Determination of the compound class and functional groups in protonated analytes via diagnostic gas-phase ion-molecule reactions.

Diagnostic gas-phase ion-molecule reactions serve as a powerful alternative to collision-activated dissociation for the structural elucidation of analytes when using tandem mass spectrometry. The use of such ion-molecule reactions has been demonstrated to provide a robust tool for the identification of specific functional groups in unknown ionized analytes, differentiation of isomeric ions, and classification of unknown ions into different compound classes. During the past several years, considerable efforts have been dedicated to exploring various reagents and reagent inlet systems for functional-group selective ion-molecule reactions with protonated analytes. This review provides a comprehensive coverage of literature since 2006 on general and predictable functional-group selective ion-molecule reactions of protonated analytes, including simple monofunctional and complex polyfunctional analytes, whose mechanisms have been explored computationally. Detection limits for experiments involving high-performance liquid chromatography coupled with tandem mass spectrometry based on ion-molecule reactions and the application of machine learning to predict diagnostic ion-molecule reactions are also discussed.

Doublecortin and JIP3 are neural-specific counteracting regulators of dynein-mediated retrograde trafficking.

Mutations in the microtubule (MT)-binding protein doublecortin (DCX) or in the MT-based molecular motor dynein result in lissencephaly. However, a functional link between DCX and dynein has not been defined. Here, we demonstrate that DCX negatively regulates dynein-mediated retrograde transport in neurons from Dcx-/y or Dcx-/y;Dclk1-/- mice by reducing dynein's association with MTs and disrupting the composition of the dynein motor complex. Previous work showed an increased binding of the adaptor protein C-Jun-amino-terminal kinase-interacting protein 3 (JIP3) to dynein in the absence of DCX. Using purified components, we demonstrate that JIP3 forms an active motor complex with dynein and its cofactor dynactin with two dyneins per complex. DCX competes with the binding of the second dynein, resulting in a velocity reduction of the complex. We conclude that DCX negatively regulates dynein-mediated retrograde transport through two critical interactions by regulating dynein binding to MTs and regulating the composition of the dynein motor complex.

A Diagnostic Nitrosamine Detection Approach for Pharmaceuticals by Using Tandem Mass Spectrometry Based on Diagnostic Gas-Phase Ion-Molecule Reactions.

N-Nitrosamines are strictly regulated in pharmaceutical products due to their carcinogenic nature. Therefore, the ability to rapidly and reliably identify the N-nitroso functionality is urgently needed. Unfortunately, not all ionized N-nitroso compounds produce diagnostic fragment ions and hence tandem mass spectrometry based on collision-activated dissociation (CAD) cannot be used to consistently identify the N-nitroso functionality. Therefore, a more reliable method was developed based on diagnostic functional-group selective ion-molecule reactions in a linear quadrupole ion trap mass spectrometer. 2-Methoxypropene (MOP) was identified as a reagent that reacts with protonated N-nitrosamines in a diagnostic manner by forming an adduct followed by the elimination of 2-propenol (CH3C(OH)═CH2]). From 18 protonated N-nitrosamine model compounds studied, 15 formed the diagnostic product ion. The lack of the diagnostic reaction for three of the N-nitrosamine model compounds was rationalized based on the presence of a pyridine ring that gets preferentially protonated instead of the N-nitroso functionality. These N-nitrosamines can be identified by subjecting a stable adduct formed upon ion-molecule reactions with MOP to CAD. Further, the ability to use ion-molecule reactions followed by CAD to differentiate protonated O-nitroso compounds with a pyridine ring from analogous N-nitrosamines was demonstrated This methodology is considered to be robust for the identification of the N-nitroso functionality in unknown analytes. Lastly, HPLC/MS2 experiments were performed to determine the detection limit for five FDA regulated N-nitrosamines.

Loss of CLOCK Results in Dysfunction of Brain Circuits Underlying Focal Epilepsy.

Because molecular mechanisms underlying refractory focal epilepsy are poorly defined, we performed transcriptome analysis on human epileptogenic tissue. Compared with controls, expression of Circadian Locomotor Output Cycles Kaput (CLOCK) is decreased in epileptogenic tissue. To define the function of CLOCK, we generated and tested the Emx-Cre; Clockflox/flox and PV-Cre; Clockflox/flox mouse lines with targeted deletions of the Clock gene in excitatory and parvalbumin (PV)-expressing inhibitory neurons, respectively. The Emx-Cre; Clockflox/flox mouse line alone has decreased seizure thresholds, but no laminar or dendritic defects in the cortex. However, excitatory neurons from the Emx-Cre; Clockflox/flox mouse have spontaneous epileptiform discharges. Both neurons from Emx-Cre; Clockflox/flox mouse and human epileptogenic tissue exhibit decreased spontaneous inhibitory postsynaptic currents. Finally, video-EEG of Emx-Cre; Clockflox/flox mice reveals epileptiform discharges during sleep and also seizures arising from sleep. Altogether, these data show that disruption of CLOCK alters cortical circuits and may lead to generation of focal epilepsy.

