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Abstract Ovarian high-grade serous carcinoma (HGSC) is a highly lethal malignancy for which early detection is a challenge and treatment of late-stage disease is ineffective. HGSC initiation involves exfoliation of fallopian tube epithelial (FTE) cells which form multicellular clusters called spheroids that colonize and invade the ovary. HGSC contains universal mutation of the tumour suppressor gene TP53. However, not all TP53 mutations are the same, as specific p53 missense mutants contain gain-of-function (GOF) properties that drive tumour formation. Additionally, the role of GOF p53 in spheroid-mediated spread is poorly understood. In this study, we developed and characterized an in vitro model of HGSC based on mutation of TP53 in mouse oviductal epithelial cells (OVE). We discovered increased bulk spheroid survival and increased anchorage-independent growth in OVE cells expressing the missense mutant p53R175H compared to OVE parental and Trp53ko cells. Transcriptomic analysis on spheroids identified decreased apoptosis signaling due to p53R175H. Further assessment of the apoptosis pathway demonstrated decreased expression of intrinsic and extrinsic apoptosis signaling molecules due to Trp53 deletion and p53R175H, but Caspase-3 activation was only decreased in spheroids with p53R175H. These results highlight this model as a useful tool for discovering early HGSC transformation mechanisms and uncover a potential anti-apoptosis GOF mechanism of p53R175H.

Neck infections are often prone to being underestimated and can manifest insidiously. The spread of infection can lead to translocation into thoracic areas, causing descending necrotizing mediastinitis (DNM). However, the application of the post-mortem approach in such cases is not well-described in the literature. A literature review was carried out according to the PRISMA methods. Nine papers were included in the final review, revealing different levels of involvement of neck layers that can be linked to different causes. Expertise with respect to the anatomy of the fasciae and spaces of the neck enables an understanding of the pathogenesis of DNM. However, a clear autoptic description was not provided in any of the articles. Therefore, we also employed a practical post-mortem approach to cases of death due to DNM. It is fundamental for pathologists to identify the exact head and neck structures involved. Providing dissectors with support from an otolaryngologist could be useful. This paper could help address such difficult cases.

Abstract High-grade serous ovarian cancer (HGSOC) is an aggressive malignancy often diagnosed at an advanced stage. Although most HGSOC patients respond initially to debulking surgery combined with cytotoxic chemotherapy, many ultimately relapse with platinum-resistant disease. Thus, improving outcomes requires new ways of limiting metastasis and eradicating residual disease. We identified previously that Liver kinase B1 (LKB1) and its substrate NUAK1 are implicated in EOC spheroid cell viability and are required for efficient metastasis in orthotopic mouse models. Here, we sought to identify additional signalling pathways altered in EOC cells due to LKB1 or NUAK1 loss-of-function. Transcriptome analysis revealed that inflammatory signalling mediated by NF-κB transcription factors is hyperactive due to LKB1-NUAK1 loss in HGSOC cells and spheroids. Upregulated NF-κB signalling due to NUAK1 loss suppresses reactive oxygen species (ROS) production and sustains cell survival in spheroids. NF-κB signalling is also activated in HGSOC precursor fallopian tube secretory epithelial cell spheroids, and is further enhanced by NUAK1 loss. Finally, immunohistochemical analysis of OVCAR8 xenograft tumors lacking NUAK1 displayed increased RelB expression and nuclear staining. Our results support the idea that NUAK1 and NF-κB signalling pathways together regulate ROS and inflammatory signalling, supporting cell survival during each step of HGSOC pathogenesis. We propose that their combined inhibition may be efficacious as a novel therapeutic strategy for advanced HGSOC.

