Search

The use of liquid chromatography coupled with mass spectrometry (LC-MS) for the characterization of oligonucleotides and nucleic acids is a powerful analytical method. Recently, hydrophilic interaction chromatography (HILIC) has been proposed as a reasonable alternative to ion-pair reversed phase separations of oligonucleotides prior to MS. A wide variety of HILIC stationary phase surface chemistries are currently available. Although their selectivity can be considerably different, few studies have compared these chemistries for LC-MS analysis of oligonucleotides. We evaluated ten different HILIC column chemistries to understand their capabilities for separating a variety of oligonucleotides. In general, we found that most columns were ineffective at separating larger ( n > 15-mer) oligonucleotides under the mobile phase and gradient conditions evaluated here. However, several stationary phases were found to be effective for separating smaller oligonucleotides such as endonuclease digestion products. Given that early eluting oligonucleotides were found to be compatible with standard electrospray ionization conditions, several different HILIC stationary phase options are available for LC-MS studies of smaller oligonucleotides including those generated in RNA modification mapping experiments.

Mass spectrometry is an important method for analysis of modified nucleosides ubiquitously present in cellular RNAs, in particular for ribosomal and transfer RNAs that play crucial roles in mRNA translation and decoding. Furthermore, modifications have effect on the lifetimes of nucleic acids in plasma and cells and are consequently incorporated into RNA therapeutics. To provide an analytical tool for sequence characterization of modified RNAs, we developed Pytheas, an open-source software package for automated analysis of tandem MS data for RNA. The main features of Pytheas are flexible handling of isotope labeling and RNA modifications, with false discovery rate statistical validation based on sequence decoys. We demonstrate bottom-up mass spectrometry characterization of diverse RNA sequences, with broad applications in the biology of stable RNAs, and quality control of RNA therapeutics and mRNA vaccines., (© 2022. The Author(s).)

Background & Aims: The liver has a unique capacity to regenerate after injury in a highly orchestrated and regulated manner. Here, we report that O-GlcNAcylation, an intracellular post-translational modification regulated by 2 enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a critical termination signal for liver regeneration following partial hepatectomy (PHX)., Methods: We studied liver regeneration after PHX on hepatocyte specific OGT and OGA knockout mice (OGT-KO and OGA-KO), which caused a significant decrease (OGT-KO) and increase (OGA-KO) in hepatic O-GlcNAcylation, respectively., Results: OGA-KO mice had normal regeneration, but the OGT-KO mice exhibited substantial defects in termination of liver regeneration with increased liver injury, sustained cell proliferation resulting in significant hepatomegaly, hepatic dysplasia, and appearance of small nodules at 28 days after PHX. This was accompanied by a sustained increase in expression of cyclins along with significant induction in pro-inflammatory and pro-fibrotic gene expression in the OGT-KO livers. RNA-sequencing studies revealed inactivation of hepatocyte nuclear 4 alpha (HNF4α), the master regulator of hepatic differentiation and a known termination signal, in OGT-KO mice at 28 days after PHX, which was confirmed by both Western blot and immunohistochemistry analysis. Furthermore, a significant decrease in HNFα target genes was observed in OGT-KO mice, indicating a lack of hepatocyte differentiation following decreased hepatic O-GlcNAcylation. Immunoprecipitation experiments revealed HNF4α is O-GlcNAcylated in normal differentiated hepatocytes., Conclusions: These studies show that O-GlcNAcylation plays a critical role in the termination of liver regeneration via regulation of HNF4α in hepatocytes., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the gold-standard technique to study RNA and its various modifications. While most research on RNA nucleosides has been focused on their biological roles, discovery of new modifications remains of interest. With state-of-the-art technology, the presence of artifacts can confound the identification of new modifications. Here, we report the characterization of a non-natural mcm 5 isoC ribonucleoside in S. cerevisiae total tRNA hydrolysate by higher-energy collisional dissociation (HCD)-based fingerprints and isotope labeling of RNA. Its discovery revealed a class of amino/imino ribonucleoside artifacts that are generated during RNA hydrolysis under ammonium-buffered mild basic conditions. We then identified digestion conditions that can reduce or eliminate their formation. These finding and method enhancements will improve the accurate detection of new RNA modifications., (© 2020 Wiley-VCH GmbH.)

