Recovery kinetics of dual AAV-mediated human otoferlin expression.

Deafness-causing deficiencies in otoferlin (OTOF) have been addressed preclinically using dual adeno-associated virus (AAV)-based approaches. However, timing of transduction, recombination of mRNA, and protein expression with dual hybrid AAV methods methods have not previously been characterized. Here, we have established an ex vivo assay to determine the kinetics of dual-AAV mediated expression of OTOF in hair cells of the mouse utricle. We utilized two different recombinant vectors that comprise DB-OTO, one containing the 5' portion of OTOF under the control of the hair cell-specific Myo15 promoter, and the other the 3' portion of OTOF. We explored specificity of the Myo15 promoter in hair cells of the mouse utricle, established dose response characteristics of DB-OTO ex vivo in an OTOF-deficient mouse model, and demonstrated tolerability of AAV1 in utricular hair cells. Furthermore, we established deviations from a one-to-one ratio of 5' to 3' vectors with little impact on recombined OTOF. Finally, we established a plateau in quantity of recombined OTOF mRNA and protein expression by 14 to 21 days ex vivo with comparable recovery timing to that in vivo model. These findings demonstrate the utility of an ex vivo model system for exploring expression kinetics and establish in vivo and ex vivo recovery timing of dual AAV-mediated OTOF expression.

Pediatric Carotid Injury after Blunt Trauma and the Necessity of CT and CTA-A Narrative Literature Review.

Background: Blunt carotid injury (BCI) in pediatric trauma is quite rare. Due to the low number of cases, only a few reports and studies have been conducted on this topic. This review will discuss how frequent BCI/blunt cerebrovascular injury (BCVI) on pediatric patients after blunt trauma is, what routine diagnostics looks like, if a computed tomography (CT)/computed tomography angiography (CTA) scan on pediatric patients after blunt trauma is always necessary and if there are any negative health effects. Methods: This narrative literature review includes reviews, systematic reviews, case reports and original studies in the English language between 1999 and 2020 that deal with pediatric blunt trauma and the diagnostics of BCI and BCVI. Furthermore, publications on the risk of radiation exposure for children were included in this study. For literature research, Medline (PubMed) and the Cochrane library were used. Results: Pediatric BCI/BCVI shows an overall incidence between 0.03 and 0.5% of confirmed BCI/BCVI cases due to pediatric blunt trauma. In total, 1.1-3.5% of pediatric blunt trauma patients underwent CTA to detect BCI/BCVI. Only 0.17-1.2% of all CTA scans show a positive diagnosis for BCI/BCVI. In children, the median volume CT dose index on a non-contrast head CT is 33 milligrays (mGy), whereas a computed tomography angiography needs at least 138 mGy. A cumulative dose of about 50 mGy almost triples the risk of leukemia, and a cumulative dose of about 60 mGy triples the risk of brain cancer. Conclusions: Given that a BCI/BCVI could have extensive neurological consequences for children, it is necessary to evaluate routine pediatric diagnostics after blunt trauma. CT and CTA are mostly used in routine BCI/BCVI diagnostics. However, since radiation exposure in children should be as low as reasonably achievable, it should be asked if other diagnostic methods could be used to identify risk groups. Trauma guidelines and clinical scores like the McGovern score are established BCI/BCVI screening options, as well as duplex ultrasound.

Organ-specific responses to atezolizumab plus bevacizumab in advanced hepatocellular carcinoma: A multicentre, retrospective study.

