Physiologyhttps://hdl.handle.net/10468/10932024-03-28T10:52:21Z2024-03-28T10:52:21Z1191A beginner's guide to gene editingHarrison, Patrick T.Hart, Stephenhttps://hdl.handle.net/10468/52552023-04-05T07:09:33Z2017-12-28T00:00:00Zdc.title: A beginner's guide to gene editing
dc.contributor.author: Harrison, Patrick T.; Hart, Stephen
dc.description.abstract: Genome editing enables precise changes to be made in the genome of living cells. The technique was originally developed in the 1980′s but largely limited to use in mice. The discovery that a targeted double stranded break (DSB) at a unique site in the genome, close to the site to be changed, could substantially increase the efficiency of editing raised the possibility of using the technique in a broader range of animal models and potentially human cells. But the challenge was to identify reagents that could create targeted breaks at a unique genomic location with minimal off-target effects. In 2005, the demonstration that programmable zinc finger nucleases (ZFNs) could perform this task, led to a number of proof-of-concept studies, but a limitation was the ease with which effective ZFNs could be produced. In 2009, the development of TAL-effector nucleases (TALENs) increased the specificity of gene editing and the ease of design and production. However, it wasn't until 2013 and the development of the CRISPR Cas9/guideRNA that gene editing became a research tool that any lab could use.
2017-12-28T00:00:00ZA framework for AI-assisted detection of Patent Ductus Arteriosus from neonatal phonocardiogramGómez-Quintana, SergiSchwarz, Christoph E.Shelevytsky, IhorShelevytska, VictoriyaSemenova, OksanaFactor, AndreeaPopovici, EmanuelTemko, Andriyhttps://hdl.handle.net/10468/134502023-04-05T06:35:13Z2021-02-05T00:00:00Zdc.title: A framework for AI-assisted detection of Patent Ductus Arteriosus from neonatal phonocardiogram
dc.contributor.author: Gómez-Quintana, Sergi; Schwarz, Christoph E.; Shelevytsky, Ihor; Shelevytska, Victoriya; Semenova, Oksana; Factor, Andreea; Popovici, Emanuel; Temko, Andriy
dc.description.abstract: The current diagnosis of Congenital Heart Disease (CHD) in neonates relies on echocardiography. Its limited availability requires alternative screening procedures to prioritise newborns awaiting ultrasound. The routine screening for CHD is performed using a multidimensional clinical examination including (but not limited to) auscultation and pulse oximetry. While auscultation might be subjective with some heart abnormalities not always audible it increases the ability to detect heart defects. This work aims at developing an objective clinical decision support tool based on machine learning (ML) to facilitate differentiation of sounds with signatures of Patent Ductus Arteriosus (PDA)/CHDs, in clinical settings. The heart sounds are pre-processed and segmented, followed by feature extraction. The features are fed into a boosted decision tree classifier to estimate the probability of PDA or CHDs. Several mechanisms to combine information from different auscultation points, as well as consecutive sound cycles, are presented. The system is evaluated on a large clinical dataset of heart sounds from 265 term and late-preterm newborns recorded within the first six days of life. The developed system reaches an area under the curve (AUC) of 78% at detecting CHD and 77% at detecting PDA. The obtained results for PDA detection compare favourably with the level of accuracy achieved by an experienced neonatologist when assessed on the same cohort.
