A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test.

OBJECTIVES: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. METHODS: The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. RESULTS: SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). CONCLUSIONS: Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.

Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use.

OBJECTIVES: During 2020, the UK's Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity. METHODS: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study. RESULTS: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen's Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen's Kappa 0.95. CONCLUSIONS: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory.

Comparing the Boxgrove and Atapuerca (Sima de los Huesos) human fossils: Do they represent distinct paleodemes?

The early Middle Pleistocene human material from Boxgrove (West Sussex, UK) consists of a partial left tibia and two lower incisors from a separate adult individual. These remains derive from deposits assigned to the MIS 13 interglacial at about 480 ka and have been referred to as Homo cf. heidelbergensis. The much larger skeletal sample from the Sima de los Huesos (Atapuerca, Spain) is dated to the succeeding MIS 12, at about 430 ka. This fossil material has previously been assigned to Homo heidelbergensis but is now placed within the Neanderthal clade. Because of the scarcity of human remains from the Middle Pleistocene and their morphological variability, this study assessed whether the Boxgrove specimens fit within the morphological variability of the homogeneous Sima de los Huesos population. Based on morphometric analyses performed against 22 lower incisors from Sima de los Huesos and published material, the data from the Boxgrove incisors place them comfortably within the range of Sima de los Huesos. Both assemblages present robust incisors distinct from the overall small recent Homo sapiens incisors, and Boxgrove also aligns closely with Homo neanderthalensis and some other European Middle Pleistocene hominins. Following morphological and cross-sectional analyses of the Boxgrove tibia compared to seven adult Sima de los Huesos specimens and a set of comparative tibiae, Boxgrove is shown to be similar to Sima de los Huesos and Neanderthals in having thick cortices and bone walls, but in contrast resembles modern humans in having a straight anterior tibial crest and a suggestion of a lateral concavity. Based on the patterns observed, there is no justification for assigning the Boxgrove and Sima de los Huesos incisors to distinct paleodemes, but the tibial data show greater contrasts and suggest that all three of these samples are unlikely to represent the same paleodeme.

Lateral decubitus dynamic CT myelography for fast cerebrospinal fluid leak localization.

Dynamic CT myelography is used to precisely localize fast spinal CSF leaks. The procedure is most commonly performed in the prone position, which successfully localizes most fast ventral leaks. We have recently encountered a small subset of patients in whom prone dynamic CT myelography is unsuccessful in localizing leaks. We sought to determine the added value of lateral decubitus dynamic CT myelography, which is occasionally attempted in our practice, in localizing the leak after failed prone dynamic CT myelography. We retrospectively identified 6 patients who underwent lateral decubitus dynamic CT myelography, which was performed in each case because their prone dynamic CT myelogram was unrevealing. Two neuroradiologists independently reviewed preprocedural spine MRI and all dynamic CT myelograms for each patient. Lateral decubitus positioning allowed for precise leak localization in all 6 patients. Five of six patients were noted to have dorsal and/or lateral epidural fluid collections on spine MRI. One patient had a single prominent diverticulum on spine MRI (larger than 6 mm), whereas the others had no prominent diverticula. Our study suggests that institutions performing dynamic CT myelography to localize fast leaks should consider a lateral decubitus study if performing the study in the prone position is unrevealing. Furthermore, the presence of dorsal and/or lateral epidural fluid collections on spine MRI may suggest that a lateral decubitus study is of higher yield and could be considered initially.

Geometric morphometric variability in the supraorbital and orbital region of Middle Pleistocene hominins: Implications for the taxonomy and evolution of later Homo.

