Heterogeneity of chemical modifications on RNA.

The chemical modifications of RNAs broadly impact almost all cellular events and influence various diseases. The rapid advance of sequencing and other technologies opened the door to global methods for profiling all RNA modifications, namely the "epitranscriptome." The mapping of epitranscriptomes in different cells and tissues unveiled that RNA modifications exhibit extensive heterogeneity, in type, amount, and in location. In this mini review, we first introduce the current understanding of modifications on major types of RNAs and the methods that enabled their discovery. We next discuss the tissue and cell heterogeneity of RNA modifications and briefly address the limitations of current technologies. With much still remaining unknown, the development of the epitranscriptomic field lies in the further developments of novel technologies.

Detection of m6A from direct RNA sequencing using a multiple instance learning framework.

RNA modifications such as m6A methylation form an additional layer of complexity in the transcriptome. Nanopore direct RNA sequencing can capture this information in the raw current signal for each RNA molecule, enabling the detection of RNA modifications using supervised machine learning. However, experimental approaches provide only site-level training data, whereas the modification status for each single RNA molecule is missing. Here we present m6Anet, a neural-network-based method that leverages the multiple instance learning framework to specifically handle missing read-level modification labels in site-level training data. m6Anet outperforms existing computational methods, shows similar accuracy as experimental approaches, and generalizes with high accuracy to different cell lines and species without retraining model parameters. In addition, we demonstrate that m6Anet captures the underlying read-level stoichiometry, which can be used to approximate differences in modification rates. Overall, m6Anet offers a tool to capture the transcriptome-wide identification and quantification of m6A from a single run of direct RNA sequencing.

Co-existent abdominoperitoneal tuberculosis with endometrial cancer: A diagnostic and surgical challenge.

The presence of abdominoperitoneal tuberculosis (APTB) complicates the diagnosis, staging and management of endometrial cancer. Lymph node involvement in APTB may mimic metastatic lymphadenopathy in patients with endometrial cancer. To our knowledge, there have only been 2 previous case reports on this topic. We will describe 3 cases of endometrial cancer co-existing with APTB. The 1st case is a 57-year-old female who underwent elective total laparoscopic hysterectomy with bilateral salpingo-oophorectomy (TLHBSO) and bilateral pelvic lymph node dissection (PLND). The final diagnosis is Stage 3C1 endometrial endometroid carcinoma with mucinous differentiation. The 2nd case is a 70-year-old female with who underwent total abdominal hysterectomy with bilateral salpingo-oophorectomy (TAHBSO) and PLND. The final diagnosis is a Stage 1A endometrioid adenocarcinoma. The 3rd case is a 63-year-old female who underwent TAHBSO and PLND and the final diagnosis was a mixed high-grade serous (90%) and endometrioid (10%) carcinoma of the endometrium. In these cases, the importance of surgical staging is emphasised to accurately stage endometrial cancer. Moreover, thorough peri-operative optimisations by a multi-disciplinary team are essential to improve the outcomes of surgery.

Identification of differential RNA modifications from nanopore direct RNA sequencing with xPore.

RNA modifications, such as N6-methyladenosine (m6A), modulate functions of cellular RNA species. However, quantifying differences in RNA modifications has been challenging. Here we develop a computational method, xPore, to identify differential RNA modifications from nanopore direct RNA sequencing (RNA-seq) data. We evaluate our method on transcriptome-wide m6A profiling data, demonstrating that xPore identifies positions of m6A sites at single-base resolution, estimates the fraction of modified RNA species in the cell and quantifies the differential modification rate across conditions. We apply xPore to direct RNA-seq data from six cell lines and multiple myeloma patient samples without a matched control sample and find that many m6A sites are preserved across cell types, whereas a subset exhibit significant differences in their modification rates. Our results show that RNA modifications can be identified from direct RNA-seq data with high accuracy, enabling analysis of differential modifications and expression from a single high-throughput experiment.

Corrigendum to "Co-existent abdominoperitoneal tuberculosis with endometrial cancer: A diagnostic and surgical challenge" [Gynecol. Oncol. Rep. 37 (2021) 100848].

[This corrects the article DOI: 10.1016/j.gore.2021.100848.].

METTL4 catalyzes m6Am methylation in U2 snRNA to regulate pre-mRNA splicing.

