Transcription of AAT•ATT Triplet Repeats in Escherichia coli Is Silenced by H-NS and IS1E Transposition

The trinucleotide repeats AAT•ATT are simple DNA sequences that potentially form different types of non-B DNA secondary structures and cause genomic instabilities in vivo.The molecular mechanism underlying the maintenance of a 24-triplet AAT•ATT repeat was examined in E. coli by cloning the repeats into the EcoRI site in plasmid pUC18 and into the attB site on the E. coli genome. Either the AAT or the ATT strand acted as lagging strand template in a replication fork. Propagations of the repeats in either orientation on plasmids did not affect colony morphology when triplet repeat transcription using the lacZ promoter was repressed either by supplementing LacI(Q)in trans or by adding glucose into the medium. In contrast, transparent colonies were formed by inducing transcription of the repeats, suggesting that transcription of AAT•ATT repeats was toxic to cell growth. Meanwhile, significant IS1E transposition events were observed both into the triplet repeats region proximal to the promoter side, the promoter region of the lacZ gene, and into the AAT•ATT region itself. Transposition reversed the transparent colony phenotype back into healthy, convex colonies. In contrast, transcription of an 8-triplet AAT•ATT repeat in either orientation on plasmids did not produce significant changes in cell morphology and did not promote IS1E transposition events. We further found that a role of IS1E transposition into plasmids was to inhibit transcription through the repeats, which was influenced by the presence of the H-NS protein, but not of its paralogue StpA.Our findings thus suggest that the longer AAT•ATT triplet repeats in E. coli become vulnerable after transcription. H-NS and its facilitated IS1E transposition can silence long triplet repeats transcription and preserve cell growth and survival

Idiopathic Ventricular Arrhythmias Originating From the Pulmonary Sinus Cusp Prevalence, Electrocardiographic/Electrophysiological Characteristics, and Catheter Ablation

AbstractBackgroundIdiopathic ventricular arrhythmias (VAs) originating from the pulmonary sinus cusp (PSC) have not been sufficiently clarified.ObjectivesThe goal of this study was to investigate the prevalence, electrocardiographic characteristics, mapping, and ablation of idiopathic VAs arising from the PSC.MethodsData were analyzed from 218 patients undergoing successful endocardial ablation of idiopathic VAs with a left bundle branch block morphology and inferior axis deviation.ResultsTwenty-four patients had VAs originating from the PSC. In the first 7 patients, initial ablation performed in the right ventricular outflow tract failed to abolish the clinical VAs but produced a small change in the QRS morphology in 3 patients. In all 24 patients, the earliest activation was eventually identified in the PSC, at which a sharp potential was observed preceding the QRS complex onset by 28.2 ± 2.9 ms. The successful ablation site was in the right cusp (RC) in 10 patients (42%), the left cusp (LC) in 8 (33%), and the anterior cusp (AC) in 6 (25%). Electrocardiographic analysis showed that RC-VAs had significantly larger R-wave amplitude in lead I and a smaller aVL/aVR ratio of Q-wave amplitude compared with AC-VAs and LC-VAs, respectively. The R-wave amplitude in inferior leads was smaller in VAs localized in the RC than in the LC but did not differ between VAs from the AC and LC.ConclusionsVAs arising from the PSC are not uncommon, and RC-VAs have unique electrocardiographic characteristics. These VAs can be successfully ablated within the PSC

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Prototype Network for Predicting Occluded Picking Position Based on Lychee Phenotypic Features

The automated harvesting of clustered fruits relies on fast and accurate visual perception. However, the obscured stem diameters via leaf occlusion lack any discernible texture patterns. Nevertheless, our human visual system can often judge the position of harvesting points. Inspired by this, the aim of this paper is to address this issue by leveraging the morphology and the distribution of fruit contour gradient directions. Firstly, this paper proposes the calculation of fruit normal vectors using edge computation and gradient direction distribution. The research results demonstrate a significant mathematical relationship between the contour edge gradient and its inclination angle, but the experiments show that the standard error projected onto the Y-axis is smaller, which is evidently more conducive to distinguishing the gradient distribution. Secondly, for the front view of occluded lychee clusters, a fully convolutional, feature prototype-based one-stage instance segmentation network is proposed, named the lychee picking point prediction network (LP3Net). This network can achieve high accuracy and real-time instance segmentation, as well as for occluded and overlapping fruits. Finally, the experimental results show that the LP3Net based on this study, along with lychee phenotypic features, achieves an average location accuracy reaching 82%, significantly improving the precision of harvesting point localization for lychee clusters

