Metabolite fingerprinting: A powerful metabolomics approach for marker identification and functional gene annotation.

Non-targeted metabolome approaches aim to detect metabolite markers related to stress, disease, developmental or genetic perturbation. In the later context, it is also a powerful means for functional gene annotation. A prerequisite for non-targeted metabolome analyses are methods for comprehensive metabolite extraction. We present three extraction protocols for a highly efficient extraction of metabolites from plant material with a very broad metabolite coverage. The presented metabolite fingerprinting workflow is based on liquid chromatography high resolution accurate mass spectrometry (LC-HRAM-MS), which provides suitable separation of the complex sample matrix for the analysis of compounds of different polarity by positive and negative electrospray ionization and mass spectrometry. The resulting data sets are then analyzed with the software suite MarVis and the web-based interface MetaboAnalyst. MarVis offers a straightforward workflow for statistical analysis, data merging as well as visualization of multivariate data, while MetaboAnalyst is used in our hands as complementary software for statistics, correlation networks and figure generation. Finally, MarVis provides access to species-specific metabolite and pathway data bases like KEGG and BioCyc and to custom data bases tailored by the user to connect the identified markers or features with metabolites. In addition, identified marker candidates can be interactively visualized and inspected in metabolic pathway maps by KEGG pathways for a more detailed functional annotation and confirmed by mass spectrometry fragmentation experiments or coelution with authentic standards. Together this workflow is a valuable toolbox to identify novel metabolites, metabolic steps or regulatory principles and pathways.

Macrophage-activating lipoprotein (MALP)-2 impairs the healing of partial tendon injuries in mice.

Tendon injuries are accounted for up to 50% of musculoskeletal injuries and often result in poor outcomes. Inflammation is a major hallmark of tendon regeneration. Therefore, we analyzed in this study whether the topical application of the pro-inflammatory mediator macrophage-activating lipoprotein (MALP)-2 improves the healing of partial tendon injuries. C57BL/6 mice underwent a partial tenotomy of the flexor digitorum longus tendon of the left hind limb, which was treated with a solution containing either 0.5 µg MALP-2 or vehicle (control). Repetitive gait analyses were performed prior to the surgical intervention as well as postoperatively on days 1, 3, 7, 14 and 36. The structural stability of the tendons was biomechanically tested on day 7 and 36. In addition, Western blot analyses were performed on isolated tendons that were treated in vitro with MALP-2 or vehicle. In both groups, partial tenotomy resulted in a pathological gait pattern during the initial postoperative phase. On day 7, the gait pattern normalized in vehicle-treated animals, but not in MALP-2-treated mice. Moreover, the tendons of MALP-2-treated mice exhibited a significantly reduced biomechanical stiffness after 7 and 36 days when compared to controls. Western blot analyses revealed a significantly higher expression of heme oxygenase (HO)-1 and lower expression of cyclin D in MALP-2-treated tendons. These findings indicate that MALP-2 delays the healing of injured tendons most likely due to increased intracellular stress and suppressed cell proliferation in this naturally bradytrophic tissue. Hence, the application of MALP-2 cannot be recommended for the treatment of tendon injuries.

The influence of nimodipine and vasopressors on outcome in patients with delayed cerebral ischemia after spontaneous subarachnoid hemorrhage.

