Involvement of AMPA receptors for Mesobuthus tamulus Pocock venom-induced depression of monosynaptic reflex in neonatal rat spinal cord in vitro

Glutamate is a putative neurotransmitter at Ia-alpha motoneuron synapse in the spinal cord and mediate the action via N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors. Since NMDA receptors are not involved in M. tamulus Pocock (MBT) venom-induced depression of spinal monosynaptic reflex (MSR), the present study was undertaken to evaluate the role of AMPA receptors in mediating the depression of MSR by MBT venom. The experiments were performed on isolated hemisected spinal cord from 4-6 day old rats. Stimulation of a dorsal root with supramaximal voltage evoked MSR and polysynaptic reflex (PSR) potentials in the corresponding segmental ventral root. Superfusion of MBT venom (0.3 microg/ml) depressed the spinal reflexes in a time-dependent manner. The maximum depression of MSR(approximately 66%) was seen at 10 min and it was 25 min for PSR (approximately 75%). The time to produce 50% depression of MSR and PSR was 6.7+/- 1.5 and 10.8 +/- 2.6 min, respectively. Pretreatment of the cords with 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX, 0.1 microM), an AMPA receptor antagonist, blocked the venom-induced depression of MSR but not PSR. The results indicate that venom-induced depression of MSR is mediated via AMPA receptors.     

RNA helicase p68 (DDX5) regulates tau exon 10 splicing by modulating a stem-loop structure at the 5' splice site

Regulation of tau exon 10 splicing plays an important role in tauopathy. One of the cis elements regulating tau alternative splicing is a stem-loop structure at the 5' splice site of tau exon 10. The RNA helicase(s) modulating this stem-loop structure was unknown. We searched for splicing regulators interacting with this stem-loop region using an RNA affinity pulldown-coupled mass spectrometry approach and identified DDX5/RNA helicase p68 as an activator of tau exon 10 splicing. The activity of p68 in stimulating tau exon 10 inclusion is dependent on RBM4, an intronic splicing activator. RNase H cleavage and U1 protection assays suggest that p68 promotes conformational change of the stem-loop structure, thereby increasing the access of U1snRNP to the 5' splice site of tau exon 10. This study reports the first RNA helicase interacting with a stem-loop structure at the splice site and regulating alternative splicing in a helicase-dependent manner. Our work uncovers a previously unknown function of p68 in regulating tau exon 10 splicing. Furthermore, our experiments reveal functional interaction between two splicing activators for tau exon 10, p68 binding at the stem-loop region and RBM4 interacting with the intronic splicing enhancer region.     

Characterization of bacteria associated with nodules of two endemic legumes of Algeria,Hedysarum naudinianum and H. perrauderianum

The root nodules of two wild legume species endemic to Algeria, Hedysarum naudinianum and He. perrauderianum, were investigated with regard to their anatomy and histology, and the identity of the associated bacteria. Both plants were found to form root nodules with regular features and well infected by rod-shaped bacteria. The culturable fraction of bacteria that could be obtained from surface-sterilized nodules included a prevailing presence of Enterobacteriaceae having 100 % 16S rDNA sequence identity with both Enterobacter cloacae and E. ludwigii. In H. perrauderianum, this taxon was the sole cultured isolate, while from H. naudinianum we also found Bacillus, Lactobacillus, Staphylococcus, Rothia, and isolates that were 100 % identical to Corynebacterium pseudodiphthericum, which is known to be an agent of respiratory and cardiac infections in humans. Whereas no culturable rhizobia and alike could be obtained on plates, PCR-based culture-independent approaches revealed in both plants the presence of a Mesorhizobium sp., which in H. perrauderianum was identical to isolates nodulating other legumes from Africa, European Mediterranean countries, and Asia, while in H. naudinianum it bore a single nucleotide polymorphism which is so far unique for any observed mesorhizobia. Data from the microsymbionts appear to suggest interesting clues to interpret the evolutionary ecology of their host plants.     

Evaluation of the Cardiotoxicity of Mitragynine and Its Analogues Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Introduction

Mitragynine is a major bioactive compound of Kratom, which is derived from the leave extracts of Mitragyna speciosa Korth or Mitragyna speciosa (M. speciosa), a medicinal plant from South East Asia used legally in many countries as stimulant with opioid-like effects for the treatment of chronic pain and opioid-withdrawal symptoms. Fatal incidents with Mitragynine have been associated with cardiac arrest. In this study, we determined the cardiotoxicity of Mitragynine and other chemical constituents isolated using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).

