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From 2001 to 2023, 17 (14%) of 120 neonates with confirmed herpes simplex virus infection tested positive for herpes simplex virus by polymerase chain reaction (PCR) from only mucosal sites without a clinical mucosal lesion. Whether mucosal PCR positivity reflects early infection that may lead to recognizable disease, transient colonization, or a false-positive PCR result remains a clinical conundrum and warrants further study., Competing Interests: Declaration of Competing Interest A.M. has received fees for participation in Advisory Boards from Janssen, Merck, Pfizer, AstraZeneca and Sanofi-Pasteur as well as research grants from NIH, Merck, and Janssen. None contributed to study design, collection, analysis, and interpretation of data, writing of the report, and decision to submit the manuscript for publication. Other authors have no conflict of interest relevant to this article to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Childhood adversities worsen physical and mental health across the lifespan. Health and social care practitioners play a key role in identifying and responding to childhood adversity, however, may be reluctant to do so due to a perceived lack of services to refer to, time pressures and a deficit of training and confidence. We aimed to (1) quantify changes in practitioner comfort and confidence to identify and respond to childhood adversity following a multimodal intervention within an integrated child and family health and social care hub and (2) to understand barriers and facilitators of practice change., Methods: Hub practitioners were surveyed about their competence and comfort to directly ask about and confidence to respond to adversity at baseline and then at six and twelve months post training. Interviews were undertaken to explore practitioner barriers and enablers of practice change. Interviews were recorded, transcribed verbatim, and analysed using reflexive thematic analysis. The theoretical domains framework was used to identify the key drivers of practice change., Results: Fifteen of 18 practitioners completed all three surveys and 70% reported increased competence and comfort to directly ask, and confidence to respond across a range of adversities over the 12-month intervention. Twenty-one practitioners completed interviews. Six themes were identified as either facilitators or barriers to practice change. Facilitator themes included (1) connection matters, (2) knowledge provides assurance, (3) confidence in ability and (4) choosing change. Barrier themes were (1) never enough time and (2) opening Pandora's box. Following analysis, key drivers of practice change were 'social influence', 'belief in capability', 'knowledge' and 'behaviour regulation' while barriers to practice change were 'environmental context and resources' and 'emotion'., Conclusions: Practitioners reported improved confidence in identifying and responding to adversity through a multimodal intervention delivered in an integrated Child and Family Hub. Changing practice requires more than just education and training. Opportunities for social connection and coaching to improve self-confidence and perceived competence are needed to overcome the fear of opening Pandora's box., (© 2024. The Author(s).)

Competing Interests: The authors have declared that no competing interests exist.

Pancreatic cancer is a significant cause of cancer-related mortality and often presents with limited treatment options. Pancreatic tumors are also notorious for their immunosuppressive microenvironment. Irreversible electroporation (IRE) is a non-thermal tumor ablation modality that employs high-voltage microsecond pulses to transiently permeabilize cell membranes, ultimately inducing cell death. However, the understanding of IRE's impact beyond the initiation of focal cell death in tumor tissue remains limited. In this study, we demonstrate that IRE triggers a unique mix of cell death pathways and orchestrates a shift in the local tumor microenvironment driven, in part, by reducing the myeloid-derived suppressor cell (MDSC) and regulatory T cell populations and increasing cytotoxic T lymphocytes and neutrophils. We further show that IRE drives induce cell cycle arrest at the G0/G1 phase in vitro and promote inflammatory cell death pathways consistent with pyroptosis and programmed necrosis in vivo . IRE-treated mice exhibited a substantial extension in progression-free survival. However, within a span of 14 days, the tumor immune cell populations reverted to their pre-treatment composition, which resulted in an attenuation of the systemic immune response targeting contralateral tumors and ultimately resulting in tumor regrowth. Mechanistically, we show that IRE augments IFN- γ signaling, resulting in the up-regulation of the PD-L1 checkpoint in pancreatic cancer cells. Together, these findings shed light on potential mechanisms of tumor regrowth following IRE treatment and offer insights into co-therapeutic targets to improve treatment strategies., Competing Interests: IA, and RD are inventors on pending and issued patents related to the work. The authors declare no additional conflicts of interest. IA and RD have had projects supported, in part, by industry funding from business entities utilizing or developing IRE-based technologies for commercial applications. Those relevant to the current study include AngioDynamics, Galvanize Therapeutics, and ManaMed Tech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Imran, Brock, Beitel-White, Powar, Orr, Aycock, Alinezhadbalalami, Salameh, Eversole, Tintera, Markov Madanick, Hendricks-Wenger, Coutermarsh-Ott, Davalos and Allen.)

Objectives: 'Healthier Wealthier Families' (HWF) seeks to reduce financial hardship in the early years by embedding a referral pathway between Australia's universal child and family health (CFH) services and financial counselling. This pilot study investigated the feasibility and short-term impacts of HWF, adapted from a successful Scottish initiative., Methods: Setting: CFH services in five sites across two states, coinciding with the COVID-19 pandemic., Participants: Caregivers of children aged 0-5 years experiencing financial hardship (study-designed screen)., Design: Mixed methods. With limited progress using a randomised trial (RCT) design in sites 1-3 (March 2020-November 2021), qualitative interviews with service providers identified implementation barriers including stigma, lack of knowledge of financial counselling, low financial literacy, research burden and pandemic disruption. This informed a simplified RCT protocol (site 4) and direct referral model (no randomisation, pre-post evaluation, site 5) (June 2021-May 2022)., Intervention: financial counselling; comparator: usual care (sites 1-4). Feasibility measures: proportions of caregivers screened, enrolled, followed up and who accessed financial counselling. Impact measures: finances (quantitative) and other (qualitative) to 6 months post-enrolment., Results: 355/434 caregivers completed the screen (60%-100% across sites). In RCT sites (1-4), 79/365 (19%-41%) reported hardship but less than one-quarter enrolled. In site 5, n=66/69 (96%) caregivers reported hardship and 44/66 (67%) engaged with financial counselling; common issues were utility debts (73%), and obtaining entitlements (43%) or material aid/emergency relief (27%). Per family, financial counselling increased income from government entitlements by an average $A6504 annually plus $A784 from concessions, grants, brokerage and debt waivers. Caregivers described benefits (qualitative) including reduced stress, practical help, increased knowledge and empowerment., Conclusions: Financial hardship screening via CFH was acceptable to caregivers, direct referral was feasible, but individual randomisation was infeasible. Larger-scale implementation will require careful, staged adaptations where CFH populations and the intervention are well matched and low burden evaluation., Trial Registration Number: ACTRN12620000154909., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Objective: To determine the adherence and safety outcomes of a 5-day antibiotic course with a "time-out" for treatment of "blood culture-negative" pneumonia in the NICU., Study Design: Prospective surveillance of all infants diagnosed with pneumonia at 7 NICUs from 8/2020-12/2021. Safety outcomes were defined a priori by re-initiation of antibiotic therapy within 14 days after discontinuation and overall and sepsis-related mortality., Results: 128 infants were diagnosed with 136 episodes of pneumonia; 88% (n = 119) were treated with 5 days of definitive antibiotic therapy. Antibiotics were restarted within 14 days in 22 (16%) of the 136 pneumonia episodes. However, only 3 (3%) of the 119 episodes of pneumonia treated for 5 days had antibiotics restarted for pneumonia. Mortality was 5% (7/128); 5 of the 7 deaths were assessed as sepsis-related., Conclusion: Adherence to the 5-day definitive antibiotic treatment for "culture-negative" pneumonia was high and the intervention seemed safe., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Objective: On 2/2019, the Neonatal Antimicrobial Stewardship Program at Nationwide Children's Hospital recommended reducing empirical antibiotic therapy for early-onset sepsis (EOS) from 48 to 24 hours with a TIME-OUT. We describe our experience with this guideline and assess its safety., Methods: Retrospective review of newborns evaluated for possible EOS at 6 NICUs from 12/2018-7/2019. Safety endpoints were re-initiation of antibiotics within 7 days after discontinuation of the initial course, positive bacterial blood or cerebrospinal fluid culture in the 7 days after antibiotic discontinuation, and overall and sepsis-related mortality., Result: Among 414 newborns evaluated for EOS, 196 (47%) received a 24 hour rule-out sepsis antibiotic course while 218 (53%) were managed with a 48 hour course. The 24-hour rule-out group were less likely to have antibiotics re-initiated and did not differ in the other predefined safety endpoints., Conclusion: Antibiotic therapy for suspected EOS may be discontinued safely within 24 hours., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