OBJECTIVE: Delayed cerebral ischemia (DCI) is a major factor contributing to the inferior outcome of patients with spontaneous subarachnoid hemorrhage (SAH). Nimodipine and induced hypertension using vasopressors are an integral part of standard therapy. Consequences of the opposite effect of nimodipine and vasopressors on blood pressure on patient outcome remain unclear. The authors report the detailed general characteristics and influence of nimodipine and vasopressors on outcome in patients with SAH. METHODS: The authors performed a 2-center, retrospective, clinical database analysis of 732 SAH patients treated between 2008 and 2016. Demographic and clinical data such as age, sex, World Federation of Neurosurgical Societies (WFNS) grade, BMI, Fisher grade, history of arterial hypertension and smoking, aneurysm location, C-reactive protein (CRP) level, and detailed dosage of vasopressors and nimodipine during the treatment period were evaluated. Clinical outcome was analyzed using the modified Rankin Scale (mRS) 6 months after treatment. Univariate and multivariate regression analyses were performed. Additionally, mean arterial pressure (MAP), age, nimodipine, and vasopressor dose cutoff were evaluated with regard to outcome. The level of significance was set at ≤ 0.05. RESULTS: Follow-up was assessed for 397 patients, 260 (65.5%) of whom achieved a good outcome (defined as an mRS score of 0-3). Univariate and multivariate analyses confirmed that nimodipine (p = 0.049), age (p = 0.049), and CRP level (p = 0.002) are independent predictors of good outcome. WFNS grade, Fisher score, hypertension, initial hydrocephalus, and total vasopressor dose showed significant influence on outcome in univariate analysis, and patient sex, smoking status, BMI, and MAP showed no significant association with outcome. A subgroup analysis of patients with milder initial SAH (WFNS grades I-III) revealed that initial hydrocephalus (p = 0.003) and CRP levels (p = 0.001) had significant influence on further outcome. When evaluating only patients with WFNS grade IV or V, age, CRP level (p = 0.011), vasopressor dose (p = 0.030), and nimodipine dose (p = 0.049) were independent predictors of patient outcome. Patients with an MAP < 93 mm Hg, a nimodipine cutoff dose of 241.8 mg, and cutoff total vasopressor dose of 523 mg had better outcomes. CONCLUSIONS: According to the authors' results, higher doses of vasopressors can safely provide a situation in which the maximum dose of nimodipine could be administered. Cutoff values of the total vasopressor dose were more than 3 times higher in patients with severe SAH (WFNS grade IV or V), while the nimodipine cutoff remained similar in patients with mild and severe SAH. Hence, it seems encouraging that a maximum nimodipine dosage can be achieved despite the need for a higher vasopressor dose in patients with SAH.

Should I Stay or Should I Go? A Prospective, Blinded Study Comparing the Diagnostic Capability of Dynamic and Stationary Pedobarography in Plantar Fasciitis.

The aim of this study was to determine the diagnostic capability of a dynamic gait analysis insole and compare its ability to detect clinical correlations to a common stationary analysis tool. Twenty-five patients with chronic plantar fasciitis were included in this prospective, blinded, diagnostic study. Conventional, stationary gait analysis on a force plate on an even surface and continuous dynamic pedobarography on a standardized course consisting of different gait tasks were performed and correlated to the disease severity. Mean patient age was 53.6 (range 41 to 68) years, with a mean pain level of 6.1 (range 4 to 10) on the Visual Analogue Scale and a calcaneodynia score of 48.7 (range 33 to 66). Significant correlations were seen between several dynamic gait values and clinical scoring: cadence (rs = 0.56, p = .004), stance time (rs = -0.6, p = .002), center-of-pressure velocity (rs = 0.44, p = .046), and double support time (rs = 0.42, p = .042). No significant correlations were seen between any force plate gait analysis values and clinical scoring. In this study setting, dynamic gait analysis was able to identify clinically relevant correlations to plantar fasciitis disease severity that standard force plate measurements could not.

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions.

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

Long-term pathological gait pattern changes after talus fractures - dynamic measurements with a new insole.

PURPOSE: The aim of the current study was to describe long-term gait changes after talus fractures, identify patterns associated with poor outcome and discuss possible treatment options based on dynamic gait analysis. METHODS: Twenty-seven patients were followed-up clinically and via gait analysis after talus fracture osteosynthesis. Continuous dynamic pedobarography with a gait analysis insole was performed on a standardized parcours consisting of different gait tasks and matched to the outcome. RESULTS: Mean follow-up was 78.3 months (range 21-150), mean AOFAS and Olerud-Molander scores 66 (range 20-100) and 54 (range 15-100). Significant correlations between fracture classification and osteoarthritis (Hawkins: rs = 0.67 / Marti-Weber: rs = 0.5) as well as several gait differences between injured and healthy foot with correlations to outcome were seen: decreased step load-integral/maximum-load; associations between centre-of-pressure displacement and outcome as well as between temporospatial measures and outcome. Overall, pressure-distribution was lateralized in patients with subtalar joint injury (Δ: 0.5765 N/cm2, p = 0.0475). CONCLUSIONS: Talus fractures lead to chronic gait changes and restricted function. Dynamic pedobarography can identify patterns associated with poor results. The observed gait patterns suggest that changes can be addressed by physical therapy and customized orthoses to improve overall outcome. The presented insole and measurement protocol are immediately feasible as a diagnostic and rehabilitation aid.