Methods and Results

The rapid delayed rectifier potassium current (I _Kr), L-type Ca²⁺ current (I _Ca,L) and action potential duration (APD) were measured by whole cell patch-clamp. The expression of KCNH2 and cytotoxicity was determined by real-time PCR and Caspase activity measurements. After significant I _Kr suppression by Mitragynine (10 µM) was confirmed in hERG-HEK cells, we systematically examined the effects of Mitragynine and other chemical constituents in hiPSC-CMs. Mitragynine, Paynantheine, Speciogynine and Speciociliatine, dosage-dependently (0.1∼100 µM) suppressed I _Kr in hiPSC-CMs by 67% ∼84% with IC₅₀ ranged from 0.91 to 2.47 µM. Moreover, Mitragynine (10 µM) significantly prolonged APD at 50 and 90% repolarization (APD50 and APD90) (439.0±11.6 vs. 585.2±45.5 ms and 536.0±22.6 vs. 705.9±46.1 ms, respectively) and induced arrhythmia, without altering the L-type Ca²⁺ current. Neither the expression,and intracellular distribution of KCNH2/Kv11.1, nor the Caspase 3 activity were significantly affected by Mitragynine.

Conclusions

Our study indicates that Mitragynine and its analogues may potentiate Torsade de Pointes through inhibition of I _Kr in human cardiomyocytes.     

The β-Prism Lectin Domain of Vibrio cholerae Hemolysin Promotes Self-assembly of the β-Pore-forming Toxin by a Carbohydrate-independent Mechanism

Vibrio cholerae cytolysin/hemolysin (VCC) is an amphipathic 65-kDa β-pore-forming toxin with a C-terminal β-prism lectin domain. Because deletion or point mutation of the lectin domain seriously compromises hemolytic activity, it is thought that carbohydrate-dependent interactions play a critical role in membrane targeting of VCC. To delineate the contributions of the cytolysin and lectin domains in pore formation, we used wild-type VCC, 50-kDa VCC (VCC⁵⁰) without the lectin domain, and mutant VCC^D617A with no carbohydrate-binding activity. VCC and its two variants with no carbohydrate-binding activity moved to the erythrocyte stroma with apparent association constants on the order of 10⁷ m⁻¹. However, loss of the lectin domain severely reduced the efficiency of self-association of the VCC monomer with the β-barrel heptamer in the synthetic lipid bilayer from ∼83 to 27%. Notably, inactivation of the carbohydrate-binding activity by the D617A mutation marginally reduced oligomerization to ∼77%. Oligomerization of VCC⁵⁰ was temperature-insensitive; by contrast, VCC self-assembly increased with increasing temperature, suggesting that the process is driven by entropy and opposed by enthalpy. Asialofetuin, the β₁-galactosyl-terminated glycoprotein inhibitor of VCC-induced hemolysis, promoted oligomerization of 65-kDa VCC to a species that resembled the membrane-inserted heptamer in stoichiometry and morphology but had reduced global amphipathicity. In conclusion, we propose (i) that the β-prism lectin domain facilitated toxin assembly by producing entropy during relocation in the heptamer and (ii) that glycoconjugates inhibited VCC by promoting its assembly to a water-soluble, less amphipathic oligomer variant with reduced ability to penetrate the bilayer.     

The Integrin β1 Subunit Regulates Paracellular Permeability of Kidney Proximal Tubule Cells

Epithelial cells lining the gastrointestinal tract and kidney have different abilities to facilitate paracellular and transcellular transport of water and solutes. In the kidney, the proximal tubule allows both transcellular and paracellular transport, while the collecting duct primarily facilitates transcellular transport. The claudins and E-cadherin are major structural and functional components regulating paracellular transport. In this study we present the novel finding that the transmembrane matrix receptors, integrins, play a role in regulating paracellular transport of renal proximal tubule cells. Deleting the integrin β1 subunit in these cells converts them from a “loose” epithelium, characterized by low expression of E-cadherin and claudin-7 and high expression of claudin-2, to a “tight” epithelium with increased E-cadherin and claudin-7 expression and decreased claudin-2 expression. This effect is mediated by the integrin β1 cytoplasmic tail and does not entail β1 heterodimerization with an α-subunit or its localization to the cell surface. In addition, we demonstrate that deleting the β1 subunit in the proximal tubule of the kidney results in a major urine-concentrating defect. Thus, the integrin β1 tail plays a key role in regulating the composition and function of tight and adherens junctions that define paracellular transport properties of terminally differentiated renal proximal tubule epithelial cells.     