This study assessed variation in the supraorbital and orbital region of the Middle Pleistocene hominins (MPHs), sometimes called Homo heidelbergensis s.l., to test whether it matched the expectations of intraspecific variation. The morphological distinctiveness and relative variation of this region, which is relatively well represented in the hominin fossil record, was analyzed quantitatively in a comparative taxonomic framework. Coordinates of 230 3D landmarks (20) and sliding semilandmarks (210) were collected from 704 specimens from species of Homo, Australopithecus, Paranthropus, Gorilla, Pan, Papio, and Macaca. Results showed that the MPHs had expected levels of morphological distinctiveness and intragroup and intergroup variation in supraorbital and orbital morphology, relative to commonly recognized non-hominin catarrhine species. However, the Procrustes distances between this group and H. sapiens were significantly higher than expected for two closely related catarrhine species. Furthermore, this study showed that variation within the MPH could be similarly well contained within existing hypodigms of H. sapiens, H. neanderthalensis, and H. erectus s.l. Although quantitative assessment of supraorbital and orbital morphology did not allow differentiation between taxonomic hypotheses in later Homo, it could be used to test individual taxonomic affiliation and identify potentially anomalous individuals. This study confirmed a complicated pattern of supraorbital and orbital morphology in the MPH fossil record and raises further questions over our understanding of the speciation of H. sapiens and H. neanderthalensis and taxonomic diversity in later Homo.

Artists on the edge of the world: An integrated approach to the study of Magdalenian engraved stone plaquettes from Jersey (Channel Islands).

The Upper Palaeolithic is characterised by the appearance of iconographic expressions most often depicting animals, including anthropomorphic forms, and geometric signs. The Late Upper Palaeolithic Magdalenian saw a flourishing of such depictions, encompassing cave art, engraving of stone, bone and antler blanks and decoration of tools and weapons. Though Magdalenian settlement exists as far northwest as Britain, there is a limited range of art known from this region, possibly associated with only fleeting occupation of Britain during this period. Stone plaquettes, flat fragments of stone engraved on at least one surface, have been found in large quantities at numerous sites spanning the temporal and geographical spread of the Magdalenian, but they have been absent so far from the archaeological record of the British Isles. Between 2015 and 2018, ten fragments of stone plaquettes extensively engraved with abstract designs were uncovered at the Magdalenian site of Les Varines, Jersey, Channel Islands. In this paper, we report detailed analyses of these finds, which provide new evidence for technologies of abstract mark-making, and their significance within the lives of people on the edge of the Magdalenian world. These engraved stone fragments represent important, rare evidence of artistic expression in what is the far northern and western range of the Magdalenian and add new insight to the wider significance of dynamic practices of artistic expression during the Upper Palaeolithic.

Taxonomic variation in the supraorbital region of catarrhine primates.

OBJECTIVES: This study aimed to test the taxonomic utility of the catarrhine supraorbital region using 3D geometric morphometrics, with the aim of establishing its potential use in elucidating the position of more debated hominin groups. MATERIALS AND METHODS: 230 3D coordinates were used to record the supraorbital morphology of two datasets: one containing 460 non-hominin catarrhine primates from species and subspecies of Gorilla, Pan, Papio, and Macaca; and the other containing 55 Pleistocene hominins from Homo, Australopithecus, and Paranthropus. Principal component analyses in tangent, form, and allometry-free shape space were used to assess differentiation of taxa, with biological distinctiveness of taxa being established using step-wise discriminant analysis with subsampling. RESULTS: Results indicated that the recorded supraorbital morphology could be used to separate non-hominin catarrhine primate genera, species, and subspecies, although accuracy was found to decrease with decreasing Linnaean rank. In addition, analyses in tangent space were found to produce the highest accuracy when classifying primates of known taxonomy. Biological distinctiveness of the middle and later Homo species was comparable to or higher than that of the non-hominin primates, and relatively lower for the earlier groups of Homo. DISCUSSION: This study indicates that the supraorbital region preserves taxonomic information that can be used to delineate between closely related groups, both within hominins and wider catarrhine primates. Therefore, this region may be used to provide insight when assessing the taxonomic affiliation of disputed hominin specimens.

External ballistics of Pleistocene hand-thrown spears: experimental performance data and implications for human evolution.

