10 mice, TLR4-deficient (both Jackson Laboratory, Bar Harbor, ME, USA), OT-II mice (from Dr. William Heath, Melbourne), and FcγR-deficient B6 mice, purchased from Taconic (Germantown, NY, USA), were used throughout the study. FcγR-deficient mice lack the γ-chain subunit of the FcγRIII and FcεRI receptors. The

deleted γ-chain is also associated with FcγRI. The deleted γ-chain subunit is essential for receptor assembly, signal transduction and cell surface expression of FcγRIII and FcεRI molecules 32. Mice were fed with OVA-free laboratory food and tap water ad libitum, and kept in a regular 12 h dark/light cycle at a temperature of 21±2°C. All experimental procedures were performed according to a protocol approved by the appropriate governmental authority and ethics committees. The mice were sensitized with OVA (10 μg, Grade VI, Sigma, Deisenhofen, Germany) or PBS absorbed to aluminium hydroxide Lenvatinib cell line (1.5 mg, Pierce

selleck inhibitor Biotechnology, Rockford, IL, USA) by i.p. injection on days 1, 14 and 21. On days 28 and 29, all mice were challenged with 1% OVA dissolved in PBS for 20 min. Allergen exposition was performed by dispersing of the relevant agent using a jet nebulizer, LC Star, 2.8 μm mass median aerodynamic diameter (Pari, Starnberg, Germany) in a closed plexiglass box, in which mice could move freely. To generate antigen-specific Th2-biased DO11.10 cells, T cells were obtained from LN and enriched and co-cultured with purified DC from BALB/c mice pulsed with G protein-coupled receptor kinase OVA323–339 peptide (Biosyntan, Berlin, Germany) in complete medium containing IL-4, IL-2, and anti-IFN-γ. Five days later, Th2-biased DO11.10 cells were quantified and 3–4×106 were adoptively transferred i.v. into BALB/c recipients 4. On three consecutive days, mice were challenged i.n. with PBS, 100 μg rabbit anti-OVA IgG (MP Biomedicals Germany, Heidelberg, Germany) (control groups), 25 μg OVA, or OVA-IC (made by mixing a 1:4 ratio of 25 μg OVA and anti-OVA IgG). Twenty-four hours after

the last challenge, lung function was analyzed and mice were dissected. Total cell counts in BALF were scored using a Neubauer chamber (Brand, Wertheim, Germany). Leukocyte subsets (eosinophils, neutrophils, macrophages or lymphocytes) were counted in BALF using cytospins (centrifuged preparations) stained with Diff-Quik (Medion Diagnostics, Düningen, Germany). A total of 400 cells were counted in each sample. Twenty-four hours after the last airway challenge, lungs were fixed with 4% formalin and embedded in paraffin. The paraffin blocks were cut into 4 μm slices and stained with hematoxilin/eosin (Merck, Darmstadt, Germany). From each mouse lung, six sections (containing hiliar structures and periphery) of the right and left lung were evaluated. Microphotographs were performed using a Nikon Eclipse 50i microscope with a Nikon Digital Sight DS-U1 Camera.

Kinase suppressor of Ras 1 (KSR1) was originally identified as a positive regulator of Ras signaling

in Caenorhabditis elegans and Drosophila and homologues were subsequently discovered in mammals 15–17. Further studies demonstrated that KSR1 is a scaffold molecule that binds critical components of the MAPK cascade and is essential for ERK activation X-396 molecular weight in a variety of cell types 18. In the immune system, KSR1 is critical for the production of pro-inflammatory cytokines by innate immune cells in response to stress signals and required for efficient activation of peripheral T cells 18, 19. Little is known, however, about the role of KSR1 in the development of T cells, although a cursory examination revealed no gross abnormalities 18. In this study, we examined the role of KSR1 in thymocyte development. As expected, KSR1 deletion resulted in impairment of

ERK activation in thymocytes following TCR stimulation. Interestingly, this diminished ERK activation had only minimal effects on T-cell development. Positive selection was normal in both KSR1−/− AND (CD4+) and HY (CD8+) TCR transgenic mice. Negative selection also appeared normal in KSR1−/− AND mice, but was slightly impaired in male HY KSR1−/− mice. Negative selection in a third model of negative selection, endogenous superantigen deletion, also appeared normal. These data indicate that a minimal amount of ERK activation may be selleck chemicals llc sufficient to sustain thymocyte maturation and that strong activation of ERK may only be required for negative selection of certain TCR expressing thymocytes. KSR1 has been shown to be required for the efficient activation of ERK in a number of cell types Cediranib (AZD2171) 18–22. We previously reported a defect in ERK activation in peripheral

