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Background HIV-exposed seronegative people who inject drugs (HESN-PWID) have been shown to have increased natural killer (NK) cell and myeloid activation when compared with control donors. Methods We investigated potential mechanisms maintaining NK activation by conducting quantitative proteome comparisons of NK cells from HESN-PWID subjects and control donors. Proteins upregulated in NK cells were measured in the plasma of HESN-PWID subjects by ELISA and further investigated for their ability to induce innate immune activation in vitro. Results The NK cell proteome comparison showed markedly higher levels of interferon-stimulated proteins and S100 proteins, including S100A14. Consistent with these results, we observed significantly higher levels of S100A14 in the plasma of HESN-PWID subjects compared with controls (P = 0.033, n = 25). In vitro, the addition of recombinant S100A14 protein significantly activated NK cells in a peripheral blood mononuclear cell mixture (P = 0.011, n = 9), but not purified NK cells alone. Treatment of purified monocytes with recombinant S100A14 protein induced secretion of TNF-alpha and led to significantly higher NK CD69 activation (P = 0.0156, n = 7) in a co-culture through a TLR4-dependent interaction. Conclusions Our study identified S100A14 as a novel protein increased within NK cells and plasma of HESN-PWID subjects with the capacity to sustain NK activation through TLR4-dependent activation of myeloid cells.

Using a mobile research facility, we enrolled 141 opioid users from a neighborhood of Philadelphia, an urban epicenter of the opioid epidemic. Nearly all (95.6%) met DSM-5 criteria for severe opioid use disorder. The prevalence of HIV infection (8.5%) was more than seven times that found in the general population of the city. Eight of the HIV-positive participants (67.0%) reported receiving antiretroviral treatment but almost all of them had unsuppressed virus (87.5%). The majority of participants (57.4%) reported symptoms consistent with major depressive disorder. Severe economic distress (60.3%) and homelessness were common (57%). Polysubstance use was nearly universal, 72.1% had experienced multiple overdoses and prior medication for opioid use disorder (MOUD) treatment episodes (79.9%), but few currently engaged in addiction care. The prevalence, multiplicity and severity of chronic health and socioeconomic problems highlight consequences of the current opioid epidemic and underscore the urgent need to develop integrated models of treatment.Utilizando un Centro de Investigación Móvil, inscribimos a 141 usuarios de opioides del vecindario de Filadelfia, un epicentro urbano de la epidemia de opioides. Casi todos (95,6%) cumplieron con los criterios del DSM-5 para el trastorno del uso severo del consumo de opioides. La prevalencia de la infección de VIH (8,5%) fue másﹶ de 7 veces superior a las encontrada en la población general de la ciudad. Ocho de los participantes con VIH positivo (67,0%) reportaron haber recibido tratamiento antirretroviral pero casi todos tuvieron virus no suprimido (87,5%). La mayoría de los participantes (57,4%) informaron síntomas compatibles con el Desorden Depresivo Mayor. La angustia severa por lo económico (60,3%) y las personas sin hogar fueron comunes (57%). El uso de múltiples sustancias fue casi universal, el 721% había experimentado múltiples sobredosis y previos medicamentos para el tratamiento del trastorno por consumo de opioides (MOUD) (79,9%), pero muy pocos estaban comprometidos con la atención a las adicciones. La prevalencia, la multiplicidad y la seriedad de los problemas de salud crónica y los problemas socioeconómicos destacan las consecuencias de la actual epidemia de opioides y subrayan la urgente necesidad de desarrollar nuevos modelos de tratamiento integrados.

Previous literature suggests that acute opioid use results in the functional impairment of the immune response, thereby decreasing resistance to viral infection. Here, we assessed if innate and adaptive immune responses are compromised ex vivo in persons who inject drugs (PWID) and whether long-term injection drug use may impact host susceptibility to in vitro HIV infection. We measured the frequency, activation state, and functional profile of NK cells, dendritic cells, and CD4+ and CD8+ T cells in low-risk PWID who do not share needles, high-risk needle-sharing PWID, and control donors who did not inject drugs. We also assessed plasma levels of inflammatory markers and CD4+ T cell susceptibility to HIV infection. We observed a significant increase in the amount of sCD14 (P = 0.0023, n = 16) and sCD163 (P = 0.0001, n = 16) in the plasma of PWID compared to controls. Evidence of constitutive activation was noted in PWID as compared to controls with increased CD69 expression in CD56dim NK cells (P = 0.0103, n = 26) and increased CD38 and HLA-DR expression in CD4+ T cells (P = 0.0355, n = 23). However, no innate or adaptive functional differences were detected between PWID and controls, including: NK cell direct or antibody-dependent cellular cytotoxicity poly-functional response, TLR-stimulated dendritic cell/NK crosstalk, CD8+ T cell response to Staphylococcal enterotoxin B or CMV/EBV/FLU peptides, or constitutive or anti-CD3/CD28-stimulated CD4+ T cell infectivity with CCR5-tropic or CXCR4-tropic HIV-1 isolates. Our data indicate that PWID who utilize opioids over as prolonged time frame can retain a functional ex vivo immune response without a measurable increase in CD4+ T cell infectivity suggesting that leukocytes from PWID are not intrinsically more susceptibility to infection with HIV than non-PWID controls.