BACKGROUND AND AIMS: Anti-programmed death 1 (PD-1) monotherapy triggers various responses by each organ. In advanced hepatocellular carcinoma (HCC), while extrahepatic lesions demonstrate objective response rates (ORR) of 20%-40%, only 10% of intrahepatic lesions respond. Although first-line atezolizumab/bevacizumab has shown survival benefits in advanced HCC, organ-specific responses remain unexplored. Therefore, we aimed to assess organ-specific responses in patients with advanced HCC receiving atezolizumab/bevacizumab. METHODS: This retrospective, multicenter, observational study included patients who received first-line atezolizumab/bevacizumab for advanced HCC. Patients with Child-Pugh class A, measurable tumour lesions and serial imaging available for response evaluation were eligible. RESULTS: Between May 2020 and June 2021, 131 patients (median age: 62) from three cancer referral institutions were included. Ninety-one had hepatitis B (69.5%), 108 were at Barcelona clinic liver cancer stage C (82.4%), and 78 had extrahepatic metastasis (59.5%). After a median follow-up of 10.1 months, median progression-free survival was 6.8 months (95% confidence interval [CI], 4.6-9.2), median overall survival remained unreached (95% CI, range unavailable) and the ORR was 29.0%. Among 270 individual tumour lesions, the liver was the most commonly involved organ (n = 158). Atezolizumab/bevacizumab induced ORR of 27.8%, 42.2%, 29.1% and 21.0% for liver, lymph nodes, lungs and other sites, respectively. The organ-specific response rate for intrahepatic tumours decreased with increasing size (35.6%: <5 cm, 15.0%: ≥ 5 cm). CONCLUSIONS: Unlike anti-PD-1 monotherapy, atezolizumab/bevacizumab demonstrated favourable responses in intrahepatic lesions, comparable to those in extrahepatic lesions, and may potentially overcome the immune-tolerant hepatic microenvironment in patients with advanced HCC.

[Plastering-A technique fallen into obscurity]. / Gipsen ­ eine in Vergessenheit geratene Technik.

Immobilization of fractures and dislocations is a basic technique in orthopedic trauma surgery care. The orthopedic surgeon should be familiar with the various materials, techniques and possible complications. Despite other techniques, the classical white plaster cast remains an integral part of orthopedic trauma surgery care. The application of such a cast must be learned as failure to observe the basic principles can result in harm to the patient. In many hospitals, the application of a plaster cast is delegated to the nursing staff according to the physician's instructions. As a result, many young medical colleagues lack the knowledge of how to apply a plaster cast. In addition to the treatment of fractures, immobilization after dislocation, inflammation and ligamentous injuries are some of the areas of application. In this article the application of a plaster cast is described based on a case study, from the indications to the execution and possible complications.

[Complications after conservative vs. operative treatment of severe thoracic trauma]. / Komplikationen nach operativer vs. konservativer Versorgung des schweren Thoraxtraumas.

BACKGROUND: Thoracic trauma is a frequent injury in the routine treatment of injured patients. Due to the increasing demographic changes a further increase is to be expected, especially after low-energy trauma. OBJECTIVE: Expected complications after conservative vs. operative treatment of various injury patterns of thoracic trauma. MATERIAL AND METHODS: Evaluation of a selective literature search regarding possible complications after thoracic trauma and formulation of instructions for action as expert recommendations. CONCLUSION: Both conservative and operative treatment of thoracic trauma have their specific complications, which have to be known to the treating physician. Lung contusions are often underestimated in the initial radiological diagnostics but often lead to relevant problems during the further course of treatment. After conservative treatment of rib fractures persistent pain, functional limitations or even relevant deformities due to secondary dislocation, can remain. There is a significant risk of overlooking or underestimating relevant injuries during the initial diagnostics which then leads to secondary complications. By far the most frequent risk of surgical treatment is an incorrect positioning of chest tubes. Overall, postoperative infections after chest trauma are relatively rare.

[Chest Tube in Thoracic Trauma - Recommendations of the Interdisciplinary Thoracic Trauma Task Group of the German Society for Thoracic Surgery (DGT) and the German Trauma Society (DGU)]. / Thoraxdrainage beim Thoraxtrauma ­ Empfehlungen der interdisziplinären Arbeitsgemeinschaft Thoraxtrauma der Deutschen Gesellschaft für Thoraxchirurgie (DGT) und der Deutschen Gesellschaft für Unfallchirurgie (DGU).