2021-02-05T00:00:00ZA method for AI assisted human interpretation of neonatal EEGGómez-Quintana, SergiO'Shea, AlisonFactor, AndreeaPopovici, EmanuelTemko, Andriyhttps://hdl.handle.net/10468/134492023-04-05T06:36:36Z2022-06-29T00:00:00Zdc.title: A method for AI assisted human interpretation of neonatal EEG
dc.contributor.author: Gómez-Quintana, Sergi; O'Shea, Alison; Factor, Andreea; Popovici, Emanuel; Temko, Andriy
dc.description.abstract: The study proposes a novel method to empower healthcare professionals to interact and leverage AI decision support in an intuitive manner using auditory senses. The methodâ s suitability is assessed through acoustic detection of the presence of neonatal seizures in electroencephalography (EEG). Neurophysiologists use EEG recordings to identify seizures visually. However, neurophysiological expertise is expensive and not available 24/7, even in tertiary hospitals. Other neonatal and pediatric medical professionals (nurses, doctors, etc.) can make erroneous interpretations of highly complex EEG signals. While artificial intelligence (AI) has been widely used to provide objective decision support for EEG analysis, AI decisions are not always explainable. This work developed a solution to combine AI algorithms with a human-centric intuitive EEG interpretation method. Specifically, EEG is converted to sound using an AI-driven attention mechanism. The perceptual characteristics of seizure events can be heard using this method, and an hour of EEG can be analysed in five seconds. A survey that has been conducted among targeted end-users on a publicly available dataset has demonstrated that not only does it drastically reduce the burden of reviewing the EEG data, but also the obtained accuracy is on par with experienced neurophysiologists trained to interpret neonatal EEG. It is also shown that the proposed communion of a medical professional and AI outperforms AI alone by empowering the human with little or no experience to leverage AI attention mechanisms to enhance the perceptual characteristics of seizure events.
2022-06-29T00:00:00ZA paradigm shift in oxygen sensing with a twist in the tale!O'Halloran, Ken D.https://hdl.handle.net/10468/37912023-04-05T06:46:00Z2016-08-30T00:00:00Zdc.title: A paradigm shift in oxygen sensing with a twist in the tale!
dc.contributor.author: O'Halloran, Ken D.
dc.description.abstract: AMP-activated protein kinase (AMPK) is pivotal to metabolic homeostasis in eukaryotes, serving as a critical energy sensor. Increased AMPK activity during oxygen deprivation (hypoxia) protects against potentially catastrophic deficits in ATP supply. Whilst the nervous system circuitry for elaboration of the complex cardiorespiratory response to hypoxia has been understood in some detail for many decades, there is continued and considerable interest in the molecular machinery underpinning the mechanism(s) of oxygen sensing. In this issue of the Biochemical Journal, Evans et al. (2016) review their recent work, which points to a pivotal role for AMPK in the transduction of cellular hypoxic stress to integrated ventilatory behaviour, critical in the defence of whole-body oxygen homeostasis. Of great surprise, there is profound blunting of the hyperventilatory response to hypoxic stress in AMPK deficient mice, with resultant dysregulated breathing arising in spite of normal peripheral oxygen sensing and appropriate sensory input to the brain! Their pointedly provocative review challenges current dogma, and in doing so raises intriguing questions that probe fundamental aspects of our understanding of the mammalian ventilatory response to hypoxic stress. The engaging review by Evans et al. (2016) is an interesting read that is sure to encourage colourful debate.
2016-08-30T00:00:00ZA trilogy of stressors in the neonatal intensive care unit: towards therapy for preterm adversityDias Casacao, Maria Luishttps://hdl.handle.net/10468/154902024-02-02T02:06:45Z2023-01-01T00:00:00Zdc.title: A trilogy of stressors in the neonatal intensive care unit: towards therapy for preterm adversity
dc.contributor.author: Dias Casacao, Maria Luis
dc.description.abstract: Premature infants are born with immature breathing network and an innate immune system that responds differently to that of infants born full-term. All infants born less than 28 weeks of gestation develop apnoea of prematurity-related symptoms, which decreases to about 20% of infants born at 34 weeks of gestation and to less than 10% of those infants born beyond 34 weeks of gestation. Premature infants are not only at risk of developing breathing disorders but also at increased risk of infection due to early life vulnerability (the earlier a baby is born, the more likely is to have health problems due to immature organ development). Gram-positive (GP) infections are the most common type of late onset infection in