from domestic products as well as current and legacy industrial processes. Many of these organic water pollutants are recalcitrant and only partially degraded using conventional water and wastewater treatment processes. In recent decades, visible light active photocatalyst has gained attention as a non-conventional alternative for the removal of organic pollutants during water treatment, including industrial wastewater and drinking water treatment. This paper reviews the current state of research on the use of visible light active photocatalysts, their modified methods, efficacy, and pilot-scale applications for the degradation of organic pollutants in water supplies and waste streams. Initially, the general mechanism of the visible light active photocatalyst is evaluated, followed by an overview of the major synthesis techniques. Because few of these photocatalysts are commercialized, particular attention was given to summarizing the different types of visible light active photocatalysts developed to the pilot-scale stage for practical application and commercialization. The organic pollutant degradation ability of these visible light active photocatalysts was found to be considerable and in many cases comparable with existing and commercially available advanced oxidation processes. Finally, this review concludes with a summary of current achievements and challenges as well as possible directions for further research. PRACTITIONER POINTS: Visible light active photocatalysis is a promising advanced oxidation process (AOP) for the reduction of organic water pollutants. Various mechanisms of photocatalysis using visible light active materials are identified and discussed. Many recent photocatalysts are synthesized from renewable materials that are more sustainable for applications in the 21st century. Only a small number of pilot-scale applications exist and these are outlined in this review.

Flow Cytometry and Fecal Indicator Bacteria Analyses for Fingerprinting Microbial Pollution in Karst Aquifer Systems.

Microbial pollution of aquifers is a persistent water quality problem globally which poses significant risks to public health. Karst aquifer systems are exceptionally vulnerable to pollution from fecal contamination sources as a result of rapid recharge of water from the surface via discrete pathways linked to highly conductive, solutionally enlarged conduits alongside strong aquifer heterogeneity. Consequently, rapid changes in microbial water quality, which are difficult to monitor with expensive and time-consuming conventional microbiological methods, are a major concern in karst environments. This study examined flow cytometric (FCM) fingerprinting of bacterial cells in groundwater together with fecal indicator bacteria (FIB) at nine separate karst springs of varying catchment size over a 14 month period in order to assess whether such a technique can provide faster and more descriptive information about microbial pollution through such karst aquifer systems. Moreover, the data have also been evaluated with respect to the potential of using turbidity as an easy-to-measure proxy indicator of microbial pollution in a novel way. We argue that FCM provides additional data from which enhanced insights into fecal pollution sources and its fate and transport in such karst catchments can be gained. We also present valuable new information on the potential and limitations of turbidity as an indicator of fecal groundwater contamination in karst. FCM has the potential to become a more widely used tool in the field of contaminant hydrogeology.

An aggregation inhibitor specific to oligomeric intermediates of Aß42 derived from phage display libraries of stable, small proteins.

The self-assembly of amyloid ß peptide (Aß) to fibrillar and oligomeric aggregates is linked to Alzheimer's disease. Aß binders may serve as inhibitors of aggregation to prevent the generation of neurotoxic species and for the detection of Aß species. A particular challenge involves finding binders to on-pathway oligomers given their transient nature. Here we construct two phage­display libraries built on the highly inert and stable protein scaffold S100G, one containing a six-residue variable surface patch and one harboring a seven-residue variable loop insertion. Monomers and fibrils of Aß40 and Aß42 were separately coupled to silica nanoparticles, using a coupling strategy leading to the presence of oligomers on the monomer beads, and they were used in three rounds of affinity selection. Next-generation sequencing revealed sequence clusters and candidate binding proteins (SXkmers). Two SXkmers were expressed as soluble proteins and tested in terms of aggregation inhibition via thioflavin T fluorescence. We identified an SXkmer with loop­insertion YLTIRLM as an inhibitor of the secondary nucleation of Aß42 and binding analyses using surface plasmon resonance technology, Förster resonance energy transfer, and microfluidics diffusional sizing imply an interaction with intermediate oligomeric species. A linear peptide with the YLTIRLM sequence was found inhibitory but at a lower potency than the more constrained SXkmer loop. We identified an SXkmer with side-patch VI-WI-DD as an inhibitor of Aß40 aggregation. Remarkably, our data imply that SXkmer-YLTIRLM blocks secondary nucleation through an interaction with oligomeric intermediates in solution or at the fibril surface, which is a unique inhibitory mechanism for a library-derived inhibitor.

