Contribution of Aquaporins to Cellular Water Transport Observed by a Microfluidic Cell Volume Sensor

Here we demonstrate that an impedance-based microfluidic cell volume sensor can be used to study the roles of aquaporin (AQP) in cellular water permeability and screen AQP-specific drugs. Human embryonic kidney (HEK-293) cells were transiently transfected with AQP3- or AQP4-encoding genes to express AQPs in plasma membranes. The swelling of cells in response to hypotonic stimulation was traced in real time using the sensor. Two time constants were obtained by fitting the swelling curves with a two-exponential function, a fast time constant associated with osmotic water permeability of AQP-expressing cells and a slow phase time constant associated mainly with water diffusion through lipid bilayers in the nontransfected cells. The AQP-expressing cells showed at least 10× faster osmotic water transport than control cells. Using the volume sensor, we examined the effects of Hg2+ and Ni2+ on the water transport via AQPs. Hg2+ inhibited the water flux in AQP3-expressing cells irreversibly, while Ni2+ blocked the AQP3 channels reversibly. Neither of the two ions blocked the AQP4 channels. The microfluidic volume sensor can sense changes in cell volume in real time, which enables perfusion of various reagents sequentially. It provides a convenient tool for studying the effect of reagents on the function and regulation mechanism of AQPs

Cellulose nanomaterials from rubberwood obtained via enzymatic hydrolysis route.

Novel strategy for the production of advanced materials from the waste generated in natural rubber exploration can improve the whole production chain (PARASHAR and CHAWLA, 2021). Lignocellulosic biomass from rubberwood has the potential for several applications, being formed by cellulose, hemicellulose, lignin, and inorganic compounds in smaller amounts. In this context, this work shows the characterization of nanomaterials obtained via enzymatic hydrolysis from bleached fibers of rubber tree. The fibers were mercerized with 5% (w/w) aqueous sodium hydroxide solution for 2h at 80 °C. Afterwards, the fibers were bleached with a solution composed of equal parts (v/v) of acetate buffer (27% by weight NaOH and 7.5% (v/v) glacial acetic acid, in distilled water) and aqueous sodium chlorite (1.7% by weight NaClO2 in water). Then, the fibers were bleached twice with a solution of equal parts (v/v) of 4% NaOH (w/w) and 24% H2O2 (v/v). The bleached fiber was submitted to enzymatic hydrolysis in sodium citrate buffer (0.1 M, pH 5), with a solids load of 15% (w/v) and an enzymatic load (Cellic Ctec 3 - Novozymes®) of 10 mg / g of biomass in a shaker incubator at 50 °C. and 200 rpm for 48h. The characterizations were made by AFM measurements (Dimension V microscope - Veeco); X-ray diffraction (XRD) using a Shimadzu 6000 diffractometer with CuK ( = 1,54 Å), at room temperature and with 2 angle between 5 and 40° (1° min-1 ); thermogravimetric analysis (TGA) in an inert atmosphere, using TA Instruments equipment, model Q500 with a heating rate of 10 °C/min from room temperature to 600 °C. The X-ray diffractograms showed that the main peaks corresponding to the crystallographic planes of Type I cellulose. The Bragg angles (2) were: 16.0°, 22.4° and 34.6°, with the highest intensity in the crystallographic plane (002). The crystallinity index, calculated according to the method proposed by Segal (1959), was 57.8% for in natura fiber, 75.1% for bleached fiber and 80.8% after hydrolysis. Analyzing the thermograms, it was observed that the Tonset of the fiber in natura was 289 °C, that of the bleached fiber was 292 °C and after the hydrolysis it was 327 °C. There was a change in the maximum degradation peak, in the final temperature of the process and in the amount of residue after the treatment. The AFM images exhibited structures rod-like shapes, with mean length of 349.9±169.6 nm and the diameter of 4.0±1.7 nm, after 48h to enzymatic hydrolysis. Our results showed that the obtained nanomaterials can be effectively extracted from the studied lignocellulosic source using the enzymatic route. The nanostructures showed high crystallinity, excellent thermal stability and high aspect ratio. Thus, nanomaterials presented important characteristics for the use in several applications, including as reinforcement

