Antithrombin III infusion improves anticoagulation in congenital diaphragmatic hernia patients on extracorporeal membrane oxygenation.

PURPOSE: Achieving effective anticoagulation during neonatal extracorporeal membrane oxygenation (ECMO) without increasing the risk of hemorrhage remains challenging. The use of antithrombin III (AT-III) for this purpose has been examined, but studies have been limited to intermittent bolus dosing. We aimed to evaluate the efficacy and safety of an institutionally developed AT-III continuous infusion protocol in neonates receiving ECMO for the treatment of congenital diaphragmatic hernia (CDH). METHODS: In this single center, retrospective study, all neonates with a CDH who received ECMO support during the study period were included. Data on anticoagulation labs and therapy, life-threatening bleeding, and circuit changes were analyzed. RESULTS: Eleven patients were divided into two groups: patients with AT-III continuous infusion (n = 5) and without (n = 6). There were no differences in the gestational age (p = 0.29), sex (p = 1.00), ECMO duration (p = 0.59), or initial AT-III levels (p = 0.76) between groups. Patients in the AT-III infusion group had on average 18.5% higher AT-III levels (p < 0.0001). Patients receiving continuous AT-III infusions spent a significantly higher percentage of ECMO time within the therapeutic range, measured using anti-Factor Xa levels (64.9±4.2% vs. 29.1±8.57%, p = 0.008), and required fewer changes to the heparin infusion rate (6.48±0.88 vs 2.38±0.36 changes/day changes/day, p = 0.005). Multivariate analysis revealed continuous infusion of AT-III did not increase the rate of intracranial or surgical bleeding (p = 0.27). CONCLUSION: AT-III as a continuous infusion in CDH neonates on ECMO provides a decreased need to modify heparin infusion and more consistent therapeutic anticoagulation without increasing the risk of life-threatening bleeding.

The Effects of the COVID-19 Pandemic on Burn Clinic.

The COVID-19 pandemic has led to anxiety and fears for the general public. It is unclear how the behavior of people with acute burns and the services available to them has changed during the pandemic. The aim of our observational study was to evaluate our clinic's experience with patients presenting with burns during the first 10 months of the COVID-19 pandemic and determine if delays in presentation and healthcare delivery exist within our burn population. Patients referred to our clinic from March 1, 2020 to December 15, 2020 were reviewed for time of presentation after injury. We defined a true delay in presentation of >5 days from date of injury to date of referral for patients who were not inpatients at our facility or received initial care elsewhere prior to referral. Of the 246 patients who were referred to our clinic, during this time period, 199 patients (80.89%) attended their appointments. Our in-person clinic volume from referrals increased in July 2020 with a sharp decrease in August 2020. Our total clinic volume decreased in 2020 from 2019 by about 14%. Referrals to our clinic decreased in 2020 from 2019 by about 34%. Video telehealth visits did not account for the decrease in visits. There was low incidence of delays in presentation to our clinic during the pandemic. Additional investigation is necessary to see if the incidence of burn injury decreased. Despite the pandemic, our clinic remained ready and open to serve the burn population.

Safety, Feasibility, and Merits of Longitudinal Molecular Testing of Multiple Metastatic Sites to Inform mTNBC Patient Treatment in the Intensive Trial of Omics in Cancer.

