Small Angle Neutron Scattering from D2O–H2O Nanodroplets and Binary Nucleation Rates in A Supersonic Nozzle

Small angle neutron scattering (SANS) experiments were used to characterize binary nanodroplets composed of D2O and H2O. The droplets were formed by expanding dilute mixtures of condensible vapor in a N2 carrier gas through a supersonic nozzle, while maintaining the onset of condensation at a fixed position in the nozzle. It is remarkable, given the small coherent scattering length density of light water, that even the pure H2O aerosol gave a scattering signal above background. The scattering spectra were analyzed assuming a log-normal distribution of droplets. On average, the geometric radius of the nanodroplets rg was rg=13 (±1) nm, the polydispersity ln σr was ln σr=0.19 (±0.07), and the number density N was N=(2±0.2)⋅1011 cm−3. The aerosol volume fractions derived from the SANS measurements are consistent with those derived from the pressure trace experiments, suggesting that the composition of the droplets was close to that of the initial condensible mixture. A quantitative analysis of the scattering spectra as a function of the isotopic composition gave further evidence that the binary droplets exhibit ideal mixing behavior. Because both the stagnation temperature T0 and the location of onset were fixed, the temperature corresponding to the maximum nucleation rate was constant at TJ max=229 (±1) K. Thus, the experiments let us estimate the isothermal peak nucleation rates as a function of the isotopic composition. The nucleation rates were found to be essentially constant with Jmax equal to (3.6±0.5)⋅1016 cm−3 s−1 at a mean supersaturation of 44 (±3)

H2O–D2O Condensation in A Supersonic Nozzle

We examined the condensation of H2O, D2O, and four intermediate mixtures (20, 40, 60, and 80 mol % D2O) in a supersonic nozzle. Because the physical and chemical properties of protonated and deuterated water are so similar, this system is ideal for studying the change in condensation behavior as a function of condensible composition. In our experiments dilute mixtures of condensible vapor in N2 are expanded from three different stagnation temperatures resulting in a broad range of onset temperatures (190–238 K) and pressures (27–787 kPa). For a fixed stagnation temperature, the partial pressure required to maintain the onset of condensation at a given location or temperature in the nozzle is consistently higher for H2O than for D2O. In contrast, the supersaturation at fixed onset temperature is usually higher for D2O than for H2O and this difference increases toward lower temperature. The partial pressure at onset for the intermediate mixtures varied linearly between the values observed for the pure components in this ideal system

Quantitative non-canonical amino acid tagging based proteomics identifies distinct patterns of protein synthesis rapidly induced by hypertrophic agents in cardiomyocytes, revealing new aspects of metabolic remodeling

Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. Pathological myocardial growth is a risk factor for cardiac failure to which faster protein synthesis is a major driving element. We aimed to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are due to alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other non-dividing primary cells. To address this, an optimized QuaNCAT LC/MS method was used to selectively quantify newly synthesized proteins in such cells. The study showed both classical (phenylephrine; PE) and more recent (insulin) pathological cardiac hypertrophic agents increased the synthesis of proteins involved in glycolysis, the Krebs cycle / beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was upregulated by signaling through mTORC1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was upregulated in rat hearts following TAC. This isoform possesses specific regulatory properties that may be involved in metabolic remodeling and as a novel candidate biomarker. Levels of translation factor eEF1 also increased during TAC, likely contributing to faster cell mass accumulation. Interestingly, PKM2 and eEF1 were not up-regulated in pregnancy or exercise induced CH, suggesting them as pathological CH specific markers. The study methods may be of utility to the examination of protein synthesis in primary cells

Long non-coding RNAs and latent HIV : a search for novel targets for latency reversal

