MINERVA: A Facile Strategy for SARS-CoV-2 Whole-Genome Deep Sequencing of Clinical Samples.

Analyzing the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from clinical samples is crucial for understanding viral spread and evolution as well as for vaccine development. Existing RNA sequencing methods are demanding on user technique and time and, thus, not ideal for time-sensitive clinical samples; these methods are also not optimized for high performance on viral genomes. We developed a facile, practical, and robust approach for metagenomic and deep viral sequencing from clinical samples. We demonstrate the utility of our approach on pharyngeal, sputum, and stool samples collected from coronavirus disease 2019 (COVID-19) patients, successfully obtaining whole metatranscriptomes and complete high-depth, high-coverage SARS-CoV-2 genomes with high yield and robustness. With a shortened hands-on time from sample to virus-enriched sequencing-ready library, this rapid, versatile, and clinic-friendly approach will facilitate molecular epidemiology studies during current and future outbreaks.

Dynamic within-host cefiderocol heteroresistance caused by blaSHV-12 amplification in pandrug-resistant and hypervirulent Klebsiella pneumoniae sequence type 11.

AIMS: Although cefiderocol (FDC) is not prescribed in China, FDC-resistant pandrug-resistant hypervirulent Klebsiella pneumoniae (PDR-hvKp) is emerging. In this study, we performed FDC susceptibility testing of clinical Kp isolates to explore the prevalence of FDC-resistant isolates and the mechanism of FDC-resistance. METHODS: We retrospectively selected 151 carbapenem-resistant Kp isolates to assess FDC susceptibility. Seven isolates harboring blaSHV-12 from two patients were enrolled for whole-genome sequencing. The antimicrobial resistance, virulence, blaSHV-12 expression, and fitness costs in different media were examined. The amplification of blaSHV-12 was further investigated by qPCR and long-read sequencing. RESULTS: The 151 isolates showed a low MIC50/MIC90 (1/4 mg/L) of FDC. The seven isolates were ST11 PDR-hvKp, and two represented FDC-resistance (MIC=32 mg/L). The IncR/IncFII plasmids of two FDC-resistant isolates harbored 6 and 15 copies of blaSHV-12, whereas four FDC-susceptible isolates carried one copy and one harbored three copies. These blaSHV-12 genes concatenated together and were located within the same 7.3 kb fragment flanked by IS26, which contributed to the increased expression and FDC resistance without fitness costs. The amplification of blaSHV-12 and FDC resistance could be induced by FDC in vitro and reversed during continuous passage. CONCLUSIONS: The amplification of blaSHV-12 and the consequent dynamic within-host heteroresistance are important concerns for the rational application of antibiotics. Long-read sequencing might be a superior way to detect resistance gene amplification rapidly and accurately.

ST218 Klebsiella pneumoniae became a high-risk clone for multidrug resistance and hypervirulence.

BACKGROUND: The occurrence of multidrug-resistant and hypervirulent Klebsiella pneumoniae (MDR-hvKp) worldwide poses a great challenge for public health. Few studies have focused on ST218 MDR-hvKp. METHODS: Retrospective genomic surveillance was conducted at the Peking University Third Hospital from 2017 and clinical information was obtained. To understand genomic and microbiological characteristics, antimicrobial susceptibility testing, plasmid conjugation and stability, biofilm formation, serum killing, growth curves and whole-genome sequencing were performed. We also assessed the clinical and microbiological characteristics of ST218 compared with ST23. RESULTS: A total of eleven ST218 Kp isolates were included. The most common infection type was lower respiratory tract infection (72.7%, 8/11) in our hospital, whereas ST23 hvKp (72.7%, 8/11) was closely associated with bloodstream infection. Notably, nosocomial infections caused by ST218 (54.5%, 6/11) was slightly higher than ST23 (36.4%, 4/11). All of the ST218 and ST23 strains presented with the virulence genes combination of iucA + iroB + peg344 + rmpA + rmpA2. Interestingly, the virulence score of ST218 was lower than ST23, whereas one ST218 strain (pPEKP3107) exhibited resistance to carbapenems, cephalosporins, ß-lactamase/inhibitors and quinolones and harbored an ~ 59-kb IncN type MDR plasmid carrying resistance genes including blaNDM-1, dfrA14 and qnrS1. Importantly, blaNDM-1 and qnrS1 were flanked with IS26 located within the plasmid that could successfully transfer into E. coli J53. Additionally, PEKP2044 harbored an ~ 41-kb resistance plasmid located within tetA indicating resistance to doxycycline. CONCLUSION: The emergence of blaNDM-1 revealed that there is great potential for ST218 Kp to become a high-risk clone for MDR-hvKp, indicating the urgent need for enhanced genomic surveillance.

