Long-Term Safety and Efficacy of Transcatheter Microwave and Radiofrequency Denervation in a Chronic Ovine Model.

BACKGROUND: Transcatheter renal denervation (RDN) has had inconsistent efficacy and concerns for durability of denervation. We aimed to investigate long-term safety and efficacy of transcatheter microwave RDN in vivo in normotensive sheep in comparison to conventional radiofrequency ablation. METHODS AND RESULTS: Sheep underwent bilateral RDN, receiving 1 to 2 microwave ablations (maximum power of 80-120 W for 240 s-480 s) and 12 to 16 radiofrequency ablations (180 s-240 s) in the main renal artery in a paired fashion, alternating the side of treatment, euthanized at 2 weeks (acute N=15) or 5.5 months (chronic N=15), and compared with undenervated controls (N=4). Microwave RDN produced substantial circumferential perivascular injury compared with radiofrequency at both 2 weeks [area 239.8 (interquartile range [IQR] 152.0-343.4) mm2 versus 50.1 (IQR, 32.0-74.6) mm2, P <0.001; depth 16.4 (IQR, 13.9-18.9) mm versus 7.5 (IQR, 6.0-8.9) mm P <0.001] and 5.5 months [area 20.0 (IQR, 3.4-31.8) mm2 versus 5.0 (IQR, 1.4-7.3) mm2, P=0.025; depth 5.9 (IQR, 1.9-8.8) mm versus 3.1 (IQR, 1.2-4.1) mm, P=0.005] using mixed models. Renal denervation resulted in significant long-term reductions in viability of renal sympathetic nerves [58.9% reduction with microwave (P=0.01) and 45% reduction with radiofrequency (P=0.017)] and median cortical norepinephrine levels [71% reduction with microwave (P <0.001) and 72.9% reduction with radiofrequency (P <0.001)] at 5.5 months compared with undenervated controls. CONCLUSIONS: Transcatheter microwave RDN produces deep circumferential perivascular ablations without significant arterial injury to provide effective and durable RDN at 5.5 months compared with radiofrequency RDN.

Intra-cardiac motion detection catheter for the early identification of acute pericardial tamponade during invasive cardiac procedures.

Objectives: To develop and test an intra-cardiac catheter fitted with accelerometers to detect acute pericardial effusion prior to the onset of hemodynamic compromise. Background: Early detection of an evolving pericardial effusion is critical in ensuring timely treatment. We hypothesized that the reduction in movement of the lateral heart border present in developing pericardial effusions could be quantified by positioning an accelerometer in a lateral cardiac structure. Methods: A "motion detection" catheter was created by implanting a 3-axis accelerometer at the distal tip of a cardiac catheter. The pericardial space of 5 adult sheep was percutaneously accessed, and pericardial tamponade was created by infusion of normal saline. The motion detection catheter was positioned in the coronary sinus. Intracardiac echocardiography was used to confirm successful creation of pericardial effusion and hemodynamic parameters were collected. Results: Statistically significant reduction in acceleration from baseline was detected after infusion of only 40 ml of normal saline (p < 0.05, ANOVA). In comparison, clinically significant change in systolic blood pressure (defined as >10% drop in baseline systolic blood pressure) occurred after infusion of 80 ml of normal saline (107 ± 22 mmHg vs. 90 ± 12 mmHg p = 0.97, ANOVA), and statistically significant change was recorded only after infusion of 200 ml (107 ± 22 mmHg vs. 64 ± 5 mmHg, p < 0.05, ANOVA). Conclusions: An intra-cardiac motion detection catheter is highly sensitive in identifying acute cardiac tamponade prior to clinically and statistically significant changes in systolic blood pressure, allowing for early detection and treatment of this potentially life-threatening complication of all modern percutaneous cardiac interventions.

Adeno-associated virus-9 reverses delayed gastric emptying of solids in diabetic mice.

