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Biochemistry and Chemical Biology
MCU encodes the pore conducting mitochondrial calcium currents
Dipayan Chaudhuri,
Yasemin Sancak,
Vamsi K Mootha,
David E Clapham ,
Massachusetts General Hospital, United States;
Howard Hughes Medical Institute, Boston Children's Hospital, United States;
Harvard Medical School, United States;
Research Article Jun 4, 2013
Cited 105
Mitochondrial calcium (Ca2+) import is a well-described phenomenon regulating cell survival and ATP production. Of multiple pathways allowing such entry, the mitochondrial Ca2+ uniporter is a highly Ca2+-selective channel complex encoded by several recently-discovered genes. However, the identity of the pore-forming subunit remains to be established, since knockdown of all the candidate uniporter genes inhibit Ca2+ uptake in imaging assays, and reconstitution experiments have been equivocal. To definitively identify the channel, we use whole-mitoplast voltage-clamping, the technique that originally established the uniporter as a Ca2+ channel. We show that RNAi-mediated knockdown of the mitochondrial calcium uniporter (MCU) gene reduces mitochondrial Ca2+ current (IMiCa), whereas overexpression increases it. Additionally, a classic feature of IMiCa, its sensitivity to ruthenium red inhibition, can be abolished by a point mutation in the putative pore domain without altering current magnitude. These analyses establish that MCU encodes the pore-forming subunit of the uniporter channel.
Mitochondria are tiny organelles, less than a micrometre across, found inside almost all eukaryotic cells. Their main function is to act as the 'power plant' of the cell, generating adenosine triphosphate or ATP, which is the source of chemical energy for cellular processes. Beyond generating ATP, mitochondria perform many other functions: they contribute to various signalling pathways; they influence cellular differentiation; and they are involved in processes related to cell death.
Mitochondria are quite distinctive in appearance—they are enclosed by two membranes, a porous outer one and a largely impermeable inner membrane. Most mitochondrial functions involve proteins that control the movement of various molecules and ions across the inner membrane. One particularly important ion that must pass through this membrane is calcium; once inside the mitochondria, these calcium ions regulate cell survival and the generation of ATP.
Although several calcium import mechanisms exist, the best-studied pathway involves a pore-forming protein complex called the mitochondrial calcium uniporter. This ion channel has an exquisite selectivity, allowing only calcium into mitochondria even when other ions outnumber it a million-fold. Previously, researchers had identified several genes that are required for the formation of the uniporter, but it had not been established which of these encodes the central pore through which the calcium ions pass. Now, Chaudhuri et al. have shown that one of these—a gene called mitochondrial calcium uniporter (MCU)—codes for the protein subunit that creates the pore.
Prior studies used optical methods or purified proteins to study genes encoding the uniporter complex, producing controversial results regarding pore identity. This study uses a much more direct assay, namely electrophysiology performed on mitochondrial inner membranes. To access the inner membrane, the authors stripped off the outer membrane from whole mitochondria, and made them expand. By using a technique called voltage-clamping, Chaudhuri et al. were able to precisely measure calcium ion movement through intact or mutated channels. This technique controls confounding factors and minimizes the effect of contaminants that can plague interpretation of data acquired by other methods. They showed that blocking the expression of the MCU gene reduced the flow of calcium ions through the uniporter, whereas increasing MCU expression increased calcium transport.
One unique feature of the mitochondrial calcium uniporter is that it can be blocked by a dye called ruthenium red. Chaudhuri et al. used this property to confirm that the MCU gene encodes the pore-forming subunit of the channel complex—they identified a single point mutation in MCU that did not affect the channel's ability to transport calcium ions, but did abolish its sensitivity to ruthenium red. Together, these results show that the MCU gene encodes the pore of the mitochondrial calcium uniporter, and should lead to further research into the physiology and structure of this channel.
Since the initial demonstration that mitochondria take up substantial amounts of cytoplasmic Ca2+ (Deluca and Engstrom, 1961), detailed studies have revealed that this uptake can sculpt the cytoplasmic Ca2+ transient (Wheeler et al., 2012), enhance ATP synthesis (Balaban, 2009), and trigger cell death (Zoratti and Szabo, 1995). Of several pathways for Ca2+ entry, a uniporter found in the inner membrane possesses the largest capacity for uptake and was shown to be a highly Ca2+-selective ion channel (Kirichok et al., 2004). However, despite this substantial progress, the identities of the genes encoding the functional uniporter were largely unknown until only recently. In the past several years, investigators from several laboratories have identified mitochondrial calcium uptake 1 (MICU1, formerly Cbara1) (Perocchi et al., 2010), mitochondrial calcium uniporter (MCU, formerly Ccdc109a) (Baughman et al., 2011; De Stefani et al., 2011), and mitochondrial calcium uniporter regulator 1 (MCUR1, formerly Ccdc90a) (Mallilankaraman et al., 2012) as potentially integral components of this channel.
These discoveries have spurred an explosion of research into mitochondrial Ca2+ transport, yet an outstanding question in this field is which, if any, of these several genes forms the pore subunit of the channel. So far, the discovery of each of these genes occurred via Ca2+ imaging assays following RNAi-mediated knockdown. Although extremely useful for screening, this technique is ill-suited for separating modulatory effects from altered expression of the channel-forming subunit itself, and each of these genes was found to inhibit Ca2+ uptake via this assay. This difficulty arises because Ca2+ imaging measures Ca2+ entry, which depends critically on (i) the number of open portals, (ii) the voltage gradient, and (iii) the Ca2+ concentration gradient driving flux, and none of these is controlled during imaging experiments. In fact, changes in Ca2+ buffers or efflux pathways can alter matrix Ca2+, and changes in pH or other divalents can alter indicator fluorescence, without reflecting a true difference in uniporter activity. Rather, the ideal method for assaying electrogenic ion transport is voltage-clamping, which allows precise control of the voltage and concentration gradient and thus accurate measurement of the changes in channel density.
Initial attempts to apply voltage-clamping focused on MCU, which possesses two transmembrane domains and highly-conserved acidic residues in a putative pore domain (Baughman et al., 2011; De Stefani et al., 2011; Bick et al., 2012). However, although MCU peptides reconstituted into lipid bilayers showed some activity in single-channel recordings (De Stefani et al., 2011), these experiments failed to recapitulate the typical single-channel behavior of the uniporter, most importantly its characteristic conductance and long-lasting openings (99% open probability) (Kirichok et al., 2004). Although such a discrepancy may be explained by the incorporation of the channel into bilayers in the absence of regulatory subunits, lipid bilayer reconstitution is also notorious for false positive findings. Since it detects the activity of single molecules, channel-like behavior can be measured from trace contaminants after even crystallography-grade purification (Accardi et al., 2004). Thus, the identity of the pore-forming subunit remains to be definitively established.
To overcome the limitations of single-channel recordings, we chose to voltage-clamp whole mitoplasts (mitochondria stripped of their outer membrane and expanded osmotically to allow access to the inner membrane) (Figure 1A). This allows electrophysiological interrogation of all the channels assembled in their native milieu in individual mitochondria. Moreover, this technique allows us to express mutated channels and examine mitochondrial Ca2+ currents (IMiCa) for altered key features. Using this method, we show here that MCU does recapitulate key features of IMiCa, and show that a single point-mutation can generate resistance to pharmacologic inhibition.
MCU expression recapitulates IMiCa.
(A) A HEK-293T cell mitoplast under differential interference contrast (far left), showing a typical figure-eight shape. The lobe bounded only by the mitochondrial inner membrane appears less dense (white arrow) than the lobe also bounded by the outer membrane (black arrow). Matrix-targeted mCherry demonstrates that the inner membrane forms a surface on both lobes (middle left). GFP-tagged mitofusin-1 (an outer membrane GTPase, middle right) reveals the outer membrane partially encapsulating only one lobe. Far right: merged image. (B) Western blots demonstrate reduced MCU expression following short-hairpin RNA-mediated knockdown of MCU but not GFP (control). ATP5B is a loading control. (C) Exemplar current traces demonstrate endogenous IMiCa in control knockdown (black trace), which is largely blocked by 100 nM RuR (red trace). Voltage ramps from −160 to +80 mV for 750 ms were delivered every 6 s from a holding potential of 0 mV. (D) Significantly reduced IMiCa after MCU knockdown. (E) Summary data, n = 6–10 per condition, error bars report SEM.
Stable transfection of HEK-293T cells with a short-hairpin RNA targeting MCU produced a substantial reduction in the protein when assayed by Western blot (Figure 1B) or quantitative real-time polymerase chain reaction (17 ± 5% transcripts remaining compared to shGFP). We isolated mitoplasts from these cells using the Kirichok protocol (Fedorenko et al., 2012; Fieni et al., 2012; Figure 1A). As expected from Ca2+-imaging experiments (Baughman et al., 2011), mitoplasts from control cells showed robust IMiCa during voltage ramps from −160 mV to +80 mV (Figure 1C). Because IMiCa features a half-saturation value (K0.5) of 20 mM [Ca2+]bath, we maximized current by recording in a 100 mM Ca2+ gluconate bath solution (Kirichok et al., 2004). Utilizing high external Ca2+ allows us to conclude that changes observed after modifying MCU expression is due to altered channel levels rather than modulation of K0.5, which might be set by accessory subunits. Other critical features of IMiCa replicated in HEK-293T cells include its strong inward-rectification and high-affinity blockade by ruthenium red (RuR, 87 ± 2% inhibition in 100 nM RuR, Figure 1C,E).
