Prokaryotic voltage-gated sodium channels are more effective than endogenous Nav1.5 channels in rescuing cardiac action potential conduction: an in silico study.

Methods to augment Na+ current in cardiomyocytes hold potential for the treatment of various cardiac arrhythmias involving conduction slowing. Because the gene coding cardiac Na+ channel (Nav1.5) is too large to fit in a single adeno-associated virus (AAV) vector, new gene therapies are being developed to enhance endogenous Nav1.5 current (by overexpression of chaperon molecules or use of multiple AAV vectors) or to exogenously introduce prokaryotic voltage-gated Na+ channels (BacNav) whose gene size is significantly smaller than that of the Nav1.5. In this study, based on experimental measurements in heterologous expression systems, we developed an improved computational model of the BacNav channel, NavSheP D60A. We then compared in silico how NavSheP D60A expression vs. Nav1.5 augmentation affects the electrophysiology of cardiac tissue. We found that the incorporation of BacNav channels in both adult guinea pig and human cardiomyocyte models increased their excitability and reduced action potential duration. When compared with equivalent augmentation of Nav1.5 current in simulated settings of reduced tissue excitability, the addition of the BacNav current was superior in improving the safety of conduction under conditions of current source-load mismatch, reducing the vulnerability to unidirectional conduction block during premature pacing, preventing the instability and breakup of spiral waves, and normalizing the conduction and ECG in Brugada syndrome tissues with mutated Nav1.5. Overall, our studies show that compared with a potential enhancement of the endogenous Nav1.5 current, expression of the BacNav channels with their slower inactivation kinetics can provide greater anti-arrhythmic benefits in hearts with compromised action potential conduction.NEW & NOTEWORTHY Slow action potential conduction is a common cause of various cardiac arrhythmias; yet, current pharmacotherapies cannot augment cardiac conduction. This in silico study compared the efficacy of recently proposed antiarrhythmic gene therapy approaches that increase peak sodium current in cardiomyocytes. When compared with the augmentation of endogenous sodium current, expression of slower-inactivating bacterial sodium channels was superior in preventing conduction block and arrhythmia induction. These results further the promise of antiarrhythmic gene therapies targeting sodium channels.

Efficacy of five nematicides against root-knot nematode when applied via single and double drip tapes in a Florida sandy soil.

BACKGROUND: The efficacy of drip-applied nematicides depends on adequate product distribution, which can be difficult in sandy soils. Three new non-fumigant nematicides (fluazaindolizine, fluensulfone, fluopyram), together with two old nematicides, oxamyl and metam potassium, were evaluated when applied via single and double drip tapes to control root-knot nematode in cucumber and squash in Florida between February 2020 and December 2022. RESULTS: Nematicide applications via double drip tapes resulted in lower root gall infection (and tend to have higher yield) as compared to a single tape for fluopyram, but no difference was noted between single and double tapes for oxamyl and fluazaindolizine. Fluensulfone response was somewhere in between and metam potassium had higher squash yield when applied with double tapes. Root-knot infection was higher in cucumber than in squash, and metam potassium had the highest yields and lowest nematode infection compared to other nematicide treatments. CONCLUSION: The benefit of double versus single drip tapes depended on the type of nematicide that was applied and was evident for nematicides that have poor water solubility like fluopyram. Some benefit was noted for metam potassium, but no or limited benefit was noted for oxamyl, fluazaindolizine and fluensulfone. © 2023 Society of Chemical Industry.

Root-knot nematode damage to a cucurbit double crop is increased by chloropicrin fumigation on the previous tomato crop.

