Fermentation of cocoa pod husks with Pleurotus salmoneo-stramineus for food applications.

Cocoa pod husks (CPHs), the major side-stream from cocoa production, were valorized through fermentation with Pleurotus salmoneo-stramineus (PSS). Considering ergosterol as a biomarker for the fungal content, the mycelium accounted for 54% of the total biomass after 8 days in submerged cultures. The crude protein content of fermented CPH (CPHF) increased from 7.3 g/100 g DM in CPH to 18.9 g/100 g DM. CPH fermentation resulted in a high biological value of 86 for the protein. The water and oil binding capacities of CPHF were 3.5 mL/g and 2.1 mL/g, respectively. The particle diameter dv,0,90 of CPHF was 373 µm as compared to 526 µm for CPH. The total dietary fiber was 73.4 g/100 g DM in CPHF and 63.6 g/100 g DM in CPH. The amount of soluble fiber was 2.3 g/100 g DM in CPHF and 10.1 g/100 g DM in CPH; the insoluble fraction accounted for 71.1 g/100 g DM and 53.6 g/100 g DM, respectively. Bread doughs with CPH or CPHF were characterized for texture, color, and farinographic properties. The dough hardness, consistency, and browning index increased with the concentration of CPH, whereas for CPHF, springiness and peak viscosities declined. We demonstrate the upcycling of CPH into nutritious and functional ingredients through PSS fermentation.

Feasibility of His bundle pacing facilitated by EASI derived 12­lead ECG.

INTRODUCTION: His bundle pacing (HBP) has become popular in recent years as a more physiological alternative to conventional right ventricular pacing. Implantation requires 12­lead ECG during surgery, which is not readily available in a standard operating room. Often but not always HBP is performed in an electrophysiology lab. EASI is a reduced lead system which enables derived 12­lead ECG. EASI derived 12­lead ECGs on modern tablet computers offer a more mobile and lightweight ECG solution which does not obstruct fluoroscopy during implantation. This case series aims to compare standard 12­lead ECG to EASI derived 12­lead ECG in patients undergoing HBP implantation. METHODS AND RESULTS: A total of 11 patients received permanent HBP guided only by fluoroscopy, a pacing system analyzer (Medtronic CareLink SmartSync Device Manager) and EASI derived 12­lead ECG (CardioSecur Pro). During the first postoperative device interrogation HBP criteria, as defined in the EHRA consensus paper on conduction system pacing, were evaluated with the EASI derived system as well as a standard 12­lead ECG and compared to each other. There was perfect agreement with regards to these criteria which lead to identical conclusions in all cases. CONCLUSION: HBP implantation can be performed with EASI derived 12­lead ECG instead of conventional 12­lead ECG. Criteria for discriminating between selective His bundle, non-selective His bundle or myocardial capture alone are clearly visible in the EASI derived ECG leading to the same conclusion when compared to standard 12­lead ECG. Compared to a conventional 12­lead ECG the EASI system offers a leaner setup with less visual obstruction on fluoroscopy.

Biocatalytic Production of Odor-Active Fatty Aldehydes from Fungal Lipids.

Odor-active fatty aldehydes are important compounds for the flavor and fragrance industry. By a coupled enzymatic reaction using an α-dioxygenase (α-DOX) and an aldehyde dehydrogenase (FALDH), scarcely available aldehydes from the biotransformation of margaroleic acid [17:1(9Z)] were characterized and have shown highly interesting odor profiles, including citrus-like, soapy, herbaceous, and savory notes. In particular, (Z)-8-hexadecenal and (Z)-7-pentadecenal exhibited notable meaty odor characteristics. Submerged cultivation of Mortierella hyalina revealed the accumulation of the above-mentioned, naturally uncommon fatty acid 17:1(9Z). Its production was significantly increased by the modulation of culture conditions, whereas the highest accumulation was observed after 4 days at 24 °C and l-isoleucine supplementation. The lipase-, α-DOX-, and FALDH-mediated biotransformation of M. hyalina lipid extract resulted in a complex aldehyde mixture with a high aldehyde yield of ∼50%. The odor qualities of the formed aldehydes were assessed by means of gas chromatography-olfactometry, and several of the obtained fatty aldehydes have been sensorially described for the first time. To assess the aldehyde mixture's potential as a flavor ingredient, a sensory evaluation was conducted. The obtained product exhibited intense citrus-like, green, and soapy odor impressions.

