Purification and total acid number reduction of raw diesel via column chromatography

1 online resource (vii, 49 p.) : illustrations (chiefly color)Includes abstract.Includes bibliographical references (p. 47-49).Crude petroleum products have impurities that are harmful to industrial equipment, vehicles, and the environment. The American Society of Testing and Materials (ASTM) is a regulatory body that controls the standards for different classes of fuel. These regulations can include acidity, sulfur content, flash point, and many more parameters. ASTM has different standards depending on the type of fuel. For example, diesel that is used in vehicles that are driven on streets is known as road going diesel and has the designated class of ASTM D975. In this research, the standards for ASTM D975 will be used. An unrefined diesel product provided by an industrial collaborator will be upgraded to reach the ASTM standards for acidity and sulfur content. Also, the industrial collaborator asked us to improve the colour and smell of the diesel. Acidity is measured by the total acid number (TAN) with the units of mg of KOH per g of diesel. The TAN value was lowered through acid-base neutralization using different conditions. These conditions include various temperatures, solid KOH, and different concentrations of aqueous KOH solutions with or without a phase transfer catalyst (PTC). A novel technique of using column chromatography was developed to purify the diesel. This technique greatly improved the colour and smell, with colour ranging from an initial brown colour to green or colourless. The odour could be converted to a petroleum like odour from a strong initial camp fire burning aroma. The column was optimized to be run without solvent and the stationary phase recycled. The column can purify up to 800 mL of diesel per 100 g of silica gel and has a recovery of 70%. The TAN value was also reduced to less than 0.03 mg of KOH per gram of diesel using this method of purification

Depolarization of sperm membrane potential is a common feature of men with subfertility and is associated with low fertilization rate at IVF

STUDY QUESTION. Are significant abnormalities in outward (K+) conductance and resting membrane potential (Vm) present in the spermatozoa of patients undertaking IVF and ICSI and if so, what is their functional effect on fertilization success? SUMMARY ANSWER. Negligible outward conductance (≈5% of patients) or an enhanced inward conductance (≈4% of patients), both of which caused depolarization of Vm, were associated with a low rate of fertilization following IVF. WHAT IS KNOWN ALREADY. Sperm-specific potassium channel knockout mice are infertile with defects in sperm function, suggesting that these channels are essential for fertility. These observations suggest that malfunction of K+ channels in human spermatozoa might contribute significantly to the occurrence of subfertility in men. However, remarkably little is known of the nature of K+ channels in human spermatozoa or the incidence and functional consequences of K+ channel defects. STUDY DESIGN, SIZE AND DURATION. Spermatozoa were obtained from healthy volunteer research donors and subfertile IVF and ICSI patients attending a hospital assisted reproductive techniques clinic between May 2013 and December 2015. In total, 40 IVF patients, 41 ICSI patients and 26 normozoospermic donors took part in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS. Samples were examined using electrophysiology (whole-cell patch clamping). Where abnormal electrophysiological characteristics were identified, spermatozoa were further examined for Ca2+ influx induced by progesterone and penetration into viscous media if sufficient sample was available. Full exome sequencing was performed to specifically evaluate potassium calcium-activated channel subfamily M α 1 (KCNMA1), potassium calcium-activated channel subfamily U member 1 (KCNU1) and leucine-rich repeat containing 52 (LRRC52) genes and others associated with K+ signalling. In IVF patients, comparison with fertilization rates was done to assess the functional significance of the electrophysiological abnormalities. MAIN RESULTS AND THE ROLE OF CHANCE. Patch clamp electrophysiology was used to assess outward (K+) conductance and resting membrane potential (Vm) and signalling/motility assays were used to assess functional characteristics of sperm from IVF and ICSI patient samples. The mean Vm and outward membrane conductance in sperm from IVF and ICSI patients were not significantly different from those of control (donor) sperm prepared under the same conditions, but variation between individuals was significantly greater (P< 0.02) with a large number of outliers (>25%). In particular, in ≈10% of patients (7/81), we observed either a negligible outward conductance (4 patients) or an enhanced inward current (3 patients), both of which caused depolarization of Vm. Analysis of clinical data from the IVF patients showed significant association of depolarized Vm (≥0 mV) with low fertilization rate (P= 0.012). Spermatozoa with electrophysiological abnormities (conductance and Vm) responded normally to progesterone with elevation of [Ca2+]i and penetration of viscous medium, indicating retention of cation channel of sperm (CatSper) channel function. LIMITATIONS, REASONS FOR CAUTION. For practical, technical, ethical and logistical reasons, we could not obtain sufficient additional semen samples from men with conductance abnormalities to establish the cause of the conductance defects. Full exome sequencing was only available in two men with conductance defects. WIDER IMPLICATIONS OF THE FINDINGS. These data add significantly to the understanding of the role of ion channels in human sperm function and its impact on male fertility. Impaired potassium channel conductance (Gm) and/or Vm regulation is both common and complex in human spermatozoa and importantly is associated with impaired fertilization capacity when the Vm of cells is completely depolarized

