Utilizing a Dual Human Transporter MDCKII-MDR1-BCRP Cell Line to Assess Efflux at the Blood Brain Barrier.

To facilitate the design of drugs readily able to cross the blood brain barrier (BBB), a Madin-Darby canine kidney (MDCK) cell line was established that over expresses both P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP), the main human efflux transporters of the BBB. Proteomics analyses indicate BCRP is expressed at a higher level than Pgp in this cell line. This cell line shows good activity for both transporters [BCRP substrate dantrolene efflux ratio (ER) 16.3 ± 0.9, Pgp substrate quinidine ER 27.5 ± 1.2], and use of selective transporter inhibitors enables an assessment of the relative contributions to overall ERs. The MDCKII-MDR1-BCRP ER negatively correlates with rat unbound brain/unbound plasma ratio, Kpuu Highly brain penetrant compounds with rat Kpuu ≥ 0.3 show ERs ≤ 2 in the MDCKII-MDR1-BCRP assay while compounds predominantly excluded from the brain, Kpuu ≤ 0.05, demonstrate ERs ≥ 20. A subset of compounds with MDCKII-MDR1-BCRP ER < 2 and rat Kpuu < 0.3 were shown to be substrates of rat Pgp using a rat transfected cell line, MDCKII-rMdr1a. These compounds also showed ERs > 2 in the human National Institutes of Health (NIH) MDCKI-MDR1 (high Pgp expression) cell line, which suggests that they are weak human Pgp substrates. Characterization of 37 drugs targeting the central nervous system in the MDCKII-MDR1-BCRP efflux assay show 36 have ERs < 2. In drug discovery, use of the MDCKII-MDR1-BCRP in parallel with the NIH MDCKI-MDR1 cell line is useful for identification of compounds with high brain penetration. SIGNIFICANCE STATEMENT: A single cell line that includes both the major human efflux transporters of the blood brain barrier (MDCKII-MDR1-BCRP) has been established facilitating the rapid identification of efflux substrates and enabling the design of brain penetrant molecules. Efflux ratios using this cell line demonstrate a clear relationship with brain penetration as defined by rat brain Kpuu.

Post-meal exercise under ecological conditions improves post-prandial glucose levels but not 24-hour glucose control.

We investigated whether post-meal walking (PMW) improved post-prandial glucose and 24h glucose control under free-living conditions among physically inactive young women. METHODS: Young women (Age: 20±1years; percent body fat: 28.2 ± 12%; BMI: 23.8 ± 4.2kg·m-1) completed a randomised crossover study to assess if PMW confers benefit. On the PMW day, women completed three bouts of brisk walks, and on the Control day they were instructed to follow normal habitual activities. Continuous glucose monitors captured post-prandial and 24h glucose, and physical activity monitors tracked physical activity throughout the study. RESULTS: PMW walking increased total daily step count (Control = 9,159 ± 2,962 steps vs. PMW = 14,611±3,891 steps, p<0.001) and activity scores (Control=33.87±1.16 METs·h vs. PMW = 36.11±1.58 METs·h, p < 0.001). PMW led to lower 3h average post-prandial glucose (main effect of condition, p=0.011) and 3h post-prandial area under curve glucose responses (main effect of condition, p = 0.027) compared to the control condition. Post hoc analysis revealed the largest decline occurred after dinner (3h average glucose Control = 7.55±1.21 mmol/L vs. PMW = 6.71 ± 0.80mmol/L, p = 0.039), when insulin sensitivity is typically diminished. Despite improvements in post-prandial glucose control, this did not translate to improvements in 24h glucose control (p > 0.05). CONCLUSION: Physically inactive and metabolically healthy young women, PMW improves post-prandial glucose but not 24h glucose control.

Outcomes Analysis of Textured Versus Smooth Tissue Expanders in Breast Reconstruction.

