Protocol for preparing Thiopropyl Sepharose resin used for capturing S-nitrosylated proteins.

S-nitrosothiol (SNO)-Resin Assisted Capture (SNO-RAC) relies on a Thiopropyl Sepharose resin to identify S-nitrosylated proteins (SNO-proteins) and sites of S-nitrosylation. Here, we present a protocol for preparing Thiopropyl Sepharose resin with efficiency of SNO-protein capture comparable to the discontinued commercial version. We describe steps for amine coupling, disulfide reduction, and generation of thiol reactive resin. We then detail quality control procedures. This resin is also suitable for Acyl-RAC assays to capture palmitoylated proteins. For complete details on the use and execution of the SNO-RAC protocol, please refer to Forrester et al.,1 Fonseca et al.,2 and Seth et al.3.

Data Science as a Core Competency in Undergraduate Medical Education in the Age of Artificial Intelligence in Health Care.

The increasingly sophisticated and rapidly evolving application of artificial intelligence in medicine is transforming how health care is delivered, highlighting a need for current and future physicians to develop basic competency in the data science that underlies this topic. Medical educators must consider how to incorporate central concepts in data science into their core curricula to train physicians of the future. Similar to how the advent of diagnostic imaging required the physician to understand, interpret, and explain the relevant results to patients, physicians of the future should be able to explain to patients the benefits and limitations of management plans guided by artificial intelligence. We outline major content domains and associated learning outcomes in data science applicable to medical student curricula, suggest ways to incorporate these themes into existing curricula, and note potential implementation barriers and solutions to optimize the integration of this content.

An optimized protocol for isolation of S-nitrosylated proteins from C. elegans.

Post-translational modification by S-nitrosylation regulates numerous cellular functions and impacts most proteins across phylogeny. We describe a protocol for isolating S-nitrosylated proteins (SNO-proteins) from C. elegans, suitable for assessing SNO levels of individual proteins and of the global proteome. This protocol features efficient nematode lysis and SNO capture, while protection of SNO proteins from degradation is the major challenge. This protocol can be adapted to mammalian tissues. For complete information on the generation and use of this protocol, please refer to Seth et al. (2019).

Electronic Data Capture Versus Conventional Data Collection Methods in Clinical Pain Studies: Systematic Review and Meta-Analysis.

BACKGROUND: The most commonly used means to assess pain is by patient self-reported questionnaires. These questionnaires have traditionally been completed using paper-and-pencil, telephone, or in-person methods, which may limit the validity of the collected data. Electronic data capture methods represent a potential way to validly, reliably, and feasibly collect pain-related data from patients in both clinical and research settings. OBJECTIVE: The aim of this study was to conduct a systematic review and meta-analysis to compare electronic and conventional pain-related data collection methods with respect to pain score equivalence, data completeness, ease of use, efficiency, and acceptability between methods. METHODS: We searched the Medical Literature Analysis and Retrieval System Online (MEDLINE), Excerpta Medica Database (EMBASE), and Cochrane Central Register of Controlled Trials (CENTRAL) from database inception until November 2019. We included all peer-reviewed studies that compared electronic (any modality) and conventional (paper-, telephone-, or in-person-based) data capture methods for patient-reported pain data on one of the following outcomes: pain score equivalence, data completeness, ease of use, efficiency, and acceptability. We used random effects models to combine score equivalence data across studies that reported correlations or measures of agreement between electronic and conventional pain assessment methods. RESULTS: A total of 53 unique studies were included in this systematic review, of which 21 were included in the meta-analysis. Overall, the pain scores reported electronically were congruent with those reported using conventional modalities, with the majority of studies (36/44, 82%) that reported on pain scores demonstrating this relationship. The weighted summary correlation coefficient of pain score equivalence from our meta-analysis was 0.92 (95% CI 0.88-0.95). Studies on data completeness, patient- or provider-reported ease of use, and efficiency generally indicated that electronic data capture methods were equivalent or superior to conventional methods. Most (19/23, 83%) studies that directly surveyed patients reported that the electronic format was the preferred data collection method. CONCLUSIONS: Electronic pain-related data capture methods are comparable with conventional methods in terms of score equivalence, data completeness, ease, efficiency, and acceptability and, if the appropriate psychometric evaluations are in place, are a feasible means to collect pain data in clinical and research settings.

Regulation of MicroRNA Machinery and Development by Interspecies S-Nitrosylation.

