Low pH immobilizes and kills human leukocytes and prevents transmission of cell-associated HIV in a mouse model

BACKGROUND: Both cell-associated and cell-free HIV virions are present in semen and cervical secretions of HIV-infected individuals. Thus, topical microbicides may need to inactivate both cell-associated and cell-free HIV to prevent sexual transmission of HIV/AIDS. To determine if the mild acidity of the healthy vagina and acid buffering microbicides would prevent transmission by HIV-infected leukocytes, we measured the effect of pH on leukocyte motility, viability and intracellular pH and tested the ability of an acidic buffering microbicide (BufferGel(®)) to prevent the transmission of cell-associated HIV in a HuPBL-SCID mouse model. METHODS: Human lymphocyte, monocyte, and macrophage motilities were measured as a function of time and pH using various acidifying agents. Lymphocyte and macrophage motilities were measured using video microscopy. Monocyte motility was measured using video microscopy and chemotactic chambers. Peripheral blood mononuclear cell (PBMC) viability and intracellular pH were determined as a function of time and pH using fluorescent dyes. HuPBL-SCID mice were pretreated with BufferGel, saline, or a control gel and challenged with HIV-1-infected human PBMCs. RESULTS: Progressive motility was completely abolished in all cell types between pH 5.5 and 6.0. Concomitantly, at and below pH 5.5, the intracellular pH of PBMCs dropped precipitously to match the extracellular medium and did not recover. After acidification with hydrochloric acid to pH 4.5 for 60 min, although completely immotile, 58% of PBMCs excluded ethidium homodimer-1 (dead-cell dye). In contrast, when acidified to this pH with BufferGel, a microbicide designed to maintain vaginal acidity in the presence of semen, only 4% excluded dye at 10 min and none excluded dye after 30 min. BufferGel significantly reduced transmission of HIV-1 in HuPBL-SCID mice (1 of 12 infected) compared to saline (12 of 12 infected) and a control gel (5 of 7 infected). CONCLUSION: These results suggest that physiologic or microbicide-induced acid immobilization and killing of infected white blood cells may be effective in preventing sexual transmission of cell-associated HIV

An extremely powerful long-lived superluminal ejection from the black hole MAXI J1820+070

Black holes in binary systems execute patterns of outburst activity where two characteristic X-ray states are associated with different behaviours observed at radio wavelengths. The hard state is associated with radio emission indicative of a continuously replenished, collimated, relativistic jet, whereas the soft state is rarely associated with radio emission, and never continuously, implying the absence of a quasi-steady jet. Here we report radio observations of the black hole transient MAXI J1820$+$070 during its 2018 outburst. As the black hole transitioned from the hard to soft state we observed an isolated radio flare, which, using high angular resolution radio observations, we connect with the launch of bi-polar relativistic ejecta. This flare occurs as the radio emission of the core jet is suppressed by a factor of over 800. We monitor the evolution of the ejecta over 200 days and to a maximum separation of 10$''$, during which period it remains detectable due to in-situ particle acceleration. Using simultaneous radio observations sensitive to different angular scales we calculate an accurate estimate of energy content of the approaching ejection. This energy estimate is far larger than that derived from state transition radio flare, suggesting a systematic underestimate of jet energetics

Physiologically based modeling of lisofylline pharmacokinetics following intravenous administration in mice

Lisofylline (LSF), is the R-(−) enantiomer of the metabolite M1 of pentoxifylline, and is currently under development for the treatment of type 1 diabetes. The aim of the study was to develop a physiologically based pharmacokinetic (PBPK) model of LSF in mice and to perform simulations in order to predict LSF concentrations in human serum and tissues following intravenous and oral administration. The concentrations of LSF in serum, brain, liver, kidneys, lungs, muscle, and gut were determined at different time points over 60 min by a chiral HPLC method with UV detection following a single intravenous dose of LSF to male CD-1 mice. A PBPK model was developed to describe serum pharmacokinetics and tissue distribution of LSF using ADAPT II software. All pharmacokinetic profiles were fitted simultaneously to obtain model parameters. The developed model characterized well LSF disposition in mice. The estimated intrinsic hepatic clearance was 5.427 ml/min and hepatic clearance calculated using the well-stirred model was 1.22 ml/min. The renal clearance of LSF was equal to zero. On scaling the model to humans, a good agreement was found between the predicted by the model and presented in literature serum LSF concentration–time profiles following an intravenous dose of 3 mg/kg. The predicted LSF concentrations in human tissues following oral administration were considerably lower despite the twofold higher dose used and may not be sufficient to exert a pharmacological effect. In conclusion, the mouse is a good model to study LSF pharmacokinetics following intravenous administration. The developed PBPK model may be useful to design future preclinical and clinical studies of this compound