PURPOSE: Patients with metastatic triple-negative breast cancer (mTNBC) have poor outcomes. The Intensive Trial of Omics in Cancer (ITOMIC) sought to determine the feasibility and potential efficacy of informing treatment decisions through multiple biopsies of mTNBC deposits longitudinally over time, accompanied by analysis using a distributed network of experts. METHODS: Thirty-one subjects were enrolled and 432 postenrollment biopsies performed (clinical and study-directed) of which 332 were study-directed. Molecular profiling included whole-genome sequencing or whole-exome sequencing, cancer-associated gene panel sequencing, RNA-sequencing, and immunohistochemistry. To afford time for analysis, subjects were initially treated with cisplatin (19 subjects), or another treatment they had not received previously. The results were discussed at a multi-institutional ITOMIC Tumor Board, and a report transmitted to the subject's oncologist who arrived at the final treatment decision in conjunction with the subject. Assistance was provided to access treatments that were predicted to be effective. RESULTS: Multiple biopsies in single settings and over time were safe, and comprehensive analysis was feasible. Two subjects were found to have lung cancer, one had carcinoma of unknown primary site, tumor samples from three subjects were estrogen receptor-positive and from two others, human epidermal growth factor receptor 2-positive. Two subjects withdrew. Thirty-four of 112 recommended treatments were accessed using approved drugs, clinical trials, and single-patient investigational new drugs. After excluding the three subjects with nonbreast cancers and the two subjects who withdrew, 22 of 26 subjects (84.6%) received at least one ITOMIC Tumor Board-recommended treatment. CONCLUSION: Further exploration of this approach in patients with mTNBC is merited.

What Happens After a Positive Screen for Depression and Posttraumatic Stress Disorder in the Outpatient Burn Clinic?

Multiple reports have demonstrated a wide prevalence of both depression and posttraumatic stress disorder (PTSD) within 1 year of burn injury. The purpose of this study is to determine outcomes of burn patients after a positive outpatient screen for depression or PTSD at an American Burn Association-verified burn center. All patients who screened positive were offered referral for psychologic and/or psychiatric counseling. Rescreening was performed with a goal of approximately 6 months. A total of 445 patients were enrolled with 91 (20.6%) screening positive for depression and 59 (13.4%) for PTSD. TBSA burned was associated with a positive screen for depression (P = .008) and PTSD (P = .012) while electrical injury was associated with a positive screen for depression (P = .029). Rescreening was done in 15.5% with 23% rescreening positive for depression and 15% for PTSD. The study validated the need for early screening and referral for psychologic and/or psychiatric counseling in this population.

How does blood loss relate to the extent of surgical wound excision?

PURPOSE: We investigated a novel system that uses image-processing algorithms to accurately measure the hemoglobin content of discarded surgical sponges to determine how blood loss relates to the excised area when current methods to minimize bleeding are employed. The system was used during 130 procedures in adult patients having wound excisions ≥1% BSA (study group). An historic group of 105 similar cases in whom visually estimated blood was determined was also evaluated. RESULTS: Surgical blood loss was less than previous estimates. The correlation between blood loss and the excised area in the study group was poor (R2=0.3988 for all patients and R2=0.1439 for excisions ≥10% BSA). Moreover, the visual estimates of blood loss in the historic group were more closely related to excised area than the accurate measurements in the study group (R2=0.6017 (historic), R2=0.3988 (study), p<0.001 for both). The mean absolute unstandardized residuals were 140.18±158.52 (historic) vs. 307.99±317.03 (study), p<0.001. CONCLUSIONS: As demonstrated in the historic group, visual estimates of blood loss tend to be more related to the size of excision than the amount of bleeding. The actual blood loss is not well correlated with the extent of excision. Clinicians should not rely on traditional blood loss estimates. Accurate measurement is needed to inform transfusion decisions and guide care.

Participant Attitudes Toward an Intensive Trial of Multiple Biopsies, Multidimensional Molecular Analysis, and Reporting of Results in Metastatic Triple-Negative Breast Cancer.

