In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis.

UnlabelledToxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δgra38 and Δgra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis.ImportanceMost intracellular pathogens reside inside a membrane-bound vacuole within their host cell that is extensively modified by the pathogen to optimize intracellular growth and avoid host defenses. In Toxoplasma, this vacuole is modified by a host of secretory GRA proteins, many of which remain unidentified. Here we demonstrate that in vivo biotinylation of proximal and interacting proteins using the promiscuous biotin ligase BirA* is a powerful approach to rapidly identify vacuolar GRA proteins. We further demonstrate that one factor identified by this approach, GRA39, plays an important role in the ability of the parasite to replicate within its host cell and cause disease

Comparing the treatment of endometriosis‐related pain by excision of endometriosis or hysterectomy: A multicentre prospective cohort study

Objective: To compare the effectiveness of endometriosis excision alone to excision plus hysterectomy, with and without bilateral oophorectomy, for endometriosis‐related symptoms. Design: Multicentre prospective cohort. Setting: Eighty‐six specialist endometriosis centres. Population: Women undergoing rectovaginal endometriosis surgery between 2009 and 2021. Methods: We performed multivariable regression with random effects for patient and centre, controlling for age, BMI, smoking, laparoscopic versus open approach and type of bowel surgery performed, with sensitivity analysis for loss to follow‐up. Main Outcome Measures: Pain scores, bowel symptoms and quality‐of‐life measures. Results: Compared to endometriosis excision alone, women undergoing hysterectomy with conservation of ovaries had greater improvement in non‐cyclical pain (MD: 1.41/10, 95% CI: 1.03–1.78, p < 0.001), dyspareunia (MD: 1.12/10, 95% CI: 0.71–1.53, p < 0.001), back pain (MD: 1.29/10, 95% CI: 0.92–1.67, p < 0.001) and quality‐of‐life scores (MD: 8.77/100, 95% CI: 5.79–11.75, p < 0.001) at 24 months post‐operatively. Women undergoing hysterectomy with bilateral oophorectomy also had greater improvement in non‐cyclical pelvic pain (MD: 2.22/10, 95% CI: 1.80–2.63, p < 0.001), dyspareunia (MD: 1.05/10, 95% CI: 0.59–1.52, p < 0.001), back pain (MD: 1.18/10, 95% CI: 0.77–1.59, p < 0.001) and quality of life (MD: 12.41/100, 95% CI: 9.07–15.74, p < 0.001) at 24 months compared to endometriosis excision alone. Compared to hysterectomy with ovarian conservation, hysterectomy with bilateral oophorectomy was associated with greater improvement in non‐cyclical pelvic pain (MD: 0.81/10, 95% CI: 0.32–1.30, p = 0.001) at 24 months and quality of life (MD: 3.74/100, 95% CI: 0.56–6.92, p = 0.021) at 12 months, although this result was sensitive to loss to follow‐up. Conclusions: Patients who undergo endometriosis excision plus hysterectomy experience greater improvement in pain and quality of life compared to those who have endometriosis excision alone. There are additional benefits of bilateral oophorectomy with hysterectomy, although its value is less clear due to loss of follow‐up

Engineering nucleotide specificity of succinyl-CoA synthetase in blastocystis: the emerging role of gatekeeper residues

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design

An approach for the identification of targets specific to bone metastasis using cancer genes interactome and gene ontology analysis

Metastasis is one of the most enigmatic aspects of cancer pathogenesis and is a major cause of cancer-associated mortality. Secondary bone cancer (SBC) is a complex disease caused by metastasis of tumor cells from their primary site and is characterized by intricate interplay of molecular interactions. Identification of targets for multifactorial diseases such as SBC, the most frequent complication of breast and prostate cancers, is a challenge. Towards achieving our aim of identification of targets specific to SBC, we constructed a 'Cancer Genes Network', a representative protein interactome of cancer genes. Using graph theoretical methods, we obtained a set of key genes that are relevant for generic mechanisms of cancers and have a role in biological essentiality. We also compiled a curated dataset of 391 SBC genes from published literature which serves as a basis of ontological correlates of secondary bone cancer. Building on these results, we implement a strategy based on generic cancer genes, SBC genes and gene ontology enrichment method, to obtain a set of targets that are specific to bone metastasis. Through this study, we present an approach for probing one of the major complications in cancers, namely, metastasis. The results on genes that play generic roles in cancer phenotype, obtained by network analysis of 'Cancer Genes Network', have broader implications in understanding the role of molecular regulators in mechanisms of cancers. Specifically, our study provides a set of potential targets that are of ontological and regulatory relevance to secondary bone cancer.Comment: 54 pages (19 pages main text; 11 Figures; 26 pages of supplementary information). Revised after critical reviews. Accepted for Publication in PLoS ON

Host-specific differences in top-expanded TCR clonotypes correlate with divergent outcomes of anti-PD-L1 treatment in responders versus non-responders

Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment; however, the responses to ICI treatment are highly variable in different individuals and the underlying mechanisms remain poorly understood. Here, we employed a mouse squamous cell carcinoma (SCC) model where tumor-bearing recipients diverged into responders (R) versus non-responders (NR) upon anti-PD-L1 treatment. We performed in-depth TCRβ sequencing with immunoSEQ platform to delineate the differences in CD8 tumor-infiltrating lymphocytes (TILs). We found that R and NR CD8 TILs both exhibited evidence of clonal expansion, suggesting activation regardless of response status. We detected no differences in clonal expansion or clonal diversity indexes between R vs. NR. However, the top expanded (&gt;1%) TCRβ clonotypes appeared to be mutually exclusive between R and NR CD8 TILs, showing a preferential expansion of distinct TCRβ clonotypes in response to the same SCC tumor in R vs. NR. Notably, the mutual exclusivity of TCR clonotypes in R vs. NR was only observed when top TCRβ clonotypes were counted, because such top-expanded clonotypes are present in the opposite outcome group at a much lower frequency. Many TCRβ sequences were detected in only one recipient at a high frequency, implicating highly individualized anti-tumor immune responses. We conclude that differences in the clonal frequency of top TCR clonotypes between R and NR CD8 TILs may be one of the factors underlying differential anti-PD-L1 responses. This notion may offer a novel explanation for variable ICI responses in different individuals, which may substantially impact the development of new strategies for personalized cancer immunotherapy

Uterine Artery Embolization in Patients with a Large Fibroid Burden: Long-Term Clinical and MR Follow-up

Uterine artery embolization (UAE) in patients with a large fibroid burden is controversial. Anecdotal reports describe serious complications and limited clinical results. We report the long-term clinical and magnetic resonance (MR) results in a large series of women with a dominant fibroid of >10 cm and/or an uterine volume of >700 cm3. Seventy-one consecutive patients (mean age, 42.5 years; median, 40 years; range, 25–52 years) with a large fibroid burden were treated by UAE between August 2000 and April 2005. Volume reduction and infarction rate of dominant fibroid and uterus were assessed by comparing the baseline and latest follow-up MRIs. Patients were clinically followed at various time intervals after UAE with standardized questionnaires. There were no serious complications of UAE. During a mean follow-up of 48 months (median, 59 months; range, 6–106 months), 10 of 71 patients (14%) had a hysterectomy. Mean volume reduction of the fibroid and uterus was 44 and 43%. Mean infarction rate of the fibroid and overall fibroid infarction rate was 86 and 87%. In the vast majority of patients there was a substantial improvement of symptoms. Clinical results were similar in patients with a dominant fibroid >10 cm and in patients with large uterine volumes by diffuse fibroid disease. In conclusion, our results indicate that the risk of serious complications after UAE in patients with a large fibroid burden is not increased. Moreover, clinical long-term results are as good as in other patients who are treated with UAE. Therefore, a large fibroid burden should not be considered a contraindication for UAE

Development and Validation of an Epitope Prediction Tool for Swine (PigMatrix) Based on the Pocket Profile Method

Background: T cell epitope prediction tools and associated vaccine design algorithms have accelerated the development of vaccines for humans. Predictive tools for swine and other food animals are not as well developed, primarily because the data required to develop the tools are lacking. Here, we overcome a lack of T cell epitope data to construct swine epitope predictors by systematically leveraging available human information. Applying the “pocket profile method”, we use sequence and structural similarities in the binding pockets of human and swine major histocompatibility complex proteins to infer Swine Leukocyte Antigen (SLA) peptide binding preferences. We developed epitope-prediction matrices (PigMatrices), for three SLA class I alleles (SLA-1*0401, 2*0401 and 3*0401) and one class II allele (SLA-DRB1*0201), based on the binding preferences of the best-matched Human Leukocyte Antigen (HLA) pocket for each SLA pocket. The contact residues involved in the binding pockets were defined for class I based on crystal structures of either SLA (SLA-specific contacts, Ssc) or HLA supertype alleles (HLA contacts, Hc); for class II, only Hc was possible. Different substitution matrices were evaluated (PAM and BLOSUM) for scoring pocket similarity and identifying the best human match. The accuracy of the PigMatrices was compared to available online swine epitope prediction tools such as PickPocket and NetMHCpan. Results: PigMatrices that used Ssc to define the pocket sequences and PAM30 to score pocket similarity demonstrated the best predictive performance and were able to accurately separate binders from random peptides. For SLA-1*0401 and 2*0401, PigMatrix achieved area under the receiver operating characteristic curves (AUC) of 0.78 and 0.73, respectively, which were equivalent or better than PickPocket (0.76 and 0.54) and NetMHCpan version 2.4 (0.41 and 0.51) and version 2.8 (0.72 and 0.71). In addition, we developed the first predictive SLA class II matrix, obtaining an AUC of 0.73 for existing SLA-DRB1*0201 epitopes. Notably, PigMatrix achieved this level of predictive power without training on SLA binding data. Conclusions: Overall, the pocket profile method combined with binding preferences from HLA binding data shows significant promise for developing T cell epitope prediction tools for pigs. When combined with existing vaccine design algorithms, PigMatrix will be useful for developing genome-derived vaccines for a range of pig pathogens for which no effective vaccines currently exist (e.g. porcine reproductive and respiratory syndrome, influenza and porcine epidemic diarrhea)