Proteome characterization of liver-kidney comorbidity after microbial sepsis.

Sepsis is a life-threatening condition that occurs when the body responds to an infection but subsequently triggers widespread inflammation and impaired blood flow. These pathologic responses can rapidly cause multiple organ dysfunction or failure either one by one or simultaneously. The fundamental common mechanisms involved in sepsis-induced multiple organ dysfunction remain unclear. Here, employing quantitative global and phosphoproteomics, we examine the liver's temporal proteome and phosphoproteome changes after moderate sepsis induced by cecum ligation and puncture. In total, 4593 global proteins and 1186 phosphoproteins according to 3275 phosphosites were identified. To characterize the liver-kidney comorbidity after sepsis, we developed a mathematical model and performed cross-analyses of liver and kidney proteome data obtained from the same set of mice. Beyond immune response, we showed the commonly disturbed pathways and key regulators of the liver-kidney comorbidity are linked to energy metabolism and consumption. Our data provide open resources to understand the communication between the liver and kidney as they work to fight infection and maintain homeostasis.

Systematic comparison of rAAV vectors manufactured using large-scale suspension cultures of Sf9 and HEK293 cells.

Recombinant adeno-associated virus (rAAV) vectors could be manufactured by plasmid transfection into human embryonic kidney 293 (HEK293) cells or baculovirus infection of Spodoptera frugiperda (Sf9) insect cells. However, systematic comparisons between these systems using large-scale, high-quality AAV vectors are lacking. rAAV from Sf9 cells (Sf9-rAAV) at 2-50 L and HEK293 cells (HEK-rAAV) at 2-200 L scales were characterized. HEK-rAAV had ∼40-fold lower yields but ∼10-fold more host cell DNA measured by droplet digital PCR and next-generation sequencing, respectively. The electron microscope observed a lower full/empty capsid ratio in HEK-rAAV (70.8%) than Sf9-rAAV (93.2%), while dynamic light scattering and high-performance liquid chromatography analysis showed that HEK-rAAV had more aggregation. Liquid chromatography tandem mass spectrometry identified different post-translational modification profiles between Sf9-rAAV and HEK-rAAV. Furthermore, Sf9-rAAV had a higher tissue culture infectious dose/viral genome than HEK-rAAV, indicating better infectivity. Additionally, Sf9-rAAV achieved higher in vitro transgene expression, as measured by ELISA. Finally, after intravitreal dosing into a mouse laser choroidal neovascularization model, Sf9-rAAV and HEK-rAAV achieved similar efficacy. Overall, this study detected notable differences in the physiochemical characteristics of HEK-rAAV and Sf9-rAAV. However, the in vitro and in vivo biological functions of the rAAV from these systems were highly comparable. Sf9-rAAV may be preferred over HEK293-rAAV for advantages in yields, full/empty ratio, scalability, and cost.

A multiomics analysis of direct interkingdom dynamics between influenza A virus and Streptococcus pneumoniae uncovers host-independent changes to bacterial virulence fitness.

BACKGROUND: For almost a century, it has been recognized that influenza A virus (IAV) infection can promote the development of secondary bacterial infections (SBI) mainly caused by Streptococcus pneumoniae (Spn). Recent observations have shown that IAV is able to directly bind to the surface of Spn. To gain a foundational understanding of how direct IAV-Spn interaction alters bacterial biological fitness we employed combinatorial multiomic and molecular approaches. RESULTS: Here we show IAV significantly remodels the global transcriptome, proteome and phosphoproteome profiles of Spn independently of host effectors. We identified Spn surface proteins that interact with IAV proteins (hemagglutinin, nucleoprotein, and neuraminidase). In addition, IAV was found to directly modulate expression of Spn virulence determinants such as pneumococcal surface protein A, pneumolysin, and factors associated with antimicrobial resistance among many others. Metabolic pathways were significantly altered leading to changes in Spn growth rate. IAV was also found to drive Spn capsule shedding and the release of pneumococcal surface proteins. Released proteins were found to be involved in evasion of innate immune responses and actively reduced human complement hemolytic and opsonizing activity. IAV also led to phosphorylation changes in Spn proteins associated with metabolism and bacterial virulence. Validation of proteomic data showed significant changes in Spn galactose and glucose metabolism. Furthermore, supplementation with galactose rescued bacterial growth and promoted bacterial invasion, while glucose supplementation led to enhanced pneumolysin production and lung cell apoptosis. CONCLUSIONS: Here we demonstrate that IAV can directly modulate Spn biology without the requirement of host effectors and support the notion that inter-kingdom interactions between human viruses and commensal pathobionts can promote bacterial pathogenesis and microbiome dysbiosis.