PURPOSE: Multidimensional molecular analysis of tumor tissue intensively over space and time can provide insight into how cancers evolve and escape treatment. Attitudes of participants in such trials have not been assessed. We explored patient views regarding an intensive study incorporating multiple biopsies, multidimensional molecular testing, and drug response predictions that are reported to the oncologist and patient. PATIENTS AND METHODS: A structured, self-administered survey was conducted among the first 15 patients enrolled in ITOMIC-001 (Intensive Trial of Omics in Cancer). Patients with metastatic triple-negative breast cancer were accrued at two sites in Washington state. Surveys containing 17 items were administered at enrollment and after the return of results. Surveys explored perceptions regarding risks, personal benefits, benefits to others, uncertainties associated with interpreting complex molecular results, concerns regarding multiple biopsies, and potential loss of confidentiality. At follow-up, three additional unique items explored patient coping. RESULTS: All participants expressed a strong desire for their experiences to benefit others, and all perceived a higher likelihood of deriving benefit than described during detailed consent discussions. Loss of confidentiality ranked lowest among patient concerns. Despite acknowledging uncertainties and risks inherent in complex molecular testing for clinical reporting, participants wanted access to findings in evaluating treatment choices, even if the best available evidence was weak. Follow-up surveys demonstrated relatively little change in attitudes, although concern about study biopsies generally declined. Study participation helped several patients cope better with their disease. CONCLUSION: In advanced breast cancer, these findings demonstrate the feasibility of engaging motivated patients in trials that navigate the uncertainties associated with intensive spatial and longitudinal multidimensional molecular testing for the purpose of advancing precision medicine.

A Distributed Network for Intensive Longitudinal Monitoring in Metastatic Triple-Negative Breast Cancer.

Accelerating cancer research is expected to require new types of clinical trials. This report describes the Intensive Trial of OMics in Cancer (ITOMIC) and a participant with triple-negative breast cancer metastatic to bone, who had markedly elevated circulating tumor cells (CTCs) that were monitored 48 times over 9 months. A total of 32 researchers from 14 institutions were engaged in the patient's evaluation; 20 researchers had no prior involvement in patient care and 18 were recruited specifically for this patient. Whole-exome sequencing of 3 bone marrow samples demonstrated a novel ROS1 variant that was estimated to be present in most or all tumor cells. After an initial response to cisplatin, a hypothesis of crizotinib sensitivity was disproven. Leukapheresis followed by partial CTC enrichment allowed for the development of a differential high-throughput drug screen and demonstrated sensitivity to investigational BH3-mimetic inhibitors of BCL-2 that could not be tested in the patient because requests to the pharmaceutical sponsors were denied. The number and size of CTC clusters correlated with clinical status and eventually death. Focusing the expertise of a distributed network of investigators on an intensively monitored patient with cancer can generate high-resolution views of the natural history of cancer and suggest new opportunities for therapy. Optimization requires access to investigational drugs.

A randomized comparison study of Aquacel Ag and Glucan II as donor site dressings with regard to healing time, cosmesis, infection rate, and patient's perceived pain: a pilot study.

This study was undertaken to compare pain, healing time, infection rate, and cosmetic outcome between Aquacel Ag (Convatec) and Glucan II (Brennan Medical) as donor site dressings. The authors performed a prospective, randomized, patient-controlled study. Eligible patients had two donor sites harvested. One site was dressed with Aquacel Ag and the other site with Glucan II. Patients were followed at set time points for 6 months to determine the rate of epithelialization, patient's perceived pain, infection rate, and the cosmetic outcome. A total of 20 patients were enrolled in the study. All patient data were collected through reepithelialization. The average time to wound healing for Aquacel Ag was 12.5 ± 2.07 days compared with Glucan II 12.7 ± 1.99 days. Perceived pain scores for each donor site were recorded. On postoperative day 5, patients reported significantly less pain with the Aquacel Ag site (Aquacel Ag 1.75 vs Glucan II 2.5, P = .02). Three donor sites showed clinical signs of infection (two Glucan II and one Aquacel Ag) prompting culture and dressing removal. There was no statistically significant difference in cosmetic outcomes of the donor sites at any time point. When comparing Aquacel Ag and Glucan II, our study has determined that there is no significant difference with regard to healing time, infection rates, and cosmetic outcomes. Both dressings are comparable with regard to ease of application and postoperative care.

Impact of simulation-based extracorporeal membrane oxygenation training in the simulation laboratory and clinical environment.

