A novel chronic in vivo oral cadmium exposure-washout mouse model for studying cadmium toxicity and complex diabetogenic effects.

Type II diabetes mellitus (T2DM) is characterized by insulin resistance, ß-cell dysfunction and hyperglycemia. In addition to well known risk factors such as lifestyle and genetic risk score, accumulation of environmental toxicants in organs relevant to glucose metabolism is increasingly recognized as additional risk factors for T2DM. Here, we describe the development of an in vivo oral cadmium (Cd) exposure model. It was shown that oral Cd exposure in drinking water followed by washout and high fat diet (HFD) in C57BL/6N mice results in islet Cd bioaccumulation comparable to that found in native human islets while mitigating the anorexic effects of Cd to achieve the same weight gain required to induce insulin resistance as in Cd naïve control mice. Inter individual variation in plasma glucose and insulin levels as well as islet Cd bioaccumulation was observed in both female and male mice. Regression analysis showed an inverse correlation between islet Cd level and plasma insulin following a glucose challenge in males but not in females. This finding highlights the need to account for inter individual target tissue Cd concentrations when interpreting results from in vivo Cd exposure models. No effect of Cd on insulin secretion was observed in islets ex vivo, highlighting differences between in vivo and ex vivo cadmium exposure models. In summary, our oral in vivo Cd exposure-washout with HFD model resulted in islet Cd bioaccumulation that is relevant in the context of environmental cadmium exposure in humans. Here, we showed that islet Cd bioaccumulation is associated with complex cadmium-mediated changes in glucose clearance and ß-cell function. The model described here will serve as a useful tool to further examine the relationship between Cd exposure, islet Cd bioaccumulation, dysglycemia and their underlying mechanisms.

Cadmium-mediated pancreatic islet transcriptome changes in mice and cultured mouse islets.

Type II diabetes mellitus (T2DM) is a multifactorial disease process that is characterized by insulin resistance and impairment of insulin-producing pancreatic islets. There is evidence that environmental exposure to cadmium contributes to the development of T2DM. The presence of cadmium in human islets from the general population and the uptake of cadmium in ß-cells have been reported. To identify cadmium-mediated changes in gene expression and molecular regulatory networks in pancreatic islets, we performed next-generation RNA-Sequencing (RNA-Seq) in islets following either in vivo (1 mM CdCl2 in drinking water) or ex-vivo (0.5 µM CdCl2) exposure. Both exposure regiments resulted in islet cadmium concentrations that are comparable to those found in human islets from the general population. 6-week in vivo cadmium exposure upregulates the expression of five genes: Synj2, Gjb1, Rbpjl, Try5 and 5430419D17Rik. Rbpjl is a known regulator of ctrb, a gene associated with diabetes susceptibility. With 18-week in vivo cadmium exposure, we found more comprehensive changes in gene expression profile. Pathway enrichment analysis showed that these secondary changes were clustered to molecular mechanisms related to intracellular protein trafficking to the plasma membrane. In islet culture, cadmium ex vivo significantly induces the expression of Mt1, Sphk1, Nrcam, L3mbtl2, Rnf216 and Itpr1. Mt1 and Itpr1 are known to be involved in glucose homeostasis. Collectively, findings reported here revealed a complex cadmium-mediated effect on pancreatic islet gene expression at environmentally relevant cadmium exposure conditions, providing the basis for further studies into the pathophysiological processes arising from cadmium accumulation in pancreatic islets.

HIV post-exposure prophylaxis in the emergency department: An updated assessment and opportunities for HIV prevention identified.