T cells in response to PMA or CD3-crosslinking 18. To determine the extent to which ERK activation in thymocytes also requires KSR1, we stimulated KSR1 WT or knockout thymocytes with PMA (Fig. 1A) or anti-CD3 (Fig. 1B) for various time points, lysed the cells and measured the level of activated ERK using an ERK phospho-specific antibody. As expected, there was a significant defect in ERK activation in KSR1−/− thymocytes downstream of both stimuli. Interestingly, we noted that the defect after PMA stimulation was reproducibly always more significant than after CD3 stimulation. We quantified the ERK activation defect using flow cytometric analysis using the phospho-ERK antibody (Fig. 1C–F). This also allowed us to measure the ERK activation defect in individual thymocyte subsets. The analysis confirmed that there is a significant ERK activation defect after PMA activation and that it is more significant than the defect after CD3 activation (Fig. 1C–F). The ERK activation defect in KSR1−/− thymocytes appeared to be greatest in CD4 and CD8 SP with a smaller but consistent defect in the DN and DP subsets.

Background: Worldwide, more people are receiving anti-TNFα for rheumatologic disorders. There have been a small but significant number of reports regarding https://www.selleckchem.com/products/Erlotinib-Hydrochloride.html its relationship with renal disease. Case Report: A 55 year old lady presented in August 2011 with polyarthralgia and diagnosed to have HLA-B27 positive arthritis. She had no evidence of renal disease at initial presentation. She was treated initially with disease modifying drugs including Prednisolone, Methotrexate, Sulfasalazine and Leflunomide. She also had a history of bronchiectasis, multinodular goitre and haemochromatosis. In March 2012 she represented with an exacerbation

of arthritis with anorexia, significant weight loss and uveitis. She was found to have microscopic haematuria and proteinuria with negative autoimmune and vasculitic screens. Her renal biopsy was suggestive of early focal segmental glomerulosclerosis and was started on ACEI. During the course of her therapy she was commenced on Etarnecept initially and later Adalimumab with improvement in her arthritis and uveitis. However she was noted to have episodic recurrent acute elevation of serum creatinine in July 2013 and again in October 2013, each time coinciding with anti-TNFα injection.

Adalimumab treatment was discontinued in view of temporal association with episodic renal dysfunction. Since then her renal functions were stabilized Navitoclax mw with reduced proteinuria. Conclusions: We presented a case of de novo glomerulonephritis with acute exacerbations related to anti-TNFα

therapy. In most cases, renal condition improves with withdrawal of therapy as is seen in our patient. High index of suspicion is required when patients are receiving these newer products for presence of renal disease. 302 ACUTE KIDNEY INJURY AND ISCHEMIC ACUTE HEPATIC FAILURE AFTER CONSUMPTION OF JAVA BARB FISH GALLBLADDER IN WEST JAVA, INDONESIA M RUDIANSYAH1, RS GONDODIPUTRO2, R BANDIARA2, AH MARTAKUSUMAH2, R SUPRIYADI2, A AFIATIN2 1Division of Nephrology & Hypertension, Department of Internal Medicine, Faculty of Medicine, Universitas Lambung next Mangkurat/Ulin Hospital Banjarmasin; 2Division of Nephrology & Hypertension, Department of Internal Medicine, Faculty of Medicine, Universitas Padjajaran/Hasan Sadikin Hospital Bandung, Indonesia Introduction: Fish gallbladder is usually consumed in Asian countries as a traditional medicine. It improves fatigueness, arthritis, and erectile dysfunction. In Tasikmalaya, West Java, Indonesia, people believe if they consume fresh fish gallbladder of Java Barb (Barbonymus gonionotus) or so called “Tawes” will improves their healthy. In some reports, eating java barb can causes systemic toxicities such as acute kidney injury and acute hepatic failure.