The ultrastructure of the pseudobranch of the euryhaline, self-fertilizing fish, Rivulus marmoratus (Cyprinodontidae) was studied with thin sectio and freeze-fracture transmission electron microscopy. In specimens raised from birth in 1% or 200% seawater, the pseudobranch contains two mitochondria-rich cell types, pseudobranchial cells and chloride cells, each of which has an extensive tubular system. Chloride cells only occur on the lateral aspects of the pseudobranch, with their apical crypts open to the environment. Apical crypt invagination to a “pit” structure and multicellular complexes occur in both salinity extremes. The ultrastructure of chloride cells in 1% and 200% SW is consistent with that described previously in the opercular epithelium, opercular skin, and gill of this species; elaboration of mitochondria and basolateral membrane accompanies increased environmental salinity (King et al.: Cell and Tissue Research 257:367–377, 1989). Pseudobranchial cells constitute the majority of the cells; they do not extend to the surface but have a more organized tubular system that is continuous with the basal membrane. These cells do not exhibit ultrastructural changes in response to increased salinity. © 1993 Wiley-Liss, Inc.

Switched excitation allows the miniaturisation of excitation circuitry for transducer integrated front ends, high channel count and portable ultrasound systems. Harmonic Reduction Pulse Width Modulation (HRPWM) provides a method to design five level switched mode excitation signals with control of instantaneous amplitude, frequency and phase plus minimised third harmonics for advanced ultrasound applications. This paper details the application of HRPWM using commercial transmit front end integrated circuits and linear array transducers. The ability of HRPWM to control the pressure of the ultrasound wave is investigated. A full scale error between desired and measured pressure of 3.5% at 4.1 MHz is demonstrated. The temporal windowing of linear frequency modulated excitation signals using HRPWM is demonstrated. Pulse compression linear imaging of a tissue phantom is demonstrated where an improvement in the −20 dB axial resolution of a nylon mono-filament target from 2.14 mm using bipolar excitation to 1.88 mm using HRPWM is shown.

The human papillomavirus (HPV) vaccine was recently approved for use in males. Certain groups, such as men who have sex with men (MSM), are at increased risk of HPV infection. The purpose of the study was to understand perceptions of HPV and the vaccine among adolescent and young adult males, both heterosexual and MSM. Seventy-six males (45 heterosexual, 31 MSM) completed a questionnaire and participated in a focus group. Overall, 42% had heard of HPV and 39% had heard of the HPV vaccine. Males had moderate to favorable attitudes toward vaccination, although intentions to vaccinate were more neutral. MSM were more knowledgeable, aware, and in control of the decision to vaccinate than heterosexual males. Increasing awareness and knowledge about HPV and the vaccine may be necessary to encourage vaccination; certain subgroups of males may be more receptive to HPV vaccination than others.

Coarse time quantization of delay profiles within ultrasound array systems can produce undesirable side lobes in the radiated beam profile. The severity of these side lobes is dependent upon the magnitude of phase quantization error--the deviation from ideal delay profiles to the achievable quantized case. This paper describes a method to improve interchannel delay accuracy without increasing system clock frequency by utilizing embedded phase-locked loop (PLL) components within commercial field-programmable gate arrays (FPGAs). Precise delays are achieved by shifting the relative phases of embedded PLL output clocks in 208-ps steps. The described architecture can achieve the necessary interelement timing resolution required for driving ultrasound arrays up to 50 MHz. The applicability of the proposed method at higher frequencies is demonstrated by extrapolating experimental results obtained using a 5-MHz array transducer. Results indicate an increase in transmit dynamic range (TDR) when using accurate delay profiles generated by the embedded-PLL method described, as opposed to using delay profiles quantized to the system clock.

Ultraviolet light and the heavy metal, cadmium, both have toxic effects on many microorganisms. In this communication we describe a method by which the bacterium Klebsiella aerogenes surmounts both problems using one biological process: the synthesis of cadmium sulfide (CdS) crystallites. These semiconductor particles absorb radiation in the UV spectral region and therefore, when K. aerogenes produces extracellular CdS material in response to environments containing cadmium ions, a photoprotective layer is formed. The effect of UVA radiation on cultures of Klebsiella aerogenes was monitored using electron microscopy, energy-dispersive X-ray analysis and electronic spectroscopy. The results show that at wavelengths 320 nm Λ 400 nm, a photoprotection period of between 4 and 6 h is induced, which eventually fails due to photodegradation of the semiconductor layer to metallic cadmium and elemental sulfur.