The latent cellular reservoir of HIV is recognized as the major barrier to cure from HIV infection. Long non-coding RNAs (lncRNAs) are more tissue and cell type-specific than protein coding genes, and may represent targets of choice for HIV latency reversal. Using two in vitro primary T-cell models, we identified lncRNAs dysregulated in latency. PVT1 and RP11-347C18.3 were up-regulated in common between the two models, and RP11-539L10.2 was down-regulated. The major component of the latent HIV reservoir, memory CD4+ T-cells, had higher expression of these lncRNAs, compared to naive T-cells. Guilt-by-association analysis demonstrated that lncRNAs dysregulated in latency were associated with several cellular pathways implicated in HIV latency establishment and maintenance: proteasome, spliceosome, p53 signaling, and mammalian target of rapamycin (MTOR). PVT1, RP11-347C18.3, and RP11-539L10.2 were down-regulated by latency reversing agents, suberoylanilide hydroxamic acid and Romidepsin, suggesting that modulation of lncRNAs is a possible secondary mechanism of action of these compounds. These results will facilitate prioritization of lncRNAs for evaluation as targets for HIV latency reversal. Importantly, our study provides insights into regulatory function of lncRNA during latent HIV infection

Upregulated Glucose Metabolism Correlates Inversely with CD8(+) T-cell Infiltration and Survival in Squamous Cell Carcinoma

Antibodies that block T-cell–regulatory checkpoints have recently emerged as a transformative approach to cancer treatment. However, the clinical efficacy of checkpoint blockade depends upon inherent tumor immunogenicity, with variation in infiltrating T cells contributing to differences in objective response rates. Here, we sought to understand the molecular correlates of tumor-infiltrating T lymphocytes (TIL) in squamous cell carcinoma (SCC), using a systems biologic approach to integrate publicly available omics datasets with histopathologic features. We provide evidence that links TIL abundance and therapeutic outcome to the regulation of tumor glycolysis by EGFR and HIF, both of which are attractive molecular targets for use in combination with immunotherapeutics

Dose-responsive gene expression in suberoylanilide hydroxamic acid-treated resting CD4+ T cells

Persistent latently infected CD4+ T cells represent a major obstacle to HIV eradication. Histone deacetylase inhibitors (HDACis) are a proposed activation therapy. However, off-target effects on expression in host immune cells are poorly understood. We hypothesized that HDACi-modulated genes would be best identified with dose-response analysis

HPV, tumour metabolism and novel target identification in head and neck squamous cell carcinoma

Background Metabolic changes in tumour cells are used in clinical imaging and may provide potential therapeutic targets. Human papillomavirus (HPV) status is important in classifying head and neck cancers (HNSCC), identifying a distinct clinical phenotype; metabolic differences between these HNSCC subtypes remain poorly understood. Methods We used RNA sequencing to classify the metabolic expression profiles of HPV+ve and HPV−ve HNSCC, performed a meta-analysis on FDG-PET imaging characteristics and correlated results with in vitro extracellular flux analysis of HPV−ve and HPV+ve HNSCC cell lines. The monocarboxylic acid transporter-1 (MCT1) was identified as a potential metabolic target and tested in functional assays. Results Specific metabolic profiles were associated with HPV status, not limited to carbohydrate metabolism. There was dominance of all energy pathways in HPV-negative disease, with elevated expression of genes associated with glycolysis and oxidative phosphorylation. In vitro analysis confirmed comparative increased rates of oxidative phosphorylation and glycolysis in HPV-negative cell lines. PET SUV(max) scores however were unable to reliably differentiate between HPV-positive and HPV-negative tumours. MCT1 expression was significantly increased in HPV-negative tumours, and inhibition suppressed tumour cell invasion, colony formation and promoted radiosensitivity. Conclusion HPV-positive and negative HNSCC have different metabolic profiles which may have potential therapeutic applications

Mixed effects of suberoylanilide hydroxamic acid (SAHA) on the host transcriptome and proteome and their implications for HIV reactivation from latency