No wound complications occurred, and all patients could resume oral intake. The cephalic vein is a more reliable recipient vein than is the internal mammary vein. The skin graft-covered pectoralis major muscle flap provides secure external coverage to prevent anastomotic leakage

even in complicated cases. Combined use of the cephalic vein and the skin graft-covered pectoralis major muscle flap is a versatile option for secondary thoracic esophageal reconstruction. © 2013 Wiley Periodicals, Inc. Microsurgery 34:319–323, 2014. “
“Treatment of recurrent carpal tunnel syndrome (CTS) is challenging, this website especially in a case with recurrent CTS and a neuroma formation. Resection of the neuroma causing the syndrome, reconstruction of the nerve gap of the median nerve, and covering up the reconstructed median nerve with well-vascularized soft tissue for prevention of CTS re-recurrence are the essential learn more procedures. We report a case of recurrent CTS with severe pain due to a neuroma-in-continuity successfully treated using a free anterolateral thigh (ALT) flap with a vascularized lateral femoral cutaneous nerve (LFCN). A 2 cm neuroma existed in the median nerve and was resected.

The nerve gap was repaired using a vascularized LFCN included in the ALT flap. The ALT flap was transferred to the wrist to cover the median nerve. The severe pain disappeared completely and the sensory and motor impairment of the median nerve improved 5 months after the free flap surgery, as the Tinel’s sign

moved distally away from the wrist and disappeared. The result of the Semmes-Weinstein test improved from 5.08 to 4.31 and she was able to flex and extend the right wrist and fingers without pain. CTS did not recur 15 months after the surgery. A free ALT flap with vascularized LFCN allows nerve reconstruction for the median nerve gap created after neuroma resection and coverage of the median nerve with well-vascularized soft tissue to prevent adhesion and CTS recurrence. © 2013 Wiley Periodicals, Inc. Microsurgery 34:145–148, 2014. “
“This report describes two incidental findings of aberrant MycoClean Mycoplasma Removal Kit branches of the radial digital nerves in the middle finger of a 52-year-old man who cut himself with a grinding machine, and in the index finger of a 45-year-old female who sustained a flexor sheath infection following a dog bite. In both patients, two equally sized radial digital nerves were found and both nerves originated from one common digital nerve. © 2010 Wiley-Liss, Inc. Microsurgery, 2010. “
“Although increasingly rare, failed microsurgical flaps are a complicated clinical problem when they occur. Review of reports of management following microsurgical flap failure offers an outline of options. A substantial number of breast and extremity patients elect abandonment of reconstruction. The majority of head and neck, breast, and extremity patients proceed to nonmicrosurgical reconstructive options.

Infants who engaged in more shared focus and turn taking looked more to the program than infants who interacted less with their caregivers. These results are discussed in terms of social mediation of coviewing during early infancy. “
“To examine key parameters of

the initial conditions in early category learning, two studies compared 5-month-olds’ object categorization between tasks involving previously unseen novel objects, and between measures within tasks. Infants in Experiment 1 participated in a visual familiarization–novelty preference (VFNP) task with two-dimensional (2D) stimulus images. Infants provided no evidence of categorization by Ivacaftor mouse either their looking or their examining even though infants in previous research systematically categorized the same objects by examining when they could handle them directly. Infants in Experiment 2 participated in a VFNP task with 3D stimulus objects that allowed visual examination of objects’ 3D instantiation while denying manual contact with the objects. Under these conditions, infants demonstrated categorization by

examining but not by looking. Focused examination appears to be a key component of young infants’ ability to form category representations of novel objects, and 3D instantiation appears to better engage such examining. “
“This study examines the relationship between various basic mental processing abilities in infancy. Two groups of 7-month-olds received the same Tipifarnib concentration delayed-response task to assess visuo-spatial working memory, but two different habituation–dishabituation tasks to assess processing speed and recognition memory. The single-stimulus group (N = 32) was familiarized with only one abstract stimulus, whereas the categorization group (N = 32) received varying exemplars of the Parvulin same kind. In the categorization group, infants high on working memory showed stronger habituation and dishabituation responses than infants scoring low in working memory. No corresponding relations were found for

the single-stimulus group. This suggests that working memory performance is systematically linked to other basic mental skills in 7-month-olds, but that corresponding relations may not get evident in any kind of habituation–dishabituation procedure. Implications for understanding the complex interplay of basic mental abilities in infancy will be discussed. “
“Adults’ processing of own-race faces differs from that of other-race faces. The presence of an “other-race” feature (ORF) has been proposed as a mechanism underlying this specialization. We examined whether this mechanism, which was previously identified in adults and in 9-month-olds, is evident at 3.5 months. Caucasian 3.