An enzymatic tandem reaction to produce odor-active fatty aldehydes.

Aldehydes represent a versatile and favored class of flavoring substances. A biocatalytic access to odor-active aldehydes was developed by conversion of fatty acids with two enzymes of the α-dioxygenase pathway. The recombinant enzymes α-dioxygenase (α-DOX) originating from Crocosphaera subtropica and fatty aldehyde dehydrogenase (FALDH) from Vibrio harveyi were heterologously expressed in E. coli, purified, and applied in a coupled (tandem) repetitive reaction. The concept was optimized in terms of number of reaction cycles and production yields. Up to five cycles and aldehyde yields of up to 26% were achieved. Afterward, the approach was applied to sea buckthorn pulp oil as raw material for the enzyme catalyzed production of flavoring/fragrance ingredients based on complex aldehyde mixtures. The most abundant fatty acids in sea buckthorn pulp oil, namely palmitic, palmitoleic, oleic, and linoleic acid, were used as substrates for further biotransformation experiments. Various aldehydes were identified, semi-quantified, and sensorially characterized by means of headspace-solid phase microextraction-gas chromatography-mass spectrometry-olfactometry (HS-SPME-GC-MS-O). Structural validation of unsaturated aldehydes in terms of double-bond positions was performed by multidimensional high-resolution mass spectrometry experiments of their Paternò-Büchi (PB) photoproducts. Retention indices and odor impressions of inter alia (Z,Z)-5,8-tetradecadienal (Z,Z)-6,9-pentadecadienal, (Z)-8-pentadecenal, (Z)-4-tridecenal, (Z)-6-pentadecenal, and (Z)-8-heptadecenal were determined for the first time. KEY POINTS: • Coupled reaction of Csα-DOX and VhFALDH yields chain-shortened fatty aldehydes. • Odors of several Z-unsaturated fatty aldehydes are described for the first time. • Potential for industrial production of aldehyde-based odorants from natural sources.

BMP9 a possible alternative drug for the recently withdrawn BMP7? New perspectives for (re-)implementation by personalized medicine.

Promotion of rhBMP2 and rhBMP7 for the routine use to support fracture healing has been hampered by high costs, safety concerns and reasonable failure rates, imposing restrictions in its clinical use. Since there is little debate regarding its treatment potential, there is rising need for a better understanding of the mode of action of these BMPs to overcome its drawbacks and promote more efficacious treatment strategies for bone regeneration. Recently, BMP9, owing to its improved osteogenic potential, is gaining attention as a promising therapeutic alternative. Our study aimed at identifying specific gene expression patterns which may predict and explain individual responses to rhBMP7 and rhBMP9 treatments. Therefore, we investigated the effect of rhBMP7 and rhBMP9 on primary human osteoblasts from 110 donors and corresponding THP-1-derived osteoclasts. This was further compared with each other and our reported data on rhBMP2 response. Based on the individual donor response, we found three donor groups profiting from rhBMP treatment either directly via stimulation of osteoblast function or viability and/or indirectly via inhibition of osteoclasts. The response on rhBMP7 treatment correlated with expression levels of the genes BAMBI, SOST, Noggin, Smad4 and RANKL, while the response of rhBMP9 correlated to the expression levels of Alk6, Endoglin, Smurf1, Smurf2, SOST and RANKL in these donors. Noteworthy, rhBMP9 treatment showed significantly increased osteogenic activity (AP activity and Smad nuclear translocation) when compared to the two clinically used rhBMPs. Based on patient's respective expression profiles, clinical application of rhBMP9 either solely or in combination with rhBMP2 and/or rhBMP7 can become a promising new approach to fit the patient's needs to promote fracture healing.

Distinct Gene Expression Patterns Defining Human Osteoblasts' Response to BMP2 Treatment: Is the Therapeutic Success All a Matter of Timing?