Compared to the control condition, IMiCa in mitoplasts from shMCU-expressing cells was markedly smaller (Figure 1D). The RuR-sensitive component of total Ca2+ current was reduced by 78 ± 14% (p<0.001, Figure 1E), with no significant difference in the RuR-insensitive residual component, suggesting that the knockdown was specific to IMiCa and not a generalized reduction in membrane conductance. Moreover, differences were not due to alterations in mitochondrial structure, as mitoplast capacitance, a surrogate for inner membrane surface area (100µm2/pF), was consistent across all conditions tested here (shGFP: 0.48 ± 0.10 pF, shMCU: 0.34 ± 0.09 pF, p>0.05).
Next, we examined if overexpression of wild-type or mutant human MCU proteins substantially changed IMiCa. We focused on an MCU mutated to encode an alanine at a highly-conserved serine in the putative pore-forming loop (S259A) (Baughman et al., 2011; Bick et al., 2012). The wild-type and mutant MCU proteins were tagged with a carboxyl-terminal FLAG epitope, and localized appropriately to mitochondria (Figure 2A,B). To confirm targeting to the inner membrane, we treated HEK-293T mitochondrial fractions with increasing concentrations of digitonin, and assayed for enzymatic digestion with proteinase K (Baughman et al., 2011; Figure 2C,D). Since the FLAG-tagged proteins were protected from digestion to a comparable extent as peptidyl-prolyl cis-trans isomerase F (PPIF, a matrix protein), whereas proteins of the outer- and inter-membrane spaces were not, the MCU constructs are appropriately targeted with their carboxyl-termini facing the mitochondrial matrix. Turning to our whole-mitoplast recordings (Figure 2E,G), we found that overexpression of wild-type MCU-Flag produced a robust increase in IMiCa of approximately 3.4-fold compared to endogenous HEK-293T currents (compare to Figure 1C,E). This enhanced IMiCa retained its sensitivity to RuR (Figure 2E,G).
MCU mutants alter IMiCa sensitivity to RuR.
(A) Confocal imaging of HEK-293T cells for FLAG-tagged MCU (left) and cytochrome C oxidase I (a mitochondrial marker, center). The merged image (right) shows colocalization. (B) As in (A) but for MCU-259A-FLAG. (C) Immunoblot analysis of proteinase K digestion after wild-type MCU-FLAG-expressing HEK-293T mitochondrial fractions are digitonin-permeabilized at the specified concentrations, confirming mitochondrial targeting. (D) Proteinase K digestion analysis for MCU-S259A-FLAG mutant. (E) Significant increase in IMiCa after MCU-FLAG overexpression. (F) Loss of RuR block in S259A mutant. (G) Summary data, n = 5–9 per condition.
Finally, we studied the S259A-MCU mutant to see if it disrupted key features of IMiCa. In Ca2+-imaging experiments, this mutant had preserved Ca2+ uptake but diminished sensitivity to uniporter inhibitors (Baughman et al., 2011). At baseline, mitoplasts from HEK-293T cells transfected with the S259A mutant had capacitances similar to mitoplasts after wild-type MCU overexpression (MCU-Flag: 0.32 ± 0.06 pF, S259A: 0.29 ± 0.10 pF, p>0.05). Moreover, S259A overexpression mirrored the increase in IMiCa seen after wild-type MCU transfection, confirming a fully-functional channel (Figure 2F,G). However, this variant displayed markedly decreased sensitivity to RuR, with minimal inhibition at 100 nM. Since overexpression occurred on a background of endogenous channels, this mutant appears to act in a dominant-negative fashion. In particular, the S295A RuR-inhibited fraction (148 ± 33 pA/pF, Figure 2G) was much less than the RuR-inhibited fraction in endogenous IMiCa (372 ± 42 pA/pF, Figure 1F). This suggests that, since the channel is an oligomer of multiple MCU subunits (Baughman et al., 2011), mutant S259A-MCU can incorporate with endogenous, wild-type subunits to form a Ca2+-conducting channel, which nonetheless has drastically-reduced RuR sensitivity.
In our study, we use the most direct assay available to measure mitochondrial Ca2+ currents—whole-mitoplast electrophysiology (Kirichok et al., 2004). By isolating mitoplasts and recording directly from all the channels embedded in the inner membrane, we measure the ensemble current and thus avoid confounding minor contaminants. This method, moreover, is the same technique that originally demonstrated that the mitochondrial Ca2+ uniporter was an ion channel, allowing us to make a direct comparison to the characteristic features of this current (IMiCa), and to determine if the genetically-modified channels could alter them.
We focused on MCU, the leading candidate for the pore-forming subunit, as it possesses two transmembrane domains and has highly-conserved acidic residues in a putative pore-like domain. First, we show that reducing MCU transcripts via knockdown, or enhancing them through overexpression, produce parallel changes in IMiCa. Second, we show that a central feature of IMiCa—its exquisite sensitivity to blockade with ruthenium red—can be abolished via a single point mutation in the pore domain. MCU subunits carrying this mutation act in a dominant-negative fashion, producing functional channels that conduct Ca2+ ions but are largely insensitive to ruthenium red. We conclude that the functional uniporter pore at the mitochondrial inner membrane is formed by MCU multimers. We believe these experiments put to rest the outstanding question of the pore identity, firmly establishing that MCU produces IMiCa.
HEK-293T were obtained from ATCC and grown in Dulbecco's modified Eagle medium with high glucose supplemented with 10% fetal bovine serum and penicillin/streptomycin (Life Technologies, Grand Island, NY). HEK-293T cells (250,000 cells per well) were infected with lentivirus expressing short hairpin RNA (for knockdown experiments) or wild-type or mutant FLAG-tagged MCU. 2 days after infection, cells were split and selected with the appropriate antibiotic. Stable HEK-293T cells lines expressing shGFP and shMCU were maintained in 2 μg/ml puromycin (Sigma, St. Louis, MO), while MCU-FLAG and S259A lines were maintained in 200 μg/ml hygromycin B (Sigma).
Vectors for expressing shRNA (pLKO.1) were obtained from the Broad Institute's RNAi Consortium (Sigma). To silence MCU, we used a hairpin targeting the 3′ UTR, with sequence 5′-GCAAGGAGTTTCTTTCTCTTT-3′ (TRCN0000133861). The control hairpin was against GFP, with sequence 5′-ACAACAGCCACAACGTCTATA-3′ (shGFP) (TRCN0000072181). The FLAG-tagged full-length human MCU cDNA (NM_138357.1) was cloned into the pLJM5 vector (Sancak et al., 2010). The MCU mutant S259A was generated with QuikChange according to the manufacturer's instructions (Agilent, Santa Clara, CA). Human Mitofusin-1 (NM_033540) was cloned out of HEK-293T cell cDNA (see Quantitative polymerase chain reaction section, below) and ligated in-frame into the pEGFP-N3 vector (Clontech, Mountain View, CA) using KpnI and BamHI restriction sites. Matrix-targeted mCherry was engineered by replacing the GFP construct of the pAcGFP1-Mito plasmid (Clontech), using BamHI and NotI restriction sites.
Quantitative polymerase chain reaction
Primers against human MCU were designed using PRIMER-Blast (NCBI). The forward primer was 5′-TTCCTGGGACATCATGGAGC-3′, and the reverse primer was 5′-TGTCTGTCTCTGGCTTCTGG-3′. For qPCR, confluent HEK-293T cells were resuspended in TriZOL reagent (Life Technologies), and total RNA was subsequently purified following the manufacturer's instructions. 2 μg of RNA was used to synthesize cDNA using SuperScript ViLo (Life Technologies) and 100 ng of this was used per well for qPCR. Reactions were run in triplicate using SYBR Green (Agilent) on an Eppendorf Mastercycler epgradient S Realplex4 qRT-PCR cycler (Eppendorf, Hauppauge, NY). Analysis compared expression in shMCU cells to shGFP cells using the 2−ΔΔCt method, normalizing to glyceraldehyde 3-phosphate dehydrogenase expression.
10 μg of protein from cell lysates or 5 μg of protein from crude mitochondrial preparations were loaded on 12% or 18% Tris-Glycine gels, proteins were transferred to PVDF membranes and then incubated with the indicated antibodies at 4°C overnight. We used antibodies against TOMM20 (sc-17764; Santa Cruz Biotechnology, Dallas, TX), TIMM23 (No. 611222; BD Biosciences, San Jose, CA), PPIF (ab110324; Abcam, Cambridge, MA), ATP5B (ab14730; Abcam), FLAG epitope (No. 2368; Cell Signaling Technology, Danvers, MA), and MCU (polyclonal chicken antibody raised against residues 41–351 of human MCU tagged with a 6His epitope; Covance, Dedham, MA).
Proteinase K protection assay
Two confluent plates of HEK-293T cells were washed with PBS and the cells scraped into 1.5-ml microcentrifuge tubes in 1 ml of final volume. The cells were lysed by passaging through a 27.5 G needle several times. Samples were spun at 800g for 10 min at 4°C to pellet nuclei and non-lysed cells. The supernatant was removed and spun separately at 8000g for 10 min at 4°C to pellet the mitochondrial fraction. Mitochondria were resuspended in a sucrose buffer (200 mM sucrose, 10 mM Tris-MOPS, 1 mM EGTA-Tris) to a final concentration of 1 mg/ml. 20 μg of mitochondria were treated with digitonin (0.01–0.4%, Sigma) or 1% Triton-X100 (Sigma) and 100 μg/ml of proteinase K for 15 min at room temperature, in a 30 μl final volume. Proteinase K was then inhibited by addition of 5 mM PMSF. 5 μg of protein was loaded to Tris-glycine gels for Western blotting.
Stably-transfected HEK-293T cells were washed in PBS, which was used as the base solution in all following steps. Cell were fixed in 4% formaldehyde, permeabilized in 0.5% Triton-X100, and blocked in 10% goat serum, 0.1% Tween-20 (Biorad, Hercules, CA). Primary antibodies were a rabbit anti-FLAG and a mouse anti-MTCO1 (a mitochondrial marker, ab14705; Abcam). Secondary antibodies were Alexa-488 conjugated to a goat anti-rabbit, and Alexa-546 conjugated to goat anti-mouse (Life Technologies). Cells were imaged on an Olympus Fluoview 1000 confocal microscope (Center Valley, PA).
For mitoplast images, HEK-293T cells were co-transfected with mt-mCherry and MFN1-GFP plasmids using Lipofectamine 2000 reagent (Life Technologies) according to the manufacturer's instructions. Mitoplasts were isolated (as described in 'Whole-mitoplast recording') 2 days following transfection, and imaged on the confocal microscope as above.