Knowledge of the structural information is essential for understanding the functional details of modified RNA. Cellular non-coding RNA such as rRNA, tRNA and even viral RNAs contain a number of post-transcriptional modifications with varied degree of diversity and density. In this chapter, we discuss the use of a combination of biochemical and analytical tools such as ribonucleases and liquid chromatography coupled with mass spectrometry approaches for characterization of modified RNA. We present the protocols and alternate strategies for obtaining confident modified sequence information to facilitate the understanding of function., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: We hypothesized that the universal adoption of closed wounds with negative pressure wound therapy (NPWT) in emergency general surgery patients would result in low superficial surgical infection (SSI) rates., Study Design: We performed a retrospective observational study using primary wound closure with external NPWT, from May 2017 to May 2018. Patients with active soft tissue infection of the abdominal wall were excluded. Data were analyzed by Fisher's exact tests and Wilcoxon-Mann-Whitney tests, with significance is set at a value of p < 0.05., Results: Eighty-five patients (53% female) with a median age of 65 years (range 19 to 98 years) underwent laparotomies. Four patients were excluded for active soft tissue infection. Wounds were classified as dirty (n = 18), contaminated (n = 52), and clean contaminated (n = 11). Median BMI was 27 kg/m 2 (interquartile range [IQR] 23.4 to 33.0 kg/m 2 ). Median antibiotic therapy was 4 days (IQR 1 to 7 days). Twenty-six patients had open abdomen management. Patient follow-up was a median of 20 days (range 14 to 120 days). Six patients (7%) developed superficial SSI requiring conversion to open wound management. No patients developed fascial dehiscence. There were no statistically significant associations between SSI and wound class (p = 0.072), antibiotic duration (p = 0.702), open abdomen management, or preoperative risk factors (p < 0.1). Overall morbidity was 38% and mortality was 6%., Conclusions: Primary closure of high risk incisions combined with NPWT is associated with acceptably low SSI rates. Due to the low morbidity and decreased cost associated with this technique, primary closure with NPWT should replace open wound management in the emergency general surgery population., (Copyright © 2018 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Research into post-transcriptional processing and modification of RNA continues to speed forward, as their ever-emerging role in the regulation of gene expression in biological systems continues to unravel. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has proven for over two decades to be a powerful ally in the elucidation of RNA modification identity and location, but the technique has not proceeded without its own unique technical challenges. The throughput of LC-MS/MS modification mapping experiments continues to be impeded by tedious and time-consuming spectral interpretation, particularly during for the analysis of complex RNA samples. RNAModMapper was recently developed as a tool to improve the interpretation and annotation of LC-MS/MS data sets from samples containing post-transcriptionally modified RNAs. Here, we delve deeper into the methodology and practice of RNAModMapper to provide greater insight into its utility, and remaining hurdles, in current RNA modification mapping experiments., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: A new proprietary negative pressure wound device has been developed to apply negative pressure therapy to closed wounds (closed-NPWT). We postulated that closed-NPWT management of contaminated and dirty wounds would lead to faster wound healing and no significant difference in wound complications., Study Design: An IRB approved, prospective randomized trial was performed. Patients were consented preoperatively, but not entered nor assigned treatment until intraoperative findings were known. Patients were randomly assigned to either open-NPWT or a wound closed with skin staples and external closed-NPWT. Primary outcome was time to complete wound healing, defined as complete epithelization of the wound. Secondary outcomes were wound complications including wound infection, seroma, and dehiscence. Statistical analysis was performed using chi-square test, Fisher exact test, t-test, and Wilcoxon Rank-Sum test with significance of p < 0.05., Results: Twenty-five closed-NPWT and 24 open-NPWT patients were analyzed. There were no significant differences in sex, mean