Influenza Causes MLKL-Driven Cardiac Proteome Remodeling During Convalescence.

RATIONALE: Patients with and without cardiovascular diseases have been shown to be at risk of influenza-mediated cardiac complications. Recent clinical reports support the notion of a direct link between laboratory-confirmed influenza virus infections and adverse cardiac events. OBJECTIVE: Define the molecular mechanisms underlying influenza virus-induced cardiac pathogenesis after resolution of pulmonary infection and the role of necroptosis in this process. METHODS AND RESULTS: Hearts from wild-type and necroptosis-deficient (MLKL [mixed lineage kinase domain-like protein]-KO) mice were dissected 12 days after initial influenza A virus (IAV) infection when viral titers were undetectable in the lungs. Immunofluorescence microscopy and plaque assays showed presence of viable IAV particles in the myocardium without generation of interferon responses. Global proteome and phosphoproteome analyses using high-resolution accurate mass-based LC-MS/MS and label-free quantitation showed that the global proteome as well as the phosphoproteome profiles were significantly altered in IAV-infected mouse hearts in a strain-independent manner. Necroptosis-deficient mice had increased survival and reduced weight loss post-IAV infection, as well as increased antioxidant and mitochondrial function, indicating partial protection to IAV infection. These findings were confirmed in vitro by pretreatment of human and rat myocytes with antioxidants or necroptosis inhibitors, which blunted oxidative stress and mitochondrial damage after IAV infection. CONCLUSIONS: This study provides the first evidence that the cardiac proteome and phosphoproteome are significantly altered post-pulmonary influenza infection. Moreover, viral particles can persist in the heart after lung clearance, altering mitochondrial function and promoting cell death without active replication and interferon responses. Finally, our findings show inhibition of necroptosis or prevention of mitochondrial damage as possible therapeutic interventions to reduce cardiac damage during influenza infections. Graphic Abstract: A graphic abstract is available for this article.

CRL4(VprBP) E3 ligase promotes monoubiquitylation and chromatin binding of TET dioxygenases.

DNA methylation at the C-5 position of cytosine (5mC) regulates gene expression and plays pivotal roles in various biological processes. The TET dioxygenases catalyze iterative oxidation of 5mC, leading to eventual demethylation. Inactivation of TET enzymes causes multistage developmental defects, impaired cell reprogramming, and hematopoietic malignancies. However, little is known about how TET activity is regulated. Here we show that all three TET proteins bind to VprBP and are monoubiquitylated by the VprBP-DDB1-CUL4-ROC1 E3 ubiquitin ligase (CRL4(VprBP)) on a highly conserved lysine residue. Deletion of VprBP in oocytes abrogated paternal DNA hydroxymethylation in zygotes. VprBP-mediated monoubiquitylation promotes TET binding to chromatin. Multiple recurrent TET2-inactivating mutations derived from leukemia target either the monoubiquitylation site (K1299) or residues essential for VprBP binding. Cumulatively, our data demonstrate that CRL4(VprBP) is a critical regulator of TET dioxygenases during development and in tumor suppression.

Kinetic Multi-omic Analysis of Responses to SARS-CoV-2 Infection in a Model of Severe COVID-19.

