Min Equilibration Research Articles

The optimization of low volume-SPME method for volatilomics analysis of exhaled breath condensate

The analysis of breath can provide insight into the metabolic state of the body. However, the collection, transport, and storage of breath samples present certain challenges. The collection of exhaled breath condensate (EBC) emerges as a promising method for breath analysis. Despite this, the volume of EBC is relatively low, necessitating a sensitive method capable of extracting analytes from a small sample volumes. Therefore, this study aimed to develop and optimize a robust and green solid-phase extraction method for the analysing volatile organic compounds (VOCs) of various polarities in a low-volume EBC using a statistical design of experiment (DoE).Based on the screening experiments and DoE, the optimal conditions were determined to be 15 min equilibration time, and 20 min of extraction at 44 °C using DVB/CAR/PDMS fibre in direct immersion (DI) mode. The method was validated for linearity, precision and accuracy, yielding satisfactory results. The optimized method was applied to quantify the concentration of selected VOCs in real samples collected from a healthy subject, as well as after consumption of coffee and smoking of an electronic cigarette. Many of the selected compounds are associated with diseases and are challenging to detect in specimens from a healthy subject, but this method can measure them as potential disease markers.The proposed method enables the analysis of VOCs is very low-volume samples (200 µL), which is particularly important for studies involving body fluids. Notably, the applied approach demonstrated that different VOCs, representing various chemical classes and polarities, can be detected and quantified.

Nanocrystalline sodium mordenite as an efficient low-concentration CO2 adsorbent

Systematic control of the crystal size and morphology of zeolites to enhance their CO2 adsorption performance is of great importance from both environmental and economic perspectives. Here we report the seeded synthesis of sodium mordenite (Na-MOR) with a Si/Al ratio of 4.9 and an average crystal length and width of 70 and 40 nm, respectively, using γ-aminobutyric acid as a crystal growth inhibitor in the absence of any organic structure-directing agent (OSDA). This nanozeolite shows an unprecedented CO2 capacity of 1.58 mmol g−1 under dynamic direct air capture (DAC) conditions at 25 °C, which is approximately 40 % greater than the value (1.15 mmol g−1) of the best zeolite adsorbent (an unidentified OSDA-directed Na-MOR (Si/Al = 5.0) zeolite with Na+ enrichment in 8-ring side pockets), mainly due to its nanocrystalline nature. It has also been characterized by much faster CO2 adsorption kinetics (ca. 5 vs. 50 min equilibration time) compared to a commercial Na-MOR zeolite with Si/Al = 5.0. Our study paves the way for developing a more efficient zeolite-based DAC adsorbent.

Enteric and Systemic Postprandial Lactate Shuttle Phases and Dietary Carbohydrate Carbon Flow

We interrogated the hypothesis of a Postprandial Lactate Shuttle (PLS) in young, healthy men and women (n=15). Participants reported to the lab (5:00 AM) following an overnight fast and had the forearm vein catheterized for primed continuous infusions of [6,6-2H]glucose and [3-13C]lactate stable isotope tracers. The contralateral hand vein was warmed and catharized for arterialized blood sampling. After a 90-minute (min) equilibration period participants underwent a 75 g oral glucose tolerance test (OGTT). Arterialized [lactate] rose from baseline (0.60sup>). We estimate carbon flow from the OGTT from the rates of appearance of glucose, lactate and GNG from lactate. At 120-min the 75 g load was accounted for as follows: 29 g as blood lactate, 24 g of glucose from hepatic glucose release, 8 g of glucose from GNG, and 14 g withheld in the liver. Because blood [lactate] and Ra rose before [glucose] and Ra, evidence of an Enteric PLS is provided. Because secondary increments in blood [lactate] and Ra coincided with those of glucose, results are interpreted to indicate presence of a larger, secondary Systemic PLS phase. Antiquated ideas of lactate production due to hypoxia in skeletal muscles need to be supplanted because glycolysis proceeds to lactate in fully aerobic tissues, and because dietary carbohydrate is processed via lactate shuttling. In humans, lactate is a major vehicle for carbohydrate carbon distribution and metabolis. NIH grant R01 AG059715. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sulforaphane improves vasodilatory reactivity in diaphragm arterioles from rats with pulmonary hypertension