N 6-methylation of 2'-O-methyladenosine (Am) in RNA occurs in eukaryotic cells to generate N6,2'-O-dimethyladenosine (m6Am). Identification of the methyltransferase responsible for m6Am catalysis has accelerated studies on the function of m6Am in RNA processing. While m6Am is generally found in the first transcribed nucleotide of mRNAs, the modification is also found internally within U2 snRNA. However, the writer required for catalyzing internal m6Am formation had remained elusive. By sequencing transcriptome-wide RNA methylation at single-base-resolution, we identified human METTL4 as the writer that directly methylates Am at U2 snRNA position 30 into m6Am. We found that METTL4 localizes to the nucleus and its conserved methyltransferase catalytic site is required for U2 snRNA methylation. By sequencing human cells with overexpressed Mettl4, we determined METTL4's in vivo target RNA motif specificity. In the absence of Mettl4 in human cells, U2 snRNA lacks m6Am thereby affecting a subset of splicing events that exhibit specific features such as 3' splice-site weakness and an increase in exon inclusion. These findings suggest that METTL4 methylation of U2 snRNA regulates splicing of specific pre-mRNA transcripts.

Atlas of quantitative single-base-resolution N6-methyl-adenine methylomes.

Various methyltransferases and demethylases catalyse methylation and demethylation of N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) but precise methylomes uniquely mediated by each methyltransferase/demethylase are still lacking. Here, we develop m6A-Crosslinking-Exonuclease-sequencing (m6ACE-seq) to map transcriptome-wide m6A and m6Am at quantitative single-base-resolution. This allows for the generation of a comprehensive atlas of distinct methylomes uniquely mediated by every individual known methyltransferase or demethylase. Our atlas reveals METTL16 to indirectly impact manifold methylation targets beyond its consensus target motif and highlights the importance of precision in mapping PCIF1-dependent m6Am. Rather than reverse RNA methylation, we find that both ALKBH5 and FTO instead maintain their regulated sites in an unmethylated steady-state. In FTO's absence, anomalous m6Am disrupts snRNA interaction with nuclear export machinery, potentially causing aberrant pre-mRNA splicing events.

Frontal Assessment Battery in Early Cognitive Impairment: Psychometric Property and Factor Structure.

BACKGROUND: The Frontal Assessment Battery (FAB) is a reliable and valid bedside tool for testing executive function in dementia. Given the increasing interest in utility of FAB as a screening tool in early cognitive impairment (ECI), there is a surprising lack of studies evaluating its psychometric property and factor structure, nor the influence of factors such as age, education and gender, in ECI. OBJECTIVES: This study aims to investigate the psychometric properties and factor structure of FAB in older adults with ECI, as well as the influence of age, gender and education. DESIGN, SETTING AND PARTICIPANTS: This is a retrospective, observational cross-sectional study with 300 community dwelling, predominantly Chinese older adults (14 normal, 130 mild cognitive impairment (MCI), and 156 mild dementia) who presented to Memory Clinic from January 2011 to December 2013. Measurements and Analysis: We collected data on demographic, cognitive, functional and behavioral evaluation. To examine the psychometric properties of FAB, we examined the concurrent, convergent, and discriminant validity; internal consistency by Cronbach's alpha; and factor structure by exploratory factor analysis. The influence of age, education and gender was examined using unadjusted and adjusted correlational analyses with CDR-SOB. We performed analysis for the whole group and for MCI subgroup. RESULTS: FAB total score decreases significantly from normal to dementia group attesting to concurrent validity. It correlated significantly with digit span backwards and Chinese Mini Mental State Examination (r=0.38 and 0.47 respectively, p<0.01) and poorly with Neuropsychiatric Inventory-Questionnaire and depression (r=0.004 and -0.02 respectively), supporting its convergent and discriminant validity. Factor analysis yielded a single-factor solution for FAB with fair Internal consistency (alpha=0.610). FAB is relatively unaffected by age, gender and education level. These good psychometric properties extend to MCI, albeit with greater influence by education level. FAB items of conceptualization and mental flexibility have good discriminatory ability between MCI and normal subjects. CONCLUSION: FAB has good concurrent, convergent and discriminant validity with fair internal consistency in ECI that is premised on a one-factor structure. It is relatively unaffected by age, gender or education. Taken together, FAB is a useful bedside screening tool for executive function in ECI.

The aminopeptidase inhibitor, z-L-CMK, is toxic and induces cell death in Jurkat T cells through oxidative stress.

