The elevations are the result of repeated release and re-uptake of Ca2+ stored in the smooth endoplasmic reticulum. Ca2+ release is primarily mediated by the phosphoinositide signaling system of the oocyte. The system is stimulated when the sperm causes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG); IP3 then binds its receptor on the surface of the endoplasmic reticulum that induces Ca2+ release. The manner in which the sperm generates IP3, the Ca2+ mobilizing second messenger, has been the subject of extensive research for a long time. The sperm factor hypothesis has eventually gained general acceptance, according to which it is a molecule from the sperm that diffuses into the ooplasm and stimulates the phosphoinositide cascade. Much evidence now indicates that the sperm-derived factor is phospholipase C-zeta (PLCζ) that cleaves PIP2 and generates IP3, eventually leading to oocyte activation. A recent addition to the candidate sperm factor list is the post-acrosomal sheath WW domain-binding protein (PAWP), whose role at fertilization is currently under debate. Ca2+ influx across the plasma membrane is also important as, in the absence of extracellular Ca2+, the oscillations run down prematurely. In pig oocytes, the influx that sustains the oscillations seems to be regulated by the filling status of the stores, whereas in the mouse other mechanisms might be involved. This work summarizes the current understanding of Ca2+ signaling in mammalian oocytes.Mammalian embryo development begins when the fertilizing sperm triggers a series of elevations in the oocyte’s intracellular free Ca Prior to fertilization, mammalian oocytes are arrested at the metaphase stage of the second meiotic cell division. The meiotic block is characteristic of the entire animal kingdom and, although it may happen at various stages of the cell cycle, its role is to prevent entry into the embryonic cell cycles without the sperm  was singled out by Lewis Victor Heilbrunn. Although the importance of Ca2+ in the contraction of skeletal muscle was demonstrated earlier  the first Ca2+ transient  and stored while attached to Ca2+-binding proteins  receptor is a protein complex of four subunits that surround the channel pore , by Ca2+ itself and in skeletal muscle by electromechanical coupling to the dihydropiridine receptor located in the plasma membrane , a cytoplasmic enzyme, cleaves phosphatidylinositol 4,5-bisphosphate (PIP2), a phosphoinositide, into IP3 and diacylglycerol (DAG)  domains, Src homology 2 (SH2) domains and constant or conserved region 2 (C2) domains. The mechanism of activation varies depending on the specific combination of regulatory domains present in each PLC isozyme. PH domains bind phosphoinositides such as PIP2 and PIP3 and thus they typically serve to target PLC to the plasma membrane where most phosphoinositides are located. In addition, they can also mediate interaction with heterotrimeric G proteins  can also trigger Ca2+ oscillations in mammalian oocytes  caused activation in sea urchin eggs  databases led to the identification and eventual amplification of a new, testis-specific PLC variety, termed PLCζ . WW domains are small functional modules found in many signaling and structural proteins and are known to mediate protein–protein interactions. They are named after their two signature tryptophan (W) residues that play an important role in the domains’ function. Interaction between WW domain-containing proteins and their ligands is important for a great number of cellular events such as transcriptional activation, cell cycle control and ubiquitin ligation  pumps and Na+/Ca2+ exchangers in the plasma membrane are also available for Ca2+ removal  were reduced in frequency and ultimately stopped upon superfusion with Ca2+-free medium (Igusa and Miyazaki 2+ spikes in fertilized mouse oocytes slows down or stops if Ca2+ is removed from the extracellular medium (Kline and Kline 2+ influx mechanism during fertilization were provided by experiments using dithiothreitol (DTT), a sulfhydryl reducing agent. In unfertilized mouse oocytes, DTT is able to stimulate the influx of divalent cations (including Ca2+), whereas in fertilized ones it increases the frequency of the Ca2+ oscillations  between the transients . 12-O-tetradecanoyl phorbol acetate (TPA) and phorbol-12-myristate-13-acetate (PMA) are phorbol esters that can very effectively stimulate PKC. When applied to mouse oocytes, they cause low amplitude Ca2+ oscillations and a variety of oocyte activation events downstream of the Ca2+ signal  and Orai proteins. STIM1 and STIM2 are transmembrane proteins that reside in the endoplasmic reticulum , relocates to regions close to the plasma membrane and activates the Ca2+ entry channels. The channel is formed by Orai proteins . Orai1, the most potent isoform, resides in the oolemma and, once stimulated by STIM1, it allows Ca2+ in the extracellular medium to flow into the cell  channel. The TRP protein serves as a Ca2+ channel in a number of cell types and is expressed in various oocytes  couples worldwide (Ledger 2+ oscillations, as media composition reportedly affects Ca2+ signaling and subsequent embryo development (Dumollard et al. 2+ signal, along with the entire mechanism that is set in motion by the factor, will ultimately lead to the development of methods to effectively activate oocytes when spermatozoa are unable to do so.A better understanding of the signaling mechanism that operates at fertilization offers major benefits. Proper activation of the oocyte’s developmental program is critical not only during fertilization under normal physiological conditions but also for the success of a number of assisted reproductive technologies. Genetically modified animals have vast potentials and one powerful technology to generate such animals is somatic cell nuclear transfer. Artificial oocyte activation is an integral part of the technology; however, our inability to properly activate the reconstructed oocyte is one of the reasons for the extreme inefficiency of nuclear transfer procedures Prather . Becausee Ledger . Althouge Ledger . Howevere Ledger  or PAWP e Ledger . PLCζ dee Ledger , while Pe Ledger . Thus, t


Moreover, Pb treatment inhibits the sodium/calcium exchanger 3 (NCX3) mRNA expression, one of the major means of calcium (Ca2+) extrusion at the plasma membrane during OPCs differentiation. Also addition of KB-R7943, NCX3 inhibitor, to simulate Pb toxicity, resulted in decreased myelin basic protein (MBP) expression and cell branching. Ca2+ response trace with Pb and KB-R7943 treatment did not drop down in the same recovery time as the control, which elevated intracellular Ca2+ concentration reducing MBP expression. In contrast, over-expression of NCX3 in Pb exposed OPCs displayed significant increase MBP fluorescence signal in positive regions and CNPase expression, which recovered OPCs differentiation to counterbalance Pb toxicity. In conclusion, Pb exposure disturbs OLs differentiation via affecting the function of NCX3 by inducing intracellular calcium overload.Lead (Pb) poisoning has always been a serious health concern, as it permanently damages the central nervous system. Chronic Pb accumulation in the human body disturbs oligodendrocytes (OLs) differentiation, resulting in dysmyelination, but the molecular mechanism remains unknown. In this study, Pb at 1 μM inhibits OLs precursor cells (OPCs) differentiation via decreasing the expression of Olig 2, CNPase proteins  Its devastating effect on central nervous system (CNS) is the most important problem [in vivo [Lead (Pb) poisoning has been a serious public health problem worldwide, as it causes permanent nervous system damage . Excessi problem . Acute P[in vivo . However[in vivo . The mol[in vivo  and seri[in vivo . Among m[in vivo . Pb dire2+) signaling in OLs differentiation and myelination. Changes in intracellular Ca2+ levels not only influence the transition of OPCs into mature myelinating OLs, but also intervene in the initiation of myelination and remyelination processes [2+ levels is crucial. As maintaining Ca2+ homeostasis is critical for the viability and function of OLs, disturbance of Ca2+ homeostasis is a hallmark of damage. However, reports assessing the mechanism of Pb induced disturbance of Ca2+ are scarce.During CNS development, OPCs need to pass through four stages to become mature myelin-forming cells. Abnormalities or injury in OLs usually leads to demyelination disease ,11,12. Arocesses . Thus, a2+ extrusion proteins. As a result, accumulation of oxidative stress occurs in the cell which subsequently interferes with intracellular Ca2+ homeostasis causing cellular damage. One of the major means of Ca2+ extrusion at the plasma membrane of many excitable and non-excitable cells is the sodium/calcium exchanger (NCX), which helps in rapid recovery from high intracellular Ca2+ concentrations and protects cells from Ca2+ overload and eventual death. Although the expression of the different NCX mRNAs in OLs has been investigated [2+ elevation followed by OLs damage has not yet been investigated.Recent studies have shown that Pb enhances the generation of reactive oxygen species and reduces the antioxidant defense system of cells ,15, thusstigated , their rNCX genes  have been reported in mammals [NCX3 in the regulation of the physiological and pathological functions of the CNS has been widely recognized [et al. reported that silencing or knocking out of NCX3, but not NCX2 and NCX1, impairs OLs differentiation [2+ homeostatic mechanisms is an important feature of Pb toxicity and NCX3 contributes to Ca2+ influx. The present study sought to determine the extent to which inhibition of NCX3 may affect Pb toxicity in OLs lineage. We assessed the functional activity of NCX3 during OLs development in primary OPCs cultures. The results demonstrated that expression of NCX3 is strongly down-regulated in the Pb-exposed OLs, which impairs OLs differentiation, resulting in dysmyelination. Furthermore, the over-expression of NCX3 reversed Pb-induced disturbances of oligodendrocytes differentiation. These findings provide an important insight into the molecular mechanism of Pb toxicity on OLs.Three different  mammals ,18. The cognized . Boscia ntiation . As dysrin vitro, MTT  assay was performed to determine the cell viability , the reaction time of MTT experiments were prolonged from 24 to 72 h B. It