BACKGROUND: Gastroparesis is defined by delayed gastric emptying (GE) without obstruction. Studies suggest targeting heme oxygenase-1 (HO1) may ameliorate diabetic gastroparesis. Upregulation of HO1 expression via interleukin-10 (IL-10) in the gastric muscularis propria is associated with reversal of delayed GE in diabetic NOD mice. IL-10 activates the M2 cytoprotective phenotype of macrophages and induces expression of HO1 protein. Here, we assess delivery of HO1 by recombinant adeno-associated viruses (AAVs) in diabetic mice with delayed GE. METHODS: C57BL6 diabetic delayed GE mice were injected with 1 × 1012 vg scAAV9-cre, scAAV9-GFP, or scAAV9-HO1 particles. Changes to GE were assessed weekly utilizing our [13 C]-octanoic acid breath test. Stomach tissue was collected to assess the effect of scAAV9 treatment on Kit, NOS1, and HO1 expression. KEY RESULTS: Delayed GE returned to normal within 2 weeks of treatment in 7/12 mice receiving scAAV9-cre and in 4/5 mice that received the scAAV9-GFP, whereas mice that received scAAV9-HO1 did not respond in the same manner and had GE that took significantly longer to return to normal (6/7 mice at 4-6 weeks). Kit, NOS1, and HO1 protein expression in scAAV9-GFP-treated mice with normal GE were not significantly different compared with diabetic mice with delayed GE. CONCLUSIONS AND INFERENCES: Injection of scAAV9 into diabetic C57BL6 mice produced a biological response that resulted in acceleration of GE independently of the cargo delivered by the AAV9 vector. Further research is needed to determine whether use of AAV mediated gene transduction in the gastric muscularis propria is beneficial and warranted.

Variable task switching in the feeding network of Aplysia is a function of differential command input.

Command systems integrate sensory information and then activate the interneurons and motor neurons that mediate behavior. Much research has established that the higher-order projection neurons that constitute these systems can play a key role in specifying the nature of the motor activity induced, or determining its parametric features. To a large extent, these insights have been obtained by contrasting activity induced by stimulating one neuron (or set of neurons) to activity induced by stimulating a different neuron (or set of neurons). The focus of our work differs. We study one type of motor program, ingestive feeding in the mollusc Aplysia californica, which can either be triggered when a single projection neuron (CBI-2) is repeatedly stimulated or can be triggered by projection neuron coactivation (e.g., activation of CBI-2 and CBI-3). We ask why this might be an advantageous arrangement. The cellular/molecular mechanisms that configure motor activity are different in the two situations because the released neurotransmitters differ. We focus on an important consequence of this arrangement, the fact that a persistent state can be induced with repeated CBI-2 stimulation that is not necessarily induced by CBI-2/3 coactivation. We show that this difference can have consequences for the ability of the system to switch from one type of activity to another.NEW & NOTEWORTHY We study a type of motor program that can be induced either by stimulating a higher-order projection neuron that induces a persistent state, or by coactivating projection neurons that configure activity but do not produce a state change. We show that when an activity is configured without a state change, it is possible to immediately return to an intermediate state that subsequently can be converted to any type of motor program.

Characterization of Morreton virus as an oncolytic virotherapy platform for liver cancers.

BACKGROUND: Morreton virus (MORV) is an oncolytic Vesiculovirus , genetically distinct from vesicular stomatitis virus (VSV). AIM: To report that MORV induced potent cytopathic effects (CPEs) in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) in vitro models. APPROACH AND RESULTS: In preliminary safety analyses, high intranasal doses (up to 10 10 50% tissue culture infectious dose [TCID 50 ]) of MORV were not associated with significant adverse effects in immune competent, non-tumor-bearing mice. MORV was shown to be efficacious in a Hep3B hepatocellular cancer xenograft model but not in a CCA xenograft HuCCT1 model. In an immune competent, syngeneic murine CCA model, single intratumoral treatments with MORV (1 × 10 7 TCID 50 ) triggered a robust antitumor immune response leading to substantial tumor regression and disease control at a dose 10-fold lower than VSV (1 × 10 8 TCID 50 ). MORV led to increased CD8 + cytotoxic T cells without compensatory increases in tumor-associated macrophages and granulocytic or monocytic myeloid-derived suppressor cells. CONCLUSIONS: Our findings indicate that wild-type MORV is safe and can induce potent tumor regression via immune-mediated and immune-independent mechanisms in HCC and CCA animal models without dose limiting adverse events. These data warrant further development and clinical translation of MORV as an oncolytic virotherapy platform.

