Transient decondensation of chromatin in liver nuclei of rats treated with tannic acid

Communicated by Ramaswamy H. Sarma     

High Temporal Resolution Parametric MRI Monitoring of the Initial Ischemia/Reperfusion Phase in Experimental Acute Kidney Injury

Ischemia/reperfusion (I/R) injury, a consequence of kidney hypoperfusion or temporary interruption of blood flow is a common cause of acute kidney injury (AKI). There is an unmet need to better understand the mechanisms operative during the initial phase of ischemic AKI. Non-invasive in vivo parametric magnetic resonance imaging (MRI) may elucidate spatio-temporal pathophysiological changes in the kidney by monitoring the MR relaxation parameters T₂* and T₂, which are known to be sensitive to blood oxygenation. The aim of our study was to establish the technical feasibility of fast continuous T₂*/T₂ mapping throughout renal I/R. MRI was combined with a remotely controlled I/R model and a segmentation model based semi-automated quantitative analysis. This technique enabled the detailed assessment of in vivo changes in all kidney regions during ischemia and early reperfusion. Significant changes in T₂* and T₂ were observed shortly after induction of renal ischemia and during the initial reperfusion phase. Our study demonstrated for the first time that continuous and high temporal resolution parametric MRI is feasible for in-vivo monitoring and characterization of I/R induced AKI in rats. This technique may help in the identification of the timeline of key events responsible for development of renal damage in hypoperfusion-induced AKI.     

Apo-parvalbumin as an intrinsically disordered protein

Recently defined family of intrinsically disordered proteins (IDP) includes proteins lacking rigid tertiary structure meanwhile fulfilling essential biological functions. Here we show that apo-state of pike parvalbumin (alpha- and beta-isoforms, pI 5.0 and 4.2, respectively) belongs to the family of IDP, which is in accord with theoretical predictions. Parvalbumin (PA) is a 12-kDa calcium-binding protein involved into regulation of relaxation of fast muscles. Differential scanning calorimetry measurements of metal-depleted form of PA revealed the absence of any thermally induced transitions with measurable denaturation enthalpy along with elevated specific heat capacity, implying the lack of rigid tertiary structure and exposure of hydrophobic protein groups to the solvent. Calcium removal from the PAs causes more than 10-fold increase in fluorescence intensity of hydrophobic probe bis-ANS and is accompanied by a decrease in alpha-helical content and a marked increase in mobility of aromatic residues environment, as judged by circular dichroism spectroscopy (CD). Guanidinium chloride-induced unfolding of the apo-parvalbumins monitored by CD showed the lack of fixed tertiary structure. Theoretical estimation of energetics of the charge-charge interactions in the PAs indicated their pronounced destabilization upon calcium removal, which is in line with sequence-based predictions of disordered protein chain regions. Far-UV CD studies of apo-alpha-PA revealed hallmarks of cold denaturation of the protein at temperatures below 20 degrees C. Moreover, a cooperative thermal denaturation transition with mid-temperature at 10-15 degrees C is revealed by near-UV CD for both PAs. The absence of detectable enthalpy change in this temperature region suggests continuous nature of the transition. Overall, the theoretical and experimental data obtained show that PA in apo-state is essentially disordered nevertheless demonstrates complex denaturation behavior. The native rigid tertiary structure of PA is attained upon association of one (alpha-PA) or two (beta-PA) calcium ions per protein molecule, as follows from calorimetric and calcium titration data.     

Isotherm of ligand adsorption on DNA at multiplicative noise

Fluctuations of the number of ligands adsorbed on macromolecules are considered in the case when the number of ligands in the solution fluctuates under the action of fluctuations of the external medium (external noise). For the case of small filling, the multiplicative type of stochastic differential equation is obtained, describing the time variation of the number of ligands adsorbed on macromolecules. The isotherm of adsorption of ligands on DNA is obtained. It is shown that at small ligand concentrations, for some relations between adsorption parameters and the intensity of the external noise, no macromolecule adsorption of ligands takes place.     

Respiratory gas exchange in the desert flea Xenopsylla ramesis (Siphonaptera: Pulicidae): response to temperature and blood-feeding

Xenopsylla ramesis is a flea species parasitizing gerbilline rodents in the deserts of the Middle East. This study was undertaken to determine metabolic requirements of the different developmental stages of the flea-life cycle as well as to investigate the metabolic response to temperature and starvation after blood feeding. A high resolution respirometry system was used to measure CO2 emission of fleas ranging in size from 0.166+/-0.006 mg (larvae) to 0.263+/-0.009 mg (adults). The free-living stages (larvae and adults) had significantly higher metabolic rates than the cocooned stages (pupae). CO2 emission rates of the larvae exceeded that of the adults by 2.6-fold and the pupae by 7.3 times. In the adults, both temperature and blood feeding significantly affected starvation-level metabolism. Metabolism was temperature dependent with an average Q10 of 2.57 for females and 2.55 for males over the temperature range of 10-30 degrees C. No consistent decline in thermal sensitivity at higher ambient temperatures was evident. Fleas that had a blood meal prior to starvation had significantly higher metabolic rates (0. 86 +/- 0.008 x 10(-3) ml mg(-1) h(-1)) than fleas, which were newly emerged unfed adults (0.56 +/- 0.1 x 10(-3) ml mg(-1) h(-1)). Water content also differed between fed (range approx. 67-69% body mass) and newly emerged adults (range approx. 73-75% of body mass). Feeding may stimulate some as yet undetermined physiological process that causes differential metabolic response in starving, fed and unfed fleas. Characteristics of gas exchange in desert-dwelling fleas are reflective of the off-host life style in the protected microenvironment of the host nest or burrow, rather than as a response to any type of environmental extreme.     