Brightness levels have been slightly adjusted in the images to improve contrast. This adjustment has been applied to each image as a whole and has not obscured or eliminated any particular feature of the image. All reagents were from Sigma unless otherwise stated.
Whole-mitoplast recording
We adapted prior methods to HEK-293T cells (Fedorenko et al., 2012; Fieni et al., 2012). Cells were grown to confluency on three 15-cm dishes. All subsequent manipulations were done using ice-cold solutions and equipment. Cells were scraped onto divalent-free PBS and spun down to pellet cells. The cell pellet was subsequently resuspended in a sucrose solution (250 mM sucrose, 5 mM HEPES, 1 mM EGTA, 0.1% BSA). The suspension was homogenized using an overhead stirrer. Centrifugation at 700g pelleted nuclei. The supernatant was transferred to a new tube and centrifuged at 8500g to pellet a crude mitochondrial fraction. This pellet was resuspended in a hypertonic mannitol solution (440 mM mannitol, 140 mM sucrose, 5 mM HEPES, 1 mM EGTA) for 10 min. The suspension was then passed through a French Pressure Cell homogenizer (Thermo Scientific, Waltham, MA) at 2000 p.s.i. to disrupt the mitochondrial outer membrane. The eluate was centrifuged at 10,500g to pellet mitoplasts. Mitoplasts were stored in a hypertonic solution (750 mM KCl, 100 mM HEPES, 1 mM EGTA) on ice and protected from light.
For recordings, mitoplasts were aliquoted into the initial bath solution (150 mM KCl, 10 mM HEPES, 1 mM EGTA) to allow them to expand for 20 min. An individual mitoplast was visualized and attached to the pipette tip with gentle suction. Pipettes were formed from borosilicate glass (Sutter, Novato, CA) with tip resistances of 20–60 MOhm. We used sodium gluconate for pipette internal solutions (150 mM sodium gluconate, 10 mM HEPES, 2 mM EDTA, with sucrose to achieve an osmolarity of 410–430 mOsmol). Electrophysiology was performed using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). Upon giga-ohm seal formation, pipette capacitance transients were cancelled, and the inner membrane was ruptured by 10–50 ms pulses (350–1000 mV). After establishing the whole-mitoplast configuration, a series of 4 ms pulses of 20 mV were recorded to allow curve fitting for capacitance measurements. Bath solutions were subsequently exchanged for a high-Ca2+ solution (100 mM calcium gluconate, 20 mM HEPES, 2 mM CaCl2, with sucrose to achieve osmolarity of 295–305 mOsms) and high-Ca2+ +100 nM RuR solution (Sigma). RuR solutions were prepared fresh for every experiment. Solution exchanges were rapid, as prolonged matrix exposures to Ca2+ caused activation of a significant leak component. Comparisons were made via the Student's t-test.
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Reviewing Editor; The University of Texas at Austin, United States
eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.
Thank you for sending your work entitled "MCU encodes the pore conducting mitochondrial calcium currents" for consideration at eLife. Your article has been favorably evaluated by a Senior editor and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.
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This work for the first time addresses structure-function relationships within the mitochondrial Ca2+ uniporter using the patch–clamp technique. Although indirect optical methods helped to identify three candidate proteins important for the activity of the mitochondrial Ca2+ uniporter (MICU1, MCU, and MCUR1), the subsequent attempts to understand the exact functions of these proteins led to little progress and produced controversial results. In this manuscript, the authors provide strong evidence that one of the candidate proteins, MCU, forms the pore of the mitochondrial Ca2+ uniporter. Since this is the first structure–function study of the MCU using the patch–clamp technique, methodologically this works sets an important precedent in the field.
Overall, the manuscript is well written and provides sufficient evidence to suggest that the MCU protein is a pore-forming subunit of the mitochondrial Ca2+ uniporter. There are two important points to be addressed:
1) The quality of the image of an 8-shaped mitoplast on Figure 1A is rather low. From this image it is not clear whether this is a real 8-shaped mitoplast or two attached swollen round-shaped mitoplasts. Also, in addition to the mitoplast(s), the DIC image contains debris that complicates our understanding. It would be useful to provide an image of higher quality.
2) Figure 1D,E and Figure 2E,F: It is important to describe in the main text or in the figure legends how these traces were obtained. Are these responses to a voltage ramp protocol? What was the exact voltage protocol used? If a ramp protocol was used, it would perhaps be somewhat misleading to say that these traces are real I/V curves (as in the legend to Figure 1).
We have replaced the mitoplast image from Figure 1 with the new one shown below.
For this image, we co-transfected HEK cells with a plasmid encoding a mitochondrial matrix-targeted mCherry fluorescent protein, as well as a plasmid encoding GFP-tagged mitofusin-1, a mitochondrial outer membrane GTPase. This image is free of debris, and demonstrates that the structure is one mitoplast. We have replaced the figure legend as follows:
"A HEK-293T cell mitoplast under differential interference contrast (far left), showing a typical figure-eight shape. The lobe bounded only by the mitochondrial inner membrane appears less dense (white arrow) than the lobe also bounded by the outer membrane (black arrow). Matrix-targeted mCherry demonstrates that the inner membrane forms a surface on both lobes (middle left). GFP-tagged mitofusin-1 (an outer membrane GTPase, middle right) reveals the outer membrane partially encapsulating only one lobe. Far right: merged image."
We have taken out the mention of I/V curves, and added the following to the figure legend to describe the voltage command protocol:
"Voltage ramps from −160 to +80 mV for 750 ms were delivered every 6 seconds from a holding potential of 0 mV."
Dipayan Chaudhuri
Cardiovascular Research Center, Massachusetts General Hospital, Boston, United States
Department of Cardiology, Howard Hughes Medical Institute, Boston Children's Hospital, Boston, United States
DC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
The authors declare that no competing interests exist.
Yasemin Sancak
Departments of Molecular Biology and Medicine, Massachusetts General Hospital, Boston, United States
Department of Systems Biology, Harvard Medical School, Boston, United States
YS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
Vamsi K Mootha
VKM, Conception and design, Drafting or revising the article
David E Clapham
Department of Neurobiology, Harvard Medical School, Boston, United States
DEC, Conception and design, Drafting or revising the article
dclapham@enders.tch.harvard.edu
National Institutes of Health (F32HL107021)
National Institutes of Health (R24DK080261)
We would like to thank Francesca Fieni, Andriy Fedorenko, and Yuriy Kirichok for assistance with the whole-mitoplast protocol, and Andrew Markhard for technical assistance.
Richard Aldrich, The University of Texas at Austin, United States
Received: March 5, 2013
Accepted: April 29, 2013
Version of Record published: June 4, 2013 (version 1)
© 2013, Chaudhuri et al.
Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.
mitoplast
MICU1
ruthenium red
MCUR1
Structure of dual BON-domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation
Jack Alfred Bryant et al.
The Gram-negative outer-membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents, and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here, we reveal DolP is a lipoprotein functionally conserved amongst Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for Escherichia coli DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds anionic phospholipids through an extensive membrane:protein interface. This interaction is essential for DolP function and is required for sub-cellular localisation of the protein to the cell division site, providing evidence of subcellular localisation of these phospholipids within the outer membrane. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.
ARL3 activation requires the co-GEF BART and effector-mediated turnover
Yasmin ElMaghloob et al.
The ADP-ribosylation factor-like 3 (ARL3) is a ciliopathy G-protein which regulates the ciliary trafficking of several lipid-modified proteins. ARL3 is activated by its guanine exchange factor (GEF) ARL13B via an unresolved mechanism. BART is described as an ARL3 effector which has also been implicated in ciliopathies, although the role of its ARL3 interaction is unknown. Here we show that, at physiological GTP:GDP levels, human ARL3GDP is weakly activated by ARL13B. However, BART interacts with nucleotide-free ARL3 and, in concert with ARL13B, efficiently activates ARL3. In addition, BART binds ARL3GTP and inhibits GTP dissociation, thereby stabilising the active G-protein; the binding of ARL3 effectors then releases BART. Finally, using live cell imaging, we show that BART accesses the primary cilium and colocalises with ARL13B. We propose a model wherein BART functions as a bona fide co-GEF for ARL3 and maintains the active ARL3GTP, until it is recycled by ARL3 effectors.
A molecular mechanism for LINC complex branching by structurally diverse SUN-KASH 6:6 assemblies
Manickam Gurusaran, Owen Richard Davies
The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex mechanically couples cytoskeletal and nuclear components across the nuclear envelope to fulfil a myriad of cellular functions, including nuclear shape and positioning, hearing, and meiotic chromosome movements. The canonical model is that 3:3 interactions between SUN and KASH proteins underlie the nucleocytoskeletal linkages provided by the LINC complex. Here, we provide crystallographic and biophysical evidence that SUN-KASH is a constitutive 6:6 complex in which two constituent 3:3 complexes interact head-to-head. A common SUN-KASH topology is achieved through structurally diverse 6:6 interaction mechanisms by distinct KASH proteins, including zinc-coordination by Nesprin-4. The SUN-KASH 6:6 interface provides a molecular mechanism for the establishment of integrative and distributive connections between 3:3 structures within a branched LINC complex network. In this model, SUN-KASH 6:6 complexes act as nodes for force distribution and integration between adjacent SUN and KASH molecules, enabling the coordinated transduction of large forces across the nuclear envelope.