Proton-gated Na(+) channels (ASIC) are new members of the epithelial sodium channel/degenerin gene family. ASIC3 mRNA has been detected in the homogenate of pulmonary tissues. However, whether ASIC3 is expressed in the apical membranes of lung epithelial cells and whether it regulates cystic fibrosis transmembrane conductance regulator (CFTR) function are not known at the present time. Using reverse transcription-PCR, we found that the ASIC3 mRNA was expressed in the human airway mucosal gland (Calu-3) and human airway epithelial (16HBE14o) cells. Indirect immunofluorescence microscopy revealed that ASIC3 was co-segregated with CFTR in the apical membranes of Calu-3 cells. Proton-gated, amiloride-sensitive short circuit Na(+) currents were recorded across Calu-3 monolayers mounted in an Ussing chamber. In whole-cell patch clamp studies, activation of CFTR channels with cAMP reduced proton-gated Na(+) current in Calu-3 cells from -154 +/- 28 to -33 +/- 16 pA (n = 5, p0.05) at -100 mV. On the other hand, cAMP-activated CFTR activity was significantly inhibited following constitutive activation of putative ASIC3 at pH 6.0. Immunoassays showed that both ASIC3 and CFTR proteins were expressed and co-immunoprecipitated mutually in Calu-3 cells. Similar results were obtained in human embryonic kidney 293T cells following transient co-transfection of ASIC3 and CFTR. Our results indicate that putative CFTR and ASIC3 channels functionally interact with each other, possibly via an intermolecular association. Because acidic luminal fluid in the cystic fibrosis airway and lung tends to stimulate ASIC3 channel expression and activity, the interaction of ASIC3 and CFTR may contribute to defective salt and fluid transepithelial transport in the cystic fibrotic pulmonary system.

The interaction of ion channels with the actin-based cytoskeleton in epithelial cells not only maintains the polarized expression of ion channels within specific membrane domains, it also functions in the intracellular trafficking and regulation of channel activity. Initial evidence supporting an interaction between epithelial ion channels and the actin-based cytoskeleton came from patch-clamp studies examining the effects of cytochalasins on channel activity. Cytochalasins were shown to either activate or inactivate epithelial ion channels. An interaction between the actin-based cytoskeleton and epithelial ion channels was further supported by the fact that the addition of monomeric or filamentous actin to excised patches had an effect on channel activity comparable to that of cytochalasins. Through the recent application of molecular and proteomic approaches, we now know that the interactions between epithelial ion channels and actin can either be direct or indirect, the latter being mediated through scaffolding or actin-binding proteins that serve as links between the channels and the actin-based cytoskeleton. This review discusses recent advances in our understanding of the interactions between epithelial ion channels and the actin-based cytoskeleton, and the roles these interactions play in regulating the cell surface expression, activity, and intracellular trafficking of epithelial ion channels.

Native amiloride-sensitive Na+ channels exhibit a variety of biophysical properties, including variable sensitivities to amiloride, different ion selectivities, and diverse unitary conductances. The molecular basis of these differences has not been elucidated. We tested the hypothesis that co-expression of δ-epithelial sodium channel (ENaC) underlies, at least in part, the multiplicity of amiloride-sensitive Na+ conductances in epithelial cells. For example, the δ-subunit may form multimeric channels with αβγ-ENaC. Reverse transcription-PCR revealed that δ-ENaC is co-expressed with αβγ-subunits in cultured human lung (H441 and A549), pancreatic (CFPAC), and colonic epithelial cells (Caco-2). Indirect immunofluorescence microscopy revealed that δ-ENaC is co-expressed with α-, β-, and γ-ENaC in H441 cells at the protein level. Measurement of current-voltage that cation selectivity ratios for the revealed relationships Na+/Li+/K+/Cs+/Ca2+/Mg2+, the apparent dissociation constant (Ki) for amiloride, and unitary conductances for δαβγ-ENaC differed from those of both αβγ- and δβγ-ENaC (n = 6). The contribution of the δ subunit to PLi/PNa ratio and unitary Na+ conductance under bi-ionic conditions depended on the injected cRNA concentration. In addition, the EC50 for proton activation, mean open and closed times, and the self-inhibition time of δαβγ-ENaC differed from those of αβγ- and δβγ-ENaC. Co-immunoprecipitation of δ-ENaC with α- and γ-subunits in H441 and transfected COS-7 cells suggests an interaction among these proteins. We, therefore, concluded that the interactions of δ-ENaC with other subunits could account for heterogeneity of native epithelial channels.

The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between alpha-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of alpha-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of alpha-, beta-, and gamma-ENaC revealed co-localization of alpha-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with alpha-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of alpha-ENaC. A direct interaction between F-actin and the COOH terminus of alpha-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC.

