Overexpression of multidrug resistance-associated protein 1 protects against cardiotoxicity by augmenting the doxorubicin efflux from cardiomyocytes

Doxorubicin is a commonly used anti-cancer drug used in treating a variety of malignancies. However, a major adverse effect is cardiotoxicity, which is dose dependent and can be either acute or chronic. Doxorubicin causes injury by DNA damage, the formation of free reactive oxygen radicals and induction of apoptosis. Our aim is to induce expression of the multidrug resistance-associated protein 1 (MRP1) in cardiomyocytes derived from human iPS cells (hiPSC-CM), to determine whether this will allow cells to effectively remove doxorubicin and confer cardioprotection. We generated a lentivirus vector encoding MRP1 (LV.MRP1) and validated its function in HEK293T cells and stem cell-derived cardiomyocytes (hiPSC-CM) by quantitative PCR and western blot analysis. The activity of the overexpressed MRP1 was also tested, by quantifying the amount of fluorescent dye exported from the cell by the transporter. We demonstrated reduced dye sequestration in cells overexpressing MRP1. Finally, we demonstrated that hiPSC-CM transduced with LV.MRP1 were protected against doxorubicin injury. In conclusion, we have shown that we can successfully overexpress MRP1 protein in hiPSC-CM, with functional transporter activity leading to protection against doxorubicin-induced toxicity.     

Investigating the inoculum dynamics of Cladosporium on the surface of raspberry fruits and in the air

Raspberry production is under threat from the emerging fungal pathogenic genus Cladosporium. We used amplicon-sequencing, coupled with qPCR, to investigate how fruit age, fruit location within a polytunnel, polytunnel location and sampling date affected the fruit epiphytic microbiome. Fruit age was the most important factor impacting the fungal microbiome, followed by sampling date and polytunnel location. In contrast, polytunnel location and fruit age were important factors impacting the bacterial microbiome composition, followed by the sampling date. The within-tunnel location had a small significant effect on the fungal microbiome and no effect on the bacterial microbiome. As fruit ripened, fungal diversity increased and the bacterial diversity decreased. Cladosporium was the most abundant fungus of the fruit epiphytic microbiome, accounting for nearly 44% of all fungal sequences. Rotorod air samplers were used to study how the concentration of airborne Cladosporium inoculum (quantified by qPCR) varied between location (inside and outside the polytunnel) and time (daytime vs. nighttime). Quantified Cladosporium DNA was significantly higher during the day than the night and inside the polytunnel than the outside. This study demonstrated the dynamic nature of epiphytic raspberry fruit microbiomes and airborne Cladosporium inoculum within polytunnels, which will impact disease risks on raspberry fruit.     

Effectiveness of knowledge brokering and recommendation dissemination for influencing healthcare resource allocation decisions: A cluster randomised controlled implementation trial