The host response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is poorly understood due to a lack of an animal model that recapitulates severe human disease. Here, we report a Syrian hamster model that develops progressive lethal pulmonary disease that closely mimics severe coronavirus disease 2019 (COVID-19). We evaluated host responses using a multi-omic, multiorgan approach to define proteome, phosphoproteome, and transcriptome changes. These data revealed both type I and type II interferon-stimulated gene and protein expression along with a progressive increase in chemokines, monocytes, and neutrophil-associated molecules throughout the course of infection that peaked in the later time points correlating with a rapidly developing diffuse alveolar destruction and pneumonia that persisted in the absence of active viral infection. Extrapulmonary proteome and phosphoproteome remodeling was detected in the heart and kidneys following viral infection. Together, our results provide a kinetic overview of multiorgan host responses to severe SARS-CoV-2 infection in vivo. IMPORTANCE The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has created an urgent need to understand the pathogenesis of this infection. These efforts have been impaired by the lack of animal models that recapitulate severe coronavirus disease 2019 (COVID-19). Here, we report a hamster model that develops severe COVID-19-like disease following infection with human isolates of SARS-CoV-2. To better understand pathogenesis, we evaluated changes in gene transcription and protein expression over the course of infection to provide an integrated multiorgan kinetic analysis of the host response to infection. These data reveal a dynamic innate immune response to infection and corresponding immune pathologies consistent with severe human disease. Altogether, this model will be useful for understanding the pathogenesis of severe COVID-19 and for testing interventions.

The 3M complex maintains microtubule and genome integrity.

CUL7, OBSL1, and CCDC8 genes are mutated in a mutually exclusive manner in 3M and other growth retardation syndromes. The mechanism underlying the function of the three 3M genes in development is not known. We found that OBSL1 and CCDC8 form a complex with CUL7 and regulate the level and centrosomal localization of CUL7, respectively. CUL7 depletion results in altered microtubule dynamics, prometaphase arrest, tetraploidy, and mitotic cell death. These defects are recaptured in CUL7 mutated 3M cells and can be rescued by wild-type, but not by 3M patient-derived CUL7 mutants. Depletion of either OBSL1 or CCDC8 results in defects and sensitizes cells to microtubule damage similarly to loss of CUL7 function. Microtubule damage reduces the level of CCDC8 that is required for the centrosomal localization of CUL7. We propose that CUL7, OBSL1, and CCDC8 proteins form a 3M complex that functions in maintaining microtubule and genome integrity and normal development.

Global Proteome and Phosphoproteome Characterization of Sepsis-induced Kidney Injury.

Sepsis-induced acute kidney injury (S-AKI) is the most common complication in hospitalized and critically ill patients, highlighted by a rapid decline of kidney function occurring a few hours or days after sepsis onset. Systemic inflammation elicited by microbial infections is believed to lead to kidney damage under immunocompromised conditions. However, although AKI has been recognized as a disease with long-term sequelae, partly because of the associated higher risk of chronic kidney disease (CKD), the understanding of kidney pathophysiology at the molecular level and the global view of dynamic regulations in situ after S-AKI, including the transition to CKD, remains limited. Existing studies of S-AKI mainly focus on deriving sepsis biomarkers from body fluids. In the present study, we constructed a mid-severity septic murine model using cecal ligation and puncture (CLP), and examined the temporal changes to the kidney proteome and phosphoproteome at day 2 and day 7 after CLP surgery, corresponding to S-AKI and the transition to CKD, respectively, by employing an ultrafast and economical filter-based sample processing method combined with the label-free quantitation approach. Collectively, we identified 2,119 proteins and 2950 phosphosites through multi-proteomics analyses. Among them, we identified an array of highly promising candidate marker proteins indicative of disease onset and progression accompanied by immunoblot validations, and further denoted the pathways that are specifically responsive to S-AKI and its transition to CKD, which include regulation of cell metabolism regulation, oxidative stress, and energy consumption in the diseased kidneys. Our data can serve as an enriched resource for the identification of mechanisms and biomarkers for sepsis-induced kidney diseases.

The mitochondrial proteins NLRX1 and TUFM form a complex that regulates type I interferon and autophagy.