Locked and loaded: engineering and arming oncolytic adenoviruses to enhance anti-tumor immune responses.

INTRODUCTION: Oncolytic adenoviruses (Ads) are promising therapeutics to enhance anti-tumor immune responses and modulate the immune-suppressive tumor microenvironment (TME). Due to their potent ability to deliver genes in vivo, oncolytic Ads have been armed with a variety of immune stimulatory payloads to boost tumor immunotherapy. AREAS COVERED: We describe current knowledge about engineering oncolytic Ads to insert transgene payloads, including methods to increase its genome capacity for transgene insertion. We also review several categories of immune stimulatory payloads that have been used in oncolytic Ads to combat different barriers to effective immunotherapy. EXPERT OPINION: We anticipate that multi-armed oncolytic Ads alone or in combination with other types of immunotherapies will greatly improve the efficacy of oncolytic virotherapy in the future by targeting several immune barriers. However, the production and testing of multiple payload-armed Ads can be complex and time-consuming due to the limitations of current tools. Given this, we should develop new tools for rapid construction of armed Ads, improve animal models or new systems to compare the efficacy of multiple payloads, and carefully monitor the immunological toxicity induced by payloads or by systemic delivery. Most importantly, we should pursue payload-arming approaches with great care to ensure safety.

Comparison of replicating and nonreplicating vaccines against SARS-CoV-2.

Most gene-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are nonreplicating vectors. They deliver the gene or messenger RNA to the cell to express the spike protein but do not replicate to amplify antigen production. This study tested the utility of replication in a vaccine by comparing replication-defective adenovirus (RD-Ad) and replicating single-cycle adenovirus (SC-Ad) vaccines that express the SARS-CoV-2 spike protein. SC-Ad produced 100 times more spike protein than RD-Ad and generated significantly higher antibodies against the spike protein than RD-Ad after single immunization of Ad-permissive hamsters. SC-Ad-generated antibodies climbed over 14 weeks after single immunization and persisted for more than 10 months. When the hamsters were challenged 10.5 months after single immunization, a single intranasal or intramuscular immunization with SC-Ad-Spike reduced SARS-CoV-2 viral loads and damage in the lungs and preserved body weight better than vaccination with RD-Ad-Spike. This demonstrates the utility of harnessing replication in vaccines to amplify protection against infectious diseases.

Recombinant Adeno-Associated Viral Vector-Mediated Gene Transfer of hTBX18 Generates Pacemaker Cells from Ventricular Cardiomyocytes.

Sinoatrial node dysfunction can manifest as bradycardia, leading to symptoms of syncope and sudden cardiac death. Electronic pacemakers are the current standard of care but are limited due to a lack of biological chronotropic control, cost of revision surgeries, and risk of lead- and device-related complications. We therefore aimed to develop a biological alternative to electronic devices by using a clinically relevant gene therapy vector to demonstrate conversion of cardiomyocytes into sinoatrial node-like cells in an in vitro context. Neonatal rat ventricular myocytes were transduced with recombinant adeno-associated virus vector 6 encoding either hTBX18 or green fluorescent protein and maintained for 3 weeks. At the endpoint, qPCR, Western blot analysis and immunocytochemistry were used to assess for reprogramming into pacemaker cells. Cell morphology and Arclight action potentials were imaged via confocal microscopy. Compared to GFP, hTBX18-transduced cells showed that hTBX18, HCN4 and Cx45 were upregulated. Cx43 was significantly downregulated, while sarcomeric α-actinin remained unchanged. Cardiomyocytes transduced with hTBX18 acquired the tapering morphology of native pacemaker cells, as compared to the block-like, striated appearance of ventricular cardiomyocytes. Analysis of the action potentials showed phase 4 depolarization and a significant decrease in the APD50 of the hTBX18-transduced cells. We have demonstrated that rAAV-hTBX18 gene transfer to ventricular myocytes results in morphological, molecular, physiological, and functional changes, recapitulating the pacemaker phenotype in an in vitro setting. The generation of these induced pacemaker-like cells using a clinically relevant vector opens new prospects for biological pacemaker development.