Pulmonary hypertension (PH) is a chronic, progressive disease characterized by pulmonary vascular remodeling, respiratory muscle and cardiac impairments, and exercise intolerance. Specifically, the diaphragm displays compromised contractile and vascular function, precluding it from meeting the increased metabolic demands of PH-induced chronic hyperpnea. Oxidative stress has been implicated in vascular dysfunction and PH pathology, therefore our objective was to target the antioxidant mediator Nrf2 (Nuclear factor erythroid 2-related factor 2) using sulforaphane (SFN) to determine whether this could improve vascular function in the diaphragm. We hypothesized that incubation with SFN would increase vasodilatory responsiveness in diaphragm arterioles from PH rats. Methods: Female Sprague-Dawley rats were administered monocrotaline (MCT; 50 mg/kg, i.p.) to induce PH. Disease progression was monitored via echocardiography. Once animals developed PH (~3 wks), the diaphragm was removed and first-order arterioles from the medial costal diaphragm were isolated, cannulated, and pressurized at 90 cmH2O. After a 60 min equilibration period and spontaneous tone development, endothelium-dependent and -independent vasorelaxation responses to cumulative doses of acetylcholine (ACh) and sodium nitroprusside (SNP) (10−9 to 10−4 M) were determined. These vessels were subsequently incubated with SFN (10 μM, 20 min), then ACh and SNP dose-response measurements were repeated. Results: All PH rats displayed morphometric and echocardiographic criteria for disease (i.e., right ventricular hypertrophy and reduced pulmonary artery acceleration time). Incubation with SFN improved endothelium-dependent and -independent vasodilation (p<0.05). Maximal vasodilatory responses with SFN were 14% and 16% higher for ACh (30±2 vs 34±3%; P=0.02) and SNP (52±2 vs 60±2%; P=0.03), respectively. However, vasodilatory reactivity remains substantially lower than that of healthy animals. Conclusions: These findings suggest that Nrf2 activation using SFN may improve vascular function in the diaphragm with PH. Future investigations will focus on chronic administration of SFN to PH rats to determine the therapeutic potential of this compound in vivo. The results of these studies provide insight into novel pharmacological strategies to improve diaphragm vascular and contractile function, with the potential to help mitigate dyspnea and exercise intolerance and enhance life quality for PH patients. Sustained Momentum for Investigators with Laboratories Established Grants (TIM & DCP); National Institute on Aging 1R15AG078060 (BJB & DCP); Ruth L. Kirschstein National Research Service Awards 1F31HL167618-01 (AGH) and 1F31HL170643-01 (KMS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cryopreservation of Yesso scallop (Patinopecten yessoensis) sperm

The germplasm of farmed Yesso scallop Patinopecten yessoensis in China has deteriorated since its introduction more than 40 years ago. The main aim of this study is to develop a sperm cryopreservation technique to assist the newly established program to manage this issue in China. This study investigated the factors important to the development of a non-programmable sperm cryopreservation technique in this species, including cryoprotectant agent [CPA; dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycine, sucrose, glucose and trehalose], equilibration time, rack height and thawing temperature. A low post-thaw sperm fertilization rate of 27.67 ± 2.52% was produced with the parameters optimized with the permeable CPAs (DMSO, PG and EG) only: 6% DMSO, 10 min equilibration time, 5 cm rack height and 30 °C thawing temperature. This rate was further improved to 44.00 ± 2.00% by adding 3% glucose into 6% DMSO. This addition had also improved the integrities of post-thaw sperm DNA, plasma membrane and acrosome, mitochondrial membrane potential and activities of some enzymes. Results from this study also showed that the post-thaw sperm morphology and ultrastructure were intensively compromised. In addition, the sperm agglutination found in the newly spawned sperm might be one of the phenomena resulted from the so-called germplasm deterioration, especially in the stock used in this study. The further exacerbation of agglutination after cryopreservation would also compromise the post-thaw sperm performance. The sperm cryopreservation technique established in this study would provide a better option to assist in managing the genetic diversity of farmed Yesso scallops in China.

EFFICIENT RECOVERY OF NICKEL (II) FROM A MIXTURE OF NICKEL (II) AND CHROMUIM (VI) IONS USING DUOLITE A7 RESIN

Separation of Ni(II) and Cr(VI) from a solution containing a mixture of both ions was conducted using adsorption technique with Duolite A7 resin as the adsorbent. The effects of ionic strength (1.0 - 5.0 M), resin dosage (1.0 - 5.0 g), temperature (30 - 50°C) and equilibration time (5 - 150 mins) on the separation of both ions were investigated. Up to 91% adsorption of Ni(II) by the resin was observed at pH 4.5. It was observed that Ni(II) ion under each of the conditions showed significantly higher binding affinity to the resin than Cr(VI). Adsorption of Ni(II) was rapid and was completed in about 10 min equilibration time. There was a continuous decrease in the adsorption of both ions as the temperature of the mixture increased. The FTIR spectra of the unloaded and loaded resinshowed that Duolite A7 is highly selective for the optimum recovery of Ni(II) from a solution containing a mixture of Ni(II) and Cr(VI) ions under the considered experimental conditions.

