Bernard T. Drumm's research while affiliated with Dundalk Institute of Technology and other places

Laboratory practicals in life science subjects are traditionally assessed by written reports which reflect disciplinary norms of documenting experimental activities. However, exclusive application of this assessment has potential to engage only a narrow range of competencies. In this study, we explored how multiple modes of laboratory assessment might affect student perceptions of learned skills in a life science module. We hypothesized that while a mixture of assessments may not impact student summative performance, it might positively influence student perceptions of different skills that varied assessments allowed them to practice. This was informed by universal design for learning (UDL) and teaching for understanding (TfU) frameworks. In our study, in a 3 rd year Bioscience programme, written reports were complemented with group presentations and online quizzes via Moodle. Anonymous surveys evaluated whether this expanded portfolio of assessments promoted awareness of, and engagement with, a broader range of practical competencies. Aspects that influenced student preferences in assessment mode included time limitations, time investment, ability to practice new skills, links with lecture material and experience of assessment anxiety. In particular, presentations were highlighted as promoting collaboration and communication and the quiz as an effective means of diversifying assessment schedules. A key takeaway from students was that while reports were important, an over reliance on them was detrimental. This study suggests that undergraduate life science students can benefit significantly from a holistic assessment strategy that complements reports with performance-based approaches that incorporate broader competencies and allow for greater student engagement and expression in undergraduate modules.

Collecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure-dependent frequency, but the identity of the lymphatic pacemaker cell is still debated. By analogy to pacemakers in the GI and lower urinary tracts, proposed cLV pacemaker cells include interstitial cells of Cajal like cells (ICLC), pericytes, as well as the lymphatic muscle (LMCs) cells themselves. Here we tested the extent to which these cell types are invested into the mouse cLV wall and if any cell type exhibited morphological and functional processes characteristic of pacemaker cells: a contiguous network; spontaneous Ca2+ transients; and depolarization-induced propagated contractions. We employed inducible Cre (iCre) mouse models routinely used to target these specific cell populations including: c-kitCreER T2 to target ICLC; PdgfrβCreERT2 to target pericytes; PdgfrαCreERTM to target CD34+ adventitial fibroblast-like cells or ICLC; and Myh11CreERT2 to target LMCs. These specific inducible Cre lines were crossed to the fluorescent reporter ROSA26mT/mG, the genetically encoded Ca2+ sensor GCaMP6f, and the light-activated cation channel rhodopsin2 (ChR2). c-KitCreER T2 labeled both a sparse population of LECs and round adventitial cells that responded to the mast cell activator compound 48-80. PdgfrβCreERT2 drove recombination in both adventitial cells and LMCs, limiting its power to discriminate a pericyte specific population. PdgfrαCreERTM labeled a large population of interconnected, oak leaf-shaped cells primarily along the adventitial surface of the vessel. Titrated induction of the smooth muscle-specific Myh11CreERT2 revealed a LMC population with heterogeneous morphology. Only LMCs consistently, but heterogeneously, displayed spontaneous Ca2+ events during the diastolic period of the contraction cycle, and whose frequency was modulated in a pressure-dependent manner. Optogenetic depolarization through the expression of ChR2 by Myh11CreERT2 , but not PdgfrαCreERTM or c-KitCreER T2 , resulted in a propagated contraction. These findings support the conclusion that LMCs, or a subset of LMCs, are responsible for mouse cLV pacemaking. The presence and functionality of proposed pacemaker cells in collecting lymphatic vessels was tested with various mouse Cre models to drive expression of a recombination reporter ROSA26mT/mG, a genetically encoded Ca2+ sensor GCaMP6f, or the optogenetic tool channel-rhodopsin2. Lymphatic CD34+ adventitial cells co-express PDGFRΑ+ while cKit+ cells are mast cells; and neither cell type demonstrated pacemaking functionality. Myh11CreERT2 identified lymphatic muscle cells which exhibited pacemaker behaviors such as pressure-dependent calcium events during diastole and propagated contraction induced by optical stimulation of channel-rhodopsin2.

Collecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure-dependent frequency, but the identity of the lymphatic pacemaker cell is still debated. By analogy to pacemakers in the GI and lower urinary tracts, proposed cLV pacemaker cells include interstitial cells of Cajal like cells (ICLC), pericytes, as well as the lymphatic muscle (LMCs) cells themselves. Here we tested the extent to which these cell types are invested into the mouse cLV wall and if any cell type exhibited morphological and functional processes characteristic of pacemaker cells: a contiguous network; spontaneous Ca2+ transients; and depolarization-induced propagated contractions. We employed inducible Cre (iCre) mouse models routinely used to target these specific cell populations including: c-kitCreER T2 to target ICLC; PdgfrβCreERT2 to target pericytes; PdgfrαCreERTM to target CD34+ adventitial fibroblast-like cells or ICLC; and Myh11CreERT2 to target LMCs. These specific inducible Cre lines were crossed to the fluorescent reporter ROSA26mT/mG, the genetically encoded Ca2+ sensor GCaMP6f, and the light-activated cation channel rhodopsin2 (ChR2). c-KitCreER T2 labeled both a sparse population of LECs and round adventitial cells that responded to the mast cell activator compound 48-80. PdgfrβCreERT2 drove recombination in both adventitial cells and LMCs, limiting its power to discriminate a pericyte specific population. PdgfrαCreERTM labeled a large population of interconnected, oak leaf-shaped cells primarily along the adventitial surface of the vessel. Titrated induction of the smooth muscle-specific Myh11CreERT2 revealed a LMC population with heterogeneous morphology. Only LMCs consistently, but heterogeneously, displayed spontaneous Ca2+ events during the diastolic period of the contraction cycle, and whose frequency was modulated in a pressure-dependent manner. Optogenetic depolarization through the expression of ChR2 by Myh11CreERT2 , but not PdgfrαCreERTM or c-KitCreER T2 , resulted in a propagated contraction. These findings support the conclusion that LMCs, or a subset of LMCs, are responsible for mouse cLV pacemaking. The presence and functionality of proposed pacemaker cells in collecting lymphatic vessels was tested with various mouse Cre models to drive expression of a recombination reporter ROSA26mT/mG, a genetically encoded Ca2+ sensor GCaMP6f, or the optogenetic tool channel-rhodopsin2. Lymphatic CD34+ adventitial cells co-express PDGFRΑ+ while cKit+ cells are mast cells; and neither cell type demonstrated pacemaking functionality. Myh11CreERT2 identified lymphatic muscle cells which exhibited pacemaker behaviors such as pressure-dependent calcium events during diastole and propagated contraction induced by optical stimulation of channel-rhodopsin2.

Collecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure-dependent frequency, but the identity of the lymphatic pacemaker cell is still debated. By analogy to pacemakers in the GI and lower urinary tracts, proposed cLV pacemaker cells include interstitial cells of Cajal like cells (ICLC), pericytes, as well as the lymphatic muscle (LMCs) cells themselves. Here we tested the extent to which these cell types are invested into the mouse cLV wall and if any cell type exhibited morphological and functional processes characteristic of pacemaker cells: a contiguous network; spontaneous Ca ²⁺ transients; and depolarization-induced propagated contractions. We employed inducible Cre (iCre) mouse models routinely used to target these specific cell populations including: c-kitCreER T2 to target ICLC; PdgfrβCreER T2 to target pericytes; PdgfrαCreER TM to target CD34 ⁺ adventitial fibroblast-like cells or ICLC; and Myh11CreER T2 to target LMCs. These specific inducible Cre lines were crossed to the fluorescent reporter ROSA26mT/mG, the genetically encoded Ca ²⁺ sensor GCaMP6f, and the light-activated cation channel rhodopsin2 (ChR2). c-KitCreER T2 labeled both a sparse population of LECs and round adventitial cells that responded to the mast cell activator compound 48-80. PdgfrβCreER T2 drove recombination in both adventitial cells and LMCs, limiting its power to discriminate a pericyte specific population. PdgfrαCreER TM labeled a large population of interconnected, oak leaf-shaped cells primarily along the adventitial surface of the vessel. Titrated induction of the smooth muscle-specific Myh11CreER T2 revealed a LMC population with heterogeneous morphology. Only LMCs consistently, but heterogeneously, displayed spontaneous Ca ²⁺ events during the diastolic period of the contraction cycle, and whose frequency was modulated in a pressure-dependent manner. Optogenetic depolarization through the expression of ChR2 by Myh11CreER T2 , but not PdgfrαCreER TM or c-KitCreER T2 , resulted in a propagated contraction. These findings support the conclusion that LMCs, or a subset of LMCs, are responsible for mouse cLV pacemaking. Impact The presence and functionality of proposed pacemaker cells in collecting lymphatic vessels was tested with various mouse Cre models to drive expression of a recombination reporter ROSA26mT/mG, a genetically encoded Ca ²⁺ sensor GCaMP6f, or the optogenetic tool channel-rhodopsin2. Lymphatic CD34 ⁺ adventitial cells co-express PDGFRΑ ⁺ while cKit ⁺ cells are mast cells; and neither cell type demonstrated pacemaking functionality. Myh11CreER T2 identified lymphatic muscle cells which exhibited pacemaker behaviors such as pressure-dependent calcium events during diastole and propagated contraction induced by optical stimulation of channel-rhodopsin2.

The gastrointestinal (GI) tract displays multiple motor patterns that move nutrients and wastes through the body. Smooth muscle cells (SMCs) provide the forces necessary for GI motility, but interstitial cells, electrically coupled to SMCs, tune SMC excitability, transduce inputs from enteric motor neurons and generate pacemaker activity that underlies major motor patterns, such as peristalsis and segmentation. The interstitial cells regulating SMCs are interstitial cells of Cajal (ICC) and PDGFRa+ cells. Together these cells form the SIP syncytium. ICC and PDGFRa+ cells express signature Ca2+-dependent conductances: ICC express Ca2+-activated Cl- channels, encoded by Ano1, that generate inward current, and PDGFRa+ cells express Ca2+-activated K+ channels, encoded by Kcnn3, that generate outward current. The open probabilities of interstitial cell conductances are controlled by Ca2+ release from the endoplasmic reticulum. The resulting Ca2+ transientsoccur spontaneously in a stochastic manner. Ca2+ transients in ICC induce spontaneous transient inward currents and spontaneous transient depolarization (STDs). Neurotransmission increases or decreases Ca2+transients, and the resulting depolarizing or hyperpolarizing responses conduct to other cells in the SIP syncytium. In pacemaker ICC, STDs activate voltage-dependent Ca2+ influx, which initiates a cluster of Ca2+ transients and sustains activation of ANO1 channels and depolarization during slow waves. Regulation of GI motility has traditionally been described as neurogenic and myogenic. Recent advances in understanding Ca2+ handling mechanisms in interstitial cells and how these mechanisms influence motor patterns of the GI tract, suggest the term myogenic should be replaced by the term, SIPgenic, as this review discusses.