hasin vitro , CNPase (a developing OLs marker), and MBP (a mature OLs marker) were assessed to identify the differentiation stage of OLs by immunofluorescence staining . This suggests that NCX3 plays a crucial role in OLs maturation in normal differentiation medium, but not NCX1 or NCX2.To understand the mechanism of Pb-induced dysmyelination and the function of staining A. In nor of OPCs B. A signNCXs, the transcripts were observed and significant reduction in NCX3 mRNA was reported  by Fluo-3 video-imaging. Firstly, intracellular Ca2+ concentrations at resting condition were recorded and distinguished using pseudo-color signals. Pseudo-color signals were observed weakly in the somatic region in normal controls , the trail of the elevated curve did not drop back down to baseline levels  were significantly decreased in Pb-treated OLs  was significantly decreased in cultured OPCs with 1 μM Pb and OLs presented immature features compared with the control group. Furthermore, to explore the molecular mechanism of Pb-induced damage on OLs, we investigated expression of NCX is a bidirectional membrane ion transporter that couples the influx/efflux of Ca2+ to the efflux/influx of Na+ and regulates the levels of intracellular Ca2+ [NCX genes  have been identified in mammals [NCX3 in the regulation of physiological and pathological functions of neural cells has been widely recognized. Evidence indicates that NCX3 silencing by RNA interference increases vulnerability of cerebellar granule neurons to Ca2+ overload [NCX3 significantly decreased the expression of MBP, however, increased the expression of NG2+ (marker for OPCs) in cultured OLs. Inhibition of NCX3 directly affects the differentiation of OPCs causing development arrest at immature stage. In addition, no alteration was observed in the level of Ki67, indicating that Pb toxicity did not influence the survivor of OPCs, but delayed its differentiation. Our study has also reported the involvement of downstream Ca2+ signaling mediated by NCX3 during OLs development. Inhibition of NCX3 notably increased intracellular Ca2+. Thus, the changes in intracellular Ca2+ levels regulated by NCX3 influences the developmental processes of OPCs into mature OLs [NCX3 was overexpressed in Pb-exposed OLs, the expression of MBP and CNPase proteins were partly recovered, indicating the crucial role of NCX3 in Pb-induced injury. It also provided evidence for therapy and prevention of Pb poisoning as well as instructions for drug development.lar Ca2+ . Three d mammals ,18. The overload . Our resture OLs . When NC2+ can occur in many cell types as a consequence of receptor overstimulation and exposure to cytotoxic agents [2+ overload resulting from dysregulation of channels or pumps, resulting in either apoptotic or necrotic cell death. Utilizing live cell imaging, we also observed that intracellular Ca2+ levels increased persistently (resulting in Ca2+ overload), without returning to baseline levels after treatment with Pb or the NCX3 blocker. Changes in Ca2+ channel expression during OLs development are functionally linked to the different developmental stages of myelination. The expression of NCX3 in Pb-exposed OLs decreased, leading to a change in Ca2+ homeostasis. These changes can then cause Ca2+ overload, which will cause OLs injury.Numerous studies have found that a lethal influx of Cac agents . OLs, li2+ ions eagerly interact with various biological molecules. However, high Ca2+ concentrations, at the same time, are harmful for biological materials, as excess Ca2+ causes the aggregation of nucleic acids and affects the expression of genes, especially transcription factors [+ cell development at embryonic and perinatal stages, which can be rescued by the addition of a transgene containing the Olig2 locus [in vitro provides further evidence that elevation of intracellular concentrations of Ca2+ resulting from down-regulation of NCX3 expression by Pb treatment inhibits expression of Olig2, reducing OPCs differentiation.Due to the special properties of flexible coordination chemistry, high affinity for carboxylate oxygen, and effects on fluidity and fusion of cellular membranes, Ca factors . The for factors . The bas factors ,34,35,36g2 locus ,38. In tNCX3 results in intracellular Ca2+ overload which, in turn, affects the expression of other genes involved in OPCs differentiation, such as the transcription factor Olig2. In contrast, overexpression of NCX3 in Pb-exposed OPCs counterbalanced injuries. Therefore, the results in the present study suggest that a critical step in the molecular mechanism of Pb toxicity is on OLs.Overall, the results of the present study suggest that in Pb-exposed OPCs, the down-regulation of d-lysine for experiments. The OPCs were induced to differentiate by OPC differentiation medium  + 1% fetal bovine serum + 5 mg/mL insulin). At the indicated time points, the cells were used for respective tests.The rat OPCs were propagated . At firs4/well) were seeded in 96-well plates and cultured for 6 h at 37 °C. Pb acetate was added in increasing concentrations (0.5–10 μm) and incubated for 24–72 h, followed by addition of 20 μL MTT  for another 4 h. then the supernatant was discarded and 150 μL DMSO was used to dissolve formazan. The OD for each well was measured at 540 nm using a SpectraMax M2e spectrophotometer .The rat OPCs cell line (1 × 10NCX3) or 1 μm Pb acetate for three days, then incubated in imaging wash buffer  containing 5 μM fluo-3 AM for 20 min at 37 °C. After incubation, the cells were maintained for at least 20 min at room temperature (RT) in fresh imaging wash buffer for complete dye de-esterification followed by qualitative evaluation of intracellular Ca2+ level in cells at the resting condition or the changes in its activity by 20 mM K+ stimulation under confocal laser scanning microscope. Fluorescence images were acquired and measured through confocal laser scanning microscope  and Fluoview image processing software . The experiment was carried out in triplets with 10 cells recorded for each experiment.The OPCs/OLs were grown and differentiated in glass-bottom dishes treated and untreated with KB-R7943 . The immunoreactivity was determined using confocal laser-scanning microscope . Morphological features of single cells were recorded using differential interference contrast (DIC) under confocal microscopy.NCX1: 5ʹ-CTGGAGCGCGAGGAAATGTTA-3ʹ and 5ʹ-GACGGGGTTCTCCAATCT-3ʹ; NCX2: 5ʹ-AGGAGGCCGCACACCTTTCC-3ʹ and 5ʹ-CAAGGCGTGGCTGGGCTCTC-3ʹ; NCX3: 5ʹ-GGCTGCACCATTGGTCTCA-3ʹ and 5ʹ-GACGGGGTTCTCCAATCT-3ʹ; Ki67: 5ʹ-AAGAGTGAGGGAATGCCTAT-3ʹ and 5ʹ-GCTTTCTTGGGAATGTCTGT-3ʹ; β-actin: 5ʹ-CGTTGACATCCGTAAAGACC-3ʹ and 5ʹ-CATCGTACTCCTGCTTGCT-3ʹ. The optical density of the bands of PCR products  was determined by Image Pro Plus image analysis system .Total RNA of cultured cells was collected using the Trizol reagent  according to the manufacture’s manual. Single strand cDNA was synthesized from the total RNA using random primer and SuperScriptase III (Life Technology). The specific primers of target genes were as follows: Protein samples were extracted from cultured cells using RIPA (Radio Immunoprecipitation Assay) lysis buffer with freshly 1% PMSF solution . These proteins were then denatured and separated using SDS-PAGE, followed by electrophoresis; the proteins were transferred to polyvinyldifluoride membranes. The membranes were incubated overnight at 4 °C with the primary antibodies and then incubated with HRP-linked secondary antibodies. Immunoreactive bands were detected using an ECL plus detection kit . The optical density of the bands  was determined by Image Pro Plus image analysis system and the protein determination was done using a Coomassie Brilliant Blue G250 .NCX3 gene was subcloned into pCMV-N-His vector. The control plasmid pCMV-N-His and pCMV-NCX3 were transfected into OPCs (4.5 × 104 cells per well) using Lipofectamine 3000 (Invitrogen) for 24 h and the cells were cultured in OPC-Differentiation Medium for three days.Rat β-actin) was also determined by Image Pro Plus image analysis system.For Statistical analysis, at least nine representative fields were randomly acquired at 20× magnification from each of the two experiments performed in triplicate. Cell counting was conducted on nine randomly-chosen fields for each sample. Quantification of immunostaining and cell counting was performed using the Image Pro Plus software. The optical density of the bands of PCR products or Western blot results  followed by Tukey’s


Omalizumab has been investigated in various other conditions including chronic urticaria (CU), perennial and seasonal allergic rhinitis (AR), pruritic bullous pemphigoid, latex allergy, peanut allergy, idiopathic anaphylaxis, hyper-IgE syndrome, chronic rhinosinusitis, interstitial cystitis, aspirin sensitivity, mastocytosis, eosinophilic gastroenteritis and atopic dermatitis. Most patients with chronic urticaria have an autoimmune cause: some patients produce IgE autoantibodies against autoantigens, such as thyroperoxidase or doublestranded DNA, whereas other patients make IgG autoantibodies against FcεRI, IgE, or both, which might chronically activate mast cells and basophils.I had a male patients with food allergy with pulmonary carsinoid tumor, aged 23. Autologous Serum Skin Test, Anti Nuclear Antibody,and hepatitis markers  were highly positive for kiwi, tometo, fish, orange The specific IgE levels were correlated with the SPTs. Total IgE level were 960 IU/L .A mass was defined on lober lobe of left lung on computerized tomography. PET CT SUVmax:6. The patient was operated. In postoperative period, he had recurrent laryngeal oedema and urticaria attacks. Omalizumab treatment planned because of the patient was resistant to antihistaminics and steroids.For the very first time, we used omalizumab in symptomatic therapy of recurrent laryngeal oedema and urticaria attacts in a patient with post operative pulmonary carsinoid tumor for eight months. During the four years of follow-up, no reccurrens was noted in carsinoid tumor. Control PET CT and CT results revealed normal findings.After omalizumab treatment, symptoms were decreased. Oral antihistaminics and mast cell stabilizing drugs used for treatment afterwards. Oral steroid was given only once.Resent sutudies show that, Pooled data analysis revealed that a causal relationship between omalizumab therapy and malignancy is unlikely.