Mechanical and biological characterization of alkaline substituted orthophosphate bone substitutes containing meta- and diphosphates.

Despite the growing knowledge on the mechanisms of fracture healing, bone defects often do not heal in a timely manner. Clinically, tricalcium phosphate (TCP) bone substitutes are used to fill bone defects and promote bone healing. However, the degradation rate of these implants is often too slow for sufficient bone replacement. The use of calcium phosphate material with the crystalline phase Ca10[K/Na](PO4)7 containing different amounts of di- and metaphosphates may overcome this problem, because these materials show an accelerated degradation. Therefore, we generated alkaline substituted Ca-P scaffolds with different amounts of ortho-, di- and metaphosphates. The degradation of these materials was analyzed in TRIS-HCl buffer solution in vitro. Moreover, we measured the compressive strength and porosity of the scaffolds by micro-CT analysis. The biocompatibility of the scaffolds was evaluated in vivo in the mouse dorsal skinfold chamber by means of intravital fluorescence microscopy and histology. We found that higher amounts of incorporated di- and metaphosphates increase the degradation rate and compressive strength of the scaffolds without inducing a stronger leukocytic inflammatory host tissue reaction after implantation. Histological analyses confirmed the good biocompatibility of the scaffolds containing di- and metaphosphates. In summary, this study demonstrates that the compressive strength and degradation rate of Ca-P scaffolds can be improved by incorporation of di- and metaphosphates without affecting their good biocompatibility. Hence, this material modification may be particularly beneficial for the treatment of metaphyseal bone defects in weight bearing locations.

A high-throughput expression screening platform to optimize the production of antimicrobial peptides.

BACKGROUND: Antimicrobial peptides (AMPs) are promising candidates for the development of novel antibiotics, but it is difficult to produce sufficient quantities for preclinical and clinical studies due to their toxicity towards microbial expression hosts. To avoid laborious trial-and-error testing for the identification of suitable expression constructs, we have developed a small-scale expression screening platform based on a combinatorial plasmid library. RESULTS: The combinatorial library is based on the Golden Gate cloning system. In each reaction, six donor plasmids (each containing one component: a promoter, fusion partner 1, fusion partner 2, protease cleavage site, gene of interest, or transcriptional terminator) were combined with one acceptor plasmid to yield the final expression construct. As a proof of concept, screening was carried out in Escherichia coli and Pichia pastoris to study the expression of three different model AMPs with challenging characteristics, such as host toxicity or multiple disulfide bonds. The corresponding genes were successfully cloned in 27 E. coli and 18 P. pastoris expression plasmids, each in a one-step Golden Gate reaction. After transformation, small-scale expression screening in microtiter plates was followed by AMP quantification using a His6 tag-specific ELISA. Depending on the plasmid features and the expression host, the protein yields differed by more than an order of magnitude. This allowed the identification of high producers suitable for larger-scale protein expression. CONCLUSIONS: The optimization of recombinant protein production is best achieved from first principles by initially optimizing the genetic construct. The unrestricted combination of multiple plasmid features yields a comprehensive library of expression strains that can be screened for optimal productivity. The availability of such a platform could benefit all laboratories working in the field of recombinant protein expression.

Synthesis and solution stability of water-soluble κ2N,κO-bis(3,5-dimethylpyrazolyl)ethanol manganese(i) tricarbonyl bromide (CORM-ONN1).

The reaction of (OC)5MnBr with bis(3,5-dimethyl-1-pyrazolyl)methane yields [{(PzMe2)2CH2}Mn(CO)3Br] (1). The use of tridentate heteroscorpionates such as bis(3,5-dimethyl-1-pyrazolyl)acetic acid and 2,2-bis(3,5-dimethyl-1-pyrazolyl)ethanol leads to the formation of mononuclear [(OC)3Mn{(PzMe2)2CH-CO2}] (2) and [(OC)3Mn{(PzMe2)2CH-CH2OH}]Br (3, CORM-ONN1). Salt-like photoCORM 3 is soluble in aqueous media up to a concentration of 200 µM, non-toxic up to an approx. 65 µM solution and releases all carbonyl ligands upon irradiation. The molecular complexes 1 and 2 are insoluble in aqueous solutions. CORM-ONN1 (3) slowly degrades in methanol yielding iCORM 4, consisting of the complex cation [{(PzMe2)2CH-CH2OH}{(PzMe2)2CH-CH2O}Mn]+ and the [Mn(CO)5]- counter anion with the cations linked to a dimeric unit by intermolecular hydrogen bridges between the alcohol and alkoxide functionalities.

