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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Thyroid function and pregnancy: before, during and beyond.

Thyroid disturbances are common in women during the reproductive years of their lives. Autoimmunity and altered iodine status together account for a high proportion of the abnormalities. Autoimmune thyroid disease is present in around 4% of young females, and up to 15% are at risk because they are thyroid antibody-positive. There is a strong relationship between thyroid immunity on the one hand and infertility, miscarriage, and thyroid disturbances in pregnancy and postpartum on the other hand. Suboptimal iodine status affects a large proportion of the world's population, and pregnancy further depletes iodine stores. There is controversy surrounding the degree to which iodine should be supplemented and the duration of supplementation. Recent studies have helped to clarify the relationship between maternal thyroid status and neuropsychological development of the child. The role of other environmental factors including smoking and selenium status is also now recognised. Universal screening for thyroid hormone abnormalities is not routinely recommended at present. However, measurement of thyroid function and autoantibodies should certainly be considered in those who are at high risk of thyroid disease and in those whose pregnancy is otherwise high risk. The practicing clinician needs to be aware of the thyroid changes which accompany pregnancy.

Acetaminophen: potentially toxic metabolite formed by human fetal and adult liver microsomes and isolated fetal liver cells.

A reactive metabolite of acetaminophen is hepatotoxic in humans when the drug is ingested in large overdoses. The ability of the human fetal and adult liver to oxidize acetaminophen by trapping the potentially toxic metabolite as a glutathione conjugate has been measured. Oxidation by fetal liver was approximately ten times slower than by adult liver. However, there was a definite increase in acetaminophen oxidation with fetal age. Isolated human fetal liver cells conjugated acetaminophen with sulfate but not with glucuronic acid. The results indicate that the human fetal liver is able to detoxify acetaminophen by conjugation. However, it also catalyzes the formation of an active metabolite of acetaminophen through oxidation. Hence the fetus remains at risk should a large dose of the drug cross into the fetal circulation.

Deletion polymorphism of the UGT2B17 gene is associated with increased risk for prostate cancer and correlated to gene expression in the prostate.

Metabolism of androgens includes glucuronidation, the major pathway of steroid elimination in several steroid target tissues. Glucuronidation is catalysed by UDP-glucuronosyltransferases (UGTs). UGT2B17 has been shown to be particularly active against androgens and is highly abundant in the prostate. Recently, we discovered that deletion of the UGT2B17 gene is associated with low or undetectable urinary testosterone levels. Here, we determined the phenotypic outcome of the deletion by quantifying the UGT2B17 mRNA expression in normal prostate tissues in individuals with different genotypes. Additionally, the frequency of UGT2B17 deletion polymorphism was studied in a Swedish population-based case-control study including 176 patients diagnosed with prostate cancer and 161 controls. We found that the individuals homozygous for the insertion allele expressed 30 times more UGT2B17 mRNA in prostate tissue than the heterozygotes. Carriers of the deletion allele had a significantly increased risk of prostate cancer (OR=2.07; 95% CI=1.32-3.25). In conclusion, these results show the UGT2B17 deletion polymorphism is associated with prostate cancer risk.

Transobturator mesh for cystocele repair: a short- to medium-term follow-up using 3D/4D ultrasound.

OBJECTIVE: Anterior colporrhaphy has been shown to have limited medium-term success rates in cystocele repair. Many clinicians use mesh implants, but their safety and efficacy are controversial. We therefore performed an external surgical audit using three- and four-dimensional pelvic floor ultrasound to study the short- to medium-term results of transobturator mesh placement. METHODS: Forty-six women who had undergone transobturator mesh anterior repair using the Perigee(TM) system were invited back for a follow-up appointment conducted by two non-surgeons. The appointment consisted of a standardized interview, clinical examination using the International Continence Society Pelvic Organ Prolapse Quantification system (ICS POP-Q) and translabial ultrasound examination. RESULTS: The mean follow-up time was 10 (range, 2-24) months. There had been no major intra- or postoperative complications. Thirty-six (78%) patients were subjectively satisfied with the outcome of the procedure. Cystocele recurrence (Stage 2 or 3) was observed in six (13%) patients. There were three (6.5%) cases of mesh erosion. On translabial ultrasound, we observed cystocele recurrence dorsal to the mesh in five women, associated with a marked change in mesh axis on Valsalva, implying dislodgment of the superior anchoring arms. The mesh was measured at a mean of 21 (range, 8.8-37.3; SD, 7.0) mm in length. CONCLUSIONS: At 10-month follow-up the Perigee procedure seems to be safe and effective for cystocele repair, with a satisfaction rate of 78%. In some women recurrence may occur due to dislodgment of the superior anchoring arms.

Does micturition improve in the squatting position?

Posture on the toilet is an important consideration during micturition. The objective of this prospective study is to evaluate the effects of posture on micturition in the lean forward and squatting positions. The participants were 54 volunteers who acted as their own controls. Uroflowmetric parameters were studied in each position. The two issues that arose from the study were 'squatability' and the differences in uroflowmetric parameters. Essentially there were no statistically significant differences found in our study population but the ability to squat in our population of volunteers was quite poor.

Prospective evaluation of the safety and efficacy of the Apogee system for treatment of vault prolapse.

This is a prospective study evaluating the efficacy and safety of the Apogee vault repair system in the management of vault prolapse. All patients who underwent this procedure between October 2004 and December 2005 in Townsville, Australia are included in the study. Preoperative and postoperative clinical assessments were done using the pelvic organ prolapse quantification (POPQ) system and patients were followed up at 6 weeks, 12 weeks, 6 months, 12 months and then biannually. Of the 35 patients operated, there were no intraoperative complications and only one patient (2.8%) had a recurrence of vault prolapse at 12 months. Nine patients (25%) had mesh exposure treated successfully with outpatient trimming and oestrogen. This medium term study shows that the Apogee procedure is safe with minimal complications for the management of vault prolapse.

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.

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.