Structures revealing mechanisms of resistance and collateral sensitivity of Plasmodium falciparum to proteasome inhibitors.

The proteasome of the malaria parasite Plasmodium falciparum (Pf20S) is an advantageous drug target because its inhibition kills P. falciparum in multiple stages of its life cycle and synergizes with artemisinins. We recently developed a macrocyclic peptide, TDI-8304, that is highly selective for Pf20S over human proteasomes and is potent in vitro and in vivo against P. falciparum. A mutation in the Pf20S ß6 subunit, A117D, confers resistance to TDI-8304, yet enhances both enzyme inhibition and anti-parasite activity of a tripeptide vinyl sulfone ß2 inhibitor, WLW-vs. Here we present the high-resolution cryo-EM structures of Pf20S with TDI-8304, of human constitutive proteasome with TDI-8304, and of Pf20Sß6A117D with WLW-vs that give insights into the species selectivity of TDI-8304, resistance to it, and the collateral sensitivity associated with resistance, including that TDI-8304 binds ß2 and ß5 in wild type Pf20S as well as WLW-vs binds ß2 and ß5 in Pf20Sß6A117D. We further show that TDI-8304 kills P. falciparum as quickly as chloroquine and artemisinin and is active against P. cynomolgi at the liver stage. This increases interest in using these structures to facilitate the development of Pf20S inhibitors that target multiple proteasome subunits and limit the emergence of resistance.

Structure-Activity Relationship Studies of Antimalarial Plasmodium Proteasome Inhibitors─Part II.

With increasing reports of resistance to artemisinins and artemisinin-combination therapies, targeting the Plasmodium proteasome is a promising strategy for antimalarial development. We recently reported a highly selective Plasmodium falciparum proteasome inhibitor with anti-malarial activity in the humanized mouse model. To balance the permeability of the series of macrocycles with other drug-like properties, we conducted further structure-activity relationship studies on a biphenyl ether-tethered macrocyclic scaffold. Extensive SAR studies around the P1, P3, and P5 groups and peptide backbone identified compound TDI-8414. TDI-8414 showed nanomolar antiparasitic activity, no toxicity to HepG2 cells, high selectivity against the Plasmodium proteasome over the human constitutive proteasome and immunoproteasome, improved solubility and PAMPA permeability, and enhanced metabolic stability in microsomes and plasma of both humans and mice.

Selective isolation of hyaluronan by solid phase adsorption to silica.

A solid phase adsorption method for selective isolation of hyaluronan (HA) from biological samples is presented. Following enzymatic degradation of protein, HA can be separated from sulfated glycosaminoglycans, other unsulfated glycosaminoglycans, nucleic acids, and proteolytic fragments by adsorption to amorphous silica at specific salt concentrations. The adsorbed HA can be released from silica using neutral and basic aqueous solutions. HA ranging in size from ∼9 kDa to MDa polymers has been purified by this method from human serum and conditioned medium of cultured cells.

Design, Synthesis, and Optimization of Macrocyclic Peptides as Species-Selective Antimalaria Proteasome Inhibitors.

With over 200 million cases and close to half a million deaths each year, malaria is a threat to global health, particularly in developing countries. Plasmodium falciparum, the parasite that causes the most severe form of the disease, has developed resistance to all antimalarial drugs. Resistance to the first-line antimalarial artemisinin and to artemisinin combination therapies is widespread in Southeast Asia and is emerging in sub-Saharan Africa. The P. falciparum proteasome is an attractive antimalarial target because its inhibition kills the parasite at multiple stages of its life cycle and restores artemisinin sensitivity in parasites that have become resistant through mutation in Kelch K13. Here, we detail our efforts to develop noncovalent, macrocyclic peptide malaria proteasome inhibitors, guided by structural analysis and pharmacokinetic properties, leading to a potent, species-selective, metabolically stable inhibitor.

Use of tafenoquine to treat a patient with relapsing babesiosis with clinical and molecular evidence of resistance to azithromycin and atovaquone.

Tafenoquine is a highly effective treatment for Babesia microti infections in animal models. An immunocompromised patient infected by a strain of B. microti that was at least partially resistant to both azithromycin and atovaquone was treated with tafenoquine. Systematic clinical studies using tafenoquine for treating other patients with babesiosis should be considered.

Relapsed Babesia microti Infection Following Allogeneic Hematopoietic Cell Transplantation in a Patient With B-cell Acute Lymphoblastic Leukemia: Case Report and Review of the Literature.

A patient with relapsed/refractory B-cell acute lymphoblastic leukemia developed babesiosis before allogeneic hematopoietic cell transplantation while on atovaquone for Pneumocystis jirovecii pneumonia prophylaxis. Despite receiving a prolonged course of atovaquone and azithromycin until whole-blood Babesia microti DNA was no longer detected by polymerase chain reaction, her post-transplant course was complicated by relapsed babesiosis. We investigate the potential host and parasite characteristics causing relapsing/persistent infection.

