Arsenic effectively improves the degradation of fluorene by Rhodococcus sp. 2021 under the combined pollution of arsenic and fluorene.

The performance of bacterial strains in executing degradative functions under the coexistence of heavy metals/heavy metal-like elements and organic contaminants is understudied. In this study, we isolated a fluorene-degrading bacterium, highly arsenic-resistant, designated as strain 2021, from contaminated soil at the abandoned site of an old coking plant. It was identified as a member of the genus Rhodococcus sp. strain 2021 exhibited efficient fluorene-degrading ability under optimal conditions of 400 mg/L fluorene, 30 °C, pH 7.0, and 250 mg/L trivalent arsenic. It was noted that the addition of arsenic could promote the growth of strain 2021 and improve the degradation of fluorene - a phenomenon that has not been described yet. The results further indicated that strain 2021 can oxidize As3+ to As5+; here, approximately 13.1% of As3+ was converted to As5+ after aerobic cultivation for 8 days at 30 °C. The addition of arsenic could greatly up-regulate the expression of arsR/A/B/C/D and pcaG/H gene clusters involved in arsenic resistance and aromatic hydrocarbon degradation; it also aided in maintaining the continuously high expression of cstA that codes for carbon starvation protein and prmA/B that codes for monooxygenase. These results suggest that strain 2021 holds great potential for the bioremediation of environments contaminated by a combination of arsenic and polycyclic aromatic hydrocarbons. This study provides new insights into the interactions among microbes, as well as inorganic and organic pollutants.

An LQT2-related mutation in the voltage-sensing domain is involved in switching the gating polarity of hERG.

BACKGROUND: Cyclic Nucleotide-Binding Domain (CNBD)-family channels display distinct voltage-sensing properties despite sharing sequence and structural similarity. For example, the human Ether-a-go-go Related Gene (hERG) channel and the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channel share high amino acid sequence similarity and identical domain structures. hERG conducts outward current and is activated by positive membrane potentials (depolarization), whereas HCN conducts inward current and is activated by negative membrane potentials (hyperpolarization). The structural basis for the "opposite" voltage-sensing properties of hERG and HCN remains unknown. RESULTS: We found the voltage-sensing domain (VSD) involves in modulating the gating polarity of hERG. We identified that a long-QT syndrome type 2-related mutation within the VSD, K525N, mediated an inwardly rectifying non-deactivating current, perturbing the channel closure, but sparing the open state and inactivated state. K525N rescued the current of a non-functional mutation in the pore helix region (F627Y) of hERG. K525N&F627Y switched hERG into a hyperpolarization-activated channel. The reactivated inward current induced by hyperpolarization mediated by K525N&F627Y can be inhibited by E-4031 and dofetilide quite well. Moreover, we report an extracellular interaction between the S1 helix and the S5-P region is crucial for modulating the gating polarity. The alanine substitution of several residues in this region (F431A, C566A, I607A, and Y611A) impaired the inward current of K525N&F627Y. CONCLUSIONS: Our data provide evidence that a potential cooperation mechanism in the extracellular vestibule of the VSD and the PD would determine the gating polarity in hERG.

The serine/threonine kinase Akt gene affects fecundity by reducing Juvenile hormone synthesis in Liposcelis entomophila (Enderlein).