Convergent effects of neuropeptides on the feeding central pattern generator of Aplysia californica.

These experiments focus on an interneuron (B63) that is part of the feeding central pattern generator (CPG) in Aplysia californica. Previous work has established that B63 is critical for program initiation regardless of the type of evoked activity. B63 receives input from a number of different elements of the feeding circuit. Program initiation occurs reliably when some are activated, but we show that it does not occur reliably with activation of others. When program initiation is reliable, modulatory neuropeptides are released. For example, previous work has established that an ingestive input to the feeding CPG, cerebral buccal interneuron 2 (CBI-2), releases feeding circuit activating peptide (FCAP) and cerebral peptide 2 (CP-2). Afferents with processes in the esophageal nerve (EN) that trigger egestive motor programs release small cardioactive peptide (SCP). Previous studies have described divergent cellular and molecular effects of FCAP/CP-2 and SCP on the feeding circuit that specify motor activity. Here, we show that FCAP/CP-2 and SCP additionally increase the B63 excitability. Thus, we show that peptides that have well-characterized divergent effects on the feeding circuit additionally act convergently at the level of a single neuron. Since convergent effects of FCAP/CP-2 and SCP are not necessary for specifying the type of network output, we ask why they might be important. Our data suggest that they have an impact during a task switch, i.e., when there is a switch from egestive to ingestive activity.NEW & NOTEWORTHY The activity of multifunctional central pattern generators (CPGs) is often configured by neuromodulators that exert divergent effects that are necessary to specify motor output. We demonstrate that ingestive and egestive inputs to the feeding CPG in Aplysia act convergently (as well as divergently). We ask why this convergence may be important and suggest that it may be a mechanism for a type of arousal that occurs during task switching.

Refined Capsid Structure of Human Adenovirus D26 at 3.4 Å Resolution.

Various adenoviruses are being used as viral vectors for the generation of vaccines against chronic and emerging diseases (e.g., AIDS, COVID-19). Here, we report the improved capsid structure for one of these vectors, human adenovirus D26 (HAdV-D26), at 3.4 Å resolution, by reprocessing the previous cryo-electron microscopy dataset and obtaining a refined model. In addition to overall improvements in the model, the highlights of the structure include (1) locating a segment of the processed peptide of VIII that was previously believed to be released from the mature virions, (2) reorientation of the helical appendage domain (APD) of IIIa situated underneath the vertex region relative to its counterpart observed in the cleavage defective (ts1) mutant of HAdV-C5 that resulted in the loss of interactions between the APD and hexon bases, and (3) the revised conformation of the cleaved N-terminal segments of pre-protein VI (pVIn), located in the hexon cavities, is highly conserved, with notable stacking interactions between the conserved His13 and Phe18 residues. Taken together, the improved model of HAdV-D26 capsid provides a better understanding of protein-protein interactions in HAdV capsids and facilitates the efforts to modify and/or design adenoviral vectors with altered properties. Last but not least, we provide some insights into clotting factors (e.g., FX and PF4) binding to AdV vectors.

Modulating Oncolytic Adenovirus Immunotherapy by Driving Two Axes of the Immune System by Expressing 4-1BBL and CD40L.