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice.

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

Blood loss in pelvic ring fractures: CT-based estimation.

BACKGROUND: The main cause of mortality after pelvic ring fractures is the acute haemorrhagic shock. The aim of the study was to estimate blood loss using CT and to correlate estimated blood loss to the mortality. METHODS: A retrospective analysis of 42 patients with AO/OTA C-type pelvic ring fractures was performed. In total 29 AO/OTA C1-type, 4 C2-type and 9 C3-type fractures were analysed. A CT-volumetric analysis was developed to estimate the early blood loss in the different compartments. The results were correlated to the mortality of the patients. RESULTS: Significant increases in interstitial free blood volume (392±502.8 vs. 888±663; P<0.05) and total blood loss (476±535 vs. 1,005±649; P<0.05) were seen when comparing C1 to C3-type fractures. Early blood loss significantly correlated to mortality. No significant difference was seen between all groups with respect to systolic blood pressure, haemoglobin concentration and the Injury Severity Score (ISS). CONCLUSIONS: In pelvic ring fractures the CT-estimated blood loss correlates to mortality.

Elucidation of the CO-Release Kinetics of CORM-A1 by Means of Vibrational Spectroscopy.

CO-releasing molecules (CORMs) are developed for investigations of the interaction between the signaling molecule carbon monoxide (CO) and cells or tissue. Prior to their application these molecules must be fully characterized with respect to their CO-release mechanism. One widely used CORM for biological application is sodium boranocarbonate (CORM-A1), which shows pH-dependent CO liberation. The complete reaction mechanism of CORM-A1 is not fully understood yet. Therefore, in this contribution time-resolved gas-phase IR spectroscopy is used to monitor the headspace above decaying CORM-A1 solutions at four different pH values (5.8 to 7.4). Borane carbonyl is found as an intermediate in the gas phase, which is formed during CORM degradation and further decays to CO. Concentration profiles of a pseudoconsecutive first-order reaction are successfully fitted to specific band areas of the measured gas-phase spectra, and the rate constants are obtained. The production of borane carbonyl is strongly pH dependent (half-lives between 5 and 106 min), whereas the decay of borane carbonyl in the gas phase is nearly constant with a half-life of about 33 min. The ratio of liberated CO molecules per CORM-A1 is determined to be 0.91±0.09, and boric acid is identified as further end product.

CORM-EDE1: A Highly Water-Soluble and Nontoxic Manganese-Based photoCORM with a Biogenic Ligand Sphere.

[Mn(CO)5Br] reacts with cysteamine and 4-amino-thiophenyl with a ratio of 2:3 in refluxing tetrahydrofuran to the complexes of the type [{(OC)3Mn}2(µ-SCH2CH2NH3)3]Br2 (1, CORM-EDE1) and [{(OC)3Mn}2(µ-SC6H4-4-NH3)3]Br2 (2, CORM-EDE2). Compound 2 precipitates during refluxing of the tetrahydrofuran solution as a yellow solid whereas 1 forms a red oil that slowly solidifies. Recrystallization of 2 from water yields the HBr-free complex [{(OC)3Mn}2(µ-S-C6H4-4-NH2)2(µ-SC6H4-4-NH3)] (3). The n-propylthiolate ligand (which is isoelectronic to the bridging thiolate of 1) leads to the formation of the di- and tetranuclear complexes [(OC)4Mn(µ-S-nPr)2]2 and [(OC)3Mn(µ-S-nPr)]4. CORM-EDE1 possesses ideal properties to administer carbon monoxide to biological and medicinal tissues upon irradiation (photoCORM). Isolated crystalline CORM-EDE1 can be handled at ambient and aerobic conditions. This complex is nontoxic, highly soluble in water, and indefinitely stable therein in the absence of air and phosphate buffer. CORM-EDE1 is stable as frozen stock in aqueous solution without any limitations, and these stock solutions maintain their CO release properties. The reducing dithionite does not interact with CORM-EDE1, and therefore, the myoglobin assay represents a valuable tool to study the release kinetics of this photoCORM. After CO liberation, the formation of MnHPO4 in aqueous buffer solution can be verified.