OBJECTIVES: Research suggests nonoccupational post exposure prophylaxis (nPEP) is under prescribed for people seeking treatment within 72 h of human immunodeficiency virus (HIV) exposures in the emergency department (ED). This study is an assessment of ED prescribers' knowledge, attitudes and practices regarding administration of HIV nPEP. METHODS: This was an anonymous survey based on literature review and modified Delphi technique. We approached 153 ED participants at work over a 4-month period from 5 hospital-based and 2 freestanding EDs. There were 152 completed surveys: 80 attendings, 27 residents, and 44 physician assistants. RESULTS: The majority of those surveyed (133/149, 89.3%) believe it is their responsibility to provide HIV nPEP in the ED. Although 91% (138/151) and 87% (132/151) of participants are willing to prescribe nPEP for IV drug use and unprotected sex, respectively, only 40% (61/152) of participants felt they could confidently prescribe the appropriate regimen. Only 25% (37/151) of participants prescribed nPEP in the last year. Participants considered time (27%), connecting patients to follow-up (26%), and cost to patients (23%), as barriers to prescribing nPEP. CONCLUSIONS: This study identified perceived barriers to administration of nPEP and missed opportunities for HIV prevention in the ED. Although most ED prescribers were willing to prescribe nPEP and felt it is their responsibility to do so, the majority of prescribers were not confident in prescribing it. The most commonly cited barriers to prescribing nPEP were time and access to follow-up care.

Pancreatic Islets Accumulate Cadmium in a Rodent Model of Cadmium-Induced Hyperglycemia.

Cadmium (Cd) is an anthropogenic as well as a naturally occurring toxicant associated with prediabetes and T2DM in humans and experimental models of Cd exposure. However, relatively few studies have examined the mechanism(s) of Cd-induced hyperglycemia. The purpose of this study was to examine the role of pancreatic islets in Cd-induced hyperglycemia. Male Sprague-Dawley rats were given daily subcutaneous doses of Cd at 0.6 mg/kg over 12 weeks. There was a resulting time-dependent increase in fasting blood glucose and altered insulin release in vitro. Islets isolated from control (saline-treated) and Cd-treated animals were incubated in low (0.5 mg/mL) or high (3 mg/mL) glucose conditions. Islets from 12 week Cd-treated animals had significantly less glucose-stimulated insulin release compared to islets from saline-treated control animals. The actual Cd content of isolated islets was 5 fold higher than the whole pancreas (endocrine + exocrine) and roughly 70% of that present in the renal cortex. Interestingly, islets isolated from Cd-treated animals and incubated in high glucose conditions contained significantly less Cd and zinc than those incubated in low glucose. These results show that within whole pancreatic tissue, Cd selectively accumulates in pancreatic islets and causes altered islet function that likely contributes to dysglycemia.

Using FluoZin-3 and fura-2 to monitor acute accumulation of free intracellular Cd2+ in a pancreatic beta cell line.

The understanding of cellular Cd2+ accumulation and toxicity is hampered by a lack of fluorescent indicators selective for intracellular free Cd2+ ([Cd2+]i). In this study, we used depolarized MIN6 mouse pancreatic beta cells as a model for evaluating [Cd2+]i detection with commercially available fluorescent probes, most of which have been traditionally used to visualize [Ca2+]i and [Zn2+]i. We trialed a panel of 12 probes including fura-2, FluoZin-3, Leadmium Green, Rhod-5N, indo-1, Fluo-5N, and others. We found that the [Zn2+]i probe FluoZin-3 and the traditional [Ca2+]i probe fura-2 responded most consistently and robustly to [Cd2+]i accumulation mediated by voltage-gated calcium channels. While selective detection of [Cd2+]i by fura-2 required the omission of Ca2+ from extracellular buffers, FluoZin-3 responded to [Cd2+]i similarly in the presence or absence of extracellular Ca2+. Furthermore, we showed that FluoZin-3 and fura-2 can be used together for simultaneous monitoring of [Ca2+]i and [Cd2+]i in the same cells. None of the other fluorophores tested were effective [Cd2+]i detectors in this model.

Community awareness of, use of and attitudes towards HIV pre-exposure prophylaxis (PrEP) among men who have sex with men in Vancouver, Canada: preparing health promotion for a publicly funded PrEP program.