INTRODUCTION: Women of child-bearing age make up an ever-increasing element of the aeromedical workforce in Australia and the UK. However, policy relating to the management of risk for pregnant employees in this sector is often missing or inadequate, with many women facing detrimental impacts on their career progression and financial well-being. For women who choose to continue flying, there is a lack of transparent guidance about the risks of flying within a helicopter in an aeromedical role. While grounding pregnant employees removes some risks, it is at the cost of autonomy and brings other adverse effects for the employee and employer. Updated reflections on this important topic will empower the audience to make informed discussions around pregnancy in aeromedical roles. TOPIC: Applying principles from literature surrounding commercial, military, and medical aviation, the risks to pregnant employees and the fetus are reviewed. These risks are complex and dynamic depending on gestation and underlying medical problems; thus, individualization of risk management is of key importance. In low-risk pregnancies, incapacitation risk is below the usual threshold adopted for safety-sensitive aviation activities. Based on available evidence we have quantified risks where possible and provide guidance on the relevant factors to consider in creating a holistic risk-management framework. The greatest unknown surrounds the risk from vibration, noise, and winching. These are reviewed and suggestions given for discussing this risk. We also highlight the need for policy providing acceptable nonflying options to remove the pressure to continue flying in pregnancy. APPLICATION: Based on a literature review we have generated a framework for understanding and assessing risk relating to pregnant employees in the aeromedical sector. This is intended for use by aeromedical organizations, pregnant employees, and their treating medical practitioners to provide rational and sensible policy and guidance. Storey HM, Austin J, Davies-White NL, Ransley DG, Hodkinson PD. Navigating pregnancy for employees in civilian rotary-wing aeromedicine . Aerosp Med Hum Perform. 2022; 93(12):866-876.

Background: Prescribed opioid doses > 100 mg oral morphine equivalent (OME) and/or co-prescribing of sedating psychoactive medications increase the risk of unintentional fatal overdose. We describe general practice encounters where opioids are prescribed and examine high-risk opioid prescribing., Methods: The 2006-2016 BEACH study data, a rolling national cross-sectional survey of randomly selected GPs, was analysed., Results: Opioid prescribing increased 2006-2007 to 2015-2016, however, this plateaued across the latter half-decade. From 2012-2016 3,897 GPs recorded 389,700 encounters and at least one opioid was prescribed at 5.2%. Opioid encounters more likely involved males, those 45-64 years, concession card holders and the socioeconomically disadvantaged. GPs more likely to prescribe opioids were 55 years or older, male, Australian graduates, and in regional and remote areas. The most common problems managed with opioids involved chronic non-cancer pain. One-in-ten opioid prescribing episodes involved high-risk doses and 11% involved co-prescription of sedating psychoactive medications. Over one-third of GPs provided other (non-pharmacological) interventions at encounters with opioid prescriptions., Conclusions: Only 5% of GP encounters involved an opioid prescription. Of concern, were: prescribing for chronic non-cancer pain, potentially high-risk opioid encounters where > 100 OME daily dose was prescribed, and/or there was co-prescription of sedating psychoactive medication. However, approximately one-in-three opioid prescribing encounters involved non-pharmacological interventions., (© 2022. The Author(s).)

A middle-aged man of average weight presented to the sleep medicine clinic for multiple episodes of nocturnal eating during night-time awakenings for the past several months. His symptoms were more characteristic of night eating syndrome rather than sleep-related eating disorder because of his recollection of the eating episodes and intake of edible substances during these episodes. He was treated with a low dose of sertraline with an initial improvement of symptoms followed by a relapse that was controlled with an increased dose. A year after initiation of therapy, his symptoms have resolved., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Irreversible electroporation (IRE) is a promising alternative therapy for the local treatment of prostate tumors. The procedure involves the direct insertion of needle electrodes into the target zone, and subsequent delivery of short but high-voltage pulses. Successful outcomes rely on adequate exposure of the tumor to a threshold electrical field. To aid in predicting this exposure, computational models have been developed, yet often do not incorporate the appropriate tissue-specific properties. This work aims to quantify electrical conductivity behavior during IRE for three types of tissue present in the target area of a prostate cancer ablation: the tumor tissue itself, the surrounding healthy tissue, and potential areas of necrosis within the tumor. Animal tissues were used as a stand-in for primary samples. The patient-derived prostate tumor tissue showed very similar responses to healthy porcine prostate tissue. An examination of necrotic tissue inside the tumors revealed a large difference, however, and a computational model showed that a necrotic core with differing electrical properties can cause unexpected inhomogeneities within the treatment region.