UFM1 founder mutation in the Roma population causes recessive variant of H-ABC.

OBJECTIVE: To identify the gene defect in patients with hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) who are negative for TUBB4A mutations. METHODS: We performed homozygosity mapping and whole exome sequencing (WES) to detect the disease-causing variant. We used a Taqman assay for population screening. We developed a luciferase reporter construct to investigate the effect of the promoter mutation on expression. RESULTS: Sixteen patients from 14 families from different countries fulfilling the MRI criteria for H-ABC exhibited a similar, severe clinical phenotype, including lack of development and a severe epileptic encephalopathy. The majority of patients had a known Roma ethnic background. Single nucleotide polymorphism array analysis in 5 patients identified one large overlapping homozygous region on chromosome 13. WES in 2 patients revealed a homozygous deletion in the promoter region of UFM1. Sanger sequencing confirmed homozygosity for this variant in all 16 patients. All patients shared a common haplotype, indicative of a founder effect. Screening of 1,000 controls from different European Roma panels demonstrated an overall carrier rate of the mutation of 3%-25%. Transfection assays showed that the deletion significantly reduced expression in specific CNS cell lines. CONCLUSIONS: UFM1 encodes ubiquitin-fold modifier 1 (UFM1), a member of the ubiquitin-like family involved in posttranslational modification of proteins. Its exact biological role is unclear. This study associates a UFM1 gene defect with a disease and sheds new light on possible UFM1 functional networks.

Postoperative observation of children after endoscopic type 1 posterior laryngeal cleft repair.

OBJECTIVES: To report the perioperative management and surgical outcomes in a large series of pediatric patients with endoscopically repaired type 1 posterior laryngeal cleft (PLC). STUDY DESIGN: Case series with chart review. SETTING: Urban, tertiary care, free-standing pediatric hospital. SUBJECTS AND METHODS: Patients who underwent endoscopic carbon dioxide laser-assisted repair of type 1 posterior laryngeal clefts between January 2006 and December 2012. Medical records were reviewed. RESULTS: Fifty-four patients (34 male) underwent repair of type 1 PLC. Median age was 25.5 months (range, 2-120 months). Indications for repair included aspiration (n = 39; 72%), chronic bronchitis (n = 13; 24%), and stridor with feeds (n = 2; 4%). No children remained intubated postoperatively. Thirty-three patients (61%) stayed in overnight observation ("Obs PLC") and 21 patients (39%) stayed in the pediatric intensive care unit ("PICU PLC") postoperatively. Between Obs PLC and PICU PLC groups, there was no significant difference in age (mean 22 vs 30 months, respectively; P = .28). Comorbidities were similar between the groups. Symptoms improved in 41 of the 54 patients (76%). No postoperative complications were noted. Two patients required revision PLC repair. The cost of admitting a patient to a lower acuity location was estimated to be 60% less per day than cost of a PICU admission. CONCLUSIONS: The endoscopic surgical repair of a type 1 PLC is successful and has a low morbidity and complication rate. Patients may be safely managed in an observation unit and without postoperative intubation. This approach achieved a marked cost reduction in postoperative care.

Dipyridamole monotherapy in schizophrenia: pilot of a novel treatment approach by modulation of purinergic signaling.

BACKGROUND: Emerging data indicate the neuromodulator adenosine may play a role in the therapeutics of schizophrenia. Adenosine A(2A) receptor stimulation exerts a functional antagonism at postsynaptic D(2) receptors. Data from animal models relevant to schizophrenia support a therapeutic effect of modulating adenosinergic transmission in the ventral striatum. One previous clinical trial showed superiority of adjunctive dipyridamole, an adenosine reuptake inhibitor, compared to placebo in ameliorating positive symptoms in schizophrenia patients. OBJECTIVES: The aim of this study was to examine the effects of dipyridamole monotherapy of 200 mg/day on positive and negative symptoms, with the goal of determining dosing for future adjunctive studies in schizophrenia. METHODS: Twenty symptomatic schizophrenia participants were randomized to a 6-week double-blind trial comparing olanzapine (20 mg/day) to dipyridamole monotherapy (200 mg/day). Thirteen participants completed the treatment phase (eight on dipyridamole; five on olanzapine). RESULTS: The olanzapine group showed a trend (p = 0.08) for superiority on BPRS total scores (mean ± SD: total BPRS score decreasing from 36.8 ± 2.3 at week 1, to 33.2 ± 5.5 at the end of the study). The mean total BPRS scores decreased from 36.4 ± 5.3 to 34.0 ± 7.7 in the dipyridamole group. CONCLUSIONS: Although these pilot data do not support a significant antipsychotic effect of dipyridamole monotherapy, the results provide some evidence for examining dipyridamole (200 mg/day) as adjunct to symptomatic antipsychotic-treated schizophrenia patients.

