Structural Basis for Regulated Proteolysis by the α-Secretase ADAM10.

Cleavage of membrane-anchored proteins by ADAM (a disintegrin and metalloproteinase) endopeptidases plays a key role in a wide variety of biological signal transduction and protein turnover processes. Among ADAM family members, ADAM10 stands out as particularly important because it is both responsible for regulated proteolysis of Notch receptors and catalyzes the non-amyloidogenic α-secretase cleavage of the Alzheimer's precursor protein (APP). We present here the X-ray crystal structure of the ADAM10 ectodomain, which, together with biochemical and cellular studies, reveals how access to the enzyme active site is regulated. The enzyme adopts an unanticipated architecture in which the C-terminal cysteine-rich domain partially occludes the enzyme active site, preventing unfettered substrate access. Binding of a modulatory antibody to the cysteine-rich domain liberates the catalytic domain from autoinhibition, enhancing enzymatic activity toward a peptide substrate. Together, these studies reveal a mechanism for regulation of ADAM activity and offer a roadmap for its modulation.

[Severe Conjunctival and Corneal Epithelial Dysplasia: A Case Series]. / Schwere konjunktivale und korneale epitheliale Dysplasie: eine Fallserie.

BACKGROUND/OBJECTIVES: Ocular surface squamous neoplasia (OSSN) are among the most frequent non-pigmented malignancies of the ocular surface. They have a wide range of histological characteristics - ranging from mild epithelial dysplasia to invasive carcinoma of the squamous cells of the cornea. They may be restricted to the conjunctiva or also involve the cornea. As there are no leading symptoms in the early stages, diagnosis may be very delayed in patients who do not receive regular ophthalmological treatment. The present case series describes clinical and histological data on OSSN and includes clinical and histological data on OSSN, including possible clinical presentations, important risk factors, special histological and cytological features and therapeutic options. METHODS: Retrospective case series of patients with histologically confirmed severe epithelial dysplasia of the conjunctiva and cornea consistent with OSSN who presented to the Department of Ophthalmology in Basel University Hospital. The analysis covered demographic data, symptoms, diagnostic testing (photo documentation, brush biopsy), treatment and cytological and/or histological material and findings. RESULTS: We report on five patients aged between 41 and 92 years at the time of diagnosis. The histological findings in all patients included severe epithelial dysplasia, but with a heterogenous clinical presentation. In all cases, the lesion started in the conjunctiva, but traversed the limbus and extended to the cornea. The primary treatment was always surgical removal. In one patient, this had to be repeated several times due to recurrent metaplasia and was complemented by subsequent mitomycin C therapy. The clinical outcome ranged between total restitution of the original state to inevitable enucleation. CONCLUSION: The clinical presentation of OSSN is highly heterogenous, so that the initial diagnosis is difficult. There are no official guidelines for treatment, so that the treatment of choice varied between clinics. Regular ophthalmological follow-ups are recommended, even after complete surgical excision. Possible relevant concomitant diseases and risk factors must be identified before therapy.

Adaptive monitoring in action-what drives arthropod diversity and composition in central European beech forests?

Recent studies suggest that arthropod diversity in German forests is declining. Currently, different national programs are being developed to monitor arthropod trends and to unravel the effects of forest management on biodiversity in forests. To establish effective long-term monitoring programs, a set of drivers of arthropod diversity and composition as well as suitable species groups have to be identified. To aid in answering these questions, we investigated arthropod data collected in four Hessian forest reserves (FR) in the 1990s. To fully utilize this data set, we combined it with results from a retrospective structural sampling design applied at the original trap locations in central European beech (Fagus sylvatica) forests. As expected, the importance of the different forest structural, vegetation, and site attributes differed largely between the investigated arthropod groups: beetles, spiders, Aculeata, and true bugs. Measures related to light availability and temperature such as canopy cover or potential radiation were important to all groups affecting either richness, composition, or both. Spiders and true bugs were affected by the broadest range of explanatory variables, which makes them a good choice for monitoring general trends. For targeted monitoring focused on forestry-related effects on biodiversity, rove and ground beetles seem more suitable. Both groups were driven by a narrower, more management-related set of variables. Most importantly, our study approach shows that it is possible to utilize older biodiversity survey data. Although, in our case, there are strong restrictions due to the long time between species and structural attribute sampling.