preterm infants. Activation of specific toll-like receptors (TLRs) is reported to modulate cardiorespiratory responses during infection and may play a key role in driving homeostatic instability observed during sepsis. Firstly, we sought to characterise the expression of TLRs in the brainstem, adrenal gland and in the diaphragm respiratory muscle in naïve rats during our developmental period of interest (postnatal day (PND) 3 and PND13). These studies demonstrated mRNA expression of these receptors at PND3. TLR expression fluctuated in early life depending on the subtype and tissue examined with a relative decrease in some of the mRNA expression at PND13; TLR1, 2, 4, 6, 9 and NOD2 in sternohyoid muscle, TLR1, 2, 4 and 6 in diaphragm muscle, TLR2 in adrenal glands and NOD2 in brainstem and spinal cord, but a relative increase in mRNA expression of CX3CR1 in brain and brainstem, TLR2 and 9, IL1R1, in brain and TLR2 and TLR9 in spinal cord. Sex differences were revealed in mRNA expression of TLR9 in brain and NOD2 and IL1R1 in brainstem with upregulation of expression in males. These results relating TLRs and postnatal development suggest a developmental regulation of the immune system. Given these results, we reasoned that oxygen dysregulation coupled with GP bacterial immune stimulation would modulate redox sensitive genes and TLR expression that could alter hormonal expression and impinge on respiratory function in a sex-specific manner. To test this, we developed a novel neonatal rat model in which male and female neonatal rats were exposed to intermittent hypoxia, normoxia and hyperoxia from PND3 for 10 days, followed by combined administration of GP bacterial proteins lipoteichoic acid (LTA) and peptidoglycan (PGN). This model sought to mimic physiological challenges encountered by infants born preterm.
Hypoxia challenges during the intermittent hypoxia and hyperoxia (IHH) protocol, induced a significant peripheral oxygen desaturation in treated animals. LTA and PGN (3mg and 5mg, respectively) evoked a significant immune response in PND13 rat pups when measured 3 hours post administration. Serum cytokine analysis revealed LTA&PGN triggered an increase in CCL2, IL-1α, IL-1ß, IL-5, G-CSF, IL-13, CCL3, Gro/KC, CXCL10, CX3CL1, CXCL2, IL-10, IFNy, leptin, VEGF, IL-17A and TNF-α serum concentration compared to vehicle. Interestingly, IL-1ß, Gro/KC, IL-10 and leptin expression were upregulated with combined IHH and LTA&PGN exposure. Respiratory function demonstrated an overall decrease in breathing frequency that was mainly impacted by LTA&PGN administration due to an increase in expiratory time. A decrease in minute ventilation was reported with LTA&PGN however, regarding the metabolic function, the ventilatory equivalent for carbon dioxide was similar across the groups, which is consistent with normal pH levels obtained. Additionally, a mild response to the GP challenge in the late periods of hypoxia were associated with decreased number of gasps with IHH. We analysed mRNA expression of TLRs and redox modulated genes using real-time polymerase chain reaction (RT-PCR) in brainstem, diaphragm muscle and adrenal glands. Brainstem gene expression was similar across groups. In adrenal glands tissue, there was an overall upregulation of TLRs mRNA expression with IHH exposure, except for TLR6. Moreover, TLR2 mRNA expression was upregulated with IHH compared to Sham groups, in males compared to females and in LTA&PGN compared to vehicle. An inverse trend from that of adrenal glands was reported in diaphragm muscle. We also analysed redox modulated proteins in serum using bioassays to detect 8-OHdG, 8-iso-PGF2α, AOPP and SOD in plasma. No differences were observed with IHH and LTA&PGN. However, sex differences were found in 8-iso-PGF2α and AOPP redox proteins, with upregulated expression in males compared to females. Finally, we sought to characterise the postnatal development in our animal model using a battery of developmental and motor assessments. These studies demonstrated a delay in pinnae detachment in IHH pups, a decrease in time to righting with IHH and an increase in motor and locomotion faults in IHH females. Tactile stimulation was decrease with IHH suggesting a delay in brainstem reflexes. IHH treated males presented with an increased expression of stress and anxiety-related behaviours illustrated by increased time spent in the corners of the open-field test with LTA&PGN and distance travelled with IHH, and decreased time spent in open arms in the elevated plus maze experiment in males compared to females. Altogether, these results suggest that early life stress can profoundly impact the expression of TLRs and redox genes in adrenal glands, impact the expression of cytokines such as leptin and alter development, motor coordination, stress levels in this novel neonatal rat model of early life stress.
