Mechanisms and significance of tissue-specific MICU regulation of the mitochondrial calcium uniporter complex.

Mitochondrial Ca2+ uptake, mediated by the mitochondrial Ca2+ uniporter, regulates oxidative phosphorylation, apoptosis, and intracellular Ca2+ signaling. Previous studies suggest that non-neuronal uniporters are exclusively regulated by a MICU1-MICU2 heterodimer. Here, we show that skeletal-muscle and kidney uniporters also complex with a MICU1-MICU1 homodimer and that human/mouse cardiac uniporters are largely devoid of MICUs. Cells employ protein-importation machineries to fine-tune the relative abundance of MICU1 homo- and heterodimers and utilize a conserved MICU intersubunit disulfide to protect properly assembled dimers from proteolysis by YME1L1. Using the MICU1 homodimer or removing MICU1 allows mitochondria to more readily take up Ca2+ so that cells can produce more ATP in response to intracellular Ca2+ transients. However, the trade-off is elevated ROS, impaired basal metabolism, and higher susceptibility to death. These results provide mechanistic insights into how tissues can manipulate mitochondrial Ca2+ uptake properties to support their unique physiological functions.

Building a temperature-sensitive ion channel.

The biophysical basis of temperature-sensitive ion channel gating has been a tough nut to crack. Chowdhury, et al. use a protein engineering approach to render a temperature-insensitive voltage-gated channel cold- or heat-responsive to reveal principles for temperature-gating and a plausible model for molecularly enabling this mode of environmental responsiveness.

Structure and mechanism of the mitochondrial Ca2+ uniporter holocomplex.

Mitochondria take up Ca2+ through the mitochondrial calcium uniporter complex to regulate energy production, cytosolic Ca2+ signalling and cell death1,2. In mammals, the uniporter complex (uniplex) contains four core components: the pore-forming MCU protein, the gatekeepers MICU1 and MICU2, and an auxiliary subunit, EMRE, essential for Ca2+ transport3-8. To prevent detrimental Ca2+ overload, the activity of MCU must be tightly regulated by MICUs, which sense changes in cytosolic Ca2+ concentrations to switch MCU on and off9,10. Here we report cryo-electron microscopic structures of the human mitochondrial calcium uniporter holocomplex in inhibited and Ca2+-activated states. These structures define the architecture of this multicomponent Ca2+-uptake machinery and reveal the gating mechanism by which MICUs control uniporter activity. Our work provides a framework for understanding regulated Ca2+ uptake in mitochondria, and could suggest ways of modulating uniporter activity to treat diseases related to mitochondrial Ca2+ overload.

Evidence supporting the MICU1 occlusion mechanism and against the potentiation model in the mitochondrial calcium uniporter complex.

The mitochondrial calcium uniporter is a Ca2+ channel that imports cytoplasmic Ca2+ into the mitochondrial matrix to regulate cell bioenergetics, intracellular Ca2+ signaling, and apoptosis. The uniporter contains the pore-forming MCU subunit, an auxiliary EMRE protein, and the regulatory MICU1/MICU2 subunits. Structural and biochemical studies have suggested that MICU1 gates MCU by blocking/unblocking the pore. However, mitoplast patch-clamp experiments argue that MICU1 does not block, but instead potentiates MCU via allosteric mechanisms. Here, we address this direct clash of the proposed MICU1 function. Supporting the MICU1-occlusion mechanism, patch-clamp demonstrates that purified MICU1 strongly suppresses MCU Ca2+ currents, and this inhibition is abolished by mutating the MCU-interacting K126 residue. Moreover, a membrane-depolarization assay shows that MICU1 prevents MCU-mediated Na+ flux into intact mitochondria under Ca2+-free conditions. Examining the observations underlying the potentiation model, we found that MICU1 occlusion was not detected in mitoplasts not because MICU1 cannot block, but because MICU1 dissociates from the uniporter complex. Furthermore, MICU1 depletion reduces uniporter transport not because MICU1 can potentiate MCU, but because EMRE is down-regulated. These results firmly establish the molecular mechanisms underlying the physiologically crucial process of uniporter regulation by MICU1.