BackgroundImplementing evidence into clinical practice is a key focus of healthcare improvements to reduce unwarranted variation. Dissemination of evidence-based recommendations and knowledge brokering have emerged as potential strategies to achieve evidence implementation by influencing resource allocation decisions. The aim of this study was to determine the effectiveness of these two research implementation strategies to facilitate evidence-informed healthcare management decisions for the provision of inpatient weekend allied health services.Methods and findingsThis multicentre, single-blinded (data collection and analysis), three-group parallel cluster randomised controlled trial with concealed allocation was conducted in Australian and New Zealand hospitals between February 2018 and January 2020. Clustering and randomisation took place at the organisation level where weekend allied health staffing decisions were made (e.g., network of hospitals or single hospital). Hospital wards were nested within these decision-making structures. Three conditions were compared over a 12-month period: (1) usual practice waitlist control; (2) dissemination of written evidence-based practice recommendations; and (3) access to a webinar-based knowledge broker in addition to the recommendations. The primary outcome was the alignment of weekend allied health provision with practice recommendations at the cluster and ward levels, addressing the adoption, penetration, and fidelity to the recommendations. The secondary outcome was mean hospital length of stay at the ward level. Outcomes were collected at baseline and 12 months later. A total of 45 clusters (n = 833 wards) were randomised to either control (n = 15), recommendation (n = 16), or knowledge broker (n = 14) conditions. Four (9%) did not provide follow-up data, and no adverse events were recorded. No significant effect was found with either implementation strategy for the primary outcome at the cluster level (recommendation versus control β 18.11 [95% CI −8,721.81 to 8,758.02] p = 0.997; knowledge broker versus control β 1.24 [95% CI −6,992.60 to 6,995.07] p = 1.000; recommendation versus knowledge broker β −9.12 [95% CI −3,878.39 to 3,860.16] p = 0.996) or ward level (recommendation versus control β 0.01 [95% CI 0.74 to 0.75] p = 0.983; knowledge broker versus control β −0.12 [95% CI −0.54 to 0.30] p = 0.581; recommendation versus knowledge broker β −0.19 [−1.04 to 0.65] p = 0.651). There was no significant effect between strategies for the secondary outcome at ward level (recommendation versus control β 2.19 [95% CI −1.36 to 5.74] p = 0.219; knowledge broker versus control β −0.55 [95% CI −1.16 to 0.06] p = 0.075; recommendation versus knowledge broker β −3.75 [95% CI −8.33 to 0.82] p = 0.102). None of the control or knowledge broker clusters transitioned to partial or full alignment with the recommendations. Three (20%) of the clusters who only received the written recommendations transitioned from nonalignment to partial alignment. Limitations include underpowering at the cluster level sample due to the grouping of multiple geographically distinct hospitals to avoid contamination.ConclusionsOwing to a lack of power at the cluster level, this trial was unable to identify a difference between the knowledge broker strategy and dissemination of recommendations compared with usual practice for the promotion of evidence-informed resource allocation to inpatient weekend allied health services. Future research is needed to determine the interactions between different implementation strategies and healthcare contexts when translating evidence into healthcare practice.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN12618000029291.

In a cluster randomized controlled implementation trial, Dr. Mitchell N Sarkies and colleagues examine the effectiveness of knowledge brokering and recommendation dissemination in influencing healthcare resource allocation decisions in Australia and New Zealand.     

Lessons from the Glass Cockpit: Innovation in Alarm Systems to Support Cognitive Work

Nurses working in the hospital setting increasingly have become overburdened by managing alarms that, in many cases, provide low information value regarding patient health. The current trend, aided by disposable, wearable technologies, is to promote patient monitoring that does not require entering a patient''s room. The development of telemetry alarms and middleware escalation devices adds to the continued growth of auditory, visual, and haptic alarms to the hospital environment but can fail to provide a more complete understanding of patient health. As we begin to innovate to both address alarm overload and improve patient management, perhaps using fundamentally different integration architectures, lessons from the aviation flight deck are worth considering. Commercial jet transport systems and their alarms have evolved slowly over many decades and have developed integration methods that account for operational context, provide multiple response protocol levels, and present a more integrated view of the airplane system state. We articulate three alarm system objectives: (1) supporting hazard management, (2) establishing context, and (3) supporting alarm prioritization. More generally, we present the case that alarm design in aviation can spur directions for innovation for telemetry monitoring systems in hospitals.