INTRODUCTION: Extracorporeal membrane oxygenation (ECMO) is a high-risk, complex therapy. Opportunities to develop teamwork skills and expertise to mitigate risks are few. Our objective was to assess whether simulation would improve technical and nontechnical skills in dealing with ECMO circuit emergencies and allow transfer of skills from the simulated setting to clinical environment. METHODS: Subjects were ECMO circuit providers who performed scenarios utilizing an infant simulator and functional ECMO circuit, followed immediately by video-assisted debriefings. Within the simulation laboratory, outcomes were timed responses, percentage of correct actions, teamwork, safety knowledge, and attitudes. Identification of latent safety threats (LSTs) was the focus of debriefings. Within the clinical setting, translation of learned skills was assessed by measuring circuit readiness and compliance with a cannulation initiation checklist. RESULTS: Nineteen subjects performed 96 simulations during enrollment. In the laboratory, there was no improvement in timed responses or percent correct actions. Teamwork (P = 0.001), knowledge (P = 0.033), and attitudes (P = 0.001) all improved compared with baseline. Debriefing identified 99 LSTs. Clinically, 26 cannulations occurred during enrollment. Median time from blood available to circuit readiness was 17 minutes (range, 5-95), with no improvement during the study. Compliance with the initiation checklist improved compared with prestudy baseline (P < 0.0001). CONCLUSIONS: Simulation-based training is an effective method to improve safety knowledge, attitudes, and teamwork surrounding ECMO emergencies. On-going training is feasible and allows identification of LSTs. Further work is needed to assess translation of learned skills and behaviors into the clinical environment.

Loss of the Synaptic Vesicle Protein SV2B results in reduced neurotransmission and altered synaptic vesicle protein expression in the retina.

The Synaptic Vesicle Protein 2 (SV2) family of transporter-like proteins is expressed exclusively in vesicles that undergo calcium-regulated exocytosis. Of the three isoforms expressed in mammals, SV2B is the most divergent. Here we report studies of SV2B location and function in the retina. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. In mice lacking SV2B, synaptic transmission at the synapse between photoreceptors and bipolar neurons was decreased, as evidenced by a significant reduction in the amplitude of the b-wave in electroretinogram recordings. Quantitative immunoblot analyses of whole eyes revealed that loss of SV2B was associated with reduced levels of synaptic vesicle proteins including synaptotagmin, VAMP, synaptophysin and the vesicular glutamate transporter V-GLUT1. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. Thus, SV2B contributes to the modulation of synaptic vesicle exocytosis and plays a significant role in regulating synaptic protein content.

If-then contingencies and the differential effects of the availability of an attractive alternative on relationship maintenance for men and women.

In 7 experiments, the causal effects of the availability of an attractive alternative (AA) relationship partner on current relationship thoughts and intentions were tested using confederates, mental simulations, and virtual reality. Men behaved consistent with traditional relationship-commitment theories, showing decreased willingness to tolerate their partner's transgressions after the availability of an AA was made salient. However, consistent with a motivated cognition approach to commitment and work on relational self-construals, women increased their tolerance when presented with the relationship threat of an alternative. Word-fragment and lexical decision data suggested that AAs may activate threat for women, and their ability to dampen threat accessibility is associated with prorelationship responses. Finally, this "if relationship is threatened, then defend the relationship" contingency was induced in men with an implementation intention induction.

PKA, germ cells, and fertility.

Temporal and spatial regulation of PKA activity are essential for vigorous sperm motility and for the resumption of meiosis in oocytes, two events required for successful fertilization. Genetic mutations in mice that affect PKA signaling in germ cells lead to infertility and illustrate the importance of this pathway in mammalian reproduction.

The differential effects of intrinsic and identified motivation on well-being and performance: prospective, experimental, and implicit approaches to self-determination theory.