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Yisrael Meir ha-Kohen Kagan (January 26, 1838 – September 15, 1933), known popularly as the Chofetz Chaim, after his book on lashon hara, who was also well known for the Mishna Berurah, his book on ritual law, was an influential Lithuanian Jewish rabbi, Halakhist, posek, and ethicist whose works continue to be widely influential in Orthodox Jewish life.

Biography
Kagan was born on 26 January 1838 in Dzienciol (), Grodno Governorate in Russian Empire (today Dzyatlava in Belarus), and died on 15 September 1933 in Raduń (), Nowogródek Voivodeship in Second Polish Republic (now in Belarus). His surname, Poupko, is not widely known. Kagan himself used the name "Kagan" (The Russian form of "Kohen") in official and legal documents. When Kagan was ten years old, his father died. His mother moved the family to Vilnius in order to continue her son's Jewish education. While in Vilnius, Kagan became a student of Rabbi Jacob Barit. Kagan's mother later remarried (Epstein) and moved to Radin. At 17, he married the daughter of his stepfather, and settled in Radin.

He served as the town rabbi of Radin for a short period. He then resigned from this position to establish a yeshiva in the city, which eventually became world-famous first being led by the notable Naftoli Trop, followed by the esteemed son-in-law of his rabbi Mendel Zaks. By all accounts, he was a modest and humble man, faithfully devoted to his Jewish faith. For a while, he had a shop selling household provisions, which he and his wife managed. From 1864 to 1869 he taught Talmud in Minsk and Vashilishok.

In 1869, Kagan formed a yeshiva in Radin. The yeshiva was a success and grew to prominence, later becoming known as "Yeshivas Chofetz Chaim of Radin". In addition to spreading Torah through his yeshiva, Kagan, who became known as "the Chofetz Chaim", was very active in Jewish causes. He traveled extensively to encourage the observance of Mitzvot and Shabbat  amongst Jews. He became one of the most influential rabbis within Orthodox Judaism during the late 19th and early 20th century, taking a central leadership role in the World Agudath Israel movement in Eastern Europe. An author of many works of musar literature, he was sometimes been associated with the Musar movement, although he did not support all aspects of that movement.

Although the anti-religious attitudes which pervaded Zionism greatly distressed him, Kagan initially refused to become personally involved in the matter and refrained from publicly denouncing the movement. When his views became known, he cautioned his students about joining the Zionists and declared its political aims as being contrary to the Torah. Kagan famously said that it would be preferable to lose a third of the amount of Torah we have throughout the world rather than join the WZO (World Zionist Organization). Elchonon Wasserman (killed in the Holocaust 6 July 1941) said that this means even if the majority of yeshivos would have to close, we would not be allowed to join the WZO. In 1903, his wife Frieda died, and two years later he married the daughter of Rabbi Hillel of Lapis, Miriam Freida, who was more than thirty years younger than him. It was from this marriage that his son Rabbi Aharon Kagan and his daughter Feigl Chaya Sacks were born. His son-in-law was Rabbi Mendel Zaks.

In 1925, it was announced that he would be leaving Warsaw with his daughter and son-in-law to permanently settle in Petach Tikvah (the Gate of Hope, in Hebrew), in Palestine. Upon discovering his plans, prominent rabbis and yeshiva deans persuaded him to remain in Radin and he died there on September 15, 1933, aged 95. He was interred in the nearby cemetery.Miriam Freida arrived in Montreal with her son Rabbi Aharon via Siberia and Japan in 1942, and died in New York in 1946.