The Geriatric Prognostic Index: a clinical prediction model for survival of older diffuse large B-cell lymphoma patients treated with standard immunochemotherapy.

The International prognostic Index (IPI) is the most widely used clinical prediction model for diffuse large B-cell lymphoma (DLBCL) patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP), but may be suboptimal in older patients. We aimed to develop and externally validate a clinical prediction model for older, RCHOP- treated DLBCL patients by examining geriatric assessment and lymphoma-related parameters in real-world cohorts. A population-based training set of 365 R-CHOP-treated DLBCL patients ≥70 years was identified through the Cancer Registry of Norway. The external test set consisted of a population-based cohort of 193 patients. Data on candidate predictors were retrieved from the Cancer Registry and through review of clinical records. Cox regression models for 2-year overall survival were used for model selection. Activities of daily living, the Charlson Comorbidity Index, age, sex, albumin, stage, Eastern Cooperative Oncology Group performance status and lactate dehydrogenase level were identified as independent predictors and combined into a Geriatric Prognostic Index (GPI). The GPI demonstrated good discrimination (optimismcorrected C-index 0.752), and identified low-, intermediate- and high-risk groups with significantly different survivals (2- year overall survival, 94%, 65%, and 25%, respectively). At external validation, the continuous and grouped GPI demonstrated good discrimination (C-index 0.727 and 0.710, respectively) and the GPI groups had significantly different survivals (2-year overall survival 95%, 65%, and 44%, respectively). Both the continuous and grouped GPI showed better discrimination than the IPI, revised-IPI and National Comprehensive Cancer Network (NCCN)-IPI (C-index 0.621, 0.583, and 0.670, respectively). In conclusion, we have developed and externally validated a GPI for older DLBCL patients treated with R-CHOP that outperformed the IPI, revised-IPI and NCCN-IPI. A web-based calculator is available at https://wide.shinyapps. io/GPIcalculator/.

RGD Peptidomimetic MMAE-Conjugate Addressing Integrin αVß3-Expressing Cells with High Targeting Index.

Small molecule-drug conjugates (SMDCs) mimicking the RGD sequence (-Arg-Gly-Asp-) with a non-peptide moiety require a pharmacophore-independent attachment site. A library of 36 sulfonamide-modified RGD mimetics with nM to pM affinity for integrin αV ß3 was synthesized and analysed via DAD mapping. The best structure of the conjugable RGD mimetic was used and a linker was attached to an aromatic ring by Negishi cross-coupling. The product retained high affinity and selectivity for integrin αV ß3 . The conjugable RGD mimetic was then attached to an enzymatically cleavable GKGEVA linker equipped with a self-immolative PABC and the antimitotic drug monomethyl auristatin E (MMAE). The resulting SMDC preferred binding to integrin αV ß3 over α5 ß1 in a ratio of 1 : 4519 (ELISA) and showed selectivity for αV ß3 -positive WM115 cells over αV ß3 -negative M21-L cells in the in vitro cell adhesion assay as well as in cell viability assays with a targeting index of 134 (M21-L/WM115).

Endosomal disentanglement of a transducible artificial transcription factor targeting endothelin receptor A.

Cell-penetrating peptides (CPPs) hold great promise for intracellular delivery of therapeutic proteins. However, endosomal entrapment of transduced cargo is a major bottleneck hampering their successful application. While developing a transducible zinc finger protein-based artificial transcription factor targeting the expression of endothelin receptor A, we identified interaction between the CPP and the endosomal membrane or endosomal entanglement as a main culprit for endosomal entrapment. To achieve endosomal disentanglement, we utilized endosome-resident proteases to sever the artificial transcription factor from its CPP upon arrival inside the endosome. Using this approach, we greatly enhanced the correct subcellular localization of the disentangled artificial transcription factor, significantly increasing its biological activity and distribution in vivo. With rational engineering of proteolytic sensitivity, we propose a new design principle for transducible therapeutic proteins, helping CPPs attain their full potential as delivery vectors for therapeutic proteins.

674 Cases of Late Postoperative Intraocular Lens Opacification of a Hydrophilic-Hydrophobic Acryl Intraocular Lens in Switzerland and Retrospective Opacification Risk Factor Assessment of 212 Cases.

