Targeted metabolomics analysis approach to unravel the biofilm formation pathways of Enterococcus faecalis clinical isolates.

AIM: (i) To characterize Enterococcus faecalis biofilm formation pathways by semi-targeted metabolomics and targeted nitrogen panel analysis of strong (Ef63) and weak (Ef 64) biofilm forming E. faecalis clinical isolates and (ii) to validate the identified metabolic markers using targeted inhibitors. METHODOLOGY: Previous proteomics profiling of E. faecalis clinical isolates with strong and weak biofilm formation revealed that differences in metabolic activity levels of small molecule, nucleotide and nitrogen compound metabolic processes and biosynthetic pathways, cofactor metabolic process, cellular amino acid and derivative metabolic process and lyase activity were associated with differences in biofilm formation. Hence, semi-targeted analysis of Ef 63, Ef 64 and ATC control strain Ef 29212 was performed by selecting metabolites that were part of both the previously identified pathways and a curated library with confirmed physical and chemical identity, followed by confirmatory targeted nitrogen panel analysis. Significantly regulated metabolites (p < .05) were selected based on fold change cut-offs of 1.2 and 0.8 for upregulation and downregulation, respectively, and subjected to pathway enrichment analysis. The identified metabolites and pathways were validated by minimum biofilm inhibitory concentration (MBIC) and colony forming unit (CFU) assays with targeted inhibitors. RESULTS: Metabolomics analysis showed upregulation of betaine, hypoxanthine, glycerophosphorylcholine, tyrosine, inosine, allantoin and citrulline in Ef 63 w.r.t Ef 64 and Ef 29212, and thesemetabolites mapped to purinemetabolism, urea cycle and aspartate metabolism pathways. MBIC and CFU assays using compounds against selected metabolites and metabolic pathways, namely glutathione against hypoxanthine and hydroxylamine against aspartate metabolism showed inhibitory effects against E. faecalis biofilm formation. CONCLUSIONS: The study demonstrated the importance of oxidative stress inducers such as hypoxanthine and aspartate metabolism pathway in E. faecalis biofilm formation. Targeted therapeutics against these metabolic markers can reduce the healthcare burden associated with E. faecalis infections.

Amino acid differences between diabetic older adults and non-diabetic older adults and their associations with cardiovascular function.

BACKGROUND: Ageing and insulin resistant states such as diabetes mellitus frequently coexist and increase the risk of cardiovascular disease development among older adults. Here we investigate metabolic differences in amino acid profiles between ageing and diabetes mellitus, and their associations with cardiovascular function. METHODS: In a group of community older adults we performed echocardiography, cardiac magnetic resonance imaging as well as cross sectional and longitudinal metabolomics profiling based on current and archived sera obtained fifteen years prior to examination. RESULTS: We studied a total of 515 participants (women 50%, n = 255) with a mean age 73 (SD = 4.3) years. Diabetics had higher alanine (562 vs 448, p < 0.0001), higher glutamate (107 vs 95, p = 0.016), higher proline (264 vs 231, p = 0.008) and lower arginine (107 vs 117, p = 0.043), lower citrulline (30 vs 38, p = 0.006) levels (µM) compared to non-diabetics. Over time, changes in amino acid profiles differentiated diabetic older adults from non-diabetic older adults, with greater accumulation of alanine (p = 0.002), proline (p = 0.008) and (non-significant) trend towards greater accumulation of glycine (p = 0.057) among the older diabetics compared to the older non-diabetics. However, independent of diabetes status, amino acids were associated with cardiovascular functions in ageing, [archived valine (p = 0.011), leucine (p = 0.011), archived isoleucine (p = 0.0006), archived serine (p = 0.008), archived glycine (p = 0.006) methionine (p = 0.003)] which were associated with impairments in E/A ratio. CONCLUSION: Markers of branched chain amino acids and one ­carbon metabolism pathways were associated with changes in cardiovascular function in older adults regardless of diabetes status. However, nitrogen handling pathways were specifically altered among older adults with diabetes. These findings broaden our understanding into specific amino acid pathways that may be altered between diabetic and non-diabetic older adults, and their relevance to cardiovascular function in ageing. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02791139.

Multi-omics tools for studying microbial biofilms: current perspectives and future directions.