INTRODUCTION: Because of concerns related to the correlation of breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) and textured implants, the use of smooth devices in breast reconstruction has been increasing. Currently, there is a paucity of literature evaluating the safety of smooth tissue expanders (STEs), which are now being used more frequently in first-stage breast reconstruction. This study sought to compare the safety and outcomes associated with STEs compared with textured tissue expanders in prosthesis-based breast reconstruction. METHODS: A single-institution retrospective review of 394 patients undergoing tissue expander-based breast reconstruction (147 smooth and 247 textured) between 2015 and 2019 was conducted. Patient demographics, comorbidities, treatment characteristics, complications, and surgical outcomes were evaluated. Data analysis was performed using Fisher exact and t tests. RESULTS: No significant difference in demographics or complication rates were identified, including rates of hematoma, seroma, wound dehiscence, delayed wound healing, infection, tissue expander malposition, nipple necrosis, mastectomy flap necrosis, reoperation, readmission, and explantation. Average follow-up was 19 and 22 months for the smooth and textured groups, respectively. No cases of BIA-ALCL were identified in either group. CONCLUSIONS: With equivocal safety profiles and no demonstrated risk in BIA-ALCL associated with STEs, this study supports the safety of using STEs compared with textured tissue expanders in prosthesis-based breast reconstruction with the advantage in preventing BIA-ALCL and concludes that there is no role for textured breast expanders.

Impact of Interindividual Differences in Plasma Fraction Unbound on the Pharmacokinetics of a Novel Syk Kinase Inhibitor in Beagle Dogs.

Inconsistencies in pharmacokinetic parameters between individual animals in preclinical studies are a common occurrence. Often such differences between animals are simply accepted as experimental variability rather than as indications of specific differences in animal phenotype that could lead to a different interpretation of the data. The fraction unbound in plasma is one factor influencing pharmacokinetic parameters and is typically determined using pooled plasma from multiple animals, making the assumption that there is limited population variance. However, this assumption is not often tested and may not hold true if there are polymorphisms affecting binding or variation in the concentrations of individual plasma proteins that could give rise to different fraction unbound phenotypes in individual animals. During profiling of a novel Syk inhibitor, AZ8399, striking interindividual differences in total plasma clearance and volume of distribution were observed between dogs consistent with differences in fraction unbound between animals. Determination of the fraction unbound showed a ∼5-fold difference in fraction unbound between the animals in the study. Broader analysis of individual dogs across a colony demonstrated a correlation between individual animal fraction unbound with total plasma clearance and volume of distribution. The concentrations of the common drug-binding proteins albumin and α1-acid glycoprotein in plasma were determined, and α1-acid glycoprotein levels were found to correlate with fraction unbound. Finally, single-nucleotide polymorphisms were identified at c.502 and c.522 of exon 5 of the dog α1-acid glycoprotein gene that may be correlated to the α1-acid glycoprotein concentration phenotype observed. SIGNIFICANCE STATEMENT: The current work demonstrates the potential for significant interindividual differences in plasma fraction unbound in beagle dogs and goes on to examine the underlying cause for the compound described. The findings suggest that the application of a population mean value of fraction unbound generated from a pooled sample may not always be appropriate and could introduce significant errors in scaling of in vitro clearance values, PBPK understanding, and interpretation of PKPD or toxicokinetic data in the context of unbound concentrations.

Focus and social contagion of environmental organization advocacy on Twitter.

Agriculture, overexploitation, and urbanization remain the major threats to biodiversity in the Anthropocene. The attention these threats garner among leading environmental nongovernmental organizations (eNGOs) and the wider public is critical in fostering the political will necessary to reverse biodiversity declines worldwide. I analyzed the advocacy of leading eNGOs on Twitter by scraping account timelines, screening content for advocacy relating to biodiversity threats and, for prevalent threats, further screening content for positive and negative emotional language with a sentiment lexicon. Twitter advocacy was dominated by the major threats of climate change and overexploitation and the minor threat of plastic pollution. The major threats of agriculture, urbanization, invasions, and pollution were rarely addressed. Content relating to overexploitation and plastic pollution was more socially contagious than other content. Increasing emotional negativity further increased social contagion, whereas increasing emotional positivity did not. Scientists, policy makers, and eNGOs should consider how narrowly focused advocacy on platforms like Twitter will contribute to effective global biodiversity conservation.