For unstable patients with chest trauma, the chest tube is the method of choice for the treatment of a relevant pneumothorax or haemothorax. In the case of a tension pneumothorax, needle decompression with a cannula of at least 5 cm length should be performed, directly followed by the insertion of a chest tube. The evaluation of the patient should be performed primarily with a clinical examination, a chest X-ray and sonography, but the gold standard of diagnostic testing is computed tomography (CT).A small-bore chest tube (e.g. 14 French) should be used in stable patients, while unstable patients should receive a large-bore drain (24 French or larger). Insertion of chest drains has a high complication rate of between 5% and 25%, and incorrect positioning of the tube is the most common complication. However, incorrect positioning can usually only be reliably detected or ruled out with a CT scan, and chest X-rays proofed to be insufficient to answer this question. Therapy should be carried out with mild suction of approximately 20 cmH2O, and clamping the chest tube before removal showed no beneficial effect. The removal of drains can be safely performed, either at the end of inspiration or at the end of expiration. In order to reduce the high complication rate, in the future the focus should be more on the education and training of medical staff members.

[Occult and Retained Haemothorax - Recommendations of the Interdisciplinary Thoracic Trauma Task Group of the German Trauma Society (DGU - Section NIS) and the German Society for Thoracic Surgery (DGT)]. / Okkulter und residueller Hämatothorax nach Thoraxtrauma ­ Empfehlungen der interdisziplinären Arbeitsgruppe Thoraxtrauma der Sektion NIS der Deutschen Gesellschaft für Unfallchirurgie (DGU) und der Deutschen Gesellschaft für Thoraxchirurgie (DGT).

The management of occult and retained haemothorax is challenging for all involved in the care of polytrauma patients in terms of diagnosis and treatment. The focus of decision making is preventing sequelae such as pleural empyema and avoiding a trapped lung. An interdisciplinary task force of the German Society for Thoracic Surgery (DGT) and the German Trauma Society (DGU) on thoracic trauma offers recommendations for post-trauma care of patients with occult and/or retained haemothorax, as based on a comprehensive literature review.

[VATS in Thorax Trauma]. / Videoassistierte Thorakoskopie (VATS) beim Thoraxtrauma.

Since the early 1990s, video-assisted thoracoscopy (VATS) has been increasingly established for a variety of indications in the treatment of patients with thoracic trauma. During this time, one premise for the use of thoracoscopy has not changed. Its use is consistently recommended only for trauma patients with stable circulation and respiration. To define the indications of VATS for use in thoracic trauma, the Pulmonary Injury Group - as part of the Working Committee for Thoracic Trauma of the German Society for Thoracic Surgery (DGT) and the German Society for Trauma Surgery (DGU) - has developed treatment recommendations based on a current literature review (based on the PRISMA Checklist/here: MEDLINE via PubMed from 1993 to 2022). In the present study, after reviewing the available literature, the indications for VATS in the care of thoracic trauma were identified, in order to formulate clinical recommendations for the use of VATS in thoracic trauma. The analysis of 1679 references identified a total of 4 randomised controlled trials (RCTs), 4 clinical trials, and 5 meta-analyses or systematic reviews and 39 reviews, which do not allow a higher level of recommendation than consensual recommendations, due to the low evidence of the available literature. Over the past 30 years, stabilisation options in the care of trauma patients have improved significantly, allowing expansion of indications for the use of VATS. Moreover, the recommendation for more than 50 years to thoracotomise trauma patients in case of an initial blood loss ≥ 1500 ml via the inserted chest drainage or in case of continuous blood loss ≥ 250 ml/h over 4 h is now only relative with today's better stabilisation measures. For unstable/non-stabilisable patients with a thoracic injury requiring emergency treatment, thoracotomy remains the method of choice, while VATS is recommended for a wide range of indications in the diagnosis and treatment of stable patients with a penetrating or blunt thoracic trauma. The indications for VATS are persistent haemothorax, treatment of injuries and haemorrhages to the lung, diaphragm, thoracic wall and other organ injuries, and in the secondary phase, treatment of thoracic sequelae of injury (empyema, persistent pulmonary fistula, infected atelectasis, etc.).

Treatment of Metastatic Colorectal Carcinoma with Bevacizumab in First-Line and beyond First Progression: The KORALLE Non-Interventional Cohort Study.