We also discuss the role of cholesterol metabolites in the direct regulation of tumor cell growth (intrinsic role), aiming to envisage an integrated view of these two aspects. Oxysterols Wnt inhibitor are generated during cholesterol metabolism through enzymatic reactions by means of cholesterol 24-hydroxylase (24S-HC), sterol 27-hydroxylase (27-HC), cholesterol 25-hydroxylase (25-HC), CYP7A1 (7α-HC), CYP3A4 (4β-HC),

and CYP11A1 (22R-HC), and through autoxidation [2-5], initiated by nonradical reactive oxygen species such as singlet O2, HOCl, and ozone (O3) or by inorganic free radical species derived from nitric oxide, superoxide, and hydrogen peroxide [5]. Some oxysterols, such as 7β-HC and 7KC, are exclusively generated by nonenzymatic cholesterol oxidation, whereas 7α-HC, 4β-HC, and 25-HC can be produced by both pathways

this website [2]. Finally, 24S-HC and 27-HC can be generated only by enzymatic cholesterol oxidation [2, 3, 5]. These cholesterol precursors, as well as desmosterol [6], can all activate LXRs [7]. LXRα (also known as NR1H3) and LXRβ (also known as NR1H2) are LXR isoforms belonging to the nuclear receptor superfamily, which comprises 48 ligand-dependent transcription factors that control metabolism, homeostasis, development, and cell growth [8]. LXRs regulate cholesterol homeostasis by modulating the expression of various genes (including the ATP-binding cassette (ABC) transporters C1 and G1, the sterol response element-binding protein-1c, and the apolipoprotein E). In particular, LXR-dependent gene expression has been associated with cholesterol efflux and the synthesis of fatty acids and triglycerides [9]. LXRβ is expressed ubiquitously, whereas LXRα is expressed in the liver, adipose tissue, adrenal glands, intestine, lungs, and cells of myelomonocytic lineage

[9]. Of note, Lxrα transcripts are upregulated in CD11c+ and CD11c− cells purified from mice treated with complete Freund’s adjuvant [10], whereas Lxrβ transcripts do not undergo transcript changes (Russo et al. unpublished observations). These results were reproduced in vitro by using ASK1 proinflammatory cytokines, such as TNF-α and IL-1β, and TLR ligands, such as LPS [10]. The transcriptional activity of LXRα and -β isoforms requires their heterodimerization with the retinoid X receptor (RXR). LXRs regulate gene expression through direct activation, ligand-independent and -dependent repression, and also by trans-repression [11]. Whereas the transcriptional activity inducing activation of target genes requires the binding of LXR–RXR heterodimers upon ligand engagement on the DNA promoter of the target genes, in the trans-repression model, LXR–RXR heterodimers have been shown to block nuclear factor κβ, signal transducer and transcription activator, and activator protein 1 induced transcription of the proinflammatory genes (COX-2, MMP9, IL-6, MCP-1, iNOS, and IL-1β) in macrophages [12, 13].

Four images of different

sectors of the section selected at random while out of focus were then focused, captured and analysed from each sample. From each image, 10 different regions were randomly selected. However, if the region was in the centre of the fibre, on an area of fibrosis, on a neuromuscular junction or if more than one measurement per fibre was selected, the region was moved slightly to the nearest fibre membrane. The measured regions included both a portion of the cytoplasm and the sarcolemma (Figure 1A). The principles of this technique are the following: when excited, fluorescent labelled antibodies bound to the proteins release photons AG-014699 manufacturer that are captured by the charge-coupled device, and converted into electrons. The number of electrons, which is directly proportional to the intensity of the fluorescence, is then mapped on to an image in MetaMorph and presented as an intensity value (Figure 1B,C). The dynamic range of the camera a 12-bit Photometrics CoolSnapHQ2 [Leica Microsystems (UK) Ltd, Milton Keynes, UK] was 0–4095 intensity units and our measurements were taken so that pixel saturation was avoided (all our intensity measurements were well

below the saturation limit). Intensity measurements of these regions were logged into a spreadsheet Decitabine nmr for data analysis. For each antibody used, 40 different measurements from each sample were taken. Each region where intensity values were measured contained a portion of the cytoplasm and of the sarcolemma, reflecting the location of the proteins of interest. For each region, the minimum intensity value recorded (representative of the cytoplasm or background intensity) was subtracted from the maximum intensity value (which corresponded to the sarcolemma) to correct each measurement for http://www.selleck.co.jp/products/PD-0332991.html background intensity. To correct for variation of sarcolemmal integrity between samples,