Suberoylanilide hydroxamic acid (SAHA) has been assessed in clinical trials as part of a “shock and kill” strategy to cure HIV-infected patients. While it was effective at inducing expression of HIV RNA (“shock”), treatment with SAHA did not result in a reduction of reservoir size (“kill”). We therefore utilized a combined analysis of effects of SAHA on the host transcriptome and proteome to dissect its mechanisms of action that may explain its limited success in “shock and kill” strategies. CD4+ T cells from HIV seronegative donors were treated with 1 µM SAHA or its solvent dimethyl sulfoxide (DMSO) for 24 hours. Protein expression and post-translational modifications were measured with iTRAQ proteomics using ultra high-precision two-dimensional liquid chromatography - tandem mass spectrometry. Gene expression was assessed by Illumina microarrays. Using limma package in the R computing environment, we identified 185 proteins, 18 phosphorylated forms, 4 acetylated forms and 2,982 genes, whose expression was modulated by SAHA. A protein interaction network integrating these 4 data types identified the HIV transcriptional repressor HMGA1 to be upregulated by SAHA at the transcript, protein and acetylated protein levels. Further functional category assessment of proteins and genes modulated by SAHA identified gene ontology terms related to NFκB signaling, protein folding and autophagy, which are all relevant to HIV reactivation. In summary, SAHA modulated numerous host cell transcripts, proteins and post-translational modifications of proteins, which would be expected to have very mixed effects on the induction of HIV-specific transcription and protein function. Proteome profiling highlighted a number of potential counter-regulatory effects of SAHA with respect to viral induction, which transcriptome profiling alone would not have identified. These observations could lead to a more informed selection and design of other HDACi with a more refined targeting profile, and prioritization of latency reversing agents of other classes to be used in combination with SAHA to achieve more potent induction of HIV expression

Gender-dependent differences in plasma matrix metalloproteinase-8 elevated in pulmonary tuberculosis.

Tuberculosis (TB) remains a global health pandemic and greater understanding of underlying pathogenesis is required to develop novel therapeutic and diagnostic approaches. Matrix metalloproteinases (MMPs) are emerging as key effectors of tissue destruction in TB but have not been comprehensively studied in plasma, nor have gender differences been investigated. We measured the plasma concentrations of MMPs in a carefully characterised, prospectively recruited clinical cohort of 380 individuals. The collagenases, MMP-1 and MMP-8, were elevated in plasma of patients with pulmonary TB relative to healthy controls, and MMP-7 (matrilysin) and MMP-9 (gelatinase B) were also increased. MMP-8 was TB-specific (p<0.001), not being elevated in symptomatic controls (symptoms suspicious of TB but active disease excluded). Plasma MMP-8 concentrations inversely correlated with body mass index. Plasma MMP-8 concentration was 1.51-fold higher in males than females with TB (p<0.05) and this difference was not due to greater disease severity in men. Gender-specific analysis of MMPs demonstrated consistent increase in MMP-1 and -8 in TB, but MMP-8 was a better discriminator for TB in men. Plasma collagenases are elevated in pulmonary TB and differ between men and women. Gender must be considered in investigation of TB immunopathology and development of novel diagnostic markers

Integrated plasma proteomics identifies tuberculosis-specific diagnostic biomarkers

Novel biomarkers to identify infectious patients transmitting Mycobacterium tuberculosis are urgently needed to control the global tuberculosis (TB) pandemic. We hypothesized that proteins released into the plasma in active pulmonary TB are clinically useful biomarkers to distinguish TB cases from healthy individuals and patients with other respiratory infections. We applied a highly sensitive non-depletion tandem mass spectrometry discovery approach to investigate plasma protein expression in pulmonary TB cases compared to healthy controls in South African and Peruvian cohorts. Bioinformatic analysis using linear modelling and network correlation analyses identified 118 differentially expressed proteins, significant through three complementary analytical pipelines. Candidate biomarkers were subsequently analysed in two validation cohorts of differing ethnicity using antibody-based proximity extension assays. TB-specific host biomarkers were confirmed. A six-protein diagnostic panel, comprising FETUB, FCGR3B, LRG1, SELL, CD14 and ADA2, differentiated patients with pulmonary TB from healthy controls and patients with other respiratory infections with high sensitivity and specificity in both cohorts. This biomarker panel exceeds the World Health Organisation Target Product Profile specificity criteria for a triage test for TB. The new biomarkers have potential for further development as near-patient TB screening assays, thereby helping to close the case-detection gap that fuels the global pandemic