The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care

De novo design of therapeutic peptides and their characterization

Doctor of PhilosophyDepartment of Anatomy and PhysiologyMajor Professor Not ListedPeptides are low-cost, flexible, and biocompatible and can be designed to serve various functions in biotechnology and medicine. Peptides can be designed such that they fold spontaneously and adopt a specific conformation under specific conditions, including when in contact with the three-dimensional structure of a protein or the two-dimensional structure of graphene. They are promising for design of functional materials for biotechnology and medical applications. I have studied peptide design for biotechnology, including peptide self-assembly on a graphene surface, and for medical applications such as cancer immunotherapy and treatment of coronavirus disease 2019 caused by SARS-CoV-2. In chapter 1, I describe my study of the self-assembly of a designed cyclic peptide on graphitic surfaces by molecular dynamics simulations. In experiments, it was found that hydrocarbon contaminants may interfere with this self-assembly, so we undertook a computational study of the behavior of these contaminants at the graphene–water interface and compared it to experimental data, as detailed in chapter 2. Peptides are also promising in medicine, particularly for inhibiting protein-protein interactions in situations where conventional small-molecule drugs can be unsuitable. Many viruses important for public health including SARS-CoV-2 and influenza enter cells by means of binding between viral proteins and cell surface proteins. The blockade of these undesirable protein-protein interactions has definite clinical significance. Another medical application where blocking protein-protein interactions is essential is the immune checkpoint blockade used in cancer immunotherapy. Immune checkpoint proteins most studied for cancer immunotherapy have flat and relatively hydrophobic interfaces that have impeded small-molecule drug development. Therefore, the application of peptide molecules that mimic the interacting surface of a natural binding protein is a promising alternative to small- molecule drugs. Immunotherapy activates the patient’s own immune system to treat cancer. When any foreign substance enters in the body, immune cells recognize it as a threat and neutralize it. But unfortunately, cancer cells often evolve to evade the immune system. Cytotoxic T-Lymphocyte Associated protein 4 (CTLA4) plays a crucial role in self-recognition and is an immune checkpoint protein that cancer cells may express to prevent attack from the immune system. Cancer cells frequently overexpress proteins of the B7 family, which allows them to evade the immune response by binding between these B7 proteins and CTLA4 on the surface of T cells. As presented in chapter 3, I have designed a 17-residue cyclic peptide targeting the CTLA4 protein that binds to it with a significant affinity. The binding activity was experimentally confirmed by the bio-layer interferometry (BLI) method. Studies performed by our collaborators showed an increase in CD8+ T cell-induced death of Lewis Lung Carcinoma (LLC) cells due to treatment with this peptide in vitro. In vivo, the designed peptide attenuated tumor growth in mouse models using orthotopic LLC cell allografts. A disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), called as COVID-19, has threatened global public health and the global economy. The WHO has reported 434 confirmed million cases and 6 million deaths. Although effective vaccines have been developed against SARS-CoV-2, many regions in the world still have a low rate of vaccination and even vaccinated individuals may experience reinfection. Deaths continue to be reported worldwide, exacerbated by continued mutation of the viral spike protein. SARS-CoV-2 enters the host cell through association of this spike protein, present on the envelope of the virus, and Angiotensin Converting Enzyme (ACE2), a protein expressed on the surface of host cells. As detailed in chapter 4, I have designed a 17-residue long peptide targeting the receptor-binding domain (RBD) of the spike protein to prevent COVID-19 infection. My designed peptide binds to the spike protein RBD with nanomolar affinity and blocks the binding site of ACE2. I have confirmed the binding activity using a microcantilever-based method and determined the dissociation constant using a BLI system. SARS-CoV-2 continues to mutate and produce variants. I have tested the binding activity of the designed peptide for the Delta variant, considered highly transmissible and declared as a variant of the concern (VOC) by the WHO. The BLI experiment revealed weaker binding of the designed peptide for the Delta variant spike protein compared to that for the original wild-type due to the mutations present in the receptor-binding domain of the spike protein

In Silico Electrophysiological Investigation of Transient Receptor Potential Melastatin-4 Ion Channel Biophysics to Study Detrusor Overactivity