INTRODUCTION: The non-interventional study (NIS) KORALLE evaluated the effectiveness and safety of bevacizumab in patients with metastatic colorectal carcinoma (mCRC) treated with bevacizumab in combination with fluoropyrimidine-based chemotherapy in the first-line setting and beyond first progression in routine clinical practice. METHODS: This prospective, multicenter NIS observed adult patients with mCRC who started first-line bevacizumab therapy. The planned maximum duration of observation per patient was 21 months. The primary effectiveness variable was progression-free survival in the first-line therapy setting (PFS-1). Secondary effectiveness variables included PFS after first progression as well as overall survival and overall response rate. All analyses were carried out descriptively for the full analysis population set (FAS). Effectiveness analyses were also assessed for predefined subgroups based on therapy goals. RESULTS: Between December 2012 and July 2016, 2,429 eligible patients were observed at 314 sites in Germany. In the first-line setting in the FAS, the median PFS-1 was 10.3 months (95% CI: 9.9; 10.8), the median overall survival was 16.9 months (95% CI: 16.3; 17.5), and the overall response rate (ORR-1) was 44.2% (95% CI: 41.6%; 46.8%). Effectiveness results of all subgroups were similar to the FAS. Overall, 80.9% of patients experienced any adverse events, 36.6% of patients experienced serious adverse events, and 8.8% of patients experienced fatal adverse events. CONCLUSION: The NIS KORALLE provided broad real-world evidence on effectiveness and safety of bevacizumab. Despite different treatment intentions, the combination of bevacizumab plus fluoropyrimidine-based chemotherapy was similarly effective in all subgroups in routine clinical practice. The safety information reported in this study is consistent with the known safety profile of bevacizumab.

Effect of surgical stabilization of rib fractures in polytrauma: an analysis of the TraumaRegister DGU®.

PURPOSE: In severely injured patients with multiple rib fractures the beneficial effect of surgical stabilization is still unknown. The existing literature shows divergent results and especially the indication and the right timing of an operation are subject of a broad discussion. The aim of this study was to determine the influence of a surgical stabilization of rib fractures (SSRF) on the outcome in a multi-center database with special regard to the duration of ventilation, intensive care and overall hospital stay. METHODS: Data from the TraumaRegister DGU® collected between 2008 and 2017 were used to evaluate patients over 16 years with severe rib fractures (AIS ≥ 3). In addition to the basic comparison a matched pair analysis of 395 pairs was carried out in order to find differences and to increase comparability. RESULTS: In total 483 patients received an operative treatment and 29,447 were treated conservatively. SSRF was associated with a significantly lower mortality rate (7.6% vs. 3.3%, p = 0.008) but a longer ventilation time and longer stay as well as in the intensive care unit (ICU) as the overall hospital stay. Both matched pair groups showed a good or very good neurological outcome according to the Glasgow Outcome Scale (GOS) in 4 of 5 cases. Contrary to the existing recommendations most of the patients were not operated within 48 h. CONCLUSIONS: In our data set, obviously most of the patients were not treated according to the recent literature and showed a delay in the time for operative care of well over 48 h. This may lead to an increased rate of complications and a longer stay at the ICU and the hospital in general. Despite of these findings patients with operative treatment show a significant lower mortality rate.

Evaluation of a standardized instrument for post hoc analysis of trauma-team-activation-criteria in 75,613 injured patients an analysis of the TraumaRegister DGU®.

