Subnormothermic Oxygenated Machine Perfusion (24 h) in DCD Kidney Transplantation.

Background: Ex vivo kidney perfusion is an evolving platform that demonstrates promise in preserving and rehabilitating the kidney grafts. Despite this, there is little consensus on the optimal perfusion conditions. Hypothermic perfusion offers limited functional assessment, whereas normothermic perfusion requires a more complex mechanical system and perfusate. Subnormothermic machine perfusion (SNMP) has the potential to combine the advantages of both approaches but has undergone limited investigation. Therefore, the present study sought to determine the suitability of SNMP for extended kidney preservation. Methods: SNMP at 22-25 °C was performed on a portable device for 24 h with porcine kidneys. Graft assessment included measurement of mechanical parameters and biochemical analysis of the perfusate using point-of-care tests. To investigate the viability of kidneys preserved by SNMP, porcine kidney autotransplants were performed in a donation after circulatory death (DCD) model. SNMP was also compared with static cold storage (SCS). Finally, follow-up experiments were conducted in a subset of human kidneys to test the translational significance of findings in porcine kidneys. Results: In the perfusion-only cohort, porcine kidneys all displayed successful perfusion for 24 h by SNMP, evidenced by stable mechanical parameters and biological markers of graft function. Furthermore, in the transplant cohort, DCD grafts with 30 min of warm ischemic injury demonstrated superior posttransplant graft function when preserved by SNMP in comparison with SCS. Finally, human kidneys that underwent 24-h perfusion exhibited stable functional and biological parameters consistent with observations in porcine organs. Conclusions: These observations demonstrate the suitability and cross-species generalizability of subnormothermic machine perfusion to maintain stable kidney perfusion and provide foundational evidence for improved posttransplant graft function of DCD kidneys after SNMP compared with SCS.

Developing a Classification System for Prioritizing Pediatric Dental Patients Needing Treatment under General Anesthesia.

PURPOSE: The aim of this study was to explore a classification system for children requiring full-mouth dental rehabilitation (FMDR) in the operating room (OR) and its association with adverse events. METHODS: Patients treated at a pediatric dental residency clinic and determined to need FMDR in the OR were classified on initial examination, based on the extent of caries, pain and the presence of a dental abscess. On the treatment date, parents were given a questionnaire concerning adverse events that occurred while waiting for treatment. Χ2 tests of independence were used to determine associations between classification (OR code) and the occurrence of adverse events. The Pearson's r test was used to determine relations among adverse events and wait time. RESULTS: The study included 82 patients (age range 2-10 years, mean 4.73 years, median 4 years). The average wait time was 55.6 days. The most common OR classification was caries in the outer third of dentin without pain or abscess, and the most common adverse event was difficulty eating or drinking. The OR code category most closely associated with negative outcomes was the presence of a dental abscess, followed by caries depth, then pain. Wait time was not associated with the occurrence of adverse events. CONCLUSIONS: These data provide evidence to support the need for a classification system for children requiring FMDR in the OR. Dental abscess, caries depth and pain were associated with adverse events.

Sex- and subtype-specific adaptations in excitatory signaling onto deep-layer prelimbic cortical pyramidal neurons after chronic alcohol exposure.

Long-term alcohol use results in behavioral deficits including impaired working memory, elevated anxiety, and blunted inhibitory control that is associated with prefrontal cortical (PFC) dysfunction. Preclinical observations demonstrate multiple impairments in GABAergic neurotransmission onto deep-layer principal cells (PCs) in the prelimbic cortex that suggest dependence-related cortical dysfunction is the product of elevated excitability in these cells. Despite accumulating evidence showing alcohol-induced changes in interneuron signaling onto PCs differ between sexes, there is limited data explicitly evaluating sex-specific ethanol effects on excitatory signaling onto deep-layer PCs that may further contribute to deficits in PFC-dependent behaviors. To address this, we conducted electrophysiological and behavioral tests in both male and female Sprague-Dawley rats to evaluate the effects of chronic ethanol exposure. Among our observations, we report a marked enhancement in glutamatergic signaling onto deep-layer PCs in male, but not female, rats after alcohol exposure. This phenomenon was furthermore specific to a sub-class of PC, sub-cortically projecting Type-A cells, and coincided with enhanced anxiety-like behavior, but no observable deficit in working memory. In contrast, female rats displayed alcohol-induced facilitation in working memory performance with no change in expression of anxiety-like behavior. Together, these results suggest fundamental differences in alcohol effects on cell activity, cortical sub-circuits, and PFC-dependent behaviors across male and female rats.