Background HIV rates are persistently disproportionate among men who have sex with men (MSM). Pre-exposure prophylaxis (PrEP) is an effective HIV prevention method, now publicly funded in British Columbia. This study assessed PrEP-related attitudes, sexual behaviour and self-reported use before public funding. METHODS: Adult MSM were recruited from January to June 2017 through a local community-based organisation's PrEP campaign website (www.getpreped.ca). Participants self-completed an anonymous online questionnaire, and were stratified into three groups: (i) HIV-positive participants; (ii) HIV-negative participants not using PrEP; and (iii) HIV-negative participants using PrEP. Descriptive, bivariate and univariate regression analyses were conducted. RESULTS: Of 249 participants, 191 (77%) were HIV-negative not using PrEP, 41 (17%) were HIV-negative using PrEP and 17 (7%) were HIV-positive. Among PrEP users, 90% used PrEP daily and all reported having recommended medical follow-up care. Among HIV-negative, non-PrEP-users, 44% said they would reduce condom use if they used PrEP and 28% were uncomfortable asking their doctor for PrEP. Interest in PrEP among non-users was associated with higher objective risk scores (i.e. HIV Incidence Risk Index for MSM), higher self-perceived risk, greater perceived PrEP effectiveness, no prescription medications insurance, open or single relationship status (vs closed) and not always using condoms (vs always). Among HIV-positive participants, 53% agreed PrEP reduced stigma for people living with HIV. All study groups perceived a greater percentage of MSM on PrEP (10%, 15%, 18%) than in their own social networks (5%, 4%, 6%). CONCLUSIONS: PrEP health promotion must consider comprehensive PrEP education; accuracy of self-perceived HIV risk and PrEP social norms; and barriers to culturally safe primary care for MSM.

A quantitative description of tubular system Ca(2+) handling in fast- and slow-twitch muscle fibres.

KEY POINTS: Current methods do not allow a quantitative description of Ca(2+) movements across the tubular (t-) system membrane without isolating the membranes from their native skeletal muscle fibre. Here we present a fluorescence-based method that allows determination of the t-system [Ca(2+) ] transients and derivation of t-system Ca(2+) fluxes in mechanically skinned skeletal muscle fibres. Differences in t-system Ca(2+) -handling properties between fast- and slow-twitch fibres from rat muscle are resolved for the first time using this new technique. The method can be used to study Ca(2+) handling of the t-system and allows direct comparisons of t-system Ca(2+) transients and Ca(2+) fluxes between groups of fibres and fibres from different strains of animals. ABSTRACT: The tubular (t-) system of skeletal muscle is an internalization of the plasma membrane that maintains a large Ca(2+) gradient and exchanges Ca(2+) between the extracellular and intracellular environments. Little is known of the Ca(2+) -handling properties of the t-system as the small Ca(2+) fluxes conducted are difficult to resolve with conventional methods. To advance knowledge in this area we calibrated t-system-trapped rhod-5N inside skinned fibres from rat and [Ca(2+) ]t-sys , allowing confocal measurements of Ca(2+) -dependent changes in rhod-5N fluorescence during rapid changes in the intracellular ionic environment to be converted to [Ca(2+) ] transients in the t-system ([Ca(2+) ]t-sys (t)). Furthermore, t-system Ca(2+) -buffering power was determined so that t-system Ca(2+) fluxes could be derived from [Ca(2+) ]t-sys (t). With this new approach, we show that rapid depletion of sarcoplasmic reticulum (SR) Ca(2+) induced a robust store-operated Ca(2+) entry (SOCE) in fast- and slow-twitch fibres, reducing [Ca(2+) ]t-sys to < 0.1 mm. The rapid activation of SOCE upon Ca(2+) release was consistent with the presence of STIM1L in both fibre types. Abruptly introducing internal solutions with 1 mm Mg(2+) and [Ca(2+) ]cyto (28 nm-1.3 µm) to Ca(2+) -depleted fibres generated t-system Ca(2+) uptake rates dependent on [Ca(2+) ]cyto with [Ca(2+) ]t-sys reaching final plateaus in the millimolar range. For the same [Ca(2+) ]cyto , t-system Ca(2+) fluxes of fast-twitch fibres were greater than that in slow-twitch fibres. In addition, simultaneous imaging of t-system and SR Ca(2+) signals indicated that both membrane compartments accumulated Ca(2+) at similar rates and that SOCE was activated early during SR Ca(2+) depletion.

Mechanisms of growth and patterns of gene expression in oxygen-deprived rice coleoptiles.