Non-coding RNAs and ferroptosis: potential implications for cancer therapy

Ferroptosis is a recently defined form of regulated cell death, which is biochemically and morphologically distinct from traditional forms of programmed cell death such as apoptosis or necrosis. It is driven by iron, reactive oxygen species, and phospholipids that are oxidatively damaged, ultimately resulting in mitochondrial damage and breakdown of membrane integrity. Numerous cellular signaling pathways and molecules are involved in the regulation of ferroptosis, including enzymes that control the cellular redox status. Alterations in the ferroptosis-regulating network can contribute to the development of various diseases, including cancer. Evidence suggests that ferroptosis is commonly suppressed in cancer cells, allowing them to survive and progress. However, cancer cells which are resistant to common chemotherapeutic drugs seem to be highly susceptible to ferroptosis inducers, highlighting the great potential of pharmacologic modulation of ferroptosis for cancer treatment. Non-coding RNAs (ncRNAs) are considered master regulators of various cellular processes, particularly in cancer where they have been implicated in all hallmarks of cancer. Recent work also demonstrated their involvement in the molecular control of ferroptosis. Hence, ncRNA-based therapeutics represent an exciting alternative to modulate ferroptosis for cancer therapy. This review summarizes the ncRNAs implicated in the regulation of ferroptosis in cancer and highlights their underlying molecular mechanisms in the light of potential therapeutic applications.Subject terms: Tumour biomarkers, Oncogenes     

Factors Contributing to the Delay in Diagnosis and Continued Transmission of Leprosy in Brazil – An Explorative,Quantitative, Questionnaire Based Study

BackgroundLeprosy is a leading cause of preventable disability worldwide. Delay in diagnosis of patients augments the transmission of infection, and allows progression of disease and more severe disability. Delays in diagnosis greater than ten years have been reported in Brazil. To reduce this delay, it is important to identify factors that hinder patients from presenting to doctors, and those that delay doctors from diagnosing patients once they have presented. This study aimed to explore factors associated with the delayed diagnosis of leprosy in Brazil.

Methodology/ Principal Findings

This is an exploratory study using a self-constructed questionnaire delivered to patients attending three leprosy referral clinics across three states in Brazil. Data were analysed to determine associations between variables and the time taken for participants to present to the health-service, and between variables and the time taken for doctors to diagnose participants once they had presented. Participants who suspected they had leprosy but feared community isolation were 10 times more likely to wait longer before consulting a doctor for their symptoms (OR 10.37, 95% CI 2.18–49.45, p = 0.003). Participants who thought their symptoms were not serious had a threefold greater chance of waiting longer before consulting than those who did (OR 3.114, 95% CI 1.235–7.856, p = 0.016). Forty-two point six per cent of participants reported initially receiving a diagnosis besides leprosy. These had a three times greater chance of receiving a later diagnosis of leprosy compared to those not misdiagnosed or not given a diagnosis (OR 2.867, 95% CI 1.288–6.384, p = 0.010).

Conclusions/ Significance

This study implies a need for patient education regarding leprosy symptoms and the reduction of stigma to encourage patients to present. The high rate of misdiagnosis reported suggests a need to increase clinician suspicion of leprosy. Further education regarding disease symptoms in medical school curriculums may be advisable.     

Evidence for an ammonium transport system in free-living and symbiotic cyanobacteria

The free-living cyanobacterium Anabaena variabilis showed a biphasic pattern of ¹⁴CH₃NH ₃ ⁺ uptake. Initial accumulation (up to 60 s) was independent of CH₃NH ₃ ⁺ metabolism, but long-term uptake was dependent on its metabolism via glutamine synthetase (GS). The CH₃NH ₃ ⁺ was converted into methylglutamine which was not further metabolised. The addition of l-methionine-dl-sulphoximine (MSX), to inhibit GS, inhibited CH₃NH ₃ ⁺ metabolism, but did not affect the CH₃NH ₃ ⁺ transport system.NH ₄ ⁺ , when added after the addition of ¹⁴CH₃NH ₃ ⁺ , caused the efflux of free CH₃NH ₃ ⁺ ; when added before ¹⁴CH₃NH ₃ ⁺ , NH ₄ ⁺ inhibited its uptake indicating that both NH ₄ ⁺ and CH₃NH ₃ ⁺ share a common transport system. Carbonylcyanide m-chlorophenylhydrazone and triphenyl-methylphosphonium both inhibited CH₃NH ₃ ⁺ accumulation indicating that the transport system was -dependent. At pH 7 and at an external CH₃NH ₃ ⁺ concentration of 30 mol dm^-3, A. variabilis showed a 40-fold intracellular accumulation of CH₃NH ₃ ⁺ (internal concentration 1.4 mmol dm^-3). Packets of the symbiotic cyanobacterium Anabaena azollae, directly isolated from the water fern Azolla caroliniana, also showed a -dependent NH ₄ ⁺ transport system suggesting that the reduced inhibitory effect of NH ₄ ⁺ on nitrogenase cannot be attributed to the absence of an NH ₄ ⁺ transport system but is probably related to the reduced GS activity of the cyanobiont.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - pH transmembrane pH difference - TPMP⁺ triphenylmethylphosphonium