The mitochondrial protein MAVS (also known as IPS-1, VISA, and CARDIF) interacts with RIG-I-like receptors (RLRs) to induce type I interferon (IFN-I). NLRX1 is a mitochondrial nucleotide-binding, leucine-rich repeats (NLR)-containing protein that attenuates MAVS-RLR signaling. Using Nlrx1(-/-) cells, we confirmed that NLRX1 attenuated IFN-I production, but additionally promoted autophagy during viral infection. This dual function of NLRX1 paralleled the previously described functions of the autophagy-related proteins Atg5-Atg12, but NLRX1 did not associate with Atg5-Atg12. High-throughput quantitative mass spectrometry and endogenous protein-protein interaction revealed an NLRX1-interacting partner, mitochondrial Tu translation elongation factor (TUFM). TUFM interacted with Atg5-Atg12 and Atg16L1 and has similar functions as NLRX1 by inhibiting RLR-induced IFN-I but promoting autophagy. In the absence of NLRX1, increased IFN-I and decreased autophagy provide an advantage for host defense against vesicular stomatitis virus. This study establishes a link between an NLR protein and the viral-induced autophagic machinery via an intermediary partner, TUFM.

Oral Microbial Species and Virulence Factors Associated with Oral Squamous Cell Carcinoma.

The human microbiome has been the focus of numerous research efforts to elucidate the pathogenesis of human diseases including cancer. Oral cancer mortality is high when compared with other cancers, as diagnosis often occurs during late stages. Its prevalence has increased in the USA over the past decade and accounts for over 40,000 new cancer patients each year. Additionally, oral cancer pathogenesis is not fully understood and is likely multifactorial. To unravel the relationships that are associated with the oral microbiome and their virulence factors, we used 16S rDNA and metagenomic sequencing to characterize the microbial composition and functional content in oral squamous cell carcinoma (OSCC) tumor tissue, non-tumor tissue, and saliva from 18 OSCC patients. Results indicate a higher number of bacteria belonging to the Fusobacteria, Bacteroidetes, and Firmicutes phyla associated with tumor tissue when compared with all other sample types. Additionally, saliva metaproteomics revealed a significant increase of Prevotella in five OSCC subjects, while Corynebacterium was mostly associated with ten healthy subjects. Lastly, we determined that there are adhesion and virulence factors associated with Streptococcus gordonii as well as from known oral pathogens belonging to the Fusobacterium genera found mostly in OSCC tissues. From these results, we propose that not only will the methods utilized in this study drastically improve OSCC diagnostics, but the organisms and specific virulence factors from the phyla detected in tumor tissue may be excellent biomarkers for characterizing disease progression.

Differences in plasma proteomes for active tuberculosis, latent tuberculosis and non-tuberculosis mycobacterial lung disease patients with and without ESAT-6/CFP10 stimulation.

BACKGROUND: Tuberculosis (TB) is one of the world's most problematic infectious diseases. The pathogen Mycobacterium tuberculosis (Mtb) is contained by the immune system in people with latent TB infection (LTBI). No overt disease symptoms occur. The environmental and internal triggers leading to reactivation of TB are not well understood. Non-tuberculosis Mycobacteria (NTM) can also cause TB-like lung disease. Comparative analysis of blood plasma proteomes from subjects afflicted by these pathologies in an endemic setting may yield new differentiating biomarkers and insights into inflammatory and immunological responses to Mtb and NTM. METHODS: Blood samples from 40 human subjects in a pastoral region of Ethiopia were treated with the ESAT-6/CFP-10 antigen cocktail to stimulate anti-Mtb and anti-NTM immune responses. In addition to those of active TB, LTBI, and NTM cohorts, samples from matched healthy control (HC) subjects were available. Following the generation of sample pools, proteomes were analyzed via LC-MS/MS. These experiments were also performed without antigen stimulation steps. Statistically significant differences using the Z-score method were determined and interpreted in the context of the proteins' functions and their contributions to biological pathways. RESULTS: More than 200 proteins were identified from unstimulated and stimulated plasma samples (UPSs and SPSs, respectively). Thirty-four and 64 proteins were differentially abundant with statistical significance (P < 0.05; Benjamini-Hochberg correction with an FDR < 0.05) comparing UPS and SPS proteomic data of four groups, respectively. Bioinformatics analysis of such proteins via the Gene Ontology Resource was indicative of changes in cellular and metabolic processes, responses to stimuli, and biological regulations. The m7GpppN-mRNA hydrolase was increased in abundance in the LTBI group compared to HC subjects. Charged multivesicular body protein 4a and platelet factor-4 were increased in abundance in NTM as compared to HC and decreased in abundance in NTM as compared to active TB. C-reactive protein, α-1-acid glycoprotein 1, sialic acid-binding Ig-like lectin 16, and vitamin K-dependent protein S were also increased (P < 0.05; fold changes≥2) in SPSs and UPSs comparing active TB with LTBI and NTM cases. These three proteins, connected in a STRING functional network, contribute to the acute phase response and influence blood coagulation. CONCLUSION: Plasma proteomes are different comparing LTBI, TB, NTM and HC cohorts. The changes are augmented following prior blood immune cell stimulation with the ESAT-6/CFP-10 antigen cocktail. The results encourage larger-cohort studies to identify specific biomarkers to diagnose NTM infection, LTBI, and to predict the risk of TB reactivation.