The serine/threonine kinase Akt is an important component of the insulin signalling pathway (ISP) in regulating insect metabolism, growth, and reproduction. The psocid Liposcelis entomophila (Enderlein) is a distasteful stored products pest for its fecundity. However, the molecular mechanism of Akt that controls vitellogenesis and oviposition in L. entomophila remains obscure. In this study, the function of the Akt gene in the female reproduction of L. entomophila (designated as LeAkt) was characterized and investigated. LeAkt contains a 1587 bp open reading frame encoding a 529 amino acid protein that possesses a conserved Pleckstrin Homology domain (PH) and a Ser/Thr-type protein kinase (S_TKc) domain. The mRNA expression of LeAkt was the highest in female adult stages and peaked for 7-day female adults. In female adult tissues, LeAkt was highly expressed in the head and the ovary, indicating that LeAkt was closely correlated with female ovarian development. LeAkt transcription level was significantly suppressed by oral feeding on artificial diets mixed with dsRNA-LeAkt. RNAi-mediated silencing of LeAkt led to a severe inhibition of vitellogenein (Vg) expression and ovarian development, together with lower fecundity and hatchability compared to that of the normal feeding group, suggesting a critical role for LeAkt in L. entomophila reproduction. Further studies revealed that LeAkt silencing significantly decreased the mRNA levels of several signalling and biosynthetic genes in the juvenile hormone (JH) signalling pathway, such as methoprene-tolerant (LeMet), krüppel homolog 1 (LeKr-h1) and JH methyltransferase (LeJHAMT), leading to a severe inhibition of JH biosynthesis in L. entomophila female adults. These results suggested that LeAkt was affecting JH synthesis, thereby influencing Vg synthesis and ultimately L. entomophila reproduction.

The sulfonamide-resistance dihydropteroate synthase gene is crucial for efficient biodegradation of sulfamethoxazole by Paenarthrobacter species.

Sulfonamide antibiotics (SAs) are serious pollutants to ecosystems and environments. Previous studies showed that microbial degradation of SAs such as sulfamethoxazole (SMX) proceeds via a sad-encoded oxidative pathway, while the sulfonamide-resistant dihydropteroate synthase gene, sul, is responsible for SA resistance. However, the co-occurrence of sad and sul genes, as well as how the sul gene affects SMX degradation, was not explored. In this study, two SMX-degrading bacterial strains, SD-1 and SD-2, were cultivated from an SMX-degrading enrichment. Both strains were Paenarthrobacter species and were phylogenetically identical; however, they showed different SMX degradation activities. Specifically, strain SD-1 utilized SMX as the sole carbon and energy source for growth and was a highly efficient SMX degrader, while SD-2 did could not use SMX as a sole carbon or energy source and showed limited SMX degradation when an additional carbon source was supplied. Genome annotation, growth, enzymatic activity tests, and metabolite detection revealed that strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation and a pathway of protocatechuate degradation. A new sulfonamide-resistant dihydropteroate synthase gene, sul918, was identified in strain SD-1, but not in SD-2. Moreover, the lack of sul918 resulted in low SMX degradation activity in strain SD-2. Genome data mining revealed the co-occurrence of sad and sul genes in efficient SMX-degrading Paenarthrobacter strains. We propose that the co-occurrence of sulfonamide-resistant dihydropteroate synthase and sad genes is crucial for efficient SMX biodegradation. KEY POINTS: • Two sulfamethoxazole-degrading strains with distinct degrading activity, Paenarthrobacter sp. SD-1 and Paenarthrobacter sp. SD-2, were isolated and identified. • Strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation. • A new plasmid-borne SMX resistance gene (sul918) of strain SD-1 plays a crucial role in SMX degradation efficiency.

Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N2.

Nitrogen (N2) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed "Dirammox" (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N2. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated that, in the presence of NADH and FAD, DnfA catalyzed the conversion of 15N-labeled hydroxylamine to 15N2. This conversion did not happen under oxygen (O2)-free conditions. Thus, we concluded that DnfA encodes a hydroxylamine oxidase. We demonstrate that DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a Km of 92.9 ± 3.0 µM for hydroxylamine and a kcat of 0.028 ± 0.001 s-1. Finally, we show that DnfA was localized in the cytoplasm and periplasm as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude that DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N2.

Alcaligenes ammonioxydans HO-1 antagonizes Bacillus velezensis via hydroxylamine-triggered population response.

