Oligopeptide-based molecular labelling of (bio)degradable polyester biomaterials.

Nowadays, a very important motivation for the development of new functional materials for medical purposes is not only their performance but also whether they are environmentally friendly. In recent years, there has been a growing interest in the possibility of labelling (bio)degradable polymers, in particular those intended for specific applications, especially in the medical sector, and the potential of information storage in such polymers, making it possible, for example, to track the ultimate environmental fate of plastics. This article presents a straightforward green approach that combines both aspects using an oligopeptide, which is an integral part of polymer material, to store binary information in a physical mixture of polymer and oligopeptide. In the proposed procedure the year of production of polymer films made of poly(l-lactide) (PLLA) and a blend of poly(1,4-butylene adipate-co-1,4-butylene terephthalate) and polylactide (PBAT/PLA) were encoded as the sequence of the appropriate amino acids in the oligopeptide (PEP) added to these polymers. The decoding of the recorded information was carried out using mass spectrometry technique as a new method of decoding, which enabled the successful retrieval and reading of the stored information. Furthermore, the properties of labelled (bio)degradable polymer films and stability during biodegradation of PLLA/PEP film under industrial composting conditions have been investigated. The labelled films exhibited good oligopeptide stability, allowing the recorded information to be retrieved from a green polymer/oligopeptide system before and after biodegradation. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay) study of the PLLA and PLLA/PBAT using the MRC-5 mammalian fibroblasts was presented for the first time.

Mass spectrometry in art conservation-With focus on paintings.

Conservation of historic artifacts has been a multidisciplinary field from its very beginning. Traditionally, it has been and still is associated with the history of art. It applies knowledge from technical and basic sciences, adapting their solutions to its goals. At present, however, a new tendency is clearly emerging-scientific research is starting to play an increasingly important role not only as a service, but also by proposing new solutions both in the traditional conservation areas and in new areas of conservation activities. The above trend opens up new perspectives for the field of preservation of our heritage but may also create new threats. Therefore, the conservators' caution in introducing new technologies should always be justified; after all, they are responsible for the effects of any activities on the historic objects. This, quite selective review, discusses application of mass spectrometry techniques for the detection of various components that are important to the conservators of our heritage with particular focus on paintings. The text also contains some basic knowledge of technical details to introduce the methodology to a broader group of professionals.

Patients with alcohol use disorder increase pain and analgesics use: A nationwide population-based cohort study.

BACKGROUND: Alcohol has dual effects on many systems, including the pain system. We will test whether and how chronic alcohol consumption enhances pain sensation to develop pain disorder. METHODS: We conducted a retrospective matched cohort study using data from the National Health Insurance Research Database (NHIRD) in Taiwan, in patients with and without alcohol use disorder (AUD). This study enrolled 19,174 individuals with AUD as study cohort and 19,174 propensity score-matched individuals without AUD as comparison cohort. The outcome was the incidence of pain disorders and the need for analgesics. The hazard ratios of pain disorders and the need for analgesics were evaluated using Cox proportional hazard regression analysis after adjusting for age, sex, index year, comorbidities, urbanization, areas of residence, and insurance premium. RESULTS: The 14 years of follow-up showed that AUD patients had a higher adjusted hazard ratio (aHR) for developing pain disorders than in non-AUD controls [aHR= 1.290, 95% confidence interval (CI): 1.045-1.591]. Besides, AUD patients had a higher risk of analgesic use (aHR = 1.081, 95% CI: 1.064-1.312), including opioids and non-opioid analgesics. Most importantly, AUD patients required more days of analgesic use, increased dose of analgesics, and higher costs of analgesics. Moreover, AUD patients had more anemia (aHR=2.772, 95% CI: 2.581-2.872), which could be a mediating factor. CONCLUSIONS: AUD patients had higher risks of developing pain disorders and subsequently increased analgesic demand. These results suggest that AUD worsened pain, and pain syndrome is correlated with the duration of chronic alcohol exposure.

Hemorphins-From Discovery to Functions and Pharmacology.

During the last three decades, a variety of different studies on bioactive peptides that are opioid receptor ligands, have been carried out, with regard to their isolation and identification, as well as their molecular functions in living organisms. Thus, in this review, we would like to summarize the present state-of-the art concerning hemorphins, methodological aspects of their identification, and their potential role as therapeutic agents. We have collected and discussed articles describing hemorphins, from their discovery up until now, thus presenting a very wide spectrum of their characteristic and applications. One of the major assets of the present paper is a combination of analytical and pharmacological aspects of peptides described by a team who participated in the initial research on hemorphins. This review is, in part, focused on the analysis of endogenous opioid peptides in biological samples using advanced techniques, description of the identification of synthetic/endogenous hemorphins, their involvement in pharmacology, learning, pain and other function. Finally, the part regarding hemorphin analogues and their synthesis, has been added.

