N type rapid inactivation in human Kv1.4 channels: functional role of a putative C-terminal helix

Voltage gated potassium channels are tetrameric membrane proteins, which have a central role in cellular excitability. Human Kv1.4 channels open on membrane depolarization and inactivate rapidly by a 'ball and chain' mechanism whose molecular determinants have been mapped to the cytoplasmic N terminus of the channel. Here we show that the other terminal end of the channel also plays a role in channel inactivation. Swapping the C-terminal residues of hKv1.4 with those from two non-inactivating channels (hKv1.1 and hKv1.2) affects the rates of inactivation, as well as the recovery of the channel from the inactivated state. Secondary structure predictions of the hKv1.4 sequence reveal a helical structure at its distal C-terminal. Complete removal or partial disruption of this helical region results in channels with remarkably slowed inactivation kinetics. The ionic selectivity and voltage-dependence of channel opening were similar to hKv1.4, indicative of an unperturbed channel pore. These results demonstrate that fast inactivation is modulated by structural elements in the C-terminus, suggesting that the process involves the concerted action of the N- and C-termini.     

Quantitative determination of cholesterol, sitosterol, and sitostanol in cultured Caco-2 cells by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

In this study, we describe a simple liquid extraction (methanol/choloroform, 1:1, v/v) method for endogenous free cholesterol and administered sterols extracted from cultured Caco-2 cells. To quantify sterol contents in Caco-2 cells, a new HPLC-APCI-MS method was developed. All the sterols were baseline separated using reversed-phase column (C8, 2.1 mm x 150 mm, 3.5 microm) and isocratic conditions (90%, v/v, methanol-water mixture containing 0.2 mM ammonium acetate). The full scan mass spectra of sterols were measured by an ion trap mass spectrometer equipped with an APCI ion source. The intense fragment ions resulting from the loss of water [M+H-H2O]+ (m/z 369, 395, 397 and 399 for cholesterol, stigmasterol, sitosterol, and sitostanol, respectively) were used for determinations. The absolute extraction recovery of sterols from the spiked cell samples were 109.7+/-26.2, 105.7+/-5.1, 109.8+/-5.0 and 99.0+/-7.0% for cholesterol, stigmasterol, sitosterol, and sitostanol, respectively. Furthermore, no significant matrix effect was observed for the sterols in the cell samples. The sample assay was based on the internal standard method using stigmasterol as an internal standard. The method was linear over the concentration ranges of 0.45-9.0 microM (cholesterol) and 0.225-7.2 microM (sitosterol and sitostanol). The within- and between-day precision was less than 7% and accuracy ranged from 93.51 to 101.77%. The lowest limit of quantitation (LLOQ) was 0.225 microM for sitosterol and sitostanol, and 0.45 microM for cholesterol. The accuracy range was 95-106% and precision was lower than 9% for all LLOQ values.     

Identification of the mass transfer mechanisms involved in the transport of human immunoglobulin-G in N,N,N',N'-ethylenediaminetetramethylenephosphonic acid-modified zirconia

Zirconia particles modified with N,N,N',N'-ethylenediaminetetramethylenephosphonic acid (EDTPA), further referred to as r_PEZ, were studied as a support material for use in chromatography. Our previous studies have demonstrated the utility of r_PEZ in the separation of immunoglobulins from biological fluids. In the present study we sought to understand the underlying factors and identify the rate-limiting mechanisms that govern the transport of biomolecules in r_PEZ. Pulse injection techniques were used to elucidate the individual mass transfer parameters. Elution profiles obtained under retained and unretained conditions were approximated by the Gaussian equation and the corresponding HETP contributions were estimated. The dependence of the HETP values on incremental salt concentration in the mobile phase was determined. Resulting data in conjunction with the equations outlined in literature were used to estimate the theoretical number of transfer units for the chromatographic separation process. Our results indicate that surface diffusion probably plays a minor role; however pore diffusion was established to be the rate limiting mechanism for immunoglobulin G adsorption to r_PEZ. The HETP based methodology may be used to estimate the rate limiting mechanisms of mass transfer for any given chromatographic system under appropriate conditions.     

Characterization of a novel lytic bacteriophage from an industrial <Emphasis Type="Italic">Escherichia coli</Emphasis> fermentation process and elimination of virulence using a heterologous CRISPR–Cas9 system

Bacterial–bacteriophage interactions are a well-studied and ecologically-important aspect of microbiology. Many commercial fermentation processes are susceptible to bacteriophage infections due to the use of high-density, clonal cell populations. Lytic infections of bacterial cells in these fermentations are especially problematic due to their negative impacts on product quality, asset utilization, and fouling of downstream equipment. Here, we report the isolation and characterization of a novel lytic bacteriophage, referred to as bacteriophage DTL that is capable of rapid lytic infections of an Escherichia coli K12 strain used for commercial production of 1,3-propanediol (PDO). The bacteriophage genome was sequenced and annotated, which identified 67 potential open-reading frames (ORF). The tail fiber ORF, the largest in the genome, was most closely related to bacteriophage RTP, a T1-like bacteriophage reported from a commercial E. coli fermentation process in Germany. To eliminate virulence, both a fully functional Streptococcus thermophilus CRISPR3 plasmid and a customized S. thermophilus CRISPR3 plasmid with disabled spacer acquisition elements and seven spacers targeting the bacteriophage DTL genome were constructed. Both plasmids were separately integrated into a PDO production strain, which was subsequently infected with bacteriophage DTL. The native S. thermophilus CRISPR3 operon was shown to decrease phage susceptibility by approximately 96%, while the customized CRISPR3 operon provided complete resistance to bacteriophage DTL. The results indicate that the heterologous bacteriophage-resistance system described herein is useful in eliminating lytic infections of bacteriophage DTL, which was prevalent in environment surrounding the manufacturing facility.     