The appearance of weaponry - technology designed to kill - is a critical but poorly established threshold in human evolution. It is an important behavioural marker representing evolutionary changes in ecology, cognition, language and social behaviours. While the earliest weapons are often considered to be hand-held and consequently short-ranged, the subsequent appearance of distance weapons is a crucial development. Projectiles are seen as an improvement over contact weapons, and are considered by some to have originated only with our own species in the Middle Stone Age and Upper Palaeolithic. Despite the importance of distance weapons in the emergence of full behavioral modernity, systematic experimentation using trained throwers to evaluate the ballistics of thrown spears during flight and at impact is lacking. This paper addresses this by presenting results from a trial of trained javelin athletes, providing new estimates for key performance parameters. Overlaps in distances and impact energies between hand-thrown spears and spearthrowers are evidenced, and skill emerges as a significant factor in successful use. The results show that distance hunting was likely within the repertoire of hunting strategies of Neanderthals, and the resulting behavioural flexibility closely mirrors that of our own species.

Development of an automated, interference-free, 2D-LC-MS/MS assay for quantification of a therapeutic mAb in human sera.

AIM: Hybrid LC-MS/MS assays are increasingly used to quantitate proteins in biological matrices. These assays involve analyte enrichment at the protein level. Although suitability has been demonstrated, they are limited by the lack of appropriate affinity reagents and may suffer from interferences caused by binding proteins or antibodies. RESULTS: An online stable isotope standards and capture by anti-peptide antibodies assay was developed, which involves tryptic digestion of a therapeutic monoclonal antibody in human serum to destroy interfering proteins followed by enrichment using high affinity peptide antibodies. The assay was validated and compared with a standard ligand-binding assay currently used for quantification. CONCLUSION: The data show that the stable isotope standards and capture by anti-peptide antibodies-2D-LC-MS/MS assay can be used as an alternative method for measurement of monoclonal antibodies in clinical samples.

Virotherapy using a novel chimeric oncolytic adenovirus prolongs survival in a human pancreatic cancer xenograft model.

INTRODUCTION: Pancreatic adenocarcinoma is an aggressive malignancy. Oncolytic adenoviruses (Ads) are modified genetically to target tumor cells while sparing normal cells. We modified the knob domain of the Ad serotype 5 with a serotype 3 knob domain and incorporated the CXCR4 promoter to regulate Ad E1A gene expression (Ad5/3-CXCR4-E1A). These modifications were made to efficiently infect and lyse pancreatic tumors. METHODS: Human pancreatic cancer lines CFPAC-1, PANC-1, AsPC-1, and BxPC-3 were obtained from the American Type Culture Collection. Efficiency of Ad infection in the cells was determined by the use of an Ad construct expressing the green fluorescence protein (GFP) marker in place of the E1A gene (Ad5/3-CXCR4-GFP) and quantified by flow cytometry. Oncolytic activity in the pancreatic cancer cells was determined with the Ad5/3-CXCR4-E1A oncolytic Ad by a crystal violet staining method. To determine the oncolytic effect in vivo, pancreatic cancer cells were implanted on the flanks of 40 SCID mice (4 groups). Tumors were injected intratumorally for 3 days with Ad5/3-CXCR4-E1A, Ad5 wild-type (a positive control), or phosphate-buffered saline (a no virus control). Tumor size, overall survival, and body condition scale score were recorded. Statistical analyses included the Kaplan-Meier survival curve, the log-rank test, and one-way analysis of variance. RESULTS: The serotype 3 fiber-modified Ad with the CXCR4 promoter (Ad5/3-CXCR4-E1A) was most efficient in infecting and lysing pancreatic cancer cells compared with an Ad containing an unmodified fiber knob (Ad5-CXCR4-E1A). Treatment of pancreatic tumor xenografts in vivo with Ad5/3-CXCR4-E1A group resulted in smaller tumors (P = .001), greater body condition scale score (P = .01), and greater survival time (P = .04) than the other treatment groups. CONCLUSION: Ad5/3-CXCR4-E1A treatment significantly prolonged survival in SCID mice pancreatic tumor xenografts. This novel construct represents a potential new therapy against pancreatic cancer.