Laser Speckle Contrast Imaging Guides Needling Treatment of Vascular Complications from Dermal Fillers.

BACKGROUND: Although laser Doppler imaging (LDI) accurately delineates a hypoperfused area to help target hyaluronidase treatment, laser speckle contrast imaging (LSCI) is more appropriate for assessing microvascular hemodynamics and has greater reproducibility than LDI. This study investigated the use of LSCI in the evaluation and treatment of six patients who developed vascular complications after facial dermal filler injections. METHODS: The areas of vascular occlusion were accurately defined in real time by LSCI and were more precise than visual inspections or photographic evidence for guiding needling and hyaluronidase treatment. RESULTS: All patients had achieved satisfactory outcomes as early as Day 2 of treatment and no procedure-related complications were reported after a median follow-up of 9.5 (7-37) days. CONCLUSION: LSCI accurately and noninvasively delineated vascular occlusions in real time among patients experiencing complications of facial dermal filler injections. Moreover, LSCI was more accurate than visual and photographic evaluations. Clinicians can use LSCI to reliably follow-up therapeutic outcomes after salvage interventions for vascular occlusions. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

The Removal, Excision, Sterilization, and Quarantine (RESQ) Method is a Feasible Alternative Treatment for Cardiac Implantable Electronic Device Infections.

Background: Current guidelines recommend that all infected cardiac implantable electronic devices (CIEDs) should be removed. However, financial or anatomical concerns can lead to management of infection with simple debridement, as opposed to complete removal. In this observational study, we report the outcomes of our modified procedure for this real-world dilemma. Methods and Results: The Quarantine (RESQ) method is characterized as follows: the removal (R) of all non-essential foreign materials, including old sutures and leads; the excision (E) of all non-viable, chronically inflamed, granulation, or scar tissue; the sterilization (S) of the remaining generator; and the quarantine (Q) of a new pocket in the sub-muscular layer for reimplantation. From a review of electronic medical records, 30 patients were selected and divided into three groups according to the intervention used: RESQ (n = 9) in group A, simple debridement (n = 16) in group B, and guideline-recommended replacement (n = 5) in group C. Patient baseline characteristics were similar between the groups. After analyzing the proportion of patients that were free from infection one year following their respective interventions, we found that group A performed better than group B (100% and 31.2% infection-free, respectively, p = 0.001), and was comparable to group C (both 100% infection-free, p = not applicable). Conclusions: The RESQ method is a feasible and beneficial alternative for selected patients with CIED infections who are unable to receive a generator replacement according to the recommended guideline.

Kimura Disease of the Thigh Treated With Surgical Excision and Dupilumab.

ABSTRACT: Kimura disease (KD) is a rare, chronic inflammatory disorder presenting with solitary or multiple masses. Treatment options include surgical excision, corticosteroids, and radiotherapy; however, optimal therapy remains to be established. Moreover, efficacy of a humanized monoclonal antibody, dupilumab (Dupixent), requires to be demonstrated. Here, we present a 36-year-old male patient with an enlarging mass in the left medial thigh and chronic eczema over the abdomen and lower legs. Kimura disease was diagnosed after surgical excision. Postoperative treatment with dupilumab was applied with an initial dose of 600 mg followed by 300 mg every 2 weeks for 8 months. No recurrence of KD was observed in the 1-year follow-up. The eczematous lesions improved greatly. To our knowledge, this is the first report of using dupilumab for treating KD.

Efficacy and safety of pectoralis muscle flap combined rectus abdominis muscle sheath fasciocutaneous flap for reconstruction of sternal infection.