Specific loss of CatSper function is sufficient to compromise fertilizing capacity of human spermatozoa

STUDY QUESTION Are significant abnormalities of CatSper function present in IVF patients with normal sperm concentration and motility and if so what is their functional significance for fertilization success?SUMMARY ANSWER Sperm with a near absence of CatSper current failed to respond to activation of CatSper by progesterone and there was fertilization failure at IVF.WHAT IS KNOWN ALREADY In human spermatozoa, Ca2+ influx induced by progesterone is mediated by CatSper, a sperm-specific Ca2+ channel. A suboptimal Ca2+ influx is significantly associated with, and more prevalent in, men with abnormal semen parameters, and is associated with reduced fertilizing capacity. However, abnormalities in CatSper current can only be assessed directly using electrophysiology. There is only one report of a CatSper-deficient man who showed no progesterone potentiated CatSper current. A CatSper 2 genetic abnormality was present but there was no information on the [Ca2+]i response to CatSper activation by progesterone. Additionally, the semen samples had indicating significant abnormalities (oligoasthenoteratozoospermia) multiple suboptimal functional responses in the spermatozoon. As such it cannot be concluded that impaired CatSper function alone causes infertility or that CatSper blockade is a potential safe target for contraception.STUDY DESIGN, SIZE, DURATION Spermatozoa were obtained from donors and subfertile IVF patients attending a hospital assisted reproductive techniques clinic between January 2013 and December 2014. In total 134 IVF patients, 28 normozoospermic donors and 10 patients recalled due to a history of failed/low fertilization at IVF took part in the study.PARTICIPANTS/MATERIALS, SETTING, METHODS Samples were primarily screened using the Ca2+ influx induced by progesterone and, if cell number was sufficient, samples were also assessed by hyperactivation and penetration into viscous media. A defective Ca2+ response to progesterone was defined using the 99% confidence interval from the distribution of response amplitudes in normozoospermic donors. Samples showing a defective Ca2+ response were further examined in order to characterize the potential CatSper abnormalities. In men where there was a consistent and robust failure of calcium signalling, a direct assessment of CatSper function was performed using electrophysiology (patch clamping), and a blood sample was obtained for genetic analysis.MAIN RESULTS AND THE ROLE OF CHANCE A total of 101/102 (99%) IVF patients and 22/23 (96%) donors exhibited a normal Ca2+ response. The mean (±SD) normalized peak response did not differ between donors and IVF patients (2.57 ± 0.68 [n = 34 ejaculates from 23 different donors] versus 2.66 ± 0.68 [n = 102 IVF patients], P = 0.63). In recall patients, 9/10 (90%) showed a normal Ca2+ response. Three men were initially identified with a defective Ca2+ influx. However, only one (Patient 1) had a defective response in repeat semen samples. Electrophysiology experiments on sperm from Patient 1 showed a near absence of CatSper current and exon screening demonstrated no mutations in the coding regions of the CatSper complex. There was no increase in penetration of viscous media when the spermatozoa were stimulated with progesterone and importantly there was failed fertilization at IVF.LIMITATIONS, REASONS FOR CAUTION A key limitation relates to working with a specific functional parameter (Ca2+ influx induced by progesterone) in fresh sperm samples from donors and patients that have limited viability. Therefore, for practical, technical and logistical reasons, some men (∼22% of IVF patients) could not be screened. As such the incidence of significant Ca2+ abnormalities induced by progesterone may be higher than the ∼1% observed here. Additionally, we used a strict definition of a defective Ca2+ influx such that only substantial abnormalities were selected for further study. Furthermore, electrophysiology was only performed on one patient with a robust and repeatable defective calcium response. This man had negligible CatSper current but more subtle abnormalities (e.g. currents present but significantly smaller) may have been present in men with either normal or below normal Ca2+ influx.WIDER IMPLICATIONS OF THE FINDINGS These data add significantly to the understanding of the role of CatSper in human sperm function and its impact on male fertility. Remarkably, these findings provide the first direct evidence that CatSper is a suitable and specific target for human male contraception