Background Gallbladder Na + and H 2 O absorption are increased prior to gallstone formation and may promote cholesterol nucleation. Na + /H + exchange (NHE) isoforms NHE2 and NHE3 are involved in gallbladder Na + transport in prairie dogs. We examined whether increased gallbladder Na + absorption observed during early gallstone formation is the result of NHE up-regulation. Materials and methods Native gallbladder and primary cultures of gallbladder epithelial cells (GBECs) harvested from prairie dogs fed nonlithogenic (CON) or 1.2% cholesterol diet for varying lengths of time to induce cholesterol-saturated bile (PreCRYS), cholesterol crystals (CRYS), or gallstones (GS) were used. NHE activity was assessed by measuring dimethylamiloride-inhibitable 22 Na + uptake under H + gradient in primary GBECs. HOE-694 was used to determine NHE2 and NHE3 contributions. NHE protein and mRNA expression were examined by Western and Northern blots, respectively. Results Gallbladder total NHE activity was 25.1 ± 1.3 nmol mg protein -1 min -1 in the control and increased during gallstone formation peaking at the PreCRYS stage (98.4 ± 3.9 nmol mg protein -1 min -1 ). There was a shift in NHE activity from NHE2 to NHE3 as the animals progressed from no stones through the PreCRYS and CRYS stages to gallstones. The increase in NHE activity was partly caused by an increased V max without any change in K Na m . Both NHE2 and NHE3 protein increased moderately during the PreCRYS stage without increases in mRNA expression. Conclusions Increased gallbladder Na + absorption observed prior to crystal formation is in part caused by an increase NHE activity which is not fully accounted for by an increase in NHE proteins and mRNA levels but may be explained by enhanced localization in the membranes and/or altered regulation of NHE.

Welcome to the fourth edition of Architectural Science Review for 2013. Seven papers have been selected which address the aims and fit within the breadth of scope in the journal. In addition Peter ...

Objective: The objectives of this study were to determine myocardial injury in patients with septic shock by measuring serum cardiac troponin I (cTnI), to evaluate relationship between elevated cTnI and myocardial dysfunction and to determine if cTnI is a predictor of outcome in these patients. Methods: Thirty-seven consecutive patients with septic shock were included in the study. Serum cTnI was measured at study entry and after 24 and 48 h. Transthoracic echocardiogram, electrocardiogram and regular biochemical and hemodynamic assessments were performed. Results: Sixteen (43%) patients had elevated serum cTnI. These patients had higher need for inotropic/vasopressor support (94% vs. 53%, p =0.018), higher APACHE II score (28 vs. 20, p =0.004), higher incidence of regional wall motion abnormalities on echocardiography (56% vs. 6%, p =0.002), lower ejection fraction (46% vs. 62%, p =0.04) and higher mortality (56% vs. 24%, p =0.04) compared to normal cTnI patients. By multiple logistic regression analysis, serum cTnI and APACHE II score were independent predictor of death and length of stay in intensive care unit. Serum cTnI, APACHE II score, anion gap and serum lactate were independent predictor of need for inotropic/vasopressor support. Receiver-operating characteristics of serum cTnI as a predictor of death in septic shock were significant. The elevated serum level of cTnI correlated with the lower left ventricular ejection fraction ( p Conclusions: Myocardial injury can be determined in patients with septic shock by serum cTnI. Serum cTnI concentration correlates with myocardial dysfunction in septic shock. High serum cTnI predicts increased severity of sepsis and higher mortality. A close monitoring of patients with septic shock and elevated levels cTnI is warranted.

Metastatic carcinoma involving the paranasal sinuses is uncommon. One hundred-sixty seven cases have been published in the literature since 1951. Symptoms, signs, and rhinoscopic and imaging findings are often nonspecific, and the diagnosis may be missed for considerable time. Therefore, a high level of suspicion is warranted in patients with known malignancies presenting with persistent or recurrent rhinosinusitis or facial complaints.

Lovastatin, an inhibitor of the rate-limiting enzyme in cholesterol biosynthesis, is widely used to treat hypercholesterolemia. We investigated the long-term effects of lovastatin alone and in combination with ursodeoxycholic acid on biliary lipid composition and gallstone dissolution. Forty-two prairie dogs were fed 1.2% cholesterol diet for 5 weeks, and cholecystectomy was performed on 6 animals to confirm gallstones. The remaining animals were maintained on a 0.4% cholesterol diet and were randomized to receive placebo, lovastatin (3.3 mg/g diet), ursodeoxycholic acid (10 mg/g), or combination of both drugs. After 10 weeks, animals underwent cholecystectomy. Dissolution response to therapy was determined, and serum and biliary lipids were measured. All treatment groups had significant reductions in serum cholesterol. Lovastatin treatment reduced both hepatic and gallbladder bile cholesterol, altered bile acid composition, and induced a 79% total response compared to placebo. Although ursodeoxycholic treatment induced a 44% response, long-term combination treatment elevated both gallbladder bile cholesterol and calcium and failed to produce an augmented response. These data suggest that lovastatin therapy alone may promote gallstone dissolution in humans.