Bioactive molecules can pass between microbiota and host to influence host cellular functions. However, general principles of interspecies communication have not been discovered. We show here in C. elegans that nitric oxide derived from resident bacteria promotes widespread S-nitrosylation of the host proteome. We further show that microbiota-dependent S-nitrosylation of C. elegans Argonaute protein (ALG-1)-at a site conserved and S-nitrosylated in mammalian Argonaute 2 (AGO2)-alters its function in controlling gene expression via microRNAs. By selectively eliminating nitric oxide generation by the microbiota or S-nitrosylation in ALG-1, we reveal unforeseen effects on host development. Thus, the microbiota can shape the post-translational landscape of the host proteome to regulate microRNA activity, gene expression, and host development. Our findings suggest a general mechanism by which the microbiota may control host cellular functions, as well as a new role for gasotransmitters.

Email Between Patient and Provider: Assessing the Attitudes and Perspectives of 624 Primary Health Care Patients.

BACKGROUND: Email between patients and their health care providers can serve as a continuous and collaborative forum to improve access to care, enhance convenience of communication, reduce administrative costs and missed appointments, and improve satisfaction with the patient-provider relationship. OBJECTIVE: The main objective of this study was to investigate the attitudes of patients aged 16 years and older toward receiving email communication for health-related purposes from an academic inner-city family health team in Southern Ontario. In addition to exploring the proportion of patients with a functioning email address and interest in email communication with their health care provider, we also examined patient-level predictors of interest in email communication. METHODS: A cross-sectional study was conducted using a self-administered, 1-page survey of attitudes toward electronic communication for health purposes. Participants were recruited from attending patients at the McMaster Family Practice in Hamilton, Ontario, Canada. These patients were aged 16 years and older and were approached consecutively to complete the self-administered survey (N=624). Descriptive analyses were conducted using the Pearson chi-square test to examine correlations between variables. A logistic regression analysis was conducted to determine statistically significant predictors of interest in email communication (yes or no). RESULTS: The majority of respondents (73.2%, 457/624) reported that they would be willing to have their health care provider (from the McMaster Family Practice) contact them via email to communicate health-related information. Those respondents who checked their personal email more frequently were less likely to want to engage in this electronic communication. Among respondents who check their email less frequently (fewer than every 3 days), 46% (37/81) preferred to communicate with the McMaster Family Practice via email. CONCLUSIONS: Online applications, including email, are emerging as a viable avenue for patient communication. With increasing utility of mobile devices in the general population, the proportion of patients interested in email communication with their health care providers may continue to increase. When following best practices and appropriate guidelines, health care providers can use this resource to enhance patient-provider communication in their clinical work, ultimately leading to improved health outcomes and satisfaction with care among their patients.

Atypical presentation of traumatic aortic injury.

Background. Blunt thoracic aorta injury (BAI) is second only to head injury as cause of mortality in blunt trauma. While most patients do not survive till arrival at the hospital, for the remainder, prompt diagnosis and treatment greatly improve outcomes. We report an atypical presentation of BAI, highlighting the diagnostic challenges of this condition in the emergency department. Case Presentation. A previously well 25-year-old male presented 15 hours after injury hemodynamically stable with delirium. There were no signs or symptoms suggestive of BAI. Sonography showed small bilateral pleural effusions. Chest radiograph showed a normal mediastinum. Eventually, CT demonstrated a contained distal aortic arch disruption. The patient underwent percutaneous endovascular thoracic aortic repair and recovered well. Conclusion. This catastrophic lesion may present with few reliable signs and symptoms; hence, a high index of suspicion is crucial for early diagnosis and definitive surgical management. This paper discusses the diagnostic utility of clinical features, injury mechanism, and radiographic modalities. Consideration of mechanism of injury, clinical features, and chest radiograph findings should prompt advanced chest imaging.

Exertional heat stroke in a marathon runner with extensive healed deep burns: a case report.

Exertional heat illness typically occurs over hours in younger athletic patients or military recruits who exercise at elevated temperatures for a sufficient period of time to cause the rate of heat production to exceed the capacity of the body to dissipate heat. Since the physiological response to exercise includes cutaneous vasodilation and sweating, any limitation of such a response can cause rapid hyperthermia and thus heat stroke. One such condition is extensive burns healed by cicatrisation of the skin where the scar and grafted skin surface do not have functional sweat glands and are unable to lose heat in response to high temperatures. The authors report one unique case of a female marathon runner with exertional heat stroke who had recovered from deep second and third degree burns over approximately 50% of her body a few years ago.

