"Evaluating the Benefits and Limits of Multiple Displacement Amplification with Whole-Genome Oxford Nanopore Sequencing".

Multiple Displacement Amplification (MDA) outperforms conventional PCR in long fragment and whole genome amplification which makes it attractive to couple with long-read sequencing of samples with limited quantities of DNA to obtain improved genome assemblies. Here, we explore the efficacy and limits of MDA for genome sequence assembly using Oxford Nanopore Technologies (ONT) rapid library preparations and minION sequencing. We successfully generated almost complete genome sequences for all organisms examined, including Cryptosporidium meleagridis, Staphylococcus aureus, Enterococcus faecium, and Escherichia coli, with the ability to generate high-quality data from samples starting with only 0.025 ng of total DNA. Controlled sheared DNA samples exhibited a distinct pattern of size-increase after MDA, which may be associated with the amplification of long, low-abundance fragments present in the assay, as well as generating concatemeric sequences during amplification. To address concatemers, we developed a computational pipeline (CADECT: Concatemer Detection Tool) to identify and remove putative concatemeric sequences. This study highlights the efficacy of MDA in generating high-quality genome assemblies from limited amounts of input DNA. Also, the CADECT pipeline effectively mitigated the impact of concatemeric sequences, enabling the assembly of contiguous sequences even in cases where the input genomic DNA was degraded. These results have significant implications for the study of organisms that are challenging to culture in vitro, such as Cryptosporidium, and for expediting critical results in clinical settings with limited quantities of available genomic DNA.

Achieving a Predictive Understanding of Antimicrobial Stress Physiology through Systems Biology.

The dramatic spread and diversity of antibiotic-resistant pathogens has significantly reduced the efficacy of essentially all antibiotic classes, bringing us ever closer to a postantibiotic era. Exacerbating this issue, our understanding of the multiscale physiological impact of antimicrobial challenge on bacterial pathogens remains incomplete. Concerns over resistance and the need for new antibiotics have motivated the collection of omics measurements to provide systems-level insights into antimicrobial stress responses for nearly 20 years. Although technological advances have markedly improved the types and resolution of such measurements, continued development of mathematical frameworks aimed at providing a predictive understanding of complex antimicrobial-associated phenotypes is critical to maximize the utility of multiscale data. Here we highlight recent efforts utilizing systems biology to enhance our knowledge of antimicrobial stress physiology. We provide a brief historical perspective of antibiotic-focused omics measurements, highlight new measurement discoveries and trends, discuss examples and opportunities for integrating measurements with mathematical models, and describe future challenges for the field.

A proteomic approach to understanding the pathogenesis of idiopathic macular hole formation.

Idiopathic macular holes (IMH) are full-thickness defects of retinal tissue that cause severe vision loss due to disruption of the anatomic fovea. Abnormal vitreous traction is involved in the formation of macular holes. Both glial cells and hyalocytes contribute to epiretinal membrane formation in IMH. In order to gain further insight into the pathophysiology of IMH, we conducted a discovery phase investigation of the vitreous proteome in four patients with macular holes and six controls using one-dimensional gel fractionation and liquid chromatography-tandem mass spectrometry analyses on an Orbitrap Elite mass spectrometer. Of a total of 5912 vitreous proteins, 32 proteins had increased and 39 proteins had decreased expression in IMH compared with controls, using a false discovery rate approach with p value < 0.001 and q value < 0.05. IMH was associated with increased expression of proteins in the complement pathway, α-2-macroglobulin, a major inducer of Müller glial cell migration, fibrinogen, and extracellular matrix proteins, and decreased expression of proteins involved in protein folding and actin filament binding. A proteomic approach revealed proteins and biological pathways that may be involved in the pathogenesis of IMH and could be targeted for future studies.

The proteome of normal human retrobulbar optic nerve and sclera.