Coleoptiles of rice (Oryza sativa) seedlings grown under water commonly elongate by up to 1 mm h(-1) to reach the atmosphere. We initially analysed this highly specialized phenomenon by measuring epidermal cell lengths along the coleoptile axis to determine elongation rates. This revealed a cohort of cells in the basal zone that elongated rapidly following emergence from the embryo, reaching 200 µm within 12 h. After filming coleoptiles in vivo for a day, kinematic analysis was applied. Eight time-sliced 'segments' were defined by their emergence from the embryo at four-hourly intervals, revealing a mathematically simple growth model. Each segment entering the coleoptile from the embryo elongated at a constant velocity, resulting in accelerating growth for the entire organ. Consistent with the epidermal cell lengths, relative rates of elongation (mm mm(-1) h(-1)) were tenfold greater in the small, newly emerged basal segments than the older distal tip segments. This steep axial gradient defined two contrasting growth zones (bases versus tips) in which we measured ATP production and protein, RNA and DNA content, and analysed the global transcriptome under steady-state normoxia, hypoxia (3% O2) and anoxia. Determination of the transcriptome revealed tip-specific induction of genes encoding TCP [Teosinte Branched1 (Tb1) of maize, Cycloidea (Cyc), and Proliferating Cell Factor (Pcf)] transcription factors, RNA helicases, ribosomal proteins and proteins involved in protein folding, whilst expression of F-box domain-containing proteins in the ubiquitin E3-SCF complex (Skp, Cullin, F-box containing complex) was induced specifically in bases under low oxygen conditions. We ascribed the sustained elongation under hypoxia to hypoxia-specific responses such as controlled suppression of photosystem components and induction of RNA binding/splicing functions, indicating preferential allocation of energy to cell extension.

Evaluation of cystatin C as an early biomarker of cadmium nephrotoxicity in the rat.

Cadmium (Cd) is a nephrotoxic environmental pollutant that causes insidious injury to the proximal tubule that results in severe polyuria and proteinuria. Cystatin C is a low molecular weight protein that is being evaluated as a serum and urinary biomarker for various types of ischemic and nephrotoxic renal injury. The objective of the present study was to determine if cystatin C might be a useful early biomarker of Cd nephrotoxicity. Male Sprague-Dawley rats were given daily injections of Cd for up to 12 weeks. At 3, 6, 9 and 12 weeks, urine samples were analyzed for cystatin C, protein, creatinine, ß2 microglobulin and kidney injury molecule-1. The results showed that Cd caused a significant increase in the urinary excretion of cystatin C that occurred 3-4 weeks before the onset of polyuria and proteinuria. Serum levels of cystatin C were not altered by Cd. Immunolabeling studies showed that Cd caused the relocalization of cystatin C from the cytoplasm to the apical surface of the epithelial cells of the proximal tubule. The Cd-induced changes in cystatin C labelling paralleled those of the brush border transport protein, megalin, which has been implicated as a mediator of cystatin C uptake in the proximal tubule. These results indicate that Cd increases the urinary excretion of cystatin C, and they suggest that this effect may involve disruption of megalin-mediated uptake of cystatin C by epithelial cells of the proximal tubule.

Effects of Maternal Exposure to Cadmium Oxide Nanoparticles During Pregnancy on Maternal and Offspring Kidney Injury Markers Using a Murine Model.

Nanoparticles (NP) are pervasive in many areas of modern life, with little known about their potential toxicities. One commercially important NP is cadmium oxide (CdO), which is used to synthesize other Cd-containing NP, such as quantum dots. Cadmium (Cd) is a well-known nephrotoxicant, but the nephrotoxic potential of CdO NP remains unknown, particularly when exposure occurs during pregnancy. Therefore, pregnant CD-1 mice were used to examine the effects of inhaled CdO NP (230 µg CdO NP/m(3)) on maternal and neonatal renal function by examining urinary creatinine and urinary biomarkers of kidney injury, including kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (NGAL). Inhalation of CdO NP by dams produced a fivefold increase in urinary Kim-1 with no marked effect on urinary creatinine levels. Kim-1 mRNA expression peaked by gestational day (GD) 10.5, and NGAL expression increased from GD 10.5 to 17.5. In addition, histological analyses revealed proximal tubular pathology at GD 10.5. Neonatal Kim-1 mRNA expression rose between postnatal days (PND) 7 and 14, with mammary glands/milk being the apparent source of Cd for offspring. These studies demonstrate that, similar to what is seen with other Cd forms, Cd associated with inhaled CdO NP results in renal injury to both directly exposed dam and offspring. As commercial uses for nanotechnology continue to expand throughout the world, risks for unintentional exposure in the workplace increase. Given the large number of women in the industrial workforce, care needs to be taken to protect these already vulnerable populations.