BACKGROUND: Bone morphogenetic proteins (BMPs) play a key role in bone formation. Local application of BMP2 (Dibotermin alfa) supports bone formation when applied to complex fractures. However, up to 33% of patients do not respond to this therapy. PURPOSE: Aiming to investigate whether inter-individual responses to BMP2 treatment can be predicted by gene expression patterns, we investigated the effect of BMP2 on primary human osteoblasts and THP-1 cell-derived osteoclasts from 110 donors. METHODS: Osteoblasts were obtained by collagenase digestion of spongy bone tissues. Osteoclasts were differentiated from THP-1 cells using the conditioned media of the osteoblasts. Viability was determined by resazurin conversion. As functional characteristics AP and Trap5B activity were measured. Gene expression levels were determined by RT-PCR in 21 of the 110 evaluated donors and visualized by electrophoresis. RESULTS: Based on our data, we could classify three response groups: (i) In 51.8% of all donors, BMP2 treatment induced osteoblast function. These donors strongly expressed the BMP2 inhibitor Noggin (NOG), the alternative BMP2 receptors repulsive guidance molecule B (RGMb) and activin receptor-like kinase 6 (Alk6), as well as the Wnt inhibitor sclerostin (SOST). (ii) In 17.3% of all donors, BMP2 treatment induced viability. In these donors, the initial high SOST expression significantly dropped with BMP2 treatment. (iii) 30.9% of all donors were not directly affected by BMP2 treatment. These donors expressed high levels of the pseudoreceptor BMP and activin membrane-bound inhibitor (BAMBI) and lacked SOST expression. In all donors, SOST expression correlated directly with receptor activator of NF-κB ligand (RANKL) expression, defining the cells' potential to stimulate osteoclastogenesis. CONCLUSIONS: Our data identified three donor groups profiting from BMP2 treatment either directly via stimulation of osteoblast function or viability and/or indirectly via inhibition of osteoclastogenesis, depending on their expression of BAMBI, SOST, NOG, and RANKL. On the basis of patients' respective expression profiles, the clinical application of BMP2 as well as its timing might be modified in order to better fit the patients' needs to promote bone formation or to inhibit bone resorption.

Induction of Anti-Tumor Immune Responses by Peptide Receptor Radionuclide Therapy with (177)Lu-DOTATATE in a Murine Model of a Human Neuroendocrine Tumor.

Peptide receptor radionuclide therapy (PRRT) is a relatively new mode of internally targeted radiotherapy currently in clinical trials. In PRRT, ionizing radioisotopes conjugated to somatostatin analogues are targeted to neuroendocrine tumors (NETs) via somatostatin receptors. Despite promising clinical results, very little is known about the mechanism of tumor control. By using NCI-H727 cells in an in vivo murine xenograft model of human NETs, we showed that (177)Lu-DOTATATE PRRT led to increased infiltration of CD86+ antigen presenting cells into tumor tissue. We also found that following treatment with PRRT, there was significantly increased tumor infiltration by CD49b+/FasL+ NK cells potentially capable of tumor killing. Further investigation into the immunomodulatory effects of PRRT will be essential in improving treatment efficacy.

Transforming growth factor ß1 inhibits bone morphogenic protein (BMP)-2 and BMP-7 signaling via upregulation of Ski-related novel protein N (SnoN): possible mechanism for the failure of BMP therapy?

BACKGROUND: Bone morphogenic proteins (BMPs) play a key role in bone formation. Consequently, it was expected that topical application of recombinant human (rh)BMP-2 and rhBMP-7 would improve the healing of complex fractures. However, up to 36% of fracture patients do not respond to this therapy. There are hints that a systemic increase in transforming growth factor ß1 (TGFß1) interferes with beneficial BMP effects. Therefore, in the present work we investigated the influence of rhTGFß1 on rhBMP signaling in primary human osteoblasts, with the aim of more specifically delineating the underlying regulatory mechanisms. METHODS: BMP signaling was detected by adenoviral Smad-binding-element-reporter assays. Gene expression was determined by reverse transcription polymerase chain reaction (RT-PCR) and confirmed at the protein level by western blot. Histone deacetylase (HDAC) activity was determined using a test kit. Data sets were compared by one-way analysis of variance. RESULTS: Our findings showed that Smad1/5/8-mediated rhBMP-2 and rhBMP-7 signaling is completely blocked by rhTGFß1. We then investigated expression levels of genes involved in BMP signaling and regulation (for example, Smad1/5/8, TGFß receptors type I and II, noggin, sclerostin, BMP and activin receptor membrane bound inhibitor (BAMBI), v-ski sarcoma viral oncogene homolog (Ski), Ski-related novel protein N (SnoN) and Smad ubiquitination regulatory factors (Smurfs)) and confirmed the expression of regulated genes at the protein level. Smad7 and SnoN were significantly induced by rhTGFß1 treatment while expression of Smad1, Smad6, TGFßRII and activin receptor-like kinase 1 (Alk1) was reduced. Elevated SnoN expression was accompanied by increased HDAC activity. Addition of an HDAC inhibitor, namely valproic acid, fully abolished the inhibitory effect of rhTGFß1 on rhBMP-2 and rhBMP-7 signaling. CONCLUSIONS: rhTGFß1 effectively blocks rhBMP signaling in osteoblasts. As possible mechanism, we postulate an induction of SnoN that increases HDAC activity and thereby reduces the expression of factors required for efficient BMP signaling. Thus, inhibition of HDAC activity may support bone healing during rhBMP therapy in patients with elevated TGFß serum levels.