Shigella, the causative agent of bacillary dysentery invades intestinal epithelial cells using a type III secretion system (T3SS). Through the injection of type III effectors, Shigella manipulates the actin cytoskeleton to induce its internalization in epithelial cells. At early invasion stages, Shigella induces atypical Ca2+ responses confined at entry sites allowing local cytoskeletal remodeling for bacteria engulfment. Global Ca2+ increase in the cell triggers the opening of connexin hemichannels at the plasma membrane that releases ATP in the extracellular milieu, favoring Shigella invasion and spreading through purinergic receptor signaling. During intracellular replication, Shigella regulates inflammatory and death pathways to disseminate within the epithelium. At later stages of infection, Shigella downregulates hemichannel opening and the release of extracellular ATP to dampen inflammatory signals. To avoid premature cell death, Shigella activates cell survival by upregulating the PI3K/Akt pathway and downregulating the levels of p53. Furthermore, Shigella interferes with pro-apoptotic caspases, and orients infected cells toward a slow necrotic cell death linked to mitochondrial Ca2+ overload. In this review, we will focus on the role of Ca2+ responses and their regulation by Shigella during the different stages of bacterial infection. Shigella, the causative agent of bacillary dysentery, invades the colonic mucosa, where it induces a strong inflammatory response responsible for massive destruction of the epithelium   at the plasma membrane (PM), the cytosolic Ca2+ concentration increases to reach a micromolar range  on the endoplasmic reticulum (ER), which are InsP3-gated Ca2+ release channels. Following GPCR stimulation, InsP3 is generated by the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C (PLC). Increase in InsP3 levels triggers the opening of InsP3Rs and Ca2+ release. The consecutive Ca2+ depletion from the ER activates Ca2+ entry across the PM, a process called store-operated Ca2+ entry (SOCE) carried out by the interaction of the Ca2+ depletion sensor stromal interaction molecule (STIMs) on the ER and the Ca2+ release-activated Ca2+ channel protein ORAI on the PM. Restoration of basal cytosolic Ca2+ concentration is rapidly achieved by extrusion across the PM by the Na+/Ca2+ exchanger or the Ca2+ ATPase PMCA and Ca2+ refilling into the ER by the Ca2+ ATPase SERCA  and the Ca2+ uniporter MCU . Increase in mitochondrial Ca2+ activates mitochondrial functions including ATP synthesis from oxidative phosphorylation. Because of their slow Ca2+ uptake rate, mitochondria also buffer cytosolic Ca2+ variations and play an important role in shaping physiological Ca2+ signals  mediated by cyclophilin D (CypD), influx of solutes into the mitochondrial matrix leading to the swelling of mitochondria and permeabilization of mitochondrial membranes. The release of the pro-apoptotic factor cytochrome C as well as ROS eventually leads to cell death by apoptosis or necrosis   or a small cluster of InsP3Rs   and the mitochondrial serine protease Omi/HtrA2 (High-Temperature Requirement). SMAC and Omi/HtrA2 then antagonize the inflammatory action of XIAP  inflammasome (Murakami et al., 2+-mediated opening of Cx-hemichannel during Shigella infection acts as a potent danger signal, triggering the release of cytokines oriented toward a proinflammatory Th17 cell response (Tran Van Nhieu et al., Extracellular ATP acts as a local endogenous danger signal, triggering a protective inflammatory host response to eradicate pathogens (Bours et al., Shigella uses Ca2+ signals from the onset of cell invasion to the death of infected cells. Local Ca2+ signals affect the dynamics of cytoskeletal reorganization to promote bacterial invasion. Global Ca2+ signals, usually associated with the induction of inflammatory signals, are not required for invasion and are involved at later stages of infection by Shigella to promote slow death. Between these two extremes of the cell infectious cycle and because of the versatility of Ca2+ signaling, processes key to Shigella virulence are likely to be regulated by Ca2+ signals, spatially and temporally regulated during the course of infection. The dual role of Ca2+-dependent calpain, involved in Shigella invasion and the orchestration of the death of infected cells illustrates this notion. How Ca2+ signals are generated and how they regulate the various processes associated with bacterial infection are key issues that cannot be ignored. Many pathogenic bacteria have been described to trigger Ca2+ responses during cell infection, involved in various key processes of infection such as bacterial adhesion, invasion, intracellular replication, or stimulating inflammation (Ruschkowski et al., 3, or ions such as Ca2+, has been seldom addressed, despite of their key role in fundamental cellular processes. The in-depth understanding of Shigella-induced signaling will implicate the integration of the diversion of the function of such second messengers during infection.We have discussed how All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heliotropium strigosum is used in traditional medicine to manage gastrointestinal pain, respiratory distress and vascular disorders. The present study was undertaken to provide scientific evidences for these folkloric uses by in vitro experimental settings.Heliotropium strigosum (Hs.Cr) was tested in vitro on isolated rabbit jejunum preparations to detect the possible presence of spasmolytic activity. Moreover, isolated rabbit tracheal and aorta preparations were used to ascertain the relaxant effects of the extract.A crude methanol extract of the + (80 mM)-induced spastic contractions in rabbit jejunum and shifted the Ca2+ concentration response curves towards right. The extract relaxed carbachol (1 μM)- as well as K+ (80 mM)-induced contractions in rabbit trachea at concentrations ranging from 0.01 to 10 mg/ml. Moreover, Hs.Cr. also relaxed (0.01-3.0 mg/ml) the phenylephrine (1 μM)- and K+ (80 mM)-induced contractions in isolated rabbit aorta.The Hs.Cr exhibited relaxant effects in rabbit jejunum in a concentration dependent manner (0.01-3.0 mg/ml). The Hs.Cr also relaxed K2+ channel blockade. This finding may provide a scientific basis for the folkloric uses of the plant.The Hs.Cr was found to exhibit spasmolytic, bronchodilator and vasorelaxant activities on isolated rabbit jejunum, trachea and aorta preparations, likely mediated through Ca Heliotropium strigosum Willd. (Boraginaceae), locally known as Kharsan, Gorakh pam and Bhangra, is a herb widely distributed throughout Pakistan [Pakistan . It growPakistan , 3. The Pakistan . The plaPakistan . FurtherPakistan .H. strigosum [H. strigosum exhibited antipyretic activity [H. strigosum is a very less-researched plant, as no detailed pharmacological studies exist.Strigosine, a pyrrolizidine alkaloid, has been isolated from trigosum . Methanotrigosum . Recentltrigosum . The ethactivity . Althoug2+ is a key regulator in the modulation of smooth muscle activity and the sensitivity to Ca2+ of the contractile elements changes depending on the environment surrounding the cell [Smooth muscle play a critical role in regulating functions of the gastrointestinal (GIT), respiratory and cardiovascular (CV) systems. Any irregularity in smooth muscle contractility ultimately results in many complication for GIT, including spasmogenicity of the small intestine, abdominal cramps and diarrhea. In CV, the irregular muscle contractility can determine hyper- and hypotension, and in respiratory system airway disorders like asthma. In all above mentioned disorders, cytosolic Cathe cell .To study the possible spasmolytic, vaso- and tracheal relaxant/constrictive effects of a plant extract, a tissue system comparable to human body is needed. Usually, isolated tissues from guinea pig, rabbit or rat are used to evaluate the effects of a drug on the GIT, the CV and the respiratory systems, as these animals shares many anatomical and physiological similarities of human organs. Isolated tissue are preferred over intact animals, as many experiments on isolated tissues permit easy of handling, broad visibility, requirement of small amounts of drug and low possibility of interferences in absorption and metabolism. In fact, rabbit jejunum, duodenum, aorta and trachea are preferred tissue strips for their availability, sensitivity, reproducibility of results and cost-effectiveness. Many studies are available in the scientific literature on isolated smooth tissue muscles of different animal models –14.H. strigosum in cardiovascular, respiratory and gastrointestinal ailments.As a part of studies aimed to validate traditional uses of medicinal plants , 16, theHeliotropium strigosum was collected from Khanewal Punjab, Pakistan in September, 2011. The plant was identified by the taxonomist, Dr. Saima Shehzadi, at the Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan where a voucher specimen (P.Fl. 591-2) was deposited.The whole plant was shade dried for 10 days. The dried material (1.200 kg) was coarsely ground and soaked in 80 % aqueous-methanol in an amber colored container. This material was retained at room temperature for 7 days with occasional shaking. The material was passed through a double layered muslin cloth and the fluid was subsequently filtered through a Watman-1 filter paper. The extraction procedure was repeated twice and the extracts were combined and evaporated under reduced pressure, to give a dark brown residue with a yield of 9.8 %. The extract was stored at -20 °C and fresh dilutions were prepared on the day of tests.All the chemicals, solvents, and drugs used were of reagent analytical grade. Acetylcholine, carbachol, verapamil chloride and potassium chloride were obtained from Sigma Chemicals Co., St Louis, MO, USA. Methanol, EDTA, glucose, magnesium chloride, magnesium sulfate, phenylephrine, potassium dihydrogen phosphate, sodium bicarbonate, sodium dihydrogen phosphate and calcium chloride were purchased from Merck, Darmstadt, Germany. Sodium chloride, ammonium hydroxide and sodium hydroxide were obtained from BDH laboratory supplies, Poole, England.ad libitum and were maintained in controlled conditions and 25 °C. The animals were subjected to overnight fasting prior the day of experiments, but had free access to water. All experiments were performed according to the rules of the Institute of Laboratory Animals Resources, Commission on Life Sciences, National Research Council [Adult rabbits (1.0–1.5 kg) of either sex used in experiments were purchased from the local market and housed in animal house of the Faculty of Pharmacy, Bahauddin Zakariya University, Multan. The animals were provided with standard feed and tap water  Council  and authHeliotropium strigosum (Hs.Cr) was tested for the possible presence of either spasmolytic or spasmogenic activity by using isolated rabbit jejunum preparations. Isolated rabbit jejunum segments of approximately 2 cm in length were suspended in isolated tissue baths containing Tyrode’s solution, at 37 °C, aerated with carbogen (95 % O2 and 5 % CO2). The composition of the Tyrode’s solution (mM) was: KCl (2.68), NaCl (136.9), MgCl2 (1.05), NaHCO3 (11.90), NaH2PO4 (0.42), CaCl2 (1.8) and glucose (5.55). A preload tension of 1 g was applied and intestinal responses were recorded through an isotonic transducer by a Power Lab Data Acquisition System  attached to a computer installed with a Lab Chart Software (Version 7).The methanolic extract of The tissues were allowed to equilibrate for at least 30 min prior to the addition of any drug. Isolated rabbit jejunum preparations exhibit spontaneous rhythmic contractions and allow testing of the antispasmodic (relaxant) effect without application of an agonist , 15. The+ (80 mM) [+ (80 mM)-induced contraction was expressed as the percent of the control contractile response.The possible mechanisms of the relaxant activity of the extract were investigated through the relaxation of the observed sustained spasmodic