Many other Jewish religious institutions throughout the world also bear his name. One American yeshiva named in his honor is the Yeshivas Rabbeinu Yisrael Meir HaKohen centered in Queens, New York founded by his great nephew, Rabbi Dovid Leibowitz, with several branches in the United States, Canada, and Israel. Kagan's teachings have inspired some English-speaking American Jews to establish the Chofetz Chaim Heritage Foundation, dedicated to the dissemination of his teachings to Jewish communities around the world. An Orthodox kibbutz in Israel, Hafetz Haim, was named in his honor.

During his lifetime, Kagan was venerated by Jews and non-Jews alike. Orthodox Jews across the world viewed him as one of the 36 righteous people, and Polish farmers were said to have lured him into their fields believing his feet would bring blessing to their crops.

According to some reports, despite his rabbinic leadership he did not hold semikhah until late in life, when he had to apply for a passport and needed formal semichah in order to honestly enter "rabbi" as his occupation. He then received his semikhah by telegraph from Rabbi Chaim Ozer Grodzinski of Wilna. According to another report, though, he received semikhah much earlier, at age 17.

Works

 Chofetz Chaim ( "Seeker/Desirer [of] Life"), his first book, (published in 1873), deals with the Biblical laws of gossip and slander (known in Hebrew as Lashon Hara, meaning "Evil tongue"). View the online edition in Hebrew here
 Shemirat HaLashon ("Guarding of the Tongue"), is a comprehensive discussion of the philosophy behind the Jewish concepts of power of speech and guarding one's speech. It also serves as an inspirational work designed to motivate the reader to be vigilant in the ethical usage of his speech and avoidance of others' unethical speech. Published in 1876. View the online edition in Hebrew here
 Mishnah Berurah ("Clarified teachings") is an important and widely used commentary, consisting of six volumes, on the Orach Chayim section of Yosef Karo's digested compilation of practical Jewish Law, the Shulchan Aruch. It combines his own elucidations and differing opinions with those of other Aharonim (post-medieval authorities.) [As found in the book by Rabbi Moses M. Yoshor "The Chafetz Chaim" on page 603 the 1st volume was published in 1884; 2nd volume in 1886; 3rd volume in 1891; 4th volume in 1898; 5th volume in 1902; 6th volume in 1907.]
 Biur Halacha ("Explanation of the Law") is a commentary tangential to the Mishna Berurah. It usually provides complex analysis of the legal rulings of earlier Jewish halachic authorities.
 Sha'ar HaTziyyun ("Gate of Distinction") serves primarily to document sources for laws and customs quoted in the Mishnah Berurah, but sometimes serves also to clarify ambiguous legal statements. The name Sha'ar HaTziyyun derives from the phrase sh'arim m'tzuyanim ba'halacha, translated as "gateways distinguished in (or marked in) Jewish Law," referring to the Torah study and scholarship that would distinguish Jewish homes. Rabbi Kagan chose the title as a pun, hinting at the distinction of the scholarship referenced in his work, but primarily referring to (as he writes on his title page) the function of Sha'ar HaTziyyun to document (mark) sources.
 Ahavat Chesed – one volume, published in 1888. On the commandment of lending money to the needy, the value of being kind to one another and various ways to do so.
 Machaneh Yisrael – one volume, on the minimum requirements of Torah observance for soldiers in the army.
 Tiferes Odom – one volume, on the importance of a Jew having a beard and peyos (sidelocks).
 Geder Olam – one volume, published in 1890. On the importance of a married Jewish woman covering her hair.
 Nidchei Yisrael – two volumes, published in 1893.
 Shem Olam – one volume, published in 1893.

 Chomas Hadas – one volume, published in 1905. On the importance of a man to study Torah, and encourage others to learn, as well as the need to create groups in every city wherein a man could acquire Torah.
 Likutei Halachos,  a halakhic digest of all sugyot on which there was no "Rif" or "Rosh"; originally intended to encourage the study of the laws of the Temple service. Five volumes: the first volume was published in 1900; 2nd volume in 1903; 3rd volume in 1913; 4th volume in 1922. There is a fifth volume called "Hashlamah" or "Completion" which was published in 1925.
 Gibores Ari – two volumes, published in 1907.
 Tohoras Yisroel – one volume, published in 1910. On the importance of women to purify themselves in the waters of a mikvah (ritual bath) in accordance with accepted halachic practice.
 Toras Kohanim – one volume, published in 1911.
 Asefas Zekenim – three volumes, published in 1913.
 Chovas Hashemiro – one volume, published in 1915.
 Toras Habayis – one volume, published in 1923.
 Zechor Lemiryom – one volume, published in 1925.
 Beis Yisroel – one volume, published in 1925.
 Sefer Hamitzvos Hakotzor – two volumes on those Biblical commandments that are applicable during the Exile, outside the land of Israel, and when the Temple is not in existence. Published in 1931.
 Tzipita Lishuah ("Have you yearned the redemption") is based on a passage from tractate Shabbat, which states that after one passes away, he is asked by the heavenly court: "Have you yearned for the redemption?" This work describes the importance of actively awaiting Moshiach every day and doing everything in our power (learning Torah and doing mitzvot) to bring the redemption. The Chofetz Chaim's expectation of the immediate redemption was so strong that he would always carry special garments to change into once the redemption begins.

References

Sources

External links

 Chofetz Chaim Heritage Foundation
 Video lecture on the Chofetz Chaim by Dr. Henry Abramson
 Many of the Chafetz Chaim's works are available online in English http://chafetzchayim.org/
 The Ohr Olam Edition 'Mishnah Berurah' (new English translation)

1838 births
1933 deaths
People from Dzyatlava
People from Slonimsky Uyezd
Belarusian Haredi rabbis
Haredi rabbis in Europe
Haredi rosh yeshivas
Polish Haredi rabbis
Kohanim writers of Rabbinic literature
Exponents of Jewish law
Authors of books on Jewish law
Anti-Zionist Haredi rabbis