Developmental effects on sleep–wake patterns in infants receiving a cow’s milk-based infant formula with an added prebiotic blend: A Randomized Controlled Trial

Background Few studies have evaluated nutritive effects of prebiotics on infant behavior state, physiology, or metabolic status. Methods In this double-blind randomized study, infants (n = 161) received cow’s milk-based infant formula (Control) or similar formula with an added prebiotic blend (polydextrose and galactooligosaccharides [PDX/GOS]) from 14–35 to 112 days of age. Infant wake behavior (crying/fussing, awake/content) and 24-h sleep–wake actograms were analyzed (Baseline, Days 70 and 112). Salivary cortisol was immunoassayed (Days 70 and 112). In a subset, exploratory stool 16S ribosomal RNA-sequencing was analyzed (Baseline, Day 112). Results One hundred and thirty-one infants completed the study. Average duration of crying/fussing episodes was similar at Baseline, significantly shorter for PDX/GOS vs. Control at Day 70, and the trajectory continued at Day 112. Latency to first and second nap was significantly longer for PDX/GOS vs. Control at Day 112. Cortisol awakening response was demonstrated at Days 70 and 112. Significant stool microbiome beta-diversity and individual taxa abundance differences were observed in the PDX/GOS group. Conclusions Results indicate faster consolidation of daytime waking state in infants receiving prebiotics and support home-based actigraphy to assess early sleep–wake patterns. A prebiotic effect on wake organization is consistent with influence on the gut–brain axis and warrants further investigation. Impact Few studies have evaluated nutritive effects of prebiotics on infant behavior state, cortisol awakening response, sleep–wake entrainment, and gut microbiome. Faster consolidation of daytime waking state was demonstrated in infants receiving a prebiotic blend in infant formula through ~4 months of age. Shorter episodes of crying were demonstrated at ~2 months of age (time point corresponding to age/developmental range associated with peak crying) in infants receiving formula with added prebiotics. Results support home-based actigraphy as a suitable method to assess early sleep–wake patterns. Prebiotic effect on wake organization is consistent with influence on the gut–brain axis and warrants further investigation

Measurement of D-s(+) and D-s(*+) production in B meson decays and from continuum e(+)e(-) annihilation at √s=10.6 GeV

This is the pre-print version of the Article. The official published version can be accessed from the links below. Copyright @ 2002 APSNew measurements of Ds+ and Ds*+ meson production rates from B decays and from qq̅ continuum events near the Υ(4S) resonance are presented. Using 20.8 fb-1 of data on the Υ(4S) resonance and 2.6 fb-1 off-resonance, we find the inclusive branching fractions B(B⃗Ds+X)=(10.93±0.19±0.58±2.73)% and B(B⃗Ds*+X)=(7.9±0.8±0.7±2.0)%, where the first error is statistical, the second is systematic, and the third is due to the Ds+→φπ+ branching fraction uncertainty. The production cross sections σ(e+e-→Ds+X)×B(Ds+→φπ+)=7.55±0.20±0.34pb and σ(e+e-→Ds*±X)×B(Ds+→φπ+)=5.8±0.7±0.5pb are measured at center-of-mass energies about 40 MeV below the Υ(4S) mass. The branching fractions ΣB(B⃗Ds(*)+D(*))=(5.07±0.14±0.30±1.27)% and ΣB(B⃗Ds*+D(*))=(4.1±0.2±0.4±1.0)% are determined from the Ds(*)+ momentum spectra. The mass difference m(Ds+)-m(D+)=98.4±0.1±0.3MeV/c2 is also measured.This work was supported by DOE and NSF (USA), NSERC (Canada), IHEP (China), CEA and CNRS-IN2P3 (France), BMBF (Germany), INFN (Italy), NFR (Norway), MIST (Russia), and PPARC (United Kingdom). Individuals have received support from the Swiss NSF, A. P. Sloan Foundation, Research Corporation, and Alexander von Humboldt Foundation

Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy

Cellular functions are fundamentally regulated by intracellular temperature, which influences biochemical reactions inside a cell. Despite the important contributions to biological and medical applications that it would offer, intracellular temperature mapping has not been achieved. Here we demonstrate the first intracellular temperature mapping based on a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy. The spatial and temperature resolutions of our thermometry were at the diffraction limited level (200 nm) and 0.18–0.58 °C. The intracellular temperature distribution we observed indicated that the nucleus and centrosome of a COS7 cell, both showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. The heat production from mitochondria was also observed as a proximal local temperature increase. These results showed that our new intracellular thermometry could determine an intrinsic relationship between the temperature and organelle function

