Improved method robustness and ruggedness in liquid chromatography-mass spectrometry by increasing the acid content of the mobile phase.

A routine multiresidue method developed for the detection and quantification of veterinary drug residues in animal-based food was used to analyze sheep (ovine) liver. Unlike when working with previously validated matrices (e.g., bovine liver), some of the analytes of interest chromatographed in the form of split- or even fully baseline separated peaks. In other cases a significantly longer retention times (tR) was observed. A detailed investigation led to the elucidation of taurocholic acid as the causative agent. This compound is present in sheep liver at significantly higher concentrations than in most other animal tissues. Taurocholic acid is a zwitterionic compound and likely acts as an ion pairing agent, which modifies the selectivity of the stationary phase in a highly spatial and dynamic way. Injecting smaller volumes of matrix extract or the use of a significantly higher formic acid concentration in the mobile phase reduced or even completely eliminated the peak splitting. A more detailed examination led to the observation that the problem is not restricted to this particular matrix and extraction procedure or the used stationary phase. In fact, a higher formic acid concentration (e.g., 1.0 % versus 0.1 %) significantly improves the peak shape of many analytes present in fortified matrix samples as well as in pure standard solutions. In addition, analytical column aging was observed as being slower with a higher formic acid concentration. Finally the peak shape of analytes interacting with the metallic parts along the flow path of the liquid chromatograph was also significantly improved. Use of 0.1 % acid in mobile phases is often taken for granted in LC-MS. Regardless of the stationary phase, a higher ionic strength better stabilizes the pH and reduces unwanted interactions, which ultimately improves the method robustness. Flow injection experiments often show that 0.1 % acid concentrations produce the highest analyte signals. Yet, the use of 1 % acid in the mobile phase often leads to narrower and therefore taller chromatographic peaks, which may lead to lower detection limits for many analytes and to an improved separation efficiency.

Metformin shows anti-neoplastic properties by inhibition of oxidative phosphorylation and glycolysis in epidermolysis bullosa-associated aggressive cutaneous squamous cell carcinoma.

BACKGROUND: While most cutaneous squamous cell carcinomas (cSCCs) are treatable, certain high-risk cSCCs, such as those in recessive dystrophic epidermolysis bullosa (RDEB) patients, are particularly aggressive. Owing to repeated wounding, inflammation and unproductive healing, RDEB patients have a 68% cumulative risk of developing life-threatening cSCCs by the age of 35, and a 70% risk of death by the age of 45. Despite aggressive treatment, cSCC represents the leading cause of premature mortality in these patients, highlighting an unmet clinical need. Increasing evidence points to a role of altered metabolism in the initiation and maintenance of cSCC, making metabolism a potential therapeutic target. OBJECTIVES: We sought to determine the feasibility of targeting tumour cell energetics as a strategy to selectively hinder the growth advantage of aggressive cSCC. METHODS: We evaluated the cell energetics profiles of RDEB-SCC cells by analysing available gene expression data against multiple gene signatures and single-gene targets linked to metabolic reprogramming. Additionally, we employed real-time metabolic profiling to measure glycolysis and respiration in these cells. Furthermore, we investigated the anti-neoplastic properties of the metformin against human and murine high-risk cSCCs in vitro and in vivo. RESULTS: Gene expression analyses highlighted a divergence in cell energetics profiles between RDEB-SCC and non-malignant RDEB keratinocytes, with tumour cells demonstrating enhanced respiration and glycolysis scores. Real-time metabolic profiling supported these data and additionally highlighted a metabolic plasticity of RDEB-SCC cells. Against this background, metformin exerted an anti-neoplastic potential by hampering both respiration and glycolysis, and by inhibiting proliferation in vitro. Metformin treatment in an analogous model of fast-growing murine cSCC resulted in delayed tumour onset and slower tumour growth, translating to a 29% increase in median overall survival. CONCLUSIONS: Our data indicate that metformin exerts anti-neoplastic properties in aggressive cSCCs that exhibit high-risk features by interfering with respiration and glycolytic processes.

[Künstliche Harnsphincter zur Behandlung von Stress-Harninkontinenz - eine oft nicht ausgelastete Behandlungsoption in Deutschland]. / Der artifizielle Schließmuskel zur Behandlung der Belastungsharninkontinenz ­ in Deutschland eine oftmals unterschätzte Therapiemethode.