PURPOSE: To report the prevalence of late postoperative opacification of a hydrophilic and hydrophobic acrylic intraocular lens (IOL) and to assess the risk factors in a subset of 212 eyes of patients referred to the University Eye Department in Basel, Switzerland. DESIGN: Retrospective case series. METHODS: A survey was performed at all large ophthalmological clinics in Switzerland regarding exchanged Lentis LS-502-1 lenses, and the number of affected eyes was counted. Moreover, consecutive patients who were referred to a tertiary clinic between September 2015 and November 2016 with Lentis LS-502-1 opacification were investigated. Peri- and postoperative charts, medical history, and topical and systemic medications were assessed. RESULTS: A total of 674 opacified Lentis LS-502-1 lenses have been reported in Switzerland, and 212 consecutive eyes of 182 patients were included in the study. All IOLs had a similar pattern of opacification with a yellowish, diffuse appearance, and most of them showed a small, paracentral, roundish area that was less affected or not at all. Arterial hypertension (73%), hypercholesterolemia (34%), and diabetes (21%) were the main associated systemic diseases, and statins (34%) and betablockers (34%) were the main treatments used. CONCLUSIONS: The prevalence of IOL opacification was 9.9%. No associated systemic eye disease or medications could be detected, which was implicated in the opacification process. The reason for opacification remains unclear, but it seems to be unrelated to the patient's state; therefore, it is attributed to primary calcification.

Rituximab-treated patients with lymphoma develop strong CD8 T-cell responses following COVID-19 vaccination.

B-cell depletion induced by anti-cluster of differentiation 20 (CD20) monoclonal antibody (mAb) therapy of patients with lymphoma is expected to impair humoral responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccination, but effects on CD8 T-cell responses are unknown. Here, we investigated humoral and CD8 T-cell responses following two vaccinations in patients with lymphoma undergoing anti-CD20-mAb therapy as single agent or in combination with chemotherapy or other anti-neoplastic agents during the last 9 months prior to inclusion, and in healthy age-matched blood donors. Antibody measurements showed that seven of 110 patients had antibodies to the receptor-binding domain of the SARS-CoV-2 Spike protein 3-6 weeks after the second dose of vaccination. Peripheral blood CD8 T-cell responses against prevalent human leucocyte antigen (HLA) class I SARS-CoV-2 epitopes were determined by peptide-HLA multimer analysis. Strong CD8 T-cell responses were observed in samples from 20/29 patients (69%) and 12/16 (75%) controls, with similar median response magnitudes in the groups and some of the strongest responses observed in patients. We conclude that despite the absence of humoral immune responses in fully SARS-CoV-2-vaccinated, anti-CD20-treated patients with lymphoma, their CD8 T-cell responses reach similar frequencies and magnitudes as for controls. Patients with lymphoma on B-cell depleting therapies are thus likely to benefit from current coronavirus disease 2019 (COVID-19) vaccines, and development of vaccines aimed at eliciting T-cell responses to non-Spike epitopes might provide improved protection.

Validation of the Dutch translation of the quality of recovery-15 scale.

BACKGROUND: The 15-item Quality of Recovery-15 (QoR-15) scale is strongly recommended as a standard patient-reported outcome measure assessing the quality of recovery after surgery and anesthesia in the postoperative period. This study aimed to validate the Dutch translation of the questionnaire (QoR-15NL). MATERIALS AND METHODS: An observational, prospective, single-centre cohort study was conducted. Patients who underwent surgery under general anesthesia completed the QoR-15NL (preoperatively (t1) and twice postoperatively (t2 and t3)) and a visual analogue scale (VAS) for general recovery at t2. A psychometric evaluation was performed to assess the QoR-15NL's validity, reliability, responsiveness, reproducibility and feasibility. RESULTS: Two hundred and eleven patients agreed to participate (recruitment rate 94%), and 165 patients were included (completion rate 78%). The QoR-15NL score correlated with the VAS for general recovery (rs = 0.59). Construct validity was further demonstrated by confirmation of expected negative associations between the QoR-15NL and duration of surgery (rs = -0.25), duration of Post Anesthesia Care Unit stay (rs = -0.31), and duration of hospital stay (rs = -0.27). The QoR-15NL score decreased significantly according to the extent of surgery. Cronbach's alpha was 0.87, split-half reliability was 0.8, and the test-retest intra-class coefficient was 0.93. No significant floor- or ceiling effect was observed. CONCLUSION: The QoR-15NL scale is a valid, easy-to-use, and reliable outcome assessment tool with high responsiveness for patient-reported quality of recovery after surgery and general anesthesia in the Dutch-speaking population. The QoR-15NL's measurement properties are comparable to the original questionnaire and other translated versions. TRIAL REGISTRATION: not applicable.