Stimulation of angiogenesis by cilostazol accelerates fracture healing in mice.

Cilostazol, a selective phosphodiesterase-3 inhibitor, is known to control cyclic adenosine monophosphate (c-AMP) and to stimulate angiogenesis through upregulation of pro-angiogenic factors. There is no information, however, whether cilostazol affects fracture healing. We, therefore, studied the effect of cilostazol on callus formation and biomechanics during fracture repair. Bone healing was analyzed in a murine femur fracture stabilized with an intramedullary screw. Radiological, biomechanical, histomorphometric, histochemical, and protein biochemical analyses were performed at 2 and 5 weeks after fracture. Twenty-five mice received 30 mg/kg body weight cilostazol p.o. daily. Controls (n=24) received equivalent amounts of vehicle. In cilostazol-treated animals radiological analysis at 2 weeks showed an improved healing with an accelerated osseous bridging compared to controls. This was associated with a significantly higher amount of bony tissue and a smaller amount of cartilage tissue within the callus. Western blot analysis showed a higher expression of cysteine-rich protein 61 (CYR61), bone morphogenetic protein (BMP)-4, and receptor activator of NF-kappaB ligand (RANKL). At 5 weeks, improved fracture healing after cilostazol treatment was indicated by biomechanical analyses, demonstrating a significant higher bending stiffness compared to controls. Thus, cilostazol improves fracture healing by accelerating both bone formation and callus remodeling.

Comparison of healing process in open osteotomy model and open fracture model: Delayed healing of osteotomies after intramedullary screw fixation.

Murine osteotomy and fracture models have become the standard to study molecular mechanisms of bone healing. Because there is little information whether the healing of osteotomies differs from that of fractures, we herein studied in mice the healing of femur osteotomies compared to femur fractures. Twenty CD-1 mice underwent a standardized open femur osteotomy. Another 20 mice received a standardized open femur fracture. Stabilization was performed by an intramedullary screw. Bone healing was studied by micro-CT, biomechanical, histomorphometric and protein expression analyses. Osteotomies revealed a significantly lower biomechanical stiffness compared to fractures. Micro-CT showed a reduced bone/tissue volume within the callus of the osteotomies. Histomorphometric analyses demonstrated also a significantly lower amount of osseous tissue in the callus of osteotomies (26% and 88% after 2 and 5 weeks) compared to fractures (50% and 100%). This was associated with a delayed remodeling. Western blot analyses demonstrated comparable BMP-2 and BMP-4 expression, but higher levels of collagen-2, CYR61 and VEGF after osteotomy. Therefore, we conclude that open femur osteotomies in mice show a markedly delayed healing when stabilized less rigidly with an intramedullary screw. This should be considered when choosing a model for studying the mechanisms of bone healing in mice.

Carbon monoxide release properties and molecular structures of phenylthiolatomanganese(I) carbonyl complexes of the type [(OC)4Mn(µ-S-aryl)]2.

Several phenylthiolatomanganese carbonyl complexes of the type [(OC)4Mn(µ-SR)]2 (R = Ph (), C6H4-4-CH3 (), C6H4-4-CF3 (), C6H4-4-F (), C6H4-4-Cl (), C6H4-4-OMe (), C6F5 (), and CH2C6H4-4-Cl ()) have been prepared via the reaction of Mn2(CO)10 with diaryldisulfane or via the reaction of [(OC)5MnBr] with arylthiols. These complexes lose two carbon monoxide molecules quite easily yielding tetranuclear [(OC)3Mn(µ3-SR)]4 (). Derivatives with fluoro-substituted aryl groups commonly form mixtures of dinuclear and tetranuclear which can quantitatively be converted to by heating of the corresponding reaction mixtures. A unique trinuclear structure is found for the mesityl derivative [(OC)4Mn(µ-SMes)]3 () which is maintained in solution as verified by IR and NMR spectroscopy. Traces of an already known dinuclear by-product of the type [(OC)3Mn(µ-SC6H3(-4-Me)-2-SC6H4-4-Me)]2 () have been structurally characterized. The suitability of [(OC)4Mn(µ-SPh)]2 () as a CO releasing molecule (CORM) for the administration of carbon monoxide has been studied. Two CO molecules are released upon dissolving in strongly Lewis basic solvents L, yielding [(OC)3Mn(L)(µ-SPh)]2, which liberates all the remaining CO molecules upon irradiation (photoCORM behavior).