Oncolytic viruses (OVs) can have utility for direct killing of cancer cells, but may also serve to activate the immune system against cancer cells. While viruses alone can serve as immune stimulators, there is great interest in arming OVs with genes encoding immune stimulatory proteins to amplify their effects. In this work, we have tested the efficacy of conditionally-replicating adenoviruses (CRAds) with and without selected immunostimulatory payloads, murine CD40L (mCD40L) or 4-1BBL (m4-1BBL), in an immune competent mouse model of melanoma. When CRAd657-m4-1BBL and CRAd657-mCD40L were injected into B16-hCAR murine melanoma tumors, both single vectors delayed tumor growth and prolong survival compared to empty CRAd657. However, combined injection of both CRAd-m4-1BBL and CRAd-mCD40L mediated significantly better control of tumor growth. All of the payloads increased immune cell infiltration into tumors and notably reduced expression of PD-1 exhaustion marker on T cells. Tumor volumes were negatively associated with total infiltrating T cell population. We found that the payloads increased immune cell infiltration into tumors with some specificities: recruitment of CD8+ T cells was higher with m4-1BBL expression, while mCD40L expression induced more CD4+ T cell infiltration. Importantly, the combination of CRAd657-m4-1BBL and CRAd657-mCD40L induced the highest immune cells/T cell infiltration and the highest anti-TRP-2 tumor-associated antigen T cell responses than empty or single gene vector. This combination also caused depigmentation in areas adjacent to the tumor sites in more animals. These data indicate that driving two axes of the immune system with combined immune stimulatory payloads can lead to improved anticancer immune responses and better tumor control in an immune competent model of cancer.

Ex Vivo and In Vivo CD46 Receptor Utilization by Species D Human Adenovirus Serotype 26 (HAdV26).

Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and HIV-1. However, its primary receptor portfolio remains controversial, potentially including sialic acid, coxsackie and adenovirus receptor (CAR), integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned on the basis of the inability to cocrystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infection in vitro and in vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domain CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wild-type and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (i.m.) injection but not after intranasal (i.n.) administration. Ad26 transduction was 10-fold lower than Ad5 transduction after intratumoral (i.t.) injection of CD46-expressing tumors. Ad26 transduction of liver was 1,000-fold lower than that ofAd5 after intravenous (i.v.) injection. These data demonstrate the use of CD46 by Ad26 in certain situations but also show that the receptor has little consequence by other routes of administration. Finally, i.v. injection of high doses of Ad26 into CD46 mice induced release of liver enzymes into the bloodstream and reduced white blood cell counts but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during coronavirus disease 2019 (COVID-19) vaccination with this serotype of adenovirus. IMPORTANCE The human species D Ad26 is being investigated as a low-seroprevalence vector for oncolytic virotherapy and gene-based vaccination against HIV-1 and SARS-CoV-2. However, there is debate in the literature about its tropism and receptor utilization, which directly influence its efficiency for certain applications. This work was aimed at determining which receptor(s) this virus uses for infection and its role in virus biology, vaccine efficacy, and, importantly, vaccine safety.

The spike protein of SARS-CoV-2 induces heme oxygenase-1: Pathophysiologic implications.

BACKGROUND: Acute kidney injury (AKI) is both a consequence and determinant of outcomes in COVID-19. The kidney is one of the major organs infected by the causative virus, SARS-CoV-2. Viral entry into cells requires the viral spike protein, and both the virus and its spike protein appear in the urine of COVID-19 patients with AKI. We examined the effects of transfecting the viral spike protein of SARS-CoV-2 in kidney cell lines. METHODS: HEK293, HEK293-ACE2+ (stably overexpressing ACE2), and Vero E6 cells having endogenous ACE2 were transfected with SARS-CoV-2 spike or control plasmid. Assessment of gene and protein expression, and syncytia formation was performed, and the effects of quercetin on syncytia formation examined. FINDINGS: Spike transfection in HEK293-ACE2+ cells caused syncytia formation, cellular sloughing, and focal denudation of the cell monolayer; transfection in Vero E6 cells also caused syncytia formation. Spike expression upregulated potentially nephrotoxic genes (TNF-α, MCP-1, and ICAM1). Spike upregulated the cytoprotective gene HO-1 and relevant signaling pathways (p-Akt, p-STAT3, and p-p38). Quercetin, an HO-1 inducer, reduced syncytia formation and spike protein expression. INTERPRETATION: The major conclusions of the study are: 1) Spike protein expression in kidney cells provides a relevant model for the study of maladaptive and adaptive responses germane to AKI in COVID-19; 2) such spike protein expression upregulates HO-1; and 3) quercetin, an HO-1 inducer, may provide a clinically relevant/feasible protective strategy in AKI occurring in the setting of COVID-19. FUNDING: R01-DK119167 (KAN), R01-AI100911 (JPG), P30-DK079337; R01-DK059600 (AA).

