ExoSTING, an extracellular vesicle loaded with STING agonists, promotes tumor immune surveillance.

Cyclic dinucleotide (CDN) agonists of the STimulator of InterferoN Genes (STING) pathway have shown immune activation and tumor clearance in pre-clinical models. However, CDNs administered intratumorally also promote STING activation leading to direct cytotoxicity of many cell types in the tumor microenvironment (TME), systemic inflammation due to rapid tumor extravasation of the CDN, and immune ablation in the TME. These result in a failure to establish immunological memory. ExoSTING, an engineered extracellular vesicle (EV) exogenously loaded with CDN, enhances the potency of CDN and preferentially activates antigen presenting cells in the TME. Following intratumoral injection, exoSTING was retained within the tumor, enhanced local Th1 responses and recruitment of CD8+ T cells, and generated systemic anti-tumor immunity to the tumor. ExoSTING at therapeutically active doses did not induce systemic inflammatory cytokines, resulting in an enhanced therapeutic window. ExoSTING is a novel, differentiated therapeutic candidate that leverages the natural biology of EVs to enhance the activity of CDNs.

A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties.

Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two "scaffold" proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily. Molecules were loaded into EVs at high density and exhibited potent in vitro activity when fused to full-length or truncated forms of PTGFRN or BASP1. Furthermore, these engineered EVs retained pharmacodynamic activity in a variety of animal models. This engineering platform provides a simple approach to functionalize EVs with topologically diverse macromolecules and represents a significant advance toward unlocking the therapeutic potential of EVs.

Development and validation of a training and assessment tool for laparoscopic radical nephrectomy. / Desarrollo y validación de una herramienta de entrenamiento y evaluación para la nefrectomía radical laparoscópica.

INTRODUCTION: Laparoscopic radical nephrectomy(LRN) is a cornerstone in managing renal cancer and small renal masses. Twenty-first century surgical training faces challenges, thus must be efficient and safe so surgeons attain relevant skills, protecting patients and operative outcomes. This study aimed to systematically develop a tool for training and assessment in LRN and validate the developed tool for use by trainee urologists. METHODS: This prospective, longitudinal, multi-institutional study was undertaken from September 2014 - June 2015. Healthcare Failure Mode and Effect Analysis was utilised for development and followed by validation where the assessment tool was distributed to five specialists to increase content validity. Four experts were observed as a multi-institutional approach. Hand-assisted, transperitoneal and retroperitoneal approaches were considered. RESULTS: The LRN Assessment Tool comprised four phases, 17 processes, 41 sub-processes. Four surgeons and operating teams were observed across four hospitals for 19.5hours (5.75h hand-assisted, 8.75h trans-peritoneal, 5h retro-peritoneal). After hazard analysis, three checklists were constructed. Those for hand-assisted LRN and transperitoneal LRN contained four phases, 20 processes, 33 sub-processes and that for retroperitoneal LRN contained four phases, 20 processes, 30 sub-processes. These were merged to form one assessment tool. The final result was a four phase LRN Assessment Tool with 17 processes, 41 sub-processes. All participants agreed the final LRN Assessment Tool included pertinent steps. CONCLUSIONS: The LRN Assessment Tool was developed using Healthcare Failure Mode and Effect Analysis risk analysis to ensure hazardous procedural sub-steps were included. Validation ascertained important processes were not overlooked. Full application through a pilot study must be undertaken.

Codeine influences the serum and urinary profile of endogenous androgens but does not interact with the excretion rate of administered testosterone.

Today's doping tests involve longitudinal monitoring of urinary steroids including the testosterone glucuronide and epitestosterone glucuronide ratio (T/E) in an Athlete Biological Passport (ABP). The aim of this study was to investigate the possible influence of short-term use of codeine on the urinary excretion of androgen metabolites included in the steroidal module of the passport prior to and after the co-administration with testosterone. The study was designed as an open study with the subjects being their own control. Fifteen healthy male volunteers received therapeutic doses of codeine (Kodein Meda) for 6 days. On Day 3, 500 mg or 125 mg of testosterone enanthate (Testoviron®-Depot) was administered. Spot urine samples were collected for 17 days, and blood samples were collected at baseline, 3, 6, and 14 days after codeine intake. The circulatory concentration of total testosterone decreased significantly by 20% after 3 days' use of codeine (p = 0.0002) and an atypical ABP result was noted in one of the subjects. On the other hand, the concomitant use of codeine and testosterone did not affect the elevated urinary T/E ratio. In 75% of the individuals, the concentration of urinary morphine (a metabolite of codeine) was above the decision limit for morphine. One of the participants displayed a morphine/codeine ratio of 1.7 after codeine treatment, indicative of morphine abuse. In conclusion, our study shows that codeine interferes with the endogenous testosterone concentration. As a result, the urinary steroid profile may lead to atypical findings in the doping test.