Persistent Gut Microbial Dysbiosis in Children with Acute Lymphoblastic Leukemia (ALL) During Chemotherapy.

Prophylactic or therapeutic antibiotic use along with chemotherapy treatment potentially has a long-standing adverse effect on the resident gut microbiota. We have established a case-control cohort of 32 pediatric and adolescent acute lymphoblastic leukemia (ALL) patients and 25 healthy siblings (sibling controls) to assess the effect of chemotherapy as well as antibiotic prophylaxis on the gut microbiota. We observe that the microbiota diversity and richness of the ALL group is significantly lower than that of the control group at diagnosis and during chemotherapy. The microbiota diversity is even lower in antibiotics-exposed ALL patients. Although the gut microbial diversity tends to stabilize after 1-year post-chemotherapy, their abundances were altered because of chemotherapy and prophylactic antibiotic treatments. Specifically, the abundances of mucolytic gram-positive anaerobic bacteria, including Ruminococcus gnavus and Ruminococcus torques, tended to increase during the chemotherapy regimen and continued to be elevated 1 year beyond the initiation of chemotherapy. This dysbiosis may contribute to the development of gastrointestinal complications in ALL children following chemotherapy. These findings set the stage to further understand the role of the gut microbiome dynamics in ALL patients and their potential role in alleviating some of the adverse side effects of chemotherapy and antibiotics use in immunocompromised children.

Self-Assembled STrap for Global Proteomics and Salivary Biomarker Discovery.

Clinical biomarkers identified by shotgun proteomics require proteins in body fluids or tissues to be enzymatically digested before being separated and sequenced by liquid chromatography-tandem mass spectrometry. How well peptide signals can be resolved and detected is largely dependent on the quality of sample preparation. Conventional approaches such as in-gel, in-solution, and filter-based digestion, despite their extensive implementation by the community, become less appealing due to their unsatisfying protein/peptide recovery rate, lengthy sample processing, and/or lowcost-effectiveness. Suspension trapping has recently been demonstrated as an ultrafast approach for proteomic analysis. Here, for the first time, we extend its application to human salivary proteome analyses. In particular, we present a simple self-assembled glass fiber filter device which can be packed with minimal difficulty, is extremely cost-effective, and maintains the same performance as commercial filters. As a proof-of-principle, we analyzed the whole saliva from 8 healthy individuals as well as a cohort of 10 subjects of oral squamous cell carcinoma (OSCC) patients and non-OSCC subjects. Label-free quantification revealed surprisingly low interindividual variability and several known markers. Our study provides the first evidence of an easy-to-use and low-cost device for clinical proteomics as well as for general proteomic sample preparation.

Characterization of Early-Phase Neutrophil Extracellular Traps in Urinary Tract Infections.