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Is It Safe to Go to the Doctor's Office For Non-COVID Care Right Now?
May 18, 2020 by laurasmith20200
Take these steps to stay healthy if you have to make a doctor's appointment right now.
The country is gradually emerging from lockdown, with almost every state rolling out phased reopening plans for schools, entertainment venues, and non-essential retailers. But what about a doctor's office, hospital, or outpatient facility for non-coronavirus care? When are they reopening, and is it risky to go right now?
During stay-at-home orders, many doctors closed their offices or outpatient spaces entirely, or they only offered telemedicine appointments. Meanwhile, hospitals have been focusing on providing lifesaving care to COVID-19 patients and other emergency cases. As a result, non-emergency treatment, such as elective surgeries and general checkups, have been postponed.
Doctor's offices and hospitals have always been thought of as safe places. But it's totally understandable to feel apprehensive about getting health care right now. Here's what experts want you to know about going back to your doctor, ER, or outpatient facility for non-COVID-19 visits and treatment.
When will my doctor reopen for non-coronavirus patients?
It depends on where you live and what kind of appointment you need. If you're waiting to schedule elective surgery, whether that goes ahead depends on your state government. "We're not moving ahead with elective surgeries until we are given authorization from the governor and Department of Health to do so," Andrew Rubin, senior vice president for clinical affairs and ambulatory care at NYU Langone Health, tells Health. "Right now, most cases that are moving forward are those that qualify as urgent or medically necessary."
Rubin says that some of the urgent or medically necessary procedures NYU Langone Health is now doing were delayed at the start of the pandemic as long as 90 days, including certain cancer procedures and surgeries, neurologic testing and treatment, and cardiac prosecutes and surgeries.
Typically, your local county health authority, in consultation with your state, will give guidance to facilities about when and how to reopen. "The 'when to open' will rest with the authorities, along with recommended or regulatory mandated guidelines, and the the 'how we will open' will be nuanced by the physicians and facility providers," former hospital CEO, health care advisor, and biomedical ethicist Michael Hunn tells Health.
After restricting their practice to emergency care, Diagnostic and Interventional Spinal Care (DISC) in Newport Beach, California, has now returned to general surgical procedures to treat urgent cases as well as people in severe pain. "Over the coming weeks, we will return to full-spectrum pain management as well," Robert S. Bray, Jr., MD, DISC founding director, tells Health.
NYC-based gynecologist Rebecca C. Brightman, MD, assistant clinical professor of obstetrics, gynecology, and reproductive medicine at the Icahn School of Medicine at Mount Sinai, opened up her private practice to patients for non-urgent matters and routine visits on May 11. "We're following guidelines outlined by the American College of Obstetricians and Gynecologists (ACOG), New York State, and the Department of ob-gyn at the Mount Sinai Healthcare system," Dr. Brightman tells Health.
"Some physicians aren't ready to see patients, and a lot of that has to do with the type of medicine they practice," adds Dr. Brightman. "If I wasn't comfortable about seeing my patients, I wouldn't."
What precautions are health care providers taking to keep patients safe?
Health care providers might be gradually getting back to normal in some parts of the country, but new procedures are in place to help reduce the risk of spreading the coronavirus. At NYU Langone Health, all providers, staff, and patients are screened for COVID-19 symptoms at the entrance of the facility, including a temperature check with a "no-touch" thermometer. All patients are required to wear a mask or face covering, and social distancing measures are enforced in waiting areas, says Rubin.
Extensive protective and preventive measures are also in place at DISC. All employees are tested on a weekly basis at minimum, and "every patient is tested several days before their surgical procedure to assure that no COVID cases are entered into the facility," says Dr. Bray.
Everyone in Dr. Brightman's office is practicing social distancing, sanitizing, and wearing masks. "We are also screening all patients, employees, physicians, couriers, and anyone else who enters the office with a questionnaire, plus we are recording their temperature," she says. "If the screening is abnormal, they won't be allowed into the office." Appointments are scheduled in a way that minimizes the number of patients in the waiting room at any given time.
What can patients do to lower their risk at a doctor's office or hospital?
Ahead of your appointment, health care staff should let you know what specific precautions you're expected to take to reduce the spread of COVID-19. Hunn says patients and visitors should take their temperature and wash their hands before they leave the house, wear a mask in the car on the way, wear gloves when necessary, and avoid touching their faces, especially their nose or eyes.
"Only show up when you need to and not early," advises Hunn. "Use doors that automatically open. Try not to touch surfaces. Protect others by remaining six feet apart. Be consistent, and don't let anyone near you who is not doing the same." Expect to wear a mask inside the doctor's office or health care facility as well, including during your appointment.
Can patients bring someone with them for support?
Many people might be keen to take a friend or family member with them to a doctor's appointment or hospital visit. But many hospitals and practices are limiting the number of visitors to maintain safety standards.
"Currently, adults may not be accompanied to their appointment unless our staff determines it is medically necessary, and children may be accompanied by two adult caretakers," says Rubin, of NYU Langone. But this is an evolving situation, so he advises checking the current visitor policies before your appointment, and calling your doctor's office with questions if needed.
When will my doctor's office return to normal?
More than ever, patients want to feel safe in their health care facility, and their physicians want safe environments in which to practice. Depending on how the COVID-19 pandemic plays out—only time will tell whether there will be a second wave of the virus, and we're still many months, if not longer, away from a vaccine—the new protocol could become the norm.
"Both patients and surgeons are now looking to more 'safe' environments like ambulatory surgery centers and outpatient clinics that can tightly control their environments and their infection control procedures," says Hunn.
DISC is one example of the ambulatory surgery center model that can care for patients in a non-COVID environment. "This type of facility in a closed environment provides a very safe available option," says Dr. Bray. "We have the capability of overnight stay, 24-hour nursing, and all necessary follow-up. And we are going to maintain our limited visiting and limited outside-world contact until this crisis is adequately resolved."
Bottom line: Like just about everything these days, going to your doctor or another medical setting as the country opens up incurs some risk. But if you have a true medical need, it's important that you get care and treatment. "Some patients with heart attacks, strokes and sports or orthopedic injuries have avoided getting the proper care, which has caused significant harm and long-term negative effects to their health," says Hunn.
If you're not in a high-risk group for the coronavirus and take all the necessary precautions before and during your visit, you can rest assured that your health care provider will be doing everything they can to keep you safe and healthy.
The information in this story is accurate as of press time. However, as the situation surrounding COVID-19 continues to evolve, it's possible that some data have changed since publication. While Health is trying to keep our stories as up-to-date as possible, we also encourage readers to stay informed on news and recommendations for their own communities by using the CDC, WHO, and their local public health department as resources.
Source: https://www.health.com/condition/infectious-diseases/coronavirus/is-it-safe-to-go-to-the-doctors-office
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You have just finished reading my post about the beautiful decorative boxes in my living room? Perhaps you should read this as well.
Before starting boxes of your own you need to consider one more thing: the outlets location, which I did not do (as I started on the wall free of them). Big mistake. My goal was to have the same height from the ceiling to the top of the box, and the bottom of the box to the floor. I also measured a distance that I liked, from the corner of the wall to the side of the box and copied the same distance between the boxes of each wall. What can possibly go wrong? My first wall looked fantastic.
On to the wall with the window: Always the same height top and bottom but a narrower version. And there is an outlet but I measured perfectly since it just lands bellow the molding for the box on the left of the window.
As for the one on the right side of the wall I have again an outlet and a switch to manage.
The outlet no problem but for the switch, I had to use a little bit of imagination.
Four boxes down, four to go. This is fun and my project is going like a breeze. But my happiness is cut short.
I must've measured wrong cause the outlet falls exactly where the molding needs to be!! But my measurements are ok. IT'S THE OUTLET !!!!!! Too close to the side of the box to go around. Argh….. What do I do? Let's put that one on hold. I might have the brain later. So the other box on that wall is perfect.
The last wall at last. Two outlets and a switch. The switch looks perfect. No juggling for that one since it's outside the box.
The first outlet on that wall was not installed at the same height as well. Almost didn't have the room to install the cover.
And the other outlet, lucky it didn't fall in the box because I would've been forced to use my imagination there too.
Now with the one I'd put aside. I even called my dad on that one but at the end it came out ok.
So the moral of the story: take into consideration your electrical outlets and switches.
As for my outlets, they are all hidden behind furnitures. But if yours are visible I suggest that you measure each of them first and making sure that they are either inside or outside your boxes. That being said, if your contractor was meticulous you won't have to worry about that.


Today we went to Turqouise jewellery store in Cappadocia and enjoy the scenery from outside the store for a while. The balloon doesn't fly again today due too the wind, but the view is great. A long journey was waiting, the trip to Bolu via Ankara. Our first stop was a salt lake that supplied 98% of Turkish household needs. The wind was blowing hard and the weather was colder than yesterday, my face felt frozen because of standing for 5 minutes outdoor.
Lunch is Turkish Kebab and soup, as usual. We went to Ankara, the capital city of Turkey. We were visiting Attaturk Museum and tomb, which is heavily guarded by the army. The security was strict in this area, the museum contains the possession of Attaturk when he was alive and the history of his life and war.
We are heading to Bolu as I write this post. We start to see snows on our way from Ankara, we were excited to finally see the snow and it really felt like Christmas. But soon enough we have too much snow, first the road are slippery, then it started to snowing heavily. The visibility of the road ahead is very narrow and we are all watching the road, helping our bus driver.
On the hotel it was still snowing, despite the weather I went with my sister outside and take some long exposure using my new Manfrotto Befree tripod.
This entry was posted in Travel, Turkey-Abu Dhabi and tagged ankara, attaturk, bolu, bolu thermal hotel, cappadocia, landscape, museum, salt lake, snow, tomb, Turkey. Bookmark the permalink.
Looks a bit like western Oklahoma!
Well, I searched for "Western Oklahoma" in Google and it does resembles the farm and fields, but not the thunder of course.