ABSTRAKT: Harninkontinenz ist in Deutschland weit verbreitet und betrifft Millionen von Frauen und Männern. Vor allem Männer, die postoperativ dauerhaft inkontinent sind, werden hierzulande trotz der ausreichenden Verfügbarkeit chirurgischer Optionen unterbehandelt. Der künstliche Schließmuskel wird seit Jahrzehnten erfolgreich zur Behandlung angeborener und erworbener Stress-Harninkontinenz bei Männern und Frauen sowie neurogener Harninkontinenz eingesetzt und wird in Form neuer Modelle weiterentwickelt. Aufgrund der guten Ergebnisse, Es gilt jetzt als Standardtherapie für Männer mit anhaltender, mittelschwerer bis schwerer Harninkontinenz. Die operationstechnische Technik ist anspruchsvoll, kann aber erlernt werden. Die meisten Komplikationen können in erfahrenen Händen deutlich reduziert werden. Patientenzufriedenheit mit künstlichen Harnsphinctern (AUS) ist hoch und korreliert mit der Kontinenzrate und nicht mit der relativ hohen Revisionsrate, weshalb diese Behandlungsoption in Deutschland zunehmend mehr Patienten mit mittelschwerer bis schwerer Harninkontinenz angeboten werden sollte. Urologen in der allgemeinen Praxis spielen in diesem Zusammenhang eine zentrale Rolle.

Optimized multimatrix calibration concept for liquid chromatography mass spectrometry-based bioanalysis methods.

In this paper, a calibration procedure for LC/MS-based bioanalysis methods, termed "A/B fortification", is proposed. The concept relies on the post-extraction fortification (B-spike) of an aliquot of the injection-ready sample extract for the determination and compensation of specific signal suppression or enhancement effects compared to matrix-free extract prepared in buffer or mobile phase. Conventional analyte recovery, observed due to the incomplete extraction of analytes from the sample or losses during a cleanup, is determined by the conventional pre-extraction fortification (A-spike) of a blank sample that belongs to the same type of matrix as the sample with the unknown analyte concentration. This approach permits a higher throughput than conventional sample fortification strategies. The results obtained by utilizing the A/B fortification concept were extensively compared against conventional methods (representative bank matrix fortification, sample fortification and internal standard). The proposed concept (based on the pre-fortification of a reference matrix and post-fortification of the sample) was found to be significantly less biased than internal standard-based techniques. The A/B fortification indicated a better accuracy than the sample fortification or representative blank matrix fortification approach and, most importantly, produced significantly fewer outliers. This was linked to the fact that in the case of the A/B fortification, the uncertainty of the subtraction of two peak areas (fortified minus unfortified sample) is reduced, because fortifications are not made prior to the extraction step but are made into the final injection-ready sample extract. Fortification into an injection-ready aliquot eliminates all sample processing-related differences (procedural errors), which can affect conventional sample fortification-based quantifications.

Does the ion mobility resolving power as provided by commercially available ion mobility quadrupole time-of-flight mass spectrometry instruments permit the unambiguous identification of small molecules in complex matrices?

Quadrupole based mass spectrometry based detection has experienced enormous improvements in terms of sensitivity over the last centuries. This development has not been equally matched with improvements in selectivity. Hence, the use of unit mass based MS/MS transitions or high resolution (HRMS) based extracted ion chromatograms is gradually becoming insufficient in the field of high sensitivity multi-residue analysis (e.g. pesticides in food). As a consequence, commercial instruments hyphenating ion mobility (IMS) with low or high resolution mass spectrometry based detection have appeared. The use of such an additional (frequently claimed to be orthogonal) dimension is intended to increase selectivity. In addition, IMS derived collision cross section (CCS) has been proposed to be used as an additional identification point for the unambiguous identification of trace compounds in complex matrices. It is the topic of this paper to investigate the benefit of using such a hyphenated technique for trace analysis of small molecules in complex matrices. The potential of CCS to serve as additional identification point has been critically evaluated. Discussed are the effect of CCS data on false detects and missing detects of analytes present at trace levels. This involves the investigation of the physical resolving power provided by HRMS, IMS and chromatography as well as the correlation among these parameters (orthogonality). It is the conclusion that currently commercially available travelling wave and linear drift tube based IMS devices with a resolving power of up to 50 permit a reduction of false detects, yet this comes at the price of a higher likelihood of missing detects. The reduction of missing detects and the use of CCS as potential confirmatory information would require IMS resolving powers above 100.

