Enhanced acoustic streaming effects via sharp-edged 3D microstructures.

Acoustofluidic micromanipulation is an important tool for biomedical research, where acoustic forces offer the ability to manipulate fluids, cells, and particles in a rapid, biocompatible, and contact-free manner. Of particular interest is the investigation of acoustically driven sharp edges, where high tip velocity magnitudes and strong acoustic potential gradients drive rapid motion. Whereas prior devices utilizing 2D sharp edges have demonstrated promise for micromanipulation activities, taking advantage of 3D structures has the potential to increase their performance and the range of manipulation activities. In this work, we investigate high-magnitude acoustic streaming fields in the vicinity of sharp-edged, sub-wavelength 3D microstructures. We numerically model and experimentally demonstrate this in fabricating parametrically configured 3D microstructures whose tip-angle and geometry influence acoustic streaming velocities and the complexity of streaming vortices, finding that the simulated and realized velocities and streaming patterns are both tunable and a function of microstructure shape. These sharp-edge interfaces hold promise for biomedical studies benefiting from precise and targeted micromanipulation.

Micro-Acoustic Holograms for Detachable Microfluidic Devices.

Acoustic microfluidic devices have advantages for diagnostic applications, therapeutic solutions, and fundamental research due to their contactless operation, simple design, and biocompatibility. However, most acoustofluidic approaches are limited to forming simple and fixed acoustic patterns, or have limited resolution. In this study,a detachable microfluidic device is demonstrated employing miniature acoustic holograms to create reconfigurable, flexible, and high-resolution acoustic fields in microfluidic channels, where the introduction of a solid coupling layer makes these holograms easy to fabricate and integrate. The application of this method to generate flexible acoustic fields, including shapes, characters, and arbitrarily rotated patterns, within microfluidic channels, is demonstrated.

Clinician perspectives on technology-enhanced in vivo exposures during prolonged exposure therapy for PTSD.

OBJECTIVE: To investigate clinicians' perceptions regarding the use of mobile technology tools during prolonged exposure (PE) therapy to allow for monitoring and enhancing in-vivo exposures (IVEs). METHODS: Clinicians with training in PE therapy (N = 32; average of 9 years of practice) completed surveys asking about their perspectives on the utility of virtually attending IVEs with patients while simultaneously having access to real-time subjective and physiological data (i.e., heart rate, galvanic skin conductance) to guide exposure exercises and assure optimal stimulus engagement. RESULTS: Findings showed clinicians to have a favorable view of applying technology devices and systems to enhance IVEs of PE therapy. Most clinicians (93.8%) believed that real-time monitoring of IVEs-particularly monitoring patients' subjective distress and completion of and duration of time in the IVE-would be useful and significantly enhance PE therapy. CONCLUSION: The positive perceptions toward integrating technology into IVEs in this study have important implications for the development and implementation of technology-enhanced PE therapy. A mobile technology system that incorporates real-time indicators of engagement (i.e., both subjective and physiological) during IVEs and allows clinicians to review recordings of, or virtually accompany, patients during IVEs has the potential to innovate and transform PE and other exposure-based treatments. Clinicians also believed that technology-enhanced IVEs may help reduce early termination from PE.

Programmable Acoustic Holography using Medium-Sound-Speed Modulation.

Acoustic holography offers the ability to generate designed acoustic fields to manipulate microscale objects. However, the static nature or large aperture sizes of 3D printed acoustic holographic phase plates limits the ability to rapidly alter generated fields. In this work， a programmable acoustic holography approach is demonstrated by which multiple discrete or continuously variable acoustic targets can be created. Here, the holographic phase plate encodes multiple images, where the desired field is produced by modifying the sound speed of an intervening fluid media. Its flexibility is demonstrated in generating various acoustic patterns, including continuous line segments, discrete letters and numbers, using this method as a sound speed indicator and fluid identification tool. This programmable acoustic holography approach has the advantages of generating reconfigurable and designed acoustic fields, with broad potential in microfluidics, cell/tissue engineering, real-time sensing, and medical ultrasound.

Sound-Speed Modifying Acoustic Metasurfaces for Acoustic Holography.