Enfoque y Contagio Social de la Promoción de Organizaciones Ambientales en Twitter Resumen La agricultura, la sobreexplotación y la urbanización siguen siendo las principales amenazas para la biodiversidad durante el Antropoceno. La atención que atraen estas amenazas entre las principales organizaciones no gubernamentales ambientales (ONGa) y el público en general es crítico para el fomento de la voluntad política necesaria para revertir las declinaciones en la biodiversidad a nivel mundial. Analicé la promoción de las principales ONGa en Twitter mediante la extracción de información de la cronología de las cuentas, la búsqueda de contenidos de promoción relacionados con las amenazas para la biodiversidad y el lenguaje emocional negativo con un léxico de opinión. La promoción en Twitter estuvo dominada por dos amenazas predominantes y una amenaza menor: el cambio climático y la sobreexplotación y la contaminación por plástico, respectivamente. Las amenazas predominantes como la agricultura, la urbanización, las invasiones y la contaminación fueron mencionadas en raras ocasiones. El contenido relacionado con la sobreexplotación y la contaminación por plástico tuvo un mayor contagio social que cualquier otro contenido. El incremento en la negatividad emocional incrementó todavía más el contagio social, mientras que el incremento en la positividad emocional no lo hizo. Los científicos, los formuladores de políticas y las ONGa deberían considerar cómo contribuirá la promoción, con un enfoque casi exclusivo en plataformas como Twitter, a la conservación efectiva de la biodiversidad en todo el mundo.

Extreme Fermi Surface Smearing in a Maximally Disordered Concentrated Solid Solution.

We show that the Fermi surface can survive the presence of extreme compositional disorder in the equiatomic alloy Ni_{0.25}Fe_{0.25}Co_{0.25}Cr_{0.25}. Our high-resolution Compton scattering experiments reveal a Fermi surface which is smeared across a significant fraction of the Brillouin zone (up to 40% of 2π/a). The extent of this smearing and its variation on and between different sheets of the Fermi surface have been determined, and estimates of the electron mean free path and residual resistivity have been made by connecting this smearing with the coherence length of the quasiparticle states.

Is Thoracodorsal Nerve Transection Needed in Latissimus Dorsi Breast Reconstruction?

PURPOSE: Latissimus dorsi (LD) breast reconstruction is of proven efficacy. Advantages of thoracodorsal nerve transection are potential prevention of muscle spasticity/movement; disadvantages are possible long-term muscle atrophy and volume loss. This study's purpose is to provide data that would support or refute nerve transection. METHODS: A retrospective study of all LD breast reconstruction patients from 2011 to 2017 was done. Total number of flaps was identified, as was thoracodorsal nerve transection. Outcomes were noted for symptomatic muscle spasticity/involuntary movement, and complications inclusive of hematoma, seroma, and capsular contracture. RESULTS: A total of 125 patients had 170 flaps. Eighty-one flaps had nerve transection; 89 did not. These cohorts had no differences in comorbidities, indications of surgery (cancer vs prophylactic), irradiation, delayed/immediate reconstruction, and use of expanders. Symptomatic muscle movement/spasticity was not significantly different: 3 (3.7%) of 78 in transection and 5 (5.6%) of 84 in nontransection (P = 0.55, χ). Incidence of seroma in the transection group was notably higher (18/81; 22% vs 12%) but not statistically significant (P = 0.09, χ). No differences existed in all other outcomes. CONCLUSIONS: Symptomatic spasticity or involuntary muscle movement occurs in a small number of patients with LD breast reconstruction and is not affected by thoracodorsal nerve transection. Movement after transection is likely due to aberrant nerve innervation and reinnervation. The absence of movement without transection is due to disruption of muscle position and origin after transfer. Seroma formation may be affected by increased axillary dissection required for nerve transection. These data do not support nerve transection, and therefore, it is not recommended.

Biomass encounter rates limit the size scaling of feeding interactions.

The rate that consumers encounter resources in space necessarily limits the strength of feeding interactions that shape ecosystems. To explore the link between encounters and feeding, we first compiled the largest available dataset of interactions in the marine benthos by extracting data from published studies and generating new data. These data indicate that the size-scaling of feeding interactions varies among consumer groups using different strategies (passive or active) to encounter different resource types (mobile or static), with filter feeders exhibiting the weakest feeding interactions. Next, we used these data to develop an agent-based model of resource biomass encounter rates, underpinned by consumer encounter strategy and resource biomass density. Our model demonstrates that passive strategies for encountering small, dispersed resources limits biomass encounter rates, necessarily limiting the strength of feeding interactions. Our model is based on generalisable assumptions, providing a framework to assess encounter-based drivers of consumption and coexistence across systems.