The leucine aminopeptidase inhibitor, benzyloxycarbonyl-leucine-chloromethylketone (z-L-CMK), was found to be toxic and readily induce cell death in Jurkat T cells. Dose-response studies show that lower concentration of z-L-CMK induced apoptosis in Jurkat T cells whereas higher concentration causes necrosis. In z-L-CMK-induced apoptosis, both the initiator caspases (-8 and -9) and effector caspases (-3 and -6) were processed to their respective subunits. However, the caspases remained intact in z-L-CMK-induced necrosis. The caspase inhibitor, z-VAD-FMK inhibited z-L-CMK-mediated apoptosis and caspase processing but has no effect on z-L-CMK-induced necrosis in Jurkat T cells. The high mobility group protein B1 (HMGB1) protein was found to be released into the culture medium by the necrotic cells and not the apoptotic cells. These results indicate that the necrotic cell death mediated by z-L-CMK at high concentrations is via classical necrosis rather than secondary necrosis. We also demonstrated that cell death mediated by z-L-CMK was associated with oxidative stress via the depletion of intracellular glutathione (GSH) and increase in reactive oxygen species (ROS), which was blocked by N-acetyl cysteine. Taken together, the results demonstrated that z-L-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. The toxic side effects in Jurkat T cells mediated by z-L-CMK are associated with oxidative stress via the depletion of GSH and accumulation of ROS.

Detergent Sclerosants Stimulate Leukocyte Apoptosis and Oncosis.

OBJECTIVE/BACKGROUND: The objective was to investigate the effects of the detergent sclerosants sodium tetradecyl sulfate (STS) and polidocanol (POL) on human leukocytes at sublytic concentrations. METHODS: Leukocytes were isolated and labelled with antibodies to assess for apoptosis and examined with confocal microscopy and flow cytometry. Isolated leukocyte count and viability was assessed using trypan blue, and propidium iodide staining. Phosphatidylserine (PS) exposure, a universal hallmark to measure cell apoptosis, was identified by flow cytometry using lactadherin. Caspases 3, 8, and 9, and Bax activation, as well as inhibitory assays with pan-caspase (Z-VAD-FMK) and Bax (BI-6C9) were assessed to determine apoptotic pathways. Porimin activation was used to assess cell permeability. RESULTS: Up to 40% of leukocytes maintained membrane integrity at sublytic concentrations (≤0.15%) of sclerosants. The remaining 60% did not maintain membrane integrity but were not completely lysed. PS exposure was increased with both STS and POL exhibiting a dose- and time-dependant trend. While activation of caspases 3, 8, and 9, as well as Bax activation, were increased in leukocytes stimulated with low concentrations of STS, only caspases 3 and 9 and Bax were increased with POL. Inhibitory assays demonstrated caspases 3, 8, and 9, and Bax inhibition at low concentrations with both STS and POL. Both agents increased the leukocyte activation of porimin at all concentrations. On confocal microscopy, stains for caspases 3, 8, and 9, and Bax were increased for both STS and POL. Porimin stain was markedly positive for both STS and POL. CONCLUSION: Both sclerosants induced leukocyte apoptosis at sublytic concentrations. STS activated both extrinsic and intrinsic pathways of apoptosis, while POL stimulated the intrinsic pathway of apoptosis only. Both agents induced oncosis. Based on these results, STS appears to have a greater effect than POL.

Detergent sclerosants are deactivated and consumed by circulating blood cells.

OBJECTIVE: To investigate the deactivating effects of circulating blood cells on the lytic activity of detergent sclerosants. METHODS: Samples of whole blood (WB), platelet-rich plasma (PRP), and isolated leukocytes were incubated with various concentrations of sodium tetradecyl sulfate (STS) or polidocanol (POL) and added to human umbilical vein endothelial cells (HUVECs), which were then counted using a fluorescent plate reader. Full blood counting was performed using a hematology analyzer. Platelet lysis and microparticle formation was assessed using lactadherin binding in flow cytometry. RESULTS: Detergent sclerosant activity was decreased in WB when compared with plasma and saline controls. The sclerosant lytic activity on endothelial cells was increased 23-fold for STS and 59-fold for POL in saline controls compared with WB. At high concentrations, sclerosants lysed erythrocytes, leukocytes, and platelets. Platelets were more sensitive to the lytic activity of sclerosants than other cell types. Neutrophils were the most susceptible of all leukocytes to the lytic activity of sclerosants. The presence of erythrocytes and leukocytes in samples decreased the lytic activity of sclerosants. Sclerosants at all concentrations induced erythrocyte-derived microparticle formation. CONCLUSIONS: Detergent sclerosants are consumed and deactivated by circulating blood cells. This deactivating effect is above and beyond the neutralizing effects of plasma proteins and contributes to the overall neutralizing effect of blood. Different blood cell types exhibited varying levels of vulnerability to the lytic activity of sclerosants with platelets being the most and erythrocytes the least vulnerable (platelets > leukocytes > erythrocytes).