Fox-2 protein regulates the alternative splicing of scleroderma-associated lysyl hydroxylase 2 messenger RNA.

OBJECTIVE: Scleroderma (systemic sclerosis [SSc]) is a complex connective tissue disorder characterized by hardening and thickening of the skin. One hallmark of scleroderma is excessive accumulation of collagen accompanied by increased levels of pyridinoline collagen crosslinks derived from hydroxylysine residues in the collagen telopeptide domains. Lysyl hydroxylase 2 (LH2), an important alternatively spliced enzyme in collagen biosynthesis, acts as a collagen telopeptide hydroxylase. Changes in the pattern of LH2 alternative splicing, favoring increased inclusion of the alternatively spliced LH2 exon 13A, thereby increasing the levels of the long transcript of LH2 (LH2[long]), are linked to scleroderma disease. This study was undertaken to examine the role played by RNA binding protein Fox-2 in regulating exon 13A inclusion, which leads to the generation of scleroderma-associated LH2(long) messenger RNA (mRNA). METHODS: Phylogenetic sequence analysis of introns flanking exon 13A was performed. A tetracycline-inducible system in T-Rex 293 cells was used to induce Fox-2 protein, and endogenous LH2(long) mRNA was determined by reverse transcriptase-polymerase chain reaction. An LH2 minigene was designed, validated, and used in Fox-2 overexpression and mutagenesis experiments. Knockdown of Fox-2 was performed in mouse embryonic fibroblasts and in fibroblasts from SSc patients. RESULTS: Overexpression of Fox-2 enhanced the inclusion of exon 13A and increased the generation of LH2(long) mRNA, whereas knockdown of Fox-2 decreased LH2(long) transcripts. Mutational analysis of an LH2 minigene demonstrated that 2 of the 4 Fox binding motifs flanking LH2 exon 13A are required for inclusion of exon 13A. In early passage fibroblasts derived from patients with scleroderma, the knockdown of Fox-2 protein significantly decreased the endogenous levels of LH2(long) mRNA. CONCLUSION: Our findings indicate that Fox-2 plays an integral role in the regulation of LH2 splicing. Knockdown of Fox-2 and other methods to decrease the levels of fibrosis-associated LH2(long) mRNA in primary scleroderma cells may suggest a novel approach to strategies directed against scleroderma.

Expression of cloned cDNAs in mammalian cells from a cryptic promoter upstream to T7 in pGEM-4Z cloning vector.

In this study, we demonstrate that pGEM-4Z can be used as a mammalian expression vector. Western blotting and Immunocytochemical analyses revealed that transfection of pGEM-4Z-containing human cathepsin L cDNA under T-7 but not under SP-6 promoter into NIH 3T3 cells resulted in a high-level expression of cathepsin L. Expression of proteins using this vector in mammalian cells was further confirmed by using luciferase reporter gene. Furthermore, NIH 3T3 cells after stable or transient transfection with pGEM-4Z containing the first exon, first intron, and rest of the human cathepsin L cDNA downstream to its T-7 promoter synthesized and secreted large quantities of cathepsin L. RNase protection assays and 5' RACE established that the cloned cathepsin L cDNA is transcribed from a cryptic promoter present in the backbone of this vector upstream to T-7 sequence. This promoter was active in cell lines derived from four different mammalian species. In NIH 3T3 cells, this cryptic promoter could transcribe structural part of the genomic DNA into a primary transcript, which was efficiently spliced into mature mRNA and translated into protein. Thus this vector is equally useful for expressing proteins from genomic DNA. This hitherto unknown property of pGEM-4Z may be useful for expression of proteins in mammalian cells besides its use in synthesis of riboprobes, DNA sequencing, and in vitro transcription coupled translation assays.

TIA nuclear proteins regulate the alternate splicing of lysyl hydroxylase 2.