SAT064 Establishment And Initial Characterization Of Two Parallel Targeted Human ZnT8 Knock-in Murine Lines For Studying The Type 2 Diabetes Risk Variant At SNP rs13266634 Of The ZnT8 Gene

Abstract Disclosure: J.C. Wang: None. C. Adelma: None. M.G. Hayes: None. M. El Muayed: None. Background: Studies showed an association between R variant at position 325 in the β-cell zinc transporter ZnT8 and an increased risk for developing type 2 diabetes mellitus (T2DM). However, the mechanism by which this variant confers an increased risk for type 2 diabetes remains unclear. Methods: We generated a murine model for studying the effect of SNP rs13266634 on β-cell physiology. This model consists of two parallel murine lines in which conditional (RIP-Cre induced) expression constructs for the human variants of SLC30A8 (ZnT8), namely R (hZnT8R325, T2DM risk allele) and W (hZnT8W325, non-risk allele) of SNPrs13266634 were inserted into the rosa26 locus via targeted integration. β-cell specific expression of each of ZnT8R325 and ZnT8W325 was induced by cross breeding with a RIP-Cre line, expressing Cre recombinase in β-cells. All mouse lines were backcrossed into a C57BL/6N background null for the mouse variant of ZnT8. Islet zinc concentrations was measured by ICP-MS following hydrolysis in HNO3. Ex-vivo Glucose Stimulated Insulin Secretion Ratio (GSISR) was measured by incubation of isolated islets in 2.8 mM glucose, followed by 16.7 mM glucose minutes after an initial 60 min equilibration phase and calculating the insulin secretion at 16.7 mM/2.8 mM glucose. Data are reported as averages ± SEM from 3 independent experiments (n=3), each with 4 technical replicates. Results: Expression of both variants was verified by mRNA PCR from islets. Levels of expression in both lines were identical, owing to the targeted integration approach. Expression of each of hZnT8R325 and ZnT8W325 resulted in restoration of islet zinc concentration to levels similar to those found in normal human islets and wild type mouse islets. This confirms proper function of the hZnT8R325 transgenes in this newly established model. hZnT8R expressing islets showed a trend to lower insulin response to ex-vivo glucose stimulation compared to hZnT8W325 expressing islets (GSISR- 1.37 ± 0.41 vs 1.97 ± 0.32 respectively), as well as Cre expressing (GSISR 1.53 ± 0.40) or inactive construct controls (GSISR 2.43 ± 0.57). In-vivo glucose stimulated insulin secretion was not significantly different between hZnT8R325 expressing mice and those expressing hZnT8W325. As previously reported by others, all mice with β-cell Cre expression (RIP-Cre activated hZnT8 expressing lines and RIP-Cre controls) showed a lower insulin secretory response to glucose compared to non-Cre expressing mice (inactive construct controls). Conclusion: The newly created murine mouse model expresses functional human homologues of the ZnT8 variants hZnT8R325 and hZnT8W325 that result in restored beta cell zinc uptake. A trend to lower insulin secretory capacity was observed in ex-vivo glucose stimulated insulin secretion experiments. This model will be valuable tool to elucidate the functional consequences of the T2DM risk variant at SNP rs13266634. Presentation: Saturday, June 17, 2023

Effect of Different Plant Extracts on Microbial Population in The Frozen African Catfish (Clarias gariepinus) Semen.

Fish sperm cryopreservation has been attempted on roughly freshwater and marine species since 1953. This study sought to assess the potential of various plant extracts to function as natural antimicrobial agents in the frozen semen of African catfish (Clarias gariepinus). Diluted sperm was packaged in 0.25ml straws and left for 10min equilibration at 4°C. Following equilibration, the straws were exposed to liquid nitrogen vapor for 10 min and plunged into the liquid nitrogen (-196°C) and then thawed in a water bath at 35°C for 20s. Sperm samples were put into sterile 1.5 ml tubes immediately after thawing and the microbial count was detected with classical microbiological culture method. In the results of microbiological analyses, these tree plant extracts especially Echinacea purpurea were found highly effective for decreasing bacterial contamination levels of African catfish (C. gariepinus) semen. These plant extracts may have the potential for antibacterial effect, and they can be useful for the dilution of semen.