The advent of omics technologies has greatly improved our understanding of microbial biology, particularly in the last two decades. The field of microbial biofilms is, however, relatively new, consolidated in the 1980s. The morphogenic switching by microbes from planktonic to biofilm phenotype confers numerous survival advantages such as resistance to desiccation, antibiotics, biocides, ultraviolet radiation, and host immune responses, thereby complicating treatment strategies for pathogenic microorganisms. Hence, understanding the mechanisms governing the biofilm phenotype can result in efficient treatment strategies directed specifically against molecular markers mediating this process. The application of omics technologies for studying microbial biofilms is relatively less explored and holds great promise in furthering our understanding of biofilm biology. In this review, we provide an overview of the application of omics tools such as transcriptomics, proteomics, and metabolomics as well as multi-omics approaches for studying microbial biofilms in the current literature. We also highlight how the use of omics tools directed at various stages of the biological information flow, from genes to metabolites, can be integrated via multi-omics platforms to provide a holistic view of biofilm biology. Following this, we propose a future artificial intelligence-based multi-omics platform that can predict the pathways associated with different biofilm phenotypes.

Characterization of Virus Capsids and Their Assembly Intermediates by Multicycle Resistive-Pulse Sensing with Four Pores in Series.

Virus self-assembly is a critical step in the virus lifecycle. Understanding how viruses assemble and disassemble provides needed insight into developing antiviral pharmaceuticals. Few tools offer sufficient resolution to study assembly intermediates that differ in size by a few dimers. Our goal is to improve resistive-pulse sensing on nanofluidic devices to offer better particle-size and temporal resolution to study intermediates and capsids generated along the assembly pathway. To increase the particle-size resolution of the resistive-pulse technique, we measured the same, single virus particles up to a thousand times, cycling them back and forth across a series of nanopores by switching the polarity of the applied potential, i.e., virus ping-pong. Multiple pores in series provide a unique multipulse signature during each cycle that improves particle tracking and, therefore, identification of a single particle and reduces the number of cycles needed to make the requisite number of measurements. With T = 3 and T = 4 hepatitis B virus (HBV) capsids, we showed the standard deviation of the particle-size distribution decreased with the square root of the number of measurements and approached discriminating particles differing in size by single dimers. We then studied in vitro assembly of HBV capsids and observed that the ensemble of intermediates shift to larger sizes over 2 days of annealing. On the contrary, assembly reactions diluted to lower dimer concentrations an hour after initiation had fewer intermediates that persisted after the 2 day incubation and had a higher ratio of T = 4 to T = 3 capsids. These reactions indicate that labile T = 4 intermediates are formed rapidly, and dependent on conditions, intermediates may be trapped as metastable species or progress to yield complete capsids.

Molecular jenga: the percolation phase transition (collapse) in virus capsids.

Virus capsids are polymeric protein shells that protect the viral cargo. About half of known virus families have icosahedral capsids that self-assemble from tens to thousands of subunits. Capsid disassembly is critical to the lifecycles of many viruses yet is poorly understood. Here, we apply a graph and percolation theory to examine the effect of removing capsid subunits on capsid stability and fragmentation. Based on the structure of the icosahedral capsid of hepatitis B virus (HBV), we constructed a graph of rhombic subunits arranged with icosahedral symmetry. Though our approach neglects dependence on energetics, time, and molecular detail, it quantitatively predicts a percolation phase transition consistent with recent in vitro studies of HBV capsid dissociation. While the stability of the capsid graph followed a gradual quadratic decay, the rhombic tiling abruptly fragmented when we removed more than 25% of the 120 subunits, near the percolation threshold observed experimentally. This threshold may also affect results of capsid assembly, which also experimentally produces a preponderance of 90 mer intermediates, as the intermediate steps in these reactions are reversible and can thus resemble dissociation. Application of percolation theory to understanding capsid association and dissociation may prove a general approach to relating virus biology to the underlying biophysics of the virus particle.

Nanofluidic Devices with 8 Pores in Series for Real-Time, Resistive-Pulse Analysis of Hepatitis B Virus Capsid Assembly.