BACKGROUND: Double-cropping is a common practice in vegetable plasticulture whereby a second crop is planted on the same plastic bed as the first crop. Root-knot nematodes (Meloidogyne spp.) are one of the major soilborne constraints in double-cropped vegetables due to nematode population build-up on the first crop. We evaluated the effect of fumigant and non-fumigant nematicides applied on the first crop, on nematode infection and yield of the second crop in 10 field trials between 2017 and 2020. Fumigants were chloropicrin (Pic100), chloropicrin +1,3-D (PicClor60), and non-fumigant nematicides were oxamyl (Vydate), fluensulfone (Nimitz), fluopyram (Velum) and fluazaindolizine (Salibro). The first crop was tomato and double crops were cucumber, squash, zucchini, and cantaloupe. RESULTS: Fumigation with chloropicrin on the first crop increased root-knot nematode damage on the double-crop at the end of the season in seven trials, while the opposite was noted in one trial, and no difference was noted in two trials. Fumigation with chloropicrin+1,3-D resulted in root-knot nematode damage less than chloropicrin but more than non-fumigated plots. Cucurbit yield was greater in non-fumigated beds in four trials, and in chloropicrin-treated beds in two trials. Fluensulfone reduced root-knot nematode damage on the second crop in five out of 10 trials. CONCLUSION: Our results indicate that chloropicrin applied on the tomato crop may lead to increased root-knot nematode damage on the double crop. More research is needed to understand the processes behind this, but it is possibly related to a reduction in natural nematode soil suppressiveness due to the broad-spectrum fungicidal activity of chloropicrin. © 2022 Society of Chemical Industry.

Engineered bacterial voltage-gated sodium channel platform for cardiac gene therapy.

Therapies for cardiac arrhythmias could greatly benefit from approaches to enhance electrical excitability and action potential conduction in the heart by stably overexpressing mammalian voltage-gated sodium channels. However, the large size of these channels precludes their incorporation into therapeutic viral vectors. Here, we report a platform utilizing small-size, codon-optimized engineered prokaryotic sodium channels (BacNav) driven by muscle-specific promoters that significantly enhance excitability and conduction in rat and human cardiomyocytes in vitro and adult cardiac tissues from multiple species in silico. We also show that the expression of BacNav significantly reduces occurrence of conduction block and reentrant arrhythmias in fibrotic cardiac cultures. Moreover, functional BacNav channels are stably expressed in healthy mouse hearts six weeks following intravenous injection of self-complementary adeno-associated virus (scAAV) without causing any adverse effects on cardiac electrophysiology. The large diversity of prokaryotic sodium channels and experimental-computational platform reported in this study should facilitate the development and evaluation of BacNav-based gene therapies for cardiac conduction disorders.

In vitro discovery of novel prokaryotic ion channel candidates for antiarrhythmic gene therapy.

Sudden cardiac death continues to have a devastating impact on public health prompting the continued efforts to develop more effective therapies for cardiac arrhythmias. Among different approaches to normalize function of ion channels and prevent arrhythmogenic remodeling of tissue substrate, cardiac cell and gene therapies are emerging as promising strategies to restore and maintain normal heart rhythm. Specifically, the ability to genetically enhance electrical excitability of diseased hearts through voltage-gated sodium channel (VGSC) gene transfer could improve velocity of action potential conduction and act to stop reentrant circuits underlying sustained arrhythmias. For this purpose, prokaryotic VGSC genes are promising therapeutic candidates due to their small size (<1kb) and potential to be effectively packaged in adeno-associated viral (AAV) vectors and delivered to cardiomyocytes for stable, long-term expression. This article describes a versatile method to discover and characterize novel prokaryotic ion channels for use in gene and cell therapies for heart disease including cardiac arrhythmias. Detailed protocols are provided for: (1) identification of potential ion channel candidates from large genomic databases, (2) candidate screening and characterization using site-directed mutagenesis and engineered human excitable cell system and, (3) candidate validation using electrophysiological techniques and an in vitro model of impaired cardiac impulse conduction.

Efficacy of nonfumigant nematicides against Meloidogyne javanica as affected by soil temperature under pasteurized and natural soil conditions.

BACKGROUND: Biotic and abiotic factors such as microbes and soil temperature can affect nematicide efficacy. Two experiments were conducted to test the effect of three soil temperatures on the efficacy of nonfumigant nematicides (fluopyram, fluensulfone, oxamyl and fluazaindolizine) against Meloidogyne javanica in pasteurized and natural soil in planta. RESULTS: The results showed that all tested nematicides were more efficacious in pasteurized than in natural soil. Temperature affected the nematicides differently with no effect of soil temperature on oxamyl and fluazaindolizine, whereas fluopyram and fluensulfone had greater efficacy at higher soil temperatures. CONCLUSION: Temperature effects were noted for some but not all nonfumigant nematicides. Fluopyram and fluensulfone were less effective when applied in cold soil, whereas oxamyl and fluazaindolizine were not affected by soil temperature. Although all nematicides resulted in almost complete control of M. javanica in pasteurized soil, this was not the case in natural soil, and much more root damage and nematode reproduction was noted in the latter. © 2021 Society of Chemical Industry.