Acoustic metasurfaces offer unique capabilities to steer and direct acoustic fields, though these are generally composed of complex 3D structures, complicating their fabrication and applicability to higher frequencies. Here, an ultrathin metasurface approach is demonstrated, wherein planarized micropillars in a discretized phase array are utilized. This subwavelength metasurface is easily produced via a single-step etching process and is suitable for megahertz-scale applications. The flexibility of this approach is further demonstrated in the production of complex acoustic patterns via acoustic holography. This metasurface approach, with models used to predict their behavior, has broad potential in applications where robust, high-frequency acoustic manipulation is required, including microfluidics, cell/tissue engineering, and medical ultrasound.

High-Flow Nasal Cannula for Apneic Oxygenation in Obese Patients for Elective Surgery: A Systematic Review and Meta-Analysis.

BACKGROUND: Obese patients desaturate rapidly during the apneic period after induction of anesthesia for elective surgery. Administration of oxygen using high-flow nasal cannulae (HFNCs) may prevent desaturation in nonobese patients compared to facemask (FM) preoxygenation. The aim of this meta-analysis was to compare the effectiveness of HFNC to FM preoxygenation techniques in reducing preintubation desaturation in obese patients undergoing elective surgery. METHODS: This study protocol was registered on PROSPERO (CRD42022309391). Adult studies that compared HFNC and FM preoxygenation in obese patients requiring general anesthesia for elective surgery were included. The primary outcome was desaturation resulting in oxygen saturation of <92% from induction of anesthesia until intubation. Secondary outcomes included the lowest arterial oxygen content before intubation expressed in mm Hg, safe apnea time expressed in seconds, the lowest oxygen saturation before intubation expressed as a percentage, patient-reported discomfort, the need for rescue ventilation, and the incidence of aspiration of gastric contents during intubation. Risk of bias was assessed using the Cochrane Collaboration tool. Certainty was assessed following the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. RESULTS: Six studies including 351 participants were eligible for analysis. There was no difference in odds of oxygen desaturation <92% between HFNC and FM (odds ratio [OR], 0.49; 95% confidence interval [CI], 0.15-1.63; P = .24). The HFNC group had a significantly longer safe apnea time (mean difference [MD], -124.20 with 95% CI, -200.47 to -47.93; P = .001). There was no difference between HFNC and FM in the lowest arterial oxygen content (MD, -23.90; 95% CI, -88.64 to 40.85; P = .47) and the lowest peripheral oxygenation saturation (MD, -0.47 with 95% CI, -5.07 to 4.12; P = .84). HFNC had a lower odd of discomfort than FM (OR, 0.13; 95% CI, 0.03-0.52; P = .004). There was no difference in the odds of aspiration of gastric contents between HFNC and FM (OR, 0.33; 95% CI, 0.01-8.21; P = .50). The risk of bias for our primary and secondary outcomes was low. The GRADE assessment for our primary outcome indicated a low level of certainty. For secondary outcomes, the GRADE assessment indicated a very low certainty for all outcomes except for patient discomfort, which was indicated as a moderate level of certainty. CONCLUSIONS: There may be no difference between HFNC and FM preoxygenation in preventing oxygen desaturation <92% or the lowest oxygen saturation before intubation. Preparation remains important to prevent and manage desaturation during induction of obese patients.

Respiration mask waveguide optimisation for maximised speech intelligibility.

Both the scarcity and environmental impact of disposable face masks, as in the COVID-19 pandemic, have instigated the recent development of reusable masks. Such face masks reduce transmission of infectious agents and particulates, but often impact a user's ability to be understood when materials, such as silicone or hard polymers, are used. In this work, we present a numerical optimisation approach to optimise waveguide topology, where a waveguide is used to transmit and direct sound from the interior of the mask volume to the outside air. This approach allows acoustic energy to be maximised according to specific frequency bands, including those most relevant to human speech. We employ this method to convert a resuscitator mask, made of silicone, into respiration personal protective equipment (PPE) that maximises the speech intelligibility index (SII). We validate this approach experimentally as well, showing improved SII when using the fabricated device. Together, this design represents a unique and effective approach to utilize and adapt available apparatus to filter air while improving the ability to communicate effectively, including in healthcare settings.