INTRODUCTION: To improve the quality of criteria for trauma-team-activation it is necessary to identify patients who benefited from the treatment by a trauma team. Therefore, we evaluated a post hoc criteria catalogue for trauma-team-activation which was developed in a consensus process by an expert group and published recently. The objective was to examine whether the catalogue can identify patients that died after admission to the hospital and therefore can benefit from a specialized trauma team mostly. MATERIALS AND METHODS: The catalogue was applied to the data of 75,613 patients from the TraumaRegister DGU® between the 01/2007 and 12/2016 with a maximum abbreviated injury score (AIS) severity ≥ 2. The endpoint was hospital mortality, which was defined as death before discharge from acute care. RESULTS: The TraumaRegister DGU® dataset contains 18 of the 20 proposed criteria within the catalogue which identified 99.6% of the patients who were admitted to the trauma room following an accident and who died during their hospital stay. Moreover, our analysis showed that at least one criterion was fulfilled in 59,785 cases (79.1%). The average ISS in this group was 21.2 points (SD 9.9). None of the examined criteria applied to 15,828 cases (average ISS 8.6; SD 5). The number of consensus-based criteria correlated with the severity of injury and mortality. Of all deceased patients (8,451), only 31 (0.37%) could not be identified on the basis of the 18 examined criteria. Where only one criterion was fulfilled, mortality was 1.7%; with 2 or more criteria, mortality was at least 4.6%. DISCUSSION: The consensus-based criteria identified nearly all patients who died as a result of their injuries. If only one criterion was fulfilled, mortality was relatively low. However, it increased to almost 5% if two criteria were fulfilled. Further studies are necessary to analyse and examine the relative weighting of the various criteria. Our instrument is capable to identify severely injured patients with increased in-hospital mortality and injury severity. However, a minimum of two criteria needs to be fulfilled. Based on these findings, we conclude that the criteria list is useful for post hoc analysis of the quality of field triage in patients with severe injury.

Geriatric Proximal Femur Fractures During the Covid-19 Pandemic - Fewer Cases, But More Comorbidities.

BACKGROUND: The COVID-19 pandemic is challenging healthcare systems worldwide. This study examines geriatric patients with proximal femur fractures during the COVID-19 pandemic, shifts in secondary disease profile, the impact of the pandemic on hospitalization and further treatment. METHODS: In a retrospective monocentric study, geriatric proximal femur fractures treated in the first six months of 2020 were analyzed and compared with the same period of 2019. Pre-traumatic status (living in a care home, under supervision of a legal guardian), type of trauma, accident mechanism, geriatric risk factors, associated comorbidities, time between hospitalization and surgery, inpatient time and post-operative further treatment of 2 groups of patients, aged 65-80 years (Group 1) and 80+ years (Group 2) were investigated. RESULTS: The total number of patients decreased (70 in 2019 vs. 58 in 2020), mostly in Group 1 (25 vs. 16) while the numbers in Group 2 remained almost constant (45 vs. 42). The percentage of patients with pre-existing neurological conditions rose in 2020. This corresponded to an increase in patients under legal supervision (29.3%) and receiving pre-traumatic care in a nursing home (14.7%). Fractures were mostly caused by minor trauma in a home environment. In 2020, total number of inpatient days for Group 2 was lower compared to Group 1 (p = 0.008). Further care differed between the years: fewer Group 1 patients were discharged to geriatric therapy (69.6% vs. 25.0%), whereas in Group 2 the number of patients discharged to a nursing home increased. CONCLUSIONS: Falling by elderly patients is correlated to geriatric comorbidities, consequently there was no change in the case numbers in this age group. Strategic measures to avoid COVID-19 infection in hospital setting could include reducing the length of hospital stays by transferring elderly patients to a nursing home as soon as possible and discharging independent, mobile patients to return home.

Long-term efficacy of interventions for actinic keratosis: protocol for a systematic review and network meta-analysis.

BACKGROUND: Actinic keratoses (AK) are common precancerous lesions of the skin due to cumulative sun exposure. A variety of interventions are available for the treatment; however, the majority of randomised controlled trials (RCTs) and meta-analyses focus on short-term efficacy outcomes. This network meta-analysis aims to investigate the long-term (> 12 months) efficacy of interventions for AK. METHODS: To identify relevant studies, we will perform a systematic literature research in MEDLINE, Embase, and CENTRAL and hand-search pertinent trial registers. Two authors will independently screen titles and abstracts for eligibility. We will include RCTs with an inter-individual (parallel arm) design. The study population includes patients with a clinical or histopathologic diagnosis of AK. Eligibility will be restricted to the following interventions: surgical approaches, cryosurgery, ablative laser treatment, topical drug treatment with 5-fluorouracil, imiquimod, ingenol mebutate, diclofenac, or photodynamic therapy. As outcomes, we will consider the following endpoints: (1) the participant complete clearance rate, (2) the participant partial clearance rate, (3) the lesion-specific clearance, (4) the mean lesion reduction per patient, and (5) the number of withdrawals due to adverse events after at least 12 months after the end of treatment. Monotherapy or placebo will serve as a comparison. Estimates of effects from individual studies will be pooled using a random-effects model. Heterogeneity will be evaluated based on I2 and chi-square test. The risk of bias will be estimated with the Cochrane Risk of Bias Tool by two review authors independently. The quality of evidence of the outcomes will be assessed with the GRADE approach. A network meta-analysis will be performed to combine direct and indirect evidence from the included RCTs. DISCUSSION: The potential of interventions to achieve a sustained clearance of AK has not been assessed to date. To investigate the long-term efficacy of interventions is important as the natural disease course is highly variable and relapses occur frequently even after initial lesion clearance. This review will help to set a framework for clinical decision making in patients with AK. SYSTEMATIC REVIEW REGISTRATION: CRD42018095903 (PROSPERO).