Integrated optical rotation detection scheme for chip-scale atomic magnetometer empowered by silicon-rich SiNx metalens.

High-performance atomic magnetometers (AMs) rely on the measurement of optical rotation, which requires a set of bulky polarization optics that limit their applications in scenarios where portability and compactness are necessary. In this study, a miniaturized AM is constructed based on a cubic 87Rb vapor cell and monolithic metalens, which provides an integrated scheme to achieve optical rotation detection induced by the circular birefringence of polarized atoms. The designed metalens achieves polarization splitting with deflection angles of ±10∘ and focusing with efficiencies of approximately 30% for orthogonal linear polarizations. The sensitivity of our compact device is ∼30 fT/Hz1/2 with a dynamic range of around ±1.45 nT. We envision that the presented approach paves the way for the chip integration of emerging atomic devices, which are in demand for applications such as biomagnetic imaging and portable atomic gyroscopes.

Reflections on appropriately liberalizing ART for groups requiring special attention in China.

PURPOSE: The continuous advancement of assisted reproductive technologies (ART) and the evolving attitudes towards marriage and fertility among the general public have led to an increasing number of groups requiring special attention (GRSA) desiring to fulfill their reproductive needs through these technologies. These groups include single women (including single mothers without children), same-sex couples, and women in high-risk occupations, among others. The purpose of this paper is to explore the feasibility of appropriately liberalizing ART for GRSA. METHODS: This paper discusses the advantages of a moderate liberalization of ART for GRSA from two perspectives: a theoretical basis and a practical significance level. It also analyzes the current constraints on liberalizing ART and presents suggestions for moderate liberalization. RESULTS: The moderate liberalization of ART can provide technical support for respecting and realizing the reproductive freedom of GRSA, which has certain theoretical and practical significance. However, it is also subject to constraints. CONCLUSION: We call for government to keep pace with the times, based on the current stage of political, economic, and social development, to further recognize and protect citizens' reproductive rights, prioritize the practical needs of the public, and explore policies and regulations for gradually loosening the restrictions on ART for GRSA.

Fusion plasmid enhanced the endemic extensively drug resistant Klebsiella pneumoniae clone ST147 harbored blaOXA-48 to acquire the hypervirulence and cause fatal infection.

BACKGROUND: Klebsiella Pneumoniae (Kp) sequence type (ST) 147 has emerged globally and spread rapidly, particularly the extensively drug resistant (XDR) isolates. However, the infections caused by this subtype is rare reported in China for now. The clinical, microbiological and genomic characteristics are unclear. METHODS: A systemic retrospective study was conducted in a Chinese tertiary hospital. Clinical information of the infection cases was collected, and whole-genome sequencing and phenotypic experiments were performed on the ST147 isolates. The resistance and virulence genes were identified, and the plasmids harboring these genes were further studied. RESULTS: Six ST147 isolates from six patients among 720 available clincial Kp isolates were detected. Notably, two isolates, PEKP4035 and PEKP4265, represented both XDR and hypervirulence by acquiring blaOXA-48, blaCTX-M-15 and key virulence genes, iucA + rmpA2, representing no fitness cost and resulting fatal infection. Four of the six ST147 isolates presented with more nucleotide differences, whereas the PEKP4035 and PEKP4265 both isolated from the intensive care unit possessed 20 single nucleotide polymorphisms among one year, indicating the prolonged survive and transmission. Interestingly, the two isolates harbored the same fused plasmid composed of sul2 and iucA + rmpA2, which might be generated by recombination of a plasmid like KpvST101_OXA-48 with the pLVPK plasmid via IS26. Besides, two ~ 70 kb plasmids conferring multiple-drug resistance were also identified among the two isolates, which presented resistance genes including blaOXA-48, blaCTX-M-16, strA and strB. Interestingly, we reported that blaCTX-M-15, a common resistance gene within ST147, has successfully transferred into the chromosome by ISEcp1. CONCLUSIONS: XDR hypervirulent ST147 Kp is emerging, suggesting enhanced surveillance is essential.