Application of the pMHC array to characterise tumour antigen specific T cell populations in leukaemia patients at disease diagnosis

Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms’ Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8–1.4 x 106). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1126-134 (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1950-958 epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients

Clinically relevant enhancement of human sperm motility using compounds with reported phosphodiesterase inhibitor activity

STUDY QUESTION: Can we identify compound(s) with reported phosphodiesterase inhibitor (PDEI) activity that could be added to human spermatozoa in vitro to enhance their motility without compromising other sperm functions? SUMMARY ANSWER: We have identified several compounds that produce robust and effective stimulation of sperm motility and, importantly, have a positive response on patient samples. WHAT IS KNOWN ALREADY: For >20 years, the use of non-selective PDEIs, such as pentoxifylline, has been known to influence the motility of human spermatozoa; however, conflicting results have been obtained. It is now clear that human sperm express several different phosphodiesterases and these are compartmentalized at different regions of the cells. By using type-specific PDEIs, differential modulation of sperm motility may be achieved without adversely affecting other functions such as the acrosome reaction (AR). STUDY DESIGN, SIZE, DURATION: This was a basic medical research study examining sperm samples from normozoospermic donors and subfertile patients attending the Assisted Conception Unit (ACU), Ninewells Hospital Dundee for diagnostic semen analysis, IVF and ICSI. Phase 1 screened 43 commercially available compounds with reported PDEI activity to identify lead compounds that stimulate sperm motility. Samples were exposed (20 min) to three concentrations (1, 10 and 100 µM) of compound, and selected candidates (n = 6) progressed to Phase 2, which provided a more comprehensive assessment using a battery of in vitro sperm function tests.  PARTICIPANTS/MATERIALS, SETTING, METHODS: All healthy donors and subfertile patients were recruited at the Medical Research Institute, University of Dundee and ACU, Ninewells Hospital Dundee (ethical approval 08/S1402/6). In Phase 1, poor motility cells recovered from the 40% interface of the discontinuous density gradient were used as surrogates for patient samples. Pooled samples from three to four different donors were utilized in order to reduce variability and increase the number of cells available for simultaneous examination of multiple compounds. During Phase 2 testing, semen samples from 23 patients attending for either routine diagnostic andrology assessment or IVF/ICSI were prepared and exposed to selected compounds. Additionally, 48 aliquots of prepared samples, surplus to clinical use, were examined from IVF (n = 32) and ICSI (n = 16) patients to further determine the effects of selected compounds under clinical conditions of treatment. Effects of compounds on sperm motility were assessed by computer-assisted sperm analysis. A modified Kremer test using methyl cellulose was used to assess sperm functional ability to penetrate into viscous media. Sperm acrosome integrity and induction of apoptosis were assessed using the acrosomal content marker PSA-FITC and annexin V kit, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: In Phase 1, six compounds were found to have a strong effect on poor motility samples with a magnitude of response of ≥60% increase in percentage total motility. Under capacitating and non-capacitating conditions, these compounds significantly (P ≤ 0.05) increased the percentage of total and progressive motility. Furthermore, these compounds enhanced penetration into a cervical mucus substitute (P ≤ 0.05). Finally, the AR was not significantly induced and these compounds did not significantly increase the externalization of phosphatidylserine (P = 0.6, respectively). In general, the six compounds maintained the stimulation of motility over long periods of time (180 min) and their effects were still observed after their removal. In examinations of clinical samples, there was a general observation of a more significant stimulation of sperm motility in samples with lower baseline motility. In ICSI samples, compounds #26, #37 and #38 were the most effective at significantly increasing total motility (88, 81 and 79% of samples, respectively) and progressive motility (94, 93 and 81% of samples, respectively). In conclusion, using a two-phased drug discovery screening approach including the examination of clinical samples, 3/43 compounds were identified as promising candidates for further study. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study and caution must be taken when extrapolating the results. Data for patients were from one assessment and thus the robustness of responses needs to be established. The n values for ICSI samples were relatively small. WIDER IMPLICATIONS OF THE FINDINGS: We have systematically screened and identified several compounds that have robust and effective stimulation (i.e. functional significance with longevity and no toxicity) of total and progressive motility under clinical conditions of treatment. These compounds could be clinical candidates with possibilities in terms of assisted reproductive technology options for current or future patients affected by asthenozoospermia or oligoasthenozoospermia