To improve the precision of resistive-pulse measurements, we have used a focused ion beam instrument to mill nanofluidic devices with 2, 4, and 8 pores in series and compared their performance. The in-plane design facilitates the fabrication of multiple pores in series which, in turn, permits averaging of the series of pulses generated from each translocation event. The standard deviations (σ) of the pulse amplitude distributions decrease by 2.7-fold when the average amplitudes of eight pulses are compared to the amplitudes of single pulses. Similarly, standard deviations of the pore-to-pore time distributions decrease by 3.2-fold when the averages of the seven measurements from 8-pore devices are contrasted to single measurements from 2-pore devices. With signal averaging, the inherent uncertainty in the measurements decreases; consequently, the resolution (mean/σ) improves by a factor equal to the square root of the number of measurements. We took advantage of the improved size resolution of the 8-pore devices to analyze in real time the assembly of Hepatitis B Virus (HBV) capsids below the pseudocritical concentration. We observe that abundances of assembly intermediates change over time. During the first hour of the reaction, the abundance of smaller intermediates decreased, whereas the abundance of larger intermediates with sizes closer to a T = 4 capsid remained constant.

Detection of late intermediates in virus capsid assembly by charge detection mass spectrometry.

The assembly of hundreds of identical proteins into an icosahedral virus capsid is a remarkable feat of molecular engineering. How this occurs is poorly understood. Key intermediates have been anticipated at the end of the assembly reaction, but it has not been possible to detect them. In this work we have used charge detection mass spectrometry to identify trapped intermediates from late in the assembly of the hepatitis B virus T = 4 capsid, a complex of 120 protein dimers. Prominent intermediates are found with 104/105, 110/111, and 117/118 dimers. Cryo-EM observations indicate the intermediates are incomplete capsids and, hence, on the assembly pathway. On the basis of their stability and kinetic accessibility we have proposed plausible structures. The prominent trapped intermediate with 104 dimers is attributed to an icosahedron missing two neighboring facets, the 111-dimer species is assigned to an icosahedron missing a single facet, and the intermediate with 117 dimers is assigned to a capsid missing a ring of three dimers in the center of a facet.

Urine tricarboxylic acid cycle metabolites and risk of end stage kidney disease in patients with type 2 diabetes.

CONTEXT: Metabolites in tricarboxylic acid (TCA) pathway have pleiotropic functions. OBJECTIVE: To study the association between urine TCA cycle metabolites and the risk for chronic kidney disease (CKD) progression in individuals with type 2 diabetes. DESIGN, SETTING AND PARTICIPANTS: A prospective study in a discovery (n = 1826) and a validation (n = 1235) cohort of type 2 diabetes in a regional hospital and a primary care facility. EXPOSURE AND OUTCOME: Urine lactate, pyruvate, citrate, alpha-ketoglutarate, succinate, fumarate and malate were measured by mass spectrometry. CKD progression was defined as a composite of sustained eGFR below 15 ml/min/1.73 m2 , dialysis, renal death or doubling of serum creatinine. RESULTS: During a median of 9.2 (IQR 8.1-9.7) and 4.0 (3.2-5.1) years of follow-up, 213 and 107 renal events were identified. Cox regression suggested that urine lactate, fumarate and malate were associated with an increased risk (adjusted hazard ratio, aHR [95% CI] 1.63 [1.16-2.28], 1.82 [1.17-2.82] and 1.49 [1.05-2.11], per SD), while citrate was associated with a low risk (aHR 0.83 [0.72-0.96] per SD) for the renal outcome after adjustment for cardio-renal risk factors. These findings were reproducible in the validation cohort. Noteworthy, fumarate and citrate were independently associated with the renal outcome after additional adjustment for other metabolites. CONCLUSION: Urine fumarate and citrate predict the risk for progression to ESKD independent of clinical risk factors and other urine metabolites. These two metabolites in TCA cycle pathway may play important roles in the pathophysiological network underpinning progressive loss of kidney function in patients with type 2 diabetes.

Lipidomics Workflow for Analyzing Lipid Profiles Using Multiple Reaction Monitoring (MRM) in Liver Homogenate of Mice with Non-alcoholic Steatohepatitis (NASH).