Modeling the Effect of Excitation on Depth of Anesthesia Monitoring in γ-Aminobutyric Acid Type A Receptor Agonist ABP-700.

BACKGROUND: γ-Aminobutyric acid type A (GABAA) receptor agonists are known to cause involuntary muscle movements. The mechanism of these movements is not known, and its relationship to depth of anesthesia monitoring is unclear. We have explored the effect of involuntary muscle movement on the pharmacokinetic-pharmacodynamic model for the GABAA receptor agonist ABP-700 and its effects on the Bispectral Index (BIS) as well as the Modified Observer's Assessment of Alertness/Sedation (MOAA/S) scores. METHODS: Observations from 350 individuals (220 men, 130 women) were analyzed, comprising 6,312 ABP-700 concentrations, 5,658 ABP-700 metabolite (CPM-acid) concentrations, 25,745 filtered BIS values, and 6,249 MOAA/S scores, and a recirculatory model developed. Various subject covariates and pretreatment with an opioid or a benzodiazepine were explored as covariates. Relationships between BIS and MOAA/S models and involuntary muscle movements were examined. RESULTS: The final model shows that the pharmacokinetics of ABP-700 are characterized by small compartmental volumes and rapid clearance. The BIS model incorporates an effect-site for BIS suppression and a secondary excitatory/disinhibitory effect-site associated with a risk of involuntary muscle movements. The secondary effect-site has a threshold that decreases with age. The MOAA/S model did not show excitatory effects. CONCLUSIONS: The GABAA receptor agonist ABP-700 shows the expected suppressive effects for BIS and MOAA/S, but also disinhibitory effects for BIS associated with involuntary muscle movements and reduced by pretreatment. Our model provides information about involuntary muscle movements that may be useful to improve depth of anesthesia monitoring for GABAA receptor agonists.

Clearance of neurotoxic peptides and proteins by meningothelial cells.

Meningothelial cells (MECs) are the cellular component of the meninges that provide physical protection to the central nervous system (CNS). Their main function is the formation of a barrier enclosing the brain including the cerebrospinal fluid (CSF). Further, MECs are involved in maintaining CSF homeostasis by clearing CSF from bacteria and apoptotic cells. Furthermore, secretion of pro- and anti-inflammatory cytokines and chemokines involves MECs in immunological processes in the CNS. We demonstrated that meningothelial Ben-Men-1 cells ingest neurotoxic peptides amyloid-ß (Aß1-40) and protein α-synuclein up to about 10-fold more efficiently compared to neuronal-like SH-SY5Y cells. Aß1-40 and α-synuclein are mainly taken up via macropinocytosis. Caveolar endocytosis in addition contributes to α-synuclein ingestion. Upon uptake, both are trafficked towards lysosomal degradation. While production of reactive oxygen species (ROS) following exposure to Aß25-35 and α-synuclein was similar between Ben-Men-1 and SH-SY5Y cells, mitochondrial function in Ben-Men-1 was significantly more robust to Aß25-35 treatment compared to neuronal-like SHSY5Y cells. Similarly, Ben-Men-1 were significantly less susceptible to Aß25-35-induced cell death than neuronal-like cells. Furthermore, co-culture with Ben-Men-1 offered significant protection to neuronal-like cells against Aß25-35-induced apoptosis. This study reveals for the first time the function of MECs as scavengers of neurotoxic Aß and α-synuclein, thereby connecting these cells to neuroprotective processes and suggesting a new mechanism and pathway for clearing neurotoxic substances from the CSF.

Ocular Pathology and Occasionally Detectable Intraocular Severe Acute Respiratory Syndrome Coronavirus-2 RNA in Five Fatal Coronavirus Disease-19 Cases.