Few studies have assessed the efficacy and safety of reconstruction of sternal infection using a pectoralis muscle flap combined with a rectus abdominis muscle (RAM) sheath fasciocutaneous flap. We report here our experience with this procedure to reconstruct the sternal defect in patients (n = 46) with a deep sternal wound infection (DSWI) after cardiac surgery. After wound reconstruction, the proportion of prolonged mechanical ventilation use and intensive care unit (ICU) stay were 17.4% (n = 8) and 21.7% (n = 10), respectively. The 30-day all-cause mortality was 15.2%; recurrence rate was 17.4%; postoperative complications were 15.2%; and median hospital stay was 31 (0-157) days. Multivariate logistic regression analysis revealed that hypertension (ß = 21.32, 95%CI 4.955-37.68, P = .014), drainage-tube use (ß = 0.944, 95%CI 0.273-1.614, P = .008), and prolonged intensive care unit stay (ß = 53.65, 95%CI 31.353-75.938, P < .001) were significantly correlated with hospital stay. In conclusion, a procedure including surgical debridement, sternal reconstruction with bilateral PM and RAM sheath flap, long-term antibiotics, and adequate drainage is a beneficial technique in the reconstruction of deep sternal wound infection after cardiac surgery. Duration of drainage tube use may be as an index for a hospital stay or wound healing.

Percutaneous Debridement of and Fibrin Glue Injection Into a Pretibial Morel-Lavallée Lesion: A Case Report and Literature Review.

ABSTRACT: The Morel-Lavallée lesion (MLL) is a posttraumatic close degloving injury, which is often underdiagnosed at first. Patients with MLLs usually present with tender and enlarging soft tissue swelling with fluctuation, decreased skin sensation, ecchymosis, or even skin necrosis hours to days after the inciting injury. The lesion can lead to intractable morbidity if it remains untreated. There is no consensus regarding the treatment for MLL at present. Here, we report an MLL in the pretibial region of a 43-year-old woman who experienced a low-energy contusion in a motorbike accident. The pretibial lesion was diagnosed using sonography and fine-needle aspiration. We successfully treated the patient by performing percutaneous debridement via a small incision and injections of fibrin after conservative treatment failed. The method we herein propose achieved the goal of open surgical debridement, providing faster recovery and a high degree of patient comfort. We reviewed the available pertinent literature and propose our own treatment protocol with the aim to establish common therapies ofMLL.

Proteolytic control of the mitochondrial calcium uniporter complex.

The mitochondrial calcium uniporter is a Ca2+-activated Ca2+ channel complex mediating mitochondrial Ca2+ uptake, a process crucial for Ca2+ signaling, bioenergetics, and cell death. The uniporter is composed of the pore-forming MCU protein, the gatekeeping MICU1 and MICU2 subunits, and EMRE, a single-pass membrane protein that links MCU and MICU1 together. As a bridging subunit required for channel function, EMRE could paradoxically inhibit uniporter complex formation if expressed in excess. Here, we show that mitochondrial mAAA proteases AFG3L2 and SPG7 rapidly degrade unassembled EMRE using the energy of ATP hydrolysis. Once EMRE is incorporated into the complex, its turnover is inhibited >15-fold. Protease-resistant EMRE mutants produce uniporter subcomplexes that induce constitutive Ca2+ leakage into mitochondria, a condition linked to debilitating neuromuscular disorders in humans. The results highlight the dynamic nature of uniporter subunit assembly, which must be tightly regulated to ensure proper mitochondrial responses to intracellular Ca2+ signals.

Near Complete Recovery of Visual Acuity After Calcium Hydroxylapatite Injection-Related Vision Loss: A Case Report and Literature Review.

Injection of fillers has gained popularity over the past decades in aesthetic treatments. Calcium hydroxylapatite (CaHA; Radiesse) was introduced in the year 2003 and received approval from the Food and Drug Administration in 2006 for the treatment of moderate-to-severe wrinkles. The properties of CaHA include biostimulation, neocollagenesis, and stability over a long period. However, similar to other fillers, CaHA is associated with the risk of complications such as ecchymosis, inflammation, local infection, skin necrosis, and vascular occlusion. Iatrogenic vision loss remains the most devastating complication related to vascular occlusion. Development of vision impairment is associated with a relatively high risk of permanent damage to vision acuity and poor prognosis. The current report presents a case of a patient who suffered from skin necrosis, vision impairment, and ophthalmoplegia after the injection of CaHA into the nasal dorsum. Significant improvement in visual acuity was observed during hospitalization after the treatment. The patient recovered to near-normal visual acuity and completely recovered from ophthalmoplegia. We aimed to discuss the current treatment employed and review the literature on CaHA-related vision loss.

Substrate selectivity in arginine-dependent acid resistance in enteric bacteria.