Seed‐specific RNAi in safflower generates a superhigh oleic oil with extended oxidative stability

Vegetable oils extracted from oilseeds are an important component of foods, but are also used in a range of high value oleochemical applications. Despite being biodegradable, nontoxic and renewable current plant oils suffer from the presence of residual polyunsaturated fatty acids that are prone to free radical formation that limit their oxidative stability, and consequently shelf life and functionality. Many decades of plant breeding have been successful in raising the oleic content to ~90%, but have come at the expense of overall field performance, including poor yields. Here, we engineer superhigh oleic (SHO) safflower producing a seed oil with 93% oleic generated from seed produced in multisite field trials spanning five generations. SHO safflower oil is the result of seed‐specific hairpin‐based RNA interference of two safflower lipid biosynthetic genes, FAD2.2 and FATB, producing seed oil containing less than 1.5% polyunsaturates and only 4% saturates but with no impact on lipid profiles of leaves and roots. Transgenic SHO events were compared to non‐GM safflower in multisite trial plots with a wide range of growing season conditions, which showed no evidence of impact on seed yield. The oxidative stability of the field‐grown SHO oil produced from various sites was 50 h at 110°C compared to 13 h for conventional ~80% oleic safflower oils. SHO safflower produces a uniquely stable vegetable oil across different field conditions that can provide the scale of production that is required for meeting the global demands for high stability oils in food and the oleochemical industry.     

Scorer and modality agreement for the detection of intervertebral disc calcification in Dachshunds

Background

The Dachshund is a chondrodystrophic breed of dog predisposed to premature degeneration and calcification, and subsequent herniation, of intervertebral discs (IVDs). This condition is heritable in Dachshunds and breeding candidates are screened for radiographically detectable intervertebral disc calcification (RDIDC), a feature of advanced disc degeneration and a prognostic factor for clinical disease. RDIDC scoring has been previously shown to be consistent within scorers; however, strong scorer effect (subjectivity) was also reported. The aim of this study was to estimate the within- and between-scorer agreement (repeatability and reproducibility, respectively) of computed tomography (CT) scanning and magnetic resonance imaging (MRI) for scoring IVD calcification, and to compare these modalities with radiographic scoring.

Results

Twenty-one Dachshund dogs were screened for IVD calcification using the three imaging modalities. Three scorers scored each case twice, independently. Repeatability was highest for radiography (95.4%), and significantly higher than for CT (90.4%) but not MRI (93.8%). Reproducibility was also highest for radiography (92.9%), but not significantly higher than for CT or MRI (89.4% and 86.4%, respectively). Overall, CT scored IVDs differently than radiography and MRI (64.8% and 62.7% agreement, respectively), while radiography and MRI scored more similarly (85.7% agreement).

Conclusions

Despite high precision for radiography, previous evidence of scorer subjectivity was confirmed, which was not generally observed with CT and MRI. The increased consistency of radiography may be related to prior scorer experience with the modality and RDIDC scoring. This study does not support replacing radiography with CT or MRI to screen for heritable IVD calcification in breeding Dachshunds; however, evaluation of dog-level precision and the accuracy of each modality is recommended.

     

Rogue Waves in Multi-Ion Cometary Plasmas

The effect of pair ions on the formation of rogue waves in a six-component plasma composed of two hot and one colder electron component, hot ions, and pair ions is studied. The kappa distribution, which provides an unambiguous replacement for a Maxwellian distribution in space plasmas, is connected with nonextensive statistical mechanics and provides a continuous energy spectrum. Hence, the colder and one component of the hotter electrons is modeled by kappa distributions and the other hot electron component, by a q-nonextensive distribution. It is found that the rogue wave amplitude is different for various pair-ion components. The magnitude, however, increases with increasing spectral index and nonextensive parameter q. These results may be useful in understanding the basic characteristics of rogue waves in cometary plasmas.     

Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope

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Effects of transient intrathoracic pressure changes (hiccups) on systemic arterial pressure

Mathew, Oommen P. Effects of transient intrathoracicpressure changes (hiccups) on systemic arterial pressure.J. Appl. Physiol. 83(2): 371-375, 1997.The purpose of the study was to determine the effect oftransient changes in intrathoracic pressure on systemic arterialpressure by utilizing hiccups as a tool. Values of systolic anddiastolic pressures before, during, and after hiccups were determinedin 10 intubated preterm infants. Early-systolic hiccups decreasedsystolic blood pressure significantly (P < 0.05) compared with control(39.38 ± 2.72 vs. 46.46 ± 3.41 mmHg) and posthiccups values,whereas no significant change in systolic blood pressure occurredduring late-systolic hiccups. Diastolic pressure immediately after thehiccups remained unchanged during both early- and late-systolichiccups. In contrast, diastolic pressure decreased significantly(P < 0.05) when hiccups occurred during diastole (both early and late). Systolic pressures of the succeeding cardiac cycle remained unchanged after early-diastolic hiccups, whereas they decreased after late-diastolic hiccups. Theseresults indicate that transient decreases in intrathoracic pressurereduce systemic arterial pressure primarily through an increase in thevolume of the thoracic aorta. A reduction in stroke volume appears tocontribute to the reduction in systolic pressure.