Antagonism is a common behavior seen between microbes in nature. Alcaligenes ammonioxydans HO-1 converts ammonia to nitrogen under aerobic conditions, which leads to the accumulation of extracellular hydroxylamine (HA), providing pronounced growth advantages against many bacterial genera, including Bacillus velezensis V4. In contrast, a mutant variant of A. ammonioxydans, strain 2-29, that cannot produce HA fails to antagonize other bacteria. In this article, we demonstrate that cell-free supernatants derived from the antagonistic HO-1 strain were sufficient to reproduce the antagonistic behavior and the efficiency of this inhibition correlated strongly with the HA content of the supernatant. Furthermore, reintroducing the capacity to produce HA to the 2-29 strain or supplementing bacterial co-cultures with HA restored antagonistic behavior. The HA-mediated antagonism was dose-dependent and affected by the temperature, but not by pH. HA caused a decline in biomass, cell aggregation, and hydrolysis of the cell wall in exponentially growing B. velezensis bulk cultures. Analysis of differential gene expression identified a series of genes modulating multicellular behavior in B. velezensis. Genes involved in motility, chemotaxis, sporulation, polypeptide synthesis, and non-ribosomal peptide synthesis were all significantly downregulated in the presence of HA, whereas autolysis-related genes showed upregulation. Taken together, these findings indicate that HA affects the population response of coexisting strains and also suggest that A. ammonioxydans HO-1 antagonize other bacteria by producing extracellular HA that, in turn, acts as a signaling molecule.

Degrading Characterization of the Newly Isolated Nocardioides sp. N39 for 3-Amino-5-methyl-isoxazole and the Related Genomic Information.

3-amino-5-methyl-isoxazole (3A5MI) is a persistent and harmful intermediate in the degradation of antibiotic sulfamethoxazole. It was accumulated in the environments day by day and has caused great environmental risks due to its refractory characteristic. Microbial degradation is economic and environmentally friendly and a promising method to eliminate this pollutant. In this study, a bacterial strain, Nocardioides sp. N39, was isolated. N39 can grow on 3A5MI as the sole carbon, nitrogen and energy resource. The effect of different factors on 3A5MI degradation by N39 was explored, including initial 3A5MI concentration, temperature, pH value, dissolved oxygen and additional carbon or nitrogen source. The degradation ability of N39 to various 3A5MI analogs was also explored. Nevertheless, the degrading ability of N39 for 3A5MI is not permanent, and long-term storage would lead to the loss of this ability. This may result from the mobile genetic elements in the bacterium according to the genomic comparison of N39 and its degrading ability-lost strain, N40. Despite this, N39 could support a lot of useful information about the degradation of 3A5MI and highlight the importance of studies about the environmental effects and potential degradation mechanism.

Dirammox Is Widely Distributed and Dependently Evolved in Alcaligenes and Is Important to Nitrogen Cycle.

Nitrogen cycle is an essential process for environmental health. Dirammox (direct ammonia oxidation), encoded by the dnfT1RT2ABCD cluster, was a novel pathway for microbial N2 production defined in Alcaligenes ammonioxydans HO-1. Here, a copy of the cluster dnfT1RT2ABCD as a whole was proved to have existed and very conserved in all Alcaligenes genomes. Phylogenetic analyses based on 16S rRNA gene sequences and amino acid sequences of DnfAs, together with G + C content data, revealed that dnf cluster was evolved associated with the members of the genus Alcaligenes. Under 20% O2 conditions, 14 of 16 Alcaligenes strains showed Dirammox activity, which seemed likely taxon-related. However, the in vitro activities of DnfAs catalyzing the direct oxidation of hydroxylamine to N2 were not taxon-related but depended on the contents of Fe and Mn ions. The results indicated that DnfA is necessary but not sufficient for Dirammox activity. The fact that members of the genus Alcaligenes are widely distributed in various environments, including soil, water bodies (both freshwater and seawater), sediments, activated sludge, and animal-plant-associated environments, strongly suggests that Dirammox is important to the nitrogen cycle. In addition, Alcaligenes species are also commonly found in wastewater treatment plants, suggesting that they might be valuable resources for wastewater treatment.

Degradation of amoxicillin by newly isolated Bosea sp. Ads-6.

