Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries

The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm2. We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate) and subsequent bioinformatic analysis (~60 seconds per plate) thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population) can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins

Localisation in single Landau bands

We consider a single-band approximation to the random Schro¨dinger operator in an external magnetic field. The random potential is taken to be constant on unit squares and i.i.d. on each square with a bounded distribution. We prove that the eigenstates corresponding to energies at the edges of the Landau band are localized. This is an important ingredient in the theory of the Quantum Hall Effect

The nature of the spectrum for a Landau hamiltonian with delta impurities

We consider a single-band approximation to the random Schr6dinger operator in an external magnetic field. The random potential consists of delta functions of random strengths situated on the sites of a regular two-dimensional lattice. We characterize the entire spectrum of this Hamiltonian when the magnetic field is sufficiently high. We show that the whole spectrum is pure point, the energy coinciding with the first Landau level in the absence of a random potential being infinitely degenerate, while the eigenfunctions corresponding to energies in the rest of the spectrum are localized

Long Cycles in a Perturbed Mean Field Model of a Boson Gas

In this paper we give a precise mathematical formulation of the relation between Bose condensation and long cycles and prove its validity for the perturbed mean field model of a Bose gas. We decompose the total density $\rho=\rho_{{\rm short}}+\rho_{{\rm long}}$ into the number density of particles belonging to cycles of finite length ($\rho_{{\rm short}}$) and to infinitely long cycles ($\rho_{{\rm long}}$) in the thermodynamic limit. For this model we prove that when there is Bose condensation, $\rho_{{\rm long}}$ is different from zero and identical to the condensate density. This is achieved through an application of the theory of large deviations. We discuss the possible equivalence of $\rho_{{\rm long}}\neq 0$ with off-diagonal long range order and winding paths that occur in the path integral representation of the Bose gas.Comment: 10 page

Stillbirth rate by maternal HIV serostatus and antiretroviral use in pregnancy in South Africa: An audit

No abstract

Clonal expansion of T memory stem cells determines early anti-leukemic responses and long-term CAR T cell persistence in patients

Low-affinity CD19 chimeric antigen receptor (CAR) T cells display enhanced expansion and persistence, enabling fate tracking through integration site analysis. Here we show that integration sites from early (1 month) and late (>3 yr) timepoints cluster separately, suggesting different clonal contribution to early responses and prolonged anti-leukemic surveillance. CAR T central and effector memory cells in patients with long-term persistence remained highly polyclonal, whereas diversity dropped rapidly in patients with limited CAR T persistence. Analysis of shared integrants between the CAR T cell product and post-infusion demonstrated that, despite their low frequency, T memory stem cell clones in the product contributed substantially to the circulating CAR T cell pools, during both early expansion and long-term persistence. Our data may help identify patients at risk of early loss of CAR T cells and highlight the critical role of T memory stem cells both in mediating early anti-leukemic responses and in long-term surveillance by CAR T cells

A primer set for the rapid isolation of scFv fragments against cell surface antigens from immunised rats

Antibody phage display is a powerful platform for discovery of clinically applicable high affinity monoclonal antibodies against a broad range of targets. Libraries generated from immunized animals offer the advantage of in vivo affinity-maturation of V regions prior to library generation. Despite advantages, few studies have described isolation of antibodies from rats using immune phage display. In our study, we describe a novel primer set, covering the full rat heavy chain variable and kappa light chain variable regions repertoire for the generation of an unbiased immune libraries. Since the immune repertoire of rats is poorly understood, we first performed a deep sequencing analysis of the V(D)J regions of VH and VLK genes, demonstrating the high abundance of IGVH2 and IGVH5 families for VH and IGVLK12 and IGVLK22 for VLK. The comparison of gene’s family usage in naïve rats have been used to validate the frequency’s distribution of the primer set, confirming the absence of PCR-based biases. The primers were used to generate and assemble a phage display library from human CD160-vaccinated rats. CD160 represents a valid therapeutic target as it has been shown to be expressed on chronic lymphocytic leukaemia cells and on the surface of newly formed vessels. We utilised a novel phage display panning strategy to isolate a high affinity pool (KD range: 0.399–233 nM) of CD160 targeting monoclonal antibodies. Subsequently, identified binders were tested for function as third generation Chimeric Antigen Receptors (CAR) T cells demonstrating specific cytolytic activity. Our novel primer set coupled with a streamlined strategy for phage display panning enable the rapid isolation and identification of high affinity antibodies from immunised rats. The therapeutic utility of these antibodies was demonstrated in CAR format

Stillbirth rate by maternal HIV serostatus and antiretroviral use in pregnancy in South Africa : an audit

The global perinatal mortality burden is high, with over 2.6 million stillbirths annually. The plurality (41%) of stillbirths occur in sub-Saharan Africa, which also has the highest HIV burden (20% prevalence) in the world. The extent to which these two phenomena are related has not been fully characterised.http://www.samj.org.zadm2022Obstetrics and Gynaecolog

A Rapamycin-Activated Caspase 9-Based Suicide Gene

Engineered T cell therapies show considerable promise in the treatment of refractory malignancies. Given the ability of engineered T cells to engraft and persist for prolonged periods along with unpredicted toxicities, incorporation of a suicide gene to allow selective depletion after administration is desirable. Rapamycin is a safe and widely available immunosuppressive pharmaceutical that acts by heterodimerization of FKBP12 with the FRB fragment of mTOR. The apical caspase caspase 9 is activated by homodimerization through its CARD domain. We developed a rapamycin-induced caspase 9 suicide gene. First, we showed that caspase 9 could be activated by a two-protein format with replacement of the CARD domain with both FRB and FKBP12. We next identified an optimal compact single-protein rapamycin caspase 9 (rapaCasp9) by fusing both FRB and FKBP12 with the catalytic domain of caspase 9. Functionality of rapaCasp9 when co-expressed with a CD19 CAR was demonstrated in vitro and in vivo

Tunable control of CAR T cell activity through tetracycline mediated disruption of protein-protein interaction

Chimeric antigen receptor (CAR) T cells are a promising form of cancer immunotherapy, although they are often associated with severe toxicities. Here, we present a split-CAR design incorporating separate antigen recognition and intracellular signaling domains. These exploit the binding between the tetracycline repressor protein and a small peptide sequence (TIP) to spontaneously assemble as a functional CAR. Addition of the FDA-approved, small molecule antibiotic minocycline, acts as an "off-switch" by displacing the signaling domain and down-tuning CAR T activity. Here we describe the optimization of this split-CAR approach to generate a CAR in which cytotoxicity, cytokine secretion and proliferation can be inhibited in a dose-dependent and reversible manner. Inhibition is effective during on-going CAR T cell activation and inhibits activation and tumor control in vivo. This work shows how optimization of split-CAR structure affects function and adds a novel design allowing easy CAR inhibition through an FDA-approved small molecule