Toxoplasmosis in Transplant Recipients, Europe, 2010-2014

Transplantation activity is increasing, leading to a growing number of patients at risk for toxoplasmosis. We reviewed toxoplasmosis prevention practices, prevalence, and outcomes for hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT; heart, kidney, or liver) patients in Europe. We collected electronic data on the transplant population and prevention guidelines/regulations and clinical data on toxoplasmosis cases diagnosed during 2010-2014. Serologic pretransplant screening of allo-hematopoietic stem cell donors was performed in 80% of countries, screening of organ donors in 100%. SOT recipients were systematically screened in 6 countries. Targeted anti-Toxoplasma chemoprophylaxis was heterogeneous. A total of 87 toxoplasmosis cases were recorded (58 allo-HSCTs, 29 SOTs). The 6-month survival rate was lower among Toxoplasma-seropositive recipients and among allo-hematopoietic stem cell and liver recipients. Chemoprophylaxis improved outcomes for SOT recipients. Toxoplasmosis remains associated with high mortality rates among transplant recipients. Guidelines are urgently needed to standardize prophylactic regimens and optimize patient management

Hidden spin-current conservation in 2d Fermi liquids

We report the existence of regimes of the two dimensional Fermi liquid that show unusual conservation of the spin current and may be tuned by varying some parameter like the density of fermions. We show that for reasonable models of the effective interaction the spin current may be conserved in general in 2d, not only for a particular regime. Low temperature spin waves propagate distinctively in these regimes and entirely new ``spin-acoustic'' modes are predicted for scattering-dominated temperature ranges. These new high-temperature propagating spin waves provide a clear signature for the experimental search of such regimes.Comment: 4 pages, no figures, revised version, accepted for pub. in the PR

Capturing, sharing and analysing biophysical data from protein engineering and protein characterization studies

Large amounts of data are being generated annually on the connection between the sequence, structure and function of proteins using site-directed mutagenesis, protein design and directed evolution techniques. These data provide the fundamental building blocks for our understanding of protein function, molecular biology and living organisms in general. However, much experimental data are never deposited in databases and is thus ‘lost’ in journal publications or in PhD theses. At the same time theoretical scientists are in need of large amounts of experimental data for benchmarking and calibrating novel predictive algorithms, and theoretical progress is therefore often hampered by the lack of suitable data to validate or disprove a theoretical assumption. We present PEAT (Protein Engineering Analysis Tool), an application that integrates data deposition, storage and analysis for researchers carrying out protein engineering projects or biophysical characterization of proteins. PEAT contains modules for DNA sequence manipulation, primer design, fitting of biophysical characterization data (enzyme kinetics, circular dichroism spectroscopy, NMR titration data, etc.), and facilitates sharing of experimental data and analyses for a typical university-based research group. PEAT is freely available to academic researchers at http://enzyme.ucd.ie/PEAT

Genotype–Phenotype Correlation in DFNB8/10 Families with TMPRSS3 Mutations

In the present study, genotype–phenotype correlations in eight Dutch DFNB8/10 families with compound heterozygous mutations in TMPRSS3 were addressed. We compared the phenotypes of the families by focusing on the mutation data. The compound heterozygous variants in the TMPRSS3 gene in the present families included one novel variant, p.Val199Met, and four previously described pathogenic variants, p.Ala306Thr, p.Thr70fs, p.Ala138Glu, and p.Cys107Xfs. In addition, the p.Ala426Thr variant, which had previously been reported as a possible polymorphism, was found in one family. All affected family members reported progressive bilateral hearing impairment, with variable onset ages and progression rates. In general, the hearing impairment affected the high frequencies first, and sooner or later, depending on the mutation, the low frequencies started to deteriorate, which eventually resulted in a flat audiogram configuration. The ski-slope audiogram configuration is suggestive for the involvement of TMPRSS3. Our data suggest that not only the protein truncating mutation p.T70fs has a severe effect but also the amino acid substitutions p.Ala306Thr and p.Val199Met. A combination of two of these three mutations causes prelingual profound hearing impairment. However, in combination with the p.Ala426Thr or p.Ala138Glu mutations, a milder phenotype with postlingual onset of the hearing impairment is seen. Therefore, the latter mutations are likely to be less detrimental for protein function. Further studies are needed to distinguish possible phenotypic differences between different TMPRSS3 mutations. Evaluation of performance of patients with a cochlear implant indicated that this is a good treatment option for patients with TMPRSS3 mutations as satisfactory speech reception was reached after implantation

Aquaporins: important but elusive drug targets.