A recently developed minimum volume, absorbent, vitrification device, the Kitasato Vitrification System gives excellent outcomes for in vitro produced bovine blastocysts

Cryopreservation of embryos is a crucial component of current assisted reproductive technologies (ART). While the ART outcomes for many species have been greatly improved by the introduction of minimum volume vitrification devices, these devices can be difficult to handle and load. To reduce this problem, we recently developed a vitrification carrier which has a highly absorbent surface so that it simply and rapidly removes excess free vitrification solution from the specimen before the cooling step. This Kitasato Vitrification System (KVS) gives excellent results for human and mouse embryo vitrification. This study aimed to determine whether the KVS would also be effective for bovine blastocyst vitrification by comparing outcomes for the control device that was the KVS without excess vitrification solution absorber. The effect of varying the length of time spent in the first equilibration solution (0–10 min) was also evaluated. Vitrification with the KVS resulted in significantly higher survival and hatching rates than with the control device loaded with the same volume of vitrification solution (survival: 98.6% vs 87.6%, hatching at 72 h post warming: 87.3% vs 66.7%, respectively). The best outcomes were obtained with a 10 min equilibration step prior to exposure to the vitrification solution for 30 s. We also evaluated the effect of embryo quality on blastocyst viability when using the KVS. Survival rates of high- and low-quality embryos were comparable but low quality embryos had significantly lower hatching rates. Overall, the results indicate that the KVS vitrification device is effective for bovine blastocyst vitrification.

Analysis of the chemotactic response of ram spermatozoa to steroid hormones

Some substances in follicular fluid may be acting as sperm attractants, and steroid hormones progesterone (P4) and estradiol (E2), both secreted by the cumulus cells, are possible candidates. We have previously observed a chemotactic effect of the ewe follicular fluid in ram spermatozoa (data not published). The presence of progesterone and estrogen receptors in ram spermatozoa suggests the involvement of these hormones in the sperm functionality, including guided movement towards the oocyte. P4 acts as a chemoattractant in human and rabbit sperm, but there is no information on its effect, or E2 effect, on ram spermatozoa. The objective of this work was to study the putative chemotactic action of P4 and E2 on ram spermatozoa, analyzing the influence of the sperm capacitation state. Swim-up selected spermatozoa from nine Rasa Aragonesa rams were incubated without (cap-TALP) or with cAMP-elevating agents (cap-Cocktail) to induce in vitro capacitation. After evaluating the sperm quality parameters in non- capacitated and incubated samples (motility, viability, capacitation state), IBIDI® µ-Slide Chemotaxis chamber (Ibidi GmbH) was loaded with swim-up, cap-TALP or cap-Cocktail sperm samples according to the manufacturer’s instructions. P4 or E2 were added to the chambers at 100 pM or 1 µM concentration each. Medium with 0.01% dimethyl sulfoxide (DMSO) was used as control treatment. After 5 min of equilibration, 200 fps videos were recorded for 3 sec with a video camera mounted on a microscope. Sperm trajectories and chemotaxis were analyzed with the OpenCASA software (Alquezar-Baeta et al., PLoS Comput Biol, 15(1), 2019), which calculates a chemotaxis index. Differences were analysed by χ² test, except for the chemotaxis index (ANOVA) using GraphPad PRISM® (versión 8.0.1). A chemotactic response was only observed in capacitated spermatozoa, as we previously described in presence of follicular fluid. P4 at both concentrations had a chemotactic effect on capacitated ram spermatozoa. However, E2 had no effect on any of the studied samples. These results reveal, for the first time, the chemotactic effect of P4 in ram spermatozoa. Grants: AGL-2017-83799-R and DGA A07-17R. S.M.-J. has a predoctoral contract from the Diputación General de Aragón (DGA).

Abstract 15591: Acute Dantrolene Treatment Reverses Arrhythmogenic Substrate and Reduces Triggers Induced by Ryanodine Receptor 2 Hyperactivity in Human Hearts