INTRODUCTION: In December 2019, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic broke out. The virus rapidly spread globally, resulting in a major world public-health crisis. The major disease manifestation occurs in the respiratory tract. However, further studies documented other systemic involvement. This study investigates histopathologic eye changes in postmortem material of coronavirus disease 2019 (COVID-19) patients. METHODS: Sections of formalin-fixed, paraffin-embedded eyes from 5 patients (10 eyes) who died of COVID-19 at the University Hospital in Basel were included. Gross examination and histological evaluation were performed by 3 independent ophthalmopathologists. Immunohistochemical staining was performed using antibodies against fibrin, cleaved caspase 3, and ACE-2. Five enucleated eyes of patients not infected with SARS-CoV-2 served as control group. All cases have been studied for presence of SARS-CoV-2 RNA by means of reverse transcription PCR and RNA in situ hybridization (ISH). The choroidal vessels of one case were analyzed with electron microscope. RESULTS: Ophthalmopathologically, 8 eyes from 4 patients displayed swollen endothelial cells in congested choroidal vessels. No further evidence of specific eye involvement of SARS-CoV-2 was found in any of the patients. In the 8 eyes with evidence of changes due to SARS-CoV-2, immunohistochemical staining demonstrated fibrin microthrombi, apoptotic changes of endothelial and inflammatory cells. In control eyes, ACE-2 was detectable in the conjunctiva, cornea, retina, and choroidea and displayed significantly lower amounts of stained cells as in COVID-19 eyes. SARS-CoV-2 RNA was detectable in both bulbi of 2/5 patients, yet ISH failed to visualize viruses. Electron microscopy showed no significant results due to the artifacts. DISCUSSION/CONCLUSION: As already described in other organs of COVID-19 patients, the ophthalmological examination revealed-microthrombi, that is, hypercoagulation and vasculopathy most probably due to endothelial damage. A possible viral spread to the endothelial cells via ACE-2 provides one pathophysiological explanation. The expression of ACE-2 receptors in the conjunctiva hints toward its susceptibility to infection. To what extend eyes, function is disrupted by SARS-CoV-2 is subject to further studies, especially in the clinic.

Platelet Microparticles Decrease Daunorubicin-Induced DNA Damage and Modulate Intrinsic Apoptosis in THP-1 Cells.

Platelets can modulate cancer through budding of platelet microparticles (PMPs) that can transfer a plethora of bioactive molecules to cancer cells upon internalization. In acute myelogenous leukemia (AML) this can induce chemoresistance, partially through a decrease in cell activity. Here we investigated if the internalization of PMPs protected the monocytic AML cell line, THP-1, from apoptosis by decreasing the initial cellular damage inflicted by treatment with daunorubicin, or via direct modulation of the apoptotic response. We examined whether PMPs could protect against apoptosis after treatment with a selection of inducers, primarily associated with either the intrinsic or the extrinsic apoptotic pathway, and protection was restricted to the agents targeting intrinsic apoptosis. Furthermore, levels of daunorubicin-induced DNA damage, assessed by measuring gH2AX, were reduced in both 2N and 4N cells after PMP co-incubation. Measuring different BCL2-family proteins before and after treatment with daunorubicin revealed that PMPs downregulated the pro-apoptotic PUMA protein. Thus, our findings indicated that PMPs may protect AML cells against apoptosis by reducing DNA damage both dependent and independent of cell cycle phase, and via direct modulation of the intrinsic apoptotic pathway by downregulating PUMA. These findings further support the clinical relevance of platelets and PMPs in AML.

The extracellular matrix composition of the optic nerve subarachnoid space.