Non-alcoholic steatohepatitis (NASH) is a condition characterized by inflammation and hepatic injury/fibrosis caused by the accumulation of ectopic fats in the liver. Recent advances in lipidomics have allowed the identification and characterization of lipid species and have revealed signature patterns of various diseases. Here, we describe a lipidomics workflow to assess the lipid profiles of liver homogenates taken from a NASH mouse model. The protocol described below was used to extract and analyze the metabolites from the livers of mice with NASH by liquid chromatography-mass spectrometry (LC-MS); however, it can be applied to other tissue homogenate samples. Using this method, over 1,000 species of lipids from five classes can be analyzed in a single run on the LC-MS. Also, partial elucidation of the identity of neutral lipid (triacylglycerides and diacylglycerides) aliphatic chains can be performed with this simple LC-MS setup. Key features Over 1,000 lipid species (sphingolipids, cholesteryl esters, neutral lipids, phospholipids, fatty acids) are analyzed in one run. Analysis of liver lipids in non-alcoholic steatohepatitis (NASH) mouse model. Normal-phase chromatography coupled to a triple quadrupole mass spectrometer.

Adverse cardiovascular and metabolic perturbations among older women: 'fat-craving' hearts.

BACKGROUND: Despite known sex-based differences in cardiovascular aging, differences in aging biology are poorly understood. We hypothesize that circulating metabolites studied cross-sectionally with cardiac aging may be associated with cardiovascular changes that distinguish cardiac aging in women. METHODS: A population-based cohort of community men and women without cardiovascular disease from Singapore underwent detailed clinical and echocardiography examinations. Cross-sectional associations between cardiac functional characteristics and metabolomics profiles were examined. RESULTS: Five hundred sixty-seven adults (48.9% women) participated. Women were younger (72 ± 4.4 years vs 73 ± 4.3 years, p = 0.022), had lower diastolic blood pressures (71 ± 11.0 mmHg vs 76 ± 11.2 mmHg, p < 0.0001, and less likely to have diabetes mellitus (18.0% vs 27.6%, p = 0.013) and smoking (3.8% vs 34.5%, p < 0.001). Body mass indices were similar (24 ± 3.8 kg/m2 vs 24 ± 3.4 kg/m2, p = 0.29), but women had smaller waist circumferences (81 ± 10.1 cm vs 85 ± 9.2 cm, p < 0.001). Women had a significantly higher E/e' ratios (10.9 ± 3.4 vs 9.9 ± 3.3, p = 0.007) and mitral A peak (0.86 ± 0.2 m/s vs 0.79 ± 0.2 m/s, p < 0.001) than men. Among women, lower E/e' ratio was associated with higher levels of C16 (OR 1.019, 95%CI 1.002-1.036, p = 0.029), C16:1 (OR 1.06, 95%CI 1.006-1.118, p = 0.028), serine (OR 1.019, 95%CI 1.002-1.036, p = 0.025), and histidine (OR 1.045, 95%CI 1.013-1.078, p = 0.006). Lower mitral A peak was associated with higher levels of histidine (OR 1.039, 95%CI 1.009-1.070, p = 0.011), isoleucine (OR 1.013, 95%CI 1.004-1.021, p = 0.004), and C20 (OR 1.341, 95%CI 1.067-1.684, p = 0.012). CONCLUSION: Impairments in diastolic functions were more frequent among older women compared to men, despite lower prevalence of vascular risk factors and preserved cardiac structure. Cardiac aging in women correlated with metabolites involved in fatty acid oxidation and tricyclic acid cycle fuelling.

Plasma Tryptophan-Kynurenine Pathway Metabolites and Risk for Progression to End-Stage Kidney Disease in Patients With Type 2 Diabetes.