Of Recommendations and Levels of Evidence: Chapter 2: Screening and assessment for unhealthy alcohol use Screening Screening for unhealthy alcohol use and appropriate interventions should be implemented in general practice (Level A), hospitals (Level B), emergency departments and community health and welfare settings (Level C). Quantity-frequency measures can detect consumption that exceeds levels in the current Australian guidelines (Level B). The Alcohol Use Disorders Identification Test (AUDIT) is the most effective screening tool and is recommended for use in primary care and hospital settings. For screening in the general community, the AUDIT-C is a suitable alternative (Level A). Indirect biological markers should be used as an adjunct to screening (Level A), and direct measures of alcohol in breath and/or blood can be useful markers of recent use (Level B). Assessment Assessment should include evaluation of alcohol use and its effects, physical examination, clinical investigations and collateral history taking (Level C). Assessment for alcohol-related physical problems, mental health problems and social support should be undertaken routinely (GPP). Where there are concerns regarding the safety of the patient or others, specialist consultation is recommended (Level C). Assessment should lead to a clear, mutually acceptable treatment plan which specifies interventions to meet the patient's needs (Level D). Sustained abstinence is the optimal outcome for most patients with alcohol dependence (Level C). Chapter 3: Caring for and managing patients with alcohol problems: interventions, treatments, relapse prevention, aftercare, and long term follow-up Brief interventions Brief motivational interviewing interventions are more effective than no treatment for people who consume alcohol at risky levels (Level A). Their effectiveness compared with standard care or alternative psychosocial interventions varies by treatment setting. They are most effective in primary care settings (Level A). Psychosocial interventions Cognitive behaviour therapy should be a first-line psychosocial intervention for alcohol dependence. Its clinical benefit is enhanced when it is combined with pharmacotherapy for alcohol dependence or an additional psychosocial intervention (eg, motivational interviewing) (Level A). Motivational interviewing is effective in the short term and in patients with less severe alcohol dependence (Level A). Residential rehabilitation may be of benefit to patients who have moderate-to-severe alcohol dependence and require a structured residential treatment setting (Level D). Alcohol withdrawal management Most cases of withdrawal can be managed in an ambulatory setting with appropriate support (Level B). Tapering diazepam regimens (Level A) with daily staged supply from a pharmacy or clinic are recommended (GPP). Pharmacotherapies for alcohol dependence Acamprosate is recommended to help maintain abstinence from alcohol (Level A). Naltrexone is recommended for prevention of relapse to heavy drinking (Level A). Disulfiram is only recommended in close supervision settings where patients are motivated for abstinence (Level A). Some evidence for off-label therapies baclofen and topiramate exists, but their side effect profiles are complex and neither should be a first-line medication (Level B). Peer support programs Peer-led support programs such as Alcoholics Anonymous and SMART Recovery are effective at maintaining abstinence or reductions in drinking (Level A). Relapse prevention, aftercare and long-term follow-up Return to problematic drinking is common and aftercare should focus on addressing factors that contribute to relapse (GPP). A harm-minimisation approach should be considered for patients who are unable to reduce their drinking (GPP). Chapter 4: Providing appropriate treatment and care to people with alcohol problems: a summary for key specific populations Gender-specific issues Screen women and men for domestic abuse (Level C). Consider child protection assessments for caregivers with alcohol use disorder (GPP). Explore contraceptive options with women of reproductive age who regularly consume alcohol (Level B). Pregnant and breastfeeding women Advise pregnant and breastfeeding women that there is no safe level of alcohol consumption (Level B). Pregnant women who are alcohol dependent should be admitted to hospital for treatment in an appropriate maternity unit that has an addiction specialist (GPP). Young people Perform a comprehensive HEEADSSS assessment for young people with alcohol problems (Level B). Treatment should focus on tangible benefits of reducing drinking through psychotherapy and engagement of family and peer networks (Level B). Aboriginal and Torres Strait Islander peoples Collaborate with Aboriginal or Torres Strait Islander health workers, organisations and communities, and seek guidance on patient engagement approaches (GPP). Use validated screening tools and consider integrated mainstream and Aboriginal or Torres Strait Islander-specific approaches to care (Level B). Culturally and linguistically diverse groups Use an appropriate method, such as the "teach-back" technique, to assess the need for language and health literacy support (Level C). Engage with culture-specific agencies as this can improve treatment access and success (Level C). Sexually diverse and gender diverse populations Be mindful that sexually diverse and gender diverse populations experience lower levels of satisfaction, connection and treatment completion (Level C). Seek to incorporate LGBTQ-specific treatment and agencies (Level C). Older people All new patients aged over 50 years should be screened for harmful alcohol use (Level D). Consider alcohol as a possible cause for older patients presenting with unexplained physical or psychological symptoms (Level D). Consider shorter acting benzodiazepines for withdrawal management (Level D). Cognitive impairment Cognitive impairment may impair engagement with treatment (Level A). Perform cognitive screening for patients who have alcohol problems and refer them for neuropsychological assessment if significant impairment is suspected (Level A)., Summary of Key Recommendations and Levels of Evidence: Chapter 5: Understanding and managing comorbidities for people with alcohol problems: polydrug use and dependence, co-occurring mental disorders, and physical comorbidities Polydrug use and dependence Active alcohol use disorder, including dependence, significantly increases the risk of overdose associated with the administration of opioid drugs. Specialist advice is recommended before treatment of people dependent on both alcohol and opioid drugs (GPP). Older patients requiring management of alcohol withdrawal should have their use of pharmaceutical medications reviewed, given the prevalence of polypharmacy in this age group (GPP). Smoking cessation can be undertaken in patients with alcohol dependence and/or polydrug use problems; some evidence suggests varenicline may help support reduction of both tobacco and alcohol consumption (Level C). Co-occurring mental disorders More intensive interventions are needed for people with comorbid conditions, as this population tends to have more severe problems and carries a worse prognosis than those with single pathology (GPP). The Kessler Psychological Distress Scale (K10 or K6) is recommended for screening for comorbid mental disorders in people presenting for alcohol use disorders (Level A). People with alcohol use disorder and comorbid mental disorders should be offered treatment for both disorders; care should be taken to coordinate intervention (Level C). Physical comorbidities Patients should be advised that alcohol use has no beneficial health effects. There is no clear risk-free threshold for alcohol intake. The safe dose for alcohol intake is dependent on many factors such as underlying liver disease, comorbidities, age and sex (Level A). In patients with alcohol use disorder, early recognition of the risk for liver cirrhosis is critical. Patients with cirrhosis should abstain from alcohol and should be offered referral to a hepatologist for liver disease management and to an addiction physician for management of alcohol use disorder (Level A). Alcohol abstinence reduces the risk of cancer and improves outcomes after a diagnosis of cancer (Level A)., (© 2021 AMPCo Pty Ltd.)

Pancreatic cancer is a significant cause of cancer-related deaths in the United States with an abysmal five-year overall survival rate that is under 9%. Reasons for this mortality include the lack of late-stage treatment options and the immunosuppressive tumor microenvironment. Histotripsy is an ultrasound-guided, noninvasive, nonthermal tumor ablation therapy that mechanically lyses targeted cells. To study the effects of histotripsy on pancreatic cancer, we utilized an in vitro model of pancreatic adenocarcinoma and compared the release of potential antigens following histotripsy treatment to other ablation modalities. Histotripsy was found to release immune-stimulating molecules at magnitudes similar to other nonthermal ablation modalities and superior to thermal ablation modalities, which corresponded to increased innate immune system activation in vivo. In subsequent in vivo studies, murine Pan02 tumors were grown in mice and treated with histotripsy. Flow cytometry and rtPCR were used to determine changes in the tumor microenvironment over time compared to untreated animals. In mice with pancreatic tumors, we observed significantly increased tumor-progression-free and general survival, with increased activation of the innate immune system 24 h posttreatment and decreased tumor-associated immune cell populations within 14 days of treatment. This study demonstrates the feasibility of using histotripsy for pancreatic cancer ablation and provides mechanistic insight into the initial innate immune system activation following treatment. Further work is needed to establish the mechanisms behind the immunomodulation of the tumor microenvironment and immune effects.