[Application and Prospect of Nanopore Sequencing Technology in Etiological Diagnosis of Blood Stream Infection].

Blood stream infection (BSI),a blood-borne disease caused by microorganisms such as bacteria,fungi,and viruses,can lead to bacteremia,sepsis,and infectious shock,posing a serious threat to human life and health.Identifying the pathogen is central to the precise treatment of BSI.Traditional blood culture is the gold standard for pathogen identification,while it has limitations in clinical practice due to the long time consumption,production of false negative results,etc.Nanopore sequencing,as a new generation of sequencing technology,can rapidly detect pathogens,drug resistance genes,and virulence genes for the optimization of clinical treatment.This paper reviews the current status of nanopore sequencing technology in the diagnosis of BSI.

Role of the fsr Quorum-Sensing System in Enterococcus faecalis Bloodstream Infection.

Enterococcus faecalis is an important intestinal colonizing bacteria and can cause various tissue infections, including invasive blood infection (BI). The annual incidence of E. faecalis BI has been estimated to be ~4.5 per 100,000, with a fatality rate that can reach 20%. However, whether bacterial colonization or invasive infections are tissue based has not been thoroughly studied. In this study, we analyzed 537 clinical isolates from 7 different tissues to identify the key genomic elements that facilitate the colonization and invasive infection of E. faecalis. Comparative genomic analysis revealed that the BI E. faecalis isolates had the largest genome size but the lowest GC content, fsr quorum-sensing system genes were enriched in the BI E. faecalis, and the fsr gene cluster could enhance biofilm formation and serum resistance ability. Our findings also provide deep insight into the genomic differences between different tissue isolates, and the fsr quorum-sensing systems could be a key factor promoting E. faecalis invasion into the blood. IMPORTANCE First, we conducted an advanced study on the genomic differences between colonizing and infecting E. faecalis, which provides support and evidence for early and accurate diagnoses. Second, we discovered that fsr was significantly associated with blood infections, which also provides additional information for studies exploring the invasiveness of E. faecalis. Most importantly, we found that fsr played an important role in both biofilm formation and serum resistance ability in E. faecalis.

Transverse light-shift in a spin-exchange relaxation-free co-magnetometer: measurement, decoupling, and suppression.

The transverse light-shift can induce non-negligible polarization error in the output signal of spin-exchange relaxation-free (SERF) co-magnetometer. In this paper, a novel method for rapid measurement of transverse light-shift based on the error of steady-state response of co-magnetometer is proposed firstly, then the sources of transverse light-shift in a compact SERF co-magnetometer is modeled and analyzed from three aspects: the non-ideal linear polarization of probe laser, the circular dichroism of the atomic spin ensembles, and the stress-induced birefringence effect of the cell wall. Furthermore, the decoupling and suppression methods of transverse light-shift based on a degree of circular polarization (DOCP) regulation scheme is presented, to realize the decoupling measurement of the transverse light-shift introduced by the whole co-magnetometer cell, and cancel it out with the non-ideal linear polarization of the probe laser. Eventually, the DOCP regulation scheme suggested in this paper achieves more than a 67% suppression ratio in transverse light-shift, and the short- and long-term performance of SERF co-magnetometer are improved due to the reduction of the coupling effect between the probe laser power and transverse field. Moreover, the measurement, decoupling and suppression methods provided in this paper also have the potential to be applied to other atomic sensors, such as the SERF magnetometers and nuclear spin co-magnetometers.

