Reconstruction of Cell Surface Densities of Ion Pumps, Exchangers, and Channels from mRNA Expression, Conductance Kinetics, Whole-Cell Calcium, and Current-Clamp Voltage Recordings, with an Application to Human Uterine Smooth Muscle Cells.

Uterine smooth muscle cells remain quiescent throughout most of gestation, only generating spontaneous action potentials immediately prior to, and during, labor. This study presents a method that combines transcriptomics with biophysical recordings to characterise the conductance repertoire of these cells, the 'conductance repertoire' being the total complement of ion channels and transporters expressed by an electrically active cell. Transcriptomic analysis provides a set of potential electrogenic entities, of which the conductance repertoire is a subset. Each entity within the conductance repertoire was modeled independently and its gating parameter values were fixed using the available biophysical data. The only remaining free parameters were the surface densities for each entity. We characterise the space of combinations of surface densities (density vectors) consistent with experimentally observed membrane potential and calcium waveforms. This yields insights on the functional redundancy of the system as well as its behavioral versatility. Our approach couples high-throughput transcriptomic data with physiological behaviors in health and disease, and provides a formal method to link genotype to phenotype in excitable systems. We accurately predict current densities and chart functional redundancy. For example, we find that to evoke the observed voltage waveform, the BK channel is functionally redundant whereas hERG is essential. Furthermore, our analysis suggests that activation of calcium-activated chloride conductances by intracellular calcium release is the key factor underlying spontaneous depolarisations.

A Systematic Review of Time and Resource Use Costs of Subcutaneous Versus Intravenous Administration of Oncology Biologics in a Hospital Setting.

BACKGROUND: The introduction of human epidermal growth factor receptor 2 (HER2)-targeted treatment options, including dual HER2 blockade, has improved the prognosis for patients with HER2-positive breast cancer (BC) substantially. However, most of these treatments are administered via the intravenous (IV) route, which can present many challenges, such as long infusion and observation times, issues associated with repeated IV access, and increased strain on time and resources of medical centers and healthcare professionals. A fixed-dose combination of pertuzumab and trastuzumab for subcutaneous (SC) injection (pertuzumab, trastuzumab, and hyaluronidase-zzxf (PHESGO®, F. Hoffmann-La Roche Ltd, Basel, Switzerland; PH FDC SC)) has been approved for use alongside chemotherapy for early-stage and metastatic HER2-positive BC. OBJECTIVES: This systematic literature review was performed to identify evidence relating to time/resource use and resulting cost differences between SC and IV administration of oncology biologics in a hospital setting, and, ultimately, to inform economic modeling and associated health technology assessment of PH FDC SC. METHODS: Electronic databases (Embase, MEDLINE, and EconLit) were searched on 9 April 2020. Additional hand searches were performed to identify publications not captured in the electronic database search. Publication screening and data extraction (study characteristics, participants, interventions, costs, and time/resource use) were carried out per the standard Cochrane review methodology. The quality of economic evidence of cost analyses was assessed using the 36-item checklist of the National Institute for Health and Care Excellence Single Technology Appraisal Specification for submission of evidence (January 2015). RESULTS: The database search identified 2,740 records, of which 237 underwent full text screening. Full text screening, prioritization of publications about patients with a cancer diagnosis, and the addition of four citations identified during the hand search resulted in 72 final included publications, relating to 71 unique studies. This included 40 publications that described the time/resource use and/or costs associated with SC versus IV trastuzumab administration for the treatment of HER2-positive BC, and 28 publications that described time/resource use and/or costs associated with rituximab SC versus IV administration for the treatment of non-Hodgkin's lymphoma/follicular lymphoma and diffuse large B-cell lymphoma. The majority of publications showed substantial time savings for preparation and administration of SC versus IV therapy, and cost savings associated with reductions in healthcare professional time and resource use for SC administration. LIMITATIONS: There was a lack of consensus between publications regarding time and cost measurements. In addition, the search was limited to publications related to anticancer drugs; the majority of the studies included were performed in European countries. CONCLUSIONS AND IMPLICATIONS: This review indicated a substantial body of evidence showing time/resource and cost savings of SC versus IV administration of oncology biologics in a hospital setting, which can be used to inform economic evaluations of PH FDC SC.