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Q: What is the purpose of this current sense resistor in my arc lighter? I have an arc lighter similar to this one:

The switch failed after a couple days (typical Chinese garbage), so I took the unit apart to replace it. Also, due to its operating frequency (~16Khz), it makes an appalling screeching sound that hurts my ears. So while I had it open, I decided I would attempt to modify it for a higher frequency.
By photographing both sides of the PCB; by using a multimeter; and by shining a light through the board, I managed to reverse-engineer it. Mostly.
My initial intention was to leave the micro in place, but insert a 555 timer between the chip and the MOSFET that drove the output coil. Its reset line would be asserted high by the chip's square wave output (by way of a diode, a resistor and a capacitor), and it would output a (thankfully inaudible) 22KHz signal to the MOSFET.¹ So far, so good.
My initial tests were promising, but in the end a pin broke off the chip and necessitated its replacement. Onto plan B then.
I figure I can just replace the (now defunct) chip with another microcontroller (specifically, a PIC or Atmel one), and write some firmware to perform the original functions, albeit at a higher frequency.
The microcontroller is unmarked, but fortuitously I have other microcontrollers with a compatible pinout. There is a problem, however. I don't understand the purpose of one of the connections on the board!
Here are the photographs I worked from. The high voltage transformer connects at the far right, and the 3.7V lithium battery at the left.² The lower image is flipped for convenience.

From those pictures, I derived the following schematic:

My understanding of the circuit is as follows:

*

*The LTC chip is responsible for charging the battery.

*Voltage on the USB connector bypasses the power switch by way of Q1 and Q2, activating the microcontroller, in order that the charging lights may illuminate in sequence.

*The microcontroller monitors the USB voltage on pin 2, and refuses to trigger if a voltage is sensed.

*Pin 3 is for detecting when the battery is fully charged, in order to display the LEDs statically lit to indicate charging is complete.

*Pin 5 is the output pin, by which the chip outputs a 60% duty-cycle 15Khz square wave³ to the transformer via a MOSFET.

So, my question is...

*

*What is the purpose of the current sense resistor R12 (measured by pin 13)?

I have no way to know the model of microcontroller used in this product, or the internal workings of its firmware. So, what can be deduced from the circuit, in terms of how pin 13 is likely to affect the microcontroller's behaviour?
EDIT: I notice there is no back-EMF clamp diode on the transformer. Should there be?

¹ Unfortunately, I discovered that the 555 drives its output low when reset is low; driving the MOSFET on continuously, except when the square wave was present. There was some smoke, and the MOSFET physically desoldered itself from the board! Fortunately I was paying attention and disconnected it immediately; and I'm pretty sure no permanent harm was done. I added an NPN transistor and the problem went away.
² Yes, those dents happened during disassembly. Oops.
³ I measured this with an oscilloscope.

A: I have seen a similar circuit used in my Gas operated water heater it also consist of an electric arc generating circuit and operates on two D type cell battery.
And have tested that circuit and I got to know that this kind of circuit need to change the Ton Time of the MOSFET to generate proper electric arc between their electrodes as fix duty cycle will not work efficiently in such case, as battery voltage will be gradually reduced Ton time should be increased.
So as I think in your case R12 provides a low resistive path for most of the current to flow to the ground and with a high resistor value like 4.7k R11 is used to sense voltage feedback so that it can vary the duty cycle accordingly.
Still, I am not 100% sure about this but you can test it with an oscilloscope and providing a variable power supply to the circuit.

A: It might be a cheap alternative for a fuse. Its bad practice but I have seen it

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Q: The moment this happens/that (soon) happens– grammaticality The moment I met her, I fell in love.
This sentence conveys the meaning that the speaker met a woman and immediately fell in love and looks to be grammatically correct to my eyes.
Now if we changed that sentence to The moment I met her, I soon fell in love., the meaning changes. It now means the speaker met this woman, and not immediately, but soon after, fell in love with her. The sentence sounds weirder than the first one, less idiomatic, but is this second sentence necessarily grammatically incorrect?
We say "the moment x happened/happens, y happened/happens." , but if we said the moment x happened, y soon happened, does the  insertion of soon render the sentence grammatically incorrect?

A: It might be grammatically correct in that the tenses agree and there's no obvious error, but semantically the sentence is nonsensical.
"The moment X happened, Y happened" states that X and Y happened pretty much simultaneously, with no time between the two actions. "Soon" means that a short but non-zero length of time has passed.
So it's either "The moment I met her, I fell in love" - which means that you fell in love at the very moment you've met her - or "After I met her, I soon fell in love", which means that it took you a short time to do so.


In this Bridges of Madison County for teens, Michelle Zink weaves a magnetic tale about summer love that stays with you long after the seasons change.
Neither Rose nor Bodhi can deny the sparks flying between them, but with the end of summer looming, they must decide if it is better to have loved and lost than never to have loved at all….
I've never read The Bridges of Madison County, nor have I seen the movie, so that similarity isn't what drew me into the book. I was intrigued by the line "summer love that stays with you long after the seasons change." There's a lot of potential in that kind of story. There are happily ever after ways the books could go and there are directions that might make my heart ache. Either way, I wanted to read this book. It was sweet and lovely and hopeful…I loved it.
Rose Darrow is an admirable character. Farm life was never what she wanted for her life. She wanted to travel, to experience life away from her little town. However, after her world is upended, she makes herself work hard on the farm. Her father is grieving, unable to work like he used to. Rose feels like it's her job to make life easier for him, to pick up where he left off. She does it without much complaint because she knows it's what's good and right. She's not entirely happy with the direction her life has taken, but she knows there's no sense in fighting it because she can't change the past, only adapt for the future.
Bohdi has been on his own for several years. He left home knowing that his home was more dangerous for him than trying to figure out how to take care of himself. He's left his old life behind and is focused on working and experiencing the world. He's got his future planned and all he wants to do is finish this last job before he begins the next part of his life. He's a hard worker who understands that most of the time, life isn't going to hand you everything you want; you've got to work for it.
Their romance has a slow build that just feels sweet. There's an instant attraction, but that's all there is between them for a while. Bohdi doesn't want attachments and Rose doesn't feel like opening herself up. But they start to work together, get to know each other. That little bit of attraction turns into a romance, albeit with a little help from some pushy friends and family. It was refreshing to read about a relationship that grows slowly, unfolding each new layer like it's something special.
There's not much I didn't like. I just wish the ending had been a little bit more. I don't mind the uncertainty of their future, so much, but the last little bit felt a little rushed and I didn't feel as content with the ending as I had hoped.
This was a wonderful contemporary novel. It had a sweet romance that was a slow simmer until just the perfect moment. There's just enough drama to make your heart beat fast, but it doesn't feel overdone or excessive. It's a great summer read.
This entry was posted in Contemporary, Young (and New) Adult and tagged Contemporary, Michelle Zink, Young Adult.


The Ingersoll caliber IN 416 automatic watch movement is found in the popular skeleton dial Ingersoll Boonville IN2705WH model. This movement is Chinese-made with 22 jewels.
With the crown pulled out one click to position 1, turn the crown counterclockwise to set the small dual time dial.
Once the dual time is set, turn the crown clockwise to set the local time.
Push the crown back in to position 0 (winding position).
The two watches use slightly different movements based on the balance wheel anchors.
These movements have dual mainsprings.


My granddaddy's grave marker, Cast Bronze. Installed at Loxely, AL cemetery.
The sculptures shown below were works that I helped construct as part of a team and were not my own works of art, except for the female figure sculpture below.


Acquisition and Sale. The acquisition (or sale) of commercial and residential real estate is a major event. We treat the occasion accordingly, and recognize the attendant goals and pitfalls from the start. The Purchase Agreement is the key element of any such transaction; it is essential that the attorney be involved before a binding agreement is signed. Title and survey matters, due diligence, financing, insurance and indemnity, environmental issues, zoning and other regulatory concerns are just a few of the matters that take center stage as the purchase process moves toward closing.
Distressed Property. Due to the current economic environment, there are many distressed pieces of real estate. There are many options that an owner has prior to foreclosure, that we can guide you through. Financial workouts, short sales, and deeds in lieu of foreclosure can all facilitated through the efforts of OSBIG.
Leasing. Real estate and contract law intersect at the junction of the real estate lease, where landlord and tenant alike must protect their real estate interests while contracting for favorable terms that meet their expectations for their respective interests in the shared property. Our experience in this area includes negotiating leases for both landlords and tenants.
Operations and Management. We assist clients with the management and operational challenges that arise during ownership of the developed property. These challenges include: land usage disputes, construction disputes, regulatory requirements, easements, and the daily operating challenges from contractors, tenants, and local governments.
Tax Deferred  Exchanges. A tax-deferred exchange is a method by which a property owner trades one or more relinquished properties for one or more replacement properties of "like-kind", while deferring the payment of federal income taxes and some state taxes on the transaction. The theory behind Section 1031 is that when a property owner has reinvested the sale proceeds into another property, the economic gain has not been realized in a way that generates funds to pay any tax. In other words, the taxpayer's investment is still the same, only the form has changed (e.g. vacant land exchanged for apartment building). Therefore, it would be unfair to force the taxpayer to pay tax on a "paper" gain. We facilitate these transactions for our clients.