Self-determination theory research has demonstrated that intrinsic and identified self-regulations are associated with successful adaptation. However, few distinctions are typically made between these regulations and their outcomes. In the present studies, the associations between intrinsic and identified motivations and outcomes of psychological well-being and academic performance are compared in educational settings. In Study 1, intrinsic self-regulation predicted psychological well-being, independent of academic performance. In contrast, identified regulation predicted academic performance. Additionally, the more that students demonstrated an identified academic regulation, the more that their psychological well-being was contingent on performance. In Study 2a, priming intrinsic self-regulation led to greater psychological well-being 10 days later. In Study 2b, an implicit measure of identified regulation predicted academic performance 6 weeks later. Results indicate the need to address important distinctions between intrinsic and identified regulations.

Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy.

Voltage-gated sodium channels (Na(V)) are critical for initiation of action potentials. Heterozygous loss-of-function mutations in Na(V)1.1 channels cause severe myoclonic epilepsy in infancy (SMEI). Homozygous null Scn1a-/- mice developed ataxia and died on postnatal day (P) 15 but could be sustained to P17.5 with manual feeding. Heterozygous Scn1a+/- mice had spontaneous seizures and sporadic deaths beginning after P21, with a notable dependence on genetic background. Loss of Na(V)1.1 did not change voltage-dependent activation or inactivation of sodium channels in hippocampal neurons. The sodium current density was, however, substantially reduced in inhibitory interneurons of Scn1a+/- and Scn1a-/- mice but not in their excitatory pyramidal neurons. An immunocytochemical survey also showed a specific upregulation of Na(V)1.3 channels in a subset of hippocampal interneurons. Our results indicate that reduced sodium currents in GABAergic inhibitory interneurons in Scn1a+/- heterozygotes may cause the hyperexcitability that leads to epilepsy in patients with SMEI.

Haploinsufficiency at the protein kinase A RI alpha gene locus leads to fertility defects in male mice and men.

Carney complex (CNC) is a familial multiple neoplasia syndrome characterized by spotty skin pigmentation, cardiac and cutaneous myxomas, and endocrine tumors. CNC is inherited as an autosomal dominant trait and is transmitted with greater frequency by women vs. men. Nearly two thirds of CNC patients are heterozygous for inactivating mutations in the gene encoding the protein kinase A (PKA) type I alpha regulatory subunit (RI alpha), PRKAR1. We report here that male mice heterozygous for the Prkar1a gene have severely reduced fertility. Sperm from Prkar1a heterozygous mice are morphologically abnormal and reduced in number. Genetic rescue experiments reveal that this phenotype results from elevated PKA catalytic activity in germ cells as early as the pachytene stage of spermatogenesis. Consistent with this defect in the male mutant mice, sperm from CNC patients heterozygous for PRKAR1A mutations were also found to be morphologically aberrant and decreased in number. We conclude that unregulated PKA activity in male meiotic or postmeiotic germ cells leads to structural defects in mature sperm and results in reduced fertility in mice and humans, contributing to the strikingly reduced transmission of PRKAR1A inactivating mutations by male patients with CNC.

Microarray-based detection and typing of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) is the most economically important veterinary pathogen because of its highly infectious nature and the devastating effects the virus has on the livestock industry. Rapid diagnostic methods are needed for detection and typing of FMDV serotypes and differentiation from other viruses causing vesicular diseases. We developed a microarray-based test that uses a FMD DNA chip containing 155 oligonucleotide probes, 35-45 base pair (bp) long, virus-common and serotype-specific, designed from the VP3-VP1-2A region of the genome. A set of two forward primers and one reverse primer were also designed to allow amplification of approximately 1100 bp of target sequences from this region. The amplified target was labelled with Alexa-Fluor 546 dye and applied to the FMD DNA chip. A total of 23 different FMDV strains representing all seven serotypes were detected and typed by the FMD DNA chip. Microarray technology offers a unique capability to identify multiple pathogens in a single chip.

The role of protein kinase A anchoring via the RII alpha regulatory subunit in the murine immune system.