The aquaporins (AQPs) are a family of small, integral membrane proteins that facilitate water transport across the plasma membranes of cells in response to osmotic gradients. Data from knockout mice support the involvement of AQPs in epithelial fluid secretion, cell migration, brain oedema and adipocyte metabolism, which suggests that modulation of AQP function or expression could have therapeutic potential in oedema, cancer, obesity, brain injury, glaucoma and several other conditions. Moreover, loss-of-function mutations in human AQPs cause congenital cataracts (AQP0) and nephrogenic diabetes insipidus (AQP2), and autoantibodies against AQP4 cause the autoimmune demyelinating disease neuromyelitis optica. Although some potential AQP modulators have been identified, challenges associated with the development of better modulators include the druggability of the target and the suitability of the assay methods used to identify modulators

Slow GABAA mediated synaptic transmission in rat visual cortex

<p>Abstract</p> <p>Background</p> <p>Previous reports of inhibition in the neocortex suggest that inhibition is mediated predominantly through GABA_Areceptors exhibiting fast kinetics. Within the hippocampus, it has been shown that GABA_Aresponses can take the form of either fast or slow response kinetics. Our findings indicate, for the first time, that the neocortex displays synaptic responses with slow GABA_Areceptor mediated inhibitory postsynaptic currents (IPSCs). These IPSCs are kinetically and pharmacologically similar to responses found in the hippocampus, although the anatomical specificity of evoked responses is unique from hippocampus. Spontaneous slow GABA_AIPSCs were recorded from both pyramidal and inhibitory neurons in rat visual cortex.</p> <p>Results</p> <p>GABA_Aslow IPSCs were significantly different from fast responses with respect to rise times and decay time constants, but not amplitudes. Spontaneously occurring GABA_Aslow IPSCs were nearly 100 times less frequent than fast sIPSCs and both were completely abolished by the chloride channel blocker, picrotoxin. The GABA_Asubunit-specific antagonist, furosemide, depressed spontaneous and evoked GABA_Afast IPSCs, but not slow GABA_A-mediated IPSCs. Anatomical specificity was evident using minimal stimulation: IPSCs with slow kinetics were evoked predominantly through stimulation of layer 1/2 apical dendritic zones of layer 4 pyramidal neurons and across their basal dendrites, while GABA_Afast IPSCs were evoked through stimulation throughout the dendritic arborization. Many evoked IPSCs were also composed of a combination of fast and slow IPSC components.</p> <p>Conclusion</p> <p>GABA_Aslow IPSCs displayed durations that were approximately 4 fold longer than typical GABA_Afast IPSCs, but shorter than GABA_B-mediated inhibition. The anatomical and pharmacological specificity of evoked slow IPSCs suggests a unique origin of synaptic input. Incorporating GABA_Aslow IPSCs into computational models of cortical function will help improve our understanding of cortical information processing.</p

Comparative functional analysis of aquaporins/glyceroporins in mammals and anurans

Maintenance of fluid homeostasis is critical to establishing and maintaining normal physiology. The landmark discovery of membrane water channels (aquaporins; AQPs) ushered in a new area in osmoregulatory biology that has drawn from and contributed to diverse branches of biology, from molecular biology and genomics to systems biology and evolution, and from microbial and plant biology to animal and translational physiology. As a result, the study of AQPs provides a unique and integrated backdrop for exploring the relationships between genes and genome systems, the regulation of gene expression, and the physiologic consequences of genetic variation. The wide species distribution of AQP family members and the evolutionary conservation of the family indicate that the control of membrane water flux is a critical biological process. AQP function and regulation is proving to be central to many of the pathways involved in individual physiologic systems in both mammals and anurans. In mammals, AQPs are essential to normal secretory and absorptive functions of the eye, lung, salivary gland, sweat glands, gastrointestinal tract, and kidney. In urinary, respiratory, and gastrointestinal systems, AQPs are required for proper urine concentration, fluid reabsorption, and glandular secretions. In anurans, AQPs are important in mediating physiologic responses to changes in the external environment, including those that occur during metamorphosis and adaptation from an aquatic to terrestrial environment and thermal acclimation in anticipation of freezing. Therefore, an understanding of AQP function and regulation is an important aspect of an integrated approach to basic biological research