Feasibility and Safety of Laparoscopic Radical Colectomy for T4b Colon Cancer at a University Hospital in Vietnam.

BACKGROUND: The choice of optimal treatment strategies for T4b colon cancers has still been discussed, particularly the initiation of neoadjuvant therapy or surgery. We conducted this study to evaluate the safety and feasibility of laparoscopic multivisceral resection for T4b colon cancers. METHODS: We used the retrospective design to include all 43 patients with T4b colon cancer at a university hospital in Vietnam from March 2017 to March 2019. All patients were followed 30 days after the surgery, and information about the day of the first flatus, length of hospital stay, iatrogenic complications, postoperative morbidity, mortality, and adjuvant chemotherapy was collected. RESULTS: The mean operating time was 187 minutes (ranging from 80 to 310), the mean blood loss was 64.3 ml (5-200), and the conversion rate was 2.3%. The mean number of lymph nodes harvested was 15.5 (SD = 8.06), and 33 patients (76.7%) had at least 12 lymph nodes harvested. A total of 21 patients (48.8%) had lymph node metastases with a mean number of lymph node metastases of 1.89 (SD = 3.4). The radial resection margin was R0 in all 43 patients (100%). The median time until the first flatus and hospital stay were 3 days (2-5) and 7.1 (6-11) days, respectively. There was no mortality at 30 days postoperatively, and one patient had iatrogenic complication (2.3%). CONCLUSION: Laparoscopic radical colectomy was feasible and safe for patients with T4b colon cancer except those requiring major and complicated reconstruction.

Ion channel engineering for modulation and de novo generation of electrical excitability.

Ion channels play essential roles in regulating electrical properties of excitable tissues. By leveraging various ion channel gating mechanisms, scientists have developed a versatile set of genetically encoded tools to modulate intrinsic tissue excitability under different experimental settings. In this article, we will review how ion channels activated by voltage, light, small chemicals, stretch, and temperature have been customized to enable control of tissue excitability both in vitro and in vivo. Advantages and limitations of each of these ion channel-engineering platforms will be discussed and notable applications will be highlighted. Furthermore, we will describe recent progress on de novo generation of excitable tissues via expression of appropriate sets of engineered voltage-gated ion channels and discuss potential therapeutic implications.

Generation and customization of biosynthetic excitable tissues for electrophysiological studies and cell-based therapies.

We describe a two-stage protocol to generate electrically excitable and actively conducting cell networks with stable and customizable electrophysiological phenotypes. Using this method, we have engineered monoclonally derived excitable tissues as a robust and reproducible platform to investigate how specific ion channels and mutations affect action potential (AP) shape and conduction. In the first stage of the protocol, we combine computational modeling, site-directed mutagenesis, and electrophysiological techniques to derive optimal sets of mammalian and/or prokaryotic ion channels that produce specific AP shape and conduction characteristics. In the second stage of the protocol, selected ion channels are stably expressed in unexcitable human cells by means of viral or nonviral delivery, followed by flow cytometry or antibiotic selection to purify the desired phenotype. This protocol can be used with traditional heterologous expression systems or primary excitable cells, and application of this method to primary fibroblasts may enable an alternative approach to cardiac cell therapy. Compared with existing methods, this protocol generates a well-defined, relatively homogeneous electrophysiological phenotype of excitable cells that facilitates experimental and computational studies of AP conduction and can decrease arrhythmogenic risk upon cell transplantation. Although basic cell culture and molecular biology techniques are sufficient to generate excitable tissues using the described protocol, experience with patch-clamp techniques is required to characterize and optimize derived cell populations.

Genetically Encoded Photoactuators and Photosensors for Characterization and Manipulation of Pluripotent Stem Cells.