Exploring the relationships between rule-governed behavior and adherence to guidelines aiming to reduce the spread of COVID-19.

Research demonstrates that socially mediated consequences impact adherence to health mandates during pandemics. However, no published research has examined whether adherence varies based on the extent to which an individual relies on arbitrary social approval (i.e., displays generalized pliance). The present study explored the relationships between adherence to COVID-19 public health measures, two types of rule-following (pliance and tracking), and perceived peer behavior in a sample of adults (n = 288). Findings revealed that adherence was negatively correlated with generalized pliance and tracking was unrelated to adherence. Pliance did not moderate the relationship between peer adherence and individual adherence. Findings are discussed with reference to the need to develop easily adaptable and context sensitive measures of types of rule-following, in addition to a measure of social tracking.

Kv1.3 is the exclusive voltage-gated K+ channel of platelets and megakaryocytes: roles in membrane potential, Ca2+ signalling and platelet count.

A delayed rectifier voltage-gated K(+) channel (Kv) represents the largest ionic conductance of platelets and megakaryocytes, but is undefined at the molecular level. Quantitative RT-PCR of all known Kv alpha and ancillary subunits showed that only Kv1.3 (KCNA3) is substantially expressed in human platelets. Furthermore, megakaryocytes from Kv1.3(/) mice or from wild-type mice exposed to the Kv1.3 blocker margatoxin completely lacked Kv currents and displayed substantially depolarised resting membrane potentials. In human platelets, margatoxin reduced the P2X(1)- and thromboxaneA(2) receptor-evoked [Ca(2+)](i) increases and delayed the onset of store-operated Ca(2+) influx. Megakaryocyte development was normal in Kv1.3(/) mice, but the platelet count was increased, consistent with a role of Kv1.3 in apoptosis or decreased platelet activation. We conclude that Kv1.3 forms the Kv channel of the platelet and megakaryocyte, which sets the resting membrane potential, regulates agonist-evoked Ca(2+) increases and influences circulating platelet numbers.

Burden of selected infectious diseases covered by UK childhood vaccinations: systematic literature review.

Aim: An overview of recent epidemiology and disease burden, independent of patient age, of diphtheria, tetanus, pertussis, hepatitis B, poliomyelitis and Hemophilus influenzae invasive disease in the UK. Materials & methods: A systematic review was undertaken. Outcomes included incidence, prevalence, risk factors and cost burden. Results: 39 publications were included. Hepatitis B prevalence is high among certain risk groups. A small pertussis risk remains in pregnancy and for infants, which led to the introduction of maternal vaccination. H. influenzae invasive disease cases are limited to rare serotypes. Polio, tetanus and diphtheria are well controlled. Conclusion: The evaluated diseases are currently well controlled, thanks to a comprehensive vaccination program, with a generally low clinical and cost burden.

The clinical and humanistic burden of cutaneous T-cell lymphomas and response to conventional and novel therapies: results of a systematic review.

Objectives: To summarize the impact of relapsed/refractory primary cutaneous T-cell lymphomas (CTCL) on quality of life (QoL) and the efficacy of available treatments in two systematic reviews (SRs).Methods: Searches were performed on 16 January 2018 and 23 January 2018, respectively, in Medline, Medline in process, the Cochrane database, and EconLit. Studies reporting QoL outcomes in adults with CTCL or treatment efficacy in relapsed/refractory CTCL were included.Results: Based on 15 QoL studies, CTCL symptoms/complications negatively affect patients' physical, emotional, and social functioning. Skin problems pose considerable symptom burden, while advanced disease stage is associated with poorer QoL. CTCL negatively affects caregivers, primarily through family dynamics and relationships. The clinical efficacy SR included 72 publications covering 23 therapies. Overall response rate (ORR) ranged from 14% (belinostat) to 95% (total skin electron beam therapy). ORRs >50% were reported for several therapies including brentuximab vedotin (50-78%) and bexarotene (39-86%). Over half (13 of 23 therapies) had ORRs <30%. Median progression-free survival varied between treatments (3.5-116.4 months) and was >20 months for brentuximab vedotin and alemtuzumab.Conclusion: CTCL negatively affects patients' and caregivers' QoL. A considerable proportion of patients have no response or no sustainable response to current treatments.