Synthesis of collagen, a major component of the extracellular matrix, is increased dramatically in fibrotic conditions such as scleroderma. This overaccumulation of collagen is associated with increased pyridinoline cross-links. These cross-links are derived by the action of the alternatively spliced long form of lysyl hydroxylase 2 (LH2), a collagen telopeptide LH. As LH2 (long) is reported to be overexpressed in scleroderma fibroblasts, the regulation of LH2 splicing suggests an important step in controlling fibrosis. Using an LH2 minigene, we have compared the regulation of the alternative splicing pattern of LH2, both endogenously and in the minigene, by the RNA-binding splicing proteins TIA-1 and TIAL1 (T-cell-restricted intracellular antigens). A decrease in the ratio of LH2 (long) to LH2 (short) was observed in fibroblasts from TIAL1 knockout mice, and in HEK293 cells knocked down for TIA-1 and TIAL1. As a corollary, overexpression of TIA-1/TIAL1 in HEK293 cells resulted in an increase in LH2 (long) minigene transcripts, accompanied by a decrease in LH2 (short). In scleroderma fibroblasts, a double TIA-1/TIAL1 knockdown reduced the ratio of LH2 (long) to LH2 (short) by over fivefold compared to controls. Identification of these TIA regulatory factors therefore suggests a tool to manipulate cellular LH2 levels in scleroderma so that potential intervention therapies may be identified.

Identification of an intronic splicing enhancer essential for the inclusion of FGFR2 exon IIIc.

The ligand specificity of fibroblast growth factor receptor 2 (FGFR2) is determined by the alternative splicing of exons 8 (IIIb) or 9 (IIIc). Exon IIIb is included in epithelial cells, whereas exon IIIc is included in mesenchymal cells. Although a number of cis elements and trans factors have been identified that play a role in exon IIIb inclusion in epithelium, little is known about the activation of exon IIIc in mesenchyme. We report here the identification of a splicing enhancer required for IIIc inclusion. This 24-nucleotide (nt) downstream intronic splicing enhancer (DISE) is located within intron 9 immediately downstream of exon IIIc. DISE was able to activate the inclusion of heterologous exons rat FGFR2 IIIb and human beta-globin exon 2 in cell lines from different tissues and species and also in HeLa cell nuclear extracts in vitro. DISE was capable of replacing the intronic activator sequence 1 (IAS1), a known IIIb splicing enhancer and vice versa. This fact, together with the requirement for DISE to be close to the 5'-splice site and the ability of DISE to promote binding of U1 snRNP, suggested that IAS1 and DISE belong to the same class of cis-acting elements.

Interleukin 7 receptor alpha chain (IL7R) shows allelic and functional association with multiple sclerosis.

Multiple sclerosis is a demyelinating neurodegenerative disease with a strong genetic component. Previous genetic risk studies have failed to identify consistently linked regions or genes outside of the major histocompatibility complex on chromosome 6p. We describe allelic association of a polymorphism in the gene encoding the interleukin 7 receptor alpha chain (IL7R) as a significant risk factor for multiple sclerosis in four independent family-based or case-control data sets (overall P = 2.9 x 10(-7)). Further, the likely causal SNP, rs6897932, located within the alternatively spliced exon 6 of IL7R, has a functional effect on gene expression. The SNP influences the amount of soluble and membrane-bound isoforms of the protein by putatively disrupting an exonic splicing silencer.

Transcription of human cathepsin L mRNA species hCATL B from a novel alternative promoter in the first intron of its gene.

Cathepsin L is a lysosomal cysteine protease over-expressed in malignancy. It is very potent in degrading collagen, elastin, laminin and other components of the basement membrane and therefore, has been implicated in tumor invasion and metastasis. Two mRNA species, hCATL A and hCATL B, which contain an identical open reading frame and different 5'UTRs, were demonstrated to be encoded by the same gene located on chromosome 9q21-22. We have previously cloned and characterized the promoter responsible for the transcription of hCATL A (hCATL A promoter). However, it was not clear whether hCATL B is a splice variant of hCATL A or transcribed from a different promoter. In the present study, we demonstrate for the first time that hCATL B is transcribed from an alternate promoter (hCATL B promoter) located in the first intron of hCATL. This TATA-less promoter initiates transcription from two cytosine nucleotides present 191 and 367 bases upstream to the translation start codon. Deletion analysis revealed that the core promoter region lies upstream to these transcription initiation sites. This region contains several putative transcription factor-binding sites like AP-1, AP-4, GATA-1, Lmo2, NF-kappa B, MZF-1, NF-AT, etc. In U-87 MG cells, hCATL B promoter exhibits at least six times less activity than our previously characterized hCATL A promoter. However, this promoter is significantly more active in malignantly transformed cells as compared to its activity in untransformed cells. Thus, our results conclusively demonstrate that hCATL B mRNA is transcribed from an alternate promoter. Increased transcriptional activity from this promoter contributes to the elevated cathepsin L expression in transformed cells.