HCMV-miR-US33-5p promotes apoptosis of aortic vascular smooth muscle cells by targeting EPAS1/SLC3A2 pathway.

BACKGROUND: In patients with acute aortic dissection (AAD), increased vascular smooth muscle cell (VSMC) apoptosis has been found. Human cytomegalovirus (HCMV)-miR-US33-5p was significantly increased in the plasma of patients with AAD. However, the roles of miR-US33-5p in human aortic VSMC (HA-VSMC) apoptosis remain to be elucidated. METHODS: In the current study, cell apoptosis was analyzed by flow cytometry, cell proliferation by CCK-8 assay, and differentially expressed genes by RNA sequencing. Luciferase reporter assay was used for binding analysis between miR-US33-5p and endothelial PAS domain protein 1 (EPAS1), and EPAS1 and amino acid transporter heavy chain, member 2 (SLC3A2). The enrichment degree of SLC3A2 promoter DNA was analyzed by chromatin immunoprecipitation assay. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and immunoblotting were performed for measuring messenger RNA (mRNA) and protein levels, respectively. RESULTS: It was found that HCMV infection inhibited proliferation but promoted HA-VSMC apoptosis by upregulating HCMV-miR-US33-5p. Transfection of HCMV-miR-US33-5p mimics the significant effect on several signaling pathways including integrin signaling as shown in the RNA sequencing data. Western blotting analysis confirmed that HCMV-miR-US33-5p mimics suppression of the activity of key factors of the integrin signal pathway including FAK, AKT, CAS, and Rac. Mechanistic study showed that HCMV-miR-US33-5p bound to the 3'-untranslated region of EPAS1 to suppress its expression, leading to suppression of SLC3A2 expression, which ultimately promoted cell apoptosis and inhibited cell proliferation. This was confirmed by the findings that silencing EPAS1 significantly reduced the SLC3A2 expression and inhibited proliferation and key factors of integrin signal pathway. CONCLUSIONS: HCMV-miR-US33-5p suppressed proliferation, key factors of integrin signal pathway, and EPAS1/SLC3A2 expression, but promoted HA-VSMC apoptosis. These findings highlighted the importance of HCMV-miR-US33-5p/EPAS1/SCL3A2 signaling and may provide new insights into therapeutic strategies for AAD.

Chromatin accessibility landscapes of immune cells in rheumatoid arthritis nominate monocytes in disease pathogenesis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease that involves a variety of cell types. However, how the epigenetic dysregulations of peripheral immune cells contribute to the pathogenesis of RA still remains largely unclear. RESULTS: Here, we analysed the genome-wide active DNA regulatory elements of four major immune cells, namely monocytes, B cells, CD4+ T cells and CD8+ T cells, in peripheral blood of RA patients, osteoarthritis (OA) patients and healthy donors using Assay of Transposase Accessible Chromatin with sequencing (ATAC-seq). We found a strong RA-associated chromatin dysregulation signature in monocytes, but no other examined cell types. Moreover, we found that serum C-reactive protein (CRP) can induce the RA-associated chromatin dysregulation in monocytes via in vitro experiments. And the extent of this dysregulation was regulated through the transcription factor FRA2. CONCLUSIONS: Together, our study revealed a CRP-induced pathogenic chromatin dysregulation signature in monocytes from RA patients and predicted the responsible signalling pathway as potential therapeutic targets for the disease.

Specific Redistribution of Severe Acute Respiratory Syndrome Coronavirus 2 Variants in the Respiratory System and Intestinal Tract.