Jacek Namiesnik-Analytical Chemist and Dedicated Biker: From Wine Analysis to Toxic Compounds.

Jacek Namiesnik, who died at the age of 69, was one of the most influential analytical chemists in Poland at the second half of the 20th century and the first two decades of the 21st century [...].

Rapamycin Improves Spatial Learning Deficits, Vulnerability to Alcohol Addiction and Altered Expression of the GluN2B Subunit of the NMDA Receptor in Adult Rats Exposed to Ethanol during the Neonatal Period.

Ethanol exposure during pregnancy alters the mammalian target of rapamycin (mTOR) signaling pathway in the fetal brain. Hence, in adult rats exposed to ethanol during the neonatal period, we investigated the influence of rapamycin, an mTOR Complex 1 (mTORC1) inhibitor, on deficits in spatial memory and reversal learning in the Barnes maze task, as well as the ethanol-induced rewarding effects (1.0 or 1.5 g/kg) using the conditioning place preference (CPP) paradigm. Rapamycin (3 and 10 mg/kg) was given before intragastric ethanol (5 g/kg/day) administration at postnatal day (PND)4-9 (an equivalent to the third trimester of human pregnancy). Spatial memory/reversal learning and rewarding ethanol effect were evaluated in adult (PND60-70) rats. Additionally, the impact of rapamycin pre-treatment on the expression of the GluN2B subunit of NMDA receptor in the brain was assessed in adult rats. Our results show that neonatal ethanol exposure induced deficits in spatial memory and reversal learning in adulthood, but the reversal learning outcome may have been due to spatial learning impairments rather than cognitive flexibility impairments. Furthermore, in adulthood the ethanol treated rats were also more sensitive to the rewarding effect of ethanol than the control group. Rapamycin prevented the neonatal effect of ethanol and normalized the GluN2B down-regulation in the hippocampus and the prefrontal cortex, as well as normalized this subunit's up-regulation in the striatum of adult rats. Our results suggest that rapamycin and related drugs may hold promise as a preventive therapy for fetal alcohol spectrum disorders.

The insulin-degrading enzyme as a link between insulin and neuropeptides metabolism.

We have applied a recently developed HPLC-MS enzymatic assay to investigate the cryptic peptides generated by the action of the insulin-degrading enzyme (IDE) on some neuropeptides (NPs) involved in the development of tolerance and dependence to opioids. Particularly, the tested NPs are generated from the NPFF precursor (pro-NPFF (A)): NPFF (FLFQPQRF) and NPAF (AGEGLSSPFWSLAAPQRF). The results show that IDE is able to cleave NPFF and NPAF, generating specific cryptic peptides. As IDE is also responsible for the processing of many other peptides in the brain (amyloid beta protein among the others), we have also performed competitive degradation assays using mixtures of insulin and the above mentioned NPs. Data show that insulin is able to slow down the degradation of both NPs tested, whereas, surprisingly, NPAF is able to accelerate insulin degradation, hinting IDE as the possible link responsible of the mutual influence between insulin and NPs metabolism.

LVV-hemorphin-7 (LVV-H7) plays a role in antinociception in a rat model of alcohol-induced pain disorders.

Alcohol can increase the sensitivity to painful stimulation or convert insensibility to pain at different stages. We hypothesized that chronic alcohol consumption changes the level of LVV-hemorphin-7 (abbreviated as LVV-H7, an opioid-like peptide generated from hemoglobin ß-chain), thereby affecting pain sensation. We established a chronic alcohol-exposed rat model to investigate the effects of LVV-H7. Adult male Sprague-Dawley rats were subjected to daily intraperitoneal injection of 10 % ethanol (w/v) at 0.5 g/kg for 15 days and subsequent alcohol withdrawal for 5 days. Using different pharmacological strategies to affect the LVV-H7 level, we investigated the correlation between LVV-H7 and pain-related behavior. Tail-flick and hot plate tests were employed to investigate alcohol-induced pain-related behavioral changes. The serum level of LVV-H7 was determined by ELISA. Our results showed that alcohol first induced an analgesia followed by a hyperalgesia during alcohol withdrawal, which could be driven by the quantitative change of LVV-H7. A positive correlation between the level of LVV-H7 and Δtail-flick latency (measured latency minus basal latency) confirmed this finding. Moreover, we revealed that the LVV-H7 levels were determined by the activity of cathepsin D and red blood cell/hemoglobin counts, which could be affected by alcohol. These results suggest that the deterioration of anti-nociception induced by alcohol is correlated to the decreased level of LVV-H7, and this could be due to alcohol-induced anemia. This study may help to develop LVV-H7 structure-based novel analgesics for treating alcohol-induced pain disorders and thus ameliorate the complications in alcoholics.