KRAS G12C Drug Development: Discrimination between Switch II Pocket Configurations Using Hydrogen/Deuterium-Exchange Mass Spectrometry.

KRAS G12C, the most common RAS mutation found in non-small-cell lung cancer, has been the subject of multiple recent covalent small-molecule inhibitor campaigns including efforts directed at the guanine nucleotide pocket and separate work focused on an inducible pocket adjacent to the switch motifs. Multiple conformations of switch II have been observed, suggesting that switch II pocket (SIIP) binders may be capable of engaging a range of KRAS conformations. Here we report the use of hydrogen/deuterium-exchange mass spectrometry (HDX MS) to discriminate between conformations of switch II induced by two chemical classes of SIIP binders. We investigated the structural basis for differences in HDX MS using X-ray crystallography and discovered a new SIIP configuration in response to binding of a quinazoline chemotype. These results have implications for structure-guided drug design targeting the RAS SIIP.

Structural Dynamics in Ras and Related Proteins upon Nucleotide Switching.

Structural dynamics of Ras proteins contributes to their activity in signal transduction cascades. Directly targeting Ras proteins with small molecules may rely on the movement of a conserved structural motif, switch II. To understand Ras signaling and advance Ras-targeting strategies, experimental methods to measure Ras dynamics are required. Here, we demonstrate the utility of hydrogen-deuterium exchange (HDX) mass spectrometry (MS) to measure Ras dynamics by studying representatives from two branches of the Ras superfamily, Ras and Rho. A comparison of differential deuterium exchange between active (GMPPNP-bound) and inactive (GDP-bound) proteins revealed differences between the families, with the most notable differences occurring in the phosphate-binding loop and switch II. The P-loop exchange signature correlated with switch II dynamics observed in molecular dynamics simulations focused on measuring main-chain movement. HDX provides a means of evaluating Ras protein dynamics, which may be useful for understanding the mechanisms of Ras signaling, including activated signaling of pathologic mutants, and for targeting strategies that rely on protein dynamics.

Modulating the strength of hydrogen bond acceptors to achieve low Caco2 efflux for oral bioavailability of PARP inhibitors blocking centrosome clustering.

During the lead generation and optimization of PARP inhibitors blocking centrosome clustering, it was discovered that increasing hydrogen bond acceptor (HBA) strength improved cellular potency but led to elevated Caco2 and MDR1 efflux and thus poor oral bioavailability. Conversely, compounds with lower efflux had reduced potency. The project team was able to improve the bioavailability by reducing efflux through systematic modifications to the strength of the HBA by changing the electronic properties of neighboring groups, whilst maintaining sufficient acceptor strength for potency. Additionally, it was observed that enantiomers with different potency showed similar efflux, which is consistent with the promiscuity of efflux transporters. Eventually, a balance between potency and low efflux was achieved for a set of lead compounds with good bioavailability which allowed the project to progress towards establishing in vivo pharmacokinetic/pharmacodynamic relationships.

Conformational insight into multi-protein signaling assemblies by hydrogen-deuterium exchange mass spectrometry.

Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) can provide information about proteins that can be challenging to obtain by other means. Structure/function relationships, binding interactions, and the effects of modification have all been measured with HDX MS for a diverse and growing array of signaling proteins and multiprotein signaling complexes. As a result of hardware and software improvements, receptors and complexes involved in cellular signaling-including those associated with membranes-can now be studied. The growing body of HDX MS studies of signaling complexes at membranes is particularly exciting. Recent examples are presented to illustrate what can be learned about signaling proteins with this technique.

Genetic variation, expression and ontogeny of sulfotransferase SULT2A1 in humans.

Sulfotransferases (SULTs) are enzymes involved in the metabolism of several endogenous molecules. The activity and expression exhibit inter- and intra-individual variations due to age and genetic variation. The aims of this study were to compare the gene expression of SULT2A1 in fetal and adult livers, to study the intra-individual tissue distribution, and investigate if expression is associated with a SULT2A1 copy number variation polymorphism. In contrast to other drug metabolizing enzyme systems the expression of SULT2A1 did not differ between fetal and adult liver samples and it was not affected by maternal smoking or gestational age. Gene expression in relation to sex could not be assessed as the sex of the fetuses was unknown. SULT2A1 was consistently expressed in livers and adrenals, being seven times more abundant in adrenals, but was absent in the lungs. The SULT2A1 copy number variation was proportional to gene expression in liver and adrenals. Our results show that SULT2A1 is important in the first trimester; particularly in the adrenals.