2-Amidino analogs of glycine-amiloride conjugates: inhibitors of urokinase-type plasminogen activator.

The relative non-toxicity of the diuretic amiloride, coupled with its selective inhibition of the protease urokinase plasminogen activator (uPA), makes this compound class attractive for structure-activity studies. Herein we substituted the C(2)-acylguanidine of C(5)-glycyl-amiloride with amidine and amidoxime groups. The data show the importance of maintaining C(5)-hydrophobicity. The C(5)-benzylglycine analogs containing either C(2)-acylguanidine or amidine inhibited uPA with an IC(50) ranging from 3 to 7 µM and were cytotoxic to human U87 malignant glioma cells.

Dual inhibition of sodium-mediated proton and calcium efflux triggers non-apoptotic cell death in malignant gliomas.

Malignant glioma cells maintain an elevated intracellular pH (pH(i)) within hypoxic-ischemic tumor microenvironments through persistent activation of sodium-proton transport (McLean et al., 2000). Amiloride has been reported to selectively kill human malignant glioma cell lines but not primary astrocytes (Hegde et al., 2004). While amiloride reduces pH(i) of malignant gliomas by inhibiting isoform 1 of sodium-proton exchange (NHE1), direct acidification was shown to be cytostatic rather than cytotoxic. At cytotoxic concentrations, amiloride has multiple drug targets including inhibition of NHE1 and sodium-calcium exchange. Amiloride's glioma cytotoxicity can be explained, at least in part, by dual inhibition of NHE1 and of Na(+)-dependent calcium efflux by isoform 1.1 of the sodium-calcium exchanger (NCX1.1), which increases [Ca(2+)](i) and initiates glioma cell demise. As a result of persistent NHE1 activity, cytosolic free levels of sodium ([Na(+)](i)) in U87 and C6 glioma cells are elevated 3-fold, as compared with normal astrocytes. Basal cytosolic free calcium levels ([Ca(2+)](i)) also are increased 5-fold. 2', 4'-dichlorobenzamil (DCB) inhibits the sodium-dependent calcium transporter (NCX1.1) much more potently than NHE1. DCB was employed in a concentration-dependent fashion in glioma cells to selectively inhibit the forward mode of NCX1.1 at ≤1µM, while dually inhibiting both NHE1 and NCX1.1 at ≥20µM. DCB (1µM) was not cytotoxic to glioma cells, while DCB (20µM) further increased basal elevated levels of [Ca(2+)](i) in glioma cells that was followed by cell demise. Cariporide and SEA0400 are more selective inhibitors of NHE1 and NCX1.1 than amiloride or DCB, respectively. Individually, Cariporide and SEA0400 are not cytotoxic, but in combination induced glioma cell death. Like amiloride, the combination of Cariporide and SEA0400 produced glioma cell death in the absence of demonstrable caspase activation.

Detection of agar, by analysis of sugar markers, associated with Bacillus anthracis spores, after culture.

Detection of small quantities of agar associated with spores of Bacillus anthracis could provide key information regarding its source or growth characteristics. Agar, widely used in growth of bacteria on solid surfaces, consists primarily of repeating polysaccharide units of 3,6-anhydro-l-galactose (AGal) and galactose (Gal) with sulfated and O-methylated galactoses present as minor constituents. Two variants of the alditol acetate procedure were evaluated for detection of potential agar markers associated with spores. The first method employed a reductive hydrolysis step, to stabilize labile anhydrogalactose, by converting to anhydrogalactitol. The second eliminated the reductive hydrolysis step simplifying the procedure. Anhydrogalactitol, derived from agar, was detected using both derivatization methods followed by gas chromatography-mass spectrometry (GC-MS) analysis. However, challenges with artifactual background (reductive hydrolysis) or marker destruction (hydrolysis) respectively lead to the use of an alternative agar marker. A minor agar component, 6-O-methyl galactose (6-O-M gal), was readily detected in agar-grown but not broth-grown bacteria. Detection was optimized by the use of gas chromatography-tandem mass spectrometry (GC-MS-MS). With appropriate choice of sugar marker and analytical procedure, detection of sugar markers for agar has considerable potential in microbial forensics.