Inhibitors of neuropeptide peptidases engaged in pain and drug dependence.

Owing to a broad spectrum of functions performed by neuropeptides, this class of signaling molecules attracts an increasing interest. One of the key steps in the regulation of biological activity of neuropeptides is proteolytic conversion or degradation by proteinases that change or terminate biological activity of native peptides. These enzymes, in turn, are regulated by inhibitors, which play integral role in controlling many metabolic pathways. Thus, the search for selective inhibitors and detailed knowledge on the mechanisms of binding of these substances to enzymes, could be of importance for designing new pharmacological approaches. The aim of this review is to summarize the current knowledge on the inhibitors of enzymes that convert selected groups of neuropeptides, such as dynorphins, enkephalins, substance P and NPFF fragments. The importance of these substances in pathophysiological processes involved in pain and drug addiction, have been discussed. This article is part of the special issue on Neuropeptides.

Synthesis of the Novel Covalent Cysteine Proteases Inhibitor with Iodoacetic Functional Group.

This work presents the synthesis of the novel covalent inhibitor of cysteine proteases where epoxide has been replaced by the iodoacetyl functional group. The molecule, similar in action to E-64 and DCG-04, the commonly applied inhibitors, is additionally biotinylated and contains tyrosyl iodination sites. The Fmoc solid phase synthesis has been applied. Conjugation of iodoacetic acid with the peptide was optimized by testing different conjugation agents. The purity of the final product was verified by mass spectrometry and its bioactivity was tested by incubation with a model cysteine protease-staphopain C. Finally, it was shown that the synthesized inhibitor binds to the protein at the ratio of 1:1. More detailed analysis by means of tandem mass spectrometry proved that the inhibitor binds to the cysteine present in the active site of the enzyme.

Changes in Protein Glycosylation as a Result of Aptamer Interactions with Cancer Cells.

PURPOSE: Based on the recent aptamer-related breast cancer studies, which indicate the therapeutic role of specific oligonucleotide sequences, experiments have been designed in an attempt to unravel the molecular targets of this mechanism. This article describes the study on glycoproteome changes in breast cancer cells as a result of their interactions with aptamers. EXPERIMENTAL DESIGN: Aberrations in protein glycosylation play an important role in tumorigenesis and influence cancer progression, metastasis, immunoresponse, and chemoresistance, therefore this study is focused on the identification of the alterations in glycan expression on the surface of proteins as a potential and innovative tool for biomedical applications of aptamers in cancer treatment. RESULTS: Two proteins, kinesin-like protein (KI13B) and proliferating cell nuclear antigen (PCNA), have been identified that carry N-glycan epitopes after conjugation with aptamer sequences. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple features of aptamers as an alternative to protein antibodies are utilized for various biomedical applications ranging from biomarker discovery, bioimaging, targeted therapy, drug delivery, and drug pharmacokinetics and biodistribution. Frequently, aptamers bind to their target molecules and modulate their function. Such therapeutic aptamers can modify the biological pathways for treatment of many types of diseases, such as cancer.

2D Electrophoretic pattern of bovine placental proteins during early-mid pregnancy.

The Placenta, like every tissue, possesses its own characteristic protein profile, which may change within the course of pregnancy. These changes can be used for the elucidation of the mechanisms related to both physiology of pregnancy and pathological events. The aim of the study was to describe proteinergic profiles of maternal and fetal parts of bovine placenta during early-mid pregnancy by the use of 2D electrophoresis and MALDI TOF/TOF MS identification to evaluate dynamics of the possible changes necessary for placentation. Placental samples were collected from six pregnant cows (3-5 months) in the local abattoir. Placentomes were separated, and proteins were extracted and subjected to 2D electrophoresis and MALDI TOF/TOF identification. Out of 907 spots identified by the statistical analysis of gels, 54 were identified. Out of this number, 36 spots were significantly different between examined samples. Moreover, the obtained patterns differed between maternal and fetal parts of the placenta with regard to the intensity of staining, suggesting quantitative differences in protein content. These preliminary results are unique for this period of pregnancy. Such data are important for further experiments to obtain full protein profiles necessary to understand biochemical mechanisms underlying the attachment between fetal and maternal parts of the placenta during placentation. Moreover, the outcomes may help in elucidating pregnancy biomarkers in the future.