Safety, tolerability, pharmacokinetics, and immunogenicity of a human monoclonal antibody targeting the G glycoprotein of henipaviruses in healthy adults: a first-in-human, randomised, controlled, phase 1 study.

BACKGROUND: The monoclonal antibody m102.4 is a potent, fully human antibody that neutralises Hendra and Nipah viruses in vitro and in vivo. We aimed to investigate the safety, tolerability, pharmacokinetics, and immunogenicity of m102.4 in healthy adults. METHODS: In this double-blind, placebo-controlled, single-centre, dose-escalation, phase 1 trial of m102.4, we randomly assigned healthy adults aged 18-50 years with a body-mass index of 18·0-35·0 kg/m2 to one of five cohorts. A sentinel pair for each cohort was randomly assigned to either m102.4 or placebo. The remaining participants in each cohort were randomly assigned (5:1) to receive m102.4 or placebo. Cohorts 1-4 received a single intravenous infusion of m102.4 at doses of 1 mg/kg (cohort 1), 3 mg/kg (cohort 2), 10 mg/kg (cohort 3), and 20 mg/kg (cohort 4), and were monitored for 113 days. Cohort 5 received two infusions of 20 mg/kg 72 h apart and were monitored for 123 days. The primary outcomes were safety and tolerability. Secondary outcomes were pharmacokinetics and immunogenicity. Analyses were completed according to protocol. The study was registered on the Australian New Zealand Clinical Trials Registry, ACTRN12615000395538. FINDINGS: Between March 27, 2015, and June 16, 2016, 40 (52%) of 77 healthy screened adults were enrolled in the study. Eight participants were assigned to each cohort (six received m102.4 and two received placebo). 86 treatment-emergent adverse events were reported, with similar rates between placebo and treatment groups. The most common treatment-related event was headache (12 [40%] of 30 participants in the combined m102.4 group, and three [30%] of ten participants in the pooled placebo group). No deaths or severe adverse events leading to study discontinuation occurred. Pharmacokinetics based on those receiving m102.4 (n=30) were linear, with a median half-life of 663·3 h (range 474·3-735·1) for cohort 1, 466·3 h (382·8-522·3) for cohort 2, 397·0 h (333·9-491·8) for cohort 3, and 466·7 h (351·0-889·6) for cohort 4. The elimination kinetics of those receiving repeated dosing (cohort 5) were similar to those of single-dose recipients (median elimination half-time 472·0 [385·6-592·0]). Anti-m102.4 antibodies were not detected at any time-point during the study. INTERPRETATION: Single and repeated dosing of m102.4 were well tolerated and safe, displayed linear pharmacokinetics, and showed no evidence of an immunogenic response. This study will inform future dosing regimens for m102.4 to achieve prolonged exposure for systemic efficacy to prevent and treat henipavirus infections. FUNDING: Queensland Department of Health, the National Health and Medical Research Council, and the National Hendra Virus Research Program.

Differentiation Syndrome, a Side Effect From the Therapy of Acute Promyelocytic Leukemia.

Differentiation Syndrome is a complication of all-trans retinoic acid (ATRA) therapy in patients with acute promyelocytic leukemia (APML). It appears clinically as acute end-organ damage with peripheral edema, hypotension, acute renal failure, and interstitial pulmonary infiltrates. When symptoms develop, physicians are recommended to stop ATRA therapy to minimize complications and reduce mortality immediately. This case report describes a 67-year-old male who was diagnosed with acute promyelocytic leukaemia after he developed episodes of hematuria and easy bruising at home. After beginning a treatment regime of ATRA, steroids, and arsenic, the patient began to have symptoms of differentiation syndrome.