IL-1α reversibly inhibits skeletal muscle ryanodine receptor. a novel mechanism for critical illness myopathy?

Critical illness myopathies in patients with sepsis or sustained mechanical ventilation prolong intensive care treatment and threaten both patients and health budgets; no specific therapy is available. Underlying pathophysiological mechanisms are still patchy. We characterized IL-1α action on muscle performance in "skinned" muscle fibers using force transducers and confocal Ca(2+) fluorescence microscopy for force/Ca(2+) transients and Ca(2+) sparks. Association of IL-1α with sarcoplasmic reticulum (SR) release channel, ryanodine receptor (RyR) 1, was investigated with coimmunoprecipitation and confocal immunofluorescence colocalization. Membrane integrity was studied in single, intact fibers challenged with IL-1α. IL-1α reversibly stabilized Mg(2+) inhibition of Ca(2+) release. Low Mg(2+)-induced force and Ca(2+) transients were reversibly abolished by IL-1α. At normal Mg(2+), IL-1α reversibly increased caffeine-induced force and Ca(2+) transients. IL-1α reduced SR Ca(2+) leak via RyR1, as judged by (1) increased SR Ca(2+) retention, (2) increased IL-1α force transients being reproduced by 25 µM tetracaine, and (3) reduced Ca(2+) spark frequencies by IL-1α or tetracaine. Coimmunoprecipitation confirmed RyR1/IL-1 association. RyR1/IL-1 immunofluorescence patterns perfectly colocalized. Long-term, 8-hour IL-1α challenge of intact muscle fibers compromised membrane integrity in approximately 50% of fibers, and confirmed intracellular IL-1α deposition. IL-1α exerts a novel, specific, and reversible interaction mechanism with the skeletal muscle RyR1 macromolecular release complex without the need to act via its membrane IL-1 receptor, as IL-1R membrane expression levels were not detectable in Western blots or immunostaining of single fibers. We present a potential explanation of how the inflammatory mediator, IL-1α, may contribute to muscle weakness in critical illness.

Activation and propagation of Ca2+ release from inside the sarcoplasmic reticulum network of mammalian skeletal muscle.

Skeletal muscle fibres are large and highly elongated cells specialized for producing the force required for posture and movement. The process of controlling the production of force within the muscle, known as excitation-contraction coupling, requires virtually simultaneous release of large amounts of Ca(2+) from the sarcoplasmic reticulum (SR) at the level of every sarcomere within the muscle fibre. Here we imaged Ca(2+) movements within the SR, tubular (t-) system and in the cytoplasm to observe that the SR of skeletal muscle is a connected network capable of allowing diffusion of Ca(2+) within its lumen to promote the propagation of Ca(2+) release throughout the fibre under conditions where inhibition of SR ryanodine receptors (RyRs) was reduced. Reduction of cytoplasmic [Mg(2+)] ([Mg(2+)]cyto) induced a leak of Ca(2+) through RyRs, causing a reduction in SR Ca(2+) buffering power argued to be due to a breakdown of SR calsequestrin polymers, leading to a local elevation of [Ca(2+)]SR. The local rise in [Ca(2+)]SR, an intra-SR Ca(2+) transient, induced a local diffusely rising [Ca(2+)]cyto. A prolonged Ca(2+) wave lasting tens of seconds or more was generated from these events. Ca(2+) waves were dependent on the diffusion of Ca(2+) within the lumen of the SR and ended as [Ca(2+)]SR dropped to low levels to inactivate RyRs. Inactivation of RyRs allowed re-accumulation of [Ca(2+)]SR and the activation of secondary Ca(2+) waves in the persistent presence of low [Mg(2+)]cyto if the threshold [Ca(2+)]SR for RyR opening could be reached. Secondary Ca(2+) waves occurred without an abrupt reduction in SR Ca(2+) buffering power. Ca(2+) release and wave propagation occurred in the absence of Ca(2+)-induced Ca(2+) release. These observations are consistent with the activation of Ca(2+) release through RyRs of lowered cytoplasmic inhibition by [Ca(2+)]SR or store overload-induced Ca(2+) release. Restitution of SR Ca(2+) buffering power to its initially high value required imposing normal resting ionic conditions in the cytoplasm, which re-imposed the normal resting inhibition on the RyRs, allowing [Ca(2+)]SR to return to endogenous levels without activation of store overload-induced Ca(2+) release. These results are discussed in the context of how pathophysiological Ca(2+) release such as that occurring in malignant hyperthermia can be generated.