OBJECTIVES/GOALS: Obesity is associated with gut dysbiosis, inflammation, and increased intestinal permeability. Probiotic consumption may reverse these outcomes. The goal of this study is to evaluate the evidence linking probiotic consumption to changes in intestinal permeability in subjects with overweight or obesity. METHODS/STUDY POPULATION: Articles were searched in Pubmed, Web of Science, and CAB Direct through February 2022 using search terms: intestinal permeability, overweight or obesity, and probiotic supplementation. 694 articles were exported, and 289 duplicates were identified. Titles and abstract were screened in the 405 remaining references by two investigators to determine eligibility. Eligible studies had data extracted on study participant characteristics, probiotic strain used, probiotic dosage, length of intervention, and intestinal barrier outcomes. Results were summarized in tabular form based on intestinal permeability response to probiotics. Quality of the studies was assessed based on Cochrane–Risk of Bias’ tool (RoB2). RESULTS/ANTICIPATED RESULTS: Thirteen eligible studies were identified. Probiotic genera included Akkermansia, Bifidobacterium, Lactobacillus, Streptococcus, Lactococcus, and Bacillus. Single strain probiotics were used in 3 studies, while the other 10 used multi-strain formulas. Dosage and length of probiotic supplementation ranged from 2.4 x 10^7 to 5 x 10^10 CFU/person/day and 3 to 26 weeks, respectively. The most widely used gut permeability outcomes were serum lipopolysaccharide (LPS) (n=10) and mixed sugar solution consumption with urine analysis (n=6). Five of the 10 studies reported decreases in serum LPS following probiotic consumption, while the other 5 showed no effect. Urinary lactulose/mannitol ratio decreased in 1 of the 4 studies, and urinary lactulose percent decreased in 2 studies. DISCUSSION/SIGNIFICANCE: Probiotic supplementation may be remediating an obesity-induced increase in intestinal permeability as evidenced from the effect on serum LPS and mixed sugar solution assays. However, additional studies are needed to further clarify which strain of probiotic bacteria is most effective and the optimal intervention length in subjects with obesity.

Neck infections are often prone to being underestimated and can manifest insidiously. The spread of infection can lead to translocation into thoracic areas, causing descending necrotizing mediastinitis (DNM). However, the application of the post-mortem approach in such cases is not well-described in the literature. A literature review was carried out according to the PRISMA methods. Nine papers were included in the final review, revealing different levels of involvement of neck layers that can be linked to different causes. Expertise with respect to the anatomy of the fasciae and spaces of the neck enables an understanding of the pathogenesis of DNM. However, a clear autoptic description was not provided in any of the articles. Therefore, we also employed a practical post-mortem approach to cases of death due to DNM. It is fundamental for pathologists to identify the exact head and neck structures involved. Providing dissectors with support from an otolaryngologist could be useful. This paper could help address such difficult cases. [ABSTRACT FROM AUTHOR]

School students are increasingly using apps for health-related purposes, either on their own or when recommended by psychologists or counsellors, as apps offer a way to assist students to change their behaviour. However, there is a growing need for psychologists and counsellors to be able to evaluate the quality and usefulness of such apps to effect behaviour change. This study was therefore undertaken to identify methods by which school psychologists and counsellors could evaluate health-related apps for clinical use or research purposes. After examining 15 studies of apps that met the inclusion criteria, it was clear that researchers used a number of taxonomies to evaluate the apps. There were seven taxonomies identified, of which five were generalisable to all health conditions, with the behaviour change technique (BCT) taxonomy being the most comprehensive, containing 13 key behaviour strategies. Despite the utility of the taxonomies to identify the amount of behaviour change content within the apps, it was difficult to determine how the behaviour change strategies were measured, thus reducing the ability to predict app effectiveness. Approaches to improving methods by which apps can be developed and evaluated are proposed.

Abstract Background Although low-grade serous ovarian cancer (LGSC) is rare, case-fatality rates are high as most patients present with advanced disease and current cytotoxic therapies are not overly effective. Recognizing that these cancers may be driven by MAPK pathway activation, MEK inhibitors (MEKi) are being tested in clinical trials. LGSC respond to MEKi only in a subgroup of patients, so predictive biomarkers and better therapies will be needed. Methods We evaluated a number of patient-derived LGSC cell lines, previously classified according to their MEKi sensitivity. Two cell lines were genomically compared against their matching tumors samples. MEKi-sensitive and MEKi-resistant lines were compared using whole exome sequencing and reverse phase protein array. Two treatment combinations targeting MEKi resistance markers were also evaluated using cell proliferation, cell viability, cell signaling, and drug synergism assays. Results Low-grade serous ovarian cancer cell lines recapitulated the genomic aberrations from their matching tumor samples. We identified three potential predictive biomarkers that distinguish MEKi sensitive and resistant lines: KRAS mutation status, and EGFR and PKC-alpha protein expression. The biomarkers were validated in three newly developed LGSC cell lines. Sub-lethal combination of MEK and EGFR inhibition showed drug synergy and caused complete cell death in two of four MEKi-resistant cell lines tested. Conclusions KRAS mutations and the protein expression of EGFR and PKC-alpha should be evaluated as predictive biomarkers in patients with LGSC treated with MEKi. Combination therapy using a MEKi with EGFR inhibition may represent a promising new therapy for patients with MEKi-resistant LGSC.