MINIATURIZATION IN MASS SPECTROMETRY.

Expectations for continuous miniaturization in mass spectrometry are not declining for years. Portable instruments are highly welcome by the industry, science, space agencies, forensic laboratories, and many other units. All are striving for the small, cheap, and as good as possible instruments. This review describes the recent developments of miniature mass spectrometers and also provides selected applications where these devices are used. Upcoming perspectives of further development are also discussed. @ 2019 John Wiley & Sons Ltd. Mass Spec Rev.

IDE Degrades Nociceptin/Orphanin FQ through an Insulin Regulated Mechanism.

Insulin-degrading enzyme (IDE) was applied to catalyze hydrolysis of Nociceptin/Orphanin 1-16 (OFQ/N) to show the involvement of the enzyme in degradation of neuropeptides engaged in pain transmission. Moreover, IDE degradative action towards insulin (Ins) was inhibited by the OFQ/N fragments, suggesting a possible regulatory mechanism in the central nervous system. It has been found that OFQ/N and Ins affect each other degradation by IDE, although in a different manner. Indeed, while the digestion of OFQ/N is significantly affected by the presence of Ins, the kinetic profile of the Ins hydrolysis is not affected by the presence of OFQ/N. However, the main hydrolytic fragments of OFQ/N produced by IDE exert inhibitory activity towards the IDE-mediated Ins degradation. Here, we present the results indicating that, besides Ins, IDE cleaves neuropeptides and their released fragments act as inhibitors of IDE activity toward Ins. Having in mind that IDE is present in the brain, which also contains Ins receptors, it cannot be excluded that this enzyme indirectly participates in neural communication of pain signals and that neuropeptides involved in pain transmission may contribute to the regulation of IDE activity. Finally, preliminary results on the metabolism of OFQ/N, carried out in the rat spinal cord homogenate in the presence of various inhibitors specific for different classes of proteases, show that OFQ/N proteolysis in rat spinal cord could be due, besides IDE, also to a cysteine protease not yet identified.

Detection of legal highs in the urine of methadone-treated patient by LC-MS.

Urine tests are the commonly accepted methods to control abstinence and adherence to treatment of patients who undergo methadone maintenance treatment (MMT). Depending on various national guidelines and accessibility of techniques, only selected psychoactive substances are routinely tested in urine of MMT patients. In general, they belong to the few groups of compounds: THC, cocaine, amphetamines, opiates, PCP and benzodiazepines. It is, however, well known that patients enrolled in such replacement programmes take psychoactive substances that are not routinely detected by the toxicology laboratories, to escape unexpected tests. Here, we report semiquantitative detection of legal highs taken by the MMT patient, using high-pressure liquid chromatography coupled to the flowing atmospheric pressure afterglow ion source (LC-FAPA-MS). To demonstrate effectivity of this technique, the data were confirmed by quantitative analysis using LC-ESI-MS/MS. In the analysed sample of MMT patient, a mixture of psychoactive compounds was found, namely 3-MMC (3-methylmethcathinone), pentedrone and methcathinone and determined at the concentrations of 670; 50 and 0.2 µg/mL, respectively. Such fast analytical technique may be useful for the efficient control of substances taken intentionally by MMT patients.

The kisspeptin derivative kissorphin reduces the acquisition, expression, and reinstatement of ethanol-induced conditioned place preference in rats.

Research has shown that opioids are involved in the rewarding effects of ethanol. Neuropeptide FF (NPFF) has been described as an anti-opioid peptide because, in many cases, it inhibits opioid and ethanol effects in rodents. Kissorphin (KSO) is a new peptide derived from kisspeptin-10 with structural similarities to NPFF. This peptide possesses NPFF-like biological activity in vitro. The aim of the current study was to investigate whether KSO (Tyr-Asn-Trp-Asn-Ser-Phe-NH2) influences the acquisition, expression, and reinstatement of ethanol-induced conditioned place preference (ethanol-CPP) in rats. The ethanol-CPP was established (conditioning for 5 days) by intraperitoneal (i.p.) administration of ethanol (1 g/kg, 20%, w/v) using an unbiased procedure. After that, one group of rats was used in final post-conditioning testing (expression of CPP) and the other group received a priming injection of ethanol after 10 days of extinction (reinstatement of CPP). Our experiments showed that KSO, given intravenously (i.v.) at the doses of 1, 3, and 10 nmol before every ethanol administration, inhibited the acquisition and, given acutely before the post-conditioning test or before the priming dose of ethanol, inhibited the expression and reinstatement of ethanol-CPP, respectively, in a dose-dependent manner. KSO given by itself neither induced place preference nor aversion and did not alter locomotor activity and coordination of rats. These results suggest that KSO can alter rewarding/motivational effects of ethanol. These data suggest this peptide possesses an anti-opioid character.