Urocortin 2 stimulates nitric oxide production in ventricular myocytes via Akt- and PKA-mediated phosphorylation of eNOS at serine 1177.

Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca(2+)-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A (PKA) reduced Ucn2-induced phosphorylation of eNOS but did not affect the increase in phosphorylation of Akt. Conversely, direct receptor-independent elevation of cAMP via forskolin increased phosphorylation of eNOS but not of Akt. Ucn2 increased intracellular NO concentration ([NO]i), [cGMP], [cAMP], and cell shortening. Inhibition of eNOS suppressed the increases in [NO]i and cell shortening. When both PI3K-Akt and cAMP-PKA signaling were inhibited, the Ucn2-induced increases in [NO]i and cell shortening were attenuated. Thus, in rabbit ventricular myocytes, Ucn2 causes activation of cAMP-PKA, PI3K-Akt, and MEK1/2-ERK1/2 signaling. The MEK1/2-ERK1/2 pathway is not required for stimulation of NO signaling in these cells. The other two pathways, cAMP-PKA and PI3K-Akt, converge on eNOS phosphorylation at Ser1177 and result in pronounced and sustained cellular NO production with subsequent stimulation of cGMP signaling.

Cardiac alternans and intracellular calcium cycling.

Cardiac alternans refers to a condition in which there is a periodic beat-to-beat oscillation in electrical activity and the strength of cardiac muscle contraction at a constant heart rate. Clinically, cardiac alternans occurs in settings that are typical for cardiac arrhythmias and has been causally linked to these conditions. At the cellular level, alternans is defined as beat-to-beat alternations in contraction amplitude (mechanical alternans), action potential duration (APD; electrical or APD alternans) and Ca(2+) transient amplitude (Ca(2+) alternans). The cause of alternans is multifactorial; however, alternans always originate from disturbances of the bidirectional coupling between membrane voltage (Vm ) and intracellular calcium ([Ca(2+) ]i ). Bidirectional coupling refers to the fact that, in cardiac cells, Vm depolarization and the generation of action potentials cause the elevation of [Ca(2+) ]i that is required for contraction (a process referred to as excitation-contraction coupling); conversely, changes of [Ca(2+) ]i control Vm because important membrane currents are Ca(2+) dependent. Evidence is mounting that alternans is ultimately caused by disturbances of cellular Ca(2+) signalling. Herein we review how two key factors of cardiac cellular Ca(2+) cycling, namely the release of Ca(2+) from internal stores and the capability of clearing the cytosol from Ca(2+) after each beat, determine the conditions under which alternans occurs. The contributions from key Ca(2+) -handling proteins (i.e. surface membrane channels, ion pumps and transporters and internal Ca(2+) release channels) are discussed.

Treating Diphenhydramine Overdose: A Literature Review of Currently Available Treatment Methods.

From 2019 to 2020, antihistamines were found in 15% of all US drug overdose deaths, often co-administered with fentanyl, with 3.6% of overdose deaths due to antihistamines alone. The most common antihistamine found in all these reported deaths is diphenhydramine, a ubiquitous, over-the-counter and clinically important medication. Currently, there is no antidote for diphenhydramine overdose. This review summarizes the adverse health effects and current emergency medicine treatments for diphenhydramine. Several emergency medicine case reports are reviewed, and the efficacy and outcomes of a variety of treatments are compared. The treatments reviewed include the more traditional antihistamine overdose therapeutics physostigmine and sodium bicarbonate, as well as newer ones such as donepezil, dexmedetomidine, and lipid emulsion therapy. We conclude that more study is needed to determine the ideal therapeutic approach to treating antihistamine overdoses.