Modulation of T-cell responses has played a key role in treating cancers and autoimmune diseases. Therefore, understanding how different receptors on T cells impact functional outcomes is crucial. The influence of B7-H7 (HHLA2) and CD28H (TMIGD2) on T-cell activation remains controversial. Here we examined global transcriptomic changes in human T cells induced by B7-H7. Stimulation through TCR with OKT3 and B7-H7 resulted in modest fold changes in the expression of select genes; however, these fold changes were significantly lower than those induced by OKT3 and B7-1 stimulation. The transcriptional changes induced by OKT3 and B7-H7 were insufficient to provide functional stimulation as measured by evaluating T-cell proliferation and cytokine production. Interestingly, B7-H7 was coinhibitory when simultaneously combined with TCR and CD28 stimulation. This inhibitory activity was comparable to that observed with PD-L1. Finally, in physiological assays using T cells and APCs, blockade of B7-H7 enhanced T-cell activation and proliferation, demonstrating that this ligand acts as a break signal. Our work defines that the transcriptomic changes induced by B7-H7 are insufficient to support full costimulation with TCR signaling and, instead, B7-H7 inhibits T-cell activation and proliferation in the presence of TCR and CD28 signaling.

The long-term ecological success of compensatory freshwater wetland projects has come into question based on follow-up monitoring studies over the past few decades. Given that wetland restoration may require many years to decades to converge to desired outcomes, long-term monitoring of successional patterns may increase our ability to fully evaluate success of wetland mitigation projects or guide adaptive management when needed. In Portsmouth, New Hampshire a 4 ha wetland was constructed in an abandoned gravel quarry as off-site compensatory mitigation for impacts to a scrub-shrub swamp associated with property expansion. Building upon prior evaluations from 1992 and 2002, we conducted a floral survey in 2020 to compare results with prior surveys to document vegetation successional trends over time. In addition, we monitored the avian community throughout the growing season as a measure of habitat quality. The plant community mirrored documented successional trends of freshwater wetland restoration projects as native hydrophytes dominated species composition. Plant species composition stabilized as the rate of turnover, the measurement of succession, declined by nearly half after 17 years. Researchers should consider long-term monitoring of specific sites to better understand successional patterns of created wetlands as we documented long time frames required for the development of scrub-shrub swamps, red maple swamps, and sedge meadows. High species richness was attributed to beaver activity, topographic heterogeneity from Carex stricta tussocks, and the seed bank from the application of peat from the original wetland. Habitat heterogeneity of open water, herbaceous cover, and woody vegetation supports a diverse avian community including 11 wetland dependent species. Although the mitigation project has not created the full area of lost scrub-shrub swamp after 35 years, it has developed a structurally complex habitat and diverse avian community that effectively provides the functions and values of the impacted system., Competing Interests: The authors have declared that no competing interests exist.

New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients' anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.

Objective: Tissue electroporation is achieved by applying a series of electric pulses to destabilize cell membranes within the target tissue. The treatment volume is dictated by the electric field distribution, which depends on the pulse parameters and tissue type and can be readily predicted using numerical methods. These models require the relevant tissue properties to be known beforehand. This study aims to quantify electrical and thermal properties for three different tissue types relevant to current clinical electroporation., Methods: Pancreatic, brain, and liver tissue were harvested from pigs, then treated with IRE pulses in a parallel-plate configuration. Resulting current and temperature readings were used to calculate the conductivity and its temperature dependence for each tissue type. Finally, a computational model was constructed to examine the impact of differences between tissue types., Results: Baseline conductivity values (mean 0.11, 0.14, and 0.12 S/m) and temperature coefficients of conductivity (mean 2.0, 2.3, and 1.2 % per degree Celsius) were calculated for pancreas, brain, and liver, respectively. The accompanying computational models suggest field distribution and thermal damage volumes are dependent on tissue type., Conclusion: The three tissue types show similar electrical and thermal responses to IRE, though brain tissue exhibits the greatest differences. The results also show that tissue type plays a role in the expected ablation and thermal damage volumes., Significance: The conductivity and its changes due to heating are expected to have a marked impact on the ablation volume. Incorporating these tissue properties aids in the prediction and optimization of electroporation-based therapies.

Background and Objectives: Electroporation is the phenomenon by which cell membrane permeability to ions and macromolecules is increased when the cell is briefly exposed to high electric fields. In electroporation-based treatments, such exposure is typically performed by delivering high voltage pulses across needle electrodes in tissue. For a given tissue and pulsing protocol, an electric field magnitude threshold exists that must be overreached for treatment efficacy. However, it is hard to preoperatively infer the treatment volume because the electric field distribution intricately depends on the electrodes' positioning and length, the applied voltage, and the electric conductivity of the treated tissues. For illustrating such dependencies, we have created EView (https://eview.upf.edu), a web platform that estimates the electric field distribution for arbitrary needle electrode locations and orientations and overlays it on 3D medical images., Methods: A client-server approach has been implemented to let the user set the electrode configuration easily on the web browser, whereas the simulation is computed on a dedicated server. By means of the finite element method, the electric field is solved in a 3D volume. For the sake of simplicity, only a homogeneous tissue is modeled, assuming the same properties for healthy and pathologic tissues. The non-linear dependence of tissue conductivity on the electric field due to the electroporation effect is modeled. The implemented model has been validated against a state of the art finite element solver, and the server has undergone a heavy load test to ensure reliability and to report execution times., Results: The electric field is rapidly computed for any electrode and tissue configuration, and alternative setups can be easily compared. The platform provides the same results as the state of the art finite element solver (Dice = 98.3 ± 0.4%). During the high load test, the server remained responsive. Simulations are computed in less than 2 min for simple cases consisting of two electrodes and take up to 40 min for complex scenarios consisting of 6 electrodes., Conclusions: With this free platform we provide expert and non-expert electroporation users a way to rapidly model the electric field distribution for arbitrary electrode configurations., Competing Interests: Declaration of Competing Interest Dr. Davalos has patents in the area of electroporation and may receive royalties. He is also a co-founder of a startup company developing electroporation-based therapies for neurological disorders. Natalie Beitel-White has a pending patent in electroporation and may receive royalties., (Copyright © 2020. Published by Elsevier B.V.)