IR spectroscopic methods for the investigation of the CO release from CORMs.

Carbon monoxide (CO) is a toxic gas for mammals, and despite this fact, it is naturally produced in these organisms and has been proven to be beneficial in medical treatments, too. Therefore, CO-releasing molecules (CORMs) are intensively developed to administer and dose CO for physiological applications. Nearly all of these compounds are metal carbonyl complexes, which have been synthesized and investigated. However, for most of these CORMs, the exact reaction mechanisms of CO release is not completely elucidated, although it is of utmost importance. The widely used myoglobin assay for testing the CO release has several disadvantages, and therefore, different methods have to be applied to characterize CORMs. In this work, different setups of IR absorption spectroscopy are used to analyze and quantify the CO release during the decay of various CORMs: IR spectroscopy of the gas phase is applied to follow the CO liberation, and attenuated total reflection (ATR) IR spectroscopy is used to record the decay of the metal carbonyl. IR spectroscopy supported by DFT calculations yields valuable insights in the CO release reaction mechanism. The focus is set on two different CORMs: CORM-2 (Ru2(CO)(6)Cl(4)) and on the photoactive CORM-S1 (photoCORM [Fe(CO)2(SCH2CH2NH2)2]). Our results indicate that the CO liberation from CORM-2 strongly depends on sodium dithionite, which is required for the commonly applied myoglobin assay and that CORM-S1 loses all its bound CO molecules upon irradiation with blue light.

Enhanced signal intensity in matrix-free laser desorption ionization mass spectrometry by chemical modification of bionanostructures from diatom cell walls.

RATIONALE: Laser desorption ionization for mass spectrometric measurements (LDI MS) is supported by nanostructured materials. This technique helps to overcome known limitations of matrix-assisted laser desorption/ionization (MALDI) and especially avoids interfering signals caused by matrix components. LDI can be supported by bionanostructures from the cell walls of diatoms. We explore how ionization efficiency can be improved by chemical modification of the cell walls. METHODS: We introduce procedures to chemically modify these nanopatterned silicate structures using perfluorooctyldimethylchlorosilane or pentafluorophenylpropyldimethylchlorosilane. Using a conventional MALDI-MS instrument we compare ionization using the novel materials with that of unmodified cell walls. The functionalized bionanomaterial is comprehensively evaluated for the use in LDI MS using a broad range of analytes and two commercial drugs. RESULTS: Chemical modifications lead to materials that support LDI significantly better than unmodified diatom cell walls. LDI signal intensity was up to 25-fold increased using the modified preparations. No interfering signals in the lower molecular weight range down to m/z 100 were observed, demonstrating the suitability of the method for small analytes. Crude solutions of commercial drugs, such as Aspirin complex(®) and IbuHEXAL(®) could be directly investigated without additional sample preparation. CONCLUSIONS: Chemically modified diatom cell walls represent a powerful tool to support ionization in LDI MS. The lack of background signals in the low molecular weight region of the mass spectra allows also the investigations of small analytes.

Light-triggered CO release from nanoporous non-wovens.

The water insoluble and photoactive CO releasing molecule dimanganese decacarbonyl (CORM-1) has been non-covalently embedded into poly(l-lactide-co-d/l-lactide) fibers via electrospinning to enable bioavailability and water accessibility of CORM-1. SEM images of the resulting hybrid non-wovens reveal a nanoporous fiber morphology. Slight CO release from the CORM-1 in the electrospinning process induces nanoporosity. IR spectra show the same set of carbonyl bands for the CORM-1 precursor and the non-woven. When the material was exposed to light (365-480 nm), CO release from the incorporated CORM-1 was measured via heterogeneous myoglobin assay, a portable CO electrode and an IR gas cuvette. The CO release rate was wavelength dependent. Irradiation at 365 nm resulted in four times faster release than at 480 nm. 3.4 µmol of CO per mg non-woven can be generated. Mouse fibroblast 3T3 cells were used to show that the hybrid material is non-toxic in the darkness and strongly photocytotoxic when light is applied.