Indian health care professionals' attitude towards spiritual healing and its role in alleviating stigma of psychiatric services.

Persons with mental illnesses in India and rest of developing world continue to consult religious/spiritual (R/S) healers or traditional, complementary and alternative medicine (TCAM) professionals prior to seeking psychiatric services that are devoid of spiritual components of care. We aim to understand TCAM and allopathic professionals' perspectives on patients' R/S needs within mental health services, cross-sectional study was conducted at five TCAM and two allopathic tertiary care hospitals in three different Indian states; 393 participants completed RSMPP, a self-administered, semi-structured survey questionnaire. Perspectives of TCAM and allopathic health professionals on role of spirituality in mental health care were compared. Substantial percentage, 43.7 % TCAM and 41.3 % allopathic, of participants believe that their patients approach R/S or TCAM practitioners for severe mental illness; 91.2 % of TCAM and 69.7 % of allopaths were satisfied with R/S healers (p = 0.0019). Furthermore, 91.1 % TCAM and 73.1 % allopaths (p = 0.000) believe that mental health stigma can be minimized by integrating with spiritual care services. Overall, 87 % of TCAM and 73 % of allopaths agreed to primary criterion variable: 'spiritual healing is beneficial and complementary to psychiatric care.' A quarter of allopaths (24.4 %) and 38 % of TCAM physicians reportedly cross-refer their grieving patients to religious/TCAM healer and psychiatrist/psychologist, respectively; on logistic regression, significant (p < 0.05) predictors were clinical interactions/references to r/s healers. Providing spiritual care within the setup of psychiatric institution will not only complement psychiatric care but also alleviate stigma against mental health services. Implications on developing spiritual care services like clinical chaplaincy are discussed.

Perspectives of Indian traditional and allopathic professionals on religion/spirituality and its role in medicine: basis for developing an integrative medicine program.

Allopathic medical professionals in developed nations have started to collaborate with traditional, complementary, and alternative medicine (TCAM) to enquire on the role of religion/spirituality (r/s) in patient care. There is scant evidence of such movement in the Indian medical community. We aim to understand the perspectives of Indian TCAM and allopathic professionals on the influence of r/s in health. Using RSMPP (Religion, Spirituality and Medicine, Physician Perspectives) questionnaire, a cross-sectional survey was conducted at seven (five TCAM and two allopathic) pre-selected tertiary care medical institutes in India. Findings of TCAM and allopathic groups were compared. Majority in both groups (75% of TCAM and 84.6% of allopathic practitioners) believed that patients' spiritual focus increases with illness. Up to 58% of TCAM and allopathic respondents report patients receiving support from their religious communities; 87% of TCAM and 73% of allopaths believed spiritual healing to be beneficial and complementary to allopathic medical care. Only 11% of allopaths, as against 40% of TCAM, had reportedly received 'formal' training in r/s. Both TCAM (81.8%) and allopathic (63.7%) professionals agree that spirituality as an academic subject merits inclusion in health education programs (p = 0.0003). Inclusion of spirituality in the health care system is a need for Indian medical professionals as well as their patients, and it could form the basis for integrating TCAM and allopathic medical systems in India.

Therapeutic targeting of oncogenic K-Ras by a covalent catalytic site inhibitor.

We report the synthesis of a GDP analogue, SML-8-73-1, and a prodrug derivative, SML-10-70-1, which are selective, direct-acting covalent inhibitors of the K-Ras G12C mutant relative to wild-type Ras. Biochemical and biophysical measurements suggest that modification of K-Ras with SML-8-73-1 renders the protein in an inactive state. These first-in-class covalent K-Ras inhibitors demonstrate that irreversible targeting of the K-Ras guanine-nucleotide binding site is potentially a viable therapeutic strategy for inhibition of Ras signaling.

Anterior compartment mesh: a descriptive study of mesh anchoring failure.