Advances in electronics and transducer fabrication have provided the ultrasound system designer with the opportunity for integrating electronics into the transducer head. Switched mode circuits are miniaturizable, low loss and as such are suited to array excitation. Without careful excitation signal design switched mode waveforms lack combined time varying amplitude and harmonic control. Existing modulation schemes have provided either amplitude or harmonic control. The proposed harmonic reduction pulse width modulation (HRPWM) provides a method of creating a quinary (5-level) switched waveform with both time-varying amplitude control and third harmonic cancellation. HRPWM achieves waveform control through matching the amplitude of energy contained at the fundamental frequency with the desired amplitude, and third harmonic cancellation through the use of a non-linear modulating waveform. The resulting HRPWM excitation signals use multiple bipolar low voltage pulses for the control of signals below 50% magnitude, and a five-level pulse for amplitudes above 50%. In simulations, HRPWM shows an excitation signal third harmonic power of −34.75 dB, a reduction of 19 dB from other 5-level modulation schemes, and a ultrasound pressure waveform third harmonic power of −64.19 dB, a reduction of 19.9 dB from other 5-level modulation schemes, and 25 dB lower than bipolar switched excitation.

Switched-mode operation allows the miniaturization of excitation circuitry but suffers from high harmonic distortion. This paper presents a method of phase-inversion-based selective harmonic elimination (PI-SHE) and the use of multiple switching levels. PI-SHE is shown to enable multiples of any selected harmonic to be eliminated through controlled timing of the transition between different excitation voltage levels. Multiples of the third harmonic are shown to be eliminated in three-level tone waveforms. In addition, multiples of the fifth harmonic are shown to be eliminated using five-level tone waveforms. A method of calculating the expected amplitude of each harmonic is presented. The application of PI-SHE in linear frequency-modulated (LFM) excitation is proposed. A heuristic derivation of the spectral properties of multilevel switched LFM waveforms is presented. The performance of the proposed PI-SHE method is confirmed through experimental measurement of the harmonics present in an ultrasound wave using two, three, and five levels for both tone and LFM excitation. The proposed method of controlling harmonics through the use of multilevel switched excitation is especially suitable for applications in which portability, high channel counts, and precise harmonic control are required.

The renal epithelial cell line A6, derived from Xenopus laevis, expresses epithelial Na+channels (ENaCs) and serves as a model system to study hormonal regulation and turnover of ENaCs. Our previous studies suggest that the α-subunit of Xenopus ENaC (α- xENaC) is detectable as 150- and 180-kDa polypeptides, putative immature and mature α-subunit heterodimers. The 150- and 180-kDa α- xENaC were present in distinct fractions after sedimentation of A6 cell lysate through a sucrose density gradient. Two anti-α- xENaC antibodies directed against distinct domains demonstrated that only 180-kDa α- xENaC was expressed at the apical cell surface. The half-life of cell surface-expressed α- xENaC was 24–30 h, suggesting that once ENaC matures and is expressed at the plasma membrane, its turnover is similar to that reported for mature cystic fibrosis transmembrane conductance regulator. No significant changes in apical surface expression of α- xENaC were observed after treatment of A6 cells with aldosterone for 24 h, despite a 5.3-fold increase in short-circuit current. This lack of change in surface expression is consistent with previous observations in A6 cells and suggests that aldosterone regulates ENaC gating and increases channel open probability.

Gallbladder Na+ absorption is linked to gallstone formation in prairie dogs. Na+/H+ exchange (NHE) is one of the major Na+ absorptive pathways in gallbladder. In this study, we measured gallbladder Na+/H+ exchange and characterized the NHE isoforms expressed in prairie dogs. Na+/H+ exchange activity was assessed by measuring amiloride-inhibitable transepithelial Na+ flux and apical 22Na+ uptake using dimethylamiloride (DMA). HOE-694 was used to determine NHE2 and NHE3 contributions. Basal JNams was higher than JNasm with JNanet absorption. Mucosal DMA inhibited transepithelial Na+ flux in a dose-dependent fashion, causing JNams equal to JNasm and blocking JNanet absorption at 100 μm. Basal 22Na+ uptake rate was 10.9 ± 1.0 μmol · cm−2· hr−1 which was inhibited by ∼43% by mucosal DMA and ∼30% by mucosal HOE-694 at 100 μm. RT-PCR and Northern blot analysis demonstrated expression of mRNAs encoding NHE1, NHE2 and NHE3 in the gallbladder. Expression of NHE1, NHE2 and NHE3 polypeptides was confirmed using isoform-specific anti-NHE antibodies. These data suggest that Na+/H+ exchange accounts for a substantial fraction of gallbladder apical Na+ entry and most of net Na+ absorption in prairie dogs. The NHE2 and NHE3 isoforms, but not NHE1, are involved in gallbladder apical Na+ uptake and transepithelial Na+ absorption.