Introduction: Ryanodine receptor 2 (RyR2) hyperactivity is commonly observed in structural heart diseases that are a result of ischemia or heart failure. It results in abnormal calcium handling and calcium leak that can trigger arrhythmias. Dantrolene, a RyR inhibitor, is used to prevent calcium leak from RyR1 in skeletal muscles. Hypothesis: Suppressing RyR2 hyperactivity with acute dantrolene treatment can reduce arrhythmia burden in human hearts. Methods: Human donor hearts that were not used in transplantation were obtained and 1 cm 3 cubes were isolated from the left ventricle. Slices at 400 μm thickness were generated from these cubes, pinned and perfused with Tyrode’s solution in a recording tissue chamber. Pseudo-ECGs were recorded with ECG electrodes and the slices were optically mapped to measure conduction velocity (CV), action potential duration (APD) and calcium transient duration (CaTD). After an initial 30 min equilibration period, baseline optical recordings were obtained. Slices were then treated with 1) Isoproterenol (Iso, 250 nM) to stimulate β-adrenergic receptors, 2) Caffeine (200 μM) for RyR2 receptor activation and 3) Dantrolene (10 μM), sequentially. Dual optical recordings, Vm and Ca, were obtained after each treatment. Results: Substrate: Iso reduced APD (425±36 vs 394±43, p=0.03), as expected, and addition of Caffeine restored APD to baseline. More importantly, addition of Dantrolene prolonged APD even further (460±90, p=0.04). CaTD was also similarly reduced by Iso (445±48 vs 387±25, p=0.04) and restored to baseline by Caffeine+Iso. No significant changes in CV were measured under any condition. Trigger: In slices where PVCs were inducible, Caffeine+Iso increased occurrence of PVCs (20 vs 53 PVCs/min) while addition of Dantrolene reduced PVC incidence (18 PVCs/min). Conclusions: Dantrolene treatment, acutely reversed proarrhythmogenic substrate and reduced triggers and could therefore be a suitable approach for anti-arrhythmic therapy in patients with structural heart disease. Additionally, human LV slice preparation is useful in antiarrhythmic drug studies.

Rapid Removal of Cr(VI) from Aqueous Solution Using Polycationic/Di-Metallic Adsorbent Synthesized Using Fe3+/Al3+ Recovered from Real Acid Mine Drainage

The mining of valuable minerals from wastewater streams is attractive as it promotes a circular economy, wastewater beneficiation, and valorisation. To this end, the current study evaluated the rapid removal of aqueous Cr(VI) by polycationic/di-metallic Fe/Al (PDFe/Al) adsorbent recovered from real acid mine drainage (AMD). Optimal conditions for Cr(VI) removal were 50 mg/L initial Cr(VI), 3 g PDFe/Al, initial pH = 3, 180 min equilibration time and temperature = 45 °C. Optimal conditions resulted in ≥95% removal of Cr(VI), and a maximum adsorption capacity of Q = 6.90 mg/g. Adsorption kinetics followed a two-phase pseudo-first-order behaviour, i.e., a fast initial Cr(VI) removal (likely due to fast initial adsorption) followed by a slower secondary Cr(VI) removal (likely from Cr(VI) to Cr(III) reduction on the surface). More than 90% of adsorbed Cr(VI) could be recovered after five adsorption–desorption cycles. A reaction mechanism involving a rapid adsorption onto at least two distinct surfaces followed by slower in situ Cr(VI) reduction, as well as adsorption-induced internal surface strains and consequent internal surface area magnification, was proposed. This study demonstrated a rapid, effective, and economical application of PDFe/Al recovered from bona fide AMD to treat Cr(VI)-contaminated wastewater.

Humidity and measurement of volatile propofol using MCC-IMS (EDMON)

The bedside Exhaled Drug MONitor – EDMON measures exhaled propofol in ppbv every minute based on multi-capillary column – ion mobility spectrometry (MCC-IMS). The MCC pre-separates gas samples, thereby reducing the influence of the high humidity in human breath. However, preliminary analyses identified substantial measurement deviations between dry and humid calibration standards. We therefore performed an analytical validation of the EDMON to evaluate the influence of humidity on measurement performance. A calibration gas generator was used to generate gaseous propofol standards measured by an EDMON device to assess linearity, precision, carry-over, resolution, and the influence of different levels of humidity at 100% and 1.7% (without additional) relative humidity (reference temperature: 37°C). EDMON measurements were roughly half the actual concentration without additional humidity and roughly halved again at 100% relative humidity. Standard concentrations and EDMON values correlated linearly at 100% relative humidity (R²=0.97). The measured values were stable over 100min with a variance ≤ 10% in over 96% of the measurements. Carry-over effects were low with 5% at 100% relative humidity after 5min of equilibration. EDMON measurement resolution at 100% relative humidity was 0.4 and 0.6 ppbv for standard concentrations of 3 ppbv and 41 ppbv. The influence of humidity on measurement performance was best described by a second-order polynomial function (R²≥0.99) with influence reaching a maximum at about 70% relative humidity. We conclude that EDMON measurements are strongly influenced by humidity and should therefore be corrected for sample humidity to obtain accurate estimates of exhaled propofol concentrations.

Arterial to end-tidal CO2 gradients during isocapnic hyperventilation.