Reducing Postsurgical Wound Complications: A Critical Review.

GENERAL PURPOSE: To provide information on risk factors for surgical site infections (SSIs) and actions to mitigate that risk. TARGET AUDIENCE: This continuing education activity is intended for surgeons, surgical teams, physicians, physician assistants, nurse practitioners, and nurses with an interest in skin and wound care. LEARNING OBJECTIVES/OUTCOMES: After participating in this educational activity, the participant should be better able to:1. Identify modifiable risk factors associated with the development of SSIs.2. Select steps to mitigate the risks for and morbidity from SSIs. ABSTRACT: Given the current reimbursement structure, the avoidance of a surgical site infection (SSI) is crucial. Although many risk factors are associated with the formation of an SSI, a proactive and interprofessional approach can help modify some factors. Postoperative strategies also can be applied to help prevent an SSI. If an SSI becomes a chronic wound, there are recommended guidelines and strategies that can foster healing.

On the context-dependent scaling of consumer feeding rates.

The stability of consumer-resource systems can depend on the form of feeding interactions (i.e. functional responses). Size-based models predict interactions - and thus stability - based on consumer-resource size ratios. However, little is known about how interaction contexts (e.g. simple or complex habitats) might alter scaling relationships. Addressing this, we experimentally measured interactions between a large size range of aquatic predators (4-6400 mg over 1347 feeding trials) and an invasive prey that transitions among habitats: from the water column (3D interactions) to simple and complex benthic substrates (2D interactions). Simple and complex substrates mediated successive reductions in capture rates - particularly around the unimodal optimum - and promoted prey population stability in model simulations. Many real consumer-resource systems transition between 2D and 3D interactions, and along complexity gradients. Thus, Context-Dependent Scaling (CDS) of feeding interactions could represent an unrecognised aspect of food webs, and quantifying the extent of CDS might enhance predictive ecology.

KDM4B is a master regulator of the estrogen receptor signalling cascade.

The importance of the estrogen receptor (ER) in breast cancer (BCa) development makes it a prominent target for therapy. Current treatments, however, have limited effectiveness, and hence the definition of new therapeutic targets is vital. The ER is a member of the nuclear hormone receptor superfamily of transcription factors that requires co-regulator proteins for complete regulation. Emerging evidence has implicated a small number of histone methyltransferase (HMT) and histone demethylase (HDM) enzymes as regulators of ER signalling, including the histone H3 lysine 9 tri-/di-methyl HDM enzyme KDM4B. Two recent independent reports have demonstrated that KDM4B is required for ER-mediated transcription and depletion of the enzyme attenuates BCa growth in vitro and in vivo. Here we show that KDM4B has an overarching regulatory role in the ER signalling cascade by controlling expression of the ER and FOXA1 genes, two critical components for maintenance of the estrogen-dependent phenotype. KDM4B interacts with the transcription factor GATA-3 in BCa cell lines and directly co-activates GATA-3 activity in reporter-based experiments. Moreover, we reveal that KDM4B recruitment and demethylation of repressive H3K9me3 marks within upstream regulatory regions of the ER gene permits binding of GATA-3 to drive receptor expression. Ultimately, our findings confirm the importance of KDM4B within the ER signalling cascade and as a potential therapeutic target for BCa treatment.

The lysine demethylase, KDM4B, is a key molecule in androgen receptor signalling and turnover.