Chronic ethanol exposure alters prelimbic prefrontal cortical Fast-Spiking and Martinotti interneuron function with differential sex specificity in rat brain.

Chronic ethanol exposure results in numerous neurobiological adaptations that promote deficits in medial prefrontal cortical (mPFC) function associated with blunted inhibitory control and elevated anxiety during withdrawal. Studies exploring alcohol dependence-related changes in this region have largely investigated adaptations in glutamatergic signaling, with inhibitory neurotransmission remaining relatively understudied. To address this, we used biochemical and electrophysiological methods to evaluate the effects of ethanol on the activity of deep-layer prelimbic mPFC Fast-Spiking (FS) and Martinotti interneurons after chronic ethanol exposure in male and female rats. We report that chronic alcohol exposure significantly impairs FS neuron excitability in both males and females. Interestingly, we observed a marked sex difference in the baseline activity of Martinotti cells that furthermore displayed differential sex-specific responses to alcohol exposure. In addition, alcohol effects on Martinotti neuron excitability negatively correlated with hyperpolarization-activated currents mediated by hyperpolarization-activated cyclic nucleotide gated (HCN) channels, indicative of a causal relationship. Analysis of HCN1 protein expression also revealed a substantial sex difference, although no effect of ethanol on HCN1 protein expression was observed. Taken together, these findings further elucidate the complex adaptations that occur in the mPFC after chronic ethanol exposure and reveal fundamental differences in interneuron activity between sexes. Furthermore, this disparity may reflect innate differences in intracortical microcircuit function between male and female rats, and offers a tenable circuit-level explanation for sex-dependent behavioral responses to alcohol.

Chronic Ethanol Exposure and Withdrawal Impair Synaptic GABAA Receptor-Mediated Neurotransmission in Deep-Layer Prefrontal Cortex.

BACKGROUND: The prefrontal cortex (PFC) acts as an integrative hub for the processing of cortical and subcortical input into meaningful efferent signaling, permitting complex associative behaviors. PFC dysfunction is consistently observed with ethanol (EtOH) dependence and is a core component of the pathology of alcohol use disorders in current models of addiction. While intracortical gamma-aminobutryric acid (GABA)ergic neurotransmission is understood to be essential for maintaining coordinated network activity within the cortex, relatively little is known regarding functional GABAergic adaptations in PFC during EtOH dependence. METHODS: In the present study, male and female (> postnatal day 60) Sprague-Dawley rats were administered EtOH (5.0 g/kg; intragastric gavage) for 14 to 15 consecutive days. Twenty-four hours after the final administration, animals were sacrificed and brains extracted for electrophysiological recordings of isolated GABAA receptor-mediated currents or analysis of GABAA receptor subunit protein expression in deep-layer PFC neurons. RESULTS: Chronic EtOH exposure significantly attenuated activity-dependent spontaneous GABAA receptor-mediated inhibitory postsynaptic current (IPSC) frequency with no effect on amplitude. Furthermore, analysis of IPSC decay kinetics revealed a significant enhancement of IPSC decay time that was associated with decrements in expression of the α1 GABAA receptor subunit, indicative of further impaired phasic inhibition. These phenomena occurred irrespective of neuron projection destination and sex. Based on previous observations by our laboratory of an epigenetic mechanism for EtOH-induced changes in cortical GABAA receptor subunit expression, the histone deacetylase inhibitor Trichostatin A was administered to water- and EtOH-exposed animals, and prevented EtOH-induced changes in spontaneous IPSC frequency, IPSC decay kinetics, and GABAA receptor subunit expression. CONCLUSIONS: Taken together, these results demonstrate that chronic EtOH exposure impairs synaptic inhibitory neurotransmission in deep-layer pyramidal neurons of the medial PFC in both male and female rats. These maladaptations occur in neurons projecting to numerous regions implicated in the sequelae of EtOH dependence, offering a mechanistic link between the manifestation of PFC dysfunction and negative affective states observed with extended consumption.