age, BMI, smoking history, steroid use, comorbidities, or indication for surgery in the 2 groups. One patient in the open-NPWT group and 2 patients in the closed-NPWT group developed a wound infection (p = 1.0). Four open-NPWT and 3 closed-NPWT patients died from complications unrelated to the wound. Wound healing occurred at a median of 48 days (range 6 to 126 days) for the open-NPWT group vs a median of 7 days (range 6 to 12 days) for the closed-NPWT group (p < 0.0001)., Conclusions: Wound healing was significantly faster in contaminated and dirty wounds when managed with closed-NPWT. There was no difference in wound complications between the 2 treatment groups. This approach shows promise for closed management of contaminated and dirty wounds and warrants additional prospective studies with larger patient groups., (Copyright © 2017 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: Perforated appendicitis is associated with an increased morbidity and length of stay. "Fast track" protocols have demonstrated success in shortening hospitalization without increasing morbidity for a variety of surgical processes. This study evaluates a fast track pathway for perforated appendicitis., Methods: In 2013, a treatment pathway for perforated appendicitis was adopted by the Acute Care Surgery Service for patients having surgical management of perforated appendicitis. Interval appendectomy was excluded. Patients were treated initially with intravenous antibiotics and transitioned to oral antibiotics and dismissed when medically stable and tolerating oral intake. A retrospective review of patients managed on the fast track pathway was undertaken to analyze length of stay, morbidity, and readmissions., Results: Thirty-four males and twenty-one females with an average age of 46.8 years underwent laparoscopic appendectomy for perforated appendicitis between January 2013 and December 2014. Pre-existing comorbidities included hypertension 42%, diabetes mellitus 11%, COPD 5% and heart disease 2%. No patient had conversion to open appendectomy. Average length of stay was 2.67 days and ranged from 1 to 12 days (median 2 days). Postoperative morbidity was 20% and included abscess (6 patients), prolonged ileus (3 patients), pneumonia (1 patient), and congestive heart failure (1 patient). Five patients were readmitted for abscess (3 patients), congestive heart failure (1 patient), and pneumonia (1 patient)., Conclusion: A fast track pathway for perforated appendicitis produced shorter length of stay and acceptable postoperative morbidity and readmission. This offers the potential for significant cost savings over current national practice patterns., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Common duct stones can be diagnosed by magnetic resonance cholangiopancreatography (MRCP), endoscopic ultrasound (EUS)/ERCP, and intraoperative cholangiogram (IOC). In 2015, our group adopted a standard approach of preoperative EUS/ERCP followed by laparoscopic cholecystectomy for patients with an admission bilirubin >4.0 mg/dL. For bilirubin <4.0 mg/dL, laparoscopic cholecystectomy with IOC was the initial procedure. Postoperative EUS/ERCP with endoscopic sphincterotomy was pursued for positive IOC. Exclusions included clinical suspicion of malignancy and surgically altered anatomy making endoscopic management impractical., Study Design: A retrospective comparison of protocol and pre-protocol (baseline) patients was performed, looking at patient demographics, presence of pancreatitis, common duct stone risk factors, comorbidities, length of hospitalization, and postoperative morbidity. Statistical analysis was performed with t-test, chi-square, and Wilcoxon rank-sum test with significance at p < 0.05., Results: There were 56 patients in each group, with a mean ± SD age of 50.5 ± 20.88 years and 49.3 ± 20.92 years, respectively (p = NS). There were no significant differences between baseline and protocol patients with respect to individual and cumulative preoperative comorbidities, pancreatitis, elevation of liver function tests, bilirubin, common duct size, and postoperative morbidity. There were fewer endoscopies (22 vs 35; p = 0.014), and shorter length of stay in protocol patients (2.8 days vs 3.8 days; p = 0.025)., Conclusions: Protocol-driven management of patients with suspected common duct stones reduced the number of endoscopies and length of hospitalization, with no change in postoperative morbidity. This approach has the potential to decrease endoscopy-related morbidity and overall cost without affecting quality of care., (Copyright © 2017 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