An acidic environment changes cyclin D1 localization and alters colony forming ability in gliomas.

The human glioma cell lines, U87 and T98G, were evaluated for their ability to survive and form colonies in an acidic environment of pH(ext) 6.0. In contrast to U87, which showed an 80-90% survival rate, only 40% of T98G cells survived 6 days at pH(ext) 6.0 and lost their colony forming ability when returned to a normocidic environment. Although both U87 and T98G cells maintain an intracellular pH (pH(i)) of 7.0 at pH(ext) 6.0 and arrest mostly in G1 phase of the cell cycle, only T98G demonstrated a major loss of cyclin D1 that was prevented by the proteasome inhibitor MG132. Colony forming ability was restored by stably transfecting T98G cells with a cyclin D1-expressing plasmid. Both U87 and T98G cells demonstrated increased cytoplasmic localization of cyclin D1 during exposure at pH(ext) 6.0. Upon prolonged (24 h) incubation at pH(ext) 6.0, nuclear cyclin D1 was nearly absent in T98G in contrast to U87 cells. Thus, an acidic environment triggers cytoplasmic localization and proteasomal degradation of cyclin D1.

Short-term safety and tolerability of a once-daily fixed-dose abacavir-lamivudine combination versus twice-daily dosing of abacavir and lamivudine as separate components: findings from the ALOHA study.

STUDY OBJECTIVE: To evaluate the short-term (12 wks) safety and tolerability of a once-daily, fixed-dose abacavir-lamivudine combination versus twice-daily dosing of the separate components, both with background antiretroviral therapy. DESIGN: Phase IIIB, randomized, open-label, parallel-group, multicenter study. SETTING: One hundred forty-six human immunodeficiency virus (HIV) clinics. PATIENTS: Six hundred eighty antiretroviral therapy-naïve patients with HIV type 1 RNA greater than 1000 copies/ml at baseline. INTERVENTION: Patients were randomly assigned in a 2:1 manner to receive either abacavir 600 mg-lamivudine 300 mg once/day or abacavir 300 mg twice/day and lamivudine 150 mg twice/day. Subjects were stratified based on choice of third or fourth antiretroviral drug (nucleoside reverse transcriptase inhibitor [NRTI], NNRTI, or protease inhibitor), assigned before randomization. MEASUREMENTS AND MAIN RESULTS: The primary end point was occurrence of grades 2-4 adverse events and serious adverse events; abacavir hypersensitivity reactions were considered serious adverse events. Baseline characteristics were similar between the once-daily (455 patients) and twice-daily (225 patients) groups. The rates of all grades 2-4 adverse events were similar: once-daily 33% (150 patients), twice-daily 31% (69). A slightly larger proportion of patients in the twice-daily group experienced drug-related grades 2-4 adverse events: once-daily 10% (47), twice-daily 16% (36). Rates of all serious adverse events (once-daily 11% [49], twice-daily 10% [22]) and drug-related serious adverse events (once-daily 5% [21], twice-daily 8% [17]) were similar. The rate of suspected abacavir hypersensitivity reaction was 5.3% (once-daily 4.4% [20], twice-daily 7.1% [16]), with a higher rate for the NNRTI stratum of the twice-daily group (8.6% [10]) than in any other stratum (once-daily, NNRTI 4.3% [10]; twice-daily, protease inhibitor 5.6% [6]; once-daily, protease inhibitor 4.6% [10]). CONCLUSION: In the short-term, the rates of adverse events in the once-daily and twice-daily groups appeared to be similar. The rate of suspected abacavir hypersensitivity reaction in the once-daily group was lower than the rate in the twice-daily group.

Identification of a second collagen-like glycoprotein produced by Bacillus anthracis and demonstration of associated spore-specific sugars.