Isocapnic hyperventilation (ICHV) is occasionally used to maintain the end-expired CO2 partial pressure (PETCO2) when the inspired CO2 (PICO2) rises. Whether maintaining PETCO2 with ICHV during an increase of the PICO2 also maintains arterial PCO2 (PaCO2) remains poorly documented. 12 ASA PS I-II subjects undergoing a robot-assisted radical prostatectomy (RARP) (n = 11) or cystectomy (n = 1) under general endotracheal anesthesia with sevoflurane in O2/air (40% inspired O2) were enrolled. PICO2 was sequentially increased from 0 to 0.5, 1.0, 1.5 and 2% by adding CO2 to the inspiratory limb of the circle system, while increasing ventilation to a target PETCO2 of 4.7-4.9% by adjusting respiratory rate during controlled mechanical ventilation. Pa-ETCO2 gradients were determined after a 15min equilibration period at each PICO2 level and compared using ANOVA. Mean (standard deviation) age, height, and weight were 66 (6) years, 171 (6) cm, and 75 (8) kg, respectively. Capnograms were normal and hemodynamic parameters remained stable. PETCO2 could be maintained within 4.7-4.9% in all subjects at all times except in 1 subject with 1.5% PICO2 and 5 subjects with 2.0% PICO2; data from the one subject in whom both 1.5 and 2.0% PICO2 resulted in PETCO2 > 5.1% were excluded from analysis. Pa-ETCO2 gradients did not change when PICO2 increased. The effect of a modest rise of PICO2 up to 1.5% on PETCO2 during RARP can be readily overcome by increasing ventilation without altering the Pa-ETCO2 gradients. At higher PICO2, airway pressures may become a limiting factor, which requires further study.

Microscale Purification with Direct Charged Aerosol Detector Quantitation Using Selective Online One- or Two-Dimensional Liquid Chromatography.

The pharmaceutical industry is increasingly faced with challenging separations of complex crude reaction mixtures at the microscale that require the adoption of new platforms for rapid target isolation, impurity determination, and quantitation. In this study, we describe an online microscale one- or two-dimensional liquid chromatography (1D/2D-LC) system with heart-cutting and multi (triple) detector triggering in either dimension to address this need. The advantages of charged aerosol detection (CAD) are discussed for the direct quantitation of limited quantity samples, without utilizing a second analytical instrument or gradient compensation pump. In addition to the significant time and cost savings, there is no minimum recovery requirement that exists when compared to gravimetric methods for accurate microscale quantitation. This platform has been successfully used to purify 0.5-5.0 mg scale reactions in 96- or 384-well reaction plates with a gradient time of 4 min per sample. Separations performed in both dimensions are complete in less than 12 min, including trapping and column equilibration time. The isolated arrays of small-quantity investigational compounds at a high purity enable rapid exploration of chemical reaction parameters and synthetic route scouting for biological target validation.

Monocrotaline‐induced Pulmonary Hypertension Impairs Diaphragm Vasomotor Function

INTRODUCTIONPulmonary hypertension (PH) is a chronic and progressive disease characterized by high arterial blood pressure and compensatory vascular remodeling in the pulmonary circulation. Mounting evidence suggests that the effects of PH extend beyond the pulmonary circulation. In humans and animals, PH induces diaphragm contractile dysfunction and diaphragm muscle atrophy. While the mechanistic bases for the PH‐induced diaphragm dysfunction is relatively unknown, alterations in the vasomotor control of the diaphragm resistance vasculature (i.e. arterioles) may play a key role. Therefore, we tested the hypothesis that PH increases the vasoconstrictor responsiveness and impairs the vasorelaxation of diaphragm arterioles.METHODSAdult female Sprague‐Dawley rats (n=8) were divided into healthy control (HC; n=2), ovariectomized (OVX; n=3), and PH + OVX (n=3). PH + OVX animals were given a single intraperitoneal injection of monocrotaline (MCT; 50mg/kg) to induce PH. Disease progression was then monitored via echocardiography. Once animals developed PH (~3 weeks post injection), the diaphragm was removed and first‐order arterioles (1A) from the medial costal diaphragm were isolated, cannulated, and pressurized at 90 cmH2O. After a 60 min equilibration period and spontaneous tone development, vasoconstrictor responses to potassium chloride (KCl) (10‐100 mM) and endothelium‐dependent and ‐independent vasorelaxation responses to cumulative doses of acetylcholine (ACh) and sodium nitroprusside (SNP) (10‐9 to 10‐4 M) were determined.RESULTSSpontaneous tone (%) development was not different across all three groups (HC: 36 ± 9%; OVX: 33 ± 9%; PH + OVX: 39 ± 2%; P>0.05). Compared to control vessels, maximal percent vasoconstriction with KCl was decreased with OVX (67 ± 2 versus 50 ± 1%; P<0.05). Maximal vasoconstriction (%) was reduced with PH + OVX (36 ± 4%) compared to vessels from both healthy control and OVX groups (P<0.05). Ovariectomy had no effect on endothelium‐dependent vasodilation responses to ACh versus intact control animals (P>0.05). The maximal ACh‐mediated vasodilation with PH + OVX (29 ± 9%) was decreased versus both healthy (45 ± 6%; P<0.05) and OVX animals (41 ± 4%; P<0.05). Across all three groups, there were no differences in percent relaxation with SNP (P>0.05).CONCLUSIONThese findings suggest that estrogen plays a role in the maintenance of K+ channel activity and/or the contribution of K+ channels to diaphragm vasomotor tone, and that PH further disrupts K+ channel function in diaphragm arterioles. In addition, the absence of estrogen in OVX animals had no effect on endothelial function whereas PH + OVX impaired endothelium‐dependent vasorelaxation of diaphragm resistance vessels. These findings suggest that PH, at least in the rodent model utilized herein, elicits endothelial dysfunction in the diaphragm vasculature and provide a potential mechanism for the impaired diaphragm contractile dysfunction in PH patients.