Development of a Highly Selective Plasmodium falciparum Proteasome Inhibitor with Anti-malaria Activity in Humanized Mice.

Plasmodium falciparum proteasome (Pf20S) inhibitors are active against Plasmodium at multiple stages-erythrocytic, gametocyte, liver, and gamete activation stages-indicating that selective Pf20S inhibitors possess the potential to be therapeutic, prophylactic, and transmission-blocking antimalarials. Starting from a reported compound, we developed a noncovalent, macrocyclic peptide inhibitor of the malarial proteasome with high species selectivity and improved pharmacokinetic properties. The compound demonstrates specific, time-dependent inhibition of the ß5 subunit of the Pf20S, kills artemisinin-sensitive and artemisinin-resistant P. falciparum isolates in vitro and reduces parasitemia in humanized, P. falciparum-infected mice.

Impact of a Multidimensional Technical Skills Training Session Before Obstetrics and Gynaecology Clerkship Rotation on Performance and Exposure.

OBJECTIVE: This study sought to assess the impact of an obstetrics and gynaecology (OBGYN) multidimensional technical skills training session on medical student self-perceived confidence, objective performance, clinical exposure, and interest in OBGYN. METHODS: OBGYN clerkship students were divided into control (i.e., traditional curriculum) and intervention (i.e., additional pre-rotation simulation training in vaginal delivery, suturing and knot tying, speculum and bimanual examinations, and intrapartum cervical examination) groups. Both groups underwent an objective structured clinical examination (OSCE) after the rotation and completed pre- and post-rotation questionnaires to assess confidence, clinical exposure, and interest in OBGYN. The primary outcomes were self-reported confidence and OSCE scores, and the secondary outcomes were clinical exposure and interest in OBGYN. RESULTS: A total of 148 students participated. Both groups reported least confidence with vaginal delivery and cervical examinations before the rotation. There was improved self-confidence across all skills after the rotation, with the intervention group demonstrating greater improvement for vaginal delivery (3.36 vs. 3.10; P < 0.05) and cervical examination (3.14 vs. 2.86; P < 0.05). The intervention group also demonstrated a trend towards higher OSCE scores for all skills, but only speculum and bimanual examinations reached statistical significance. The intervention increased exposure from "0 to 5" to "6 to 10" for vaginal delivery and suturing. There was no difference in interest in OBGYN between the two groups. CONCLUSION: A technical skills training session before OBGYN clerkship is feasible and has the potential to increase students' confidence, OSCE performance, and hands-on procedural exposure. The optimal combination of skills to be included still needs to be defined.

Impact of an HPV Education and Vaccination Campaign among Canadian University Students.

BACKGROUND: Uptake of HPV vaccination among university students remains low despite risky sexual practices and increased prevalence of high-risk HPV genotypes. The study objective was to determine the level of knowledge related to HPV and cervical cancer among university students and to subsequently develop a targeted education and vaccination campaign to increase uptake. METHODS: Phase I was a pilot project in which participants were recruited as part of Cervical Cancer Awareness Week 2015 at two universities, one site immediately offering vaccination and the other not. A self-administered questionnaire was used to collect demographic information from participants and evaluate their baseline knowledge related to HPV and the risks of cervical cancer, in addition to determining barriers to vaccination and future willingness to be vaccinated. Data was compiled and analyzed using descriptive statistics of means and percentages. In phase II, which followed 1 year after, a targeted education and vaccination campaign was designed based on lessons learned from phase I, and vaccination uptake was reevaluated after 1 year. RESULTS: In phase I, 56 participants responded to a questionnaire related to HPV knowledge and cervical cancer. Among these, 29 students were vaccinated in a 2-day resident-run clinic. Overall, 63% felt they were not at risk of cervical cancer, though 88% knew HPV was the cause of cervical cancer. The three barriers identified to previous vaccination were lack of access to a doctor or a nurse (25%), financial reasons (25%), and low self-perceived risk (7%). There was a 50% three-dose completion rate in phase I. Based on this information, the education campaign in phase II was expanded in the subsequent year through social media, email communication, information booths, and individual solicitation. A total of 151 students were approached for individual solicitation and education. Among these, 64 students were vaccinated on site, including five men. Most importantly, there were 18 walk-ins resulting directly from the education initiatives and person-to-person solicitation. Subsequently, in 2016, 502 students were vaccinated at the McGill student health clinic and 455 at Concordia University. CONCLUSION: HPV vaccination rates in university students are readily increased through educational campaigns, of which person-to-person solicitation proved to be the most fruitful in this study. Identifying barriers to vaccination can guide future initiatives to maximize impact.