Our knowledge of pluripotent stem cell biology has advanced considerably in the past four decades, but it has yet to deliver on the great promise of regenerative medicine. The slow progress can be mainly attributed to our incomplete understanding of the complex biologic processes regulating the dynamic developmental pathways from pluripotency to fully-differentiated states of functional somatic cells. Much of the difficulty arises from our lack of specific tools to query, or manipulate, the molecular scale circuitry on both single-cell and organismal levels. Fortunately, the last two decades of progress in the field of optogenetics have produced a variety of genetically encoded, light-mediated tools that enable visualization and control of the spatiotemporal regulation of cellular function. The merging of optogenetics and pluripotent stem cell biology could thus be an important step toward realization of the clinical potential of pluripotent stem cells. In this review, we have surveyed available genetically encoded photoactuators and photosensors, a rapidly expanding toolbox, with particular attention to those with utility for studying pluripotent stem cells.

Engineering prokaryotic channels for control of mammalian tissue excitability.

The ability to directly enhance electrical excitability of human cells is hampered by the lack of methods to efficiently overexpress large mammalian voltage-gated sodium channels (VGSC). Here we describe the use of small prokaryotic sodium channels (BacNav) to create de novo excitable human tissues and augment impaired action potential conduction in vitro. Lentiviral co-expression of specific BacNav orthologues, an inward-rectifying potassium channel, and connexin-43 in primary human fibroblasts from the heart, skin or brain yields actively conducting cells with customizable electrophysiological phenotypes. Engineered fibroblasts ('E-Fibs') retain stable functional properties following extensive subculture or differentiation into myofibroblasts and rescue conduction slowing in an in vitro model of cardiac interstitial fibrosis. Co-expression of engineered BacNav with endogenous mammalian VGSCs enhances action potential conduction and prevents conduction failure during depolarization by elevated extracellular K+, decoupling or ischaemia. These studies establish the utility of engineered BacNav channels for induction, control and recovery of mammalian tissue excitability.

Influence of repeated infusion of capsaicin-contained red pepper sauce on esophageal secondary peristalsis in humans.

BACKGROUND: The transient receptor potential vanilloid 1 has been implicated as a target mediator for heartburn perception and modulation of esophageal secondary peristalsis. Our aim was to determine the effect of repeated esophageal infusion of capsaicin-contained red pepper sauce on heartburn perception and secondary peristalsis in healthy adults. METHODS: Secondary peristalsis was performed with mid-esophageal injections of air in 15 healthy adults. Two separate protocols including esophageal infusion with saline and capsaicin-contained red pepper sauce and 2 consecutive sessions of capsaicin-contained red pepper sauce were randomly performed. KEY RESULTS: After repeated infusion of capsaicin-contained red pepper sauce, the threshold volume to activate secondary peristalsis was significantly increased during slow (p < 0.001) and rapid air injections (p = 0.004). Acute infusion of capsaicin-contained red pepper sauce enhanced heartburn perception (p < 0.001), but the intensity of heartburn perception was significantly reduced after repeated capsaicin-contained red pepper sauce infusion (p = 0.007). Acute infusion of capsaicin-contained red pepper sauce significantly increased pressure wave amplitudes of distal esophagus during slow (p = 0.003) and rapid air injections (p = 0.01), but repeated infusion of capsaicin-contained red pepper sauce significantly decreased pressure wave amplitude of distal esophagus during slow (p = 0.0005) and rapid air injections (p = 0.003). CONCLUSIONS & INFERENCES: Repeated esophageal infusion of capsaicin appears to attenuate heartburn perception and inhibit distension-induced secondary peristalsis in healthy adults. These results suggest capsaicin-sensitive afferents in modulating sensorimotor function of secondary peristalsis in human esophagus.

Dibenzocyclooctadiene Lignans from Roots and Stems of Kadsura coccinea.

Ten dibenzocyclooctadiene lignans were obtained from the ethereal soluble fraction of the dried roots and stems of KADSURA COCCINEA. Two of them were new compounds, named kadsutherin ( 8) and isokadsuranin ( 10). Their structures were elucidated on the basis of chemical and spectral analysis.