In conclusion, these studies specifically target the gaps in knowledge of the pathophysiological alterations experienced in prematurely born babies who present with impaired breathing function and contract GP infection as mimicked in this novel animal model. The results presented disclose novel insights on the physiology and hormonal alterations that these babies could face in similar conditions, with potential to positively contribute to the field of study by enlightening future targets of research. Future directions relying on the spatial characterisation of TLRs and leptin receptors and possible long-term influences on behavioural performance would also be helpful to better characterise the model.
2023-01-01T00:00:00ZAcetylated microtubules are essential for touch sensation in miceMorley, Shane J.https://hdl.handle.net/10468/46892023-04-04T07:18:06Z2016-01-01T00:00:00Zdc.title: Acetylated microtubules are essential for touch sensation in mice
dc.contributor.author: Morley, Shane J.
dc.description.abstract: The sense of touch depends upon the transformation of mechanical energy into electrical signals by peripheral sensory neurons and associated cells in the skin. This conversion is thought to be mediated by a complex of proteins in which ion channels such as Piezo2 function as mechanotransducers. However, how mechanical energy is transmitted into mechanosensitive ion channel opening, and how cellular components such as the cytoskeleton influence this process, is largely unknown. Here we show that mice lacking the tubulin acetyltransferase, Atat1, in sensory neurons display profound deficits in their ability to detect mechanical touch and pain. In the absence of Atat1, behavioural responses to innocuous and noxious mechanical stimuli are strongly reduced in multiple assays while sensitivity of mice to thermal stimuli is unaltered. In ex vivo skin-nerve preparations, the mechanosensitivity of all low- and high- threshold mechanoreceptor subtypes innervating the skin is substantially decreased in Atat1 conditional knockout mice. In cultured dorsal root ganglion neurons, both slowly- and rapidly- adapting mechanically- activated currents are absent or reduced upon Atat1 deletion with no effect on other neuronal functions. We establish that this broad loss of mechanosensitivity is dependent upon the acetyltransferase activity of Atat1, and that by mimicking α-tubulin acetylation genetically by substituting the lysine amino acid for a structurally similar glutamine, mechanosensitivity can be restored in Atat1- deficient sensory neurons. Finally, we demonstrate that acetylated microtubules localize to a prominent band under the membrane of sensory neuron cell bodies and axons, and in the absence of Atat1 and acetylated α-tubulin, cultured sensory neurons display significant reductions in their membrane elasticity. Our results indicate that the microtubule cytoskeleton is an essential component of the mammalian mechanotransduction complex and that by influencing cellular stiffness, α-tubulin acetylation can tune mechanical sensitivity across the full range of mechanoreceptor subtypes.
2016-01-01T00:00:00ZActive peer-mentored learning can improve student understanding of physiological concepts in an undergraduate journal clubDrumm, Bernard T.Rae, Mark G.Ward, Sean M.https://hdl.handle.net/10468/83242023-04-05T07:08:15Z2019-07-15T00:00:00Zdc.title: Active peer-mentored learning can improve student understanding of physiological concepts in an undergraduate journal club
dc.contributor.author: Drumm, Bernard T.; Rae, Mark G.; Ward, Sean M.
dc.description.abstract: One of the most ubiquitous active learning modalities in the biological sciences at third level is the journal club. Journal club can promote several beneficial learning outcomes for students such as gaining critical reading skills to evaluate the scientific literature, improving scientific literacy, serving as an introduction to new concepts and techniques and improving communication skills. However, it can be difficult for instructors who facilitate journal club to gauge student audiences' understanding of topics being related by presenters. At the University of Nevada, Reno School of Medicine, international life sciences undergraduate students enrolled in our research program undergo a 12-month placement in selected research laboratories within the medical school in order to develop an understanding of basic medical scientific research and physiological concepts. As such, an integral component of this program is participation in regular journal club sessions which we had assumed helped students to develop such an understanding. However as we had never empirically assessed if this was the case or not, the aim of the current study was to determine if student understanding could be improved by complementing the standard journal club with peer-mentored workshop presentations. Data from this case study suggest that by allowing students to undergo peer-mentored learning in conjunction with journal club, student understanding of physiological concepts, as well as student confidence in presenting and communication, increases.