Voltage-dependent Ca2+ currents contribute to spontaneous Ca2+ waves in rabbit corpus cavernosum myocytes.

INTRODUCTION: Corpus cavernosum myocytes generate spontaneous tone that contributes to penile detumescence. It is essential to elucidate how tone is generated to fully understand the processes involved in erection. Tissue experiments have shown that blockers of voltage-dependent Ca2+ channels (VDCCs) reduce tone. However, there is also a widespread belief that these channels are poorly expressed in this tissue. Furthermore, it is unclear how VDCC would interact with recently described intracellular Ca2+ waves, which initiate contractions. AIMS: (i) To directly examine VDCC currents in freshly isolated corpus cavernosum myocytes; and (ii) to study the relationship between VDCC and intracellular Ca2+ waves. MAIN OUTCOME MEASURES: VDCC and cytosolic Ca2+ were measured using patch clamp and confocal microscopy. Methods. Male New Zealand white rabbits were euthanized and corpus cavernosum myocytes dispersed enzymatically for patch clamp recording and confocal Ca2+ imaging (using fluo-4AM). RESULTS: Isolated myocytes developed robust VDCC that could be separated into two components. One activated at -45 mV, reversed at +40 mV, inactivated with a V(1/2) of -27 mV and was enhanced by Ba2+. This component was blocked with nifedipine, but not Ni2+ or mibefradil. The other component inactivated with a V(1/2) of -87 mV, was unchanged in Ba2+, and was blocked by Ni2+ or mibefradil, but not nifedipine. Even though Ni2+ had no effect on intracellular Ca2+ waves, nifedipine blocked them, although localized Ca2+ events remained. CONCLUSIONS: At least two VDCC are expressed in rabbit corpus cavernousum myocytes. One may be designated L-type Ca2+ current, whereas the other is a putative T-type current. The L-current facilitates conversion of local Ca2+ events into global Ca2+ waves, whereas the putative T-current plays little part in this process. These results provide a new basis for understanding the role of L-type Ca2+ current in generating detumescent tone in the corpus cavernosum.

A High-Throughput Electrophysiology Assay Identifies Inhibitors of the Inwardly Rectifying Potassium Channel Kir7.1.

Kir7.1 is an inwardly rectifying potassium channel that has been implicated in controlling the resting membrane potential of the myometrium. Abnormal uterine activity in pregnancy plays an important role in postpartum hemorrhage, and novel therapies for this condition may lie in manipulation of membrane potential. This work presents an assay development and screening strategy for identifying novel inhibitors of Kir7.1. A cell-based automated patch-clamp electrophysiology assay was developed using the IonWorks Quattro (Molecular Devices, Sunnyvale, CA) system, and the iterative optimization is described. In total, 7087 compounds were tested, with a hit rate (>40% inhibition) of 3.09%. During screening, average Z' values of 0.63 ± 0.09 were observed. After chemistry triage, lead compounds were resynthesized and activity confirmed by IC50 determinations. The most potent compound identified (MRT00200769) gave rise to an IC50 of 1.3 µM at Kir7.1. Compounds were assessed for selectivity using the inwardly rectifying potassium channel Kir1.1 (ROMK) and hERG (human Ether-à-go-go Related Gene). Pharmacological characterization of known Kir7.1 inhibitors was also carried out and analogues of VU590 tested to assess selectivity at Kir7.1.