New therapeutic strategies and paradigms are direly needed for the treatment of cancer. While the surgical removal of tumors is favored in most cancer treatment plans, resection options are often limited based on tumor localization. Over the last two decades, multiple tumor ablation strategies have emerged as promising stand-alone or combination therapeutic options for patients. These strategies are often employed to treat tumors in areas where surgical resection is not possible or where chemotherapeutics have proven ineffective. The type of cell death induced by the ablation modality is a critical aspect of therapeutic success that can impact the efficacy of the treatment and systemic anti-tumor immune system responses. Electroporation-based ablation technologies include electrochemotherapy, irreversible electroporation, and other modalities that rely on pulsed electric fields to create pores in cell membranes. These pores can either be reversible or irreversible depending on the electric field parameters and can induce cell death either alone or in combination with a therapeutic agent. However, there have been many controversial findings among these technologies as to the cell death type initiated, from apoptosis to pyroptosis. As cell death mechanisms can impact treatment side effects and efficacy, we review the main types of cell death induced by electroporation-based treatments and summarize the impact of these mechanisms on treatment response. We also discuss potential reasons behind the variability of findings such as the similarities between cell death pathways, differences between cell-types, and the variation in electric field strength across the treatment area., (Copyright © 2020 Brock, Beitel-White, Davalos and Allen.)

New methods of tumor ablation have shown exciting efficacy in pre-clinical models but often demonstrate limited success in the clinic. Due to a lack of quality or quantity in primary malignant tissue specimens, therapeutic development and optimization studies are typically conducted on healthy tissue or cell-line derived rodent tumors that don't allow for high resolution modeling of mechanical, chemical, and biological properties. These surrogates do not accurately recapitulate many critical components of the tumor microenvironment that can impact in situ treatment success. Here, we propose utilizing patient-derived xenograft (PDX) models to propagate clinically relevant tumor specimens for the optimization and development of novel tumor ablation modalities. Specimens from three individual pancreatic ductal adenocarcinoma (PDAC) patients were utilized to generate PDX models. This process generated 15-18 tumors that were allowed to expand to 1.5 cm in diameter over the course of 50-70 days. The PDX tumors were morphologically and pathologically identical to primary tumor tissue. Likewise, the PDX tumors were also found to be physiologically superior to other in vitro and ex vivo models based on immortalized cell lines. We utilized the PDX tumors to refine and optimize irreversible electroporation (IRE) treatment parameters. IRE, a novel, non-thermal tumor ablation modality, is being evaluated in a diverse range of cancer clinical trials including pancreatic cancer. The PDX tumors were compared against either Pan02 mouse derived tumors or resected tissue from human PDAC patients. The PDX tumors demonstrated similar changes in electrical conductivity and Joule heating following IRE treatment. Computational modeling revealed a high similarity in the predicted ablation size of the PDX tumors that closely correlate with the data generated with the primary human pancreatic tumor tissue. Gene expression analysis revealed that IRE treatment resulted in an increase in biological pathway signaling associated with interferon gamma signaling, necrosis and mitochondria dysfunction, suggesting potential co-therapy targets. Together, these findings highlight the utility of the PDX system in tumor ablation modeling for IRE and increasing clinical application efficacy. It is also feasible that the use of PDX models will significantly benefit other ablation modality testing beyond IRE., (Copyright © 2020 Brock, Beitel-White, Coutermarsh-Ott, Grider, Lorenzo, Ringel-Scaia, Manuchehrabadi, Martin, Davalos and Allen.)

Pulsed electric field treatment modalities typically utilize multiple pulses to permeabilize biological tissue. This electroporation process induces conductivity changes in the tissue, which are indicative of the extent of electroporation. In this study, we characterized the electroporation-induced conductivity changes using all treatment pulses instead of solely the first pulse as in conventional conductivity models. Rabbit liver tissue was employed to study the tissue conductivity changes caused by multiple, 100 μs pulses delivered through flat plate electrodes. Voltage and current data were recorded during treatment and used to calculate the tissue conductivity during the entire pulsing process. Temperature data were also recorded to quantify the contribution of Joule heating to the conductivity according to the tissue temperature coefficient. By fitting all these data to a modified Heaviside function, where the two turning points ( E 0 , E 1 ) and the increase factor ( A ) are the main parameters, we calculated the conductivity as a function of the electric field ( E ), where the parameters of the Heaviside function ( A and E 0 ) were functions of pulse number ( N ). With the resulting multi-factor conductivity model, a numerical electroporation simulation can predict the electrical current for multiple pulses more accurately than existing conductivity models. Moreover, the saturating behavior caused by electroporation can be explained by the saturation trends of the increase factor A in this model. The conductivity change induced by electroporation has a significant increase at about the first 30 pulses, then tends to saturate at 0.465 S/m. The proposed conductivity model can simulate the electroporation process more accurately than the conventional conductivity model. The electric field distribution computed using this model is essential for treatment planning in biomedical applications utilizing multiple pulsed electric fields, and the method proposed here, relating the pulse number to the conductivity through the variables in the Heaviside function, may be adapted to investigate the effect of other parameters, like pulse frequency and pulse width, on electroporation., (Copyright © 2020 Zhao, Zheng, Beitel-White, Liu, Yao and Davalos.)

High-frequency irreversible electroporation (H-FIRE) has emerged as an alternative to conventional irreversible electroporation (IRE) to overcome the issues associated with neuromuscular electrical stimulation that appear in IRE treatments. In H-FIRE, the monopolar pulses typically used in IRE are replaced with bursts of short bipolar pulses. Currently, very little is known regarding how the use of a different waveform affects the cell death dynamics and mechanisms. In this study, human pancreatic adenocarcinoma cells were treated with a typical IRE protocol and various H-FIRE schemes with the same energized time. Cell viability, membrane integrity and Caspase 3/7 activity were assessed at different times after the treatment. In both treatments, we identified two different death dynamics (immediate and delayed) and we quantified the electric field ranges that lead to each of them. While in the typical IRE protocol, the electric field range leading to a delayed cell death is very narrow, this range is wider in H-FIRE and can be increased by reducing the pulse length. Membrane integrity in cells suffering a delayed cell death shows a similar time evolution in all treatments, however, Caspase 3/7 expression was only observed in cells treated with H-FIRE.

The complement system consists of a series of soluble and cell-surface proteins that serve numerous roles in innate immunity, development, and homeostasis. Despite its many functions, the central event in the complement system is the proteolytic activation of the 185 kDa complement component 3 (C3) into its opsonin and anaphylatoxin fragments known as C3b (175 kDa) and C3a (10 kDa), respectively. The C3 protein is comprised of thirteen separate structural domains, several of which undergo extensive structural rearrangement upon activation to C3b. In addition to this, the C-terminal C345c domain found in C3, C3b, and the terminal degradation product, C3c (135 kDa), appears to adopt multiple conformations relative to the remainder of the molecule. To facilitate various structure/function studies, we designed two C3 analogs that could be activated to a C345c-less, C3c-like state following treatment with Tobacco Etch Virus (TEV) protease. We generated stably transfected Chinese Hamster Ovary (CHO) cell lines that secrete approximately 1.5 mg of the highest-expressing C3 analog per liter of conditioned culture medium. We purified this C3 analog by sequential immobilized metal ion affinity and size exclusion chromatographies, activated the protein by digestion with TEV protease, and purified the resulting C3c analog by a final size exclusion chromatography. The conformations and activities of our C3 and C3c analogs were assessed by measuring their binding profiles to known C3/b/c ligands by surface plasmon resonance. Together, this work demonstrates the feasibility of producing a C3 analog that can be site-specifically activated by an exogenous proteolytic enzyme., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Irreversible electroporation (IRE) is a focal ablation therapy that uses high voltage, short electrical pulses to destroy tumor tissue. The success of treatment directly depends on exposure of the entire tumor to a lethal electric field magnitude. However, this exposure is difficult to predict ahead of time and it is challenging for clinicians to determine optimal treatment parameters. One method clinicians rely upon for the cessation of pulse delivery is to monitor the resistance value of the tissue, as the cells within the tissue will undergo changes during electroporation. This work presents a computational model which incorporates human pancreatic tumor conductivity, and compares predicted and measured output currents from IRE treatments of human patients. The measured currents vary widely from patient to patient, suggesting there may areas of high local conductivity in the treatment area.