2019-07-15T00:00:00ZAcute hypoxia-induced diaphragm dysfunction is prevented by antioxidant pre-treatmentO'Leary, Andrew J.https://hdl.handle.net/10468/38952023-04-04T07:04:06Z2016-01-01T00:00:00Zdc.title: Acute hypoxia-induced diaphragm dysfunction is prevented by antioxidant pre-treatment
dc.contributor.author: O'Leary, Andrew J.
dc.description.abstract: Diaphragm weakness is a strong predictor of poor outcome in patients. Acute hypoxia is a feature of respiratory conditions such as acute respiratory distress syndrome and ventilator-associated lung injury. However, the effects of acute hypoxia on the diaphragm are largely unknown despite the potential clinical relevance. C57BL6/J mice were exposed to 8hr of hypoxia (FiO2 = 0.10) or normoxia. A separate group of mice were administered N-acetyl cysteine (NAC; 200mg/kg, I.P.) immediately prior to acute hypoxia exposure. Ventilation was assessed using whole-body plethysmography. O2 consumption and CO2 production were measured as indices of metabolism. Diaphragm muscle contractile performance was determined ex-vivo. Gene expression was examined at 1, 4, and 8 hrs using qRT-PCR. Protein/phosphoprotein content was assessed using a sandwich immunoassay. Proteasome activity was measured using a spectrophotometric assay. Acute hypoxia decreased diaphragm force and fatigue. Ventilation during acute hypoxia was initially increased during the first 10 minutes, but quickly returned to normoxic levels for the duration of gas exposure. Metabolism was reduced by acute hypoxia, and gene expression driving mitochondrial uncoupling was increased. Acute hypoxia increased atrophic signalling, but not proteasome activity. Acute hypoxia increased hypertrophic and hypoxia protein signalling. NAC pre-treatment prevented the acute hypoxia-induced diaphragm weakness. Diaphragm weakness is reported in mechanically ventilated patients, which is primarily attributed to inactivity of the muscle, although this is controversial. The potential role of hypoxia in the development and/or exacerbation of ICU-related weakness is unclear. These data reveals that acute hypoxia is sufficient to cause diaphragm muscle weakness, likely relates to hypoxic stress. Muscle weakness was prevented by antioxidant supplementation, independent of the hypoxia-induced hypometabolic state. These findings highlight a potentially critical role for hypoxia in diaphragm muscle dysfunction observed in patients with acute respiratory diseases, and the potential benefits of NAC in preventing acute hypoxia-induced diaphragm dysfunction.
2016-01-01T00:00:00ZAlterations of haemodynamic parameters in spontaneously hypertensive rats by Aristolochia ringens Vahl. (Aristolochiaceae)Aigbe, Flora RuthMunavvar, Abdul Sattar ZubaidRathore, HassanEseyin, OlorunfemiPei, Yen PeiAkhtar, SafiaChohan, AshfaqJin, HuiKhoo, JooliTan, SamualLazhari, MohammedAfzar, SheryarAhmed, FiazAdeyemi, Olufunmilayo OlaideJohns, Edward J.https://hdl.handle.net/10468/39082023-04-05T07:09:35Z2017-02-01T00:00:00Zdc.title: Alterations of haemodynamic parameters in spontaneously hypertensive rats by Aristolochia ringens Vahl. (Aristolochiaceae)
dc.contributor.author: Aigbe, Flora Ruth; Munavvar, Abdul Sattar Zubaid; Rathore, Hassan; Eseyin, Olorunfemi; Pei, Yen Pei; Akhtar, Safia; Chohan, Ashfaq; Jin, Hui; Khoo, Jooli; Tan, Samual; Lazhari, Mohammed; Afzar, Sheryar; Ahmed, Fiaz; Adeyemi, Olufunmilayo Olaide; Johns, Edward J.