To successfully colonize the human gut, enteric bacteria must activate acid resistance systems to survive the extreme acidity (pH 1.5-3.5) of the stomach. The antiporter AdiC is the master orchestrator of the arginine-dependent system. Upon acid shock, it imports extracellular arginine (Arg) into the cytoplasm, providing the substrate for arginine decarboxylases, which consume a cellular proton ending up in a C-H bond of the decarboxylated product agmatine (Agm(2+)). Agm(2+) and the "virtual" proton it carries are exported via AdiC subsequently. It is widely accepted that AdiC counters intracellular acidification by continuously pumping out virtual protons. However, in the gastric environment, Arg is present in two carboxyl-protonation forms, Arg(+) and Arg(2+). Virtual proton pumping can only be achieved by Arg(+)/Agm(2+) exchange, whereas Arg(2+)/Agm(2+) exchange would produce no net proton movement. This study experimentally asks which exchange AdiC catalyzes, an issue previously unapproachable due to the absence of a reconstituted system mimicking the situation of bacteria in the stomach. Here, using an oriented liposome system able to hold a three-unit pH gradient, we demonstrate that Arg/Agm exchange by AdiC is strongly electrogenic with positive charge moved outward, and thus that AdiC mainly mediates Arg(+)/Agm(2+) exchange to support effective virtual proton pumping. Further experiments reveal a mechanistic surprise--that AdiC selects Arg(+) against Arg(2+) on the basis of gross valence, rather than by local scrutiny of protonation states of the carboxyl group, as had been suggested by Arg-bound AdiC crystal structures.

Substrate selectivity in glutamate-dependent acid resistance in enteric bacteria.

The bacterial antiporter GadC plays a central role in the glutamate (Glu)-dependent acid resistance system, which protects enteric bacteria against the extreme acidity of the human stomach. Upon acid shock, GadC imports Glu into the cytoplasm, where Glu decarboxylases consume a cytoplasmic proton, which ends up as a "virtual" proton in the decarboxylated product γ-aminobutyric acid (GABA) and is then exported via GadC. It was therefore proposed that GadC counters intracellular acidification by continually pumping out virtual protons. This scenario, however, is oversimplified. In gastric environments, GadC encounters substrates in multiple carboxyl protonation forms (outside: Glu(-), Glu(0), Glu(+); inside: GABA(0), GABA(+)). Of the six possible combinations of antiport partners, Glu(+)/GABA(0) results in proton influx, Glu(0)/GABA(0) and Glu(+)/GABA(+) are proton neutral, and Glu(-)/GABA(0), Glu(-)/GABA(+), or Glu(0)/GABA(+) lead to proton extrusion. Which of these exchanges does GadC catalyze? To attack this problem, we developed an oriented GadC liposome system holding a three-unit inward pH gradient to mimic the conditions facing bacteria in the stomach. By assessing the electrogenicity of substrate transport, we demonstrate that GadC selectively exchanges Glu(-) or Glu(0) with GABA(+), resulting in effective proton extrusion of >0.9 H(+) per turnover to counter proton invasion into acid-challenged bacteria. We further show that GadC selects among protonated substrates using a charge-based mechanism, rather than directly recognizing the protonation status of the carboxyl groups. This result paves the way for future work to identify the molecular basis of GadC's substrate selectivity.

An Arrhythmia classification approach via deep learning using single-lead ECG without QRS wave detection.