Amoxicillin (AMX), one of the micro-amount hazardous pollutants, was frequently detected in environments, and of great risks to environments and human health. Microbial degradation is a promising method to eliminate pollutants. In this study, an efficient AMX-degrading strain, Ads-6, was isolated and characterized. Strain Ads-6, belonging to the genus Bosea, was also able to grow on AMX as the sole carbon and nitrogen source, with a removal of ~60% TOC. Ads-6 exhibited strong AMX-degrading ability at initial concentrations of 0.5-2 mM and pH 6-8. Addition of yeast extract could significantly enhance its degrading ability. Many degradation intermediates were identified by HPLC-MS, including new ones such as two phosphorylated products which were firstly defined in AMX degradation. A new AMX degradation pathway was proposed accordingly. Moreover, the results of comparative transcriptomes and proteomes revealed that ß-lactamase, L, D-transpeptidase or its homologous enzymes were responsible for the initial degradation of AMX. Protocatechuate branch of the beta-ketoadipate pathway was confirmed as the downstream degradation pathway. These results in the study suggested that Ads-6 is great potential in biodegradation of antibiotics as well as in the bioremediation of contaminated environments.

Genetic Foundations of Direct Ammonia Oxidation (Dirammox) to N2 and MocR-Like Transcriptional Regulator DnfR in Alcaligenes faecalis Strain JQ135.

Ammonia oxidation is an important process in both the natural nitrogen cycle and nitrogen removal from engineered ecosystems. Recently, a new ammonia oxidation pathway termed Dirammox (direct ammonia oxidation, NH3→NH2OH→N2) has been identified in Alcaligenes ammonioxydans. However, whether Dirammox is present in other microbes, as well as its genetic regulation, remains unknown. In this study, it was found that the metabolically versatile bacterium Alcaligenes faecalis strain JQ135 could efficiently convert ammonia into N2 via NH2OH under aerobic conditions. Genetic deletion and complementation results suggest that dnfABC is responsible for the ammonia oxidation to N2 in this strain. Strain JQ135 also employs aerobic denitrification, mainly producing N2O and trace amounts of N2, with nitrite as the sole nitrogen source. Deletion of the nirK and nosZ genes, which are essential for denitrification, did not impair the capability of JQ135 to oxidize ammonia to N2 (i.e., Dirammox is independent of denitrification). Furthermore, it was also demonstrated that pod (which encodes pyruvic oxime dioxygenase) was not involved in Dirammox and that AFA_16745 (which was previously annotated as ammonia monooxygenase and is widespread in heterotrophic bacteria) was not an ammonia monooxygenase. The MocR-family transcriptional regulator DnfR was characterized as an activator of the dnfABC operon with the binding motif 5'-TGGTCTGT-3' in the promoter region. A bioinformatic survey showed that homologs of dnf genes are widely distributed in heterotrophic bacteria. In conclusion, this work demonstrates that, besides A. ammonioxydans, Dirammox occurs in other bacteria and is regulated by the MocR-family transcriptional regulator DnfR. IMPORTANCE Microbial ammonia oxidation is a key and rate-limiting step of the nitrogen cycle. Three previously known ammonia oxidation pathways (i.e., nitrification, anaerobic ammonia oxidation [Anammox], and complete ammonia oxidation [Comammox]) are mediated by autotrophic microbes. However, the genetic foundations of ammonia oxidation by heterotrophic microorganisms have not been investigated in depth. Recently, a previously unknown pathway, termed direct ammonia oxidation to N2 (Dirammox), has been identified in the heterotrophic bacterium Alcaligenes ammonioxydans HO-1. This paper shows that, in the metabolically versatile bacterium Alcaligenes faecalis JQ135, the Dirammox pathway is mediated by dnf genes, which are independent of the denitrification pathway. A bioinformatic survey suggests that homologs of dnf genes are widely distributed in bacteria. These findings enhance the understanding of the molecular mechanisms of heterotrophic ammonia oxidation to N2.