Outside the operating room: How a robotics program changed resource utilization on the inpatient Ward.

OBJECTIVE: To analyze the changes in the composition of the gynecologic oncology inpatient ward following the implementation of a robotic surgery program and its impact on inpatient resource utilization and costs. METHODS: Retrospective review of the medical charts of patients admitted onto the gynecologic oncology ward the year prior to and five years after the implementation of robotics. The following variables were collected: patient characteristics, hospitalization details (reason for admission and length of hospital stay), and resource utilization (number of hospitalization days, consultations, and imaging). RESULTS: Following the introduction of robotic surgery, there were more admissions for elective surgery yet these accounted for only 21% of the inpatient ward in terms of number of hospital days, compared to 36% prior to the robotic program. This coincided with a sharp increase in the overall number of patients operated on by a minimally invasive approach (15% to 76%, p<0.0001). The cost per surgical admission on the inpatient ward decreased by 59% ($9827 vs. $4058) in the robotics era. The robotics program contributed to a ward with higher proportion of patients with complex comorbidities (Charlson≥5: RR 1.06), Stage IV disease (RR 1.30), and recurrent disease (RR 1.99). CONCLUSION: Introduction of robotic surgery allowed for more patients to be treated surgically while simultaneously decreasing inpatient resource use. With more patients with non-surgical oncological issues and greater medical complexity, the gynecologic oncology ward functions more like a medical rather than surgical ward after the introduction of robotics, which has implications for hospital-wide resource planning.

The benefits of other treatments than in vitro fertilization to aid conception in minimal and mild endometriosis.

The treatment of minimal or mild endometriosis prior to non-in-vitro fertilization (IVF) assisted reproduction to improve pregnancy outcomes is controversial. Ovulation suppression may be offered to women who do not wish to conceive to suppress advancement of the disease. There is little evidence to suggest improvements in fertility associated ovarian suppression prior to non-IVF infertility treatments. The use of intrauterine insemination without ovulation induction offers little benefit, with low pregnancy rates in most studies. Surgical ablation seems to improve outcomes when other care will not be delivered. Although controversial, surgical ablation before ovulation induction may offer benefit but further studies would be helpful. Ovulation induction seems to increase pregnancy rates and either letrozole or clomiphene citrate should be considered as first line options. If pregnancy does not occur with three months of ovulation induction, based on dropping success rates with further cycles of ovarian stimulation, IVF should be offered.

Nonmalignant Sequelae of Unconfined Morcellation at Laparoscopic Hysterectomy or Myomectomy.

The objective of this study was to evaluate nonmalignant sequelae of unconfined morcellation at hysterectomy and myomectomy. We performed a systematic review following the PRISMA statement key words of "morcellation, uterine leiomyoma, uterine fibroid, laparoscopic myomectomy, laparoscopic total hysterectomy, and laparoscopic supracervical hysterectomy" and their combination. Fifty-one articles met the inclusion criteria: 11 articles were related to endometriosis, adenomyosis, and endometrial hyperplasia; 30 articles parasitic myoma; and 9 disseminated peritoneal leiomyomatosis (DPL) and 1 DPL and endometriosis. We found that laparoscopic hysterectomy or myomectomy with unconfined morcellation is associated with the risk of iatrogenic endometriosis (1.4%), adenomyosis (0.57%), parasitic myoma (0.9%), and rarely DPL. Our study showed that benign sequelae of uterine or myoma morcellation could be found in up to 1% of cases. This is much higher than the prevalence of uterine sarcoma after morcellation. Benign conditions have less consequences than malignancy, yet they are more common and might require another operation. Accordingly, if morcellation is required, confined morcellation should be considered.

Should we diagnose and treat minimal and mild endometriosis before medically assisted reproduction?

The treatment of minimal or mild endometriosis prior to assisted reproduction (ranging from intrauterine insemination to in vitro fertilization [IVF]) to improve the likelihood of success is controversial. Ovulation suppression is commonly used in endometriosis to decrease pain, however, there is little evidence to suggest improvements in fertility associated with this technique. Moreover, current evidence is sparse and does not support ovarian suppression prior to intrauterine insemination with or without ovulation induction, while there is some evidence favoring ovarian suppression with gonadotropin releasing hormone agonists prior to IVF to improve pregnancy rates. However, the majority of studies were performed in women with moderate to severe endometriosis. There is currently conflicting evidence regarding surgical ablation or removal of endometriomas prior to IVF, and its outcome on pregnancy rates. This review highlights the paucity of data in the management of endometriosis prior to assisted reproductive technologies and suggests that further studies are needed.

Hybrid intravascular ultrasound and optical coherence tomography catheter for imaging of coronary atherosclerosis.