Conversion of mammalian cell culture media waste to microbial fermentation feed efficiently supports production of recombinant protein by Escherichia coli.

Deriving new value from waste streams through secondary processes is a central aim of the circular bioeconomy. In this study we investigate whether chemically defined spent media (CDSM) waste from cell culture bioprocess can be recycled and used as a feed in secondary microbial fermentation to produce new recombinant protein products. Our results show that CDSM supplemented with 2% glycerol supported a specific growth rate of E. coli cultures equivalent to that achieved using a nutritionally rich microbiological media (LB). The titre of recombinant protein produced following induction in a 4-hour expression screen was approximately equivalent in the CDSM fed cultures to that of baseline, and this was maintained in a 16-hr preparative fermentation. To understand the protein production achieved in CDSM fed culture we performed a quantitative analysis of proteome changes in the E. coli using mass spectrometry. This analysis revealed significant upregulation of protein synthesis machinery enzymes and significant downregulation of carbohydrate metabolism enzymes. We conclude that spent cell culture media, which represents 100s of millions of litres of waste generated by the bioprocessing industry annually, may be valorized as a feed resource for the production of recombinant proteins in secondary microbial fermentations. Data is available via ProteomeXchange with identifier PXD026884.

Analysis of oxygen isotopes of inorganic phosphate (δ18Op) in freshwater: A detailed method description for obtaining oxygen isotopes of inorganic phosphate in environmental water samples.

The ability to identify the origin of phosphorus and understand processes controlling P cycling is essential for designing effective mitigation and restoration of eutrophic freshwater ecosystems. The oxygen isotope composition of orthophosphate (δ18Op) has significant potential as a tracer for P entering freshwater ecosystems. However, methods of analysis of δ18Op are still in their preliminary stages and have proven challenging to implement for new practitioners. In order to achieve progress in developing the application of δ18Op signatures as a tracing tool, there is a need to eliminate the methodological challenges involved in accurately determining δ18Op. This protocol article describes the various steps needed to concentrate and isolate orthophosphate in freshwater samples into an adequately pure Ag3PO4 analyte, without isotopic alteration during processing. The protocol compiles the disperse experiences from previous studies, combined with our own experience. The twofold aim of the paper is toprovide a baseline for an increasing standardisation of the silver phosphate purification method associated with analysis of the oxygen isotope composition of orthophosphate (δ18Op), and to foster new research in the applicability of δ18Op signatures for P source tracing in catchment science.

Collagen X Marker Levels are Decreased in Individuals with Achondroplasia.

Collagen X marker (CXM) is a degradation fragment of collagen type X. It is a real-time biomarker of height velocity with established norms. Plasma C-type natriuretic peptide (CNP) and NTproCNP levels have also been found to correlate with growth velocity in the general population and are elevated in individuals with achondroplasia compared with age- and sex-matched controls. Collagen X marker levels in people with fibroblast growth factor receptor 3 (FGFR3)-opathies have never been systematically measured. The objective of this study was to measure CXM in a population of dwarfism caused by FGFR3-opathies. Using the same cohort in which CNP and NTproCNP levels were previously measured, archived serum aliquots from 63 children with achondroplasia, six with hypochondroplasia, and two with thanatophoric dysplasia had CXM concentrations measured. Results were plotted against age- and sex-specific norms, and standard deviation scores were plotted for comparison between clinical diagnoses. CXM levels were significantly decreased (p < 0.0001) in children with achondroplasia compared with age- and sex-matched controls. Temporal patterns of change in CXM levels were sex-dependent. As the FGFR3 pathway was more constitutively active, CXM levels decreased. New tools are emerging to study impact of skeletal dysplasia on growth plate regulation and function.

Urinary C3 levels associated with sepsis and acute kidney injury-A pilot study.