Metabolic perturbations mediated by propionyl-CoA accumulation in organs of mouse model of propionic acidemia.

Propionic acidemia (PA) is an autosomal recessive metabolic disorder after gene encoding propionyl-CoA carboxylase, Pcca or Pccb, is mutated. This genetic disorder could develop various complications which are ascribed to dysregulated propionyl-CoA metabolism in organs. However, the effect of attenuated PCC on propionyl-CoA metabolism in different organs remains to be fully understood. We investigated metabolic perturbations in organs of Pcca-/-(A138T) mice (a mouse model of PA) under chow diet and acute administration of [13C3]propionate to gain insight into pathological mechanisms of PA. With chow diet, the metabolic alteration is organ dependent. l-Carnitine reduction induced by propionylcarnitine accumulation only occurs in lung and liver of Pcca-/- (A138T) mice. [13C3]Propionate tracing data demonstrated that PCC activity was dramatically reduced in Pcca-/-(A138T) brain, lung, liver, kidney, and adipose tissues, but not significantly changed in Pcca-/-(A138T) muscles (heart and skeletal muscles) and pancreas, which was largely supported by PCCA expression data. The largest expansion of propionylcarnitine in Pcca-/-(A138T) heart after acute administration of propionate indicated the vulnerability of heart to high circulating propionate. The overwhelming propionate in blood also stimulated ketone production from the increased fatty acid oxidation in Pcca-/-(A138T) liver by lowering malonyl-CoA, which has been observed in cases where metabolic decompensation occurs in PA patients. This work shed light on organ-specific metabolic alternations under varying severities of PA.

Unlocking loxP to Track Genome Editing In Vivo.

The development of CRISPR-associated proteins, such as Cas9, has led to increased accessibility and ease of use in genome editing. However, additional tools are needed to quantify and identify successful genome editing events in living animals. We developed a method to rapidly quantify and monitor gene editing activity non-invasively in living animals that also facilitates confocal microscopy and nucleotide level analyses. Here we report a new CRISPR "fingerprinting" approach to activating luciferase and fluorescent proteins in mice as a function of gene editing. This system is based on experience with our prior cre recombinase (cre)-detector system and is designed for Cas editors able to target loxP including gRNAs for SaCas9 and ErCas12a. These CRISPRs cut specifically within loxP, an approach that is a departure from previous gene editing in vivo activity detection techniques that targeted adjacent stop sequences. In this sensor paradigm, CRISPR activity was monitored non-invasively in living cre reporter mice (FVB.129S6(B6)-Gt(ROSA)26Sortm1(Luc)Kael/J and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J, which will be referred to as LSL-luciferase and mT/mG throughout the paper) after intramuscular or intravenous hydrodynamic plasmid injections, demonstrating utility in two diverse organ systems. The same genome-editing event was examined at the cellular level in specific tissues by confocal microscopy to determine the identity and frequency of successfully genome-edited cells. Further, SaCas9 induced targeted editing at efficiencies that were comparable to cre, demonstrating high effective delivery and activity in a whole animal. This work establishes genome editing tools and models to track CRISPR editing in vivo non-invasively and to fingerprint the identity of targeted cells. This approach also enables similar utility for any of the thousands of previously generated loxP animal models.

The Complement of Projection Neurons Activated Determines the Type of Feeding Motor Program in Aplysia.