The optic nerve is a white matter tract that conveys visual information to the brain. The sclera comprises the white, protective outer layer of the eye. A characterization of the proteome of normal human retrobulbar optic nerve and sclera may facilitate studies of the eye. We conducted a proteomic analysis of optic nerve and sclera from five adults. Proteins were fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. We identified 2711 non-redundant proteins in retrobulbar optic nerve and 1945 non-redundant proteins in sclera. Optic nerve proteins included proteins expressed by oligodendrocytes (laminin, proteolipid protein, fibronectin), myelin proteins (myelin basic protein, myelin-associated glycoprotein), and paranodal structural proteins (ankyrin ß, spectrin). Sclera included 18 collagen protein chains, small leucine-rich proteoglycans (decorin, biglycan, lumican, keratocan, prolargin, fibromodulin, mimecan), non-collagenous glycoproteins (fibronectin, vitronectin, laminin), extracellular matrix proteins (thrombospondins 1-4, dystroglycan, transgelins 1-3), and integrins alpha-V, alpha-1 and 2, beta-1, -2, and -5. Twenty-one unambiguous alternative splicing protein isoforms were identified in optic nerve and ten unambiguous alternative splicing protein isoforms were identified in sclera. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD001581.

Defining the proteome of human iris, ciliary body, retinal pigment epithelium, and choroid.

The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false-positive rates of <0.1% and <1%, respectively. Forty-three unambiguous protein isoforms were identified in iris, ciliary body, and RPE/choroid. Four "missing proteins" were identified in ciliary body based on ≥2 proteotypic peptides. The mass spectrometric proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194.

Changes in oxidized lipids drive the improvement in monocyte activation and vascular disease after statin therapy in HIV.

BACKGROUND: Oxidative stress plays a significant role in atherosclerosis development. HIV infection has been linked with heightened cardiovascular disease risk. HMG-CoA reductase inhibitors may reduce oxidative stress and subsequently subclinical vascular disease in HIV. DESIGN/METHODS: This is a randomized, placebo-controlled trial to evaluate the effect of rosuvastatin in HIV-infected adults on stable antiretroviral therapy with low-density lipoprotein less than 130  mg/dl and increased inflammation or T-cell activation on subclinical vascular disease. Changes over 48 weeks in oxidative stress markers, oxidized low-density lipoprotein (oxLDL) and F2-isoprostane/creatinine ratio (F2-IsoP/Cr), were compared between groups. Spearman correlation and multivariable linear regression were used to evaluate relationships between changes in markers of oxidative stress, inflammation and monocyte activation and carotid intima media thickness (CIMT). RESULTS: One hundred and forty-seven adults enrolled (72 to rosuvastatin and 75 to placebo). In the rosuvastatin group, oxLDL decreased significantly over 24 weeks compared to placebo [mean absolute change in log-oxLDL for rosuvastatin -0.2  ±  0.468 log U/l (P < 0.001 within-group) vs. placebo -0.018  ±  0.456 log U/l (P = 0.83 within-group); P = 0.004 between groups] and this change was linked with changes in soluble CD14 and proportion of patrolling monocytes (CD14dimCD16). Although oxLDL levels increased after initially declining and were not different from placebo at week 48, the early improvement in oxLDL was associated with improved CIMT at week 48. Changes in F2-IsoP/Cr were not significant between groups. CONCLUSION: Rosuvastatin decreases oxLDL levels early after initiation and is associated with decreased monocyte activation. Early improvement in oxLDL is linked with improved CIMT in treated HIV infection.

Low Plasma Klotho Concentrations and Decline of Knee Strength in Older Adults.

BACKGROUND: Although the "anti-aging hormone" klotho is associated with sarcopenia in mice, the relationship between klotho and muscle strength in older adults is not well known. METHODS: Plasma klotho concentrations were measured in 2,734 older adults, aged 71-80 years, who participated in the Health, Aging and Body Composition Study, a prospective observational cohort study conducted in Memphis, TN and Pittsburgh, PA. Knee extension strength was measured using isokinetic dynamometry at baseline and follow-up 2 and 4 years later. Knee extension strength was normalized for weight. RESULTS: At baseline, participants in the highest tertile of plasma klotho had higher knee extension strength (ß = .72, standard error [SE] = .018, p < .0001) compared with those in the lowest tertile in a multivariable linear regression model adjusting for age, sex, race, smoking, study site, C-reactive protein, interleukin-6, and diabetes. Participants in the highest tertile of plasma klotho at baseline had less of a decline in knee strength over 4 years of follow-up (ß = -.025, SE = .011, p = .02) compared with those in the lowest tertile in a multivariable linear regression model adjusting for the same covariates above. CONCLUSIONS: Plasma klotho concentrations were an independent predictor of changes in knee strength over time in older adults. Further studies are needed to identify the biological mechanisms by which circulating klotho could modify skeletal muscle strength.