[Urinary bladder cancer as a late sequela of spinal cord injury : Decision-making aids for assessment of this causal association]. / Harnblasenkarzinom als Spätfolge einer Querschnittlähmung : Entscheidungshilfe für eine Zusammenhangsbegutachtung.

BACKGROUND: There is to date no convincing literature that has assessed the association between traumatic spinal cord injury (SCI) and the later development of urinary bladder cancer. The aim of this work is to present medical experts as well as the national accident insurance and the social courts decision-making aids based on the latest medical scientific knowledge, for assessment of this causal association. MATERIALS AND METHODS: A study conducted between April 1998 and March 2017 in the BG Trauma Hospital Hamburg forms the basis for the decision-making aids. Urinary bladder cancer was diagnosed in 32 out of 6432 treated outpatient and inpatient SCI patients. Furthermore, relevant published literature was taken into consideration for the decision-making aids. RESULTS: It was found that urinary bladder cancer in SCI patients occurs at a considerably younger age as compared to the general population, more frequently shows muscle invasive carcinoma with a higher grade at first diagnosis and a higher proportion of the more aggressive squamous cell carcinoma than that of the general population. Correspondingly, the survival time is extremely unfavorable. For medical experts a matrix was compiled where the various influencing factors, either for or against the recognition of an association between SCI and urinary bladder cancer, were weighted according to their relevance. CONCLUSION: The results showed that urinary bladder cancer in SCI patients differs considerably from that of able-bodied patients. These differences drastically shorten the survival time. A study on patients with spina bifida, i.e., a congenital spinal cord disorder, corroborates these observations. They indicate histopathological differences that have so far been intangible.

Immunotherapeutics for head and neck squamous cell carcinoma stem cells.

Cancer stem cell (CSC)-related therapy resistance has become a new obstacle to the successful application of cancer treatment and head and neck squamous cell carcinoma (HNSCC) is no exception to this finding. Head and neck squamous cell carcinoma is highly immune-suppressive, and recently the immune suppression and invasion of HNSCC-CSCs have been characterized. These characteristics have received research and clinical attention because they would enable the stratification of patients into specific cancer subtypes and, consequently, the establishment of new therapeutic approaches with improved efficacy. This review discusses the feasibility of CSC-targeted strategies and their incorporation with nanotechnology to improve the efficacy of cancer immunotherapy.

[Use of synthetic slings and mesh implants in the treatment of female stress urinary incontinence and prolapse : Statement of the Working Group on Urological Functional Diagnostics and Female Urology of the Academy of the German Society of Urology]. / Einsatz von synthetischen Band- und Netzimplantaten bei der Behandlung von Belastungsinkontinenz und Descensus genitalis der Frau : Stellungnahme des Arbeitskreises Urologische Funktionsdiagnostik und Urologie der Frau der Akademie der Deutschen Gesellschaft für Urologie.

Due to a safety alert issued by the US Food and Drug Administration (FDA) in 2011 for transvaginal mesh implants to treat female prolapse as a result of numerous reports of complications such as infection, chronic pain, dyspareunia, vaginal erosion, shrinkage and erosion into other organs nearly all industrial products have been withdrawn from the market in the meantime. The United Kingdom, Australia, and New Zealand extended warnings and prohibitions even on the implantation of midurethral slings (TVT, TOT). In view of these current international controversies regarding the use of implanted materials for the treatment of stress incontinence and prolapse and the lack of clear guidelines for the use of biomaterials, the opinion of the Working Group on Urological Functional Diagnostics and Female Urology should provide clarity. The Opinion is based on the SCENIHR Report of the "European Commission's Scientific Committee on Emerging and Newly Identified Health Risks", the "Consensus Statement of the European Urology Association and the European Urogynaecological Association on the Use of Implanted Materials for Treating Pelvic Organ Prolapse and Stress Urinary Incontinence" and in compliance with relevant EAU and national guidelines and the opinion of the Association for Urogynaecology and Plastic Pelvic Floor Reconstruction (AGUB eV). In addition, recommendations are given for the future handling of implants of slings and meshes for the treatment of stress incontinence and prolapse from a urologic viewpoint.