Interactions between viruses and the host antibody immune response are critical in the development and control of disease, and antibodies are also known to interfere with the efficacy of viral vector-based gene delivery. The adeno-associated viruses (AAVs) being developed as vectors for corrective human gene delivery have shown promise in clinical trials, but preexisting antibodies are detrimental to successful outcomes. However, the antigenic epitopes on AAV capsids remain poorly characterized. Cryo-electron microscopy and three-dimensional image reconstruction were used to define the locations of epitopes to which monoclonal fragment antibodies (Fabs) against AAV1, AAV2, AAV5, and AAV6 bind. Pseudoatomic modeling showed that, in each serotype, Fabs bound to a limited number of sites near the protrusions surrounding the 3-fold axes of the T=1 icosahedral capsids. For the closely related AAV1 and AAV6, a common Fab exhibited substoichiometric binding, with one Fab bound, on average, between two of the three protrusions as a consequence of steric crowding. The other AAV Fabs saturated the capsid and bound to the walls of all 60 protrusions, with the footprint for the AAV5 antibody extending toward the 5-fold axis. The angle of incidence for each bound Fab on the AAVs varied and resulted in significant differences in how much of each viral capsid surface was occluded beyond the Fab footprints. The AAV-antibody interactions showed a common set of footprints that overlapped some known receptor-binding sites and transduction determinants, thus suggesting potential mechanisms for virus neutralization by the antibodies.

Adeno-associated virus serotype 9 (AAV9) has enhanced capsid-associated tropism for cardiac muscle and the ability to cross the blood-brain barrier compared to other AAV serotypes. To help identify the structural features facilitating these properties, we have used cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction (cryo-reconstruction) and X-ray crystallography to determine the structure of the AAV9 capsid at 9.7- and 2.8-Å resolutions, respectively. The AAV9 capsid exhibits the surface topology conserved in all AAVs: depressions at each icosahedral two-fold symmetry axis and surrounding each five-fold axis, three separate protrusions surrounding each three-fold axis, and a channel at each five-fold axis. The AAV9 viral protein (VP) has a conserved core structure, consisting of an eight-stranded, β-barrel motif and the αA helix, which are present in all parvovirus structures. The AAV9 VP differs in nine variable surface regions (VR-I to -IX) compared to AAV4, but at only three (VR-I, VR-II, and VR-IV) compared to AAV2 and AAV8. VR-I differences modify the raised region of the capsid surface between the two-fold and five-fold depressions. The VR-IV difference produces smaller three-fold protrusions in AAV9 that are less "pointed" than AAV2 and AAV8. Significantly, residues in the AAV9 VRs have been identified as important determinants of cellular tropism and transduction and dictate its antigenic diversity from AAV2. Hence, the AAV9 VRs likely confer the unique infection phenotypes of this serotype.

Nicotinic acetylcholine receptor (nAChR) subtypes are key drug targets, but it is challenging to pharmacologically differentiate between them because of their highly similar sequence identities. Furthermore, α-conotoxins (α-CTXs) are naturally selective and competitive antagonists for nAChRs and hold great potential for treating nAChR disorders. Identifying selectivity-enhancing mutations is the chief aim of most α-CTX mutagenesis studies, although doing so with traditional docking methods is difficult due to the lack of α-CTX/nAChR crystal structures. Here, we use homology modeling to predict the structures of α-CTXs bound to two nearly identical nAChR subtypes, α3β2 and α3β4, and use free-energy perturbation (FEP) to re-predict the relative potency and selectivity of α-CTX mutants at these subtypes. First, we use three available crystal structures of the nAChR homologue, acetylcholine-binding protein (AChBP), and re-predict the relative affinities of twenty point mutations made to the α-CTXs LvIA, LsIA, and GIC, with an overall root mean square error (RMSE) of 1.08 ± 0.15 kcal/mol and an R2 of 0.62, equivalent to experimental uncertainty. We then use AChBP as a template for α3β2 and α3β4 nAChR homology models bound to the α-CTX LvIA and re-predict the potencies of eleven point mutations at both subtypes, with an overall RMSE of 0.85 ± 0.08 kcal/mol and an R2 of 0.49. This is significantly better than the widely used molecular mechanics—generalized born/surface area (MM-GB/SA) method, which gives an RMSE of 1.96 ± 0.24 kcal/mol and an R2 of 0.06 on the same test set. Next, we demonstrate that FEP accurately classifies α3β2 nAChR selective LvIA mutants while MM-GB/SA does not. Finally, we use FEP to perform an exhaustive amino acid mutational scan of LvIA and predict fifty-two mutations of LvIA to have greater than 100X selectivity for the α3β2 nAChR. Our results demonstrate the FEP is well-suited to accurately predict potency- and selectivity-enhancing mutations of α-CTXs for nAChRs and to identify alternative strategies for developing selective α-CTXs.