The extent of SARS-CoV-2 infection throughout the United States population is currently unknown. High quality serology is key to avoiding medically costly diagnostic errors, as well as to assuring ...properly informed public health decisions. Here, we present an optimized ELISA-based serology protocol, from antigen production to data analyses, that helps define thresholds for IgG and IgM seropositivity with high specificities. Validation of this protocol is performed using traditionally collected serum as well as dried blood on mail-in blood sampling kits. Archival (pre-2019) samples are used as negative controls, and convalescent, PCR-diagnosed COVID-19 patient samples serve as positive controls. Using this protocol, minimal cross-reactivity is observed for the spike proteins of MERS, SARS1, OC43 and HKU1 viruses, and no cross reactivity is observed with anti-influenza A H1N1 HAI. Our protocol may thus help provide standardized, population-based data on the extent of SARS-CoV-2 seropositivity, immunity and infection.
The SARS-CoV-2 spike trimer is the primary antigen for several serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Until stable cell lines are developed to ...increase the titer of this secreted protein in mammalian cell culture, the low yield of spike protein produced from transient transfection of HEK293 cells will be a limiting factor for these assays. To improve the yield of spike protein and support the high demand for antigens in serology assays, we investigated several recombinant protein expression variables by altering the incubation temperature, harvest time, chromatography strategy, and final protein manipulation. Through this investigation, we developed a simplified and robust purification strategy that consistently yields 5 mg of protein per liter of expression culture for two commonly used forms of the SARS-CoV-2 spike protein. We show that these proteins form well-behaved stable trimers and are consistently functional in serology assays across multiple protein production lots.
•Improved yields of SARS-CoV-2 spike protein through modification of expression and purification parameters.•Yields of greater than 5 mg/l obtained for VRC spike under optimal conditions.•Spike protein quality was validated by QC methods to ensure utility in serology assays.
A majority of human therapeutic antibody candidates show pharmacokinetic properties suitable for clinical use, but an unexpectedly fast antibody clearance is sometimes observed that may limit the ...clinical utility. Pharmacokinetic data in cynomolgus monkeys collected for a panel of 52 antibodies showed broad distribution of target-independent clearance values (2.4-61.3 mL/day/kg), with 15 (29%) having clearance > 10 mL/day/kg. Alteration in the interaction with the recycling FcRn receptor did not account for the faster than expected clearance observed for the antibodies; off-target binding was presumed to account for the fast clearance. We developed an assay based on ELISA detection of non-specific binding to baculovirus particles that can identify antibodies having increased risk for fast clearance. This assay can be used during lead generation or optimization to identify antibodies with increased risk of having fast clearance in both humans and cynomolgus monkeys, and thus increase the likelihood of obtaining a suitable drug candidate.
This review serves as a synopsis of multimodality imaging in cardiac amyloidosis (CA), which is a disease characterized by deposition of misfolded protein fragments in the heart. It emphasizes and ...summarizes the diagnostic possibilities and their prognostic values. In general, echocardiography is the first diagnostic tool in patients with an identified systemic disease or unclear left ventricular hypertrophy. Several echocardiographic parameters will raise suspicion and lead to further testing. Cardiac magnetic resonance and scintigraphy with bone avid radiotracers are crucial for diagnosis of CA and even enable a distinction between different subtypes. The subject is illuminated with established guidelines and innovative recent publications to further improve early diagnosis of cardiac amyloidosis in light of current treatment options.
tRNAs are synthesized as immature precursors, and on their way to functional maturity, extra nucleotides at their 5′ ends are removed by an endonuclease called RNase P. All RNase P enzymes ...characterized so far are composed of an RNA plus one or more proteins, and tRNA 5′ end maturation is considered a universal ribozyme-catalyzed process. Using a combinatorial purification/proteomics approach, we identified the components of human mitochondrial RNase P and reconstituted the enzymatic activity from three recombinant proteins. We thereby demonstrate that human mitochondrial RNase P is a protein enzyme that does not require a
trans-acting RNA component for catalysis. Moreover, the mitochondrial enzyme turns out to be an unexpected type of patchwork enzyme, composed of a tRNA methyltransferase, a short-chain dehydrogenase/reductase-family member, and a protein of hitherto unknown functional and evolutionary origin, possibly representing the enzyme's metallonuclease moiety. Apparently, animal mitochondria lost the seemingly ubiquitous RNA world remnant after reinventing RNase P from preexisting components.