Intracellular cAMP may inhibit T cell activation and proliferation via activation of the cAMP-dependent protein kinase, PKA. PKA signaling is maintained through interactions of the regulatory subunit with A-kinase anchoring proteins (AKAPs). We demonstrated that T cells contain AKAPs and now ask whether PKA anchoring to AKAPs via the RIIalpha regulatory subunit is necessary for cAMP-mediated inhibition of T cell activation. We studied the immune systems of mice lacking the RIIalpha regulatory subunit of PKA (-/-) and the ability of cells isolated from these mice to respond to cAMP. Dissection of spleen and thymus from wild-type (WT) and -/- mice, single cell suspensions generated from these organs, and flow cytometry analysis illustrate that the gross morphology, cell numbers, and cell populations in the spleen and thymus of the -/- mice are similar to WT controls. In vitro, splenocytes from -/- mice respond to anti-CD3/anti-CD28 and PMA/ionomycin stimulation and produce IL-2 similar to WT. Cytokine analysis revealed no significant difference in Th1 or Th2 differentiation. Finally, equivalent frequencies of CD8(+) IFN-gamma producing effector cells were stimulated upon infection of WT or -/- mice with Listeria monocytogenes. These data represent the first study of the role of RIIalpha in the immune system in vivo and provide evidence that T cell development, homeostasis, and the generation of a cell-mediated immune response are not altered in the RIIalpha -/- mice, suggesting either that RIIalpha is not required for normal immune function or that other proteins are able to compensate for RIIalpha function.

Comparative PRKAR1A genotype-phenotype analyses in humans with Carney complex and prkar1a haploinsufficient mice.

Carney complex (CNC) is a familial multiple neoplasia syndrome characterized by cardiac and extracardiac myxomas in the setting of spotty skin pigmentation and endocrinopathy. We previously identified PRKAR1A (regulatory subunit 1alpha of protein kinase A) mutations in CNC. Mutational analyses of the PRKAR1A gene in 51 unrelated CNC probands now detect mutations in 65%. All mutations, except for one unique missense mutation, lead to PRKAR1A haploinsufficiency. Therefore, we studied the consequences of prkar1a haploinsufficiency in mice. Although we did not observe cardiac myxomas or altered pigmentation in prkar1a(+/-) mice, we did observe some phenotypes similar to CNC, including altered heart rate variability. Moreover, prkar1a(+/-) mice exhibited a marked propensity for extracardiac tumorigenesis. They developed sarcomas and hepatocellular carcinomas. Sarcomas were frequently associated with myxomatous differentiation. Tumors from prkar1a(+/-) mice did not exhibit prkar1a loss of heterozygosity. Thus, we conclude that although PRKAR1A haploinsufficiency does predispose to tumorigenesis, distinct secondary genetic events are required for tumor formation.

Sperm-specific protein kinase A catalytic subunit Calpha2 orchestrates cAMP signaling for male fertility.

An unusual cAMP signaling system mediates many of the events that prepare spermatozoa to meet the egg. Its components include the atypical, bicarbonate-stimulated, sperm adenylyl cyclase and a cAMP-dependent protein kinase (PKA) with the unique catalytic subunit termed Calpha(2) or C(s). We generated mice that lack Calpha(2) to determine its importance in the events downstream of cAMP production. Male Calpha(2) null mice produce normal numbers of sperm that swim spontaneously in vitro. Thus, Calpha(2) has no required role in formation of a functional flagellum or the initiation of motility. In contrast, we find that Calpha(2) is required for bicarbonate to speed the flagellar beat and facilitate Ca(2+) entry channels. In addition, Calpha(2) is needed for the protein tyrosine phosphorylation that occurs late in the sequence of sperm maturation and for a negative feedback control of cAMP production, revealed here. Consistent with these specific defects in several important sperm functions, Calpha(2) null males are infertile despite normal mating behavior. These results define several crucial roles of PKA in sperm cell biology, bringing together both known and unique PKA-mediated events that are necessary for male fertility.