OBJECTIVE: To demonstrate the feasibility of imaging human coronary atherosclerosis using a novel hybrid intravascular ultrasound (IVUS) and optical coherence tomography (OCT) imaging catheter. BACKGROUND: IVUS and OCT have synergistic advantages and recent studies involving both modalities suggest the use of a hybrid imaging catheter may offer improved guidance of coronary interventions and plaque characterization. METHODS: A 1.3 m custom hybrid IVUS-OCT imaging probe was built within a 4F catheter using a 42 MHz ultrasound transducer and an OCT imaging fiber. Coplanar images were simultaneously acquired ex vivo by both modalities in 31 arterial segments from 11 cadaveric human coronaries. IVUS and OCT images were acquired at 250 µm intervals, of which 13 of the arterial segments were selected as representative of a diverse set of pathological findings. The selected segments were then imaged with either digital X-ray or micro-CT, processed for histological analysis and compared with the corresponding IVUS and OCT images. RESULTS: Images of human coronary atherosclerosis using the hybrid IVUS-OCT catheter demonstrated a range of vascular pathologies that were confirmed on histology. The anticipated synergistic advantages of each modality were qualitatively apparent, including the deeper tissue penetration of IVUS and the superior contrast, resolution and near-field image quality of OCT. CONCLUSIONS: Preliminary ex vivo images using a hybrid IVUS-OCT catheter demonstrated feasibility in using the device for intracoronary imaging of atherosclerosis. Future studies will include in vivo imaging and larger samples sizes to enable quantitative comparisons of tissue characterization and feature identification using hybrid imaging catheters versus standalone IVUS and OCT imaging techniques. © 2012 Wiley Periodicals, Inc.

Waking up the next morning: surgeons' emotional reactions to adverse events.

CONTEXT: The adverse patient event is an inherent component of surgical practice, but many surgeons are unprepared for the profound emotional responses these events can evoke. This study explored surgeons' reactions to adverse events and their impact on subsequent judgement and decision making. METHODS: Using a constructivist grounded theory approach, we conducted 20 semi-structured, 60-minute interviews with surgeons across subspecialties, experience levels, and sexes to explore surgeons' recollections of reactions to adverse events. Further interviews were conducted with six general surgeons to explore more immediate reactions after 28 adverse events. Data coding was both inductive, developing a new framework based on emergent themes, and deductive, using an existing framework for care providers' reactions to adverse events. RESULTS: Surgeons expressed feeling unique and alone in the depths of their reactions to adverse events and consistently described four phases of response, each containing cognitive and emotive components, following such events. The initial phase (the kick) involved feelings of failure ('Am I good enough?') experienced with a significant physiological response. This was shortly followed by a second phase (the fall), during which the surgeon experienced a sense of chaos and assessed the extent of his or her contribution to the event ('Was it my fault?'). During the third phase (the recovery), the surgeon reflected on the adverse event ('What can I learn?') and experienced a sense of 'moving on'. In the fourth phase (the long-term impact), the surgeon experienced the prolonged and cumulative effects of these reactions on his or her own personal and professional identities. Surgeons also described an effect on their clinical judgement, both for the case in question (minimisation) and future cases (overcompensation). CONCLUSIONS: Surgeons progress through a series of four phases following adverse events that are potentially caused by or directly linked to surgeon error. The framework provided by this study has implications for teaching, surgeon wellness and surgeon error.

"First, do no harm": balancing competing priorities in surgical practice.

PURPOSE: To explore surgeons' perceptions of the factors that influence their intraoperative decision making, and implications for professional self-regulation and patient safety. METHOD: Semistructured interviews were conducted with 39 academic surgeons from various specialties at four hospitals associated with the University of Toronto Faculty of Medicine. Purposive and theoretical sampling was performed until saturation was achieved. Thematic analysis of the transcripts was conducted using a constructivist grounded-theory approach and was iteratively elaborated and refined as data collection progressed. A preexisting theoretical professionalism framework was particularly useful in describing the emergent themes; thus, the analysis was both inductive and deductive. RESULTS: Several factors that surgeons described as influencing their decision making are widely accepted ("avowed," or in patients' best interests). Some are considered reasonable for managing multiple priorities external to the patient but are not discussed openly ("unavowed," e.g., teaching pressures). Others are actively denied and consider the surgeon's best interests rather than the patient's ("disavowed," e.g., reputation). Surgeons acknowledged tension in balancing avowed factors with unavowed and disavowed factors; when directly asked, they found it difficult to acknowledge that unavowed and disavowed factors could lead to patient harm. CONCLUSIONS: Some factors that are not directly related to the patient enter into surgeons' intraoperative decision making. Although these are probably reasonable to consider within "real-world" practice, they are not sanctioned in current patient care constructs or taught to trainees. Acknowledging unavowed and disavowed factors as sources of pressure in practice may foster critical self-reflection and transparency when discussing surgical errors.