contractions following the exposure to high concentration of K (80 mM) . The ext (80 mM) . The obs2+-free Tyrode’s solution to which EDTA (0.1 mM) was added in order to remove calcium from the tissues. This bath solution was further replaced with K+-rich and Ca2+-free Tyrode’s solution, having the following composition (mM): KCl (50), NaCl (91.04), MgCl2 (1.05), NaHCO3 (11.90), NaH2PO4 (0.42), glucose (5.55) and EDTA (0.1). Subsequent to an incubation period of 30 min, cumulative Ca2+ concentrations were applied to the tissue bath to obtain control calcium dose-response curves (DRCs). On achievement of the super-imposable control calcium dose-response curves , the tissues were then washed and allowed to equilibrate with the plant extract for 1 h and then the concentration response curves of Ca2+ were recorded and compared to the control curves. The DRCs of Ca2+ were recorded in the presence of different concentrations of the plant extracts in tissue bath.The calcium channel blocking effect of the test substances were confirmed by previously reported methods , 15, 19.3 (25.0), CaCl2 (2.5), KCl (4.7), KH2PO4 (1.3), MgSO4 (1.2) and glucose (11.7). The trachea was cleaned free from the surrounding fatty tissues and rings of 2-3 mm width containing 2-3 cartilages were prepared. Each ring was opened by a longitudinal incision on the ventral side opposite to the smooth muscles layer to form a strip with smooth muscles layer in middle and cartilages on both sides. These tracheal preparations were mounted in 20 ml organ bath containing Krebs solution being maintained at 37 °C and aerated with carbogen. A preload tension of 1 g was applied and tissue preparations were allowed to be equilibrated for 1 h prior to any challenge by the extract. Tissue preparations were stabilized by repeated applications of carbachol (1 μM) until constant responses were recorded. The carbachol (1 μM) and high K+(80 mM)-induced sustained contractions were subsequently used for testing of different doses of the test material in a cumulative fashions. The isometric responses were recorded through a Power Lab Data Acquisition System  attached to a computer with a Lab Chart Software (Version 7). The standard drug with Ca2+ channel blocking effect, verapamil, was tested on high K+(80 mM)- and carbachol- induced spastic contractions in order to confirm the possible mechanism of action.The rabbit tracheas were dissected out and kept in a Krebs solution of the following composition (mM): NaCl (118.2), NaHCO+ (80 mM) or phenylephrine (1 μM) depending upon the protocol of the experiment. The vasorelaxant/vasoconstrictive effects of the extract were studied in a cumulative manner. Changes in isometric tension of aortic rings were obtained via a force-displacement transducer  coupled to a Power Lab data acquisition system  and a computer running Lab Chart software (version 7).The effect of Hs.Cr on systemic vascular resistance was assessed on isolated rabbit aorta preparations. Rabbits of either sex were sacrificed by a blow on the back of head and descending thoracic aorta was dissected out and kept in the normal Krebs solution. It was then cut vertically in 2-3 mm width segments. Each isolated tissue segment was then hung in a tissue organ bath  containing Kreb’s solution aerated with carbogen at 37 °C. A preload tension of 2 g was applied to each preparation and allowed to equilibrate for 1 h. After equilibration, tissue was stabilized by repeated exposure to K50 values with 95 % CI were calculated by using the computer software “Graphpad Prism Program” (version 5.0), San Diego CA, USA. Concentration response curves were analyzed by the non-linear regression of sigmoid response curve (variable slope). The statistic applied was the student’s t- test and p <0.05 was considered as significant.In isolated tissue experiments, data were expressed as the mean ± SEM and the EC50 value was 0.381 mg/ml    Fig. a. Simila+ (80 mM)-induced contractions in isolated rabbit jejunum preparations with an EC50 value of 0.243 mg/ml  -induced contractions with an EC50 value of 0.041 μM   at a concentration range of 0.1-0.3 mg/ml in a manner similar to verapamil -induced contractions in isolated rabbit tracheal preparation with respective EC50 values of 3.26 mg/ml  and 1.17 mg/ml  -induced contractions in isolated rabbit tracheal preparations with respective EC50 valuess of 0.114  and 0.048 μM  - and K+(80 mM)-induced contractions in isolated rabbit aorta preparations with respective EC50 values of 0.831 mg/ml  and 0.493 mg/ml  -induced contractions, with respective EC50 values of 0.90 μM  and 0.43 μM  - and KH. strigosum in smooth isolated muscle by constructing dose-response curves. The application of the extract to the spontaneous contractions of isolated rabbit jejunum preparations resulted in a relaxation of the contractile effect, evident by the decrease in the amplitude as well as the frequency of the contractions. The spontaneous contractions and relaxation of intestinal tissues is based on the phenomenon of alternative depolarization and repolarization. The maximal depolarization results from an action potential generation by the rapid inward Ca2+ movement through voltage dependant L-type Ca2+ channels (VDLCs) [2+ concentration [In this study we observed the effects of a methanolic extract of  (VDLCs) . Severalntration .+(80 mM)-induced contractions in isolated rabbit jejunum to account for the observed relaxant effect on spontaneous contractions in the same system. The preparations, after exposure to K+(>30 mM), are liable to be contracted due to the opening of VDLCs, the rapid influx of extracellular Ca2+ and an increase in cytosolic Ca2+ [+(80 mM)-induced contractions; hence it is possible to hypothesize that it cause the blockade of Ca2+ channels [2+ channel blocking activity was confirmed further by the rightward shift of calcium CRCs in isolated rabbit jejunum preparations in a manner similar to verapamil. This drug is a well known member of Ca2+ channel blockers, exerting an inhibitory effect on Ca2+ influx [The Hs.Cr was applied to Klic Ca2+ . The extchannels . The pro+ influx , 24.+(80 mM)-induced contractions in isolated rabbit trachea. It relaxed both carbachol (1 μM)- and K+(80 mM)-induced contractions and this non-specific relaxant effect is presumed to be mediated through Ca2+ channel blocking activity [To assess the effectiveness of Hs.Cr in respiratory disorders, it was applied on CCh (1 μM)- and Kactivity .+(80 mM), as these preparations have been used to investigate the nature of Ca2+ channel blocking activity. The K+(80 mM)-induced contractions are outcome of Ca2+ channels opening, influx of extra-cellular Ca2+ and Ca2+ release from endoplasmic reticulum. Phenylephrine- induced contractions are mediated through the activation of α-adrenergic receptors and the subsequent Ca2+ influx through the receptor operated Ca2+ channels (ROCs). Hs.Cr relaxed both phenylephrine and K+(80 mM)-induced contractions in a manner comparable to verapamil. The observed nonspecific relaxant effect is likely to be mediated through a Ca2+ channel blocking mechanism. The Ca2+ channel blockers are reported to provide relief in respiratory congestions and such activity of the Hs.Cr may provide a scientific basis for the traditional use of the plant in respiratory stress.The Hs.Cr was also tested on isolated rabbit aorta pre-treated with phenylephrine (1 μM)- and KH. strigosum in gastrointestinal spasms, respiratory distress and vasospastic disorders, mediated possibly through the blockade of voltage-dependent calcium channels or the release of intracellular Ca2+. However, more detailed studies are required to establish the safety, efficacy and toxicity of this plant and to isolate the bioactive constituents.This study provides a rationale for the folkloric use of


Rearranging furniture in elementary school classrooms encourages classroom activities. In elementary schools in Indonesia and some other developing countries, usually only one style of furniture is used for all children, and the furniture is heavy and oversized for younger children. This affects their ability to carry it. The objective of this study is to investigate the effects of elementary school furniture weight and children’s age on performance of three carrying tasks , from the ergonomics point of view. A total of 42 schoolchildren  participated in this study. Two types of Japanese chairs , one type of Indonesian chair , and two types of desks (height: 58 cm and 68 cm) were used. Indonesian chairs took significantly longer time to carry than the two Japanese chairs, and there was a significant negative relationship between age and task time for Chairs B and C, but not Chair A. Success rates for lifting and turning the chair declined as age decreased and chair weight increased, but were not significantly influenced by desk height. Success rates for carrying a chair and desk together significantly decreased with heavier furniture. Children aged six showed an extremely low success rate in almost all conditions. In conclusion, children’s ability to carry furniture is affected by their age and furniture characteristics, especially weight. In order to encourage classroom activities in elementary school, school furniture should be of appropriate weight. Supervision for younger children is required during classroom furniture arrangement. It has long been noted that elementary schools should provide desks and chairs that are easy for students to move and carry. School furniture  that is easy to move and carry could help to improve the quality of education –4. PreviIncreasing the weight that one carries increases the physiological cost of walking, and the load on leg muscles . StudiesSome solutions have been proposed with regard to this issue. Breithecker  introducIn developed countries such as Australia, Japan, and Scotland, elementary school chairs and desks are lighter, and may be easily carried and moved by all children ; howeverIn developing countries such as Nigeria, Malaysia, Indonesia, and Vietnam , the weiTaking all of the above finding into account, it is necessary to propose a weight guideline for elementary school furniture that is proportional to children’s body dimensions at certain ages. The present study investigated the effects of elementary school furniture weight and children’s age on performance of three tasks—carrying a chair, carrying both a chair and a desk, and lifting a chair onto a desk. This study also compared the difficulty of these tasks for Indonesian and Japanese furniture.Healthy Indonesian and Japanese children (N = 42) who could understand the experimenters’ instructions participated in this study. The Indonesian children included 6 boys and 11 girls, and the Japanese children included 12 boys and 13 girls. Their ages ranged between 6.0 and 9.9 years and they were categorized into four age-range groups: 6 (6.0–6.9), 7 (7.0–7.9), 8 (8.0–8.9), and 9 (9–9.9) years. In both Indonesia and Japan, children go to elementary school for six years, starting at age 6. The age range of our participants corresponded to the lower and middle grades (first to fourth year) of elementary school. Before the experiment, participants’ parents signed informed consent forms. This study was approved by the ethics committee of the Faculty of Design, Kyushu University, Japan .Representative furniture from Indonesian and Japanese public elementary schools were used as the main equipment in this experiment, including one Indonesian chair, two Japanese chairs, and one Japanese desk.Two Japanese (Chairs A and B) and one Indonesian (Chair C) chairs were used to create three different chair weight conditions . In JapaIn contrast, in Indonesia, there is no precise standard for school furniture design for each age. Accordingly, the ordinary elementary school chair had a seat height and weight of 420 mm and 5 kg, respectively . This isOnly a Japanese elementary school desk was used in this experiment . This deThe experiment was conducted in a large flat space over six days in February and March 