Multiple projection neurons are often activated to initiate behavior. A question that then arises is, what is the unique functional role of each neuron activated? We address this issue in the feeding system of Aplysia. Previous experiments identified a projection neuron [cerebral buccal interneuron 2 (CBI-2)] that can trigger ingestive motor programs but only after it is repeatedly stimulated, i.e., initial programs are poorly defined. As CBI-2 stimulation continues, programs become progressively more ingestive (repetition priming occurs). This priming results, at least in part, from persistent actions of peptide cotransmitters released from CBI-2. We now show that in some preparations repetition priming does not occur. There is no clear seasonal effect; priming and non-priming preparations are encountered throughout the year. CBI-2 is electrically coupled to a second projection neuron, cerebral buccal interneuron 3 (CBI-3). In preparations in which priming does not occur, we show that ingestive activity is generated when CBI-2 and CBI-3 are coactivated. Programs are immediately ingestive, i.e., priming is not necessary, and a persistent state is not induced. Our data suggest that dynamic changes in the configuration of activity can vary and be determined by the complement of projection neurons that trigger activity.

An Anticipatory Circuit Modification That Modifies Subsequent Task Switching.

Modulators are generally expected to establish a network configuration that is appropriate for the current circumstances. We characterize a situation where the opposite is apparently observed. A network effect of a peptide modulator is counterproductive in that it tends to impede rather than promote the creation of the configuration that is appropriate when the modulator is released. This raises a question: why does release occur? We present data that strongly suggest that it impacts task switching. Our experiments were conducted in an Aplysia feeding network that generates egestive and ingestive motor programs. Initial experiments focused on egestive activity and the neuron B8. As activity becomes egestive, there is an increase in synaptic drive to B8 and its firing frequency increases (Wang et al., 2019). We show that, as this occurs, there is also a persistent current that develops in B8 that is outward rather than inward. Dynamic clamp introduction of this current decreases excitability. When there is an egestive-ingestive task switch in Aplysia, negative biasing is observed (i.e., a bout of egestive activity has a negative impact on a subsequent attempt to initiate an ingestive response) (Proekt et al., 2004). Using an in vitro analog of negative biasing, we demonstrate that the outward current that develops during egestive priming plays an important role in establishing this phenomenon. Our data suggest that, although the outward current induced as activity becomes egestive is counterproductive at the time, it plays an anticipatory role in that it subsequently impacts task switching.SIGNIFICANCE STATEMENT In this study, we identify a peptide-induced circuit modification (induction of an outward current) that does not immediately promote the establishment of a behaviorally appropriate network configuration. We ask why this might occur, and present data that strongly suggest that it plays an important role during task switching. Specifically, our data suggest that the outward current we characterize plays a role in the negative biasing that is seen in the mollusc Aplysia when there is a transition from egestive to ingestive activity. It is possible that the mechanism that we describe operates in other species. A negative effect of egestion on subsequent ingestion is observed throughout the animal kingdom.

Suppression-Replacement KCNQ1 Gene Therapy for Type 1 Long QT Syndrome.

BACKGROUND: Type 1 long QT syndrome (LQT1) is caused by loss-of-function variants in the KCNQ1-encoded Kv7.1 potassium channel α-subunit that is essential for cardiac repolarization, providing the slow delayed rectifier current. No current therapies target the molecular cause of LQT1. METHODS: A dual-component suppression-and-replacement (SupRep) KCNQ1 gene therapy was created by cloning a KCNQ1 short hairpin RNA and a short hairpin RNA-immune KCNQ1 cDNA modified with synonymous variants in the short hairpin RNA target site, into a single construct. The ability of KCNQ1-SupRep gene therapy to suppress and replace LQT1-causative variants in KCNQ1 was evaluated by means of heterologous expression in TSA201 cells. For a human in vitro cardiac model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from 4 patients with LQT1 (KCNQ1-Y171X, -V254M, -I567S, and -A344A/spl) and an unrelated healthy control. CRISPR-Cas9 corrected isogenic control iPSC-CMs were made for 2 LQT1 lines (correction of KCNQ1-V254M and KCNQ1-A344A/spl). FluoVolt voltage dye was used to measure the cardiac action potential duration (APD) in iPSC-CMs treated with KCNQ1-SupRep. RESULTS: In TSA201 cells, KCNQ1-SupRep achieved mutation-independent suppression of wild-type KCNQ1 and 3 LQT1-causative variants (KCNQ1-Y171X, -V254M, and -I567S) with simultaneous replacement of short hairpin RNA-immune KCNQ1 as measured by allele-specific quantitative reverse transcription polymerase chain reaction and Western blot. Using FluoVolt voltage dye to measure the cardiac APD in the 4 LQT1 patient-derived iPSC-CMs, treatment with KCNQ1-SupRep resulted in shortening of the pathologically prolonged APD at both 90% and 50% repolarization, resulting in APD values similar to those of the 2 isogenic controls. CONCLUSIONS: This study provides the first proof-of-principle gene therapy for complete correction of long QT syndrome. As a dual-component gene therapy vector, KCNQ1-SupRep successfully suppressed and replaced KCNQ1 to normal wild-type levels. In TSA201 cells, cotransfection of LQT1-causative variants and KCNQ1-SupRep caused mutation-independent suppression and replacement of KCNQ1. In LQT1 iPSC-CMs, KCNQ1-SupRep gene therapy shortened the APD, thereby eliminating the pathognomonic feature of LQT1.