BACKGROUND Pericardial cyst is a rare benign mass of the mediastinum. More than two-thirds of pericardial cysts are located in the right cardiophrenic angle and less than one-third in the left ...cardiophrenic angle. Most cases are asymptomatic and discovered incidentally during to thoracic imaging such as chest X-ray, CT scans, and transthoracic echocardiograms. When pericardial cysts present with symptoms, they are often persistent and non-specific and include chest pain, dyspnea, and persistent cough. The optimal management of pericardial cysts is unclear, and no large studies regarding safety, efficacy, and long-term follow-up exist. Management strategies include cyst resection with sternotomy, thoracotomy or video-assisted thoracic surgery, cyst aspiration, and sclerosis after aspiration. The optimal mode of follow-up for asymptomatic cases is also unclear. Here, we present a case of a large pericardial cyst in the left cardiophrenic angle in a middle-aged Danish woman with persistent and unresolved dyspnea and chest pain. CASE REPORT A 57-year-old woman was referred for transthoracic echocardiography because of year-long cough and left-sided chest pain, which were exacerbated in the supine position. The echocardiography revealed a large cyst-like structure over the left ventricle. A cardiac CT scan and MRI scan were performed, confirming the presence of a large pericardial cyst with no communication with the pericardium. The cyst was surgically removed via thoracotomy. CONCLUSIONS Pericardial cysts should be considered as a rare differential diagnosis, giving rise to common cardio-pulmonary symptoms such as chest pain, dyspnea, and cough.
Antibody–drug conjugates (ADCs) are designed to combine the exquisite specificity of antibodies to target tumor antigens with the cytotoxic potency of chemotherapeutic drugs. In addition to the ...general chemical stability of the linker, a thorough understanding of the relationship between ADC composition and biological disposition is necessary to ensure that the therapeutic window is not compromised by altered pharmacokinetics (PK), tissue distribution, and/or potential organ toxicity. The six-transmembrane epithelial antigen of prostate 1 (STEAP1) is being pursued as a tumor antigen target. To assess the role of ADC composition in PK, we evaluated plasma and tissue PK profiles in rats, following a single dose, of a humanized anti-STEAP1 IgG1 antibody, a thio-anti-STEAP1 (ThioMab) variant, and two corresponding thioether-linked monomethylauristatin E (MMAE) drug conjugates modified through interchain disulfide cysteine residues (ADC) and engineered cysteines (TDC), respectively. Plasma PK of total antibody measured by enzyme-linked immunosorbent assay (ELISA) revealed ∼45% faster clearance for the ADC relative to the parent antibody, but no apparent difference in clearance between the TDC and unconjugated parent ThioMab. Total antibody clearances of the two unconjugated antibodies were similar, suggesting minimal effects on PK from cysteine mutation. An ELISA specific for MMAE-conjugated antibody indicated that the ADC cleared more rapidly than the TDC, but total antibody ELISA showed comparable clearance for the two drug conjugates. Furthermore, consistent with relative drug load, the ADC had a greater magnitude of drug deconjugation than the TDC in terms of free plasma MMAE levels. Antibody conjugation had a noticeable, albeit minor, impact on tissue distribution with a general trend toward increased hepatic uptake and reduced levels in other highly vascularized organs. Liver uptakes of ADC and TDC at 5 days postinjection were 2-fold and 1.3-fold higher, respectively, relative to the unmodified antibodies. Taken together, these results indicate that the degree of overall structural modification in anti-STEAP1-MMAE conjugates has a corresponding level of impact on both PK and tissue distribution.
The following contribution examines the question of the existence or nonexistence of so-called functional immunity or immunity ratione materiae for crimes under international law in relation to ...national (in particular German) prosecuting authorities and courts. This matter is of utmost relevance to the daily work of the German Federal Public Prosecutor General (Generalbundesanwalt beim Bundesgerichtshof) because the office carries the main responsibility for investigating and bringing to trial crimes under international law in Germany.
The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this ...process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as “membrane-distal” and estimate to comprise ∼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional flycasting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.
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