dc.description.abstract: Aristolochia ringens Vahl. (Aristolochiaceae (AR); 馬兜鈴 mǎ dōu líng) is used traditionally in Nigeria for the management of various disorders including oedema. Preliminary investigation revealed its modulatory effect on the cardiovascular system. This study was aimed at investigating the effect of the aqueous root extract of A. ringens (AR) on haemodynamic parameters of spontaneously hypertensive rats (SHRs). The effect of oral subacute (21 days) and intravenous acute exposure of SHRs to the extract were assessed using tail cuff and carotid artery canulation methods respectively. In the latter, the effect of chloroform, butanol and aqueous fractions of AR were also evaluated. The extract significantly reduced systolic and diastolic blood pressures in SHRs, with peak reductions of 20.3% and 26.7% respectively at 50 mg/kg by the 21st day of oral subacute exposure. Upon intravenous exposure, AR (50 mg/kg) reduced systolic and diastolic blood pressure by as much as 53.4 ± 2.2 and 49.2 ± 2.8 mmHg respectively. A dose-dependent reduction in heart rate, significant at 25 and 50 mg/kg was also observed. Hexamethonium (20 mg/kg) and atropine (1 mg/kg) inhibited the extract's reduction of systolic blood pressure, diastolic blood pressure and heart rate significantly. The extract's butanol fraction produced the greatest systolic and diastolic blood pressures reduction of 67.0 ± 3.8 and 68.4 mmHg respectively at 25 mg/kg and heart rate reduction of 40 ± 7 beats per minute at 50 mg/kg. HPLC analysis revealed the presence of 4-hydroxybenzoic acid and quercetin in AR. The extract's alterations of haemodynamic parameters in this study show that it has hypotensive effect on spontaneously hypertensive rats.
2017-02-01T00:00:00ZAn efficient and cost-effective method of generating postnatal (P2–5) mouse primary hippocampal neuronal culturesKaar, AidanMorley, Shane J.Rae, Mark G.https://hdl.handle.net/10468/46042023-04-05T07:09:32Z2017-05-22T00:00:00Zdc.title: An efficient and cost-effective method of generating postnatal (P2–5) mouse primary hippocampal neuronal cultures
dc.contributor.author: Kaar, Aidan; Morley, Shane J.; Rae, Mark G.
dc.description.abstract: Background: Primary culture of postnatal central neurons is a widely used methodology for applications such as the investigation of neuronal development, protein trafficking/distribution and cellular signalling. However, successful production and maintenance of such cultures, particularly from postnatal animals, can be challenging. In attempting to surmount these difficulties, several disparate culturing methodologies have been developed. Such methodologies are centred on the identification and optimisation of critical steps and, as such, the protocols and reagents utilised can differ quite markedly from protocol to protocol, often with the suggestion that the use of a (usually expensive) proprietary reagent(s), lengthy substrate preparation and/or cell isolation techniques is/are necessary for successful culture preparation. New method: Herein, we present a simple and inexpensive protocol for the preparation of primary hippocampal neurons from postnatal (2–5 day old) mice, which remain viable for experimental use for over one month. Results: Neurons cultured using this method follow well established developmental norms and display typical responses to standard physiological stimuli such as depolarisation and certain pharmacological agents. Comparison with existing methods/conclusion: By using a novel trituration technique, simplified methodology and non-proprietary reagents, we have developed a reliable protocol that enables the cost effective and efficient production of high quality postnatal mouse hippocampal cultures. This method, if required, can also be utilised to prepare neurons both from other regions of the brain as well as from other species such as rat.
2017-05-22T00:00:00Z