A Consensus-Based Criterion Standard for the Requirement of a Trauma Team.

BACKGROUND: Trauma team activation (TTA) represents a considerable expenditure of trauma centre resources. It is mainly triggered by field triage criteria. The overall quality of the criteria may be evaluated based on the rate of over- and undertriage. However, there is no gold standard that defines which adult patients truly require a trauma team. The objective of this study was to develop consensus-based criteria defining the necessity for a trauma team. METHODS: A consensus group was formed by trauma specialists experienced in emergency and trauma care with a specific interest in field triage and having previously participated in guideline development. A literature search was conducted to identify criteria that have already been used or suggested. The initial list of criteria was discussed in two Delphi round and two consensus conferences. The entire process of discussion and voting was highly standardized and extensively documented, resulting in a final list of criteria. RESULTS: Initially 95 criteria were identified. This was subsequently reduced to 20 final criteria to appropriately indicate the requirement for attendance of a trauma team. The criteria address aspects related to injury severity, admission to an intensive care unit, death within 24 h, need for specified invasive procedures, need for surgical and/or interventional radiological procedures, and abnormal vital signs within a defined time period. CONCLUSIONS: The selected criteria may be applied as a tool for research and quality control concerning TTA. However, future studies are necessary to further evaluate for possible redundancy in criteria that may allow for further reduction in criteria.

The presynaptic ribbon maintains vesicle populations at the hair cell afferent fiber synapse.

The ribbon is the structural hallmark of cochlear inner hair cell (IHC) afferent synapses, yet its role in information transfer to spiral ganglion neurons (SGNs) remains unclear. We investigated the ribbon's contribution to IHC synapse formation and function using KO mice lacking RIBEYE. Despite loss of the entire ribbon structure, synapses retained their spatiotemporal development and KO mice had a mild hearing deficit. IHCs of KO had fewer synaptic vesicles and reduced exocytosis in response to brief depolarization; a high stimulus level rescued exocytosis in KO. SGNs exhibited a lack of sustained excitatory postsynaptic currents (EPSCs). We observed larger postsynaptic glutamate receptor plaques, potentially compensating for the reduced EPSC rate in KO. Surprisingly, large-amplitude EPSCs were maintained in KO, while a small population of low-amplitude slower EPSCs was increased in number. The ribbon facilitates signal transduction at physiological stimulus levels by retaining a larger residency pool of synaptic vesicles.

Phosphoinositol-4,5-Bisphosphate Regulates Auditory Hair-Cell Mechanotransduction-Channel Pore Properties and Fast Adaptation.