Certain carbohydrates (rhamnose, 3-O-methyl rhamnose, and galactosamine) have been demonstrated to be present in Bacillus anthracis spores but absent in vegetative cells. Others have demonstrated that these spore-specific sugars are constituents of the glycoprotein BclA. In the current work, spore extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A second collagen-like glycoprotein, BclB, was identified in B. anthracis. The protein moiety of this glycoprotein was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and the carbohydrate components by gas chromatography-mass spectrometry and tandem mass spectrometry. Spore-specific sugars were also demonstrated to be components of BclB.

Large particles are responsible for elevated bacterial marker levels in school air upon occupation.

Muramic acid (Mur) is found in bacterial peptidoglycan (PG) whereas 3-hydroxy fatty acids (3-OH FAs) are found in Gram-negative bacterial lipopolysaccharide (LPS). Thus Mur and 3-OH FAs serve as markers to assess bacterial levels in indoor air. An initial survey, in a school, demonstrated that the levels of dust, PG and LPS (pmol m(-3)) were each much higher in occupied rooms than in the same rooms when unoccupied. In each instance, the Mur content of dust was increased and the hydroxy fatty acid distribution changed similarly suggesting an alteration in the bacterial population. Here, findings are compared with results from two additional schools. Follow-up aerosol monitoring by particle size was also performed for the first time for all 3 schools. The particle size distribution was shown to be quite different in occupied versus unoccupied schoolrooms. Within individual classrooms, concentrations of airborne particles [greater-than-or-equal]0.8 [micro sign]m in diameter, and CO(2) were correlated. This suggests that the increased levels of larger particles are responsible for elevation of bacterial markers during occupation. Release of culturable and non-culturable bacteria or bacterial aggregates from children (e.g. from flaking skin) might explain this phenomenon.

Amiloride peptide conjugates: prodrugs for sodium-proton exchange inhibition.

Inhibition of the sodium-proton exchanger (NHE) plays an important role in reducing tissue damage during ischemic reperfusion injury; however, pharmacological inhibitors of NHE have restricted access to acutely ischemic tissues because of severely compromised tissue perfusion. We describe the syntheses, characterization, and NHE inhibitory activities of a novel class of amiloride derivatives where peptides are conjugated to the amiloride C(5) amino group. These new peptide-C(5)-amiloride conjugates are inactive; however, peptide residues were chosen such that selective cleavage by neutral endopeptidase 24.11 (enkephalinase) liberates an amino acid-C(5)-amiloride conjugate that inhibits NHE in a glial cell line. These results confirm the feasibility of using peptide-amiloride conjugates as NHE inhibitor prodrugs. We envision the design of analogous peptide-amiloride prodrugs that can be administered prior to ischemic events and subsequently activated by endopeptidases selectively expressed by ischemic tissues.

Monomeric and polymeric gram-negative peptidoglycan but not purified LPS stimulate the Drosophila IMD pathway.

Insects depend solely upon innate immune responses to survive infection. These responses include the activation of extracellular protease cascades, leading to melanization and clotting, and intracellular signal transduction pathways inducing antimicrobial peptide gene expression. In Drosophila, the IMD pathway is required for antimicrobial gene expression in response to gram-negative bacteria. The exact molecular component(s) from these bacteria that activate the IMD pathway remain controversial. We found that highly purified LPS did not stimulate the IMD pathway. However, lipid A, the active portion of LPS in mammals, activated melanization in the silkworm Bombyx morii. On the other hand, the IMD pathway was remarkably sensitive to polymeric and monomeric gram-negative peptidoglycan. Recognition of peptidoglycan required the stem-peptide sequence specific to gram-negative peptidoglycan and the receptor PGRP-LC. Recognition of monomeric and polymeric peptidoglycan required different PGRP-LC splice isoforms, while lipid A recognition required an unidentified soluble factor in the hemolymph of Bombyx morii.

Evaluation of the methyl ester O-methyl acetate derivative of muramic acid for the determination of peptidoglycan in environmental samples by ion-trap GC-MS-MS.