New therapies are needed to eradicate androgen resistant, prostate cancer. Prostate cancer usually metastasizes to bone where the concentration of calcium is high, making Ca 2+ a promising toxin. Ionophores can deliver metal cations into cells, but are currently too toxic for human use. We synthesized a new rotaxane (CEHR2) that contains a benzyl 15-crown-5 ether as a blocking group to efficiently bind Ca 2+ . CEHR2 transfers Ca 2+ from an aqueous solution into CHCl 3 to greater extent than alkali metal cations and Mg 2+ . It also transfers Ca 2+ to a greater extent than CEHR1, which is a rotaxane with an 18-crown-6 ether as a blocking group. CEHR2 was more toxic against the prostate cancer cell lines PC-3, 22Rv1, and C4-2 than CEHR1. This project demonstrates that crown ether rotaxanes can be designed to bind a targeted metal cation, and this selective cation association can result in enhanced toxicity., Competing Interests: The authors declare no competing financial interest.

Introduction: Despite numerous health benefits, less than half of American adults engage in regular physical activity. Exercise videogames (EVG) may be a practical and attractive alternative to traditional forms of exercise. However there is insufficient research to determine whether EVG play alone is sufficient to produce prolonged engagement in physical activity or improvements in cardiovascular fitness and overall health risk. The goal of the present study is to test the efficacy of exercise videogames to increase time spent in moderate to vigorous physical activity (MVPA) and to improve cardiovascular risk indices among adults., Methods: Wii Heart Fitness is a rigorous 3-arm randomized controlled trial with adults comparing three 12-week programs: (1) supervised EVGs, (2) supervised standard exercise, and (3) a control condition. Heart rate is monitored continuously throughout all exercise sessions. Assessments are conducted at baseline, end of intervention (week 12), 6 and 9 months. The primary outcome is time spent in MVPA physical activity. Secondary outcomes include changes in cardiovascular fitness, body composition, blood lipid profiles and maintenance of physical activity through six months post-treatment. Changes in cognitive and affective constructs derived from Self Determination and Social Cognitive Theories will be examined to explain the differential outcomes between the two active treatment conditions., Conclusion: The Wii Heart Fitness study is designed to test whether regular participation in EVGs can be an adequate source of physical activity for adults. This study will produce new data on the effect of EVGs on cardiovascular fitness indices and prolonged engagement with physical activity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: Lipid peroxides and their reactive aldehyde derivatives (LPPs) have been linked to obesity-related pathologies, but whether they have a causal role has remained unclear. Glutathione peroxidase 4 (GPx4) is a selenoenzyme that selectively neutralizes lipid hydroperoxides, and human gpx4 gene variants have been associated with obesity and cardiovascular disease in epidemiological studies. This study tested the hypothesis that LPPs underlie cardio-metabolic derangements in obesity using a high fat, high sucrose (HFHS) diet in gpx4 haploinsufficient mice (GPx4(+/-)) and in samples of human myocardium., Methods: Wild-type (WT) and GPx4(+/-) mice were fed either a standard chow (CNTL) or HFHS diet for 24 weeks, with metabolic and cardiovascular parameters measured throughout. Biochemical and immuno-histological analysis was performed in heart and liver at termination of study, and mitochondrial function was analyzed in heart. Biochemical analysis was also performed on samples of human atrial myocardium from a cohort of 103 patients undergoing elective heart surgery., Results: Following HFHS diet, WT mice displayed moderate increases in 4-hydroxynonenal (HNE)-adducts and carbonyl stress, and a 1.5-fold increase in GPx4 enzyme in both liver and heart, while gpx4 haploinsufficient (GPx4(+/-)) mice had marked carbonyl stress in these organs accompanied by exacerbated glucose intolerance, dyslipidemia, and liver steatosis. Although normotensive, cardiac hypertrophy was evident with obesity, and cardiac fibrosis more pronounced in obese GPx4(+/-) mice. Mitochondrial dysfunction manifesting as decreased fat oxidation capacity and increased reactive oxygen species was also present in obese GPx4(+/-) but not WT hearts, along with up-regulation of pro-inflammatory and pro-fibrotic genes. Patients with diabetes and hyperglycemia exhibited significantly less GPx4 enzyme and greater HNE-adducts in their hearts, compared with age-matched non-diabetic patients., Conclusion: These findings suggest LPPs are key factors underlying cardio-metabolic derangements that occur with obesity and that GPx4 serves a critical role as an adaptive countermeasure.