Freezing Protocol Optimization for Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm under Field Conditions.

Simple SummaryThe germplasm banks of wild species, such as Iberian red deer, are not widespread, mainly due to the difficulties of collecting and cryopreserving reproductive cells. Optimal freezing protocols under field conditions could be a breakthrough for these species. In this study, epididymal sperm was evaluated using two methods of sperm storage during refrigeration (tube and straw); four equilibration periods (0, 30, 60, and 120 min); and four methods of freezing (cryopreservation in liquid nitrogen vapors in a tank (control) or box, freezing in dry ice, or freezing over a metallic plate). The results showed that samples stored in straws during refrigeration produced less apoptotic spermatozoa and more viable spermatozoa withactive mitochondria. A long equilibration period (120 min) yielded a higher percentage of acrosomal integrity. Moreover, there was no difference in sperm quality between freezing in liquid nitrogen vapors in a tank or box. However, a worse quality was obtained when the samples were cryopreserved in dry ice or over a metallic plate compared to the control. Creating germplasm banks of wild species, such as the Iberian red Deer (Cervus elaphus hispanicus) can be challenging. One of the main difficulties is the obtention and cryopreservation of good-quality reproductive cells when the spermatozoa are obtained from epididymides after death. To avoid a loss of seminal quality during transport, developing alternative methods for cooling and freezing sperm samples under field conditions is necessary. The objective of this study was to evaluate the effects of different durations of equilibrium and different techniques of cooling and freezing on Iberian red deer epididymal sperm quality after thawing to optimize the processing conditions in this species. Three experiments were carried out: (I) evaluation of refrigeration in straws or tubes of 15 mL; (II) study of equilibration period (0, 30, 60, or 120 min); and (III) comparison of four freezing techniques (liquid nitrogen vapor in a tank (C), liquid nitrogen vapor in a polystyrene box (B), dry ice (DY), and placing straws on a solid metallic plate floating on the surface of liquid nitrogen (MP)). For all experiments, sperm motility and kinematic parameters, acrosomal integrity, sperm viability, mitochondrial membrane potential, and DNA integrity were evaluated after thawing. All statistical analyses were performed by GLM-ANOVA analysis. Samples refrigerated in straws showed higher values (p ≤ 0.05) for mitochondrial activity and lower values (p ≤ 0.05) for apoptotic cells. Moreover, the acrosome integrity showed significant differences (p ≤ 0.05) between 0 and 120 min, but not between 30 and 60 min, of equilibration. Finally, no significant differences were found between freezing in liquid nitrogen vapors in a tank or in a box, although there was a low quality after thawing when the samples were cryopreserved in dry ice or by placing straws on a solid metallic plate floating on the surface of liquid nitrogen. In conclusion, under field conditions, it would be possible to refrigerate the sperm samples by storing them in straws with a 120 min equilibration period and freezing them in liquid nitrogen vapors in a tank or box.

