Single- and duplex TaqMan-quantitative PCR for determining the copy numbers of integrated selection markers during site-specific mutagenesis in Toxoplasma gondii by CRISPR-Cas9.

Herein, we developed a single and a duplex TaqMan quantitative PCR (qPCR) for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker for pyrimethamine resistance in Toxoplasma gondii knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted mdhfr-ts and of the wild-type (WT) dhfr-ts (wtdhfr-ts) which is present as single copy gene in Toxoplasma and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the dhfr-ts fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of dhfr-ts copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp dhfr-ts fragment and the T. gondii 529-bp repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by dhfr-ts amplification is equal to the number of tachyzoites determined by amplification of the Toxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of mdhfr-ts, the calculated ratio is 2. We then applied both approaches to test T. gondii RH mutants in which the major surface antigen (SAG1) was disrupted through insertion of mdhfr-ts using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated mdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of T. gondii KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of mdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in Toxoplasma. TaqMan qPCR tools may contribute to more frequent use of WT Toxoplasma strains during functional genomics

Inhomogeneous Superconductivity in Condensed Matter and QCD

Inhomogeneous superconductivity arises when the species participating in the pairing phenomenon have different Fermi surfaces with a large enough separation. In these conditions it could be more favorable for each of the pairing fermions to stay close to its Fermi surface and, differently from the usual BCS state, for the Cooper pair to have a non zero total momentum. For this reason in this state the gap varies in space, the ground state is inhomogeneous and a crystalline structure might be formed. This situation was considered for the first time by Fulde, Ferrell, Larkin and Ovchinnikov, and the corresponding state is called LOFF. The spontaneous breaking of the space symmetries in the vacuum state is a characteristic feature of this phase and is associated to the presence of long wave-length excitations of zero mass. The situation described here is of interest both in solid state and in elementary particle physics, in particular in Quantum Chromo-Dynamics at high density and small temperature. In this review we present the theoretical approach to the LOFF state and its phenomenological applications using the language of the effective field theories.Comment: RevTex, 83 pages, 26 figures. Submitted to Review of Modern Physic

A haemagglutination test for rapid detection of antibodies to SARS-CoV-2

Serological detection of antibodies to SARS-CoV-2 is essential for establishing rates of seroconversion in populations, and for seeking evidence for a level of antibody that may be protective against COVID-19 disease. Several high-performance commercial tests have been described, but these require centralised laboratory facilities that are comparatively expensive, and therefore not available universally. Red cell agglutination tests do not require special equipment, are read by eye, have short development times, low cost and can be applied at the Point of Care. Here we describe a quantitative Haemagglutination test (HAT) for the detection of antibodies to the receptor binding domain of the SARS-CoV-2 spike protein. The HAT has a sensitivity of 90% and specificity of 99% for detection of antibodies after a PCR diagnosed infection. We will supply aliquots of the test reagent sufficient for ten thousand test wells free of charge to qualified research groups anywhere in the world

Two doses of SARS-CoV-2 vaccination induce robust immune responses to emerging SARS-CoV-2 variants of concern

The extent to which immune responses to natural infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and immunization with vaccines protect against variants of concern (VOC) is of increasing importance. Accordingly, here we analyse antibodies and T cells of a recently vaccinated, UK cohort, alongside those recovering from natural infection in early 2020. We show that neutralization of the VOC compared to a reference isolate of the original circulating lineage, B, is reduced: more profoundly against B.1.351 than for B.1.1.7, and in responses to infection or a single dose of vaccine than to a second dose of vaccine. Importantly, high magnitude T cell responses are generated after two vaccine doses, with the majority of the T cell response directed against epitopes that are conserved between the prototype isolate B and the VOC. Vaccination is required to generate high potency immune responses to protect against these and other emergent variants

Holocene Sedimentation on the Vietnamese Shelf: From Source to Sink

25 sediment cores from modern water depths between 21 m and 169 m and two river samples from the Mekong and Red River have been investigated in order to characterize sedimentary processes, stratigraphy, provenance, distribution and sediment budget during the Late Pleistocene and Holocene on the Vietnamese Shelf. A stratigraphy, based on 45 AMS 14C dates was developed. The sediments could be assigned to 3 areas which are characterzied by different sedimentological conditions resulting in varying sedimentation rates (Northern Shelf 10 cm/kyr, Central Shelf 50 - 100 cm/kyr, Southern Shelf 5 - 10 cm/kyr). Investigations of major- and trace element as well as Sr- and Nd-isotopic composition of shelf and river sediments showed, that the sediments on the Vietnamese Shelf are mainly delivered by the small mountainous rivers and the Mekong. Together with denudation data taken from the literature a conceptual sediment buget for the Vietnamese Shelf was calculated. It shows that the small mountainous rivers of central Vietnam deliver the equivalent of ca. 25 - 60 % of the amount of sediment transported by the Mekong