The meninges not only surround the brain and the spinal cord but also the optic nerve. Meningeal-derived extracellular matrix (ECM) is a crucial component of the pial basement membrane, glia limitans and important for maintenance of optic nerve axon integrity, homeostasis and retinal ganglion cell health. To get closer insight into optic nerve meningeal-derived ECM composition, we performed proteomic analysis of the sheep optic nerve subarachnoid space (SAS). Candidate components were confirmed in cultures of primary human meningothelial cells (phMECs) and human optic nerve samples. Sheep optic nerve SAS samples were analysed by LC-MS, identified proteins were matched to their human orthologs and filtered using gene lists representing all major ECM components. To validate these findings digital droplet PCR (ddPCR) to evaluate mRNA expression of all candidate components identified was performed on cultures of phMECs. In addition, one protein per major ECM group was stained on human optic nerve sections and on phMEC cultures. Employing LC-MS, 1273 proteins were identified and subjected to bioinformatic analysis. Gene ontology analysis revealed six out of forty-four collagen types (1A1, 1A2, 3A1, 6A2, 6A3 and 14A1), three out of eleven laminin subunits (A4, B2, C1) and six out of twenty-seven hyaluronan binding proteins (CD44, versican (VCAN), C1q binding protein, neurocan (NCAN), brevican (BCAN) and hyalaluronan proteoglycan link protein 2 (HAPLN2)) were present in our cohort. DdPCR in phMEC cell culture confirmed presence of all candidate components except NCAN, BCAN and HAPLN2. Immunohistochemistry (IHC) staining on human optic nerve sections and immunofluorescence (IF) staining on in vitro cultured phMECs showed strong immunopositivity for collagen-typeI-α1 (COL1A1), lamininγ1 (LAMC1), and VCAN. Fibronectin (FN1) was exclusively present in cultures of phMECs. Using a combined bioinformatics and immunohistological approach, we describe the ECM composition of the optic nerve subarachnoid space. As this space plays an important role in maintaining optic nerve function, a better understanding of ECM composition in this delicate environment might be key to further pathophysiological insight into optic nerve degeneration and associated disorders.

High throughput sequencing reveals high specificity of TNFAIP3 mutations in ocular adnexal marginal zone B-cell lymphomas.

The majority of ocular adnexal (OA) lymphomas (OAL) are extranodal marginal zone lymphomas (MZL). First high throughput sequencing (HTS) studies on OA-MZL showed inconsistent results and the distribution of mutations in reactive lymphoid lesions of this anatomic region has not yet been sufficiently addressed. We characterized OAL and lymphoid lesions of the OA by targeted HTS. The study included 34 OA-MZL, 11 chronic conjunctivitis, five mature small cell B-cell lymphomas spreading to the OA, five diseases with increase of IgG4+ plasma cells, three Burkitt lymphomas (BL), three diffuse large B-cell lymphomas (DLBCL), three mantle cell lymphomas, three idiopathic orbital inflammations/orbital pseudo tumors (PT), and three OA lymphoid hyperplasia. All cases were negative for Chlamydia. The mutational number was highest in BL and lowest in PT. The most commonly (and exclusively) mutated gene in OA-MZL was TNFAIP3 (10 of 34 cases). Altogether, 20 out of 34 patients harbored mutually exclusive mutations of either TNFAIP3, BCL10, MYD88, ATM, BRAF, or NFKBIE, or nonexclusive mutations of IRF8, TNFRSF14, KLHL6, and TBL1XR1, all encoding for NK-κB pathway compounds or regulators. Thirteen patients (38%) had, to a great part, mutually exclusive mutations of chromatin modifier-encoding genes: KMT2D, CREBBP, BCL7A, DNMT3A, EP300, or HIST1H1E. Only four patients harbored co-occurring mutations of genes encoding for NK-κB compounds and chromatin modifiers. Finally, PTEN, KMT2D, PRDM1, and HIST1H2BK mutations were observable in reactive lymphoid lesions too, while such instances were devoid of NF-κB compound mutations and/or mutations of acetyltransferase-encoding genes. In conclusion, 80% of OA-MZL display mutations of either NK-κB compounds or chromatin modifiers. Lymphoid lesions of the OA bearing NF-κB compound mutations and/or mutations of acetyltransferase-encoding genes highly likely represent lymphomas.

Quantifying natural disturbances using a large-scale dendrochronological reconstruction to guide forest management.

