Rate and irregularity of electrical activation during atrial fibrillation affect myocardial NGF expression via different signalling routes

An irregular ventricular response during atrial fibrillation (AF) has been shown to mediate an increase in sympathetic nerve activity in human subjects. The molecular mechanisms remain unclear. This study aimed to investigate the impact of rate and irregularity on nerve growth factor (NGF) expression in cardiomyocytes, since NGF is known to be the main contributor to cardiac sympathetic innervation density. Cell cultures of neonatal rat ventricular myocytes were electrically stimulated for 48 h with increasing rates (0, 5 and 50 Hz) and irregularity (standard deviation (SD) = 5%, 25% and 50% of mean cycle length). Furthermore, we analyzed the calcineurin-NFAT and the endothelin-1 signalling pathways as possible contributors to NGF regulation during arrhythmic stimulation. We found that the increase of NGF expression reached its maximum at the irregularity of 25% SD by 5 Hz (NGF: 5 Hz 0% SD = 1 vs. 5 Hz 25% SD = 1.57, P < 0.05). Specific blockade of the ET-A receptor by BQ123 could abolish this NGF increase (NGF: 5 Hz 25% SD + BQ123 = 0.66, P < 0.05). High frequency electrical field stimulation (HFES) with 50 Hz decreased the NGF expression in a significant manner (NGF: 50 Hz = 0.55, P < 0.05). Inhibition of calcineurin-NFAT signalling with cyclosporine-A or 11R-VIVIT abolished the HFES induced NGF down-regulation (NGF: 50 Hz + CsA = 1.14, P < 0.05). In summary, this study reveals different signalling routes of NGF expression in cardiomyocytes exposed to increasing rates and irregularity. Whether this translates into different degrees of NGF expression and possibly neural sympathetic growth in various forms of ventricular rate control during AF remains to be elucidated in further studies.     

New antiprotozoal agents: Synthesis and biological evaluation of different 4-(7-chloroquinolin-4-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone derivatives

In an endeavor to develop efficacious antiprotozoal agents 4-(7-chloroquinolin-4-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone derivatives (5–14) were synthesized, characterized and biologically evaluated for antiprotozoal activity. The compounds were screened in vitro against the HM1: IMSS strain of Entamoeba histolytica and NF54 chloroquine-sensitive strain of Plasmodium falciparum. Among the synthesized compounds six exhibited promising antiamoebic activity with IC₅₀ values (0.14–1.26 μM) lower than the standard drug metronidazole (IC₅₀ 1.80 μM). All nine compounds exhibited antimalarial activity (IC₅₀ range: 1.42–19.62 μM), while maintaining a favorable safety profile to host red blood cells. All the compounds were less effective as an antimalarial and more toxic (IC₅₀ range: 14.67–81.24 μM) than quinine (IC₅₀: 275.6 ± 16.46 μM) against the human kidney epithelial cells. None of the compounds exhibited any inhibitory effect on the viability of Anopheles arabiensis mosquito larvae.     

Identifying people at high risk for developing sleep apnea syndrome (SAS): a cross-sectional study in a Pakistani population

Background  

Obstructive Sleep Apnea (OSA) is associated with many cardiovascular and psychiatric diseases. Day-time sleepiness is a common consequence of sleep apnea and correlates with road-traffic accidents (RTA). Pakistan has a high prevalence of factors which predispose an individual to OSA and death from RTAs are a huge burden. However there is a dearth of prevalence studies in this regard. We aim to understand local relevance of the disease and estimate the prevalence of individuals high-risk for OSA.     

High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

Background

Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods.

Results

A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point.

Conclusions

Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.     