Hyperinsulinemia reduces osteoblast activity in vitro via upregulation of TGF-ß.

Affecting more than 230,000,000 patients, diabetes mellitus is one of the most frequent metabolic disorders in developed countries. Among other complications, diabetic patients have an increased fracture risk and show delayed fracture healing. During the disease progression, these patients' blood glucose and insulin levels vary significantly. Thus, the aim of this study was to analyze the effects of glucose and insulin on primary human osteoblasts. Although, in the presence of insulin and glucose, proliferation of osteoblasts was increased (1.2- to 1.7-fold), their alkaline phosphatase activity and, consequently, production of mineralized matrix were significantly reduced down to 55 % as compared to control cells (p < 0.001). Interestingly, the observed effects were mainly due to stimulation with insulin. Increase in glucose did not alter osteoblasts' function significantly but further enhanced the effects of insulin. Expression of active and total transforming growth factor beta (TGF-ß) was increased by glucose and insulin. Stimulation with both glucose and insulin induced gene expression changes (e.g., osteocalcin, Runx2, Satb2, or Stat1) comparable to treatment with recombinant TGF-ß(1), further indicating osteoblasts' dysfunction. Inhibition of TGF-ß signaling completely abolished the negative effects of glucose and insulin. In summary, glucose and insulin treatment causes osteoblast dysfunction, which is accompanied by an increased TGF-ß expression. Blocking TGF-ß signaling abrogates the functional loss observed in glucose- and insulin-treated osteoblasts, thus identifying TGF-ß as a key regulator. Therefore, increased TGF-ß expression during diabetes may be a feasible pathogenic mechanism underlying poor bone formation in uncontrolled diabetes mellitus.

Peptide receptor radionuclide therapy with Y-DOTATOC and (177)Lu-DOTATOC in advanced neuroendocrine tumors: results from a Danish cohort treated in Switzerland.

AIM: Limited therapeutic options have highlighted the demand for new treatment modalities for patients with advanced neuroendocrine tumors (NET). Promising results of initial studies have warranted the implementation of peptide receptor radionuclide therapy (PRRT) in clinical practice. However, this treatment option still needs clinical evaluation. METHODS: In this study, we evaluated the PRRT treatment response of 69 Danish patients with NET mainly originating from the gastroenteropancreatic system. Fifty-six patients (81%) were referred for PRRT to the Department of Nuclear Medicine, University Hospital Basel, Switzerland, between 2004 and 2008 due to progression assessed by the referring physicians. However, when retrospectively evaluated, only 42 of the 69 patients (61%) had progression according to RECIST (Response Evaluation Criteria in Solid Tumors). Most patients were treated with 9°Y-DOTATOC. RESULTS: Based on RECIST, a complete response was observed in 5 patients (7.4%), a partial response in 11 patients (16.2%) and stable disease in 42 patients (61.8%). Progressive disease after completed therapy was observed in 10 patients (14.7%). The median progression-free survival was 29 months (95% CI: 22-36 months). Pancreatic NET seemed to respond better to PRRT than small intestinal carcinoid tumors (p = 0.03). The overall frequency of serious adverse events was low. CONCLUSION: Implementation of PRRT in clinical routine has provided a valuable new therapeutic option for the treatment of advanced NET. We suggest that PRRT may advance from second- or third-line to first- or second-line therapy in inoperable/unresectable NET patients.