Histone deacetylases mediate GABAA receptor expression, physiology, and behavioral maladaptations in rat models of alcohol dependence.

Alcohol use disorders are chronic debilitating diseases characterized by severe withdrawal symptoms that contribute to morbidity and relapse. GABAA receptor (GABAAR) adaptations have long been implicated in the chronic effects of alcohol and contribute to many withdrawal symptoms associated with alcohol dependence. In rodents, GABAAR hypofunction results from decreases in Gabra1 expression, although the underlying mechanism controlling Gabra1 expression after chronic ethanol exposure is still unknown. We found that chronic ethanol exposure using either ethanol gavage or two-bottle choice voluntary access paradigms decreased Gabra1 expression and increased Hdac2 and Hdac3 expression. Administration of the HDAC inhibitor trichostatin A (TSA) after chronic ethanol exposure prevents the decrease in Gabra1 expression and function as well as the increase in Hdac2 and Hdac3 expression in both the cortex and the medial prefrontal cortex (mPFC). Chronic ethanol exposure and withdrawal, but not acute ethanol exposure or acute withdrawal, cause a selective upregulation of HDAC2 and HDAC3 associated with the Gabra1 promoter that accompanies a decrease in H3 acetylation of the Gabra1 promoter and the reduction in GABAAR α1 subunit expression. TSA administration prevented each of these molecular events as well as behavioral manifestations of ethanol dependence, including tolerance to zolpidem-induced loss of righting reflex, reduced open-arm time in the elevated plus maze, reduced center-time and locomotor activity in the open-field assay, and TSA reduced voluntary ethanol consumption. The results show how chronic ethanol exposure regulates the highly prominent GABAAR α1 subunit by an epigenetic mechanism that represents a potential treatment modality for alcohol dependence.

Associations between patients' understanding of periodontal disease, treatment compliance, and disease status.

OBJECTIVE: The aim of the present study was to investigate the associations between patients' understanding of periodontal disease, their individual compliance to homecare, self-reported periodontal disease, and how they have been educated regarding their oral health. METHOD AND MATERIALS: A cross-sectional study was performed by distributing questionnaires to patients at the University of Alberta Dental Clinic. A survey consisting of 45 questions was constructed in order to quantify patients' understanding of periodontal disease (section 1), where they received their information, how often they have been instructed by dental practitioners, their homecare habits (section 2), and their self-reported periodontal status (section 3). RESULTS: In total, 286 surveys were analyzed from participants (92 males; 191 females; 3 other), with an age range of 15-87 years (mean 42.6 ± 15.1). Correctly answered questions ranged from 39.5% to 95.8%, the highest related to smoking and lowest regarding gingival pockets. Only 42.0% and 42.7% understood the purpose of periodontal probing and the difference between gingivitis and periodontitis respectively. Of the participants, 13.3% and 54.2% had never been shown how to clean their teeth or counselled regarding dietary habits from dental professionals respectively, and only 24.8% had been shown more than four times how to clean their teeth. A moderate association was found between sections 1 (knowledge) and 2 (habits/instruction), between sections 1 and 3 (self-reported health), and between sections 2 and 3. CONCLUSION: Moderate association between knowledge, homecare habits, instruction from dental professionals, and self-reported periodontal health was found. Patient understanding is lacking in certain areas and there seems to be a lack of patient education from dental professionals. As dental professionals improve patient instruction, there may be an improvement in patient compliance to homecare and periodontal health.

The Impact of Altitude on Sleep-Disordered Breathing in Children Dwelling at High Altitude: A Crossover Study.