Label-free electronic detection of bio-toxins using aligned carbon nanotubes.

A facile route for sensitive label-free detection of bio-toxins using aligned single walled carbon nanotubes is described. This approach involves patterning of a catalyst on the surface of a quartz substrate using a sub-100 µm stripe-patterned polydimethylsiloxane stamp for aligned carbon nanotube generation followed by fabrication of field effect transistor (FET). Atomic force microscopy, field emission scanning electron microscopy and Raman spectroscopy are employed to characterize the synthesized nanotubes. Unlike previous reports, the adopted approach enables direct electronic detection of bio-toxins with sensitivities comparable to ELISA. As a proof of concept, the fabricated FET responds to nM concentration levels (with a LOD of ∼2 nM) of epsilon toxin produced by Clostridium perfringens and a prominent food toxin. This facile approach could be customized to detect other classes of toxins and biomarkers upon appropriate functionalization of the aligned carbon nanotubes. Finally, we demonstrate the use of the FET-platform for detection of toxin in more complex matrices such as orange juice.

Evaluation of family-centred practices in the early intervention programmes for infants and young children in Singapore with Measure of Processes of Care for Service Providers and Measure of Beliefs about Participation in Family-Centred Service.

BACKGROUND: The primary purpose of this study was to report on an evaluation of the perceptions and beliefs of service providers towards family-centred practices in 11 early intervention programmes for infants and young children in Singapore. METHODS: The Measure of Processes of Care for Service Providers (MPOC-SP) and Measure of Beliefs about Participation in Family-Centred Service (MBP-FCS) were administered to 213 service providers made up of teachers, therapists, psychologists and social workers providing centre-based therapy to children with special needs who were below the age of 6 years. RESULTS: Exploratory factor analyses were performed with both scales. Nineteen of the 27 MPOC-SP items were retained and supported the original four-factor structure model. The exploratory factor analyses on MBP-FCS provided a less satisfactory outcome. Fourteen of the 28 items were retained and these loaded onto four factors. The two factors relating to Beliefs about benefits of FCS and Beliefs about the absence of negative outcomes from FCS failed to emerge as separate factors. Further multiple regressions indicated that more direct work with families and positive self-efficacy in implementing FCS contributed significantly to explaining service providers' positive perception towards family-centred practice in service delivery. CONCLUSIONS: This is the first time MPOC-SP and MBP-FCS were administered to a population in an Asian context. While MBP-FCS would benefit from further development work on its construct, MPOC-SP offered important insights into service providers' perspectives about family-centred practices that would have useful implications for professional and service development.

Electrically tunable white-color electroluminescence from Si-implanted silicon nitride thin film.

Visible electroluminescence (EL) with two composite bands, i.e., a violet band and a green-yellow band has been observed from Si-implanted silicon nitride thin films. By varying the intensity ratio of the two composite EL bands in terms of the injection current, strong white-color EL can be achieved at certain injection currents (e.g., ~265 mA/cm(2)). The observed transition in EL color from violet to white under different injection conditions is studied based on the understanding that the violet band is originated from silicon nitride matrix while the green-yellow band is related to the implanted Si. The Si-implanted silicon nitride thin film offers the possibility of electrically tunable white-light Si-based light emitters.

Aligned carbon nanotubes on quartz substrate for liquid gated biosensing.

A facile and high performance biosensing platform using aligned carbon nanotubes on quartz substrate is reported in this communication. Single walled carbon nanotubes are grown on quartz substrates by a chemical vapor deposition process and are characterized with field emission scanning electron microscopy and atomic force microscopy in order to verify the quality of the material. The quartz substrate is then directly used as a biosensor in a field effect transistor configuration. In order to demonstrate the sensing capabilities of the fabricated sensor devices, electronic detection of prostate specific antigen, a potential cancer biomarker, is carried out by adopting liquid gated configuration. A conductivity change due to the specific binding of target antigen with the immobilized receptor antibody demonstrates the sensing capabilities of the fabricated device. Sub-nM detection sensitivities have been obtained using the adopted direct immunoassay approach, which shows that the device responds to clinically relevant concentration regimes.

Detection of the p53 response in zebrafish embryos using new monoclonal antibodies.