Intrahost analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic sequences identified 2 viral haplotypes comprised of 3 genetically linked mutations from the respiratory and intestinal tracts of a patient with coronavirus disease 2019. Spatiotemporal data suggest that this patient initially had dual infection of 2 SARS-CoV-2 variants, which subsequently redistributed into the 2 systems.

Broad and Differential Animal Angiotensin-Converting Enzyme 2 Receptor Usage by SARS-CoV-2.

The COVID-19 pandemic has caused an unprecedented global public health and economic crisis. The origin and emergence of its causal agent, SARS-CoV-2, in the human population remains mysterious, although bat and pangolin were proposed to be the natural reservoirs. Strikingly, unlike the SARS-CoV-2-like coronaviruses (CoVs) identified in bats and pangolins, SARS-CoV-2 harbors a polybasic furin cleavage site in its spike (S) glycoprotein. SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as its receptor to infect cells. Receptor recognition by the S protein is the major determinant of host range, tissue tropism, and pathogenesis of coronaviruses. In an effort to search for the potential intermediate or amplifying animal hosts of SARS-CoV-2, we examined receptor activity of ACE2 from 14 mammal species and found that ACE2s from multiple species can support the infectious entry of lentiviral particles pseudotyped with the wild-type or furin cleavage site-deficient S protein of SARS-CoV-2. ACE2 of human/rhesus monkey and rat/mouse exhibited the highest and lowest receptor activities, respectively. Among the remaining species, ACE2s from rabbit and pangolin strongly bound to the S1 subunit of SARS-CoV-2 S protein and efficiently supported the pseudotyped virus infection. These findings have important implications for understanding potential natural reservoirs, zoonotic transmission, human-to-animal transmission, and use of animal models.IMPORTANCE SARS-CoV-2 uses human ACE2 as a primary receptor for host cell entry. Viral entry mediated by the interaction of ACE2 with spike protein largely determines host range and is the major constraint to interspecies transmission. We examined the receptor activity of 14 ACE2 orthologs and found that wild-type and mutant SARS-CoV-2 lacking the furin cleavage site in S protein could utilize ACE2 from a broad range of animal species to enter host cells. These results have important implications in the natural hosts, interspecies transmission, animal models, and molecular basis of receptor binding for SARS-CoV-2.

Molecular characterization of two novel NDM-1-producing atypical enteroaggregative Escherichia coli isolates from patients.

To investigate NDM-1-producing atypical Enteroaggregative Escherichia coli (aEAEC) of sequence type 349 from hospitalized patients, the isolates 13ZX28 and 13ZX36 were subjected to antimicrobial susceptibility testing, conjugation and whole genome sequencing. Only one single nucleotide mutation was detected in chromosomes despite different plasmid profiles. Both isolates were positive for blaNDM-1 mediating resistance to carbapenem. A novel plasmid p13ZX28-272 (~272-kb) from 13ZX28 encodes blaNDM-1. Interestingly, its sequence was identical to the two plasmids p13ZX36-200 (~200-kb) and p13ZX36-70 (~70-kb) from 13ZX36. Formation of the former episome possibly involved homologous recombination through a 4948-bp large fragment located on each of the two latter plasmids. Furthermore, plasmid p13ZX28-272 could be resolved into a ~ 98-kb daughter plasmid by IS26 rearrangement following conjugation. The plasticity of the plasmids is recognized, which warrants further investigation to evaluate the underlying public health risk and understand how antibiotic selection pressure drives this process.

The crosstalk network of XIST/miR-424-5p/OGT mediates RAF1 glycosylation and participates in the progression of liver cancer.