Structural Organization and Protein-Protein Interactions in Human Adenovirus Capsid.

Human adenoviruses (HAdVs) are large (150 MDa), complex, nonenveloped dsDNA viruses that cause self-limiting respiratory, ocular and enteric infections. They are significant health hazard in young, elderly and immuno-compromised populations. Moreover, various adenoviruses (AdVs) of mammalian origin are being used as vectors in gene, vaccine and cancer therapies. Multiple copies of at least 13 different proteins, all in all ~2800 protein molecules, come together to form an adenovirus virion packaging the ~36 Kbp geome. The details of structural organization of the adenovirus capsid and underlying network of protein-protein interactions provide clues into designing the modified and novel adenovirus vectors with desired functionalities and/or targeting specificities. The advancements in 3D structure determination by cryo-electron microscopy (cryo-EM) in the past decade have enabled unveiling of the complex organization of adenovirus architecture at near atomic resolution. Specifically, these studies revealed the structures and the network of interactions involving cement/minor proteins in stabilizing the AdV icosahedral architecture, which appear to be mostly conserved among human adenoviruses. In this chapter, we describe the current state of knowledge on the structure and organization of human adenoviruses.

Expression of ACE2 in the Intact and Acutely Injured Kidney.

Background: The actions of angiotensin-converting enzyme 2 (ACE2) oppose those of the renin-angiotensin-aldosterone system. ACE2 may be a cytoprotectant in some tissues. This study examined ACE2 expression in models of AKI. Methods: ACE2 mRNA and protein expression and ACE2 activity were assessed in murine ischemic AKI. Renal ACE2 mRNA expression was evaluated in LPS-induced AKI in wild-type (C57BL/6J) mice, in heme oxygenase-1+/+ and heme oxygenase-1-/- mice, and after unilateral ureteral obstruction (UUO) in wild-type mice. The effect of sex and age on renal ACE2 protein expression was also assessed. Results: In ischemic AKI, ACE2 mRNA and protein expression and ACE2 activity were reduced as compared with such indices in the intact kidney. In ischemic AKI, ACE2, which, in health, is prominently expressed in the tubular epithelium, especially proximal tubules, is decreased in expression in these segments. Decreased ACE2 expression in AKI did not reflect reduced GFR, because ACE2 mRNA expression was unaltered after UUO. LPS induced renal ACE2 mRNA expression in wild-type mice, but this effect did not occur in heme oxygenase-1-deficient mice. In ischemic and LPS-induced AKI, renal expression of the Mas receptor was increased. In the intact kidney, renal ACE2 protein expression decreased in female mice as compared with male mice, but was unaltered with age. Conclusion: We conclude that renal ACE2 expression is decreased in ischemic AKI, characterized by decreased GFR and abundant cell death, but is upregulated in LPS-induced AKI, an effect requiring heme oxygenase-1. Determining the significance of ACE2 expression in experimental AKI merits further study. We suggest that understanding the mechanism underlying ACE2 downregulation in AKI may offer insights relevant to COVID-19: ACE2 expression is downregulated after ACE2 mediates SARS-CoV-2 cellular entry; such downregulation is proinflammatory; and AKI commonly occurs and determines outcomes in COVID-19.