The androgen receptor (AR) is a key molecule involved in prostate cancer (PC) development and progression. Post-translational modification of the AR by co-regulator proteins can modulate its transcriptional activity. To identify which demethylases might be involved in AR regulation, an siRNA screen was performed to reveal that the demethylase, KDM4B, may be an important co-regulator protein. KDM4B enzymatic activity is required to enhance AR transcriptional activity; however, independently of this activity, KDM4B can enhance AR protein stability via inhibition of AR ubiquitination. Importantly, knockdown of KDM4B in multiple cell lines results in almost complete depletion of AR protein levels. For the first time, we have identified KDM4B to be an androgen-regulated demethylase enzyme, which can influence AR transcriptional activity not only via demethylation activity but also via modulation of ubiquitination. Together, these findings demonstrate the close functional relationship between AR and KDM4B, which work together to amplify the androgen response. Furthermore, KDM4B expression in clinical PC specimens positively correlates with increasing cancer grade (P < 0.001). Consequently, KDM4B is a viable therapeutic target in PC.

Deubiquitinating enzyme Usp12 is a novel co-activator of the androgen receptor.

The androgen receptor (AR), a member of the nuclear receptor family, is a transcription factor involved in prostate cell growth, homeostasis, and transformation. AR is a key protein in growth and development of both normal and malignant prostate, making it a common therapeutic target in prostate cancer. AR is regulated by an interplay of multiple post-translational modifications including ubiquitination. We and others have shown that the AR is ubiquitinated by a number of E3 ubiquitin ligases, including MDM2, CHIP, and NEDD4, which can result in its proteosomal degradation or enhanced transcriptional activity. As ubiquitination of AR causes a change in AR activity or stability and impacts both survival and growth of prostate cancer cells, deubiquitination of these sites has an equally important role. Hence, deubiquitinating enzymes could offer novel therapeutic targets. We performed an siRNA screen to identify deubiquitinating enzymes that regulate AR; in that screen ubiquitin-specific protease 12 (Usp12) was identified as a novel positive regulator of AR. Usp12 is a poorly characterized protein with few known functions and requires the interaction with two cofactors, Uaf-1 and WDR20, for its enzymatic activity. In this report we demonstrate that Usp12, in complex with Uaf-1 and WDR20, deubiquitinates the AR to enhance receptor stability and transcriptional activity. Our data show that Usp12 acts in a pro-proliferative manner by stabilizing AR and enhancing its cellular function.

Fortune favours the bold: a higher predator reduces the impact of a native but not an invasive intermediate predator.

Emergent multiple predator effects (MPEs) might radically alter predictions of predatory impact that are based solely on the impact of individuals. In the context of biological invasions, determining if and how the individual-level impacts of invasive predators relates to their impacts in multiple-individual situations will inform understanding of how such impacts might propagate through recipient communities. Here, we use functional responses (the relationship between prey consumption rate and prey density) to compare the impacts of the invasive freshwater mysid crustacean Hemimysis anomala with a native counterpart Mysis salemaai when feeding on basal cladoceran prey (i) as individuals, (ii) in conspecific groups and (iii) in conspecific groups in the presence of a higher fish predator, Gasterosteus aculeatus. In the absence of the higher predator, the invader consumed significantly more basal prey than the native, and consumption was additive for both mysid species - that is, group consumption was predictable from individual-level consumption. Invaders and natives were themselves equally susceptible to predation when feeding with the higher fish predator, but an MPE occurred only between the natives and higher predator, where consumption of basal prey was significantly reduced. In contrast, consumption by the invaders and higher predator remained additive. The presence of a higher predator serves to exacerbate the existing difference in individual-level consumption between invasive and native mysids. We attribute the mechanism responsible for the MPE associated with the native to a trait-mediated indirect interaction, and further suggest that the relative indifference to predator threat on the part of the invader contributes to its success and impacts within invaded communities.

Wounds in patients with HIV.

Highly active antiretroviral therapy has dramatically reduced morbidity and mortality among patients who are HIV-positive. A retrospective review of the authors' data separated subjects into cohorts based on HIV status and matched them for age and gender. The authors' data reveal a higher fraction of venous ulcers compared with a lower fraction of pressure ulcers in the seropositive population.

Perioperative use of bispectral (BIS) monitor for a pressure ulcer patient with locked-in syndrome (LIS).