Estimates of historical disturbance patterns are essential to guide forest management aimed at ensuring the sustainability of ecosystem functions and biodiversity. However, quantitative estimates of various disturbance characteristics required in management applications are rare in longer-term historical studies. Thus, our objectives were to (1) quantify past disturbance severity, patch size, and stand proportion disturbed and (2) test for temporal and subregional differences in these characteristics. We developed a comprehensive dendrochronological method to evaluate an approximately two-century-long disturbance record in the remaining Central and Eastern European primary mountain spruce forests, where wind and bark beetles are the predominant disturbance agents. We used an unprecedented large-scale nested design data set of 541 plots located within 44 stands and 6 subregions. To quantify individual disturbance events, we used tree-ring proxies, which were aggregated at plot and stand levels by smoothing and detecting peaks in their distributions. The spatial aggregation of disturbance events was used to estimate patch sizes. Data exhibited continuous gradients from low- to high-severity and small- to large-size disturbance events. In addition to the importance of small disturbance events, moderate-scale (25-75% of the stand disturbed, >10 ha patch size) and moderate-severity (25-75% of canopy disturbed) events were also common. Moderate disturbances represented more than 50% of the total disturbed area and their rotation periods ranged from one to several hundred years, which is within the lifespan of local tree species. Disturbance severities differed among subregions, whereas the stand proportion disturbed varied significantly over time. This indicates partially independent variations among disturbance characteristics. Our quantitative estimates of disturbance severity, patch size, stand proportion disturbed, and associated rotation periods provide rigorous baseline data for future ecological research, decisions within biodiversity conservation, and silviculture intended to maintain native biodiversity and ecosystem functions. These results highlight a need for sufficiently large and adequately connected networks of strict reserves, more complex silvicultural treatments that emulate the natural disturbance spectrum in harvest rotation times, sizes, and intensities, and higher levels of tree and structural legacy retention.

Habitat-tree protection concepts over 200 years.

The protection and sustainable management of habitat trees is an integral part of modern forest nature conservation concepts such as retention forestry. Bats, cavity-nesting birds, arboreal marsupials, and many different saproxylic species depend on habitat trees and their great variety of microhabitats and old-growth characteristics. With a focus on insights from temperate forests, we traced the development of habitat-tree protection over 200 years. The idea was first conceptualized by foresters and natural scientists in the early 19th century. At that time, utilitarian conservation aimed to protect cavity trees that provided roosts and nesting holes for insectivorous bats and birds. By the second half of the 19th century, habitat-tree protection was well known to foresters and was occasionally implemented. Knowledge of the protection of large old trees, a special kind of habitat tree, for sociocultural and aesthetic reasons developed similarly. But, many foresters of that time and in the following decades fundamentally rejected protection of habitat trees for economic reasons. Beginning in the 1970s, forest conservation and integrative forest management became increasingly important issues worldwide. Since then, the protection of habitat trees has been implemented on a large scale. Long-term views on the development of conservation concepts are important to inform the implementation of conservation today. In particular, historical analyses of conservation concepts allow the testing of long-term conservation outcomes and make it possible to study the resilience of conservation approaches to changing social or ecological conditions. We encourage all conservation ecologists to assess the practical and conceptual impact of the initial ideas that led to modern conservation concepts in terms of long-term biodiversity conservation.

Conceptos de Protección de Árboles Hábitat en los Últimos 200 Años Resumen La protección y el manejo sustentable de los árboles hábitat son una parte integral de los conceptos modernos de conservación de la naturaleza del bosque, conceptos como la silvicultura de retención. Los murciélagos, las aves que anidan en cavidades, los marsupiales arbóreos y muchas especies saproxílicas diferentes dependen de los árboles hábitat y su gran variedad de microhábitats y características de bosque maduro. Rastreamos el desarrollo de la protección de árboles hábitat en los últimos 200 años con un enfoque de conocimiento a partir de los bosques templados. La idea primero la conceptualizaron silvicultores y científicos naturales en el siglo XIX. En aquel tiempo, la conservación utilitarista buscaba proteger los árboles con cavidades que proporcionaban perchas y agujeros para anidar a los murciélagos y las aves insectívoras. Para la segunda mitad del siglo XIX, la protección de los árboles hábitat era muy conocida por los silvicultores y ocasionalmente la implementaban. El conocimiento de la protección de los grandes árboles maduros, un tipo especial de árbol hábitat, por razones socioculturales y estéticas tuvieron un desarrollo similar a la protección. Aún así, muchos de los silvicultores de esa época y de las décadas subsecuentes fundamentalmente rechazaron la protección de los árboles hábitat por razones económicas. A principios de la década de 1970, la conservación de los bosques y el manejo integrativo de los bosques se convirtieron en temas cada vez más importantes en todo el mundo. Desde entonces, la protección de los árboles hábitat se ha implementado a gran escala. Las perspectivas a largo plazo sobre el desarrollo de los conceptos de conservación son de importancia para informar la implementación de la conservación hoy en día. Particularmente, los análisis históricos sobre los conceptos de conservación permiten el ensayo de resultados de conservación a largo plazo y posibilitan el estudio de la resiliencia de las estrategias de conservación ante las cambiantes condiciones ecológicas y sociales. Alentamos a todos los ecologistas de la conservación a evaluar el impacto práctico y conceptual de las ideas iniciales que derivaron en los conceptos modernos de conservación en términos de la conservación a largo plazo de la biodiversidad.

Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis.

We present a structure of the ∼650-kDa functional replisome of bacteriophage T7 assembled on DNA resembling a replication fork. A structure of the complex consisting of six domains of DNA helicase, five domains of RNA primase, two DNA polymerases, and two thioredoxin (processivity factor) molecules was determined by single-particle cryo-electron microscopy. The two molecules of DNA polymerase adopt a different spatial arrangement at the replication fork, reflecting their roles in leading- and lagging-strand synthesis. The structure, in combination with biochemical data, reveals molecular mechanisms for coordination of leading- and lagging-strand synthesis. Because mechanisms of DNA replication are highly conserved, the observations are relevant to other replication systems.

Opposite acute potassium and sodium shifts during transplantation of hypothermic machine perfused donor livers.

Liver transplantation is frequently associated with hyperkalemia, especially after graft reperfusion. Dual hypothermic oxygenated machine perfusion (DHOPE) reduces ischemia/reperfusion injury and improves graft function, compared to conventional static cold storage (SCS). We examined the effect of DHOPE on ex situ and in vivo shifts of potassium and sodium. Potassium and sodium shifts were derived from balance measurements in a preclinical study of livers that underwent DHOPE (n = 6) or SCS alone (n = 9), followed by ex situ normothermic reperfusion. Similar measurements were performed in a clinical study of DHOPE-preserved livers (n = 10) and control livers that were transplanted after SCS only (n = 9). During DHOPE, preclinical and clinical livers released a mean of 17 ± 2 and 34 ± 6 mmol potassium and took up 25 ± 9 and 24 ± 14 mmol sodium, respectively. After subsequent normothermic reperfusion, DHOPE-preserved livers took up a mean of 19 ± 3 mmol potassium, while controls released 8 ± 5 mmol potassium. During liver transplantation, blood potassium levels decreased upon reperfusion of DHOPE-preserved livers while levels increased after reperfusion of SCS-preserved liver, delta potassium levels were -0.77 ± 0.20 vs. +0.64 ± 0.37 mmol/L, respectively (P = .002). While hyperkalemia is generally anticipated during transplantation of SCS-preserved livers, reperfusion of hypothermic machine perfused livers can lead to decreased blood potassium or even hypokalemia in the recipient.

Pretransplant sequential hypo- and normothermic machine perfusion of suboptimal livers donated after circulatory death using a hemoglobin-based oxygen carrier perfusion solution.

Ex situ dual hypothermic oxygenated machine perfusion (DHOPE) and normothermic machine perfusion (NMP) of donor livers may have a complementary effect when applied sequentially. While DHOPE resuscitates the mitochondria and increases hepatic adenosine triphosphate (ATP) content, NMP enables hepatobiliary viability assessment prior to transplantation. In contrast to DHOPE, NMP requires a perfusion solution with an oxygen carrier, for which red blood cells (RBC) have been used in most series. RBC, however, have limitations and cannot be used cold. We, therefore, established a protocol of sequential DHOPE, controlled oxygenated rewarming (COR), and NMP using a new hemoglobin-based oxygen carrier (HBOC)-based perfusion fluid (DHOPE-COR-NMP trial, NTR5972). Seven livers from donation after circulatory death (DCD) donors, which were initially declined for transplantation nationwide, underwent DHOPE-COR-NMP. Livers were considered transplantable if perfusate pH and lactate normalized, bile production was ≥10 mL and biliary pH > 7.45 within 150 minutes of NMP. Based on these criteria five livers were transplanted. The primary endpoint, 3-month graft survival, was a 100%. In conclusion, sequential DHOPE-COR-NMP using an HBOC-based perfusion fluid offers a novel method of liver machine perfusion for combined resuscitation and viability testing of suboptimal livers prior to transplantation.