[Advances in bioremediation of polycyclic aromatic hydrocarbons contaminated soil].

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent pollutants that are widely distributed in the environment. Due to their stable structure and poor degradability, PAHs exhibit carcinogenic, teratogenic, and mutagenic toxicity to the ecological environment and organisms, thus increasing attentions have been paid to their removals and remediation. Green, safe and economical technologies are widely used in the bioremediation of PAHs-contaminated soil. This article summarizes the present status of PAHs pollution in soil of China from the aspects of origin, migration, fate, and pollution level. Meanwhile, the types of microorganisms and plants capable of degrading PAHs, as well as the underlying mechanisms, are summarized. The features of three major bioremediation technologies, i.e., microbial remediation, phytoremediation, and joint remediation, are compared. Analysis of the interaction mechanisms between plants and microorganisms, selection and cultivation of stress-resistant strains and plants, as well as safety and efficacy evaluation of practical applications, are expected to become future directions in this field.

Novel Alcaligenes ammonioxydans sp. nov. from wastewater treatment sludge oxidizes ammonia to N2 with a previously unknown pathway.

Heterotrophic nitrifiers are able to oxidize and remove ammonia from nitrogen-rich wastewaters but the genetic elements of heterotrophic ammonia oxidation are poorly understood. Here, we isolated and identified a novel heterotrophic nitrifier, Alcaligenes ammonioxydans sp. nov. strain HO-1, oxidizing ammonia to hydroxylamine and ending in the production of N2 gas. Genome analysis revealed that strain HO-1 encoded a complete denitrification pathway but lacks any genes coding for homologous to known ammonia monooxygenases or hydroxylamine oxidoreductases. Our results demonstrated strain HO-1 denitrified nitrite (not nitrate) to N2 and N2 O at anaerobic and aerobic conditions respectively. Further experiments demonstrated that inhibition of aerobic denitrification did not stop ammonia oxidation and N2 production. A gene cluster (dnfT1RT2ABCD) was cloned from strain HO-1 and enabled E. coli accumulated hydroxylamine. Sub-cloning showed that genetic cluster dnfAB or dnfABC already enabled E. coli cells to produce hydroxylamine and further to 15 N2 from (15 NH4 )2 SO4 . Transcriptome analysis revealed these three genes dnfA, dnfB and dnfC were significantly upregulated in response to ammonia stimulation. Taken together, we concluded that strain HO-1 has a novel dnf genetic cluster for ammonia oxidation and this dnf genetic cluster encoded a previously unknown pathway of direct ammonia oxidation (Dirammox) to N2 .

Candidatus Kaistella beijingensis sp. nov., Isolated from a Municipal Wastewater Treatment Plant, Is Involved in Sludge Foaming.

Biological foaming (or biofoaming) is a frequently occurring problem in wastewater treatment plants (WWTPs) and is attributed to the overwhelming growth of filamentous bulking and foaming bacteria (BFB). Biological foaming has been intensively investigated, with BFB like Microthrix and Skermania having been identified from WWTPs and implicated in foaming. Nevertheless, studies are still needed to improve our understanding of the microbial diversity of WWTP biofoams and how microbial activities contribute to foaming. In this study, sludge foaming at the Qinghe WWTP of China was monitored, and sludge foams were investigated using culture-dependent and culture-independent microbiological methods. The foam microbiomes exhibited high abundances of Skermania, Mycobacterium, Flavobacteriales, and Kaistella. A previously unknown bacterium, Candidatus Kaistella beijingensis, was cultivated from foams, its genome was sequenced, and it was phenotypically characterized. Ca. K. beijingensis exhibits hydrophobic cell surfaces, produces extracellular polymeric substances (EPS), and metabolizes lipids. Ca. K. beijingensis abundances were proportional to EPS levels in foams. Several proteins encoded by the Ca. K. beijingensis genome were identified from EPS that was extracted from sludge foams. Ca. K. beijingensis populations accounted for 4 to 6% of the total bacterial populations in sludge foam samples within the Qinghe WWTP, although their abundances were higher in spring than in other seasons. Cooccurrence analysis indicated that Ca. K. beijingensis was not a core node among the WWTP community network, but its abundances were negatively correlated with those of the well-studied BFB Skermania piniformis among cross-season Qinghe WWTP communities. IMPORTANCE Biological foaming, also known as scumming, is a sludge separation problem that has become the subject of major concern for long-term stable activated sludge operation in decades. Biological foaming was considered induced by foaming bacteria. However, the occurrence and deterioration of foaming in many WWTPs are still not completely understood. Cultivation and characterization of the enriched bacteria in foaming are critical to understand their genetic, physiological, phylogenetic, and ecological traits, as well as to improve the understanding of their relationships with foaming and performance of WWTPs.