High-resolution mass spectrometry-based multi-residue method covering relevant steroids, stilbenes and resorcylic acid lactones in a variety of animal-based matrices.

A new, relatively simple sample processing and detection workflow has been developed for the quantification and confirmation of banned growth-promoting substances in a wide variety of animal-based food products. The method covers all required compounds (belonging to the so-called A1, A3, A4 and B2f groups as termed by the relevant EU legislation) which are currently monitored by the official European community surveillance programs. The sample processing includes a thermal sample denaturation step, intended to prevent undesirable side-reactions during the following enzymatic deconjugation of covalently bound analytes. A pH-adjusted dual liquid/liquid-extraction produces sufficient clean extracts for a wide range of matrices (urine, muscle, liver, serum, full blood). The method has been validated using two hybrid quadrupole high-resolution mass spectrometers (Orbitrap and time-of-flight technology-based instruments). Full-scan data acquisition, interlaced with targeted modes (unit mass isolation of the precursors, followed by collision-induced fragmentation), produces sufficiently sensitive and selective detection of the analytes within all the validated matrices. The proposed method is an alternative to currently used methods that are restricted to a limited set of analytes and matrices.

[Medical quality standards for selected urological aids and devices : Consensus for patients with neurogenic urinary bladder dysfunction]. / Medizinische Qualitätsstandards ausgewählter urologischer Hilfsmittel : Konsens für Patienten mit neurogenen Harnblasenfunktionsstörungen.

BACKGROUND: Using the CE mark of therapeutic appliances is, on its own, not sufficient enough for their appropriate and effective application. In order to treat the patient successfully, not jeopardizing the success of the treatment, medical quality criteria for therapeutic appliances care are necessary to acceptably compensate for a patient's disabilities. OBJECTIVES: Medical quality criteria are formulated for the most frequently used urological aids and devices, considering hygienic requirements, international literature and the practical experience of physicians and nurses with regard to the care of patients with neurogenic urinary bladder dysfunction. METHODS: An expert group of urologists, surgeons, rehabilitation physicians and nurses has developed medical quality criteria via a structured consensus procedure. Developing these criteria, the group has taken into account current jurisprudence, the current resource directory of neurourological relevant aids, data from international literature and hygiene requirements. RESULTS: Medical quality requirements are discussed and defined for selected groups of urological devices (single use catheters, indwelling catheters, external catheters, urine bags, templates and diapers as well as devices for the electrostimulation of nerves). CONCLUSION: The presented quality requirements offer the possibility to stabilize quality of care with neurourological relevant therapeutic appliances. The catalogue of therapeutic appliances must be urgently updated. Urinal catheters for single use must be classified as an individual product group. Devices for anterior root stimulation and neuromodulation must be included in the resource directory. The incontinence severity classification needs to be reviewed.

HPV vaccination of immunocompromised hosts.

It is well-established that immunocompromised people are at increased risk of HPV-related disease compared with those who are immunocompetent. Prophylactic HPV sub-unit vaccines are safe and immunogenic in immunocompromised people and it is strongly recommended that vaccination occur according to national guidelines. When delivered to immunocompromised populations, HPV vaccines should be given as a 3-dose regimen.

[Artificial urinary sphincter in women-too uncommon?] / Artifizieller Schließmuskel der Frau ­ zu selten?

The artificial urinary sphincter (AUS) is a well-known standard in the therapy of male incontinence, but not in females. This review highlights options, results, and a critical appraisal of the use of AUS in women.

[Urodynamic studies prior to urinary incontinence surgery : What is useful?] / Urodynamik vor operativer Inkontinenztherapie : Was ist sinnvoll?

Surgery is often necessary after failure of conservative therapy for urinary incontinence. Guidelines recommend urodynamic studies before surgery. A distinction is made between non-invasive (uroflowmetry) and invasive methods (cystometry and pressure-flow study, if necessary as combined videourodynamics, as well as urethral pressure profile). All examinations serve to objectify and quantify the symptoms, to correctly assign symptoms to the pathophysiology and anatomy as well as to identify risk factors, which often have a significant influence on the success of surgical therapy. Given appropriate experience, complications and often significant sequelae of bladder dysfunction affecting the patient's quality of life and life expectancy can be recognized. Urodynamic studies are performed to help narrow down potential diagnoses, to develop therapeutic strategies, and to obtain prognostic parameters. The following article is intended to provide some support.