Muramic acid (MA) is a unique amino sugar that is a constituent of the peptidoglycan (PG) present in prokaryotic cell walls. MA can serve as a marker for quantifying bacterial load, e.g. in indoor environments, by using gas chromatography-tandem mass spectrometry (GC-MS-MS). We demonstrated recently that the methyl ester O-methyl acetate (MMA) derivative can be used to detect MA in house dust by ion-trap GC-MS-MS. However, since the MMA derivative is not formed from free MA quantification was not optimal. Here we report 1) significant improvements in sample preparation of the MMA derivative and 2) an evaluation of the performance of derivative, using for comparison the alditol acetate derivative, the gold standard in quantitative trace analysis of MA in complex matrices. The MMA derivative was analysed using an MS instrument with internal ionization and the alditol acetate derivative was analysed using an instrument with external ionization. 13C-labelled cyanobacteria, containing MA in their PG, were used as the internal standard. A linear relationship was found between the two methods in studies on 27 parallel samples of airborne dust from school classes collected on filters. Although the analytical sensitivity of the MMA derivatives was somewhat slightly lower than of the alditol acetate derivative, this may be due to differences in yield of derivative, sample clean-up efficiency, or different performance of the GC columns or MS instruments. However preparation of the MMA derivative is quick and compatible with preparation of methyl esters of 3-hydroxy fatty acids (used as markers of Gram negative endotoxin) allowing the levels of both markers to be determined in the same dust sample. In conclusion, the MMA procedure can be used to determine MA in environmental samples with good reproducibility provided the concentration of the 13C-labelled MA internal standard in the cyanobacteria is first determined with an alternative method.

Perinecrotic glioma proliferation and metabolic profile within an intracerebral tumor xenograft.

Intracerebral C6 glioma xenografts spontaneously develop centrally located necrotic regions that are bordered by densely packed neoplastic cells. Proliferative and metabolic heterogeneity in these perinecrotic regions were assessed by bromodeoxyuridine (BrdU) incorporation, by immunocytological and by histochemical analyses. The borders of necrotic regions are comprised of glioma cells that express increased levels of the type 1 glucose transporter (GLUT-1) with rare cells having incorporated BrdU. By contrast, BrdU-positive glioma cells are located immediately adjacent to GLUT-1-positive cells bordering areas of necrosis. BrdU-positive glioma cells are also scattered throughout poorly vascularized, central regions of the tumor and are present at the highly vascularized tumor periphery. GLUT-1 expression increased considerably when C6 glioma cells were grown for 48 h under either the acidotic conditions of pH 6.8 or under hypoxic conditions. The perinecrotic GLUT-1-positive glioma cells in poorly vascularized, centrally located tumor regions demonstrated a 75% reduction in glycogen content and negligible glycogenolytic capacity, when compared with normal brain white matter. Cytochrome c oxidase (COX) and lactate dehydrogenase (LDH) maintained 50% enzymatic activity compared to controls, while succinate dehydrogenase (SDH) activity was 25% of control values. Based upon these findings, a metabolic model is proposed in which GLUT-1-positive perinecrotic cells are growth arrested and predominantly rely upon non-oxidative glycolysis. It is further postulated that BrdU-positive, GLUT-1-negative glioma cells within the poorly vascularized, central tumor region convert glucose-6-phosphate to nucleotide precursors for DNA replication.

Carbohydrates and glycoproteins of Bacillus anthracis and related bacilli: targets for biodetection.

The spore is the form released in a bioterrorism attack. There is a real need for definition of new targets for Bacillus anthracis that might be incorporated into emerging biodetection technologies. Particularly of interest are macromolecules found in B. anthracis that are (1) spore-specific, (2) readily accessible on the spore surface and (3) distinct from those present in related organisms. One of the few biochemical methods to identify the spores of B. anthracis is based on the presence of rhamnose and 3-O-methyl rhamnose as determined by gas chromatography-mass spectrometry. Related organisms additionally contain 2-O-methyl rhamnose and fucose. Carbohydrates and glycoproteins of the B. cereus group of organisms and the related B. subilis group are reviewed here. It is hypothesized that the spore-specific carbohydrate is a component of the newly described glycoprotein of the exosporium of B. anthracis. Further work to define the protein and carbohydrate components of the glycoprotein of B. anthracis could be highly useful in developing new technologies for rapid biodetection.