Enhanced electrical activity in detrusor smooth muscle (DSM) cells is a key factor in detrusor overactivity which causes overactive bladder pathological disorders. Transient receptor potential melastatin-4 (TRPM4) channels, which are calcium-activated cation channels, play a role in regulating DSM electrical activities. These channels likely contribute to depolarizing the DSM cell membrane, leading to bladder overactivity. Our research focuses on understanding TRPM4 channel function in the DSM cells of mice, using computational modeling. We aimed to create a detailed computational model of the TRPM4 channel based on existing electrophysiological data. We employed a modified Hodgkin-Huxley model with an incorporated TRP-like current to simulate action potential firing in response to current and synaptic stimulus inputs. Validation against experimental data showed close agreement with our simulations. Our model is the first to analyze the TRPM4 channel’s role in DSM electrical activity, potentially revealing insights into bladder overactivity. In conclusion, TRPM4 channels are pivotal in regulating human DSM function, and TRPM4 channel inhibitors could be promising targets for treating overactive bladder

Atomically resolved interfacial water structures on crystalline hydrophilic and hydrophobic surfaces

[EN] Hydration layers are formed on hydrophilic crystalline surfaces immersed in water. Their existence has also been predicted for hydrophobic surfaces, yet the experimental evidence is controversial. Using 3D-AFM imaging, we probed the interfacial water structure of hydrophobic and hydrophilic surfaces with atomic-scale spatial resolution. We demonstrate that the atomic-scale structure of interfacial water on crystalline surfaces presents two antagonistic arrangements. On mica, a common hydrophilic crystalline surface, the interface is characterized by the formation of 2 to 3 hydration layers separated by approximately 0.3 nm. On hydrophobic surfaces such as graphite or hexagonal boron nitride (h-BN), the interface is characterized by the formation of 2 to 4 layers separated by about 0.5 nm. The latter interlayer distance indicates that water molecules are expelled from the vicinity of the surface and replaced by hydrocarbon molecules. This creates a new 1.5-2 nm thick interface between the hydrophobic surface and the bulk water. Molecular dynamics simulations reproduced the experimental data and confirmed the above interfacial water structures. This journal isEuropean Research Council ERC-AdG-340177, the Ministerio de Ciencia, Innovación y Universidades (PID2019- 106801GB-I00; MAT2016-76507-R) and European Commission Marie Sklodowska-Curie grant agreement No. 721874. J.C. acknowledges financial support by the US National Science Foundation under Grant No. CHE-1726332 and DMR-1945589.Peer reviewe

Meningeal osteochondroma simulating meningioma with metaplastic change: a rare golf-ball-like lesion of non-meningothelial mesenchymal origin

Non-meningothelial mesenchymal tumors of the central nervous system (CNS), including those originating from the meninges, histologically correspond to tumors of soft tissue or bone. These individual entities arising from the meninges are rare, and probably have their origin in the multipotent primitive mesenchymal stem cells of the dura. Though it is a common bone tumor, the meningeal origin of osteochondroma has only very rarely been reported. We describe a case of a 35-year-old female with a well-demarcated, golf-ball-like osteochondroma of meningeal origin which was enucleated en bloc on craniotomy. Such a lesion can resemble a meningioma that exhibits metaplastic (osseous) change on imaging. However, provided that there is clinico-radiological awareness of such tumors, magnetic resonance imaging (MRI) can guide the way to this rare differential diagnosis, as it reflects the pathologic appearance of osteochondroma and allows the thickness of the cartilage cap to be estimated in order to check for rare malignant change. Complete excision along with the cartilage cap usually offers a favorable prognosis without recurrence

Coexistence of central nucleus, cores, and rods: Diagnostic relevance

Background: Congenital myopathies (CMs) though considered distinct disorders, simultaneous occurrence of central nucleus, nemaline rods, and cores in the same biopsy are scarcely reported. Objective: A retrospective reassessment of cases diagnosed as CMs to look for multiple pathologies missed, if any, during the initial diagnosis. Materials and Methods: Enzyme histochemical, and immunohistochemical-stained slides from 125 cases diagnosed as congenital myopathy were reassessed. Results: The study revealed 15 cases (12%) of congenital myopathy with more than one morphological feature. Central nucleus with cores (n = 11), central nucleus, nemaline rods and cores (n = 3), and nemaline rods with cores (n = 1). 4/11 cases were diagnosed as centronuclear myopathy (CNM) in the first instance; in addition, cores were revealed on reassessment. Discussion: The prevalence of CMs of all neuromuscular disorders is approximately 6 in 100,000 live births, with regional variations. Three main defined CMs include centro nuclear myopathy (CNM), nemaline rod myopathy (NRM), and central core disease (CCD). However, they are more diverse with overlapping clinical and histopathological features, thus broadening the spectra within each category of congenital myopathy. Conclusion: Identification of cases with overlap of pathological features has diagnostic relevance