we performed the same measurements on serial sections stained with a β-spectrin antibody. The spectrin intensity values obtained for the control samples were set as the standard to calculate normalization factors. For each of the antibodies, the minimum intensity value was subtracted from the maximum, then these values (one per each of the 40 fibres analysed) were normalized with the β-spectrin measurements and plotted on a graph. Data are presented in scatter plots and summarized as a ratio of the control. Statistical analysis of the data was performed using one-way analysis of the variance. We compared muscle sections taken from a normal control, a DMD patient, a BMD patient and a manifesting carrier, using two dystrophin antibodies (Dys2 and P7). We also studied in parallel the intensity of dystrophin-associated complex proteins (ASG, BDG) and UTR (Figure 2A).

The disease clinically presents with lower motor neurone signs of progressive weakening and wasting of the voluntary muscles, and upper motor neurone signs of spasticity and hyper-reflexia, with death usually resulting from respiratory failure within 3–5 years of onset [44–46]. At a cytopathological level, mitochondrial dysmorphology is noticeably present, with swollen and vacuolated mitochondria populating motor neurones, muscles and intramuscular nerves [47–49]. Additionally, axonal accumulation of phosphorylated

neurofilaments and somatic formation of ubiquitin-immunoreactive proteinaceous and TAR DNA binding protein-43 (TDP-43) inclusions are all seen in degenerating motor find more neurones [44,46,50,51]. Despite many years of intense research, the aetiology of the disease remains largely ambiguous, with

the majority of cases being sporadic. Several pathogenic processes have been implicated as being causal or contributory to the disease, including oxidative stress, defective axonal transport, glutamatergic excitotoxicity, proteasome this website dysfunction, mitochondrial dysfunction and aberrant functioning of surrounding glial cells (reviewed by [52]). In up to 10% of cases, ALS has a familial origin; mutations in several genes have been identified and implicated in the pathogenesis of the disease. However, both clinically and pathologically, familial and sporadic forms of ALS are usually indistinguishable, leading to speculation that similar pathogenic processes are responsible for both forms of the disease [44,53]. In support of this, mutations in some genes cause or contribute to both familial and sporadic forms of ALS [44,46,52]. Twenty per cent of all familial cases of ALS are caused by autosomal dominant mutations in the gene encoding superoxide dismutase 1 (SOD1) [54]. This ubiquitous enzyme mediates the conversion of a superoxide

anion, derived from oxidative phosphorylation, into hydrogen peroxide, an imperative role in antioxidant defence. However, there is no correlation between SOD1 enzymatic activity and disease onset [46,55]. Montelukast Sodium It has been proposed that ALS results from a toxic gain of function of mutant SOD1 (mSOD1) [46]. Most of the current knowledge concerning the pathogenic process of familial ALS (FALS) in vivo has derived from studies of transgenic mouse models, expressing a number of different SOD1 mutations (reviewed by [53]). Furthermore, as human pathology is almost indistinguishable between sporadic ALS (SALS) and FALS, this mSOD1 mouse model is also used to understand the pathogenesis of SALS. Mitochondria are central to the aetiology of ALS and correlate with mitochondrial involvement in several other neurodegenerative disorders [18,45].

For example, at 6 or 7 years after transplantation,

Keene et al. [46] demonstrated grafted cell survival, as shown by the various striatal markers found within the grafted Olaparib manufacturer tissue. However, basic markers of cell cytoarchitecture such as haematoxylin & eosin and Nissl reveal that grafted cells depict a morphology very different from host cells [43]. Cells within p-zones are ballooned, vacuolated, lack structural cytoplasmic integrity and even stain positively for apoptotic markers such as caspase-3. When identical immunohistological stainings are compared between the reports by Keene and Cicchetti, and those published for the 6- [22] and 18-month post-transplantation cases [42], it is apparent that grafted striatal projection neurones exhibit a much weaker staining and that they lack dendritic extensions almost completely