The bispectral (BIS) monitor uses brain electroencephalographic data to measure the depth of sedation and pharmacological response during anaesthetic procedures. In this case, the BIS monitor was used for another purpose, to demonstrate postoperatively to the nursing staff that a patient with history of locked-in syndrome (LIS), who underwent pressure ulcer debridement, had periods of wakefulness and apparent sensation, even with his eyes closed. Furthermore, as patients with LIS can feel pain, despite being unable to move, local block or general anaesthesia should be provided for sharp surgical debridement and other painful procedures. This use of the BIS has shown that as a general rule, the staff should treat the patient as though he might be awake and sensate even if he does not open his eyes or move his limbs. The goal of this study was to continuously monitor pain level and communicate these findings to the entire wound team, i.e. anaesthesiologists, surgeons and nurses.

Less is more: limiting narcotic prescription quantities for common orthopedic procedures.

BACKGROUND: Clinicians are now appreciating that the perception of pain is a multifaceted, biopsychosocial construct. Expectation of postsurgical pain is part of this construct and should be considered preoperatively. It is our belief that by establishing reasonable expectations with preoperative teaching, we can minimize narcotic use and lessen untoward issues that can potentially follow. With this goal in mind, we have been using a comprehensive pre- and postoperative program for our outpatient orthopedic surgery patients for the last 5 years, which includes physical, pharmacologic, and simple sport psychological techniques. MATERIALS AND METHODS: We reviewed postoperative prescription narcotic purchases in 133 consecutive surgical patients during the last year (2013). All patients were given a prescription postoperatively for 10 hydrocodone 5-mg/acetaminophen 500-mg tablets, with 1 refill. We then contacted the patients' pharmacies to assess the actual amount purchased. RESULTS: Data were available for 100 patients. Of these, 62 patients had undergone "simple" arthroscopies and 38 had had "open" procedures, including 25 anterior cruciate ligament reconstructions, 4 tibial tubercle osteotomies, and various other surgeries. Of the 62 arthroscopies, 24 patients (39%) refilled their prescriptions, with 4 patients (6%) needing > 1 refill. Of the 38 open procedures, 16 patients (42%) refilled their medications, 2 (5%), more than once. Thus, 89% of patients required ≤ 20 narcotic tablets after undergoing common orthopedic operations. No patient needed chronic narcotic medication. DISCUSSION: Pain is a complex issue and patient expectation of postoperative pain is one aspect that can potentially affect the amount of narcotics used. By preparing the patient both physically and psychologically, we believe the amount of narcotics used postoperatively can be decreased without affecting pain control. As a result, the multiple possible detriments of having more narcotics available than actually necessary would be lessened. By limiting the overall number of narcotic tablets prescribed, decreased use by the patient when such a medication may no longer be appropriate, and minimized use by others in the household who might have access to it would decrease.

Overcoming osimertinib resistance with AKT inhibition in EGFRm-driven Non-Small-Cell-Lung-Cancer with PIK3CA/PTEN alterations.

PURPOSE: Osimertinib is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) indicated for the treatment of EGFR mutated (EGFRm)-driven lung adenocarcinomas. Osimertinib significantly improves progression-free survival in first-line treated patients with EGFRm advanced NSCLC. Despite the durable disease control, the majority of patients receiving osimertinib eventually develop disease progression. EXPERIMENTAL DESIGN: ctDNA profiling analysis on-progression plasma samples from patients treated with osimertinib in both first (Phase 3, FLAURA trial) and second-line trials (Phase 3, AURA3 trial) revealed a high prevalence of PIK3CA/AKT/PTEN alterations. In vitro and in vivo evidence using CRISPR engineered NSCLC cell lines and PXD models support a functional role for PIK3CA and PTEN mutations in the development of osimertinib resistance. RESULTS: These alterations are functionally relevant as EGFRm NSCLC cells with engineered PIK3CA/AKT/PTEN alterations develop resistance to osimertinib and can be re-sensitized by treatment with the combination of osimertinib and the AKT inhibitor capivasertib. Moreover, xenograft and PDX in vivo models with PIK3CA/AKT/PTEN alterations display limited sensitivity to osimertinib relative to models without alteration, and in these double mutant models capivasertib and osimertinib combination elicits an improved anti-tumor effect versus osimertinib alone. CONCLUSIONS: Together, this approach offers a potential treatment strategy for patients with EGFRm-driven NSCLC that have a sub-optimal response, or develop resistance, to osimertinib through PIK3CA/AKT/PTEN alterations.