Thermodynamics of Adsorption on Graphenic Surfaces from Aqueous Solution

Adsorption of organic molecules from aqueous solution to the surface of carbon nanotubes or graphene is an important process in many applications of these materials. Here we use molecular dynamics simulation, supplemented by analytical chemistry, to explore in detail the adsorption thermodynamics of a diverse set of aromatic compounds on graphenic materials, elucidating the effects of the solvent, surface coverage, surface curvature, defects, and functionalization by hydroxy groups. We decompose the adsorption free energies into entropic and enthalpic components and find that different classes of compoundssuch as phenols, benzoates, and alkylbenzenescan easily be distinguished by the relative contributions of entropy and enthalpy to their adsorption free energies. Overall, entropy dominates for the more hydrophobic compounds, while enthalpy plays the greatest role for more hydrophilic compounds. Experiments and independent simulations using two different force field frameworks (CHARMM and Amber) support the robustness of these conclusions. We determine that concave curvature is generally associated with greater adsorption affinity, more favorable enthalpy, and greater contact area, while convex curvature reduces both adsorption enthalpy and contact area. Defects on the graphene surfaces can create concave curvature, resulting in localized binding sites. As the graphene surface becomes covered with aromatic solutes, the affinity for adsorbing an additional solute increases until a complete monolayer is formed, driven by more favorable enthalpy and partially canceled by less favorable entropy. Similarly, hydroxylation of the surface leads to preferential adsorption of the aromatic solutes to remaining regions of bare graphene, resulting in less favorable adsorption entropy, but compensated by an increase in favorable enthalpic interactions

Feline sarcomatoid renal cell carcinoma with peritoneal carcinomatosis and effusion

A 9-y-old, castrated male, domestic medium-hair cat diagnosed previously with chronic kidney disease developed anorexia and vomiting. Ultrasonography revealed abdominal effusion and a left renal perihilar mass. Cytologic evaluation of the peritoneal fluid and mass identified atypical epithelioid cells suspected to be of renal epithelial or possible mesothelial origin. Immunohistochemical (IHC) evaluation of a formalin-fixed, paraffin-embedded peritoneal fluid cell block indicated both pancytokeratin and vimentin expression in the atypical epithelioid cell population. With scanning electron microscopic evaluation, similar epithelioid cells lacked the cell-surface microvilli expected of mesothelium, supporting an antemortem diagnosis of probable carcinoma. On postmortem examination, the left kidney was effaced by an infiltrative neoplasm with myriad similar nodules throughout the peritoneum. The neoplasm was composed primarily of polygonal-to-spindle-shaped cells with strong vimentin and weak pancytokeratin cytoplasmic immunolabeling. Further IHC characterization with PAX8, CK18, KIT, napsin A, SMA, desmin, CD18, and claudin 5 was performed. Histologic and IHC findings supported a diagnosis of sarcomatoid renal cell carcinoma with peritoneal carcinomatosis. An in vitro cell culture line of neoplastic cells harvested from the primary tumor was successfully established for future research endeavors

A Water Extract from Chlorella sorokiniana Cell Walls Stimulates Growth of Bone Marrow Cells and Splenocytes

A water extract derived from the isolated cell walls of Chlorella sorokiniana (C. sorokiniana, Chlorella water extract, CWE) was analyzed for the presence of lipopolysaccharide (LPS)-related material via the Limulus amebocyte lysate (LAL) assay and evaluated for its growth stimulation effect on the bone marrow cells and splenocytes in vitro cell cultures. The extract contained low levels of LPS-related material, and a mass spectrum suggested that the extract contained many components, including a low level of a lipid A precursor, a compound known as lipid X, which is known to elicit a positive response in the LAL assay. Treatment with the CWE dose- and time-dependently stimulated the growth of mouse bone marrow cells (BMCs) and splenocytes (SPLs). Treatment with the CWE also increased specific BMC subpopulations, including antigen-presenting cells (CD19+ B cells, 33D1+ dendritic cells and CD68+ macrophages), and CD4+ and CD8+ T cells, but decreased the number of LY6G+ granulocytes. Treatment with the CWE also increased cytokine mRNA associated with T cell activation, including TNFα, IFNγ, and granzyme B in human lymphoblasts. The present study indicates that the cell wall fraction of C.sorokiniana contains an LPS-like material and suggests a candidate source for the bioactivity that stimulates growth of both innate and adaptive immune cells