Arrhythmia, a frequently encountered and life-threatening cardiac disorder, can manifest as a transient or isolated event. Traditional automatic arrhythmia detection methods have predominantly relied on QRS-wave signal detection. Contemporary research has focused on the utilization of wearable devices for continuous monitoring of heart rates and rhythms through single-lead electrocardiogram (ECG), which holds the potential to promptly detect arrhythmias. However, in this study, we employed a convolutional neural network (CNN) to classify distinct arrhythmias without QRS wave detection step. The ECG data utilized in this study were sourced from the publicly accessible PhysioNet databases. Taking into account the impact of the duration of ECG signal on accuracy, this study trained one-dimensional CNN models with 5-s and 10-s segments, respectively, and compared their results. In the results, the CNN model exhibited the capability to differentiate between Normal Sinus Rhythm (NSR) and various arrhythmias, including Atrial Fibrillation (AFIB), Atrial Flutter (AFL), Wolff-Parkinson-White syndrome (WPW), Ventricular Fibrillation (VF), Ventricular Tachycardia (VT), Ventricular Flutter (VFL), Mobitz II AV Block (MII), and Sinus Bradycardia (SB). Both 10-s and 5-s ECG segments exhibited comparable results, with an average classification accuracy of 97.31%. It reveals the feasibility of utilizing even shorter 5-s recordings for detecting arrhythmias in everyday scenarios. Detecting arrhythmias with a single lead aligns well with the practicality of wearable devices for daily use, and shorter detection times also align with their clinical utility in emergency situations.

Mechanisms of dual modulatory effects of spermine on the mitochondrial calcium uniporter complex.

The mitochondrial Ca2+ uniporter mediates the crucial cellular process of mitochondrial Ca2+ uptake, which regulates cell bioenergetics, intracellular Ca2+ signaling, and cell death initiation. The uniporter contains the pore-forming MCU subunit, an EMRE protein that binds to MCU, and the regulatory MICU1 subunit, which can dimerize with MICU1 or MICU2 and under resting cellular [Ca2+] occludes the MCU pore. It has been known for decades that spermine, which is ubiquitously present in animal cells, can enhance mitochondrial Ca2+ uptake, but the underlying mechanisms remain unclear. Here, we show that spermine exerts dual modulatory effects on the uniporter. In physiological concentrations of spermine, it enhances uniporter activity by breaking the physical interactions between MCU and the MICU1-containing dimers to allow the uniporter to constitutively take up Ca2+ even in low [Ca2+] conditions. This potentiation effect does not require MICU2 or the EF-hand motifs in MICU1. When [spermine] rises to millimolar levels, it inhibits the uniporter by targeting the pore region in a MICU-independent manner. The MICU1-dependent spermine potentiation mechanism proposed here, along with our previous finding that cardiac mitochondria have very low MICU1, can explain the puzzling observation in the literature that mitochondria in the heart show no response to spermine.

Deep convolutional neural network for rib fracture recognition on chest radiographs.

Introduction: Rib fractures are a prevalent injury among trauma patients, and accurate and timely diagnosis is crucial to mitigate associated risks. Unfortunately, missed rib fractures are common, leading to heightened morbidity and mortality rates. While more sensitive imaging modalities exist, their practicality is limited due to cost and radiation exposure. Point of care ultrasound offers an alternative but has drawbacks in terms of procedural time and operator expertise. Therefore, this study aims to explore the potential of deep convolutional neural networks (DCNNs) in identifying rib fractures on chest radiographs. Methods: We assembled a comprehensive retrospective dataset of chest radiographs with formal image reports documenting rib fractures from a single medical center over the last five years. The DCNN models were trained using 2000 region-of-interest (ROI) slices for each category, which included fractured ribs, non-fractured ribs, and background regions. To optimize training of the deep learning models (DLMs), the images were segmented into pixel dimensions of 128 × 128. Results: The trained DCNN models demonstrated remarkable validation accuracies. Specifically, AlexNet achieved 92.6%, GoogLeNet achieved 92.2%, EfficientNetb3 achieved 92.3%, DenseNet201 achieved 92.4%, and MobileNetV2 achieved 91.2%. Discussion: By integrating DCNN models capable of rib fracture recognition into clinical decision support systems, the incidence of missed rib fracture diagnoses can be significantly reduced, resulting in tangible decreases in morbidity and mortality rates among trauma patients. This innovative approach holds the potential to revolutionize the diagnosis and treatment of chest trauma, ultimately leading to improved clinical outcomes for individuals affected by these injuries. The utilization of DCNNs in rib fracture detection on chest radiographs addresses the limitations of other imaging modalities, offering a promising and practical solution to improve patient care and management.

Structure and function of the human mitochondrial MRS2 channel.