Background: Despite promising treatments for breast cancer, mortality rates remain high and treatments for metastatic disease are limited. High-frequency irreversible electroporation (H-FIRE) is a novel tumor ablation technique that utilizes high-frequency bipolar electric pulses to destabilize cancer cell membranes and induce cell death. However, there is currently a paucity of data pertaining to immune system activation following H-FIRE and other electroporation based tumor ablation techniques., Methods: Here, we utilized the mouse 4T1 mammary tumor model to evaluate H-FIRE treatment parameters on cancer progression and immune system activation in vitro and in vivo., Findings: H-FIRE effectively ablates the primary tumor and induces a pro-inflammatory shift in the tumor microenvironment. We further show that local treatment with H-FIRE significantly reduces 4T1 metastases. H-FIRE kills 4T1 cells through non-thermal mechanisms associated with necrosis and pyroptosis resulting in damage associated molecular pattern signaling in vitro and in vivo. Our data indicate that the level of tumor ablation correlates with increased activation of cellular immunity. Likewise, we show that the decrease in metastatic lesions is dependent on the intact immune system and H-FIRE generates 4T1 neoantigens that engage the adaptive immune system to significantly attenuate tumor progression., Interpretation: Cell death and tumor ablation following H-FIRE treatment activates the local innate immune system, which shifts the tumor microenvironment from an anti-inflammatory state to a pro-inflammatory state. The non-thermal damage to the cancer cells and increased innate immune system stimulation improves antigen presentation, resulting in the engagement of the adaptive immune system and improved systemic anti-tumor immunity., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Endotoxins are complex molecules and one of the most challenging impurities requiring separation in biopharmaceutical protein purification processes. Usually these contaminants are cleared during the downstream process, but if endotoxin interacts with the target protein it becomes difficult to remove. In the present study we identified a detergent, octyl-β-D-1-thioglucopyranoside (OTG), that disrupted endotoxin-protein interactions. The integration of this detergent into washes on several chromatography media was demonstrated to provide a separation tool for decreasing endotoxin from target proteins. This study also examined the mechanism of OTG endotoxin-protein disruption through phase modification incubation and chromatographic studies. The non-ionic OTG wash was shown to break both hydrophobic and electrostatic interactions between the endotoxin and protein. This mechanism contrasts with the breaking of hydrophobic interactions by washing with known endotoxin decreasing Triton X-100 detergent. The difference in mechanisms likely results in the ability of OTG to decrease endotoxin to levels less than those resulting from a detergent wash such as Triton X-100. Finally, we show the impact of the OTG endotoxin removal tool on the biopharmaceutical industry. While maintaining monomer purity and activity levels, endotoxin removal from a fusion protein allowed for decreased background levels in a T cell functional assay. The lowered baseline of T cell responses allowed for more effective detection of molecular interaction with the cells. The detergent wash can be used to both decrease the overall level of endotoxin in a purified protein solution and to enable more effective screening of lead candidate molecules., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Irreversible electroporation (IRE) is a cancer therapy that uses short, high-voltage electrical pulses to treat tumors. Due to its predominantly non-thermal mechanism and ability to ablate unresectable tumors, IRE has gained popularity in clinical treatments of both liver and pancreatic cancers. Existing computational models use electrical properties of animal tissue that are quantified a priori to predict the area of treatment in three dimensions. However, the changes in the electrical properties of human tissue during IRE treatment are so far unexplored. This work aims to improve models by characterizing the dynamic electrical behavior of human liver and pancreatic tissue. Fresh patient samples of each tissue type, both normal and tumor, were collected and IRE pulses were applied between two parallel metal plates at various voltages. The electrical conductivity was determined from the resistance using simple relations applicable to cylindrical samples. The results indicate that the percent change in conductivity during IRE treatments varies significantly with increasing electric field magnitudes. This percent change versus applied electric field behavior can be fit to a sigmoidal curve, as proposed in prior studies. The generic conductivity data from human patients from this work can be input to computational software using patient-specific geometry, giving clinicians a more accurate and personalized prediction of a given IRE treatment.

Neutrophils contain high levels of chymotrypsin-like serine proteases (NSPs) within their azurophilic granules that have a multitude of functions within the immune system. In response, the pathogen Staphylococcus aureus has evolved three potent inhibitors (Eap, EapH1, and EapH2) that protect the bacterium as well as several of its secreted virulence factors from the degradative action of NSPs. We previously showed that these so-called EAP domain proteins represent a novel class of NSP inhibitors characterized by a non-covalent inhibitory mechanism and a distinct target specificity profile. Based upon high levels of structural homology amongst the EAP proteins and the NSPs, as well as supporting biochemical data, we predicted that the inhibited complex would be similar for all EAP/NSP pairs. However, we present here evidence that EapH1 and EapH2 bind the canonical NSP, Neutrophil Elastase (NE), in distinct orientations. We discovered that alteration of EapH1 residues at the EapH1/NE interface caused a dramatic loss of affinity and inhibition of NE, while mutation of equivalent positions in EapH2 had no effect on NE binding or inhibition. Surprisingly, mutation of residues in an altogether different region of EapH2 severely impacted both the NE binding and inhibitory properties of EapH2. Even though EapH1 and EapH2 bind and inhibit NE and a second NSP, Cathepsin G, equally well, neither of these proteins interacts with the structurally related, but non-proteolytic granule protein, azurocidin. These studies expand our understanding of EAP/NSP interactions and suggest that members of this immune evasion protein family are capable of diverse target recognition modes., (© 2017 The Protein Society.)

Heusler compounds XY 2 Z with 24 valence electrons per formula unit are potential thermoelectric materials, given their thermal and chemical stability and their relatively earth-abundant constituent elements. We present results on the 24-electron compound TiFe 2 Sn here. First principles calculations on this compound suggest semiconducting behavior. A relatively flat conduction band that could be associated with a high Seebeck coefficient upon electron doping is found. A series of compounds have been prepared and characterized using a combination of synchrotron x-ray and neutron diffraction studies to understand the effects of site order/disorder phenomena and n-type doping. Samples fabricated by a three step processing approach were subjected to high temperature Seebeck and electrical resistivity measurements. Ti:Fe anti-site disorder is present in the stoichiometric compound and these defects are reduced when starting Ti-rich compositions are employed. Additionally, we investigate control of the Seebeck coefficient through the introduction of carriers through the substitution of Sb on the Sn site in these intrinsically p-type materials.