Study Objectives: Sleep-disordered breathing (SDB) is prevalent among children and is associated with adverse health outcomes. Worldwide, approximately 250 million individuals reside at altitudes higher than 2000 meters above sea level (masl). The effect of chronic high-altitude exposure on children with SDB is unknown. This study aims to determine the impact of altitude on sleep study outcomes in children with SDB dwelling at high altitude. Methods: A single-center crossover study was performed to compare results of high-altitude home polysomnography (H-PSG) with lower altitude laboratory polysomnography (L-PSG) in school-age children dwelling at high altitude with symptoms consistent with SDB. The primary outcome was apnea-hypopnea index (AHI), with secondary outcomes including obstructive AHI; central AHI; and measures of oxygenation, sleep quality, and pulse rate. Results: Twelve participants were enrolled, with 10 included in the final analysis. Median altitude was 1644 masl on L-PSG and 2531 masl on H-PSG. Median AHI was 2.40 on L-PSG and 10.95 on H-PSG. Both obstructive and central respiratory events accounted for the difference in AHI. Oxygenation and sleep fragmentation were worse and pulse rate higher on H-PSG compared to L-PSG. Conclusions: These findings reveal a clinically substantial impact of altitude on respiratory, sleep, and cardiovascular outcomes in children with SDB who dwell at high altitude. Within this population, L-PSG underestimates obstructive sleep apnea and central sleep apnea compared to H-PSG. Given the shortage of high-altitude pediatric sleep laboratories, these results suggest a role for home sleep apnea testing for children residing at high altitude.

Automated Visual Fin Identification of Individual Great White Sharks.

This paper discusses the automated visual identification of individual great white sharks from dorsal fin imagery. We propose a computer vision photo ID system and report recognition results over a database of thousands of unconstrained fin images. To the best of our knowledge this line of work establishes the first fully automated contour-based visual ID system in the field of animal biometrics. The approach put forward appreciates shark fins as textureless, flexible and partially occluded objects with an individually characteristic shape. In order to recover animal identities from an image we first introduce an open contour stroke model, which extends multi-scale region segmentation to achieve robust fin detection. Secondly, we show that combinatorial, scale-space selective fingerprinting can successfully encode fin individuality. We then measure the species-specific distribution of visual individuality along the fin contour via an embedding into a global 'fin space'. Exploiting this domain, we finally propose a non-linear model for individual animal recognition and combine all approaches into a fine-grained multi-instance framework. We provide a system evaluation, compare results to prior work, and report performance and properties in detail.

Phenotype-dependent inhibition of glutamatergic transmission on nucleus accumbens medium spiny neurons by the abused inhalant toluene.

Abused inhalants are voluntarily inhaled at high concentrations to produce intoxicating effects. Results from animal studies show that the abused inhalant toluene triggers behaviors, such as self-administration and conditioned place preference, which are commonly associated with addictive drugs. However, little is known about how toluene affects neurons within the nucleus accumbens (NAc), a brain region within the basal ganglia that mediates goal-directed behaviors and is implicated in the development and maintenance of addictive behaviors. Here we report that toluene inhibits a component of the after-hyperpolarization potential, and dose-dependently inhibits N-methyl-D-aspartate (NMDA)-mediated currents in rat NAc medium spiny neurons (MSN). Moreover, using the multivariate statistical technique, partial least squares discriminative analysis to analyze electrophysiological measures from rat NAc MSNs, we show that toluene induces a persistent depression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-mediated currents in one subtype of NAc MSNs, and that the electrophysiological features of MSN neurons predicts their sensitivity to toluene. The CB1 receptor antagonist AM281 blocked the toluene-induced long-term depression of AMPA currents, indicating that this process is dependent on endocannabinoid signaling. The neuronal identity of recorded cells was examined using dual histochemistry and shows that toluene-sensitive NAc neurons are dopamine D2 MSNs that express preproenkephalin mRNA. Overall, the results from these studies indicate that physiological characteristics obtained from NAc MSNs during whole-cell patch-clamp recordings reliably predict neuronal phenotype, and that the abused inhalant toluene differentially depresses excitatory neurotransmission in NAc neuronal subtypes.