The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy.

Abnormal uterine activity in pregnancy causes a range of important clinical disorders, including preterm birth, dysfunctional labour and post-partum haemorrhage. Uterine contractile patterns are controlled by the generation of complex electrical signals at the myometrial smooth muscle plasma membrane. To identify novel targets to treat conditions associated with uterine dysfunction, we undertook a genome-wide screen of potassium channels that are enriched in myometrial smooth muscle. Computational modelling identified Kir7.1 as potentially important in regulating uterine excitability during pregnancy. We demonstrate Kir7.1 current hyper-polarizes uterine myocytes and promotes quiescence during gestation. Labour is associated with a decline, but not loss, of Kir7.1 expression. Knockdown of Kir7.1 by lentiviral expression of miRNA was sufficient to increase uterine contractile force and duration significantly. Conversely, overexpression of Kir7.1 inhibited uterine contractility. Finally, we demonstrate that the Kir7.1 inhibitor VU590 as well as novel derivative compounds induces profound, long-lasting contractions in mouse and human myometrium; the activity of these inhibitors exceeds that of other uterotonic drugs. We conclude Kir7.1 regulates the transition from quiescence to contractions in the pregnant uterus and may be a target for therapies to control uterine contractility.

Spatial heterogeneity enhances and modulates excitability in a mathematical model of the myometrium.

The muscular layer of the uterus (myometrium) undergoes profound changes in global excitability prior to parturition. Here, a mathematical model of the myocyte network is developed to investigate the hypothesis that spatial heterogeneity is essential to the transition from local to global excitation which the myometrium undergoes just prior to birth. Each myometrial smooth muscle cell is represented by an element with FitzHugh-Nagumo dynamics. The cells are coupled through resistors that represent gap junctions. Spatial heterogeneity is introduced by means of stochastic variation in coupling strengths, with parameters derived from physiological data. Numerical simulations indicate that even modest increases in the heterogeneity of the system can amplify the ability of locally applied stimuli to elicit global excitation. Moreover, in networks driven by a pacemaker cell, global oscillations of excitation are impeded in fully connected and strongly coupled networks. The ability of a locally stimulated cell or pacemaker cell to excite the network is shown to be strongly dependent on the local spatial correlation structure of the couplings. In summary, spatial heterogeneity is a key factor in enhancing and modulating global excitability.

Effects of female steroid hormones on A-type K+ currents in murine colon.

Idiopathic constipation is higher in women of reproductive age than postmenopausal women or men, suggesting that female steroid hormones influence gastrointestinal motility. How female hormones affect motility is unclear. Colonic motility is regulated by ion channels in colonic myocytes. Voltage-dependent K(+) channels serve to set the excitability of colonic muscles. We investigated regulation of Kv 4.3 channel expression in response to acute or chronic changes in female hormones. Patch clamp experiments and quantitative PCR were used to compare outward currents and transcript expression in colonic myocytes from male, non-pregnant, pregnant and ovariectomized mice. Groups of ovariectomized mice received injections of oestrogen or progesterone to investigate the effects of hormone replacement. The capacitance of colonic myocytes from non-pregnant females was larger than in males. Net outward current density in male and ovariectomized mice was higher than in non-pregnant females and oestrogen-treated ovariectomized mice. Current densities in late pregnancy were lower than in female controls. Progesterone had no effect on outward currents. A-type currents were decreased in non-pregnant females compared with ovariectomized mice, and were further decreased by pregnancy or oestrogen replacement. Kv 4.3 transcripts did not differ significantly between groups; however, expression of the potassium channel interacting protein KChIP1 was elevated in ovariectomized mice compared with female controls and oestrogen-treated ovariectomized mice. Delayed rectifier currents were not affected by oestrogen. In the mouse colon, oestrogen suppresses A-type currents, which are important for regulating excitability. These observations suggest a possible link between female hormones and altered colonic motility associated with menses, pregnancy and menopause.