Malfunctions in airway smooth muscle Ca2+-signalling leads to airway hyperresponsiveness in asthma and chronic obstructive pulmonary disease. Ca2+-release from intracellular stores is important in mediating agonist-induced contractions, but the role of influx via l-type Ca2+ channels is controversial. We re-examined roles of the sarcoplasmic reticulum Ca2+ store, refilling of this store via store-operated Ca2+ entry (SOCE) and l-type Ca2+ channel pathways on carbachol (CCh, 0.1-10 µM)-induced contractions of mouse bronchial rings and intracellular Ca2+ signals of mouse bronchial myocytes. In tension experiments, the ryanodine receptor (RyR) blocker dantrolene (100 µM) reduced CCh-responses at all concentrations, with greater effects on sustained rather than initial components of contraction. 2-Aminoethoxydiphenyl borate (2-APB, 100 μM), in the presence of dantrolene, abolished CCh-responses, suggesting the sarcoplasmic reticulum Ca2+ store is essential for contraction. The SOCE blocker GSK-7975A (10 µM) reduced CCh-contractions, with greater effects at higher (e.g. 3 and 10 µM) CCh concentrations. Nifedipine (1 µM), abolished remaining contractions in GSK-7975A (10 µM). A similar pattern was observed on intracellular Ca2+-responses to 0.3 µM CCh, where GSK-7975A (10 µM) substantially reduced Ca2+ transients induced by CCh, and nifedipine (1 µM) abolished remaining responses. When nifedipine (1 µM) was applied alone it had less effect, reducing tension responses at all CCh concentrations by 25% - 50%, with greater effects at lower (e.g. 0.1 and 0.3 µM) CCh concentrations. When nifedipine (1 µM) was examined on the intracellular Ca2+-response to 0.3 µM CCh, it only modestly reduced Ca2+ signals, while GSK-7975A (10 µM) abolished remaining responses. In conclusion, Ca2+-influx from both SOCE and l-type Ca2+ channels contribute to excitatory cholinergic responses in mouse bronchi. The contribution of l-type Ca2+ channels was especially pronounced at lower doses of CCh, or when SOCE was blocked. This suggests l-type Ca2+ channels might be a potential target for bronchoconstriction under certain circumstances.

We investigated effects of TMEM16A blockers benzbromarone, MONNA, CaCCinhA01 and Ani9 on isometric contractions in mouse bronchial rings and on intracellular calcium in isolated bronchial myocytes. Separate concentrations of carbachol (0.1-10 μM) were applied for 10 min periods to bronchial rings, producing concentration-dependent contractions that were well maintained throughout each application period. Benzbromarone (1 μM) markedly reduced the contractions with a more pronounced effect on their sustained component (at 10 min) compared to their initial component (at 2 min). Iberiotoxin (0.3 μM) enhanced the contractions, but they were still blocked by benzbromarone. MONNA (3 μM) and CaCCinhA01 (10 μM) had similar effects to benzbromarone, but were less potent. In contrast, Ani9 (10 μM) had no effect on carbachol-induced contractions. Confocal imaging revealed that benzbromarone (0.3 μM), MONNA (1 μM) and CaCCinhA01 (10 μM) increased intracellular calcium in isolated myocytes loaded with Fluo-4AM. In contrast, Ani9 (10 μM) had no effect on intracellular calcium. Benzbromarone and MONNA also increased calcium in calcium-free extracellular solution, but failed to do so when intracellular stores were discharged with caffeine (10 mM). Caffeine was unable to cause further discharge of the store when applied in the presence of benzbromarone. Ryanodine (100 μM) blocked the ability of benzbromarone (0.3 μM) to increase calcium, while tetracaine (100 μM) reversibly reduced the rise in calcium induced by benzbromarone. We conclude that benzbromarone and MONNA caused intracellular calcium release, probably by opening ryanodine receptors. Their ability to block carbachol contractions was likely due to this off-target effect.