Solvent Extraction and Separation of Silver(I) in Real Sample Using Hexaacetato Calix[6]arene

Abstract: In this study, silver(I) was extracted at pH 11.5 into organic phase using 0.0001 M hexa-p-t-butyl-hexaacetato calix[6]arene solution in xylene after a five min equilibration period. Silver(I) was back extracted with 1 M HNO3 and determined photometrically. Log-log plot gives 1:1 stoichiometry of extracted species indicating that one ligand reacts with one mole of silver(I). The values of ∆G, ∆H and ∆S were determined by studying the extraction equilibrium at different temperatures. Quantitative separation of binary and synthetic mixtures of silver from associate elements was effectively carried out. The extraction and separation of silver(I) from real and multicomponent samples were also studied. The RSD was ≤0.5%. Keywords: Solvent extraction, Acetyl derivative, Calix[6]arene, Separation, Silver(I).

Separation of platinum group metals from model chloride solution using phosphonium-based ionic liquid

• Liquid-liquid separation of PGMs’ from HCl medium using Cyphos IL 101 was studied. • Increasing acid and total Cl – ion concentration inhibited the extraction efficiency. • Poorly extracted Rh(III) efficiently scrubbed from organic phase in high acid solution. • Outer sphere complexation of [PtCl 6 ] 2− and [PdCl 4 ] 2− with ionic liquid was observed. • Quantitative stripping of Pt(IV) with NaSCN and Pd(II) with CH 4 N 2 S was achieved. The present study dealt with liquid–liquid separation of PGMs (Pt, Pd, and Rh) from a model chloride-based leach solution using an undiluted phosphonium-based ionic liquid, Cyphos IL 101. Based on the parametric optimization studies, 30 min equilibration of both phases at an organic-to-aqueous volume ratio of 1 showed the highest PGMs’ extraction of > 99% Pt(IV) and Pd(II), and ~ 22% Rh(III). Depending upon the HCl and total Cl − ions concentrations, species like [PtCl 6 ] 2− and [PdCl 4 ] 2− exhibited remarkable ability for ion-pairing with Cyphos IL 101 unlike the higher charge density of [RhCl 6 ] 3− which significantly lowered down to be < 5% at an aqueous feed of 0.1 mol.L −1 HCl. Thermodynamic properties with negative changes in enthalpy and entropy indicated that the PGMs’ extraction process exhibit exothermic behaviour with the formation of outer -sphere complexes in the ionic liquid phase. With the supports of experimental results and spectral analysis, the possible structure of Pt(IV)-Pd(II)-loaded ionic liquid could also be revealed. The poorly extracted Rh(III) was efficiently scrubbed by contacting with 5.0 mol.L −1 HCl solution, leaving the organic phase free from Rh(III). The selectivity in back-extraction of Pt(IV) and Pd(II) was achieved by first contacting the loaded ionic liquid with 1 × 10 −1 mol.L −1 NaSCN in two stages, which exhibiting ~ 99% efficacy of Pt(II) stripping. Subsequently, Pt(IV)-depleted organic phase was stripped with 1 × 10 −2 mol.L −1 CH 4 N 2 S that yielded > 99% Pd(II) recovery in a single contact. Results of extraction-stripping cycles that were showing similar efficiency of PGMs extractability clearly indicated the stability and reusability of ionic liquid.

Is catalase an effective additive to alleviate oxidative stress during cryopreservation of zebrafish sperm at the repository level?

The goal of this study was to investigate whether supplementation of cryoprotective medium with catalase (CAT), an antioxidation enzyme, is efficient for zebrafish sperm cryopreservation from the viewpoint of high-throughput genetic repository operations. Three cryoprotectants (10%, v/v), dimethylacetamide (DMA), dimethylformamide (DMF), and methanol were used. The objectives were to evaluate the effects of CAT on sperm motility, plasma membrane integrity, and concentration for: 1) fresh sperm at equilibration up to 60 min; 2) post-thaw sperm after cooling at 10, 20, and 40 °C/min), and 3) post-thaw fertilization and embryo survival rates. Catalase addition did not improve sperm motility, regardless of the cryoprotectants added. After 10-min exposure to DMA or methanol, membrane integrity was significantly decreased (70–75%) compared to controls. With catalase, sperm cells maintained membrane integrity and after 50 min equilibration, cell concentrations were maintained with CAT compared to cryoprotectant-only test groups. However, after cryopreservation and thawing, CAT did not affect the outcome of motility, membrane integrity, cell concentration, fertilization, or embryo survival assays. Analysis of cooling rates also indicated that CAT did not affect 3-hpf fertilization or 24-hpf survival rates. Overall, addition of CAT could provide some protection of sperm from oxidative stress before freezing, but not after thawing. We propose that decisions concerning routine use of CAT for repositories, especially those handling tens of thousands of frozen samples per year, would depend on whether efficient high-throughput operation, or specific research questions are programmatic goals.