BACKGROUND: Liver cancer is a major public health concern, but the mechanistic actions of biomarkers contributing to liver cancer remain to be determined. In this study, we aimed to investigate the regulatory cascade of microRNA-424-5p (miR-424-5p), X-inactive-specific transcript (XIST) and O-GlcNAc transferase (OGT) in liver cancer. METHODS: Differentially expressed miRNAs and target genes related to liver cancer were predicted by bioinformatics analyses, and their expression was determined in liver tissues of patients with liver cancer and liver cancer cells. The RNA immunoprecipitation (RIP), RNA pull-down and dual luciferase reporter assay were used to examine the binding affinity among XIST and miR-424-5p and OGT. Then, gain- and loss-of-function assays were conducted to evaluate the effects of the XIST/miR-424-5p/OGT axis on malignant phenotypes. A nude mouse model of liver cancer was further established for in vivo substantiation. RESULTS: XIST and OGT were up-regulated in liver cancer tissues and cells, responsible for poor prognosis in patients with liver cancer, while miR-424-5p was down-regulated. XIST competitively bound to miR-424-5p to increase OGT expression. XIST silencing inhibited malignant phenotypes of liver cancer cells, while miR-424-5p down-regulation negated its effect. miR-424-5p suppressed RAF1 glycosylation by negatively regulating OGT expression and promoted its ubiquitination/degradation. Furthermore, XIST knockdown inhibited tumour growth and metastasis in nude mice, while ectopic OGT reversed its effect. CONCLUSION: These results reveal a novel mechanism by which the interaction of XIST/miR-424-5p/OGT participates in the malignancy and metastasis of liver cancer.

Silver-catalyzed decarboxylative radical allylation of α,α-difluoroarylacetic acids for the construction of CF2-allyl bonds.

An efficient silver-catalyzed method of decarboxylative radical allylation of α,α-difluoroarylacetic acids to build CF2-allyl bonds has been developed. Using allylsulfone as an allyl donor, α,α-difluorine substituted arylacetic acids bearing various functional groups are successfully allylated to access a series of 3-(α,α-difluorobenzyl)-1-propylene compounds in moderate to excellent yields in aqueous CH3CN solution under the mild conditions. Experimental studies disclosed that the α-fluorine substitution of arylacetic acid has a great influence on free radical activity and reactivity.

Prevalence of elder abuse and victim-related risk factors during the COVID-19 pandemic in China.

BACKGROUND: With the accelerated aging of the Chinese population, elder abuse has become a serious social problem. As COVID-19 has had a very large impact on economic development and lifestyle in China, it has also affected elder abuse. The purpose of this study is to estimate the prevalence of elder abuse in China during the COVID-19 pandemic, and to identify changes in risk factors for elder abuse in the context of COVID-19. METHODS: We designed a cross-sectional study. In Hunan Province, a face-to-face questionnaire survey was conducted among elderly people over 65 years of age. To ensure the consistency of the measurement standards, we used the elder abuse questionnaire from the "Third Survey on Chinese Women's Social Status." According to related research, we selected 10 victim-related risk factors as independent variables. A logistic regression model was established to analyze the relationship between the independent variables and the four kinds of abuse. RESULTS: We collected 10,362 samples from Hunan Province. During the COVID-19 pandemic, the prevalence of financial abuse and neglect was significantly higher than that in 2010. Income had a significant impact on the four types of abuse. The lower the income was, the greater the risk of abuse. Moreover, factors such as an older age, being a woman, a lower cognitive ability, and not having a cohabiting spouse increased the possibility of abuse. The greater the number of children was, the greater the risks of physical abuse, financial abuse, and elder neglect. Seniors with higher education levels, those who frequently participated in social activities, and those with religious beliefs were less likely to suffer abuse. CONCLUSIONS: During the COVID-19 epidemic, the prevalence of elder abuse in China has increased, which may be related to economic instability and social distancing measures. Increasing the income of the elderly and giving them more social support are important measures to reduce the prevalence of elder abuse.

Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli Cultured from Cattle Farms in Xinjiang Uygur Autonomous Region, China, During 2016-2017.