Acute kidney injury (AKI) is an abrupt deterioration of renal function often caused by severe clinical disease such as sepsis, and patients require intensive care. Acute-phase parameters for systemic inflammation are well established and used in routine clinical diagnosis, but no such parameters are known for AKI and inflammation at the local site of tissue damage, namely the nephron. Therefore, we sought to investigate complement factors C3a/C3 in urine and urinary sediment cells. After the development of a C3a/C3-specific mouse monoclonal antibody (3F7E2), urine excretion from ICU sepsis patients was examined by dot blot and immunoblotting. This C3a/C3 ELISA and a C3a ELISA were used to obtain quantitative data over 24 hours for 6 consecutive days. Urine sediment cells were analyzed for topology of expression. Patients with severe infections (n = 85) showed peak levels of C3a/C3 on the second day of ICU treatment. The majority (n = 59) showed C3a/C3 levels above 20 µg/ml at least once in the first 6 days after admission. C3a was detectable on all 6 days. Peak C3a/C3 levels correlated negatively with peak C-reactive protein (CRP) levels. No relationship was found between peak C3a/C3 with peak leukocyte count, age, or AKI stage. Analysis of urine sediment cells identified C3a/C3-producing epithelial cells with reticular staining patterns and cells with large-granular staining. Opsonized bacteria were detected in patients with urinary tract infections. In critically ill sepsis patients with AKI, urinary C3a/C3 inversely correlated with serum CRP. Whether urinary C3a/C3 has a protective function through autophagy, as previously shown for cisplatin exposure, or is a by-product of sepsis caused by pathogenic stimuli to the kidney must remain open in this study. However, our data suggest that C3a/C3 may function as an inverse acute-phase parameter that originates in the kidney and is detectable in urine.

Variability in fluvial suspended and streambed sediment phosphorus fractions among small agricultural streams.

Agriculture is a major source of sediment and particulate phosphorus (P) inputs to freshwaters. Distinguishing between P fractions in sediment can aid in understanding its eutrophication risk. Although streams and rivers are important parts of the P cycle in agricultural catchments, streambed sediment and especially fluvial suspended sediment (FSS) and its P fractions are less studied. To address this knowledge gap, seasonal variations in FSS P fractions and their relation to water quality and streambed sediment were examined in three Swedish agricultural headwater catchments over 2 yr. Sequential fractionation was used to characterize P fractions in both streambed sediment and FSS. All catchments had similar annual P losses (0.4-0.8 kg ha-1 ), suspended solids (124-183 mg L-1 ), and FSS total P concentrations (1.15-1.19 mg g-1 ). However, distribution of P fractions and the dominant P fractions in FSS differed among catchments (p < .05), which was most likely dependent on differences in catchment geology, clay content, external P sources, and flow conditions. The most prominent seasonal pattern in all catchments was found for iron-bound P, with high concentrations during low summer flows and low concentrations during winter high flows. Streambed sediment P fractions were in the same concentration ranges as in FSS, and the distribution of the fractions differed between catchments. This study highlights the need to quantify P fractions, not just total P in FSS, to obtain a more complete understanding of the eutrophication risk posed by agricultural sediment losses.

Comorbidities in type 2 diabetes patients with and without atherosclerotic cardiovascular disease: a retrospective database analysis.

Objective: The objective of this study was to describe the pattern of comorbidities in patients with type 2 diabetes mellitus with and without atherosclerotic cardiovascular disease.Methods: This was a retrospective, cross-sectional analysis of the IQVIA Commercial Data Delivery database. Patients were ≥18 years on their last encounter between 1 October 2014 and 30 September 2015 and had either a type 2 diabetes mellitus diagnosis or a prescription for an oral diabetes medication. Atherosclerotic cardiovascular disease was confirmed by diagnosis codes. Comorbidities were identified using diagnosis codes, clinical measurements, and/or medication use.Results: A total of 1,522,526 type 2 diabetes mellitus patients were included in the analysis, 25% of whom had atherosclerotic cardiovascular disease. The most common comorbidities were hypertension, hyperlipidemia, overweight/obesity, chronic kidney disease, congestive heart failure, and neuropathy. These were present, respectively, in the following percentages of patients with and without cardiovascular disease: 98.3 and 91.0%, 94.8 and 78.5%, 80.5 and 80.6%, 38.5 and 18.9, 20.2, and 4.3%, and 13.7 and 8.6%. Thus, the frequencies of hyperlipidemia, chronic kidney disease, and congestive heart failure were notably higher in patients with cardiovascular disease. This trend held true for patients grouped by sex, age, and race.Conclusions: Patients with type 2 diabetes mellitus and atherosclerotic cardiovascular disease have different rates of certain comorbidities compared to those without atherosclerotic cardiovascular disease.