Comprehensive Analysis Reveals Dynamic and Evolutionary Plasticity of Rab GTPases and Membrane Traffic in Tetrahymena thermophila

Cellular sophistication is not exclusive to multicellular organisms, and unicellular eukaryotes can resemble differentiated animal cells in their complex network of membrane-bound structures. These comparisons can be illuminated by genome-wide surveys of key gene families. We report a systematic analysis of Rabs in a complex unicellular Ciliate, including gene prediction and phylogenetic clustering, expression profiling based on public data, and Green Fluorescent Protein (GFP) tagging. Rabs are monomeric GTPases that regulate membrane traffic. Because Rabs act as compartment-specific determinants, the number of Rabs in an organism reflects intracellular complexity. The Tetrahymena Rab family is similar in size to that in humans and includes both expansions in conserved Rab clades as well as many divergent Rabs. Importantly, more than 90% of Rabs are expressed concurrently in growing cells, while only a small subset appears specialized for other conditions. By localizing most Rabs in living cells, we could assign the majority to specific compartments. These results validated most phylogenetic assignments, but also indicated that some sequence-conserved Rabs were co-opted for novel functions. Our survey uncovered a rare example of a nuclear Rab and substantiated the existence of a previously unrecognized core Rab clade in eukaryotes. Strikingly, several functionally conserved pathways or structures were found to be associated entirely with divergent Rabs. These pathways may have permitted rapid evolution of the associated Rabs or may have arisen independently in diverse lineages and then converged. Thus, characterizing entire gene families can provide insight into the evolutionary flexibility of fundamental cellular pathways

Visualization of Glutamine Transporter Activities in Living Cells Using Genetically Encoded Glutamine Sensors

Glutamine plays a central role in the metabolism of critical biological molecules such as amino acids, proteins, neurotransmitters, and glutathione. Since glutamine metabolism is regulated through multiple enzymes and transporters, the cellular glutamine concentration is expected to be temporally dynamic. Moreover, differentiation in glutamine metabolism between cell types in the same tissue (e.g. neuronal and glial cells) is often crucial for the proper function of the tissue as a whole, yet assessing cell-type specific activities of transporters and enzymes in such heterogenic tissue by physical fractionation is extremely challenging. Therefore, a method of reporting glutamine dynamics at the cellular level is highly desirable. Genetically encoded sensors can be targeted to a specific cell type, hence addressing this knowledge gap. Here we report the development of Föster Resonance Energy Transfer (FRET) glutamine sensors based on improved cyan and yellow fluorescent proteins, monomeric Teal Fluorescent Protein (mTFP)1 and venus. These sensors were found to be specific to glutamine, and stable to pH-changes within a physiological range. Using cos7 cells expressing the human glutamine transporter ASCT2 as a model, we demonstrate that the properties of the glutamine transporter can easily be analyzed with these sensors. The range of glutamine concentration change in a given cell can also be estimated using sensors with different affinities. Moreover, the mTFP1-venus FRET pair can be duplexed with another FRET pair, mAmetrine and tdTomato, opening up the possibility for real-time imaging of another molecule. These novel glutamine sensors will be useful tools to analyze specificities of glutamine metabolism at the single-cell level

The Lysosome and Intracellular Signalling.

In addition to being the terminal degradative compartment of the cell's endocytic and autophagic pathways, the lysosome is a multifunctional signalling hub integrating the cell's response to nutrient status and growth factor/hormone signalling. The cytosolic surface of the limiting membrane of the lysosome is the site of activation of the multiprotein complex mammalian target of rapamycin complex 1 (mTORC1), which phosphorylates numerous cell growth-related substrates, including transcription factor EB (TFEB). Under conditions in which mTORC1 is inhibited including starvation, TFEB becomes dephosphorylated and translocates to the nucleus where it functions as a master regulator of lysosome biogenesis. The signalling role of lysosomes is not limited to this pathway. They act as an intracellular Ca2+ store, which can release Ca2+ into the cytosol for both local effects on membrane fusion and pleiotropic effects within the cell. The relationship and crosstalk between the lysosomal and endoplasmic reticulum (ER) Ca2+ stores play a role in shaping intracellular Ca2+ signalling. Lysosomes also perform other signalling functions, which are discussed. Current views of the lysosomal compartment recognize its dynamic nature. It includes endolysosomes, autolysosome and storage lysosomes that are constantly engaged in fusion/fission events and lysosome regeneration. How signalling is affected by individual lysosomal organelles being at different stages of these processes and/or at different sites within the cell is poorly understood, but is discussed