Healthcare, and the hospital setting in particular, has experienced rapid growth of auditory, visual, and haptic alarms. These alarms can be notoriously unreliable or can focus on narrowly defined changes to the patient''s state.1 Further, this alarm proliferation has led nursing staff to become increasingly overburdened and distressed by managing alarms.2 Current alarm system architectures do not effectively integrate meaningful data that support increased patient status awareness and management.3 In contrast, commercial jet transports, over many decades, have developed integration methods that account for operational context, provide multiple response protocol levels, and present a more integrated view of airplane state to support operational decision making. Similar methods for advanced control rooms in nuclear power generation have been reviewed by Wu and Li.4In healthcare, The Joint Commission (TJC) and hospital quality departments have generated guidance that further elevates the need to address the industry''s “alarm problem.” In 2014, TJC issued an accreditation requirement (National Patient Safety Goal 06.01.01) titled, “Reduce patient harm associated with clinical alarm systems.”5 This requirement continues to be included in the 2020 requirements for accreditation.From the authors'' perspective, this requirement is leading to solutions that will not effectively support performance of essential tasks and is moving away from the types of innovations that are being sought in aviation and other settings. For example, healthcare administrators advocate categorizing alarms into high-priority (“run”), medium-priority (“walk”), and low-priority (“shuffle”) alarms independent of unit context, hospital context, situational context, and historical patient context.6 In addition, each alarm category is assigned a minimum response time. When nurses do not meet response time targets, administrators may add staff (“telemetry monitor watchers”), increase the volume of alarms, escalate alarms to other staff to respond, increase the “startling” nature of alarms to better direct attention, and benchmark average response times by individual nurse identifiers. Although well intentioned, these approaches can sometimes add to the alarm overload problem by creating more alarms and involving more people in alarm response.The authors, who have investigated human performance in several operational settings, believe that a need exists to reflect more broadly on the role of alarms in understanding and managing a system (be it an aircraft or a set of patients in a hospital department). Most alarms in hospitals signal when a variable is outside a prespecified range that is determined from the patient population (e.g., high heart rate), when a change in cardiac rhythm occurs (e.g., ventricular fibrillation [V-fib]), or when a problem occurs with the alarm system (e.g., change battery). These alarms support shifts in attention when the event being alarmed requires an action by a nurse and when the relative priority of the response is clear in relation to competing demands.Certain alarms are useful for other purposes, such as aiding situation awareness about planned, routine tasks (e.g., an expected event of high heart rate has occurred, which indicates that a staff member is helping a patient to the bathroom). Increasingly, secondary alarm notification systems (SANSs), otherwise known as middleware escalation systems, are incorporating communications through alarms, such as patient call systems, staff emergency broadcasts, and demands for “code blue” teams to immediately go to a patient''s bedside.Thus, alarms are used to attract attention (i.e., to orient staff to an important change). However, from a cognitive engineering perspective, we believe alarms can also be used to support awareness, prioritization, and decision making. That is, the current siloed approach to alarm presentation in healthcare, which is driven by technology, impedes the ability to properly understand and appreciate the implications of alarms. Understanding the meaning and implications of alarms can best be achieved when they are integrated via a system interface that places the alarm in the broader context of system state. We hope that sharing our insights can spur both design and alarm management innovations for bedside telemetry monitoring devices and related middleware escalation systems and dashboards.In this article, we provide insights from human factors research, and from the integrated glass cockpit in particular, to prompt innovation with clinical alarm systems. To draw lessons from aviation and other domains, we conducted a series of meetings among three human factors engineers with expertise in alarm design in healthcare, aviation, nuclear power generation, and military command and control domains. In the process, we identified differences in the design, use, and philosophies for managing alarms in different domains; defined alarm systems; clarified common elements in the “alarm problem” across these domains; articulated objectives for an alarm system that supports a human operator in controlling a complex process (i.e., supervisory control); and identified levels of alarm system maturity. Based on these activities, we assert that:

1. Clinical alarm systems fail to reduce unnecessary complexity compared with the integrated glass cockpit.
2. Aviation and clinical alarm systems share core objectives.
3. The challenges with aviation and clinical alarm systems are similar, including where alarm systems fall short of their objectives.
4. We can demarcate levels in the process of alarm system evolution, largely based on alarm reliability, system integration, and how system state is described. The higher levels point the way for innovation in clinical alarm systems.

     

Maternal Piwi regulates primordial germ cell development to ensure the fertility of female progeny in Drosophila

In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the Drosophila early embryo. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.     

Synthesis and in vitro evaluation of SG3227, a pyrrolobenzodiazepine dimer antibody-drug conjugate payload based on sibiromycin

A novel pyrrolobenzodiazepine dimer payload, SG3227, was rationally designed based on the naturally occurring antitumour compound sibiromycin. SG3227 was synthesized from a dimeric core in an efficient fashion. An unexpected room temperature Diels-Alder reaction occurred during the final step of the synthesis and was circumvented by use of an iodoacetamide conjugation moiety in place of a maleimide. The payload was successfully conjugated to trastuzumab and the resulting ADC exhibited potent activity against a HER2-expressing human cancer cell line in vitro.