The zebrafish has many advantages as a vertebrate model organism and has been extensively used in the studies of development. Its potential as a model in which to study tumour suppressor and oncogene function is now being realized. Whilst in situ hybridization of mRNA has been well developed in this species to study gene expression, antibody probes are in short supply. We have, therefore, generated a panel of anti-zebrafish p53 monoclonal antibodies and used these to study the p53 response in zebrafish embryos. By immunohistochemistry, we show that the exposure of zebrafish embryos to p53-activating agents such as R-roscovitine and gamma-irradiation results in the accumulation of p53 protein in the gut epithelium, liver and pancreas. A combination of R-roscovitine and gamma-irradiation results in massive p53 induction, not only in the pharyngeal arches, gut region and liver but also in brain tissues. Induction of apoptosis and expression of p53 response genes are seen in regions that correspond to sites of p53 protein accumulation. In contrast, although zebrafish tp53(M214K) mutant embryos showed a similar accumulation of p53 protein, a complete lack of a downstream p53-dependent response was observed. In this system the p53 gene is identified as a p53-responsive gene itself. Our results demonstrate that zebrafish p53 protein can readily be induced in embryos and detected using these new antibody tools, which will increase the usefulness of zebrafish as a model in compound-based screening for novel drugs in cancer research.

Intraoperative cholangiography facilitates simple transcystic clearance of ductal stones in units without expertise for laparoscopic bile duct surgery.

BACKGROUND: In the absence of facilities and expertise for laparoscopic bile duct exploration (LBDE), most patients with suspected ductal calculi undergo preoperative endoscopic duct clearance. Intraoperative cholangiography (IOC) is not performed at the subsequent laparoscopic cholecystectomy. This study aimed to investigate the rate of successful duct clearance after simple transcystic manipulations. METHODS: This prospective study investigated 1,408 patients over 13 years in a unit practicing single-session management of biliary calculi. For the great majority, IOC was attempted. Abnormalities were dealt with by flushing of the duct, glucagon injection, Dormia basket trawling, choledochoscopic transcystic exploration, or choledochotomy. RESULTS: Of 1,056 cholangiograms performed (75%), 287 were abnormal (27.2%). Surgical trainees, operating under supervision, successfully performed 24% of all cholangiograms. Of 396 patients admitted with biliary emergencies, 94.1% had abnormal cholangiograms. Of the 287 patients with abnormal IOCs, 9.4% required no intervention, 18% were clear after glucagon and flushing, and 13% were cleared using Dormia basket trawling under fluoroscopy. A total of 95 patients required formal LBDE, and 2 required postoperative endoscopic retrograde cholangiopancreatography (ERCP). No postoperative ERCP for retained stones was required after simple transcystic manipulation. Eight conversions occurred, one during a transcystic exploration. Follow-up evaluation continued for as long as 6 years in some cases. Two patients had recurrent stones after LBDE and a clear postoperative tube cholangiogram. CONCLUSION: In this series, 10% of the abnormal cholangiograms occurred in patients without preoperative risk factors for bile duct stones. Altogether, 88 IOCs (31%) were cleared after either simple flushing or trawling with a Dormia basket. Formal LBDE was not required for 40% of abnormal cholangiograms. Simple transcystic manipulations to clear the bile ducts justify the use of routine IOC in units without laparoscopic biliary expertise.

Detection of betanodavirus in juvenile barramundi, Lates calcarifer (Bloch), by antigen capture ELISA.

Betanodavirus infection of fish has been responsible for mass mortalities in aquaculture hatcheries worldwide. Betanodaviruses possess a bipartite single-stranded RNA genome consisting of the 3.1 kb RNA1 encoding an RNA-dependent RNA polymerase and the B2 protein, while the 1.4 kb RNA2 encodes the viral nucleocapsid protein, alpha. A panel of six monoclonal antibodies against the alpha protein of greasy grouper nervous necrosis virus (GGNNV) was developed for use in diagnostics. All antibodies reacted with native and recombinant alpha in immunoblot and indirect immunofluorescence assays. Each of the monoclonal antibodies reacted against discrete regions of the alpha protein, though none reacted with the extreme C-terminal region of the protein. One of the monoclonal antibodies, specific for the K151-T246 region of alpha, was used for the development of an antigen capture ELISA. In this assay we could detect 10(3)-10(4) TCID(50) units of virus derived from infected tissue culture supernatants. Head tissue extracts prepared from experimentally infected barramundi, Lates calcarifer, juveniles were assayed for GGNNV using the antigen capture assay and a clear increase in alpha antigen was detected from 5 to 15 days post-challenge. The assay thus represents a useful method for field-based detection of betanodavirus in fish hatcheries.