Most outbreaks of Shiga toxin-producing Escherichia coli (STEC) are attributed to consumption of contaminated foodstuffs including beef and dairy products. In this study, we evaluated the prevalence of non-O157 STEC cultured from beef and dairy cattle and collected in Xinjiang Uygur Autonomous Region in China. Results identified 67 non-O157 STEC recovered from the 793 samples including beef cattle (10.28%, 43/418) and dairy cattle (6.40%, 24/375). A total of 67 non-O157 STEC was sequenced allowing for in silico analyses of their serotypes, virulence genes, and identification of the corresponding multilocus sequence types (STs). Twenty-one O serogroups and nine H serotypes were identified and the dominant serotype identified was O22:H8. One stx1 subtype (stx1a) and four stx2 subtypes (2a, 2b, 2c, and 2d) were found in the 67 non-O157 STEC isolates. The results revealed that stx1a+stx2a-positive STEC isolates were predominant (32.83%, 22/67), followed by stx1a+stx2d (29.85%, 20/67) and stx2a alone (17.91%, 12/67). Non-O157 STEC isolates carried virulence genes ehxA (98.51%), subA (53.73%), and cdtB (17.91%). Of the four adherence-associated genes tested, eaeA was absent, whereas lpfA and iha were present in 67 and 55 non-O157 STEC isolates, respectively. The STEC isolates were divided into 48 pulsed-field gel electrophoresis patterns and 10 STs, and ST446 (O22:H8) was the dominant clone (22.38%). Our results revealed that there was a high genetic diversity among non-O157 STEC isolated from beef and dairy cattle, some of which have potential to cause human diseases.

Polymeric Membrane Electrodes Using Calix[4]pyrrole Bis/Tetra-Phosphonate Cavitands as Ionophores for Potentiometric Acetylcholine Sensing with High Selectivity.

A handful of bis/tetra-phosphonate calix[4]pyrroles with recognition sites embedding in hydrophobic cavitands were evaluated for the first time as ionophores for polymeric membrane Ach+-selective electrodes. Highly selective potentiometric Ach+ could be achieved over its analogues, especially for Ch+, which differs only by an acetate tail from Ach+. The superior performance of the proposed ISEs might be ascribed to a dual-site binding mode, in which the trimethylammonium head and acetate tail were accommodated by the phosphonate group-bridged aryl walls and the bowl-shaped aromatic cavity, through cation-π/charge-dipole interaction and the convergent four N-H···O hydrogen bonds, respectively. To gain more insight into the performance of the proposed ISEs, the cation-ionophore complex constants in the membrane phase were determined, and the binding affinity trend correlates well with the selectivity pattern. These results suggest that conformational preorganization of the ionophores and complementary weak interactions do change the selectivity of the ionophores. Studies on the influence of the sample solution pH demonstrated that the developed ISEs can be employed in a wide pH range of 4.0-9.6 with a fast response (<60 s), good reversibility, and long lifetime. Optimizing the membrane components, such as ionophores, lipophilic additives, and plasticizers, yielded ISEs, showing Nernstian responses to Ach+ with improved linear ranges and detection limits (a slope of -59.5 mV/dec in the linear range of 1 × 10-6-1 × 10-2 M with a detection limit of 3 × 10-7 M), which led to the success of the determination of Ach+ in spiked urine and milk samples.

SMAtool reveals sequences and structural principles of protein-RNA interaction.

Protein binding events on RNA are highly related to RNA secondary structure, which affects post-transcriptional regulation and translation. However, it remains challenging to describe the association between RNA secondary structure and protein binding events. Here, we present Structure Motif Analysis tool (SMAtool), a pipeline that integrates RNA secondary structure and protein binding site information to profile the binding structure preference of each protein. As an example of applying SMAtool, we extracted the RNA-structure and binding site information respectively from the DMS-seq and eCLIP-seq data of the K562 cell-line, and used SMAtool to analyze the structure motif of each RNA binding protein (RBP). This new approach provided results consistent with X-ray crystallography data from the protein data bank (PDB) database, demonstrating that it can help researchers investigate the structure preference of RBP, and understand the role of RNA secondary structure in gene expression. Availability and implementation: https://github.com/QuKunLab/SMAtool.