Diurnal Evapotranspiration and Its Controlling Factors of Alpine Ecosystems during the Growing Season in Northeast Qinghai-Tibet Plateau

It is generally believed that evapotranspiration at night is too miniscule to be considered. Thus, few studies focus on the nocturnal evapotranspiration (ETN) in alpine region. In this study, based on the half-hour eddy and meteorological data of the growing season (from May to September) in 2019, we quantified the ETN of alpine desert (AD), alpine meadow (AM), alpine meadow steppe (AMS), and alpine steppe (AS) in the Qinghai Lake Basin and clarified the different response of evapotranspiration to climate variables in daytime and nighttime with the variation of elevation. The results show that: (1) ETN accounts for 9.88~15.08% of total daily evapotranspiration and is relatively higher in AMS (15.08%) and AD (12.13%); (2) in the daytime, net radiation (Rn), temperature difference (TD), vapor pressure difference (VPD), and soil moisture have remarkable influence on evapotranspiration, and Rn and VPD are more important at high altitudes, while TD is the main factor at low altitudes; (3) in the nighttime, VPD and wind speed (WS) control ETN at high altitudes, and TD and WS drive ETN at low altitudes. Our results are of great significance in understanding ETN in the alpine regions and provide reference for further improving in the evapotranspiration estimation model

Effects of <i>hns</i> gene on the growth of AAT•ATT repeats-carrying strains in chromosome.

<p>Significant filamentous cells were observed in JM83<i>Δhns</i> -AAT and JM83<i>Δhns</i> -ATT,but cannot be seen with JM83 -AAT and JM83 -ATT, nor be seen with JM83(pAAT₂₄), JM83(pATT₂₄), JM83<i>Δhns</i> (pAAT₂₄), JM83<i>Δhns</i>(pATT₂₄) and JM83(pUC18), JM83<i>Δhns</i> (pUC18) (data not shown).</p

Characterization of the IS1E insertion positions.

<p>A) a. Transposition of IS1E elements into plasmids, plasmid isolated from the healthy colonies, and b. Restriction digestion of the plasmid DNA with transposons. Lanes 1, molecular weight (pBR322/BstN1); Lane 2 and 3, pAAT₂₄ and pATT₂₄ without IS1E transposition, digested by <i>Pst</i>I; Lane 4, pAAT₂₄, with IS1E transposition, digested by <i>Pst</i>I; Lane 5, pAAT ₂₄ with IS1E transposition, digested by <i>Eco</i>R1;Lane 6 and 7, pAAT₂₄ with IS1E transposition, digested by <i>Pst</i>I and <i>EcoR</i>I respectively; Lane 8 and 9 pAAT₂₄ with IS1E transposition, digested by using <i>Pvu</i>II. B) Mapping the IS1E inserting elements in the promoter and the AAT repeats region by DNA sequencing, the positions of the mostly recognized sites were shown.</p

The α–complementation assay for the role of IS1E insertions.

<p>a) α-complementation by an AAT•ATT repeats free pAAT24IS1E-A plasmid, and b) α-complementation by pUC18 plasmid (For detail see text).</p

IS1E transposition into AAT•ATT repeats.

<p>A) Plasmids of AAT orientation propagated in LB medium were recovered and digested by <i>Pst</i> I (lane 1), <i>Eco</i>R I (lane 2); and propagated in LB medium in the presence of IPTG, and were digested by <i>Pst</i>I (lane 3), and <i>Eco</i>RI (lane 4); Plasmids of ATT orientation propagated in LB medium with or without IPTG, digested by <i>Pst</i> I (lane 5), <i>Eco</i>R I (lane 6); <i>Pst</i> I (lane 7 with IPTG induction) and <i>Eco</i>R I (lane 8 with IPTG induction). M1 and M2 are DNA molecular weights; B) Schematic illustration of the directions of IS1E transposition into the AAT•ATT repeats in light of the transcription of either AAT or ATT orientation.</p