[Surgical procedures in patients treated with platelet function inhibitors]. / Therapie mit Thrombozytenfunktionshemmern bei operativen Eingriffen.

The platelet function inhibitors (PFI) acetylsalicylic acid (ASA) and clopidogrel are widely used in a broad spectrum of atherothrombotic diseases, either as mono- or dual antiplatelet therapy. Platelet function is inhibited for the whole lifespan of platelets (10 days). In case of surgical procedures the bleeding risk under continued antiplatelet therapy has to be balanced against the risk of ischemic complications due to withdrawal of antiplatelet therapy. Especially after stent implantation, the high risk and unfavorable prognosis of stent thrombosis have to be considered. Whereas surgical procedures with a low bleeding risk may be performed with continued antiplatelet therapy, there is a need for partial or total discontinuation of antiplatelet therapy in surgical procedures with higher bleeding risks.

[Pulmonary consequences of marijuana smoking]. / Pulmonale konsekvenser af hashrygning.

Based on previously published studies, this review describes the pulmonary consequences of marijuana smoking. Smoking of marijuana is significantly associated with chronic bronchitis (cough and phlegm), but it has not been firmly established whether it also leads to a reduction in lung function. Both epidemiological studies and case reports suggest that regular smokers of marijuana have a higher risk of developing malignancies in both the upper and lower airways. Smoking of marijuana contaminated with fungus spores has been reported to lead to pulmonary aspergillus infections in immunocompromised patients, and sharing of marijuana water pipes has been associated with transmission of tuberculosis.

Proarrhythmic effect of pacemaker stimulation in patients with implanted cardioverter-defibrillators.

BACKGROUND: We sought to determine the potential of right ventricular VVI backup pacing to induce ventricular tachyarrhythmias in patients with implanted cardioverter-defibrillators. METHODS AND RESULTS: All consecutive patients presenting exclusively with pacemaker-induced tachycardias (PITs) were included in a prospective study using a crossover protocol. Patients were randomized to either group 1 (augmentation of the baseline frequency of the pacemaker to 60 bpm) or group 2 (pacemaker turned off) and were followed up for 1 year and then crossed over to the other programming, looking for reoccurrence of PIT. Of 150 consecutive patients, 39 (26%) had PIT, 13 of them exclusively (8.6%). Forty of 1063 analyzed tachyarrhythmias of all the patients were PIT (3%). Before inclusion in the study, the patients had 2.7+/-0.9 PITs in 11+/-6.5 months with their pacemakers programmed empirically at 42.3 bpm. During the study phase, no PIT occurred while the pacemaker was turned off, whereas programming to 60 bpm led to the recurrence of PIT in 5 of 6 patients (1.4+/-0.6 per patient). At the end of the study, 9 patients underwent a prolonged follow-up with their pacemakers turned off, resulting in spontaneous episodes of ventricular tachycardia/fibrillation in 5 patients, but PITs were no longer observed. CONCLUSIONS: This crossover protocol proves the potential proarrhythmic effect of pacemaker stimulation in implantable cardioverter-defibrillator patients. Resulting PITs led to clinical symptoms and antitachycardia therapy by the implantable cardioverter-defibrillator. Thus, in patients presenting with PIT but without a pacemaker indication, the pacemaker feature should be turned off, or, alternatively, the longest possible escape interval should be programmed.

An interaction between ATP and high K+: mutual impairment of ATP- and high K(+)-evoked [Ca2+]i increase in NG 108-15 cells.

The interaction between ATP- and high K(+)-evoked increase in intracellular free calcium concentration ([Ca2+]i) was investigated to gain an insight into the mechanism of interaction of ATP with voltage-sensitive calcium channels. [Ca2+]i was measured in the neuronal model, neuroblastoma x glioma hybrid cells (NG 108-15), using the fluorescence indicator fura-2. In the presence of 1.8 mM extracellular Ca2+, ATP induced a rapid, concentration-dependent increase in [Ca2+]i. High K+ (50 mM) evoked a [Ca2+]i rise from 109 +/- 11 nM to 387 +/- 81 nM (n = 16). The application of either of these two [Ca2+]i-increase provoking agents in sequence with the other caused impairment of the latter effect. The mutual desensitization of the responses to ATP and high K+ strongly suggests that both agents rely at least in part on the same source of Ca2+ for elevation of [Ca2+]i in NG 108-15 cells.