2013. Participants performed three different tasks: 1) carrying a chair, 2) turning and lifting a chair on a desk, and 3) carrying both a chair and a desk together. Before each task, we gave participants brief instructions about how to complete each task, and time for practice. To prevent injury, participants wore non-slip work gloves and shoes. In addition, we instructed them to stop the task immediately if they felt it was impossible or dangerous to continue. During the task, an adult stood beside the participants for safety.Participants performed tasks from lighter/smaller to heavier/larger furniture, in order to prevent injury, and to allow participants to judge easily whether they could perform the next task level. After each task, an experimenter ensured that factors such as pain, fatigue, lack of motivation, etc. would not influence performance of the next condition. During each task, participants’ movements were recorded using a digital video camera  at 30 frames per second.This task involved bringing each chair  from the start to finish line (a 3 m distance). A chair was placed sideways behind the start line, and participants sat on the chair to wait for the start command. After we gave a signal to start, they stood up from the chair, raised the chair with their hands, carried it toward the finish line, and put it down on the floor beyond the finish line. Before tasks, we instructed them that they 1) could hold any part of the chair, 2) should not push or slide the chair on the floor, and 3) should walk at an ordinary speed during the task.This task involved turning a chair upside down and putting it on a desk. In this task, three chair weights  and two desk heights  were employed.A chair was placed in front of the desk as indicated in This task involved moving a desk with a chair, which was placed upside down on top of the desk , from thA desk was placed facing forward behind the start line. First, the participant stood in front of the desk, and held the sides of the desk top with both hands while waiting for the start command. After the start command, participants carried the desk and chair to the finish line, and placed it on the floor beyond the finish line. Before the task, participants were instructed to walk at a normal speed and to push or slide the desk across the floor.Before the experiment, height, weight, body mass index (BMI), and grip strength were measured. Grip strength was measured for both hands using a digital handgrip dynamometer .The time taken to complete each task (task time) was measured using frame-by-frame playback of the recorded video. In Task 1, the start time was the moment when one chair leg bottom was raised off the floor, and finish time was when one chair leg bottom touched the floor. For Task 2, the start time was when one chair leg bottom was lifted, and the finish time was when the chair’s seat was placed on the table. Lastly, for Task 3, the start time was when one leg bottom of the desk and chair was raised, and finish time was when one was placed on the floor.In the task involving lifting and turning a chair onto the desk, we judged that children were successful if they could complete the task without dropping the chair, without stopping for a while in the middle of the task, and without putting themselves in danger. Dangerous situations were identified as those in which children carried the chair with an unstable grip and they were at risk of dropping it.Statistical analyses were performed using IBM SPSS Version 21.0 (2012) for Windows, Chicago, USA. Descriptive results were presented as means and standard deviations. Two-way analysis of variance (ANOVA) was used to identify differences in physical characteristics related to gender and nationality. Pearson correlation coefficients were calculated to analyze the relationship among children’s physical characteristics, between children’s physical characteristics and task time, and between chair type and task time. A repeated measures one-way ANOVA was employed to compare task time of the three chair types in the chair-moving task, followed by post-hoc pairwise Bonferroni-corrected comparison tests for significant differences among chair types. The exact chi-square test was used to compare success rates among task conditions for the lifting and turning task and the moving a chair and desk together task , 25. TheAll participants successfully completed the task of carrying a chair for all chair conditions. In both desk height conditions, the exact chi-square test revealed significant differences in success rates of lifting and turning a chair among the three chair conditions (p < 0.01). Chairs A and B showed success rates over 60%, whereas Chair C showed a lower success rate (around 20%) for both desk height conditions . In contFor the task that involved carrying both a chair and a desk, the exact chi-square test showed a significant effect of task condition on success rate (p < 0.01). Success rate decreased with increasing total weight of the chair and desk . Fig 13 The present study found no significant differences in physical measurements between genders. In previous research, gender differences have been regarded as an important factor in designing the size of elementary school furniture . HoweverThis study compared Japanese and Indonesian chairs, used in their respective countries, and involved children of Japanese and Indonesian ethnicity. Given that Japanese children are familiar with Japanese chairs, they would be expected to perform better with Japanese chairs; the same would be true for Indonesian children and Indonesian chairs. Therefore, to ensure equality, this study included children who were of Japanese and Indonesian ethnicity. Previous research has found significant effects of ethnic group on anthropological measurements in children , 32. EthChairs are frequently carried for class activities in elementary schools. In the present study, the task time of Chair C was significantly higher than for the other two chairs . The proFurthermore, the present study showed a negative relationship between participant age and task time for Chairs B and C, but not Chair A . In otheOn the other hand, some studies recommend that the weight of carried items should be proportional to children’s body weight –7. In adChairs are sometimes lifted and turned upside down in elementary schools in some countries , 42. WheThe success rate for this task was strongly influenced by participant age  for bothOn the other hand, among children aged 7 to 9, success rates depended on chair type as well as age. All children aged 9 succeeded at the task for Chairs A and B, but some did not for Chair C. These failures were mainly due to strength limitations, unlike for children aged 6. Children’s muscle strength increases with age , which iThe present study failed to show significant effects of desk height on success rates for lifting and turning a chair. Some previous studies have demonstrated an effect of destination height when humans manually lift a load from the floor , 45. ThuIn the current study, the success rate of carrying a chair and desk together decreased as age decreased, and as total weight of the chair and desk increased . FurnituChildren aged 6 showed a very low success rate for this task (approximately 22%), which was much different from those aged 7 and over . As descThe present study provided useful information regarding weight guidelines of elementary school furniture for promoting classroom activities . HoweverThe findings of the present study support the need for lighter chairs to encourage more dynamic class activities. However, decreasing the weight of furniture, while still manufacturing durable and strong products, may be difficult and unaffordable for developing countries. Frail chairs may lead to injuries due to lack of quality control with regard to safety. Therefore, future research could alternatively focus on the key parts of the chair, which are usually held by children, as an effective alternative strategy to improve ease of chair transport.The findings of the present study indicate that children’s carrying of a chair, carrying both a chair and a desk, and lifting and turning a chair onto a desk, are strongly influenced by not only children’s age, but also features of the chair, especially weight. Based on the findings of the present study, the weight guideline of furnitures for elementary school children aged 6–9 was proposed . Decreas


Oryctolagus cuniculus) is an important experimental animal for studying human diseases, such as hypercholesterolemia and atherosclerosis. Despite this, genetic information and RNA expression profiling of laboratory rabbits are lacking. Here, we characterized the whole-genome variants of three breeds of the most popular experimental rabbits, New Zealand White (NZW), Japanese White (JW) and Watanabe heritable hyperlipidemic (WHHL) rabbits. Although the genetic diversity of WHHL rabbits was relatively low, they accumulated a large proportion of high-frequency deleterious mutations due to the small population size. Some of the deleterious mutations were associated with the pathophysiology of WHHL rabbits in addition to the LDLR deficiency. Furthermore, we conducted transcriptome sequencing of different organs of both WHHL and cholesterol-rich diet (Chol)-fed NZW rabbits. We found that gene expression profiles of the two rabbit models were essentially similar in the aorta, even though they exhibited different types of hypercholesterolemia. In contrast, Chol-fed rabbits, but not WHHL rabbits, exhibited pronounced inflammatory responses and abnormal lipid metabolism in the liver. These results provide valuable insights into identifying therapeutic targets of hypercholesterolemia and atherosclerosis with rabbit models.The rabbit ( Oryctolagus cuniculus) is an important experimental animal model for biomedical science. Rabbits are not only the most-used animal for the production of antibodies, but also they are widely used for studying a variety of human diseases, such as infectious disease, neoplasms, ophthalmic disease, Alzheimer’s disease, and respiratory diseaseLDLR) in Watanabe heritable hyperlipidemic (WHHL) rabbits can lead to spontaneous hypercholesterolemia and atherosclerosis, even when they are on a normal chow diet4LDLR deficiency as a cause for human familial hypercholesterolemia8The European rabbit . Although both WHHL and Chol-fed rabbits exhibit hypercholesterolemia, their lipoprotein profiles are quite different; WHHL rabbits have increased levels of LDL-cholesterol accompanied by low HDLs, while Chol-fed rabbits have increased hepatically and intestinally derived remnant lipoproteins, called β-VLDL  of the DEGs , which is consistent with the notion that atherosclerosis is a chronic inflammatory disease initiated by lipid deposition in the arterial wallFor a more detailed model-by-tissue comparison, we selected DEGs observed in at least one condition and performed clustering of the conditions based on the logthe DEGs . The resR = −0.09, z-score = 4.69). Furthermore, the DEGs mediating inflammatory responses were identified in the Chol-fed but not in the WHHL rabbits , farnesoid X receptor (NR1H4), SREBF1, SREBF2, PPARA and PPARGABCA1 (FDR = 5.87 × 10−8), ABCG1 (FDR = 9.74 × 10−5), PLTP (FDR = 0.05) and LPL (FDR = 3.89 × 10−12), the target genes responsible for cholesterol transport and lipoprotein remodeling, were significantly up-regulated in the livers of Chol-fed rabbits  was also significantly overexpressed, which could trigger the ubiquitylation and degradation of LDLRSREBF2 is a transcriptional factor responsible for cholesterol synthesis and is activated by low cellular cholesterol levelsSREBF2 (FDR = 0.05) and its target genes, FDFT1 (FDR = 0.02) and CYP51A1 (FDR = 0.02), were significantly repressed in the livers of Chol-fed rabbits  along with its target genes, such as FABP4 (FDR = 0.04) and ACSL4 (FDR = 0.09), suggesting the occurrence of active fatty-acid uptake and storage  and catalogued millions of variants for these breeds. We also performed