OBJECTIVES: To define types of support failure after anterior compartment mesh placement and to investigate any association with predictors of recurrence. METHODS: This was a retrospective study on patients ≥ 3 months after anterior mesh placement. All patients underwent a standardized interview, clinical examination using the International Continence Society Pelvic Organ Prolapse Quantification system (ICS POP-Q) and three-dimensional/four-dimensional (3D/4D) translabial ultrasound. Mesh failure was defined as recurrence of anterior/central compartment prolapse seen on ultrasound. Failures were classified as anterior, global and apical. Their association with hiatal area on Valsalva maneuver and levator avulsion was tested. RESULTS: Three hundred and one patients were seen initially, of whom five were excluded because of missing data, leaving 296. Mean follow-up was 1.8 (range, 0.3-5.6) years. Mean age was 65 (range, 32-88) years. One hundred and thirty-nine (47%) women were fitted with a Perigee mesh, 66 (22%) with an Anterior Prolift mesh and 91 (31%) with an Anterior Elevate mesh. Recurrent symptoms (lump/drag) were reported in 65 (22%), a recurrent cystocele was noted in 128 clinically (43%) and in 105 on ultrasound (35%). Avulsion was diagnosed in 117 patients (40%). Mean hiatal area on Valsalva was 33.3 (range, 14.1-60.0) cm2. Mesh failure was diagnosed in 112 patients (38%), comprising global failure in 81 (27%), apical failure in 23 (8%) and anterior failure in eight (3%). Apical and global failures were significantly associated with hiatal area, associations that remained after controlling for potential confounders. CONCLUSIONS: Mesh failure, i.e. anterior or central compartment recurrent prolapse, was noted in 38% of patients on average 1.8 years after placement of anterior compartment mesh. Global and apical failures together constituted 93% of all mesh failures, both types of failure being significantly associated with hiatal area on Valsalva maneuver.

Is levator avulsion a predictor of cystocele recurrence following anterior vaginal mesh placement?

OBJECTIVE: Levator avulsion has been shown to be a predictor of cystocele recurrence following anterior colporrhaphy. The aim of this study was to determine if levator avulsion is a risk factor for prolapse recurrence following anterior colporrhaphy with mesh. METHODS: This was a retrospective analysis of data obtained from three surgical audits for subjective and objective outcomes following anterior colporrhaphy with mesh. Recurrence was defined as cystocele ≥ Stage 2 on the prolapse quantification system of the International Continence Society; symptoms of vaginal lump/bulge; or cystocele on ultrasound, defined as maximum bladder descent to ≥ 10 mm below the symphysis pubis. Levator avulsion was diagnosed using tomographic ultrasound imaging. RESULTS: Two hundred and nine patients were followed up at a mean of 2.2 years (range, 3 months to 5.6 years) after anterior vaginal mesh placement. 24% (51/209) had recurrent prolapse symptoms, 33% (68/209) clinical cystocele recurrence ≥ Stage 2, and 26% (54/209) a recurrent cystocele on ultrasound. Twenty-eight out of 80 (35%) women with levator avulsion had significant sonographic cystocele recurrence (odds ratio (OR), 2.24 (95% confidence interval (CI), 1.13-4.43)). This finding was confirmed after adjusting for potential predictors of prolapse recurrence on multivariate logistic regression (OR, 2.13 (95% CI, 1.04-4.39); P = 0.04). CONCLUSION: Levator avulsion doubles the risk of cystocele recurrence after anterior colporrhaphy with transobturator mesh.

The sacrospinous ligament: conveniently effective or effectively convenient?

The sacrospinous ligament has been used for over 50 years as a convenient structure for treating vaginal vault and more recently, uterine prolapse. The procedure has evolved over the years and its efficacy has been hotly debated with invariable comparisons made to abdominal sacral colpopexy. Mesh surgery has introduced a newer dimension to the debate. This review is an attempt to clarify the anatomy, reflect on various techniques and offer a critique on the current 'status' of the sacrospinous ligament.

Microscopic alterations of vaginal tissue in women with pelvic organ prolapse.

The aim of this study was to identify any histological changes between the prolapsed and non-prolapsed vaginal skin to enable us to answer the broader question of whether to excise or re-suture the prolapsed tissue during vaginal reconstructive surgery. Two tissue samples were taken from 20 women who underwent prolapse surgery, one prolapsed and another non-prolapsed tissue. The samples were analysed blind histologically by a pathologist. Our study showed statistically significant differences in three tissue components namely myofibroblast differentiation (0.047), elastin (0.048) and collagen (0.095), depending on whether the tissue was prolapsed or non-prolapsed. However, the study had certain limitations and practical difficulties faced in histological approach of tissue analysis. Histology alone is an insufficient indicator to answer our question and definitive understanding of the prolapsed tissue may result from a randomised study on a larger study population.