Markers of the p53 pathway further refine molecular profiling in high-risk endometrial cancer: A TransPORTEC initiative.

BACKGROUND: The morphological classification of high-risk endometrial cancer is of limited prognostic value. Recent attempts to stratify tumours according to molecular signatures have shown considerable promise. Here we attempted to further refine molecular classifications using markers of the p53 pathway. METHODS: We analysed the expression of p53 as well as three downstream markers of the p53 pathway, p21, mdm2 and phospho-p63 (pp63), by immunohistochemistry in a series of 114 endometrial cancers (86 endometrioid, 28 non-endometrioid subtype) with high-risk features (such as high tumour grade and deep myometrial invasion) and correlated results with clinical outcome. The Cancer Genome Atlas (TCGA) data were used to analyse TP63 mutations and copy-number alterations using cBioPortal. TP53 was silenced in two endometrial cancer cell lines to study its effect on p21 and p63. RESULTS: About half of the tumours showed a p53 mutant phenotype and there was a strong negative correlation with p21 expression. Being marker positive for pp63 or mdm2 was associated with a significantly increased likelihood of dying, [hazard ratios 5.93 (95% CI 2.37-7.27) and 7.48 (95% CI 3.04-9.39), respectively]. These findings were seen in both p53 wildtype and p53 mutant tumours. Only 11% of TCGA endometrial cancers had a functional TP63 alteration. Upon silencing of TP53, p21 expression was decreased in one cell line, but no effects on p63 were observed. CONCLUSION: Markers of the p53 pathway improve stratification of endometrial cancers and provide novel insights into the role of this pathway in the disease.

Predicting Outcome in Subarachnoid Hemorrhage (SAH) Utilizing the Full Outline of UnResponsiveness (FOUR) Score.

BACKGROUND: Existing scoring systems for aneurysmal subarachnoid hemorrhage (SAH) patients fail to accurately predict patient outcome. Our goal was to prospectively study the Full Outline of UnResponsiveness (FOUR) score as applied to newly admitted aneurysmal SAH patients. METHODS: All adult patients presenting to Health Sciences Center in Winnipeg from January 2013 to July 2015 (2.5 year period) with aneurysmal SAH were prospectively enrolled in this study. All patients were followed up to 6 months. FOUR score was calculated upon admission, with repeat calculation at 7 and 14 days. The primary outcomes were: mortality, as well as dichotomized 1- and 6-month Glasgow Outcome Scale (GOS) and modified Rankin Scale (mRS) values. RESULTS: Sixty-four patients were included, with a mean age of 54.2 years (range 26-85 years). The mean FOUR score upon admission pre- and post-external ventricular drain (EVD) was 10.3 (range 0-16) and 11.1 (range 3-16), respectively. There was a statistically significant association between pre-EVD FOUR score (total, eye, respiratory and motor sub-scores) with mortality, 1-month GOS, and 6-month GOS/mRS (p < 0.05 in all). The day 7 total, eye, respiratory, and motor FOUR scores were associated with mortality, 1-month GOS/mRS, and 6-month GOS/mRS (p < 0.05 in all). The day 14 total, eye, respiratory, and motor FOUR scores were associated with 6-month GOS (p < 0.05 in all). The day 7 cumulative FOUR score was associated with the development of clinical vasospasm (p < 0.05). CONCLUSIONS: The FOUR score at admission and day 7 post-SAH is associated with mortality, 1-month GOS/mRS, and 6-month GOS/mRS. The FOUR score at day 14 post-SAH is associated with 6-month GOS. The brainstem sub-score was not associated with 1- or 6-month primary outcomes.

[Neuro-urological diagnosis and therapy of lower urinary tract dysfunction in patients with spinal cord injury : S2k Guideline of the German-Speaking Medical Society of Paraplegia (DMGP), AWMF register no. 179/001]. / Neuro-urologische Diagnostik und Therapie bei Funktionsstörungen des unteren Harntrakts nach einer Rückenmarkschädigung : S2k-Leitlinie der Deutschsprachigen Medizinischen Gesellschaft für Paraplegie (DMGP), AWMF-Register Nr: 179/001.