The therapeutic use of microbubbles for targeted drug or gene delivery is a highly active area of research. Phospholipid-encapsulated microbubbles typically have a polydisperse size distribution over the 1 to 10 μm range and can be functionalized for molecular targeting and loaded with drug-carrying liposomes. Sonoporation through the generation of shear stress on the cell membrane by microbubble oscillations is one mechanism that results in pore formation in the cell membrane and can improve drug delivery. A microbubble oscillating at its resonant frequency would generate maximum shear stress on a membrane. However, because of the polydisperse nature of phospholipid microbubbles, a range of resonant frequencies would exist in a single population. In this study, the use of linear chirp excitations was compared with equivalent duration and acoustic pressure tone excitations when measuring the sonoporation efficiency of targeted microbubbles on human colorectal cancer cells. A 3 to 7 MHz chirp had the greatest sonoporation efficiency of 26.9 ± 5.6%, compared with 16.4 ± 1.1% for the 1.32 to 3.08 MHz chirp. The equivalent 2.2- and 5-MHz tone excitations have efficiencies of 12.8 ± 2.1% and 15.6 ± 1.1%, respectively, which were all above the efficiency of 4.1 ± 3.1% from the control exposure.

The mammalian urinary bladder exhibits transepithelial Na+absorption that contributes to Na+gradients established by the kidney. Electrophysiological studies have demonstrated that electrogenic Na+absorption across the urinary bladder is mediated in part by amiloride-sensitive Na+channels situated within the apical membrane of the bladder epithelium. We have used a combination of in situ hybridization, Northern blot analysis, and immunocytochemistry to examine whether the recently cloned epithelial Na+channel (ENaC) is expressed in the rat urinary bladder. In situ hybridization and Northern blot analyses indicate that α-, β-, and γ-rat ENaC (rENaC) are expressed in rat urinary bladder epithelial cells. Quantitation of the levels of α-, β-, and γ-rENaC mRNA expression in rat urinary bladder, relative to β-actin mRNA expression, indicates that, although comparable levels of α- and β-rENaC subunits are expressed in the urinary bladder of rats maintained on standard chow, the level of γ-rENaC mRNA expression is 5- to 10-fold lower than α- or β-rENaC mRNA. Immunocytochemistry, using an antibody directed against α-rENaC, revealed that ENaCs are predominantly localized to the luminal membrane of the bladder epithelium. Together, these data demonstrate that ENaC is expressed in the mammalian urinary bladder and suggest that amiloride-sensitive Na+transport across the apical membrane of the mammalian urinary bladder epithelium is mediated primarily by ENaC.

A method of output pressure control for ultrasound transducers using switched excitation is described. The method generates width-modulated square-wave pulse sequences that are suitable for driving ultrasound transducers using MOSFETs or similar devices. Sequences are encoded using an optimized level-shifted, carrier-comparison, pulse-width modulation (PWM) strategy derived from existing PWM theory, and modified specifically for ultrasound applications. The modifications are: a reduction in carrier frequency so that the smallest number of pulses are generated and minimal switching is necessary; alteration of a linear carrier form to follow a trigonometric relationship in accordance with the expected fundamental output; and application of frequency modulation to the carrier when generating frequency-modulated, amplitude- tapered signals. The PWM method permits control of output pressure for arbitrary waveform sequences at diagnostic frequencies (approximately 5 MHz) when sampled at 100 MHz, and is applicable to pulse shaping and array apodization. Arbitrary waveform generation capability is demonstrated in simulation using convolution with a transducer's impulse response, and experimentally with hydrophone measurement. Benefits in coded imaging are demonstrated when compared with fixed-width square-wave (pseudo-chirp) excitation in coded imaging, including reduction in image artifacts and peak side-lobe levels for two cases, showing 10 and 8 dB reduction in peak side-lobe level experimentally, compared with 11 and 7 dB reduction in simulation. In all cases, the experimental observations correlate strongly with simulated data.

Conducting ultrasound measurements in a nuclear environment necessitates a non-invasive and non-intrusive transducer design, capable of operating through thick walled stainless steel vessels. Decommissioning of legacy infrastructure requires transducers capable of retro fitting. Achieving spatial measurements across vessel or pipe cross-sections requires a tomographic approach, typically using multiple invasive or intrusive point source transducers with a fanbeam profile enabling point to multipoint measurements. Non-invasive point source transducers are unable to create fan beams within the vessel. Differing path lengths within the vessel wall combined with the ultrasonic velocity mismatch between the wall and vessel contents creates a plane wave ultrasound beam within the vessel which is unsuitable for tomography. The objective of this study is to create a non-invasive transducer array with the spatial diversity to allow point to multipoint measurement. A non-intrusive array design methodology, overcoming the inherent focusing effects of curved inner pipe surface, is proposed and illustrated through the design, manufacture and testing of an 18 virtual point tomographic array on a stainless steel 6" pipe with 18mm thick walls. The pipe transfers a slurry of corroded Magnox nuclear fuel rods during decommissioning activities. A point to multi-point transducer arrangement is achieved through the creation of virtual sources, located on the inner surface of the pipe wall. Transducers are arranged at discrete angles around the virtual point such that refraction at the inner pipe surface creates multiple beams, each directed at individual, virtual points, to create point to multipoint measurements. The transducer array is constructed using an electrical discharge machined (EDM) stainless steel support with random 1"3 piezo-composite transducers. Finite element verification of the design PZFlex and the manufactured array is experimentally characterized.