deep mRNA sequencing of the aorta, liver, heart and kidney from the rabbits and provided the most comprehensive transcriptome assembly to date. The completion of rabbit genome and transcriptome information will facilitate many facets of rabbit studies to investigate human diseases and also drive the generation of transgenic and knock-out rabbitsLDLR functional deficiency, some of the deleterious mutations could also play a role in the pathogenesis of hypercholesterolemia and atherosclerosis in WHHL rabbits. For example, a high-frequency deleterious mutation was predicted at a quite conserved site on ALDH2, which was previously reported to play a cardio-protective role via both the elimination of toxic aldehydes under oxidative stress and the bioactivation of nitroglycerin to nitric oxideALDH2, can reduce myocardial infarct size induced by acute ischemiaApoe knockout miceThe whole-genome sequences of the three experimental rabbit breeds were consistent with their breeding history. Particularly, the severe bottleneck during artificial selection of WHHL rabbits gave rise to many deleterious mutations with high frequency in the breed. In addition to the Although rabbits are frequently used to study human atherosclerosis, gene expression profiling of aortic and liver lesions has not been systemically elucidated. We conducted transcriptome sequencing of these two tissues of WHHL and Chol-fed NZW rabbits and compared them with those of control rabbits. Quantification of the transcriptome allowed us to identify more than 2,000 DEGs in aortic atherosclerotic lesions of WHHL and Chol-fed rabbits, as well as more than 1,000 DEGs in the livers of Chol-fed rabbits. We found that many inflammation-related genes were upregulated in aortic atherosclerotic lesions of both WHHL and Chol-fed rabbits, which is consistent with the notion that atherosclerosis is essentially evoked by inflammationIn conclusion, we have successfully characterized the whole-genome variants and transcriptome profiles of the three breeds of laboratory rabbits. In a future study, we will investigate whether these deleterious mutations and DEGs play any roles in the pathogenesis of various disorders in the hyperlipidemic rabbits. These resources will be vital in identifying therapeutic targets for the treatment of hyperlipidemia and atherosclerosis.n = 10). For the transcriptome profiling, we also used NZW rabbits fed with a cholesterol-rich diet containing 0.3% cholesterol and 3% soybean oil for 16 weeks, which is commonly used for studying atherosclerosis. The liver, kidney, heart and aortic arch were collected for extracting total RNA for transcriptomic analysis (n = 4). All animal experiments were performed with the approval of the Animal Care and Use Committee of the University of Yamanashi, Kobe University and University of Michigan. The methods were carried out in accordance with relevant and approved guidelines and regulations.NZW rabbits were purchased from Covance Com., USA, and JW rabbits were purchased from Japan SLC Inc.  and WHHL rabbits were provided by Kobe University School of Medicine, Japan. The rabbits were fed a normal chow diet. Animals were euthanized by overdose injection of pentobarbital solution. We collected liver tissue for extracting DNA for genome sequencing  according to the manufacturer’s manual. DNA quality and integrity were controlled by the A260/280 ratio, agarose gel electrophoresis using Qubit 2.0  and a Bioanalyzer 2100 . For sequencing the library preparation, 1 μg of genomic DNA was sheared to fragments of 300–400 bp, end-repaired, A-tailed and ligated to Illumina sequencing adapters. The ligated products were selected for a length of 400–500 bp on a 2% agarose gel and amplified by LM-PCR. The library was sequenced on an Illumina HiSeq2000 with 2 × 100 bp paired-end mode, which was controlled by HiSeq Control Software. Total RNA sample quality and integrity were controlled with a Bioanalyzer 2100 . For the library preparation, 3 μg total RNA was captured by Dynabeads Oligo , sheared to fragments of 200 bp, and reverse transcribed by the SuperScriptIII cDNA Synthesis Kit . cDNA was end-repaired, A-tailed and ligated to Illumina sequencing adapters and amplified by PCR. Library preparation was performed with the TruSeq RNA LT V2 Sample Prep Kit . The sequencing library was qualified by Qubit 2.0  and a Bioanalyzer 2100  and then sequenced on an Illumina HiSeq2000 with 2 × 100 bp paired-end mode, which was controlled by HiSeq Control Software.http://sift-db.bii.a-star.edu.sg/, v2014-10-07)Raw sequencing reads were processed by in-house scripts to filter low quality reads and trim low quality bases  at the 3′ end of reads. Remaining reads were aligned to the rabbit reference genome, oryCun2 by BWA-MEM (v0.7.4)π, Watterson’s θ and Tajima’s D were calculated using packages in Bioperl (v1.6.1)Genetic relationships between all pairwise rabbits were estimated by KINGPrior genes involved in cardiovascular diseases were retrieved from QIAGEN’s Ingenuity Pathway Knowledge Base (accessed 2014-09-28), which is a data repository of manually reviewed biological interactions and functional annotations. In addition, cardiovascular disease-associated genes provided by the NHGRI GWAS Catalog2-fold change of normalized counts. Hierarchical clustering was performed with the heatmap.2 function in R (http://www.r-project.org/).Quality of RNA sequencing reads were checked by the NGS QC Toolkit (v2.3.2)http://www.ingenuity.com/products/ipa, accessed 2014-11-26). Human annotations of the rabbit orthologs were used for analysis of the data sets. IPA’s default cutoffs were adopted for activation state prediction . The regulation network of lipid metabolism in the liver was visualized by Cytoscape (v3.2.0)Function, pathway and regulator enrichment of DEGs were analyzed through the use of QIAGEN’s Ingenuity Pathway Analysis .


Deletions in the long arm of chromosome 5 or loss of the whole chromosome are rare in childhood Acute Myeloid Leukemia (AML) patients. It is also unknown if the wide variety of breakpoints have diverging implications in the patient’s outcome. Despite -5/5q- abnormalities have usually been described as a poor prognostic feature, however, the low frequency of -5/5q- in pediatric AML patients limits a full knowledge about this cytogenetic and clinical category, which is an intriguing factor for further research and new findings. Here, we report an AML child showing an uncommon deletion in 5q associated with 2 new abnormalities involving chromosome 2 within a complex karyotype well-characterized by several molecular cytogenetic approaches. Our work stimulates upcoming studies with more detailed descriptions about 5q abnormalities to better define its role in the stratification risk of such cytogenetic subgroup in childhood AML. The knowledge of more complete descriptions of such cases harboring 5q- abnormalities, obtained through the application of molecular cytogenetic approaches, are necessary to precisely stratify the risk of this rare subgroup in childhood AML.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


The ability to use the information gleaned to gain insight into the function of ion channels and Ca2+ handling proteins in a more complex system, e.g., the intact heart, has remained a challenge. We have developed laser scanning fluorescence microscopy-based approaches to monitor, at the sub-cellular to multi-cellular level in the immobilized, Langendorff-perfused mouse heart, dynamic changes in [Ca2+]i and Vm. This article will review the use of single- or dual-photon laser scanning microscopy [Ca2+]i imaging in conjunction with transgenic reporter technology to (a) interrogate the extent to which transplanted, donor-derived myocytes or cardiac stem cell-derived de novo myocytes are capable of forming a functional syncytium with the pre-existing myocardium, using entrainment of [Ca2+]i transients by the electrical activity of the recipient heart as a surrogate for electrical coupling, and (b) characterize the Ca2+ handling phenotypes of cellular implants. Further, we will review the ability of laser scanning fluorescence microscopy in conjunction with a fast-response voltage-sensitive to resolve, on a subcellular level in Langendorff-perfused mouse hearts, Vm dynamics that typically occur during the course of a cardiac action potential. Specifically, the utility of this technique to measure microscopic-scale voltage gradients in the normal and diseased heart is discussed.Studies in isolated cardiomyocytes have provided tremendous information at the cellular and molecular level concerning regulation of transmembrane voltage (V Because isolated myocytes are disconnected from their neighboring myocytes as well as their non-myocytes (which are important modulators of cardiomyocyte function), this profoundly impacts on fundament physiological properties. For example, cardiomyocyte action potential properties are very likely to be different in intact muscle compared to single cells. The peak of the action potential is typically more positive in the isolated myocyte, because there is less passive outward current to oppose the depolarizing effect of locally activating inward sodium current. Multicellular preparations may thus be valuable for electrophysiological characterizations, specifically those related to action potential properties and propagation. In addition, such preparations would enable the study of key issues about myocyte Ca2+ regulation, including the conditions required for Ca2+ wave initiation and propagation from cell-to-cell. Overall, cardiac physiology/pathophysiology mandates the development of assays capable of resolving dynamic events with cellular/subcellular resolution in intact cardiac tissue.Studies in single isolated cardiomyocytes have provided important information at the cellular and molecular level concerning the electrical properties and Ca2+ release phenomena and the propagation of Ca2+ waves in isolated rat papillary muscles iontophoretically loaded with the calcium indicator fluo-3  to measure spatiotemporal dispersion of electrical and Ca2+ signals at the microscopic scale in normal and diseased heart.In their pioneering study, Wier and co-workers developed a confocal laser scanning microscopy-based technique to monitor local sarcoplasmic reticulum (SR) Ca2+]i) dynamics in the intact, Langendorff-perfused mouse heart, using two-photon laser scanning microscopy (TPLSM) in conjunction with calcium-sensitive fluorescent dyes  in strongly scattering tissue , indicating that SR Ca2+ release activated by propagating depolarization is highly synchronized among neighboring cardiomyocytes in situ, at least within the temporal resolution of the system (line-scan speed = 1.33 ms/line). The line-scan image  as a function of time confirm entrainment of [Ca2+]i transients in all three cardiomyocytes by stimulus-evoked propagating action potentials. Further, superimposition of normalized [Ca2+]i transients reveal acceleration of [Ca2+]i decay kinetics with an increase in pacing rate  were observed to occur between stimulated [Ca2+]i transients as shown in Figure 2+ waves have previously been shown to result from propagating activation of neighboring clusters of ryanodine receptors in the SR membrane. Annihilation of Ca2+ waves by colliding, action potential-induced transients is thought to be due to ryanodine receptor refractoriness in the wake of a wave of calcium release  of in situ cardiomyocytes in the presence of cytochalasin D are at least qualitatively similar to those seen in isolated mouse ventricular cardiomyocytes in the absence of excitation-contraction uncouplers but under otherwise comparable experimental conditions , supporting the notion that cytochalasin D not only effectively uncouples contraction from excitation but also retains basic properties of action potential-evoked [Ca2+]i transients of cardiomyocytes. Previous studies by the Salama laboratory  is not feasible because of its adverse effects on electrical excitability , Annine-6plus—loaded hearts