[43,45], pointing to a rather unhealthy morphology. In contrast, various subclasses of striatal interneurones including NADPH-d-, ChAT-, parvalbumin- and calretinin-positive cells, show a better long-term survival, suggesting a degeneration or neuronal sparing pattern similar to that observed with HD pathology [42,43,46]. Although there may be signs of degeneration selleck chemicals within the grafted tissue, ingrowth of host-derived TH fibres can be observed, suggesting connections and interactions between the host and donor cells [43,46]. These results are in accordance with earlier animal model studies as well as transplanted PD patients [55,56]. Such TH innervation was not found in the 10-year post-transplantation case depicting cysts and mass lesions [45], suggesting that TH innervation of grafted tissue is not a random process. However, DARPP-32-

and calbindin-positive for cells within the grafts do not appear to cross the graft–host interface, suggesting a limited connectivity of the graft with the host brain [46]. One study reported the presence of cortical glutamatergic input onto the grafted striatal cells, using both immunohistochemistry and transmission electron microscopy [43]. Moreover, a notable microglial and astrocytic gliosis was observed in the vicinity of grafted tissue 9–10 years after transplantation [43,44], while such a response was found to be less intense in the graft than in the host at earlier intervals (6 and 7 years) [46]. Finally, elements associated to vasculature and vasculature network, such as endothelial cells and capillaries [stained with Von Willebrand factor (vWF)], pericytes [using platelet derived growth factor receptor-beta (PDGFR-β) as a marker] and larger-calibre blood vessels [detected with the α-smooth muscle actin (α-SMA) marker], demonstrated poor revascularization of the grafted tissue [44].

For bioinformatics analyses, the JCVI annotation service (JCVI, Rockville, MD, USA) was used as well as the antiSmash 2.0 server,[36, 37] the NCBI BLAST® server and the PKS/NRPS tool[38] to predict and annotate putative biosynthetic gene clusters. Burkholderia learn more gladioli was grown in potato dextrose broth for 4 days. To achieve a

large surface area to volume ratio 30 Fernbach flasks were filled with 500 ml PDB (Difco™, Becton, Dickinson and Company, Heidelberg, Germany) medium and sterilised. Flasks were inoculated with 2.5 ml bacteria suspension (24 h grown in PDB at 30 °C and 110 rpm orbital shaking) and incubated at 28 °C for 4 days. The cultures were extracted with ethyl acetate (1 : 1) and concentrated under reduced pressure. Burkholderia gladioli was cultivated under

various conditions to monitor secondary metabolite production. The following media were used: nutrient agar and nutrient broth, both according to DSMZ (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) protocol, a previously described rich liquid medium for secondary metabolite production[39] and MGY liquid medium consisting of yeast extract (1.25 g l−1) and M9 salts (50×, part A: 350 g l−1 K2HPO4; 100 g l−1 KH2PO4; part B: 29.4 g l−1 tri-Na-citrate-dihydrate; 50 g l−1 (NH4)2SO4; 5 g l−1 MgSO4) and glycerol (10 g l−1). All cultures were incubated at 30 °C and liquid cultures were shaken at 110 rpm in baffled flasks. Bacterial–fungal cocultivation was performed on 94 mm petri dishes containing 20 ml PDA at 30 °C. KPT-330 supplier The fungus was inoculated on a small spot on the agar plate by transferring a small inoculation loop of spore and hyphae material from a sporulating plate. At the same time, a small inoculation loop containing bacteria from a well growing agar plate was streaked out on the other half of the plate. PH indicator

containing PDA agar plates were supplemented with a 0.06 g ml−1 Litmus solution (1 : 30; Merck, Darmstadt, Germany) and incubated at 30 °C for 7 days. Analytical HPLC was performed on a Shimadzu DNA ligase LC-10Avp series HPLC system consisting of an autosampler, high-pressure pumps, column oven and PDA. HPLC conditions: C18 column (Eurospher 100-5, 250 × 4.6 mm, Knauer GmbH Berlin, Germany) and gradient elution (MeCN/0.1% (v/v) TFA 0.5/99.5 in 30 min to MeCN/0.1% (v/v) TFA 100/0, MeCN 100% for 10 min), flow rate 1 ml min−1; injection volume: 20 μl. Compounds were quantified by integrating the peak area (UV max, Shimadzu Deutschland GmbH, Duisburg, Germany) using Shimadzu Class-VP software (version 6.14 SP1). LC-MS measurements were performed using an Exactive Orbitrap High Performance Benchtop LC-MS with an electrospray ion source and an Accela HPLC system (Thermo Fisher Scientific, Bremen, Germany). HPLC conditions: C18 column (Betasil C18 3 μm 150 × 2.1 mm, Thermo Fisher Scientific, Bremen, Germany) and gradient elution (MeCN/0.