The damage and tolerance mechanisms of Phaffia rhodozyma mutant strain MK19 grown at 28 °C.

BACKGROUND: Phaffia rhodozyma has many desirable properties for astaxanthin production, including rapid heterotrophic metabolism and high cell densities in fermenter culture. The low optimal temperature range (17-21 °C) for cell growth and astaxanthin synthesis in this species presents an obstacle to efficient industrial-scale astaxanthin production. The inhibition mechanism of cell growth at > 21 °C in P. rhodozyma have not been investigated. RESULTS: MK19, a mutant P. rhodozyma strain grows well at moderate temperatures, its cell growth was also inhibited at 28 °C, but such inhibition was mitigated, and low biomass 6 g/L was obtained after 100 h culture. Transcriptome analysis indicated that low biomass at 28 °C resulted from strong suppression of DNA and RNA synthesis in MK19. Growth inhibition at 28 °C was due to cell membrane damage with a characteristic of low mRNA content of fatty acid (f.a.) pathway transcripts (acc, fas1, fas2), and consequent low f.a. CONTENT: Thinning of cell wall and low mannose content (leading to loss of cell wall integrity) also contributed to reduced cell growth at 28 °C in MK19. Levels of astaxanthin and ergosterol, two end-products of isoprenoid biosynthesis (a shunt pathway of f.a. biosynthesis), reached 2000 µg/g and 7500 µg/g respectively; ~2-fold higher than levels at 21 or 25 °C. Abundance of ergosterol, an important cell membrane component, compensated for lack of f.a., making possible the biomass production of 6 g/L for MK19 at 28 °C. CONCLUSIONS: Inhibition of growth of P. rhodozyma at 28 °C results from blocking of DNA, RNA, f.a., and cell wall biosynthesis. In MK19, abundant ergosterol made possible biomass production 6 g/L at 28 °C. Significant accumulation of astaxanthin and ergosterol indicated an active MVA pathway in MK19 at 28 °C. Strengthening of the MVA pathway can be a feasible metabolic engineering approach for enhancement of astaxanthin synthesis in P. rhodozyma. The present findings provide useful mechanistic insights regarding adaptation of P. rhodozyma to 28 °C, and improved understanding of feasible metabolic engineering techniques for industrial scale astaxanthin production by this economically important yeast species.

Eleutheroside B, a selective late sodium current inhibitor, suppresses atrial fibrillation induced by sea anemone toxin II in rabbit hearts.