HIV-1 Expressing the Envelopes of Transmitted/Founder or Control/Reference Viruses Have Similar Infection Patterns of CD4 T-Cells in Human Cervical Tissue Ex Vivo

Recently, it was found that 80% of sexual HIV-1 transmissions are established by a single virion/viral genome. To investigate whether the transmitted/founder (T/F) viruses have specific biological properties favoring sexual transmission, we inoculated human cervical tissue explants with isogenic HIV-1 viruses encoding Env sequences from T/F and control reference (C/R) HIV-1 variants as well as with full length T/F HIV-1 and compared their replication efficiencies, T cell depletion, and the activation status of infected cells. We found that all the HIV-1 variants were capable of transmitting infection to cervical tissue ex vivo and in this system preferentially replicate in activated CD4 T cells and deplete these cells. There was no difference in the biological properties of T/F and C/R HIV-1 variants as evaluated in ex vivo cervical tissue.     

Microbicides: still a long road to success

The development of efficient microbicides, the topically applied compounds that protect uninfected individuals from acquiring HIV-1, is a promising strategy to contain HIV-1 epidemics. Such microbicides should of course possess anti-HIV-1 activity, but they should also act against other genital pathogens, which facilitate HIV-1 transmission. The new trend in microbicide strategy is to use drugs currently used in HIV-1 therapy. The success of this strategy is mixed so far and is impaired by our limited knowledge of the basic mechanisms of HIV-1 transmission as well as by the inadequacy of the systems in which microbicides are tested in preclinical studies.     

Recoverin as a redox-sensitive protein

Recoverin is a member of the neuronal calcium sensor (NCS) family of EF-hand calcium binding proteins. In a visual cycle of photoreceptor cells, recoverin regulates activity of rhodopsin kinase in a Ca2+-dependent manner. Like all members of the NSC family, recoverin contains a conserved cysteine (Cys38) in nonfunctional EF-hand 1. This residue was shown to be critical for activation of target proteins in some members of the NCS family but not for interaction of recoverin with rhodopsin kinase. Spectrophotometric titration of Ca2+-loaded recoverin gave 7.6 for the pKa value of Cys38 thiol, suggesting partial deprotonation of the thiol in vivo conditions. An ability of recoverin to form a disulfide dimer and thiol-oxidized monomer under mild oxidizing conditions was found using SDS-PAGE in reducing and nonreducing conditions and Ellman's test. Both processes are reversible and modulated by Ca2+. Although formation of the disulfide dimer takes place only for Ca2+-loaded recoverin, accumulation of the oxidized monomer proceeds more effectively for apo-recoverin. The Ca2+ modulated susceptibility of the recoverin thiol to reversible oxidation may be of potential importance for functioning of recoverin in photoreceptor cells.     

Extracellular vesicles released from the filarial parasite Brugia malayi downregulate the host mTOR pathway

We have previously shown that the microfilarial (mf) stage of Brugia malayi can inhibit the mammalian target of rapamycin (mTOR; a conserved serine/threonine kinase critical for immune regulation and cellular growth) in human dendritic cells (DC) and we have proposed that this mTOR inhibition is associated with the DC dysfunction seen in filarial infections. Extracellular vesicles (EVs) contain many proteins and nucleic acids including microRNAs (miRNAs) that might affect a variety of intracellular pathways. Thus, EVs secreted from mf may elucidate the mechanism by which the parasite is able to modulate the host immune response during infection. EVs, purified from mf of Brugia malayi and confirmed by size through nanoparticle tracking analysis, were assessed by miRNA microarrays (accession number {"type":"entrez-geo","attrs":{"text":"GSE157226","term_id":"157226"}}GSE157226) and shown to be enriched (>2-fold, p-value<0.05, FDR = 0.05) for miR100, miR71, miR34, and miR7. The microarray analysis compared mf-derived EVs and mf supernatant. After confirming their presence in EVs using qPCR for these miRNA targets, web-based target predictions (using MIRPathv3, TarBAse and MicroT-CD) predicted that miR100 targeted mTOR and its downstream regulatory protein 4E-BP1. Our previous data with live parasites demonstrated that mf downregulate the phosphorylation of mTOR and its downstream effectors. Additionally, our proteomic analysis of the mf-derived EVs revealed the presence of proteins commonly found in these vesicles (data are available via ProteomeXchange with identifier PXD021844). We confirmed internalization of mf-derived EVs by human DCs and monocytes using confocal microscopy and flow cytometry, and further demonstrated through flow cytometry, that mf-derived EVs downregulate the phosphorylation of mTOR in human monocytes (THP-1 cells) to the same degree that rapamycin (a known mTOR inhibitor) does. Our data collectively suggest that mf release EVs that interact with host cells, such as DC, to modulate host responses.