Quick, accurate, smart: 3D computer vision technology helps assessing confined animals' behaviour

Mankind directly controls the environment and lifestyles of several domestic species for purposes ranging from production and research to conservation and companionship. These environments and lifestyles may not offer these animals the best quality of life. Behaviour is a direct reflection of how the animal is coping with its environment. Behavioural indicators are thus among the preferred parameters to assess welfare. However, behavioural recording (usually from video) can be very time consuming and the accuracy and reliability of the output rely on the experience and background of the observers. The outburst of new video technology and computer image processing gives the basis for promising solutions. In this pilot study, we present a new prototype software able to automatically infer the behaviour of dogs housed in kennels from 3D visual data and through structured machine learning frameworks. Depth information acquired through 3D features, body part detection and training are the key elements that allow the machine to recognise postures, trajectories inside the kennel and patterns of movement that can be later labelled at convenience. The main innovation of the software is its ability to automatically cluster frequently observed temporal patterns of movement without any pre-set ethogram. Conversely, when common patterns are defined through training, a deviation from normal behaviour in time or between individuals could be assessed. The software accuracy in correctly detecting the dogs' behaviour was checked through a validation process. An automatic behaviour recognition system, independent from human subjectivity, could add scientific knowledge on animals' quality of life in confinement as well as saving time and resources. This 3D framework was designed to be invariant to the dog's shape and size and could be extended to farm, laboratory and zoo quadrupeds in artificial housing. The computer vision technique applied to this software is innovative in non-human animal behaviour science. Further improvements and validation are needed, and future applications and limitations are discussed.</p

Partial Deletion of Chromosome 8 β-defensin Cluster Confers Sperm Dysfunction and Infertility in Male Mice

β-defensin peptides are a family of antimicrobial peptides present at mucosal surfaces, with the main site of expression under normal conditions in the male reproductive tract. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. We show here that homozygous deletion of a cluster of nine β-defensin genes (DefbΔ9) in the mouse results in male sterility. The sperm derived from the mutants have reduced motility and increased fragility. Epididymal sperm isolated from the cauda should require capacitation to induce the acrosome reaction but sperm from the mutants demonstrate precocious capacitation and increased spontaneous acrosome reaction compared to wild-types but have reduced ability to bind the zona pellucida of oocytes. Ultrastructural examination reveals a defect in microtubule structure of the axoneme with increased disintegration in mutant derived sperm present in the epididymis cauda region, but not in caput region or testes. Consistent with premature acrosome reaction, sperm from mutant animals have significantly increased intracellular calcium content. Thus we demonstrate in vivo that β-defensins are essential for successful sperm maturation, and their disruption leads to alteration in intracellular calcium, inappropriate spontaneous acrosome reaction and profound male infertility

On-demand electrical switching of antibody-antigen binding on surfaces

The development of stimuli-responsive interfaces between synthetic materials and biological systems is providing the unprecedented ability to modulate biomolecular interactions for a diverse range of biotechnological and biomedical applications. Antibody–antigen binding interactions are at the heart of many biosensing platforms, but no attempts have been made yet to control antibody–antigen binding in an on-demand fashion. Herein, a molecular surface was designed and developed that utilizes an electric potential to drive a conformational change in surface bound peptide moiety, to give on-demand control over antigen–antibody interactions on sensor chips. The molecularly engineered surfaces allow for propagation of conformational changes from the molecular switching unit to a distal progesterone antigen, resulting in promotion (ON state) or inhibition (OFF state) of progesterone antibody binding. The approach presented here can be generally applicable to other antigen–antibody systems and meets the technological needs for in situ long-term assessment of biological processes and disease monitoring on-demand