Neutrophils have an important role in the antimicrobial defense and resolution of urinary tract infections (UTIs). Our research suggests that a mechanism known as neutrophil extracellular trap (NET) formation is a defense strategy to combat pathogens that have invaded the urinary tract. A set of human urine specimens with very high neutrophil counts had microscopic evidence of cellular aggregation and lysis. Deoxyribonuclease I (DNase) treatment resulted in disaggregation of such structures, release of DNA fragments and a proteome enriched in histones and azurophilic granule effectors whose quantitative composition was similar to that of previously described in vitro-formed NETs. The effector proteins were further enriched in DNA-protein complexes isolated in native PAGE gels. Immunofluorescence microscopy revealed a flattened morphology of neutrophils associated with decondensed chromatin, remnants of granules in the cell periphery, and myeloperoxidase co-localized with extracellular DNA, features consistent with early-phase NETs. Nuclear staining revealed that a considerable fraction of bacterial cells in these structures were dead. The proteomes of two pathogens, Staphylococcus aureus and Escherichia coli, were indicative of adaptive responses to early-phase NETs, specifically the release of virulence factors and arrest of ribosomal protein synthesis. Finally, we discovered patterns of proteolysis consistent with widespread cleavage of proteins by neutrophil elastase, proteinase 3 and cathepsin G and evidence of citrullination in many nuclear proteins.

S-Trap, an Ultrafast Sample-Preparation Approach for Shotgun Proteomics.

The success of shotgun proteomic analysis depends largely on how samples are prepared. Current approaches (such as those that are gel-, solution-, or filter-based), although being extensively employed in the field, are time-consuming and less effective with respect to the repetitive sample processing, recovery, and overall yield. As an alternative, the suspension trapping (S-Trap) filter has been commercially available very recently in the format of a single or 96-well filter plate. In contrast to the conventional filter-aided sample preparation (FASP) approach, which utilizes a molecular weight cut-off (MWCO) membrane as the filter and requires hours of processing before digestion-ready proteins can be obtained, the S-Trap employs a three-dimensional porous material as filter media and traps particulate protein suspensions with the subsequent depletion of interfering substances and in-filter digestion. Due to the large (submicron) pore size, each centrifugation cycle of the S-Trap filter only takes 1 min, which significantly reduces the total processing time from approximately 3 h by FASP to less than 15 min, suggesting an ultrafast sample-preparation approach for shotgun proteomics. Here, we comprehensively evaluate the performance of the individual S-Trap filter and 96-well filter plate in the context of global protein identification and quantitation using whole-cell lysate and clinically relevant sputum samples.

The antiobesity factor WDTC1 suppresses adipogenesis via the CRL4WDTC1 E3 ligase.

WDTC1/Adp encodes an evolutionarily conserved suppressor of lipid accumulation. While reduced WDTC1 expression is associated with obesity in mice and humans, its cellular function is unknown. Here, we demonstrate that WDTC1 is a component of a DDB1-CUL4-ROC1 (CRL4) E3 ligase. Using 3T3-L1 cell culture model of adipogenesis, we show that disrupting the interaction between WDTC1 and DDB1 leads to a loss of adipogenic suppression by WDTC1, increased triglyceride accumulation and adipogenic gene expression. We show that the CRL4(WDTC) (1) complex promotes histone H2AK119 monoubiquitylation, thus suggesting a role for this complex in transcriptional repression during adipogenesis. Our results identify a biochemical role for WDTC1 and extend the functional range of the CRL4 complex to the suppression of fat accumulation.

A Method to Generate and Analyze Modified Myristoylated Proteins.

Covalent lipid modification of proteins is essential to their cellular localizations and functions. Engineered lipid motifs, coupled with bio-orthogonal chemistry, have been utilized to identify myristoylated or palmitoylated proteins in cells. However, whether modified proteins have similar properties as endogenous ones has not been well investigated mainly due to lack of methods to generate and analyze purified proteins. We have developed a method that utilizes metabolic interference and mass spectrometry to produce and analyze modified, myristoylated small GTPase ADP-ribosylation factor 1 (Arf1). The capacities of these recombinant proteins to bind liposomes and load and hydrolyze GTP were measured and compared with the unmodified myristoylated Arf1. The ketone-modified myristoylated Arf1 could be further labeled by fluorophore-coupled hydrazine and subsequently visualized through fluorescence imaging. This methodology provides an effective model system to characterize lipid-modified proteins with additional functions before applying them to cellular systems.