Tissue harmonic imaging provides better spatial resolution by producing the image with the second harmonic component generated by nonlinear propagation in tissue. However, the spectral overlap between the transmitted signal and received second harmonic signal results in image artifacts and reduces the image quality. Conventional filtering techniques such as a bandpass filter can separate the second harmonic and fundamental components at the expense of reduced bandwidth.

The Na+/H+ exchanger isoform NHE1 is localized to the basolateral membrane of renal and intestinal epithelia. We examined the plasma membrane distribution, biosynthesis, and cell surface delivery of NHE1 in A6 epithelia. NHE1 was localized to the basolateral membrane. Studies of NHE1 biosynthesis with a pulse-chase protocol demonstrated that a core glycosylated, endoglycosidase H-sensitive, 90-kDa NHE1 was present 0-5 h into the chase period and that mature 110-kDa NHE1 was present 1-24 h into the chase period. Studies of plasma membrane delivery of newly synthesized NHE1 demonstrated that the 90-kDa NHE1 was detected at both apical and basolateral membranes 2-5 h into the chase period. The 110-kDa NHE1 was observed at the basolateral membrane 5-24 h into the chase period. These results suggest that NHE1 is expressed primarily at the basolateral membrane of A6 cells, that core glycosylated NHE1 is delivered to the plasma membrane in a nonpolarized manner, and that nature 110-kDa NHE1 is delivered to the basolateral membrane.

The therapeutic application of microbubbles is a highly active area of research, since they can be used for drug or gene delivery. Phospholipid encapsulated microbubbles typically have a polydisperse size distribution over the 1-10 μm range and can be functionalised for molecular targeting as well as loaded with drug-carrying liposomes. The generation of shear stress on the cell membrane by microbubble oscillations is one mechanism that results in pore formation in the cell membrane. A microbubble oscillating at its resonant frequency would generate maximum shear stress on a membrane. However, due to the polydisperse nature of phospholipid microbubbles, a range of resonant frequencies would exist in a single population. Thus, the use of a linear modulated ultrasound pulse, or “chirp”, to excite a microbubble population could improve the sonoporation efficiency.

The high velocities encountered in major vessels impede potential drug-free thombolysis treatments such as Histotripsy by lowering the gas concentration that is known to promote inertial cavitation and soft tissue fragmentation. The ”ultrasonic tap” can retain microbubbles in a highly localised region, against the vessel wall. Phospholipid microbubbles flowing through a 4 mm wall less vessel at a depth of 30 mm within a tissue mimicking phantom were successfully accumulated at a single location. The same ultrasound medical probe was used to emit the acoustic field required to trap the microbubbles, acquire B-Mode frames and induce inertial cavitation for concentrations of 106 and 107 MB/ml. Inertial cavitation was measured through 17 mm of tissue mimicking material with similar frequency dependent attenuation to human tissue.

The second harmonic generation in medical ultrasound is either caused by tissue or ultrasound contrast agents. The conventional signal processing techniques cannot separate the harmonic response from microbubbles and tissue. The second order spectral analysis, commonly known as the frequency analysis, is the most common way of evaluating the microbubble response. Although frequency analysis can estimate the power spectrum effectively, it suppresses the phase relation between the frequency components. In this study, bispectral analysis is used to evaluate the microbubble response and separate the second harmonic generated by tissue and microbubbles via the phase coupling between fundamental and harmonic components.

This paper provides a simulation study into the use of a novel method of ultrasound array excitation referred to as randomized excitation. This technique provides a solution for temporal ultrasound power/intensity control across an ultrasound array. This technique particularly differs from other previously reported solutions as elements are randomly switched, spatially across the array and temporally in time. Simulations show this excitation scheme to be capable of producing fine temporal control of the transmitted ultrasound power at the focal region. This paper covers the basics of randomized excitation implementation, simulation results and future potential applications of randomized excitation.

In an ultrasound system, a digital beamformer is a critical component, which beamforms the data in a desired direction. A 96 channel parameterized beamforming system is designed using VHDL tools. Incoming sampled signals are delayed using FIFO chains and receive apodization is applied using a single multiplier on a sample by sample basis. Each delayed channel data is then summed using a pipelined adder tree to give a beamformed line. The design is evaluated using a 96 channel hardware research platform (UARP), but can easily be extended to more than 96 channels for different imaging techniques. It supports variable channel count, aperture sizes and delays. It increases system frame rate as compared to PC based beamforming with just another 15% logic utilization and 3% memory resources using custom FPGA board, making it viable for real-time processing and measurements.