OBJECTIVE: We sought to study the associations between plasma metabolites in the tryptophan-kynurenine pathway and the risk of progression to end-stage kidney disease (ESKD) in patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: Plasma tryptophan, kynurenine, 3-hydroxykynurenine, kynurenic acid, and xanthurenic acid concentrations were measured in discovery (n = 1,915) and replication (n = 346) cohorts. External validation was performed in Chronic Renal Insufficiency Cohort (CRIC) participants with diabetes (n = 1,312). The primary outcome was a composite of incident ESKD (progression to estimated glomerular filtration rate [eGFR] <15 mL/min/1.73 m2, sustained dialysis, or renal death). The secondary outcome was annual eGFR decline. RESULTS: In the discovery cohort, tryptophan was inversely associated with risk for ESKD, and kynurenine-to-tryptophan ratio (KTR) was positively associated with risk for ESKD after adjustment for clinical risk factors, including baseline eGFR and albuminuria (adjusted hazard ratios [HRs] 0.62 [95% CI 0.51, 0.75] and 1.48 [1.20, 1.84] per 1 SD). High levels of kynurenic acid and xanthurenic acid were associated with low risks of ESKD (0.74 [0.60, 0.91] and 0.74 [0.60, 0.91]). Consistently, high levels of tryptophan, kynurenic acid, and xanthurenic acid were independently associated with a slower eGFR decline, while a high KTR was predictive of a faster eGFR decline. Similar outcomes were obtained in the replication cohort. Furthermore, the inverse association between kynurenic acid and risk of ESKD was externally validated in CRIC participants with diabetes (adjusted HR 0.78 [0.65, 0.93]). CONCLUSIONS: Accelerated catabolism of tryptophan in the kynurenine pathway may be involved in progressive loss of kidney function. However, shunting the kynurenine pathway toward the kynurenic acid branch may potentially slow renal progression.

Sensitive ex vivo human skin transdermal assay testing method with mass spectrometric analysis for cosmetics application.

BACKGROUND: Cosmetics manufacturers are focused on cosmetic delivery systems into the skin, but the level of diffusion of the systems in the skin tissues is not well understood. The current methods, such as Franz diffusion, assess analyte diffusion in the whole skin or artificial membranes, which has limitations for understanding skin delivery systems. AIMS: Our study aimed to create a transdermal delivery method which is based on dermal-epidermal separation of human skin, allowing us to assess each layer of skin separately for its efficacy. MATERIALS AND METHODS: During the experiment, resveratrol was used as the target analyte by applying it to the skin and then separating it into dermis and epidermis. Each layer is treated individually and subjected to a high-resolution mass spectrometry analysis to detect resveratrol levels. As a result, the efficiency of resveratrol diffusion in the dermal and epidermal layers of the skin can be evaluated. RESULTS: We found that resveratrol was detected in both the dermal and epidermal layers using our method. CONCLUSIONS: Hence, we developed a sensitive method for transdermal delivery testing that can be used to evaluate skin delivery systems for cosmetic or pharmaceutical purposes.

Amino acid profile of skeletal muscle loss in type 2 diabetes: Results from a 7-year longitudinal study in asians.

AIMS: Little is known about pathophysiology of sarcopenia in diabetes. We aimed to study amino acid profile associated with skeletal muscle mass loss longitudinally in Type 2 Diabetes Mellitus (T2DM). METHODS: This is a prospective study of 1140 patients aged 56.6 ± 10.6 years from the SMART2D cohort. Skeletal muscle mass was measured using bio-impedance analysis at baseline and follow-up. Amino acids were measured by mass spectrometry. RESULTS: Over a period of up to 7.9 years, 43.9% experienced skeletal muscle mass loss. Lower baseline valine, leucine and isoleucine levels were associated with decreased skeletal muscle mass index (SMI) with corresponding coefficient 0.251(95 %CI 0.009 to 0.493), 0.298(95 %CI 0.051 to 0.544)) and 0.366(95 %CI 0.131 to 0.600). Higher baseline valine, leucine, isoleucine, alanine and tryptophan levels were associated with reduced odds of muscle mass loss with corresponding odds ratio (OR)0.797 (95 %CI 0.690 to 0.921), 0.825 (95 %CI 0.713 to 0.955), 0.826 (95 %CI 0.718-0.950), 0.847 (95 %CI 0.739-0.969) and 0.835 (95 %CI 0.720-0.979). CONCLUSION: The branched-chain amino acids valine, leucine and isoleucine were positively associated with change in SMI and reduced odds of muscle mass loss longitudinally. Further studies should be conducted to elucidate the pathophysiological mechanisms underlying the relationship between these amino acids and muscle mass loss in T2DM.

Exacerbation of cardiovascular ageing by diabetes mellitus and its associations with acyl-carnitines.