Molecular motor tug-of-war regulates elongasome cell wall synthesis dynamics in Bacillus subtilis.

Most rod-shaped bacteria elongate by inserting new cell wall material into the inner surface of the cell sidewall. This is performed by class A penicillin binding proteins (PBPs) and a highly conserved protein complex, the elongasome, which moves processively around the cell circumference and inserts long glycan strands that act as barrel-hoop-like reinforcing structures, thereby giving rise to a rod-shaped cell. However, it remains unclear how elongasome synthesis dynamics and termination events are regulated to determine the length of these critical cell-reinforcing structures. To address this, we developed a method to track individual elongasome complexes around the entire circumference of Bacillus subtilis cells for minutes-long periods using single-molecule fluorescence microscopy. We found that the B. subtilis elongasome is highly processive and that processive synthesis events are frequently terminated by rapid reversal or extended pauses. We found that cellular levels of RodA regulate elongasome processivity, reversal and pausing. Our single-molecule data, together with stochastic simulations, show that elongasome dynamics and processivity are regulated by molecular motor tug-of-war competition between several, likely two, oppositely oriented peptidoglycan synthesis complexes associated with the MreB filament. Altogether these results demonstrate that molecular motor tug-of-war is a key regulator of elongasome dynamics in B. subtilis, which likely also regulates the cell shape via modulation of elongasome processivity.

Effects of cadmium on the sub-cellular localization of ß-catenin and ß-catenin-regulated gene expression in NRK-52E cells.

The E-cadherin/ß-catenin complex is a structural component of adherens-type junctions in epithelial cells. Moreover, ß-catenin acts as an intracellular signaling molecule that can influence the expression of a variety of genes that regulate apoptosis and cell cycle control. Cadmium (Cd) is an environmental toxicant that causes renal dysfunction and disrupts cadherin-dependent cell-cell adhesion in various types of epithelial cells. In this study, we examined the effects of Cd on the subcellular localization of ß-catenin, the cadherin/ß-catenin complex and ß-catenin-mediated gene transcription in rat proximal tubule NRK-52E cells. Exposure to 5-10 µM Cd for 4 h caused the NRK cells to separate from each other without killing the cells or causing them to detach from the growing surface. This effect was associated with the loss of ß-catenin and E-cadherin from the cell-cell contacts and apparent changes in the accumulation of ß-catenin in the nuclear cell subfraction. The expression of the ß-catenin-sensitive gene, c-jun was significantly increased in cells exposed to 5 µM Cd. However, there was no change in the expression of several other ß-catenin-regulated genes including: c-myc, cyclin D1 and matrilysin. Additional studies utilizing the TOPFLASH ß-catenin reporter gene construct showed that Cd caused a 2-3 fold increase in the expression of the luciferase reporter gene. Overall, these results indicate that Cd disrupts the cadherin/ß-catenin complex in NRK-52E cells, but this effect leads to only partial activation of ß-catenin-mediated gene transcription.

Dermatologic conditions in dancers: a statewide survey.

Performing artists, such as dancers, singers, actors and musicians, rely on their physical bodies to successfully execute their artforms. However, literature regarding dermatologic conditions that impact dancers is lacking. An anonymous REDCap® secure survey was distributed by email to Dance Majors, Dance Minors, and Dance Instructors/Professors at five Virginia undergraduate institutions. Responses regarding demographics, style of dance, and dermatological diseases were recorded over a 2 month period. When asked about developing skin disease, 57 (59%) of survey participants reported experiencing skin diseases, such as acne, eczema, hyperhidrosis, and plantar warts. When asked about skin diseases exacerbated or believed to be caused from dancing, 56 (59%) reported blisters, callouses, skin splitting, nail/foot infection, ingrown nails, and floor burns. This study demonstrates two main findings: dancing may exacerbate current skin disorders and some skin conditions may be caused by dancing. Additionally, the common practice of dancing barefoot likely contributes to the development of certain skin conditions. Limitations include sample size, response bias, and lack of validation of the survey.