Described is a fractional harmonic excitation (FHE) technique suitable for imaging using ultrasound bipolar squarewave excitations. The technique differs from conventional imaging techniques as the transducer is excited using a harmonic within the excitation signal, as opposed to using the fundamental signal. As a consequence, the fundamental frequency of the FHE signal is designed at a fraction (1/3) of the intended output frequency. The advantages of the technique include a reduction in required output switching frequency and artificial extension of transmitter bandwidth. The technique is also capable of generating frequency modulated `chirp' signals. Disadvantages of the technique include reduced output pressure due to excitation by a signal harmonic and potential for transducer heating. The work demonstrates that exciting the transducer in this fashion can produce coded and non-coded images. This is verified by imaging two non-destructive test phantoms using a custom imaging system (UARP, University of Leeds, UK).

The toad urinary bladder and epithelial cell lines derived from the urinary bladder, including TBM, serve as model systems for the study of transepithelial Na+ transport. We examined biochemical characteristics of epithelial Na+ channels in toad urinary bladder and TBM cells and their cellular localization in the urinary bladder. The radiolabeled amiloride analogue [3H]benzamil bound to a single class of high-affinity binding sites in membrane vesicles from toad urinary bladder with a dissociation constant (Kd) of 10 nM. Photoactive benzamil analogues specifically labeled a 135,000-Da polypeptide in toad urinary bladder and TBM cells. A monoclonal anti-Na+ channel antibody directed against the amiloride-binding component of the channel specifically recognized a 135,000-Da polypeptide in TBM cells. Polyclonal anti-Na+ channel antibodies generated against purified bovine epithelial Na+ channel specifically recognized a 235,000-Da polypeptide in toad urinary bladder and localized Na+ channels to the apical plasma membrane of urinary bladder epithelial cells. The biochemical characteristics and the cellular localization of epithelial Na+ channels in toad urinary bladder are similar to those previously described in mammalian kidney and in the A6 cell line.

The transforming growth factor β (TGFB) superfamily proteins bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), are essential for mammalian fertility. Recent in vitro evidence suggests that the proregions of mouse BMP15 and GDF9 interact with their mature proteins after secretion. In this study, we have actively immunized mice against these proregions to test the potential in vivo roles on fertility. Mice were immunized with either N- or C-terminus proregion peptides of BMP15 or GDF9, or a full-length GDF9 proregion protein, each conjugated to keyhole limpet hemocyanin (KLH). For each immunization group, ovaries were collected from ten mice for histology after immunization, while a further 20 mice were allowed to breed and litter sizes were counted. To link the ovulation and fertility data of these two experimental end points, mice were joined during the time period identified by histology as being the ovulatory period resulting in to the corpora lutea (CL) counted. Antibody titers in sera increased throughout the study period, with no cross-reactivity observed between BMP15 and GDF9 sera and antigens. Compared with KLH controls, mice immunized with the N-terminus BMP15 proregion peptide had ovaries with fewer CL (PPPP

In ultrasound harmonic imaging with chirp coded excitation, the axial resolution can be improved by increasing the excitation signal bandwidth. However, increasing the bandwidth will cause overlapping between the received nonlinear second harmonic chirp component (SHCC) and the fundamental component. For the spectrally overlapping harmonics, signal decoding using the second harmonic matched filter (SHMF) typically produces higher range sidelobes level (RSLL), which reduces the image contrast. A multi-pulse detection scheme such as pulse inversion can be used to extract the overlapped SHCC; however it is susceptible to motion artifacts and reduced system frame-rate. In this study, the fractional Fourier transform (FrFT) is proposed with chirp coded excitation for the extraction of the overlapped SHCC. The experimental results indicate at least a 13 dB improvement in the RSLL of the FrFT filtered compressed SHCC when compared with the unfiltered compressed SHCC.

Switched excitation is a convenient alternative to high frequency linear power amplifiers for multi-channel array systems. Unfortunately, switched mode excitation signals often contain harmonics that are undesirable in applications such as coded harmonic imaging. Harmonics can be reduced in switched excitation with the addition of intermediary voltage levels and careful control of switching timing. This work discusses two, three, five and nine level switched mode excitations suitable for Linear Frequency Modulated (LFM) coded excitation. Simulations in MATLAB compare two LFM signals with a central frequency 0.5 MHz, duration of 100 μs and bandwidths of 25% and 100% to an ideal `analogue' case. Simulated results shows harmonic cancellation in the switched mode signals. For the nine level case, all harmonics below the 11th are cancelled. Experimental measurements are also obtained using the UARP system loaded with a 0.5 MHz transducer. Experimental results show cancellation of harmonics to -40 dB below the fundamental, however an increase in the third harmonic is apparent when comparing nine levels to five levels. To further verify harmonic reduction, calculations of the Total Harmonic Distortion are provided for each switched mode excitation up to the ninth harmonic.

Acoustic radiation force has become a significant factor in the research to enhance the targeting of microbubbles for molecular imaging and drug delivery. In this study microbubbles are flown through an opposing radiation force funnel and the impact on their path is compared to the theory. The experiments demonstrate that the behavior of the lipid microbubbles is in accordance with the simulation. Although the force required to stop microbubbles flowing at 10 mm/s is in the order of 100s of nN, large clusters are successfully held against the flow.