Cigarette smoking is a major risk factor for chronic obstructive pulmonary disease and is presumed to be central to the altered responsiveness to recurrent infection in these patients. We examined the effects of smoke priming underlying the exacerbated response to viral infection in mice. Lack of interleukin-33 (IL-33) signaling conferred complete protection during exacerbation and prevented enhanced inflammation and exaggerated weight loss. Mechanistically, smoke was required to upregulate epithelial-derived IL-33 and simultaneously alter the distribution of the IL-33 receptor ST2. Specifically, smoke decreased ST2 expression on group 2 innate lymphoid cells (ILC2s) while elevating ST2 expression on macrophages and natural killer (NK) cells, thus altering IL-33 responsiveness within the lung. Consequently, upon infection and release, increased local IL-33 significantly amplified type I proinflammatory responses via synergistic modulation of macrophage and NK cell function. Therefore, in COPD, smoke alters the lung microenvironment to facilitate an alternative IL-33-dependent exaggerated proinflammatory response to infection, exacerbating disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Magnetic resonance imaging (MRI) is the leading imaging technique for disease diagnostics, providing high resolution, three-dimensional images noninvasively. MRI contrast agents are designed to improve the contrast and sensitivity of MRI. However, current clinically used MRI contrast agents have relaxivities far below the theoretical upper limit, which largely prevent advancing molecular imaging of biomarkers with desired sensitivity and specificity. This review describes current progress in the development of a new class of protein-based MRI contrast agents (ProCAs) with high relaxivity using protein design to optimize the parameters that govern relaxivity. Further, engineering with targeting moiety allows these contrast agents to be applicable for molecular imaging of prostate cancer biomarkers by MRI. The developed protein-based contrast agents also exhibit additional in vitro and in vivo advantages for molecular imaging of disease biomarkers, such as high metal-binding stability and selectivity, reduced toxicity, proper blood circulation time, and higher permeability in tumor tissue in addition to improved relaxivities., (© 2014 Wiley Periodicals, Inc.)

Epidermal growth factor receptor (EGFR) and HER2 are major prognosis biomarkers and drug targets overexpressed in various types of cancer cells. There is a pressing need to develop MRI contrast agents capable of enhancing the contrast between normal tissues and tumors with high relaxivity, capable of targeting tumors, and with high intratumoral distribution and minimal toxicity. In this review, we first discuss EGFR signaling and its role in tumor progression as a major drug target. We then report our progress in the development of protein contrast agents with significant improvement of both r1 and r2 relaxivities, pharmacokinetics, in vivo retention time, and in vivo dose efficiency. Finally, we report our effort in the development of EGFR-targeted protein contrast agents with the capability to cross the endothelial boundary and with good tissue distribution across the entire tumor mass. The noninvasive capability of MRI to visualize spatially and temporally the intratumoral distribution as well as quantify the levels of EGFR and HER2 would greatly improve our ability to track changes of the biomarkers during tumor progression, monitor treatment efficacy, aid in patient selection, and further develop novel targeted therapies for clinical application.

Magnetic resonance imaging (MRI) has emerged as a leading diagnostic technique in clinical and preclinical settings. However, the application of MRI to assess specific disease markers for diagnosis and monitoring drug effect has been severely hampered by the lack of desired contrast agents with high relaxivities, and optimized in vivo retention time. We have reported the development of protein-based MRI contrast agents (ProCA1) by rational design of Gd(3+) binding sites into a stable protein resulting in significantly increased longitudinal (r(1)) and transverse (r(2)) relaxivities compared to Gd-DTPA. Here, we report a further improvement of protein contrast agents ProCA1 for in vivo imaging by protein modification with various sizes of polyethylene glycol (PEG) chain. PEGylation results in significant increases of both r(1) and r(2) relaxivities (up to 200%), and these high relaxivities persist even at field strengths up to 9.4 T. In addition, our experimental results demonstrate that modified contrast agents have significant improvement of in vivo MR imaging and biocompatibilities including dose efficiency, protein solubility, blood retention time and decreased immunogenicity. Such improvement can be important to the animal imaging and pre-clinical research at high or ultra-high field where there is an urgent need for molecular imaging probes and optimized contrast agent., (Published by Elsevier Inc.)

Children in the terminal stage of cancer may experience intractable pain despite the use of high doses of opioids. The resultant sedating effect of the opioids limits the child's ability to communicate and participate in activities, thereby negatively affecting quality of life. Ketamine, an intravenous (IV) anesthetic with analgesic properties, when used in low doses, may be useful in managing pediatric cancer pain at the end of life. Ketamine can prevent the development of opioid tolerance and provide additional analgesia without an increase in sedating effects. At the authors' institution, 2 children with end-stage cancer were started on continuous infusion low-dose ketamine to help achieve adequate pain control and allow the children to be home and interactive for the last weeks of their lives. Each case illustrates the complexities of achieving and maintaining adequate pain control and promoting care of the child and family in a setting that is most appropriate for them.

Convection-enhanced delivery (CED) permits the homogeneous distribution of therapeutic agents throughout localized regions of the brain parenchyma without causing tissue damage as occurs with bolus injection. Here, we examined whether CED infusion of the N-type calcium channel antagonists omega-conotoxin GVIA (omega-CTX-G) and omega-conotoxin MVIIA (omega-CTX-M) can attenuate kindling measures in fully amygdala-kindled rats. Rats were implanted with a combination infusion cannula-stimulating electrode assembly into the right basolateral amygdala. Fully kindled animals received infusions of vehicle, omega-CTX-G (0.005, 0.05, and 0.5 nmol), omega-CTX-M (0.05, 0.15, and 0.5 nmol), proteolytically inactivated omega-CTX-M (0.5 nmol), or carbamazepine (500 nmol) into the stimulation site. CED of omega-CTX-G and omega-CTX-M over a 20-min period resulted in a dose-dependent increase in the afterdischarge threshold and a decrease in the afterdischarge duration and behavioral seizure score and duration during a period of 20 min to 1 week after the infusion, indicating an inhibitory effect on the triggering and expression of kindled seizures. The protective effects of omega-conotoxins reached a maximum at 48 h postinfusion, and then they gradually resolved over the next 5 days. In contrast, carbamazepine was active at 20 min but not at 24 h after the infusion, whereas CED of vehicle or inactivated omega-CTX-M had no effect. Except for transient tremor in some rats receiving the highest toxin doses, no adverse effects were observed. These results indicate that local CED of high-molecular-weight presynaptic N-type calcium channel blockers can produce long-lasting inhibition of brain excitability and that they may provide prolonged seizure protection in focal seizure disorders.