BACKGROUND: Most patients with spinal cord injury (SCI) suffer from neurogenic lower urinary tract dysfunction (NLUTD), bowel dysfunction and sexual dysfunction. If these remain untreated, severe medical complications and serious limitations (restrictions) in quality of life are imminent. OBJECTIVES: In the long term, there are considerable differences in the treatment results of highly specialized centers versus other treatment facilities. MATERIALS AND METHODS: Against this background, a consensus-based guideline, according to the AWMF (Association of the Scientific Medical Societies in Germany) criteria (S2k), was developed by the neuro-urology working group of the DMPG (German-Speaking Medical Society of Paraplegia). RESULTS: The guideline defines the principles and objectives of the neuro-urological care of patients with SCI and discusses in detail the principles of diagnosis and therapy of NLUTD. The need for video-urodynamic studies as a basis for the classification of the NLUTD and as a foundation for the development of a treatment strategy is emphasized. Both conservative and surgical therapy options and their indications are explained in detail. Possible complications and their prevention in the long-term course of SCI are presented with a particular consideration of the specific features of urinary tract infections and autonomic dysreflexia. Finally, the principles of the provision of urological appliances are discussed. CONCLUSIONS: The presented S2k guideline provides the current standards in the neuro-urological care of patients with NLUTD due to SCI. Their consistent implementation both in the acute and chronic phase as well as in the context of lifelong surveillance of SCI patients should prevent the impending complications of NLUTD.

[Objective and subjective requirement of aids and appliances in patients with neurogenic lower urinary tract dysfunction : Multicenter study to determinate the daily necessity of urological aids and appliances]. / Objektiver und subjektiver Hilfsmittelbedarf bei Patienten mit neurogenen Harnblasenfunktionsstörungen : Multicenterstudie zur Ermittlung des täglichen Bedarfs an urologischen Hilfsmitteln.

BACKGROUND: The provision of urological appliances for patients with neurogenic lower urinary tract dysfunction (NLUTD) is essential. Hitherto existing standard guidelines for the estimation of monthly material requirements are based solely on estimates. OBJECTIVE: The goal of this work was to define the objective and subsequently subjective requirements for urological appliances on a scientifically validated basis. MATERIALS AND METHODS: Data concerning bladder management and daily consumption of urological appliances for patients with NLUTD were collected through a standardized survey at six different centers in Germany during the period of October to December 2014 and statistically evaluated. RESULTS: In all, 767 patient records were analyzed: 543 men and 221 woman (N/A = 3). The daily disposable catheter consumption of 577 patients who exclusively used intermittent catheterization was 5.13. Patients who used other means of bladder emptying (n = 31) in addition to catheterization consumed on average 3.17 catheters. The margin of deviation was larger for children. Of the 608 patients with intermittent catheterization, 94 (15.5 %) required additional paddings as absorbent aids (on average 2.29 paddings per day), 34 patients (5.6 %) additionally used pants (2.55 per day) and 46 patients (7.6 %) utilized condom catheters (3.81 per day) between catheterization. Among all surveyed patients, 126 (16.4 %) used paddings (5.03 per day) and 51 patients (6.6 %) pants (3.03 per day). Of all male respondents 82 (15.1 %) used condom catheters (2.80 urinary sheaths per day). CONCLUSION: Applying twice the standard deviation of the mean as a measure of assessing the objective requirement of urological appliances and aids for adult patients with NLUTD allows the following daily thresholds to be defined: 1-9 disposable catheters, 0-7 urinary sheaths, 1-9 paddings and 0-7 pants. These thresholds can serve as a basis for estimating the subjective need. They allow for a scientifically validated benchmark for an economically feasible and patient-tailored supply with urological aids and appliances. Individually required appliances and aids have to be recognized. Verifiable quality standards need to be developed.

[Not Available]. / Therapie der weiblichen OAB.

The syndrome of overactive bladder (OAB) with the symptoms of frequency, urgency and nocturia is often seen. After the diagnosis has been made by careful exclusion of other conditions, there are different symptomatic treatment strategies available. These include drug treatment as well as minimally invasive local surgical treatments and treatments which intervene with the control of the bladder by modulating central nervous areas. Alternative methods such as acupuncture may help in individual cases; the placebo effect is high and there is a lack of controlled studies.