Have you got a port wine stain birthmark on your face or body that is bothering you? While some can be covered up with makeup or clothing, if you're unable to do so it may be time to consider treating them. They will not disappear by themselves or reduce in size over time. And as you get older the lesions can become raised and thickened. A Port wine stain birthmark can't be completely removed, but they can be treated so their appearance fades.
When you decide to have your birthmark removed it's important to visit a reputable medical clinic that has highly trained and experienced Cosmetic Doctors and Cosmetic Nurses performing this treatment. Led by Dr Gavin Chan, since 2005 our team of Cosmetic Doctors and Cosmetic Nurses have effectively treated thousands of people with port wine stains.
What are port wine stain birthmarks?
Port wine stains (also called capillary vascular malformations) are birthmarks that occur because of a congenital malformation of the capillaries and veins of the skin. They appear as a reddish/purple colour and have the appearance of wine stains. In some cases raised nodules can form within the affected area.
Port wine stains are usually present at birth and affect 0.3 to 0.5% of the population. People with the condition can experience significant psychological distress, particularly if the face is affected. Although the stains commonly appear on the face, they can occur on other parts of the body.
Port wine stains are caused by a disturbance in the behaviour of blood vessels. The blood fills the blood vessels, causing what looks like a stain under the skin. Because the nerve control of blood flow is deficient, the blood flow is always at maximum level.
The typical red appearance occurs because the capillaries within the port-wine stain remain wide open. This is called a vascular birthmark. It's important to note that port-wine stains could not have been prevented, are nobody's fault, and are not hereditary.
What is the best way to treat port wine stains?
The first step of treatment is to have a consultation with our highly trained and experienced medical practitioners to assess and confirm the diagnosis. This will exclude any underlying medical problems including Sturge-Webber syndrome. It is important to note that port wine stains can often lead to increased pressure in the eye, resulting in glaucoma.
The best way to treat them is with lasers, which are attracted to the blood present in the affected area. To lighten the stain, most patients require multiple laser treatments. Complete removal is often not possible. While laser treatment can be commenced in early childhood, we only treat the adult population.
The traditional laser to remove them uses a process called selective photothermolysis. With a wavelength of 595nm this laser is highly attracted to the haemoglobin (blood). Selective treatment of the stain occurs because the it preferentially takes up the heat energy from the laser.
In recent years, port wine stains have been successfully treated with the 532nm and 1064nm wavelengths of the Gemini laser. The primary laser wavelength used in the treatment is the 532nm wavelength which is highly attracted to the haemoglobin. The 1064nm wavelength is also attracted to the haemoglobin, but not to such a high degree. An advantage of using the Gemini laser is that the head has a cooled-tip to chill the top surface of the skin. This cooling effect reduces damage to the skin surface layer as the laser passes through it.
After your treatment, the area that you had treated may feel hot for a few hours. Over the next few days this area will look like your skin has been sun burnt. Some patients initially experience a darkening of the stain, which may take up to 7 days to fade.
If your normal skin has been affected by the laser treatment, a blister or scab may develop. After laser treatment, swelling is normal but will subside after several days. The incidence of scarring after port wine laser treatment is very rare. It is important to note that darker skins can be more affected by laser treatment than light or fair skins.
After your procedure, it's important to use skincare that soothes and nourishes your skin. Dermaceutic Panthenol Ceutic soothes, nourishes and relieves skin dryness and irritation after your laser treatment. This hypoallergenic formula effectively combines 5% Panthenol and 1% Bisabolol with nourishing ingredients including Vitamin E, Shea butter and beeswax. Suitable for dry or hypersensitive skin, Dermaceutic Panthenol Ceutic can also be used on the lips and cuticles.
If you're unsure about which products will suit your skin, our highly experienced and trained Skin Therapists can advise and tailor a skincare regime to suit. Book your skin consultation by calling us on 1300 863 824.
It is important to note that the treatment of port wine stains will vary from person to person. During your initial consultation we will discuss with you what is a realistic and achievable outcome and what to expect from your treatment. Since 2005, led by Dr Gavin Chan our highly trained and experienced Cosmetic Doctors and Cosmetic Nurses have successfully treated thousands of patient. Call us on 1300 863 824 to book your consultation in or fill in the enquiry form below.


It's hard to believe that just last month this newsletter was full of excitement about Founders' Day, back-to-school and announcements about Blitz, our new K-9 Police Officer. Looking back, we now know that as we celebrated Leland's birthday and the fireworks lit up the skies over our Town, Hurricane Florence was preparing to descend on this region, bringing a wrath of wind, rain and flooding.
Hurricane Florence broke many trees and several records, but she has not, and will not, break our spirit. I am so proud of our first responders, staff, citizens, businesses, civic groups, churches, and volunteers who are working together to help those in need and start the long process of rebuilding. We had – and still have – groups from all over the state (Greensboro and Charlotte), all over the country (Missouri, Pennsylvania, Louisiana, Ohio and more), and all over the world (Canada, Israel) coming here to help us recover.
Leland hosted Missouri Task Force 1, a FEMA Urban Search & Rescue Task Force from Boone County Missouri, who worked alongside our Fire/Rescue, Police and Emergency Operations staff to respond to emergencies during Hurricane Florence and in the aftermath. Together these teams evacuated and rescued hundreds of adults, children and pets from rising floodwaters. We can't thank them enough for being here with us and saving so many lives. It is because of the unbelievably heroic work of MO-TF1 and our first responders that I can report not one life was lost in Leland due to Hurricane Florence.
Thank you, Leland, for showing the world how we truly are a community of neighbors helping neighbors. Together we will work to bring our Town back to be stronger and better than ever!
WWAY TV: Denied FEMA assistance? This might be why.
Help your commute, your health and your environment!
The Go Coast Commuter Challenge is a competition to encourage commuters to bike, walk, carpool, and use transit as much as possible. Sign up and download the "My Open Road" app to log your trips, gain points to earn prizes from local businesses. Visit wmpo.org/tdm for details.
Join us in Leland Municipal Park for our FREE concert series!
Port City Shakedown plays crowd-pleasing Motown, soul, funk, top 40 and old-school hip-hop in a set designed to get you movin' and groovin'! Here's more about the band.
Bring a blanket or a lawn chair, beverages, and your friends and family. La Bella Airosa food truck will be on site selling food.
Bring a blanket, lawn chairs, and a picnic, and gather with your children at sunset to enjoy a family night out watching aspiring musician Miguel, confronted with his family's ancestral ban on music, enter the Land of the Dead to find his great-great-grandfather, a legendary singer.
Concessions will be available for purchase. No pets, alcohol or smoking (including e-cigarettes).
Join us in Leland Municipal Park for our FREE concert series and be prepared to dance to the blues, soul and R&B sounds of 5-piece band Painted Man. Here's more about the band.
Bring a blanket or a lawn chair, beverages, and your friends and family! Poor Piggy's food truck will be on site selling food.
Dress up the family, deck out a pumpkin, and join us for this popular free event!
Area businesses and community organizations decorate vehicles and pass out goodies to trick-or-treaters of all ages. Enter a vehicle here.
Show off your creative and crafty side this Halloween with an entry in the Trunk or Treat Pumpkin Contest! Bring a carved and/or decorated pumpkin to Trunk or Treat and try for a prize! Free to enter, rules and registration are here.
Join us as we celebrate the works of Kathy Irvine & Yifenn Strickland at the opening reception for their exhibition at the LCAC Gallery.
This will be a fun filled night featuring two fantastic local bands, The Baby Boomer Band and The Mango Band, performing your favorite party music from the 60's, 70's and 80's!
Browse through the new C.A.P.E. Magazine for a wide selection of art and craft classes at the Leland Cultural Arts Center and events happening in Leland Parks. Find it on our website or pick up a copy at the Leland Cultural Arts Center or Leland Town Hall. Register for classes in person at the LCAC or online here.


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