Years ago gastrointestinal motility was thought to be due to interactions between enteric nerves and smooth muscle cells (SMCs) in the tunica muscularis. Thus, regulatory mechanisms controlling motility were either myogenic or neurogenic. Now we know that populations of interstitial cells, c-Kit+ (interstitial cells of Cajal or ICC), and PDGFRα+ cells (formerly “fibroblast-like” cells) are electrically coupled to SMCs, forming the SIP syncytium. Pacemaker and neurotransduction functions are provided by interstitial cells through Ca2+ release from the endoplasmic reticulum (ER) and activation of Ca2+-activated ion channels in the plasma membrane (PM). ICC express Ca2+-activated Cl− channels encoded by Ano1. When activated, Ano1 channels produce inward current and, therefore, depolarizing or excitatory effects in the SIP syncytium. PDGFRα+ cells express Ca2+-activated K+ channels encoded by Kcnn3. These channels generate outward current when activated and hyperpolarizing or membrane-stabilizing effects in the SIP syncytium. Inputs from enteric and sympathetic neurons regulate Ca2+ transients in ICC and PDGFRα+ cells, and currents activated in these cells conduct to SMCs and regulate contractile behaviors. ICC also serve as pacemakers, generating slow waves that are the electrophysiological basis for gastric peristalsis and intestinal segmentation. Pacemaker types of ICC express voltage-dependent Ca2+ conductances that organize Ca2+ transients, and therefore Ano1 channel openings, into clusters that define the amplitude and duration of slow waves. Ca2+ handling mechanisms are at the heart of interstitial cell function, yet little is known about what happens to Ca2+ dynamics in these cells in GI motility disorders.

In this paper, we outline a case study describing the incorporation of active learning and flipped classroom techniques in a renal physiology module in 1st year medical school. The module was redesigned over a 2-year period within the teaching for understanding (TfU) framework (generative topics, understanding goals, performances of understanding and ongoing assessment) to include more active learning exercises (clicker response systems centered on clinically relevant problem sets and classroom assessment techniques – CATs), which culminated in flipping the classroom entirely during the 2nd year. The goal, was to evaluate student perceptions of the flipped classroom model and to reflect on the use of active learning generally. In the 1st year, clicker response systems were favorably received by students, however the anonymous nature of the clicker configuration meant it was not possible to track progress of individual students. CATs revealed that content areas without active learning exercises were often deemed the most unclear by students. Student feedback indicated that the flipped classroom model in the 2nd year was positively received, with students noting it encouraged them to attend classes more regularly and they believed it assisted in developing collaborative learning and knowledge application.

Penile detumescence is maintained by tonic contraction of corpus cavernosum smooth muscle cells (CCSMC), but the underlying mechanisms have not been fully elucidated. The purpose of this study was to characterize the mechanisms underlying activation of TMEM16A Ca2+ -activated Cl- channels in freshly isolated murine CCSMC. Male C57BL/6 mice aged 10-18 weeks were euthanized via intraperitoneal injection of sodium pentobarbital (100 mg.kg-1 ). Whole-cell patch clamp, pharmacological, and immunocytochemical experiments were performed on isolated CCSM. Tension measurements were performed in whole tissue. TMEM16A expression in murine corpus cavernosum was confirmed using immunocytochemistry. Isolated CCSMC developed spontaneous transient inward currents (STICs) under voltage clamp and spontaneous transient depolarizations (STDs) in current clamp mode of the whole cell, perforated patch clamp technique. STICs reversed close to the predicted Cl- equilibrium potential and both STICs and STDs were blocked by the TMEM16A channel blockers, Ani9 and CaCC(inh)-A01. These events were also blocked by GSK7975A (ORAI inhibitor), cyclopiazonic acid (CPA, sarcoplasmic reticulum [SR] Ca2+- ATPase blocker), tetracaine (RyR blocker), and 2APB (IP3 R blocker), suggesting that they were dependent on Ca2+ release from intracellular Ca2+ stores. Nifedipine (L-type Ca2+ channel blocker) did not affect STICs, but reduced the duration of STDs. Phenylephrine induced transient depolarizations and transient inward currents which were blocked by Ani9. Similarly, phenylephrine induced phasic contractions of intact corpus cavernosum muscle strips and these events were also inhibited by Ani9. This study suggests that contraction of CCSM is regulated by activation of TMEM16A channels and therefore inhibition of these channels could lead to penile erection.