Changes in plasma membrane Ca-ATPase and stromal interacting molecule 1 expression levels for Ca(2+) signaling in dystrophic mdx mouse muscle.

The majority of the skeletal muscle plasma membrane is internalized as part of the tubular (t-) system, forming a standing junction with the sarcoplasmic reticulum (SR) membrane throughout the muscle fiber. This arrangement facilitates not only a rapid and large release of Ca(2+) from the SR for contraction upon excitation of the fiber, but has also direct implications for other interdependent cellular regulators of Ca(2+). The t-system plasma membrane Ca-ATPase (PMCA) and store-operated Ca(2+) entry (SOCE) can also be activated upon release of SR Ca(2+). In muscle, the SR Ca(2+) sensor responsible for rapidly activated SOCE appears to be the stromal interacting molecule 1L (STIM1L) isoform of STIM1 protein, which directly interacts with the Orai1 Ca(2+) channel in the t-system. The common isoform of STIM1 is STIM1S, and it has been shown that STIM1 together with Orai1 in a complex with the partner protein of STIM (POST) reduces the activity of the PMCA. We have previously shown that Orai1 and STIM1 are upregulated in dystrophic mdx mouse muscle, and here we show that STIM1L and PMCA are also upregulated in mdx muscle. Moreover, we show that the ratios of STIM1L to STIM1S in wild-type (WT) and mdx muscle are not different. We also show a greater store-dependent Ca(2+) influx in mdx compared with WT muscle for similar levels of SR Ca(2+) release while normal activation and deactivation properties were maintained. Interestingly, the fiber-averaged ability of WT and mdx muscle to extrude Ca(2+) via PMCA was found to be the same despite differences in PMCA densities. This suggests that there is a close relationship among PMCA, STIM1L, STIM1S, Orai1, and also POST expression in mdx muscle to maintain the same Ca(2+) extrusion properties as in the WT muscle.

Longitudinal and transversal propagation of excitation along the tubular system of rat fast-twitch muscle fibres studied by high speed confocal microscopy.

Mammalian skeletal muscle fibres possess a tubular (t-) system that consists of regularly spaced transverse elements which are also connected in the longitudinal direction. This tubular network provides a pathway for the propagation of action potentials (APs) both radially and longitudinally within the fibre, but little is known about the actual radial and longitudinal AP conduction velocities along the tubular network in mammalian skeletal muscle fibres. The aim of this study was to track AP propagation within the t-system network of fast-twitch rat muscle fibres with high spatio-temporal resolution when the t-system was isolated from the surface membrane. For this we used high speed confocal imaging of AP-induced Ca(2+) release in contraction-suppressed mechanically skinned fast-twitch fibres where the t-system can be electrically excited in the absence of the surface membrane. Supramaximal field pulses normally elicited a synchronous AP-induced release of Ca(2+) along one side of the fibre axis which propagated uniformly across the fibre. In some cases up to 80 or more adjacent transverse tubules failed to be excited by the field pulse, while adjacent areas responded with normal Ca(2+) release. In these cases a continuous front of Ca(2+) release with an angle to the scanning line was observed due to APs propagating longitudinally. From these observations the radial/transversal and longitudinal AP conduction velocities along the tubular network deeper in the fibre under our conditions (19 ± 1°C) ranged between 8 and 11 µm ms(-1) and 5 to 9 µm ms(-1), respectively, using different methods of estimation. The longitudinal propagation of APs appeared to be markedly faster closer to the edge of the fibre, in agreement with the presence of dense longitudinal connections immediately below the surface of the fibre and more sparse connections at deeper planes within the fibre. During long trains of closely spaced field pulses the AP-elicited Ca(2+) releases became non-synchronous along the fibre axis. This is most likely caused by local tubular K(+) accumulation that produces local depolarization and local slowing of AP propagation. Longitudinally propagating APs may reduce such inhomogeneities by exciting areas of delayed AP onset. Clearly, the longitudinal tubular pathways within the fibre for excitation are used as a safety mechanism in situations where a local depolarization obstructs immediate excitation from the sarcolemma. Results obtained from this study also provide an explanation for the pattern of contractures observed in rippling muscle disease.