Membrane proteins, such as ion channels, interact dynamically with their lipid environment. Phosphoinositol-4,5-bisphosphate (PIP2) can directly or indirectly modify ion-channel properties. In auditory sensory hair cells of rats (Sprague Dawley) of either sex, PIP2 localizes within stereocilia, near stereocilia tips. Modulating the amount of free PIP2 in inner hair-cell stereocilia resulted in the following: (1) the loss of a fast component of mechanoelectric-transduction current adaptation, (2) an increase in the number of channels open at the hair bundle's resting position, (3) a reduction of single-channel conductance, (4) a change in ion selectivity, and (5) a reduction in calcium pore blocking effects. These changes occur without altering hair-bundle compliance or the number of functional stereocilia within a given hair bundle. Although the specific molecular mechanism for PIP2 action remains to be uncovered, data support a hypothesis for PIP2 directly regulating channel conformation to alter calcium permeation and single-channel conductance.SIGNIFICANCE STATEMENT How forces are relayed to the auditory mechanoelectrical transduction (MET) channel remains unknown. However, researchers have surmised that lipids might be involved. Previous work on bullfrog hair cells showed an effect of phosphoinositol-4,5-bisphosphate (PIP2) depletion on MET current amplitude and adaptation, leading to the postulation of the existence of an underlying myosin-based adaptation mechanism. We find similar results in rat cochlea hair cells but extend these data to include single-channel analysis, hair-bundle mechanics, and channel-permeation properties. These additional data attribute PIP2 effects to actions on MET-channel properties and not motor interactions. Further findings support PIP2's role in modulating a fast, myosin-independent, and Ca2+-independent adaptation process, validating fast adaptation's biological origin. Together this shows PIP2's pivotal role in auditory MET, likely as a direct channel modulator.

Identification of new channels by systematic analysis of the mitochondrial outer membrane.

The mitochondrial outer membrane is essential for communication between mitochondria and the rest of the cell and facilitates the transport of metabolites, ions, and proteins. All mitochondrial outer membrane channels known to date are ß-barrel membrane proteins, including the abundant voltage-dependent anion channel and the cation-preferring protein-conducting channels Tom40, Sam50, and Mdm10. We analyzed outer membrane fractions of yeast mitochondria and identified four new channel activities: two anion-preferring channels and two cation-preferring channels. We characterized the cation-preferring channels at the molecular level. The mitochondrial import component Mim1 forms a channel that is predicted to have an α-helical structure for protein import. The short-chain dehydrogenase-related protein Ayr1 forms an NADPH-regulated channel. We conclude that the mitochondrial outer membrane contains a considerably larger variety of channel-forming proteins than assumed thus far. These findings challenge the traditional view of the outer membrane as an unspecific molecular sieve and indicate a higher degree of selectivity and regulation of metabolite fluxes at the mitochondrial boundary.

Small Molecules for Early Endosome-Specific Patch Clamping.

To resolve the subcellular distribution of endolysosomal ion channels, we have established a novel experimental approach to selectively patch clamp Rab5 positive early endosomes (EE) versus Rab7/LAMP1-positive late endosomes/lysosomes (LE/LY). To functionally characterize ion channels in endolysosomal membranes with the patch-clamp technique, it is important to develop techniques to selectively enlarge the respective organelles. We found here that two small molecules, wortmannin and latrunculin B, enlarge Rab5-positive EE when combined but not Rab7-, LAMP1-, or Rab11 (RE)-positive vesicles. The two compounds act rapidly, specifically, and are readily applicable in contrast to genetic approaches or previously used compounds such as vacuolin, which enlarges EE, RE, and LE/LY. We apply this approach here to measure currents mediated by TRPML channels, in particular TRPML3, which we found to be functionally active in both EE and LE/LY in overexpressing cells as well as in endogenously expressing CD11b+ lung-tissue macrophages.

Separating mitochondrial protein assembly and endoplasmic reticulum tethering by selective coupling of Mdm10.

The endoplasmic reticulum-mitochondria encounter structure (ERMES) connects the mitochondrial outer membrane with the ER. Multiple functions have been linked to ERMES, including maintenance of mitochondrial morphology, protein assembly and phospholipid homeostasis. Since the mitochondrial distribution and morphology protein Mdm10 is present in both ERMES and the mitochondrial sorting and assembly machinery (SAM), it is unknown how the ERMES functions are connected on a molecular level. Here we report that conserved surface areas on opposite sides of the Mdm10 ß-barrel interact with SAM and ERMES, respectively. We generated point mutants to separate protein assembly (SAM) from morphology and phospholipid homeostasis (ERMES). Our study reveals that the ß-barrel channel of Mdm10 serves different functions. Mdm10 promotes the biogenesis of α-helical and ß-barrel proteins at SAM and functions as integral membrane anchor of ERMES, demonstrating that SAM-mediated protein assembly is distinct from ER-mitochondria contact sites.