The proteome of human retina.

The retina is a delicate tissue that detects light, converts photochemical energy into neural signals, and transmits the signals to the visual cortex of the brain. A detailed protein inventory of the proteome of the normal human eye may provide a foundation for new investigations into both the physiology of the retina and the pathophysiology of retinal diseases. To provide an inventory, proteins were extracted from five retinas of normal eyes and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed in duplicate using LC-MS/MS on an Orbitrap Elite mass spectrometer. A total of 3436 nonredundant proteins were identified in the human retina, including 20 unambiguous protein isoforms, of which eight have not previously been demonstrated to exist at the protein level. The proteins identified in the retina included most of the enzymes involved in the visual cycle and retinoid metabolism. One hundred and fifty-eight proteins that have been associated with age-related macular degeneration were identified in the retina. The MS proteome database of the human retina may serve as a valuable resource for future investigations of retinal biology and disease. All MS data have been deposited in the ProteomeXchange with identifier PXD001242 (http://proteomecentral.proteomexchange.org/dataset/PXD001242).

Dietary intake of advanced glycation end products did not affect endothelial function and inflammation in healthy adults in a randomized controlled trial.

When food is heated to high temperatures, the characteristic "browning" generates advanced glycation end products (AGEs). AGEs are associated with an increased risk of cardiovascular disease, diabetes, and other adverse outcomes. Whether dietary AGEs are absorbed and are harmful to human health remains highly controversial. The objective of this study was to compare the effects of a diet high or low in AGEs on endothelial function, circulating AGEs, inflammatory mediators, and circulating receptors for AGEs in healthy adults. A randomized, parallel-arm, controlled dietary intervention was conducted for 6 wk with 24 healthy adults, aged 50-69 y, that compared isocaloric, food-equivalent diets that were prepared at either high or mild temperatures. Peripheral arterial tonometry, serum and urine carboxymethyl-lysine (CML), inflammatory mediators (interleukin-6, C-reactive protein, vascular adhesion molecule-1, and tumor necrosis factor-α receptors I and II), soluble receptor for AGEs, and endogenous secretory receptor for AGEs were measured at baseline and after 6 wk of dietary intervention. In the low-AGE diet group, the following changed from baseline to 6 wk (mean ± SE): serum CML from 763 ± 24 to 679 ± 29 ng/mL (P = 0.03) and urine CML from 1.37 ± 1.47 to 0.77 ± 2.01 µg/mL creatinine (P = 0.02). There were no significant changes in serum and urinary CML concentrations from baseline to follow-up in the high-AGE diet group. A high- or low-AGE diet had no significant impact on peripheral arterial tonometry or any inflammatory mediators after 6 wk of dietary intervention. In healthy middle-aged to older adults, consumption of a diet high or low in AGEs for 6 wk had no impact on endothelial function and inflammatory mediators, 2 precursors of cardiovascular disease.

Klotho in the cerebrospinal fluid of adults with and without Alzheimer's disease.

The aging-suppressor gene klotho encodes a single-pass transmembrane protein that is predominantly secreted by the choroid plexus of the brain and in the kidney. Klotho-deficient mice develop multiple aging phenotypes, including impaired cognition. Klotho concentrations have not been described in the CSF of humans. We measured klotho in the CSF of 20 older adults with Alzheimer's disease and in 20 older and 20 younger adults with normal cognition. In 10 adults, aged 38-87 years, CSF klotho measurements were made at baseline and every 6h up to 18-30 h later. Mean (95% confidence interval [C.I.]) CSF klotho in men versus women were 899 (814, 983) and 716 (632, 801) pg/mL, respectively (P=0.002). Mean (95% C.I.) CSF klotho in older adults with and without Alzheimer's disease were 664 (603, 725) and 776 (705, 828) pg/mL, respectively (P=0.02), adjusting for sex. Mean (95% C.I.) klotho in older versus younger adults were 766 (658, 874) and 992 (884, 1100) pg/mL, respectively (P=0.005), adjusting for sex. In the longitudinal study of CSF klotho, no significant circadian fluctuations were found in CSF klotho levels. This study suggests that CSF klotho concentrations are lower in females compared with males, in Alzheimer's disease, and in older versus younger adults.