Structural mass spectrometry: rapid methods for separation and analysis of peptide natural products

A significant challenge in natural product discovery is the initial discrimination of discrete secondary metabolites alongside functionally similar primary metabolic cellular components within complex biological samples. A property that has yet to be fully exploited for natural product identification and characterization is the gas-phase collision cross section, or, more generally, the mobility-mass correlation. Peptide natural products possess many of the properties that distinguish natural products, as they are frequently characterized by a high degree of intramolecular bonding and possess extended and compact conformations among other structural modifications. This report describes a rapid structural mass spectrometry technique based on ion mobility-mass spectrometry for the comparison of peptide natural products to their primary metabolic congeners using mobility-mass correlation. This property is empirically determined using ion mobility-mass spectrometry, applied to the analysis of linear versus modified peptides, and used to discriminate peptide natural products in a crude microbial extract. Complementary computational approaches are utilized to understand the structural basis for the separation of primary metabolism derived linear peptides from secondary metabolite cyclic and modified cyclic species. These findings provide a platform for enhancing the identification of secondary metabolic peptides with distinct mobility-mass ratios within complex biological samples.     

Antipsychotics increase vesicular glutamate transporter 2 (VGLUT2) expression in thalamolimbic pathways

Recently the two vesicular-glutamate-transporters VGLUT1 and VGLUT2 have been cloned and characterized. VGLUT1 and VGLUT2 together label all glutamatergic neurons, but because of their distinct expression patterns in the brain they facilitate our ability to define between a VGLUT1-positive cortical and a VGLUT2-positive subcortical glutamatergic systems. We have previously demonstrated an increased cortical VGLUT1 expression as marker of antidepressant activity. Here, we assessed the effects of different psychotropic drugs on brain VGLUT2 mRNA and protein expression. The typical antipsychotic haloperidol, and the atypicals clozapine and risperidone increased VGLUT2 mRNA selectively in the central medial/medial parafascicular, paraventricular and intermediodorsal thalamic nuclei; VGLUT2 protein was accordingly amplified in paraventricular and ventral striatum and in prefrontal cortex. The antidepressants fluoxetine and desipramine and the sedative anxiolytic diazepam had no effect. These results highlight the implication of thalamo-limbic glutamatergic pathways in the action of antipsychotics. Increased VGLUT2 expression in these neurons might constitute a marker for antipsychotic activity and subcortical glutamate neurotransmission might be a possible novel target for future generation antipsychotics.     

What women with ovarian cancer think and know about genetic testing

ObjectivesFew women with ovarian cancer undergo genetic testing for the Breast and Ovarian Cancer susceptibility genes, BRCA1 and BRCA2. With the prospect of BRCA-directed therapeutics, we investigated ovarian cancer patients' knowledge and willingness to undergo genetic testing.MethodsAll ovarian cancer patients seen in the Gynecology Center of a cancer center and a private clinic were asked to complete an anonymous questionnaire regarding knowledge and willingness to undergo BRCA testing. Women who had prior genetic testing were asked not to participate. Data was analyzed using Fisher's exact test.ResultsTwo-hundred and thirty seven ovarian cancer patients voluntarily completed the questionnaire. Fifty-five percent (131/237) of participants had not heard of BRCA testing. Of Caucasian respondents, 51% were unaware of BRCA testing, compared to 70% of Hispanic and 88% of African American respondents (p = 0.008). Awareness was correlated with education (p < 0.001). Eighty-nine percent of participants were willing to be tested if it would directly affect their therapy and 86.9% would be tested to benefit their family. Seventy-four percent of patients would pay 20% of the cost of testing, only 25.1% would pay in full.ConclusionsA majority of women with ovarian cancer are not aware of the availability of BRCA testing. This lack of awareness is more profound in minorities. Despite lack of knowledge, most patients would undergo testing if it would impact their care. However, cost may be a barrier. Given the willingness of patients to undergo testing and the possibility of targeted therapy, clinicians who care for these patients should work to make appropriate genetic counseling referrals.     

Complications of sling surgery among female Medicare beneficiaries

OBJECTIVE: To analyze Medicare claims data to determine short-term complications after sling surgery among female beneficiaries aged 65 years and over. METHODS: We analyzed the 1999-2001 Medicare Public Use Files provided by the Centers for Medicare and Medicaid Services on a 5% national random sample of beneficiaries. Women undergoing sling procedures between January 1, 1999, and July 31, 2000, (the index period) were identified by Physicians Current Procedural Terminology Coding System (4th edition) and tracked for 12 months. Main outcome measures were complications as identified by International Classification of Diseases (9th revision) (ICD-9) diagnosis codes and Current Procedural Terminology procedure codes in the first postoperative year. RESULTS: A total of 1,356 sling procedures were performed during the index period. In the 3 months after the procedure, 12.5% of women developed surgical or urologic complications, and 33.6% were diagnosed with urinary tract infections. Within 1 year of the procedure, 6.9% of subjects had a new diagnosis of outlet obstruction, and 8.0% underwent treatments to manage outlet obstruction. There was a high incidence of new diagnoses of urge incontinence (15.2%) and treatment of pelvic prolapse (23.2%). Both cystoscopy and urodynamic testing, which may serve as indicators of possible complications, were performed frequently during the first year after surgery (32.4% and 30.5%, respectively). Patient race, age, and comorbidity each had a significant influence on outcomes. CONCLUSION: Complication rates within 1 year after sling surgery among Medicare beneficiaries were found to be higher than those reported in the clinical literature. The high rates of postoperative urinary tract infections, prolapse, and outlet obstruction suggest the need for quality improvement measures in the management of women with incontinence and pelvic prolapse. LEVEL OF EVIDENCE: III.     