Disruption of S2-M4 linker coupling reveals novel subunit-specific contributions to N-methyl-d-aspartate receptor function and ethanol sensitivity.

The N-methyl-d-aspartate (NMDA) receptor is a glutamatergic ion channel and is a known site of ethanol action. Evidence suggests that ethanol inhibits NMDA receptor activity by reducing channel open probability and mean open time potentially via interaction with specific residues within the transmembrane (M) domains 3 and 4 of GluN subunits. Recent models of NMDAR function demonstrate that extracellular residues near the M domains are key regulators of gating, suggesting that they may contribute to ethanol sensitivity. To test this, we substituted cysteines at key positions in GluN1 and GluN2 M3-S2 and S2-M4 regions previously shown to affect channel open probability and mean open time similar to ethanol treatment. Although crosslinking of these domains was predicted to restrict linker domain movement and occlude ethanol inhibition, only intra-GluN1 M1:M4 linker-crosslinked receptors showed a decrease in ethanol sensitivity. For the converse experiment, we expressed NMDARs with glycine substitutions in the S2-M4 domain of GluN subunits to enhance M4 flexibility and recorded currents in response to ethanol. Glycine substitution in the GluN1 S2-M4 region significantly decreased glutamate potency of GluN1(A804G)/GluN2A receptors, while GluN1(A804G)/GluN2B receptors exhibited no change in glutamate sensitivity. In contrast, GluN1/GluN2B(S811G) receptors showed a 10-fold increase in glutamate potency while GluN1/GluN2A(S810G) receptors showed no change. Surprisingly, while S2-M4 glycine substitutions modulated ethanol sensitivity, this was observed only in receptors that did not display a change in agonist potency. Overall, these results suggest that S2-M4 linkers strongly influence receptor function and modestly impact ethanol efficacy in a subunit- and receptor subtype-dependent manner.

Dephosphorylation of GluN2B C-terminal tyrosine residues does not contribute to acute ethanol inhibition of recombinant NMDA receptors.

N-methyl-d-aspartate (NMDA) receptors are ion channels activated by the neurotransmitter glutamate and are highly expressed by neurons. These receptors are critical for excitatory synaptic signaling and inhibition of NMDA receptors leads to impaired cognition and learning. Ethanol inhibits NMDA currents at concentrations associated with intoxication and this action may underlie some of the behavioral effects of ethanol. Although numerous sites and mechanisms of action have been tested, the manner in which ethanol inhibits NMDA receptors remains unclear. Recent findings in the literature suggest that ethanol, via facilitation of tyrosine phosphatase activity, may dephosphorylate key tyrosine residues in the C-terminus of GluN2B subunits resulting in diminished channel function. To directly test this hypothesis, we engineered GluN2B mutants that contained phenylalanine in place of tyrosine at three different sites and transiently expressed them with the GluN1 subunit in human embryonic kidney (HEK) cells. Whole-cell patch clamp electrophysiology was used to record glutamate-activated currents in the absence and presence of ethanol (10-600 mM). All mutants were functional and did not differ from one another with respect to current amplitude, steady-state to peak ratio, or magnesium block. Analysis of ethanol dose-response curves showed no significant difference in IC50 values between wild-type receptors and Y1252F, Y1336F, Y1472F or triple Y-F mutants. These findings suggest that dephosphorylation of C-terminal tyrosine residues does not account for ethanol inhibition of GluN2B receptors.

Sex and dose-related differences in methylphenidate adolescent locomotor sensitization and effects on brain-derived neurotrophic factor.

