First-in-human real-time AI-assisted instrument deocclusion during augmented reality robotic surgery.

The integration of Augmented Reality (AR) into daily surgical practice is withheld by the correct registration of pre-operative data. This includes intelligent 3D model superposition whilst simultaneously handling real and virtual occlusions caused by the AR overlay. Occlusions can negatively impact surgical safety and as such deteriorate rather than improve surgical care. Robotic surgery is particularly suited to tackle these integration challenges in a stepwise approach as the robotic console allows for different inputs to be displayed in parallel to the surgeon. Nevertheless, real-time de-occlusion requires extensive computational resources which further complicates clinical integration. This work tackles the problem of instrument occlusion and presents, to the authors' best knowledge, the first-in-human on edge deployment of a real-time binary segmentation pipeline during three robot-assisted surgeries: partial nephrectomy, migrated endovascular stent removal, and liver metastasectomy. To this end, a state-of-the-art real-time segmentation and 3D model pipeline was implemented and presented to the surgeon during live surgery. The pipeline allows real-time binary segmentation of 37 non-organic surgical items, which are never occluded during AR. The application features real-time manual 3D model manipulation for correct soft tissue alignment. The proposed pipeline can contribute towards surgical safety, ergonomics, and acceptance of AR in minimally invasive surgery.

A rapid Focus-Forming Assay for quantification of infectious adenoviral vectors.

Currently available methods to titrate adenoviral vectors (AdV) in the absence of a gene reporter such as GFP, are either time-consuming or not very reproducible. A Focus-Forming Assay (FFA) for quantification of infectious AdV particles followed by automated focus counting was developed using new monoclonal antibodies (mAbs) against the human adenovirus type 5. Briefly, in this method, 96-well plates of HEK293A cells were infected with 2-fold dilutions of AdV at seeding time. Forty eight hours post-infection, the cells were fixed with methanol. The cells were then incubated with each mAb followed by a FITC conjugated anti-mouse antibody. The plates were scanned and positive cells counted using an automated fluorescence microscopy system. The results of the FFA were compared with the plaque assay and the TCID50 assay. The titer of six different recombinant AdV were compared using the FFA along with a commercial kit. The results were similar, but in contrast to the commercial kit for which the stained cells are counted manually, the software automatically counts the positives cells in the FFA. The automatic counting of positive cells makes the FFA a more precise and reliable assay compared to the commercial kit for titration of AdV.

A High-Throughput Method for Characterizing Novel Chimeric Antigen Receptors in Jurkat Cells.

Chimeric antigen receptor (CAR) development involves extensive empirical characterization of antigen-binding domain (ABD)/CAR constructs for clinical suitability. Here, we present a cost-efficient and rapid method for evaluating CARs in human Jurkat T cells. Using a modular CAR plasmid, a highly efficient ABD cloning strategy, plasmid electroporation, short-term co-culture, and flow-cytometric detection of CD69, this assay (referred to as CAR-J) evaluates sensitivity and specificity for ABDs. Assessing 16 novel anti-CD22 single-chain variable fragments derived from mouse monoclonal antibodies, CAR-J stratified constructs by response magnitude to CD22-expressing target cells. We also characterized 5 novel anti-EGFRvIII CARs for preclinical development, identifying candidates with varying tonic and target-specific activation characteristics. When evaluated in primary human T cells, tonic/auto-activating (without target cells) EGFRvIII-CARs induced target-independent proliferation, differentiation toward an effector phenotype, elevated activity against EGFRvIII-negative cells, and progressive loss of target-specific response upon in vitro re-challenge. These EGFRvIII CAR-T cells also showed anti-tumor activity in xenografted mice. In summary, CAR-J represents a straightforward method for high-throughput assessment of CAR constructs as genuine cell-associated antigen receptors that is particularly useful for generating large specificity datasets as well as potential downstream CAR optimization.

Phosphatidylinositol 4-kinase III beta is essential for replication of human rhinovirus and its inhibition causes a lethal phenotype in vivo.

Human rhinovirus (HRV) is the predominant cause of the common cold, but more importantly, infection may have serious repercussions in asthmatics and chronic obstructive pulmonary disorder (COPD) patients. A cell-based antiviral screen against HRV was performed with a subset of our proprietary compound collection, and an aminothiazole series with pan-HRV species and enteroviral activity was identified. The series was found to act at the level of replication in the HRV infectious cycle. In vitro selection and sequencing of aminothiazole series-resistant HRV variants revealed a single-nucleotide mutation leading to the amino acid change I42V in the essential HRV 3A protein. This same mutation has been previously implicated in resistance to enviroxime, a former clinical-stage antipicornavirus agent. Enviroxime-like compounds have recently been shown to target the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIß). A good correlation between PI4KIIIß activity and HRV antiviral potency was found when analyzing the data over 80 compounds of the aminothiazole series, covering a 750-fold potency range. The mechanism of action through PI4KIIIß inhibition was further demonstrated by small interfering RNA (siRNA) knockdown of PI4KB, which reduced HRV replication and also increased the potency of the PI4KIIIß inhibitors. Inhibitors from two different structural classes with promising pharmacokinetic profiles and with very good selectivity for PI4KIIIß were used to dissociate compound-related toxicity from target-related toxicity. Mortality was seen in all dosing groups of mice treated with either compound, therefore suggesting that short-term inhibition of PI4KIIIß is deleterious.

Identification of a lipid kinase as a host factor involved in hepatitis C virus RNA replication.

A functional screen of an adenovirus-delivered shRNA library that targets approximately 4500 host genes was performed to identify cellular factors that regulate hepatitis C virus (HCV) sub-genomic RNA replication. Seventy-three hits were further examined by siRNA oligonucleotide-directed knockdown, and silencing of the PI4KA gene was demonstrated to have a significant effect on the replication of a HCV genotype 1b replicon. Using transient siRNA oligonucleotide transfections and stable shRNA knockdown clones in HuH-7 cells, the PI4KA gene was shown to be essential for the replication of all HCV genotypes tested (1a, 1b and 2a) but not required for bovine viral diarrhea virus (BVDV) RNA replication.

The R1 subunit of herpes simplex virus ribonucleotide reductase protects cells against apoptosis at, or upstream of, caspase-8 activation.

The R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, which in addition to its C-terminal reductase domain possesses a unique N-terminal domain of about 400 amino acids, is thought to have an additional, as yet unknown, function. Here, we report that the full-length HSV-2 R1 has an anti-apoptotic function able to protect cells against death triggered by expression of R1(Delta2-357), an HSV-2 R1 subunit with its first 357 amino acids deleted. We further substantiate the R1 anti-apoptotic activity by showing that its accumulation at low level could completely block apoptosis induced by TNF-receptor family triggering. Activation of caspase-8 induced either by TNF or by Fas ligand expression was prevented by the R1 protein. As HSV R1 did not inhibit cell death mediated by several agents acting via the mitochondrial pathway (Bax overexpression, etoposide, staurosporine and menadione), it is proposed that it functions to interrupt specifically death receptor-mediated signalling at, or upstream of, caspase-8 activation. The N-terminal domain on its own did not exhibit anti-apoptotic activity, suggesting that both domains of R1 or part(s) of them are necessary for this new function. Evidence for the importance of HSV R1 in protecting HSV-infected cells against cytokine-induced apoptosis was obtained with the HSV-1 R1 deletion mutants ICP6Delta and hrR3. These results show that, in addition to its ribonucleotide reductase function, which is essential for virus reactivation, HSV R1 could contribute to virus propagation by preventing apoptosis induced by the immune system.