The origins of fractures in Martian boulders are unknown. Here, using Mars Exploration Rover 3D data products, we obtain orientation measurements for 1,857 cracks visible in 1,573 rocks along the Spirit traverse and find that Mars rock cracks are oriented in statistically preferred directions similar to those compiled herein for Earth rock cracks found in mid-latitude deserts. We suggest that Martian directional cracking occurs due to the preferential propagation of microfractures favourably oriented with respect to repeating geometries of diurnal peaks in sun-induced thermal stresses. A numerical model modified here with Mars parameters supports this hypothesis both with respect to the overall magnitude of stresses as well as to the times of day at which the stresses peak. These data provide the first direct field and numerical evidence that insolation-related thermal stress potentially plays a principle role in cracking rocks on portions of the Martian surface.

For development to proceed normally, animal eggs must undergo a maturation process that ultimately depends on phosphorylations of key regulatory proteins. To analyze the kinases that mediate these phosphorylations, eggs of marine nemertean worms have been treated with pharmacological modulators of intracellular signaling pathways and subsequently probed with immunoblots employing phospho-specific antibodies. This article both reviews such analyses and compares them with those conducted on mammals, while focusing on how egg maturation in nemerteans is affected by signaling pathways involving cAMP, mitogen-activated protein kinases, Src-family kinases, protein kinase C isotypes, AMP-activated kinase, and the Cdc2 kinase of maturation-promoting factor.

Pain survey was administered to 77 patients undergoing nerve conduction (NC) and needle electromyographic (EMG) procedures. The patient was asked to rate the pain immediately after the electrodiagnostic study was completed. Statistical analyses were performed to determine if there were relationships between pain and any of the technologic and physiologic variables. The findings revealed no significant correlation between age and pain from the NC or needle EMG procedure. The body mass index (BMI) also showed no correlation to pain from the procedures. Also, there was no correlation between pain and the EMG needle gauge or length. Pain from needle EMG was correlated with NC pain, and EMG was perceived as more painful than the NC procedure. Multiple regression analysis showed that age, sex, and needle size were not significantly related to pain from the EMG examination. Again, the age, sex, and BMI showed no significant relationship to pain from the NC test. From this survey, the individual's age, sex, habitus, and size of the EMG needle (to some extent) do not seem to influence significantly the perception of pain during electrodiagnostic procedures.

Sensory nerve conduction (NC) studies that were performed on patients with Guillain-Barre syndrome (GBS) were reviewed. These were compared with the sensory NC findings in patients with peripheral neuropathy of the axonal-loss type (axonopathies) and chronic inflammatory demyelinating polyneuropathy (CIDP). In GBS, the sural sensory nerve frequently showed normal NC findings compared to the median and ulnar sensory nerves. The reverse pattern was seen in axonopathies and CIDP, in which the sural nerve showed more abnormalities than the median and ulnar nerves. This pattern of sensory NC abnormalities may assist the examiner in differentiating GBS from other types of peripheral polyneuropathy.