The human Mitochondrial RNA Splicing 2 protein (MRS2) has been implicated in Mg2+ transport across mitochondrial inner membranes, thus playing an important role in Mg2+ homeostasis critical for mitochondrial integrity and function. However, the molecular mechanisms underlying its fundamental channel properties such as ion selectivity and regulation remain unclear. Here, we present structural and functional investigation of MRS2. Cryo-electron microscopy structures in various ionic conditions reveal a pentameric channel architecture and the molecular basis of ion permeation and potential regulation mechanisms. Electrophysiological analyses demonstrate that MRS2 is a Ca2+-regulated, non-selective channel permeable to Mg2+, Ca2+, Na+ and K+, which contrasts with its prokaryotic ortholog, CorA, operating as a Mg2+-gated Mg2+ channel. Moreover, a conserved arginine ring within the pore of MRS2 functions to restrict cation movements, likely preventing the channel from collapsing the proton motive force that drives mitochondrial ATP synthesis. Together, our results provide a molecular framework for further understanding MRS2 in mitochondrial function and disease.

Transumbilical Silicone Implants Breast Augmentation (TUSBA).

Transumbilical breast augmentation with pre-filled silicone implants has been performed previously, but technical challenges remain to accommodate more implant options and dissection planes. We aimed to demonstrate the feasibility of transumbilical breast augmentation using various types of pre-filled silicone implants (TUSBA), and its applicability for subglandular, subfascial, dual-plane implantation. In the early stage, TUSBA was primarily performed using endoscope-assisted blunt dissection, and later converted to full endoscopy dissection to achieve better results. Endoscope was used to confirm the pocket and check bleeding for both groups. For endoscope-assisted group, surgical techniques were modified from conventional TUBA. In full endoscopy TUSBA, the entire dissection process was performed under endoscopic monitoring. Preliminary data of patients undergoing TUSBA from June 2016 to April 2021 were retrospectively reviewed. Breast implants with smooth, textured or nanotextured surface properties and round or anatomical shapes were used, with sizes up to 500 mL. Seventy-four patients with mean age 36.4 years (range: 21-55 years) were enrolled in this study. Follow-up ranged from 1 month to 4 years and 6 months (mean: 15.6 months). No excessive postoperative pain in breast or abdomen was reported. Surgery outcomes were aesthetically pleasing in both groups. In the endoscope-assisted group, 3 (4.6%) required major revisional procedures. No revision was required in the full endoscopy group. TUSBA with various types of silicone implants is feasible, and accommodable to all dissection planes. Full endoscopy technique is helpful in reducing the higher complication rate.

Sided functions of an arginine-agmatine antiporter oriented in liposomes.

The arginine-dependent extreme acid resistance system helps enteric bacteria survive the harsh gastric environment. At the center of this multiprotein system is an arginine-agmatine antiporter, AdiC. To maintain cytoplasmic pH, AdiC imports arginine and exports its decarboxylated product, agmatine, resulting in a net extrusion of one "virtual proton" in each turnover. The random orientation of AdiC in reconstituted liposomes throws up an obstacle to quantifying its transport mechanism. To overcome this problem, we introduced a mutation, S26C, near the substrate-binding site. This mutant exhibits substrate recognition and pH-dependent activity similar to those of the wild-type protein but loses function completely upon reaction with thiol reagents. The membrane-impermeant MTSES reagent can then be used as a cleanly sided inhibitor to silence those S26C-AdiC proteins whose extracellular portion projects from the external side of the liposome. Alternatively, the membrane-permeant MTSEA and membrane-impermeant reducing reagent, TCEP, can be used together to inhibit proteins in the opposite orientation. This approach allows steady-state kinetic analysis of AdiC in a sided fashion. Arginine and agmatine have similar Michaelis-Menten parameters for both sides of the protein, while the extracellular side selects arginine over argininamide, a mimic of the carboxylate-protonated form of arginine, more effectively than does the cytoplasmic side. Moreover, the two sides of AdiC have different pH sensitivities. AdiC activity increases to a plateau at pH 4 as the extracellular side is acidified, while the cytoplasmic side shows an optimal pH of 5.5, with further acidification inhibiting transport. This oriented system allows more precise analysis of AdiC-mediated substrate transport than has been previously available and permits comparison to the situation experienced by the bacterial membrane under acid stress.