Black licorice ingestion: Yet another confounding agent in patients with melena.

We describe an 80-year-old woman with atrial fibrillation, anti-coagulated with warfarin, who on two separate occasions developed black tarry stools and an elevated international normalized ratio (INR) after eating a pound of Black Licorice. During her most recent episode, her hematocrit was 14 (baseline 34) and her INR was 5.5 (baseline 2.1). She was advised to restrict licorice consumption, and a follow-up INR two weeks later was 1.2. Black Licorice is derived from the root of the plant, Glycyrrhiza glabra. The components of its extract inhibit the P450 system enzymes that metabolize Warfarin, inhibit thrombin, and prolong fibrinogen clotting times. Hence, the anti-thrombotic activity and inhibition of warfarin metabolism might synergistically amplify anti-coagulation. The presence of Black Licorice in the stool can also mimic melena and confound its clinical presentation. Health care providers should caution patients who are at risk for bleeding or on warfarin to avoid black licorice due to an elevated risk of gastrointestinal bleeding.

I-125 brachytherapy for choroidal melanoma photographic and angiographic abnormalities: the Collaborative Ocular Melanoma Study: COMS Report No. 30.

OBJECTIVES: (1) To summarize the protocol used for grading features of postradiation abnormalities from fundus photographs and fluorescein angiograms of patients enrolled in the Collaborative Ocular Melanoma Study (COMS); (2) to document the prevalence of features of interest in the posterior pole of these eyes during 8 years of follow-up; and (3) to investigate baseline patient, tumor, and treatment characteristics associated with posterior pole features. DESIGN: Observational case series within a randomized, multicenter clinical trial. PARTICIPANTS: We evaluated 650 patients who were assigned to and received iodine-125 brachytherapy in the COMS for medium-sized tumors. METHODS: Color fundus photographs and fluorescein angiograms were taken at baseline and 2, 5, and 8 years; 30 features were graded according to a standard protocol. MAIN OUTCOME MEASURES: Prevalence at selected time intervals of fundus photographic features associated with retinopathy and optic neuropathy. RESULTS: The percentage of patients with >/=1 feature of interest was 49.2% at baseline, 84.4% at 2 years, 91.2% at 5 years, and 90.7% at 8 years. The most frequent findings across all follow-up examinations were macular microaneurysms (75.6% of examinations), macular angiographic leakage (75.1%), and optic disc hyperfluorescence (62.8%). The median number of features present increased significantly with each follow-up to a maximum of 7 features at 8 years. The prevalence of neovascularization of the disc at 5 years was 5.2%. The prevalence of optic neuropathy at 5 years was 27.4%. Prognostic factors for more prevalent and severe posterior pole abnormalities were diabetes, tumor location close to both optic nerve and foveal avascular zone, and greater dose of radiation to the foveola and optic nerve head. CONCLUSIONS: The amount and severity of retinopathy and optic neuropathy after iodine-125 brachytherapy increased through 8 years of follow-up. Assessment of photographs and angiograms taken in accord with a standard protocol provided reliable estimates of rates of development of features of retinopathy and optic neuropathy in eyes treated using the COMS brachytherapy protocol. Our findings support earlier reports that tumor factors in addition to radiation treatment may contribute to posterior pole abnormalities. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any materials discussed in this article.

Magnetic resonance imaging of rectal cancer.

Magnetic resonance imaging (MRI)is a useful modality for the evaluation of rectal cancer, providing superior anatomic/pathologic visualization when compared with endorectal ultrasound (EUS) and computed tomography (CT). Preoperative MRI is useful for tissue characterization and tumor staging, which determines the surgical approach and need for neoadjuvant/adjuvant therapy. Important prognostic factors include the circumferential resection margin (CRM), T and N stages, and extent of local invasion. Postoperative MRI to assess the extent of tumor recurrence enables early resection, which can greatly prolong survival. MRI criteria for local recurrence include T2 hyperintensity, early dynamic rim enhancement, and nodular morphology. Future research in MRI of rectal cancer is geared toward developing optimal imaging techniques including high-resolution MRI, whole-body scans, and parallel imaging; imaging of lymph nodes by MR lymphography; and response to therapy using diffusion/perfusion-weighted MR and functional imaging.