The sensory nerve conduction study (NCS) is a sensitive means for demonstrating the localized median nerve dysfunction at the wrist in carpal tunnel syndrome (CTS). We reviewed the sensory NCS data of 102 patients (178 median nerves) with CTS and tried to determine if there were significant differences in the findings between the index and middle fingers. There was no statistically significant difference in the amplitudes of the compound sensory nerve action potentials (CSNAPs) recorded antidromically from the index and middle fingers during median nerve stimulation at the palm and wrist. Moreover, there was no statistically significant difference in the finger-to-palm and palm-to-wrist sensory nerve conduction velocities (CVs) between the index and middle fingers. The mean CSNAP amplitudes and mean sensory CVs were comparable in values from these two fingers and they showed a high and positive correlation. It appears that the sensory nerves to the index and middle fingers are compromised to a similar degree in CTS, and neither finger will reveal significantly more sensory conduction abnormalities than the other will.

Purpose: Cryopreserved saphenous vein allografts are used for femoral-infrapopliteal bypass graft purposes when adequate autogenous vein is unavailable. Anticoagulation, immunosuppression therapy, or both have been suggested means for improving allograft patency. Immunosuppression has significant cost and morbidity and has produced variable results. Our successful treatment of luminal surface hypercoagulability associated with certain endovascular procedures prompted the use of an anticoagulation protocol prospectively to improve graft patency and limb salvage for patients receiving femoral-infrapopliteal cryopreserved saphenous vein allografts., Methods: Between September 1995 and October 1999, 24 patients (15 men and nine women) were enrolled in a prospective clinical trial for salvage of 26 severely ischemic lower limbs with femoral-infrapopliteal cryopreserved saphenous vein allograft bypass grafts. All patients were treated with a protocol (aspirin, low-dose heparin, low molecular weight dextran 40, dipyridamole, and warfarin), and no immunosuppressive agents were used. The cryopreserved saphenous vein allografts were matched to patients by ABO and Rh compatibility. Indications for revascularization were ischemic rest pain (n = 8), nonhealing ulcer (n = 13), or focal gangrene (n = 5), and no usable autogenous vein was available. Follow-up ranged from 2 to 35 months (mean, 19 months). We studied the location and type of outflow anastomosis, specific outflow vessel, morbidity, death, secondary procedures (digital/transmetatarsal amputation), and complications related to the treatment protocol. Life table analyses of primary graft patency and limb salvage were compared with other current reported data., Results: Primary graft patency with Kaplan-Meier life table analysis was 96% at 6 months, 87% at 12 months, and 82% at 18 and 24 months. There were no reoperations for acute graft occlusion. One graft underwent late segmental aneurysmal degeneration and rupture. There were no procedure-related deaths or bleeding complications. During late follow-up, anticoagulation was discontinued in three patients (12%) because of gastrointestinal bleeding. Limb salvage was 88% at 6 months and 80% at 12, 18, and 24 months. Patients returned to ambulatory status that was limited only by their other comorbidities., Conclusion: Femoral-infrapopliteal bypass graft for limb salvage with a cryopreserved saphenous vein allograft can be an acceptable alternative when autogenous vein is not available. Our treatment protocol substantially improved allograft patency and limb salvage when compared with current published data.

Sanguinarine, a benzophenanthridine alkaloid, causes a initial stimulation of frog skin short circuit current Isc when present in the mucosal bathing medium at 10(-4) M. The stimulation is accompanied by an increase in spontaneous potential difference (PD) and increase in D.C. resistance. No effects are seen with sanguinarine in the serosal bathing medium. The initial stimulation is followed by a decrease in Isc and PD, but a continued increase in resistance. In skins whose initial spontaneous PD is high, no initial stimulation in Isc and PD is seen; however, clamping these skins to a lower potential does not alter their initial inhibitory response to sanguinarine. Likewise, clamping the lower potential skins to higher potential does not alter their initial stimulatory response. Sanguinarine seems to be acting on the permeability barriers at the outer surface of the frog skin.