OBJECTIVE: To demonstrate differences in cardiovascular structure and function between diabetic and non-diabetic older adults. To investigate associations between acyl-carnitines and cardiovascular function as indexed by imaging measurements. METHODS: A community-based cohort of older adults without cardiovascular disease underwent current cardiovascular imaging and metabolomics acyl-carnitines profiling based on current and archived sera obtained fifteen years prior to examination. RESULTS: A total of 933 participants (women 56%, n=521) with a mean age 63±13 years were studied. Old diabetics compared to old non-diabetics had lower myocardial relaxation (0.8±0.2 vs 0.9±0.3, p=0.0039); lower left atrial conduit strain (12±4.3 vs 14±4.1, p=0.045), lower left atrial conduit strain rate (-1.2±0.4 vs -1.3±0.5, p=0.042) and lower ratio of left atrial conduit strain to left atrial booster strain (0.5±0.2 vs 0.7±0.3, p=0.0029). Higher levels of archived short chain acyl-carnitine were associated with present-day impairments in myocardial relaxation (C5:1; OR 1.03, p=0.011), worse left atrial conduit strain function (C5:1; OR 1.03, p=0.037). Increases in hydroxylated acyl-carnitines were associated with worse left atrial conduit strain [(C4-OH; OR 1.05, p=0.0017), (C16:2-OH; OR 1.18, p=0.037)]. Current, archived and changes in long chain acyl-carnitines were associated with cardiovascular functions [(C16; OR 1.02, p=0.002), (C20:3; OR 1.01, p=0.014), (C14:3; OR 1.12, p=0.033), (C18:1; OR 1.01, p=0.018), (C18:2; OR 1.01, p=0.028), (C20:4; OR 1.10, p=0.038)] (all p<0.05). CONCLUSION: Older diabetic adults had significant impairments in left ventricular myocardial relaxation and left atrial strain, compared to older non-diabetic adults. Short chain and long chain, di-carboxyl and hydroxylated acyl-carnitines were associated with these cardiovascular functional differences.

Evolution of Intermediates during Capsid Assembly of Hepatitis B Virus with Phenylpropenamide-Based Antivirals.

Self-assembly of virus capsids is a potential target for antivirals due to its importance in the virus lifecycle. Here, we investigate the effect of phenylpropenamide derivatives B-21 and AT-130 on the assembly of hepatitis B virus (HBV) core protein. Phenylpropenamides are widely believed to yield assembly of spherical particles resembling native, empty HBV capsids. Because the details of assembly can be overlooked with ensemble measurements, we performed resistive-pulse sensing on nanofluidic devices with four pores in series to characterize the size distributions of the products in real time. With its single particle sensitivity and compatibility with typical assembly buffers, resistive-pulse sensing is well-suited for analyzing virus assembly in vitro. We observed that assembly with B-21 and AT-130 produced a large fraction of partially complete virus particles that may be on-path, off-path, or trapped. For both B-21 and AT-130, capsid assembly was more sensitive to disruption under conditions where the interprotein association energy was low at lower salt concentrations. Dilution of the reaction solutions led to the rearrangement of the incomplete particles and demonstrated that these large intermediates may be on-path, but are labile, and exist in a frustrated dynamic equilibrium. During capsid assembly, phenylpropenamide molecules modestly increase the association energy of dimers, prevent intermediates from dissociating, and lead to kinetic trapping where the formation of too many capsids has been initiated, which results in both empty and incomplete particles.

A molecular breadboard: Removal and replacement of subunits in a hepatitis B virus capsid.

Hepatitis B virus (HBV) core protein is a model system for studying assembly and disassembly of icosahedral structures. Controlling disassembly will allow re-engineering the 120 subunit HBV capsid, making it a molecular breadboard. We examined removal of subunits from partially crosslinked capsids to form stable incomplete particles. To characterize incomplete capsids, we used two single molecule techniques, resistive-pulse sensing and charge detection mass spectrometry. We expected to find a binomial distribution of capsid fragments. Instead, we found a preponderance of 3 MDa complexes (90 subunits) and no fragments smaller than 3 MDa. We also found 90-mers in the disassembly of uncrosslinked HBV capsids. 90-mers seem to be a common pause point in disassembly reactions. Partly explaining this result, graph theory simulations have showed a threshold for capsid stability between 80 and 90 subunits. To test a molecular breadboard concept, we showed that missing subunits could be refilled resulting in chimeric, 120 subunit particles. This result may be a means of assembling unique capsids with functional decorations.