An assessment of contamination fingerprinting techniques for determining the impact of domestic wastewater treatment systems on private well supplies.

Private wells in Ireland and elsewhere have been shown to be prone to microbial contamination with the main suspected sources being practices associated with agriculture and domestic wastewater treatment systems (DWWTS). While the microbial quality of private well water is commonly assessed using faecal indicator bacteria, such as Escherichia coli, such organisms are not usually source-specific, and hence cannot definitively conclude the exact origin of the contamination. This research assessed a range of different chemical contamination fingerprinting techniques (ionic ratios, artificial sweeteners, caffeine, fluorescent whitening compounds, faecal sterol profiles and pharmaceuticals) as to their use to apportion contamination of private wells between human wastewater and animal husbandry wastes in rural areas of Ireland. A one-off sampling and analysis campaign of 212 private wells found that 15% were contaminated with E. coli. More extensive monitoring of 24 selected wells found 58% to be contaminated with E. coli on at least one occasion over a 14-month period. The application of fingerprinting techniques to these monitored wells found that the use of chloride/bromide and potassium/sodium ratios is a useful low-cost fingerprinting technique capable of identifying impacts from human wastewater and organic agricultural contamination, respectively. The artificial sweetener acesulfame was detected on several occasions in a number of monitored wells, indicating its conservative nature and potential use as a fingerprinting technique for human wastewater. However, neither fluorescent whitening compounds nor caffeine were detected in any wells, and faecal sterol profiles proved inconclusive, suggesting limited suitability for the conditions investigated.

Resolving the Interactome of the Human Macrophage Immunometabolism Regulator (MACIR) with Enhanced Membrane Protein Preparation and Affinity Proteomics.

Expression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and with the regulation of macrophage biology through unknown mechanisms. The encoded 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. To identify interactions of MACIR with proteins from all subcellular compartments, a membrane solubilization buffer is employed, that together with a high affinity EF hand based pull down method, increases the resolution of quantitative mass spectrometry analysis with significant enrichment of interactions from membrane bound nuclear and mitochondrial compartments compared to samples prepared with radioimmunoprecipitation assay buffer. A total of 63 significant interacting proteins are identified and interaction with the nuclear transport receptor TNPO1 and the trafficking proteins UNC119 homolog A and B are validated by immunoprecipitation. Mutational analysis in two candidate nuclear localization signal motifs in the MACIR amino acid sequence shows the interaction with TNPO1 is likely via a non-classical proline/tyrosine-nuclear localization signal motif (aa98-117). It is shown that employing a highly specific and high affinity pull down method that performs efficiently in this glycerol and detergent rich buffer is a powerful approach for the analysis of uncharacterized protein interactomes.

Renal Expression of Light Chain Binding Proteins.

Overproduction of human light chains (LCs) and immunoglobulins can result in various forms of renal disease such as cast nephropathy, monoclonal immunoglobulin deposition disease, LC proximal tubulopathy, AL amyloidosis, and crystal storing histiocytosis. This is caused by cellular uptake of LCs and overwhelmed intracellular transport and degradation in patients with high urine LC concentrations. LC kappa and lambda purification was evaluated by sodium dodecyl sulfate gel electrophoresis. LC and myeloma protein binding to immobilized renal proteins was measured by enzyme-linked immunosorbent assay (ELISA). The human protein microarray (HuProt™) was screened with purified kappa and lambda LC. Identified LC partners were subsequently analyzed in silico for renal expression sites using protein databases, Human Protein Atlas, UniProt, and Bgee. Binding of urinary LCs and immunoglobulins to immobilized whole renal proteins from 22 patients with myeloma or plasma cell dyscrasia was shown by ELISA. Forty lambda and 23 kappa interaction partners were identified from HuProt™ array screens, of which 21 were shared interactors. Among the total of 42 interactors, 12 represented cell surface proteins. Lambda binding signals were approximately 40% higher than kappa signals. LC interaction with renal cells and disease-causing pathologies are more complex than previously thought. It involves an extended spectrum of proteins expressed throughout the nephron, and their identification has been enabled by recently developed methods of protein analysis such as protein microarray screening. Further biochemical studies on interacting proteins are warranted to elucidate their clinical relevance.