Role of Probiotics in Oral Health Maintenance Among Patients Undergoing Fixed Orthodontic Therapy: a Systematic Review of Randomized Controlled Clinical Trials

The aim was to assess the role of probiotics in oral health maintenance among patients undergoing fixed orthodontic therapy (OT). An unrestricted search of indexed databases was performed until April 2020 using the following Mesh-terms: (1) probiotic and (2) orthodontic. Randomized controlled clinical trials (RCTs) evaluating the influence of probiotic therapy (PT) towards oral health maintenance/improvement in patients undergoing fixed OT were included. Data screening, selection, and extraction were performed; and the risk of bias was assessed using the Cochrane Collaboration’s tool. All evaluations were performed by 2 independent researchers; and disagreements were resolved via discussion. Nine RCTs were included. Eight studies reported that PT improves oral health in patients undergoing fixed OT. Seven studies showed that PT reduces the counts of oral pathogenic bacteria in the oral biofilm and/or saliva. One study reported that PT reduces halitosis in patients undergoing fixed OT. One study found that PT reduces the scores of plaque index (PI) and gingival index (GI); and one study reported that PT has no significant influence on PI and GI in patients undergoing fixed OT. One study reported that PT does not prevent the formation of white spot lesions during fixed OT. Three and 6 studies had a moderate and low risk of bias, respectively. A power analysis was performed in 4 studies. In conclusion, probiotics exhibit antimicrobial activity against oral pathogenic bacteria; however, due to the limitations of the studies assessed, further well-designed RCTs are needed.

     

Larger benthic foraminiferal assemblages and their response to Middle Eocene Climate Optimum in the Kohat Basin (Pakistan,eastern Tethys)

The Middle Eocene Climatic Optimum (MECO) at ～40 Ma is a significant global warming event associated with pronounced changes in the hydrosphere, atmosphere, and biosphere. The Kohat Formation in the Kohat Basin (eastern Tethys, Pakistan) is studied for identifying the response of larger benthic foraminifera (LBF) to MECO. The LBF assemblages in the Kohat Formation, covering from the Shallow Benthic Zones (SBZ) 15 to 17, suggest middle Lutetian to early Bartonian in age. Microfacies analyses indicate a lagoonal (inner carbonate ramp facies belt) to open marine (middle carbonate ramp facies belt) paleodepositional environment of the Kohat Formation. A distinct positive δ¹³C shift marks the stratigraphic position of the MECO in this formation. At the Peak-MECO event that is marked by the onset of the positive carbon isotope excursion (CIE), no evident compositional variation in the LBF assemblages is observed. However, significant changes in the LBF assemblages with the local first and last occurrences of some LBF genera can be observed in the Post-MECO and CIE recovery phase. These changes are verified by the sudden disappearance of Alveolina and orthophragminids and initial dominance of larger shell-size Nummulites fabianii, Heterostegina, and Linderina species accompanied by an increase in the species diversity. Here, we argue that the change in the observed LBF assemblages in the uppermost part of the Kohat Formation might be related to a larger foraminiferal turnover occurring during the Post-MECO event and corresponds to the CIE recovery phase.     

Irregular electrical activation of intrinsic cardiac adrenergic cells increases catecholamine-synthesizing enzymes

Background

Recently, increased cardiac norepinephrine levels were observed in patients who were exposed to irregular stimulation during electrophysiological testing. The molecular mechanisms remain unclear. Intrinsic cardiac adrenergic (ICA) cells are present in mammalian hearts and contain catecholamine-synthesizing enzymes sufficient to produce biologically active norepinephrine levels. Thus, we aimed to investigate the expression of catecholamine-synthesizing enzymes by ICA cells exposed to irregular pacing.

Methods

Co-cultures of cardiomyocytes and ICA cells were exposed to irregular pacing for 48 h (standard deviation (SD) = 5%, 25% and 50% of mean cycle length) at a constant rate of 5 Hz. The expression of catecholamine-synthesizing enzymes including tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) were analyzed on mRNA and protein levels.

Results

First, immunolabeling identified ICA cells presenting TH and DBH staining around the cell nucleus. Irregular pacing with 25% SD at a constant rate of 5 Hz significantly increased the expression of TH and DBH enzyme synthesis. Pharmacological approaches have shown that both metoprolol and losartan reversed the irregular pacing induced DBH increase, whereas the expression of TH was only blocked by metoprolol in a significant manner. Blockade of the endothelin-A receptor by BQ123 or the calcineurin–NFAT pathway by cyclosporine-A, 11R-VIVIT or FK506 revealed a potential role of both cascades in irregular pacing induced catecholamine-synthesizing enzyme expression.

Conclusions

ICA cells respond to irregular electrical activation with an increase in catecholamine-synthesizing enzymes. Drugs commonly used in clinical routine significantly influence the expression of TH and DBH by ICA cells via different signaling routes.