Doublecortin is expressed in articular chondrocytes.

Articular cartilage and cartilage in the embryonic cartilaginous anlagen and growth plates are both hyaline cartilages. In this study, we found that doublecortin (DCX) was expressed in articular chondrocytes but not in chondrocytes from the cartilaginous anlagen or growth plates. DCX was expressed by the cells in the chondrogenous layers but not intermediate layer of joint interzone. Furthermore, the synovium and cruciate ligaments were DCX-negative. DCX-positive chondrocytes were very rare in tissue engineered cartilage derived from in vitro pellet culture of rat chondrosarcoma, ATDC5, and C3H10T1/2 cells. However, the new hyaline cartilage formed in rabbit knee defect contained mostly DCX-positive chondrocytes. Our results demonstrate that DCX can be used as a marker to distinguish articular chondrocytes from other chondrocytes and to evaluate the quality of tissue engineered or regenerated cartilage in terms of their "articular" or "non-articular" nature.

Radiofrequency and microwave energy sources in surgical ablation of atrial fibrillation: a comparative analysis.

BACKGROUND: Due to its complexity and risk of bleeding, the Maze III procedure has been largely replaced by surgical ablation for atrial fibrillation (AF) using alternative energy sources. Radiofrequency (RF) and microwave (MW) are the most commonly used energy forms. In this study, we sought to compare these energy modalities in terms of clinical outcomes. METHODS: Two hundred five patients underwent surgical ablation of AF, from October 1999 to May 2004 at our institution via an endocardial approach. Patients were categorized into 2 groups: RF and MW. Baseline characteristics, operative details, and clinical outcomes were compared between the 2 groups. Rhythm success was defined as freedom from AF and atrial flutter as determined by postoperative electrocardiograms. RESULTS: One hundred twenty patients (58.5%) were ablated using RF, whereas 85 (41.5%) were ablated with MW. Most of the patients had persistent AF in both the RF and MW groups (85.7% versus 80.0%, respectively; P = .363). Intraoperative left atrial size was 6.4 +/- 1.7 cm for the RF group and 6.4 +/- 1.7 cm for the MW group (P = .820). Postoperative rhythm success at 6 and 12 months was 72.4% versus 71.4% (P +/- .611) and 75.0% versus 66.7% (P = .909) for the RF and MW groups, respectively. Hospital length of stay was comparable for both groups (15.4 +/- 14.0 versus 13.3 +/- 13.9 days; P = .307). Postoperative survival at 6 months, 1 year, and 3 years was 90.4%, 89.5%, and 86.1% for RF patients compared to 87.9%, 86.5%, and 84.4% for MW patients, respectively (log rank P = .490). CONCLUSIONS: RF and MW energy forms yield comparable postoperative rhythm success, hospital length of stay, and postoperative survival. Both sources are rapid, safe, and effective alternatives to "cut and sew" techniques for surgical treatment of AF.

A decade experience of cardiac retransplantation in adult recipients.

BACKGROUND: Cardiac retransplantation is considered to be the best therapeutic option for a failing cardiac allograft. However, poor outcomes with retransplantation have previously been reported, a factor that raises important ethical, logistic and financial issues given the limited organ donor supply. METHODS: Seven hundred sixty-six adult patients underwent cardiac transplantation for end-stage heart failure at our institution from 1992 to 2002. Of these, 41 (5.4%) were retransplants. Variables examined included recipient and donor demographics, indications for retransplant, comorbidities, cytomegalovirus (CMV) serology status, left ventricular assist device use before transplant, donor ischemic time, rate of early mortality (within 30 days), and post-transplantation survival rate. RESULTS: Indications for cardiac retransplant were transplant-related coronary artery disease in 37, acute rejection in 3, and other causes in 1. The mean interval between transplantation and retransplantation was 5.9 +/- 3.4 years. Baseline characteristics such as recipient age, gender, CMV serology status, and donor age were similar in the primary transplant and retransplant groups. Early mortality after transplantation was comparable between the 2 groups, but post-transplant survival was significantly lower in retransplant patients compared with primary transplants with 1-, 3-, 5-, and 7-year actuarial survival rates of 72.2%, 66.3%, 47.5%, and 40.7% vs. 85.1%, 79.2%, 72.9%, and 66.8%, respectively (p < 0.001). CONCLUSIONS: Cardiac retransplantation offers short-term outcomes similar to primary transplantation but lower long-term survival rates. Non-retransplant surgical options should also be considered in these patients. Careful patient selection and risk-assessment is necessary to govern appropriate allocation of limited donor organs.