Fisetin for COVID-19 in skilled nursing facilities: Senolytic trials in the COVID era

The burden of senescent cells (SnCs), which do not divide but are metabolically active and resistant to death by apoptosis, is increased in older adults and those with chronic diseases. These individuals are also at the greatest risk for morbidity and mortality from SARS-CoV-2 infection. SARS-CoV-2 complications include cytokine storm and multiorgan failure mediated by the same factors as often produced by SnCs through their senescence-associated secretory phenotype (SASP). The SASP can be amplified by infection-related pathogen-associated molecular profile factors. Senolytic agents, such as Fisetin, selectively eliminate SnCs and delay, prevent, or alleviate multiple disorders in aged experimental animals and animal models of human chronic diseases, including obesity, diabetes, and respiratory diseases. Senolytics are now in clinical trials for multiple conditions linked to SnCs, including frailty; obesity/diabetes; osteoporosis; and cardiovascular, kidney, and lung diseases, which are also risk factors for SARS-CoV-2 morbidity and mortality. A clinical trial is underway to test if senolytics decrease SARS-CoV-2 progression and morbidity in hospitalized older adults. We describe here a National Institutes of Health-funded, multicenter, placebo-controlled clinical trial of Fisetin for older adult skilled nursing facility (SNF) residents who have been, or become, SARS-CoV-2 rtPCR-positive, including the rationale for targeting fundamental aging mechanisms in such patients. We consider logistic challenges of conducting trials in long-term care settings in the SARS-CoV-2 era, including restricted access, consent procedures, methods for obtaining biospecimens and clinical data, staffing, investigational product administration issues, and potential solutions for these challenges. We propose developing a national network of SNFs engaged in interventional clinical trials.     

Evolution of the NBOMes: 25C- and 25B- Sold as 25I-NBOMe

Introduction

The NBOMes (N-benzyl-oxy-methyl derivatives of known 2C phenylethylamines) are a new and growing class of potent synthetic stimulants. Case reports provide the bulk of available safety and clinical data for clinicians. We report two cases of NBOMe intoxication with 25C-NBOMe (the first lab-confirmed US case) and 25B-NBOMe, respectively, both confirmed via triple quadrapole mass spectrometry.

Case Reports

Case 1: A 16-year-old girl had a generalized seizure after reported use of 25I-NBOMe. She presented with altered mental status, lower extremity rigidity, and elevated CPK (6042 U/L). Despite treatment with benzodiazepines, her lower extremity rigidity persisted and CPK peaked at 47,906 U/L. She was discharged on hospital day 8. Serum and urine specimens confirmed presence of 25C-NBOMe. Case 2: A 15-year-old boy developed bizarre behavior after reported use of 25I-NBOMe. In the ED, he had two generalized seizures and persistent muscle rigidity. CPK peaked at 429 U/L. Seizures were managed with benzodiazepines, and he was discharged within 24 h. Serum specimens revealed 25B-NBOMe.

Discussion

NBOMes are amphetamine derivatives and highly potent 5-HT_2A receptor agonists. Clinical manifestations are a product of enhanced central sympathetic and serotonergic tone. We report two cases of NBOMe intoxication in patients who believed they used 25I-NBOME, while lab confirmation proved otherwise. Whether unique clinical manifestations are specific to the NBOMe variant, dose, route of administration, or other factors is unknown. Laboratory confirmation may play a role in identifying unexpected NBOMe variants, while contributing to the epidemiologic data on these novel substances.     

Impact of Family History of Substance Abuse on Admission Opioid Dose,Depressive Symptoms,and Pain Catastrophizing in Patients with Chronic Pain

The objectives of this study were to examine association between a family history of substance abuse and admission morphine equivalent dose, depression and pain catastrophizing screening scores, as well as reported personal history of substance use. The retrospective research was completed in an interdisciplinary three-week pain rehabilitation center. The subject cohort included admissions from January through December 2014 with 351 datasets for family history of substance abuse and oral morphine equivalency and 341 for depression, pain catastrophizing and use of substances. Outcome measures included admission self-reported data on family history of substance abuse and past and current substance use, admission morphine equivalency dose, and scores on the Center for Epidemiologic Studies-Depression Scale and the Pain Catastrophizing Scale. One hundred forty-seven patients were using opioid medications on admission and those with a positive family history of substance abuse had an oral morphine equivalency (M = 92.12, SD = 95.32) compared to a negative history (M = 80.34, SD = 64.86); the difference was not statistically significant, t (120.01) =.87, p = .39. Patients with a positive family history reported higher levels of both depression, t (327.40) = 3.15, p = .002 and pain catastrophizing, t (338) = 2.76, p = .01. Those with a positive family history endorsed greater frequency of past alcohol use χ² (1, N = 326) = 6.67, p = 0.1 and marijuana use χ² (1, N = 341) = 4.23, p = .04 and past χ² (1, N = 329) = 9.90, p = .002 and current tobacco use χ² (1, N = 327) = 8.81, p = .003. Use of family history of substance abuse information may help provide data for multimodal treatments of chronic non-cancer-pain. The findings from this study can be used to guide future research.