Objective: Maspin expression is often deregulated in human cancer cells compared to their normal cells due to loss of epigenetic control. In contrast to normal human ovarian surface epithelial (HOSE) cells, ovarian carcinoma cells display a gain of maspin mRNA expression. The objective of this study was to determine whether gain of maspin expression in ovarian cancer is governed by epigenetic mechanisms., Methods: We examined the cytosine methylation and chromatin accessibility status of the maspin promoter in normal HOSE cells and ovarian carcinoma cells with real-time RT-PCR, sodium bisulfite genomic sequencing, and chromatin accessibility assays. 5-Aza-2'-deoxycytidine (5-aza-dC) was used to induce demethylation of the maspin promoter. Ad p53 was used to induce transient overexpression of wild-type p53., Results: Normal HOSE cells were maspin-negative in association with methylation of the maspin promoter. In the maspin-positive ovarian cancer cell lines, the maspin promoter was unmethylated. Increased maspin expression in ovarian carcinoma cells was accompanied by a more accessible chromatin structure in the maspin promoter. In the maspin-negative ovarian cancer cell line A222, maspin could be induced following 5-aza-dC treatment or by forced overexpression of p53., Conclusions: These results suggest that changes in cytosine methylation and chromatin accessibility play an important role in maspin expression in human ovarian carcinoma. Deregulation of maspin expression in ovarian cancer is due to loss of epigenetic control as has been shown in other cancers. This observation provides further evidence of the strict epigenetic control of the maspin gene.

The present study tested the hypothesis that mouse and human 5-hydroxytryptamine3A (5-HT3A) receptors may be differentially modulated by benzylidene analogs of anabaseine (BA) and that these analogs may be useful in assessing residues involved in receptor gating. Mouse and human wild-type and mouse and human chimeric 5-HT3A receptors expressed in Xenopus oocytes were evaluated with the two-electrode voltage clamp technique. Our previous studies demonstrated that 3-(2,4-dimethoxybenzylidene)-anabaseine (DMXBA) is an antagonist at the mouse wild-type 5-HT3A receptor, but that its metabolites 3-(2-hydroxy, 4-methoxybenzylidene)-anabaseine (2-OHMBA), 3-(2-methoxy, 4-hydroxybenzylidene)-anabaseine (4-OHMBA), and 3-(2,4-dihydroxybenzylidene)-anabaseine (2,4-DiOHBA) are partial agonists (J Pharmacol Exp Ther, 299: 1112-1117, 2001). In the human wild-type (HWT) 5-HT3A receptor, none of the BA compounds possessed partial agonist activity. BA compounds antagonized 1.5 microM 5-HT-mediated (EC50) responses in the HWT 5-HT3A receptor with a rank order of potency (IC50 in muM) of 2-OHMBA (1.5 +/- 0.1) > DMXBA (3.1 +/- 0.2) > 4-OHMBA (7.4 +/- 0.5) > 2,4-DiOHBA (12.8 +/- 0.7). In mouse receptor chimeras containing N-terminal human receptor orthologs, 2-OHMBA inhibited 5-HT-mediated (EC50) currents with IC50 values of 2.0 +/- 0.08 and 3.0 +/- 0.13 microM, respectively. In human receptor chimeras containing N-terminal mouse receptor orthologs, 2-OHMBA displayed partial agonist activities with EC50 values of 1.3 +/- 0.15 and 5.0 +/- 0.4 microM; efficacies were 43 and 57%, respectively. Thus, amino acids present in the distal one-third of the N terminus of mouse and human 5-HT3A receptors are necessary and sufficient to confer partial agonist or antagonist properties of 2-OHMBA.

In this retrospective study, matched peripheral blood and lung samples from patients on antiretroviral therapy were studied in order to investigate whether differences in mutations associated with resistance to nucleoside analogues could be detected between the lung and blood. Discordant mutation patterns in the reverse transcriptase (RT) between plasma and cell free bronchoalveolar lavage fluid (BAL-fluid) HIV-1 genomic RNA was observed in five out of seven patients on nucleoside reverse transcriptase inhibitor (NRTI) monotherapy and six out of seven on combination therapy. In the cellular compartments, DNA recovered from peripheral blood mononuclear cells (PBMCs) and cells from BAL-cells discordant HIV-1 resistance genotypes were detected in 15 out of 44 matched samples. Differences in resistant genotypes between PBMCs and BAL-cells were most pronounced in patients receiving combination antiretroviral therapy. The pattern and number of mutations in RT associated with resistance differed in the BAL-cells compared to PBMCs in four out of 12 subjects not receiving antiretroviral therapy at the time of bronchoscopy, three from 14 patients on NRTI monotherapy, five out of nine on dual combination therapy and three out of nine on HAART. The differences in the detection of resistance mutations between blood and the lung suggest that the lung is a site of replication for HIV-1., (Copyright 2004 Wiley-Liss, Inc.)

A preliminary structural analysis of Vibrio cholerae zonula occludens toxin (ZOT) was made by equilibrium denaturation and circular dichroism. ZOT is a structurally unstable protein in aqueous solution (DeltaG((H2O)) 3.82 kcal/mol), the putative intra- and extracellular domains unfold co-operatively, with complete denaturation via observed conformational intermediates. Refolding of denatured ZOT is not dependent on disulphide bridge formation. Partial refolding of a maltose binding protein-ZOT fusion did not prevent its specific binding to the ZOT receptor on Caco-2 cells. Immuno-gold labelling showed that the ZOT receptor localises to the intercellular contacts between cells in a confluent monolayer.

The use of genotyping assays for the detection and evaluation of drug resistance mutations within the polymerase gene of human immunodeficiency virus type 1 (HIV-1) is becoming increasingly relevant in the clinical management of HIV-1 infection. However, genotypic resistance assays available currently have been optimised for genetic subtype B strains of the virus and many clinical centres are presented with strains from subtypes A, C, and D. In the present report, we compare the performance of two sequence-based commercially available kits, the ViroSeq Genotyping System (Applied Biosystems, Foster City, CA) and the TruGene HIV-1 Genotyping Kit (Visible Genetics, Toronto, Ontario) against a panel of 35 virus isolates from HIV-1 Group M (subtypes A-J). Full-length consensus sequences were generated by the ViroSeq genotyping system for 26 of 31 (83.8%) of the isolates tested, in contrast to the TruGene genotyping system, which generated 16 of 30 (53%) usable sequences overall. Overall, subtype B isolates were sequenced with a greater degree of success than non-subtype B isolates. Discrepancies were found between the consensus sequences reported by each system for each sample (mean difference 1.0%; range 0.0-3.2%), but these appeared to be random and did not affect interpretation of the major resistance codons. In addition, both systems were able to amplify template RNA from low copy viral load plasma samples (10(2)-10(3) RNA copies/ml) taken from a random selection of patient samples encompassing subtypes A-C. While the availability of these genotyping systems should facilitate studies of HIV-1 drug resistance in countries in which these subtypes are prevalent, the performance against subtypes other than B needs to be improved., (Copyright 2003 Wiley-Liss, Inc.)