Eleutheroside B (EB) is the main active constituent derived from the Chinese herb Acanthopanax senticosus (AS) that has been reported to possess cardioprotective effects. In this study we investigated the effects of EB on cardiac electrophysiology and its suppression on atrial fibrillation (AF). Whole-cell recording was conducted in isolated rabbit atrial myocytes. The intracellular calcium ([Ca2+]i) concentration was measured using calcium indicator Fura-2/AM fluorescence. Monophasic action potential (MAP) and electrocardiogram (ECG) synchronous recordings were conducted in Langendorff-perfused rabbit hearts using ECG signal sampling and analysis system. We showed that EB dose-dependently inhibited late sodium current (INaL), transient sodium current (INaT), and sea anemone toxin II (ATX II)-increased INaL with IC50 values of 167, 1582, and 181 µM, respectively. On the other hand, EB (800 µM) did not affect L-type calcium current (ICaL), inward rectifier potassium channel current (IK), and action potential duration (APD). Furthermore, EB (300 µM) markedly decreased ATX II-prolonged the APD at 90% repolarization (APD90) and eliminated ATX II-induced early afterdepolarizations (EADs), delayed afterdepolarizations (DADs), and triggered activities (TAs). Moreover, EB (200 µM) significantly suppressed ATX II-induced Na+-dependent [Ca2+]i overload in atrial myocytes. In the Langendorff-perfused rabbit hearts, application of EB (200 µM) or TTX (2 µM) substantially decreased ATX II-induced incidences of atrial fibrillation (AF), ventricular fibrillation (VF), and heart death. These results suggest that augmented INaL alone is sufficient to induce AF, and EB exerts anti-AF actions mainly via blocking INaL, which put forward the basis of pharmacology for new clinical application of EB.

Isoliensinine Eliminates Afterdepolarizations Through Inhibiting Late Sodium Current and L-Type Calcium Current.

Isoliensinine (IL) extracted from lotus seed has a good therapeutic effect on cardiovascular diseases. However, its effect on ion channels of ventricular myocytes is still unclear. We used whole-cell patch-clamp techniques to detect the effects of IL on transmembrane ion currents and action potential (AP) in isolated rabbit left ventricular myocytes. IL inhibited the transient sodium current (INaT), late sodium current (INaL) enlarged by sea anemone toxin (ATX II) and L-type calcium current (ICaL) in a concentration-dependent manner without affecting inward rectifier potassium current (IK1) and delayed rectifier potassium current (IK). These inhibitory effects are mainly manifested as reduced the AP amplitude (APA) and maximum depolarization velocity (Vmax) and shortened the action potential duration (APD), but had no significant effect on the resting membrane potential (RMP). Moreover, IL significantly eliminated ATX II-induced early afterdepolarizations (EADs) and high extracellular calcium-induced delayed afterdepolarizations (DADs). These results revealed that IL effectively eliminated EADs and DADs through inhibiting INaL and ICaL in ventricular myocytes, which indicates it has potential antiarrhythmic action.

Hydroxylation at Multiple Positions Initiated the Biodegradation of Indeno[1,2,3-cd]Pyrene in Rhodococcus aetherivorans IcdP1.

Nowadays, contamination by polycyclic aromatic hydrocarbons (PAHs) has become a serious problem all over the world; in particular, high-molecular-weight PAHs (HWM PAHs, four to seven rings) are more harmful to human health and environment due to their more complex structure and metabolic pathway. Biodegradation of PAHs with six or more rings, such as indeno[1,2,3-cd]pyrene (IcdP), was rarely described. An IcdP-degrading strain, Rhodococcus aetherivorans IcdP1, was isolated from HWM PAH-contaminated soil. It could grow on and efficiently degrade various HWM PAHs, such as IcdP, benzo[a]pyrene, and benzo[j]fluoranthene. It showed highest degrading ability toward IcdP (> 70% within 10 days). The IcdP degradation was initiated by ring hydroxylation with multiple pathways, including the hydroxylation at the 1,2 and 7,8 positions, according to the relevant metabolites detected, e.g., cyclopenta[cd]pyrene-3,4-dicarboxylic acid and 2,3-dimethoxy-2,3-dihydrofluoranthene. The transcriptional patterns of the genes encoding ring-hydroxylating oxygenases (RHOs) and cytochrome P450 monooxygenases (CYP450s) under the induction of IcdP, pyrene, and benzo[b]fluoranthene (BbF) were compared to determine the key initial RHOs in the conversion of IcdP. The expression of genes encoding RHOs 1892-1894, 1917-1920, and 4740-4741 was induced strictly by IcdP, and the amino acid sequences of these proteins showed very low identities with their homologs. These results suggested that IcdP was degraded through a dioxygenation-initiated metabolism pattern, and RHOs 1892-1894, 1917-1920, and 4740-4741 responded to the initial ring cleavage of IcdP through 1,2-dihydrodiol or 7,8-dihydrodiol. The studies would contribute to the understanding of the molecular mechanism of initial degradation of IcdP.