Similar Neutrophil-Driven Inflammatory and Antibacterial Responses in Elderly Patients with Symptomatic and Asymptomatic Bacteriuria.

Differential diagnosis of asymptomatic bacteriuria (ASB) and urinary tract infection (UTI) is based on the presence of diverse symptoms, including fever (≥38.5°C), rigors, malaise, lethargy, flank pain, hematuria, suprapubic discomfort, dysuria, and urgent or frequent urination. There is consensus in the medical community that ASB warrants antibiotic treatment only for patients undergoing urological procedures that lead to mucosal bleeding, catheterized individuals whose ASB persists for more than 48 h after catheter removal, and pregnant women. Pyuria is associated with UTI and implicates host immune responses via release of antibacterial effectors and phagocytosis of pathogens by neutrophils. Such responses are not sufficiently described for ASB. Metaproteomic methods were used here to identify the pathogens and evaluate molecular evidence of distinct immune responses in cases of ASB compared to UTI in elderly patients who were hospitalized upon injury. Neutrophil-driven inflammatory responses to invading bacteria were not discernible in most patients diagnosed with ASB compared to those with UTI. In contrast, proteomic urine analysis for trauma patients with no evidence of bacteriuria, including those who suffered mucosal injuries via urethral catheterization, rarely showed evidence of neutrophil infiltration. The same enzymes contributing to the synthesis of leukotrienes LTB4 and LTC4, mediators of inflammation and pain, were found in the UTI and ASB cohorts. These data support the notion that the pathways mediating inflammation and pain in most elderly patients with ASB are not quantitatively different from those seen in most elderly patients with UTI and warrant larger clinical studies to assess whether a common antibiotic treatment strategy for elderly ASB and UTI patients is justified.

Serum quantitative proteomic analysis reveals potential zinc-associated biomarkers for nonbacterial prostatitis.

BACKGROUND: Prostatitis is one of the most common urological problems afflicting adult men. The etiology and pathogenesis of nonbacterial prostatitis, which accounts for 90-95% of cases, is largely unknown. As serum proteins often indicate the overall pathologic status of patients, we hypothesized that protein biomarkers of prostatitis might be identified by comparing the serum proteomes of patients with and without nonbacterial prostatitis. METHODS: All untreated samples were collected from subjects attending the Fangchenggang Area Male Health and Examination Survey (FAMHES). We profiled pooled serum samples from four carefully selected groups of patients (n = 10/group) representing the various categories of nonbacterial prostatitis (IIIa, IIIb, and IV) and matched healthy controls using a mass spectrometry-based 4-plex iTRAQ proteomic approach. More than 160 samples were validated by ELISA. RESULTS: Overall, 69 proteins were identified. Among them, 42, 52, and 37 proteins were identified with differential expression in Category IIIa, IIIb, and IV prostatitis, respectively. The 19 common proteins were related to immunity and defense, ion binding, transport, and proteolysis. Two zinc-binding proteins, superoxide dismutase 3 (SOD3), and carbonic anhydrase I (CA1), were significantly higher in all types of prostatitis than in the control. A receiver operating characteristic curve estimated sensitivities of 50.4 and 68.1% and specificities of 92.1 and 83.8% for CA1 and SOD3, respectively, in detecting nonbacterial prostatitis. The serum CA1 concentration was inversely correlated to the zinc concentration in expressed-prostatic secretions. CONCLUSIONS: Our findings suggest that SOD3 and CA1 are potential diagnostic markers of nonbacterial prostatitis, although further large-scale studies are required. The molecular profiles of nonbacterial prostatitis pathogenesis may lay a foundation for discovery of new therapies.