This study analyzed repeated methylphenidate (MPH) administration and its effects on brain-derived neurotrophic factor (BDNF) in the dorsal striatum and nucleus accumbens of male and female adolescent rats. In Experiment 1, rats were administered intraperitoneal (ip) saline, 1, 3, or 5 mg/kg dose of MPH every second day from postnatal day (P)33-P49. Locomotor activity was analyzed for 10 min after each administration. Results revealed that the 1 mg/kg dose of MPH produced locomotor suppression, however, the 5 mg/kg dose of MPH produced locomotor sensitization and robust behavioral activation in females as compared to males. In Experiment 2, animals were administered ip saline or the 5 mg/kg dose of MPH using an identical regimen but a 30 min behavioral test was employed. Dorsal striatum and nucleus accumbens tissue was assayed for BDNF at P50. Females demonstrated sensitization to MPH and increased locomotor activation compared to males. Interestingly, females given MPH demonstrated a significant 42% decrease of striatal BDNF whereas males administered MPH demonstrated a significant 50.4% increase of striatal BDNF compared to controls. There were no effects on accumbal BDNF. This report demonstrates robust sex differences in the behavioral response, but sex-dependent changes in striatal BDNF in response to MPH in adolescence.

Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media.

Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum.

Gestational IV nicotine produces elevated brain-derived neurotrophic factor in the mesocorticolimbic dopamine system of adolescent rat offspring.

Maternal smoking during pregnancy is associated with enduring psychopathology, such as increased likelihood of substance use, in offspring. Various animal models demonstrate that continuous nicotine exposure produces teratogenic effects in offspring, as well. In this experiment, a novel intravenous (IV) exposure model was used to determine if gestational nicotine (GN) treatment produced alterations in methamphetamine-induced sensitization and the expression of brain-derived neurotrophic factor (BDNF) in the mesocorticolimbic dopamine (DA) system of adolescent offspring. Dams were injected with IV saline or nicotine (0.05 mg/kg/injection) three times per day on gestational days 8-21. Habituation was measured on postnatal day (PND) 25-27 and baseline activity on PND 28. On PND 29-35, offspring were injected with saline or methamphetamine (0.3 mg/kg) and locomotor activity was measured after the first and seventh injections. On PND 36, brains were removed, flash frozen, and BDNF protein levels in the nucleus accumbens (NAcc), dorsal striatum (Str), frontal cortex (FC), and hippocampus (Hipp) were analyzed. GN did not affect habituation or the induction of methamphetamine-induced sensitization. Interestingly, GN, but not adolescent methamphetamine treatment, elevated levels of BDNF in the NAcc and Str; however, the GN-induced increase in BDNF in the FC was attenuated by adolescent methamphetamine treatment. Both GN and adolescent methamphetamine treatment increased BDNF in the Hipp. These findings indicate that GN exposure will result in increased levels of BDNF protein throughout the mesocorticolimbic DA system during adolescent development and suggests that methamphetamine abuse will modulate the expression of BDNF in motivational circuitries of adolescent offspring exposed to GN.

Enhanced CHO cell-based transient gene expression with the epi-CHO expression system.

Transient gene expression systems in mammalian cells continue to grow in popularity due to their capacity to produce significant amounts of recombinant protein in a rapid and scalable manner, without the lengthy time periods and resources required for stable cell line development. Traditionally, production of recombinant monoclonal antibodies for pre-clinical assessment by transient expression in CHO cells has been hampered by low titers. In this report, we demonstrate transient monoclonal antibody titers of 140 mg/l with CHO cells using the episomal-based transient expression system, Epi-CHO. Such titers were achieved by implementing an optimized transfection protocol incorporating mild-hypothermia and through screening of a variety of chemically defined and serum-free media for their ability to support elevated and prolonged viable cell densities post-transfection, and in turn, improve recombinant protein yields. Further evidence supporting Epi-CHO's capacity to enhance transgene expression is provided, where we demonstrate higher transgene mRNA and protein levels of two monoclonal antibodies and a destabilized enhanced green fluorescent protein with Epi-CHO compared to cell lines deficient in plasmid DNA replication and/or retention post-transfection. The results demonstrate the Epi-CHO system's capacity for the rapid production of CHO cell-derived recombinant monoclonal antibodies in serum-free conditions.