revealed orderly T-tubule arrangements in the wild-type myocytes in situ, whereas Gαq-overexpressing cells exhibited spatially non-uniform t-tubular disorganization with patchy loss or growth of t-tubules cellular level in the genesis of triggered arrhythmias in the heart. Improvements in (i) penetration depth of two-photon excitation microscopy via e.g., use of dyes that can be excited at wavelengths >1 μm, (ii) detector sensitivity, and (iii) speed of image acquisition will aid in accelerating discovery.Collectively, techniques have been and are being developed to monitor Ca2+ regulation of individual in situ cardiomyocytes in immobilized, Langendorff-perfused mouse heart. A current limitation of the methods presented here is the use of pharmacological excitation-contraction blockers, i.e., cytochalasin D alone or in combination with ryanodine, which can modify the Ca2+ handling and electrical properties of the cells under study. Blebbistatin is a possible alternative, but, like cytochalasin D, it only insufficiently suppresses motion during micron scale imaging  should provide valuable insights into electrophysiology and CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, some myofibers only exhibit contraction and Ca2+ transients at alternating (ALT) ends in response to alternating polarity field stimulation. Here, we present for the first time the methodology for identification of ALT myofibers in primary cultures and isolated muscles, as well as a study of their electrophysiological properties.Most cultured enzymatically dissociated adult myofibers exhibit spatially uniform (UNI) contractile responses and Ca2+ imaging, electric field stimulation, microelectrode recordings, immunostaining, and confocal microscopy to characterize the properties of action potential-induced Ca2+ transients, contractility, resting membrane potential, and staining of T-tubule voltage-gated Na+ channel distribution applied to cultured adult myofibers. Here, we show for the first time, with high temporal and spatial resolution, that normal control myofibers with UNI responses can be converted to ALT response myofibers by TTX addition or by removal of Na+ from the bathing medium, with reappearance of the UNI response on return of Na+. Our results suggest disrupted excitability as the cause of ALT behavior and indicate that the ALT response is due to local depolarization-induced Ca2+ release, whereas the UNI response is triggered by action potential propagation over the entire myofiber. Consistent with this interpretation, local depolarizing monopolar stimuli give uniform (propagated) responses in UNI myofibers, but only local responses at the electrode in ALT myofibers. The ALT responses in electrically inexcitable myofibers are consistent with expectations of current spread between bipolar stimulating electrodes, entering (hyperpolarizing) one end of a myofiber and leaving (depolarizing) the other end of the myofiber. ALT responses were also detected in some myofibers within intact isolated whole muscles from wild-type and MDX mice, demonstrating that ALT responses can be present before enzymatic dissociation.We used high-speed confocal microscopic CaWe suggest that checking for ALT myofiber responsiveness by looking at the end of a myofiber during alternating polarity stimuli provides a test for compromised excitability of myofibers, and could be used to identify inexcitable, damaged or diseased myofibers by ALT behavior in healthy and diseased muscle.The online version of this article (doi:10.1186/s13395-016-0076-8) contains supplementary material, which is available to authorized users. BriDissociated myofiber Ca2+imaging. Rhod-2 measurements were carried out on a high-speed confocal system  as previously described [+-free or low [Cl−] extracellular recording solution was used to evaluate the effects of Na+ and Cl− extracellular concentration, on electrically evoked Ca2+ signals in UNI and ALT fibers. The Na+-free solution contained (in mM): 150 NMDG, 10 HEPES, 2 CaCl2, 1 MgCl2, pH adjusted to 7.4 with CsOH. The low [Cl−] solution contained (in mM): 150 Na-CH3SO3, 10 HEPES, 2 CaCl2, 1 MgCl2, pH adjusted to 7.4 with NaOH. Excitation for rhod-2 was provided by the 532-nm line of a 100-mW diode laser, and emitted light was collected at >550 nm. The confocal imaging was conducted at a depth of ∼ 20 μm into the interior of the myofiber. In the majority of the experiments, 0.5-1 ms electrical field stimuli were applied via two parallel platinum wires positioned perpendicular to the bottom of the dish, ∼5 mm apart, to elicit action potentials . In a few experiments (where noted), a unipolar electrode was positioned about ∼ 100 μm away from the myofiber surface, either near the center or near the end the myofiber. Application of each stimulation protocol was synchronized relative to the start of confocal scan acquisition. Typically, the field stimulus was applied 100 ms after the start of the confocal scan sequence, thus providing control images before stimulation at the start of each sequence. These control images were used to determine the resting steady-state fluorescence level (F0). Average intensity of fluorescence within selected regions of interest (ROIs) within a myofiber was measured with Zeiss LSM Image Examiner . Images in line-scan (xt) mode  or field imaging (xy) mode (frame size: 512 × 512) at 60 frames/s were background corrected by subtracting an average value recorded outside the cell. The average F0 value in each ROI before electrical stimulation was used to scale Ca2+ signals in the same ROI as ΔF/F0. No attempts were made to estimate the actual cytosolic Ca2+ concentration. Ca2+ imaging experiments were carried out at room temperature, 21–23 °C. Whole muscle Ca2+imaging; FDB and EDL muscles were carefully dissected, keeping tendons at both extremities. After loading for 30 min with fluo-4-AM  in L-15BSA, the muscle was held in place with a plastic coverslip attached with vacuum grease in a petri dish  ratiometric imaging and analysis were performed as previously described . BrieflyResting membrane potential was measured using a conventional microelectrode amplifier . Microelectrodes were filled with 1 M K-acetate and resistances ranged between 12–40 MΩ, as previously described by others , 62.v1.4 , followed by incubation with Alexa488-conjugated goat anti-rabbit antibody . To test for the specificity of the primary antibody, in some dishes the primary antibody was preincubated with a Nav1.4877–891 neutralizing peptide  before labeling and used as a negative control. Antibody-labeled myofibers and antibody/peptide incubated myofibers were imaged on a Fluoview 500 Olympus LSM system, based on an IX/71 inverted microscope using a × 60 NA 1.2 water-immersion objective lens. The excitation for alexa-488 was provided by using a 488-nm laser. The emitted light for alexa-488 was collected at >510 nm. Prior fiber fixation, a computer-controlled microscope stage and field stimulation, mounted in our Fluoview 500 Olympus LSM system, were used to select, identify, and save in a file the locations of numerous ALT and UNI fibers in multiple dishes. After the immunostaining, we used the same LSM image system and computer-controlled stage to acquire unequivocally fluorescence confocal images from fibers previously identified as UNI or ALT. Confocal images of UNI and ALT myofibers were collected using the same image acquisition settings and enhancing parameters so that all images can be directly compared. Images were background corrected. Images were processed using ImageJ .Immunostaining was performed according to previously published methods . Myofibet test or with the non-parametric Mann-Whitney rank-sum test. Significance was set at P < 0.05.All data processing and statistical analysis was performed using OriginPro 8.0. All data are presented as mean ± SEM unless otherwise noted. Statistical significance was assessed using either parametric two-sample left); these myofibers exhibit a symmetric twitch contraction at both ends and concentric myofiber shortening in response to a brief  external field stimuli of alternating polarity, indicative of a propagated action potential and uniform contractile activation; (2) another subset of myofibers (5–20 %) responds differently; these myofibers exhibit local “asymmetric” contraction upon alternating polarity external stimulation ; from here on referred as to ALT myofibers; (3) some myofibers do not twitch at all in response to electrical stimulation; these myofibers represent a variable but very small fraction of the overall myofiber population and were not studied in detail in the present work.Using light microscopy and simultaneously monitoring the myofiber twitch activity in response to brief suprathreshold electrical field stimulation of alternate polarity, it is possible to distinguish three types of myofiber responses : (1) theivation;  another 2+ release is essential for muscle contraction and voluntary movements. To determine whether ALT myofibers exhibit deficits in the conformational coupling between the voltage sensors, the Cav1.1 channels, and the RyR1, the Ca2+ release channels, we monitored electrically induced Ca2+ transients using high-speed confocal microscopy. When UNI myofibers are loaded with a fluorescent Ca2+ indicator, the myofibers display a global Ca2+ transient in response to external brief stimulation of both polarities, indicating proper excitation, Ca2+ release and contraction-relaxation cycle . However, when the ALT myofibers are loaded with a fluorescent Ca2+ indicator, the myofibers display only local non-propagated Ca2+ transients at alternating ends upon external stimulation with alternating polarity pulses . As quantified in Fig. n = 12, N = 3; ALT: ΔF/F0 = 8.15 ± 1.4, n = 10, N = 3; P < 0.05). Thus, ALT fibers appear to have only local activation and disrupted action potential-induced Ca2+ transients. Consistent with this observation, the amplitude and duration of ALT, but not UNI responses, are graded with stimulus duration . These observations raised several questions as to why these ALT myofibers respond the way they do to electrical field stimulation. Are ALT myofibers partially depolarized due to enzymatically and or trituration-induced membrane damage, which would explain the ALT behavior? Do they exhibit structural alterations in the T-tubule system, the network of extensions of the surface membrane responsible for propagation of the muscle electrical impulse radially into the myofiber?Proper electrical excitation and Cacle Fig. , left. Hses Fig. , right. −-related membrane depolarization and obtain more stable records, we used K+-acetate filled electrodes out recording solution. Similar observations were made when the chloride channel blocker (9-anthracen carboxylic acid (9-AC) at 100 μM) was included in the control recording solution (data not shown). One interesting observation related to the low [Cl−]out recording was made in the ALT fibers and the longitudinal spread of the electrically evoked Ca2+ transient. In these ALT fibers, a reduction of [Cl−] in the extracellular solution produced an extended spread of the Ca2+ transient from the fiber’s end towards the middle region of the fiber. This effect is indicated with the yellow brackets in Fig. −]out recording solution .Most of the UNI (94 %) or ALT (78 %) fibers exhibited little, if any, alterations in their electrically evoked Caion Fig. . The ave+ channels different in ALT myofibers compared to UNI myofibers? An alteration in the level or distribution of Na+ channels could explain the abnormalities seen in ALT myofibers. Using indirect immunofluorescence confocal microscopy and an α subunit specific Nav1.4 antibody, we observed the localization of voltage-gated Na+ channels in both UNI and ALT myofibers. In UNI myofibers, Nav1.4 was localized in a striated pattern  imaging while simultaneously performing real-time analysis of the Ca2+ transients. In two out of ten muscles analyzed, we were able to identify alternate responses upon field stimulation of alternate