Curcumin, a Multi-Ion Channel Blocker That Preferentially Blocks Late Na+ Current and Prevents I/R-Induced Arrhythmias.

Increasing evidence shows that Curcumin (Cur) has a protective effect against cardiovascular diseases. However, the role of Cur in the electrophysiology of cardiomyocytes is currently not entirely understood. Therefore, the present study was conducted to investigate the effects of Cur on the action potential and transmembrane ion currents in rabbit ventricular myocytes to explore its antiarrhythmic property. The whole-cell patch clamp was used to record the action potential and ion currents, while the multichannel acquisition and analysis system was used to synchronously record the electrocardiogram and monophasic action potential. The results showed that 30 µmol/L Cur shortened the 50 and 90% repolarization of action potential by 17 and 7%, respectively. In addition, Cur concentration dependently inhibited the Late-sodium current (I Na.L), Transient-sodium current (I Na.T), L-type calcium current (I Ca.L), and Rapidly delayed rectifying potassium current (I Kr), with IC50 values of 7.53, 398.88, 16.66, and 9.96 µmol/L, respectively. Importantly, the inhibitory effect of Cur on I Na.L was 52.97-fold higher than that of I Na.T. Moreover, Cur decreased ATX II-prolonged APD, suppressed the ATX II-induced early afterdepolarization (EAD) and Ca2+-induced delayed afterdepolarization (DAD) in ventricular myocytes, and reduced the occurrence and average duration of ventricular tachycardias and ventricular fibrillations induced by ischemia-reperfusion injury. In conclusion, Cur inhibited I Na.L, I Na.T, I Ca.L, and I Kr; shortened APD; significantly suppressed EAD and DAD-like arrhythmogenic activities at the cellular level; and exhibited antiarrhythmic effect at the organ level. It is first revealed that Cur is a multi-ion channel blocker that preferentially blocks I Na.L and may have potential antiarrhythmic property.

Late Sodium Current in Atrial Cardiomyocytes Contributes to the Induced and Spontaneous Atrial Fibrillation in Rabbit Hearts.

Increased late sodium current (INa) induces long QT syndrome 3 with increased risk of atrial fibrillation (AF). The role of atrial late INa in the induction of AF and in the treatment of AF was determined in this study. AF parameters were measured in isolated rabbit hearts exposed to late INa enhancer and inhibitors. Late INa from isolated atrial and ventricular myocytes were measured using whole-cell patch-clamp techniques. We found that induced-AF by programmed S1S2 stimulation and spontaneous episodes of AF were recorded in hearts exposed to either low (0.1-3 nM) or high (3-10 nM) concentrations of ATX-II (n = 10). Prolongations in atrial monophasic action potential duration at 90% completion of repolarization and effective refractory period by ATX-II (0.1-15 nM) were greater in hearts paced at slow than at fast rates (n = 5-10, P < 0.05). Both endogenous and ATX-II-enhanced late INa density were greater in atrial than that in ventricular myocytes (n = 9 and 8, P < 0.05). Eleclazine and ranolazine reduced AF window and AF burden in association with the inhibition of both endogenous and enhanced atrial late INa with half maximal inhibitory concentrations (IC50) of 1.14 and 9.78, and 0.94 and 8.31 µM, respectively. The IC50s for eleclazine and ranolazine to inhibit peak INa were 20.67 and 101.79 µM, respectively, in atrial myocytes. In conclusion, enhanced late INa in atrial myocytes increases the susceptibility for AF. Inhibition of either endogenous or enhanced late INa, with increased atrial potency of drugs is feasible for the treatment of AF.