The structure of a dimeric form of SARS-CoV-2 polymerase

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Previous structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the enzyme and resolve its structure at 5.5 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 each and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs

Molecular interactions of FG nucleoporin repeats at high resolution

Proteins that contain repeat phenylalanine-glycine (FG) residues phase separate into oncogenic transcription factor condensates in malignant leukaemias, form the permeability barrier of the nuclear pore complex and mislocalize in neurodegenerative diseases. Insights into the molecular interactions of FG-repeat nucleoporins have, however, remained largely elusive. Using a combination of NMR spectroscopy and cryoelectron microscopy, we have identified uniformly spaced segments of transient β-structure and a stable preformed α-helix recognized by messenger RNA export factors in the FG-repeat domain of human nucleoporin 98 (Nup98). In addition, we have determined at high resolution the molecular organization of reversible FG–FG interactions in amyloid fibrils formed by a highly aggregation-prone segment in Nup98. We have further demonstrated that amyloid-like aggregates of the FG-repeat domain of Nup98 have low stability and are reversible. Our results provide critical insights into the molecular interactions underlying the self-association and phase separation of FG-repeat nucleoporins in physiological and pathological cell activities

The 3D structure of lipidic fibrils of alpha-synuclein

α-synuclein misfolding and aggregation into fibrils is a common feature of α-synucleinopathies, such as Parkinson’s disease, in which α-synuclein fibrils are a characteristic hallmark of neuronal inclusions called Lewy bodies. Studies on the composition of Lewy bodies extracted postmortem from brain tissue of Parkinson’s patients revealed that lipids and membranous organelles are also a significant component. Interactions between α-synuclein and lipids have been previously identified as relevant for Parkinson’s disease pathology, however molecular insights into their interactions have remained elusive. Here we present cryo-electron microscopy structures of six α-synuclein fibrils in complex with lipids, revealing specific lipid-fibril interactions. We observe that phospholipids promote an alternative protofilament fold, mediate an unusual arrangement of protofilaments, and fill the central cavities of the fibrils. Together with our previous studies, these structures also indicate a mechanism for fibril-induced lipid extraction, which is likely to be involved in the development of α-synucleinopathies. Specifically, one potential mechanism for the cellular toxicity is the disruption of intracellular vesicles mediated by fibrils and oligomers, and therefore the modulation of these interactions may provide a promising strategy for future therapeutic interventions

Simultaneous computed tomography-guided biopsy and radiofrequency ablation of solitary pulmonary malignancy in high-risk patients

Background: In recent years experience has been accumulated in percutaneous radiofrequency ablation (RFA) of lung malignancies in nonsurgical patients. Objectives: In this study, we retrospectively evaluated a simultaneous diagnostic and therapeutic approach including CT-guided biopsy followed immediately by RFA of solitary malignant pulmonary lesions. Methods: CT-guided transthoracic core needle biopsy of solitary pulmonary lesions suspicious for malignancy was performed and histology was proven based on immediate frozen sections. RFA probes were placed into the pulmonary tumors under CT guidance and the ablation was performed subsequently. The procedure-related morbidity was analyzed. Follow-up included a CT scan and pulmonary function parameters. Results: A total of 33 CT-guided biopsies and subsequent RFA within a single procedure were performed. Morbidity of CT-guided biopsy included pulmonary hemorrhage (24%) and a mild pneumothorax (12%) without need for further interventions. The RFA procedure was not aggravated by the previous biopsy. The rate of pneumothorax requiring chest tube following RFA was 21%. Local tumor control was achieved in 77% with a median follow-up of 12 months. The morbidity of the CT-guided biopsy had no statistical impact on the local recurrence rate. Conclusions: The simultaneous diagnostic and therapeutic approach including CT-guided biopsy followed immediately by RFA of solitary malignant pulmonary lesions is a safe procedure. The potential of this combined approach is to avoid unnecessary therapies and to perform adequate therapies based on histology. Taking the local control rate into account, this approach should only be performed in those patients who are unable to undergo or who refuse surgery. Copyright (C) 2012 S. Karger AG, Base

Neutralization of SARS-CoV-2 by highly potent, hyperthermostable, and mutation-tolerant nanobodies

Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein, we isolated 45 infection-blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS-CoV-2 at 17–50 pM concentration (0.2–0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X-ray and cryo-EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune-escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low-picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such “fold-promoting” nanobodies may allow for simplified production of vaccines and their adaptation to viral escape-mutations

Resection of thoracic malignancies infiltrating cardiac structures with use of cardiopulmonary bypass

Background: Only few reports exist on malignant thoracic neoplasms that require cardiopulmonary bypass during resection. We aimed to investigate the early and late clinical outcome of these patients. Methods: Patients with thoracic malignancies that underwent surgery between 2002 and 2014 were analyzed. All patients had cardiopulomonary bypass support during resection. Clinical and perioperative data was retrospectively reviewed for outcome and overall survival. Results: Fifteen patients (12 female, mean age of 55 ± 15 years, range 24 to 80 years) were identified. Eleven (8 female) were diagnosed with primary thoracic malignomas and four with metastases. Three patients died early postoperatively. Patients diagnosed with sarcoma had a significantly worse outcome than non-sarcoma patients (83.3 ± 15.2 % after 1 year, 31.3 ± 24.5 % after 5 years vs. 83.3 ± 15.2 % after 1 year, 0 ± 0 % after 5 years, p = 0.005). Conclusions: Malignancies with extension into cardiac structures or infiltration of great vessels can be resected with cardiopulmonary bypass support and tolerable risk. Carefully selected patients can undergo advanced operative procedures with an acceptable 1-year-survival, but only few patients achieved good long-term outcome

Dexamethasone-induced cisplatin and gemcitabine resistance in lung carcinoma samples treated ex vivo

Chemotherapy for lung cancer not only has severe side effects but frequently also exhibits limited, if any clinical effectiveness. Dexamethasone (DEX) and similar glucocorticoids (GCs) such as prednisone are often used in the clinical setting, for example, as cotreatment to prevent nausea and other symptoms. Clinical trials evaluating the impact of GCs on tumour control and patient survival of lung carcinoma have never been performed. Therefore, we isolated cancer cells from resected lung tumour specimens and treated them with cisplatin in the presence or absence of DEX. Cell number of viable and dead cells was evaluated by trypan blue exclusion and viability was measured by the MTT-assay. We found that DEX induced resistance toward cisplatin in all of 10 examined tumour samples. Similar results were found using gemcitabine as cytotoxic drug. Survival of drug-treated lung carcinoma cells in the presence of DEX was longlasting as examined 2 and 3 weeks after cisplatin treatment of a lung carcinoma cell line. These data corroborate recent in vitro and in vivo xenograft findings and rise additional concerns about the widespread combined use of DEX with antineoplastic drugs in the clinical management of patients with lung cancer

The Roles of Cyclin A2, B1, and B2 in Early and Late Mitotic Events

This paper presents evidence that chromatin condensation, like nuclear envelope breakdown, is brought about through the combined effects of cyclins A2 and B1, and that cyclins B1 and B2 are largely responsible for maintenance of a spindle assembly checkpoint arrest