polarity. Figure right panel) and Fig. 2+ transient time course from a UNI fiber using this stimulation protocol. Note that the Ca2+ transients of the UNI fiber shown in Fig. right panel) image and Fig. 2+ transient time course, of a FDB fiber illustrating alternate (ALT) responses to field stimulation.The above results show that ALT myofibers are present in cultures of dissociated FDB fibers. How do ALT myofibers originate? Is the presence of ALT myofibers a consequence of the enzymatic dissociation and mechanical trituration required to isolate individual myofibers? If that is the case, Cator Fig. . To circ2+ imaging, we searched for the presence of ALT responses in muscles isolated from MDX mice, widely used as an animal model of Duchenne muscular dystrophy, the most common and severe muscular dystrophy. MDX muscles were explored and stimulated with the same protocol used in wild-type muscles. In four out of eight muscles analyzed (two FDB and six EDL), we were able to identify alternate responses upon field stimulation of alternate polarity. Figure 2+ signals (right panel) and Fig. 2+ transient time course from a UNI-MDX myofiber from a FDB muscle. Note that the Ca2+ transients of the UNI-MDX myofiber shown in Fig. 2+ signals (right panel) and corresponding Ca2+ transients time course, respectively, of a MDX myofiber displaying alternate (ALT-MDX) responses to alternate polarity field stimulation. Remarkably, the Ca2+ transients of the ALT-WT  isolated whole muscles, indicating that the enzymatic treatment is not required to produce the ALT response. The ALT behavior of myofibers from intact muscle was observed in 20 % of the preparations and thus is likely to be a rare event in healthy muscle. In muscles from the MDX mice, the ALT responses were observed more frequently (observed in 50 % of the preparations). It is anticipated that ALT responses could be also detected in different muscle disease models that affect excitable properties of myofibers.Enzymatically dissociated adult skeletal muscle myofibers are routinely used in studies of muscle function such as the excitation-contraction coupling (ECC) , 27, 64 2+ or in resting membrane potential. Our data on resting Ca2+ is limited to global resting Ca2+, and one cannot rule out the possibility that changes in local Ca2+ domains, or microdomains  removal, in which uniform myofibers are “converted” into ALT myofibers, suggest that Na+ channels contribute to the development of the ALT phenotype. Similar responses have been reported for whole skeletal muscle and skinned myofibers treated with TTX and stimulated with prolonged pulses  in the bath solution indicate that this manipulation negligibly affects the excitation properties of UNI myofibers  in response to alternating field stimulation. The reduction of [Cl−] influences the excitable properties in a small percentage the ALT fibers . Also, less than 20 % of UNI and ALT fibers displayed spontaneous hyperexcitability in reduced [Cl−] solution. Another interesting observation was that the reduction of [Cl−] in ALT fibers results in an increased spread of the local Ca2+ transient in response to field stimulation when compared to the response elicited in control solution. This result indicates that Cl− channels regulate the fiber’s electrical length constant. Overall, our results indicate that Cl− channels may contribute, but are not critical, to the manifestation of the ALT behavior in response to field stimulation. Whether other ion channels important for skeletal muscle action potential could play a role in the suppressed excitation behavior of the ALT myofibers remains to be determined.Finally, we cannot conclude that the observed properties of ALT myofibers arise exclusively from the dysregulation of Nachannels , 92. Givtability  we inves2+ transients) to establish that normal behavior of skeletal muscle myofibers be implemented when choosing myofibers for physiological experiments, and recommend avoiding the use of muscle myofibers that display only alternating end local contractile activity in response to alternating polarity stimuli (unless that is the objective). On the other hand, if defective myofiber excitability is suspected under various experimental or pathological conditions, our results suggest that looking for myofibers exhibiting ALT behavior in response to alternating polarity electric field stimulation via remote bipolar electrodes can provide a useful approach for identifying myofibers with compromised excitability.Although it is clear that ALT myofibers can be present in FDB whole muscle and FDB cultures, many aspects of its origin are still being investigated. Are ALT myofibers present in vivo? Do ALT myofibers influence or underlie disease conditions? Regardless of the mechanism(s) responsible for abnormal excitability of ALT myofibers, we encourage the careful monitoring of the contractile response of the myofiber  measured in UNI myofiber (A) and at the responding end of the ALT myofiber (B). Signals were recorded using high-speed line scanning (100μs/line) to further characterize the temporal properties of the Ca2+ transients. Time course of the normalized Ca2+ transients in the UNI (C) and ALT (D) myofibers derived from panels A and B, respectively to compare the kinetics of Ca2+ transients. UNI fibers showed reproducible responses that were independent of the pulse duration, whereas ALT fibers exhibited graded and reproducible responses with duration proportional to the duration of the stimulus. E, Representative isochronical confocal xy images acquired milliseconds before stimulation and showing the spatial properties of the electrically evoked rhod-2 signals using monopolar field stimulation. Ca2+ transients were elicited by suprathreshold pulses of fixed duration (1 ms) but increasing amplitude (30–60 V) at one end of an ALT myofiber. Dashed lines are fixed in the horizontal axis in all frames and indicate the fiber’s end. The arrows mark the limit of longitudinal propagation of the Ca2+ signals. The increase in the gap between the dashed line (fiber’s end) and the arrow (Ca2+ front) with pulses of increased amplitude confirms the graded nature of these transients. Using this electrode configuration, this local Ca2+ signal appears to originate at the fiber’s end, spreads inwardly towards the fiber’s trunk and then suddenly stops without full propagation, which is opposite to the responses seen in UNI fibers  was measured in L-15 media supplemented with 20 μM BTS (N-benzyl-p-toluene sulphonamide) to minimize movements during microelectrode penetration. The phenotype (UNI or ALT) was characterized by monitoring twitch activity using an inverted microscope (×10 magnification) and 1-ms suprathreshold bipolar field stimulation. A, Distribution of RMP values and fraction of fibers population in UNI and ALT groups. B, Bar plot summary. Mean RMP upon impalement in UNI  and ALT fibers  were −62.7 ± 0.9 and −59.2 ± 1.6, respectively. N indicates number of mice per condition, and n indicates number of fibers tested. ALT fibers showed no statistically significant difference in RMP when compared to UNI myofibers . Figure S3. Montage of whole muscle for electrical stimulation and Ca2+ imaging. After loading for 30 min with Fluo4-AM (20 μM), the muscle was immobilized in a holder chamber and then field stimulated. The muscles (flexor digitorum brevis or extensor digitorum longus) were carefully dissected, avoiding unnecessary manipulation of muscle tissue, such as excessive pulling, and keeping tendons at both ends, then muscles were briefly placed on the surface of a glass-bottomed petri dish (A). Silicone grease  was applied to the plastic surface of the petri dish (arrows). After applying the grease a small piece (14 × 8 × 0.5 mm) of a plastic cover slip  was placed on top of muscles and used to gently hold muscles in place during stimulation. The plastic cover slip was secured via the contact with the vacuum grease applied to the petri dish surface (B–C). Muscles were stimulated, using platinum electrodes attached to the lid of the petri dish . (DOCX 1633 kb)ALT fibers exhibit graded responses to pulses of increased duration and amplitude. Representative Caers Fig. , d. F, SAdditional file 2:Uniform and alternate electrically induced twitch responses. Description: left, uniform electrically induced twitch responses. Representative transmitted light movie of a skeletal muscle myofiber illustrating its uniform twitch behavior in response to bipolar electrical field stimulation of alternate polarity. Right, alternate electrically induced twitch responses. Representative transmitted light movie of a skeletal muscle myofiber showing its alternate twitch behavior in response to bipolar electrical field stimulation. Twitch responses to a pair of pulses  interspaced by a 400-ms interval. High-speed time-lapse imaging was conducted for 2 s at 16.7 ms/frame using a ×10 objective magnification. (AVI 5445 kb)Additional file 3:Uniform and alternate electrically induced Ca2+ transients. Description: left, uniform electrically induced Ca2+ transients. Representative confocal time series of a skeletal muscle myofiber loaded with the Ca2+ indicator rhod-2 illustrating uniform Ca2+ transients in response to bipolar electrical field stimulation of alternate polarity. Right, alternate electrically induced Ca2+ transients. Representative confocal time series of a skeletal muscle myofiber loaded with the Ca2+ indicator rhod-2 showing its local and asymmetrical Ca2+ transients in response to bipolar electrical field stimulation of alternate polarity. Ca2+ transients were elicited by a pair of pulses  interspaced by a 400-ms interval. High-speed time-lapse imaging was conducted for 2 s at 16.7 ms/frame using a ×10 objective magnification. (AVI 2965 kb)Additional file 4:TTX treatment “converts” UNI myofibers to ALT myofibers. Description: left, pre-TTX: uniform electrically induced Ca2+ transients. Confocal time series of a skeletal muscle myofiber loaded with the Ca2+ indicator rhod-2 illustrating uniform Ca2+ transients in response to bipolar electrical field stimulation of alternate polarity and before the addition of tetrodotoxin (TTX). Right, post-TTX: alternate electrically induced Ca2+ transients. Confocal time series of same myofiber presented in left panel, illustrating asymmetric Ca2+ transients in response to bipolar electrical field stimulation of alternate polarity 5 min after the addition of TTX; 1μM. Ca2+ transients were elicited by a pair of pulses  interspaced by a 400-ms interval. High-speed time-lapse imaging was conducted for 2 s at 16.7 ms/frame using a ×10 objective magnification. (AVI 3650 kb)Additional file 5:Reduction of extracellular [Na+] “converts” UNI myofibers to ALT myofibers. Description: left, uniform electrically induced Ca2+ transients measured in physiological extracellular Na + concentration, [Na+]e. Confocal time series of a skeletal muscle myofiber loaded with the Ca2+ indicator rhod-2 illustrating uniform Ca2+ transients in response to bipolar electrical field stimulation of alternate polarity and before the addition of Na-free recording solution. Center, alternate electrically induced Ca2+ transients measured in Na+-free extracellular conditions. Confocal time series of the same myofiber displayed in left panel, illustrating asymmetric Ca2+ transients in response to bipolar electrical field stimulation of alternate polarity 5 min after the addition of the addition of Na+-free recording solution. Right, uniform electrically induced Ca2+ transients measured when myofiber is returned to recording solution with physiological extracellular [Na+]. Confocal time series of same myofiber displayed in center panel, illustrating uniform Ca2+ transients in response to bipolar electrical field stimulation of alternate polarity 5 min after the return to control solution. Ca2+ transients were elicited by a pair of pulses  interspaced by a 400-ms interval. High-speed time-lapse imaging was conducted for 2 s at 16.7 ms/frame using a ×10 objective magnification. (AVI 4415 kb)