Advances in the Study of Aptamer-Protein Target Identification Using the Chromatographic Approach.

Ever since the development of the process known as the systematic evolution of ligands by exponential enrichment (SELEX), aptamers have been widely used in a variety of studies, including the exploration of new diagnostic tools and the discovery of new treatment methods. Aptamers' ability to bind to proteins with high affinity and specificity, often compared to that of antibodies, enables the search for potential cancer biomarkers and helps us understand the mechanisms of carcinogenesis. The blind spot of those investigations is usually the difficulty in the selective extraction of targets attached to the aptamer. There are many studies describing the cell SELEX for the prime choice of aptamers toward living cancer cells or even whole tumors in the animal models. However, a dilemma arises when a large number of proteins are being identified as potential targets, which is often the case. In this article, we present a new analytical approach designed to selectively target proteins bound to aptamers. During studies, we have focused on the unambiguous identification of the molecular targets of aptamers characterized by high specificity to the prostate cancer cells. We have compared four assay approaches using electrophoretic and chromatographic methods for "fishing out" aptamer protein targets followed by mass spectrometry identification. We have established a new methodology, based on the fluorescent-tagged oligonucleotides commonly used for flow-cytometry experiments or as optic aptasensors, that allowed the detection of specific aptamer-protein interactions by mass spectrometry. The use of atto488-labeled aptamers for the tracking of the formation of specific aptamer-target complexes provides the possibility of studying putative protein counterparts without needing to apply enrichment techniques. Significantly, changes in the hydrophobic properties of atto488-labeled aptamer-protein complexes facilitate their separation by reverse-phase chromatography combined with fluorescence detection followed by mass-spectrometry-based protein identification. These comparative results of several methodological approaches confirmed the universal applicability of this method to studying aptamer-protein interactions with high sensitivity, showing superior properties compared with pull-down techniques.

The Forty-Sixth Euro Congress on Drug Synthesis and Analysis: Snapshot †.

The 46th EuroCongress on Drug Synthesis and Analysis (ECDSA-2017) was arranged within the celebration of the 65th Anniversary of the Faculty of Pharmacy at Comenius University in Bratislava, Slovakia from 5-8 September 2017 to get together specialists in medicinal chemistry, organic synthesis, pharmaceutical analysis, screening of bioactive compounds, pharmacology and drug formulations; promote the exchange of scientific results, methods and ideas; and encourage cooperation between researchers from all over the world. The topic of the conference, "Drug Synthesis and Analysis," meant that the symposium welcomed all pharmacists and/or researchers (chemists, analysts, biologists) and students interested in scientific work dealing with investigations of biologically active compounds as potential drugs. The authors of this manuscript were plenary speakers and other participants of the symposium and members of their research teams. The following summary highlights the major points/topics of the meeting.

The new kisspeptin derivative - kissorphin (KSO) - attenuates acute hyperlocomotion and sensitization induced by ethanol and morphine in mice.

Kissorphin (KSO) is a new peptide derived from kisspeptin-10. This peptide possesses neuropeptide FF (NPFF)-like biological activity in vitro; NPFF, in many cases, inhibits opioid and ethanol effects in rodents. Therefore, the current study explored the influence of KSO on acute ethanol- and morphine-induced hyperactivity, and on the development and expression of locomotor sensitization induced by these drugs. In the present study, sensitization to locomotor effects was induced by repeated exposure to ethanol (2.4 g/kg, intraperitoneally [i.p.], 1 × 4 days) or morphine (10 mg/kg, subcutaneously [s.c.], 1 × 7 days). We found that KSO (1-10 nmol/300 µL, intravenously [i.v.]) did not have an impact on locomotor activity of naïve mice. However, it reduced both acute ethanol- (10 nmol/300 µL) and morphine-induced hyperactivity (3 and 10 nmol/300 µL). Pretreatment of animals with KSO (10 nmol/300 µL), before every ethanol or morphine injection during development of sensitization or before the ethanol or morphine challenge, attenuated the development, as well as the expression of locomotor sensitization to both substances. Moreover, prior administration of the NPFF receptor antagonist RF9 (10 nmol/300 µL, i.v.) inhibited the ability of KSO (10 nmol/300 µL) to reduce the expression of ethanol and morphine sensitization. KSO given alone, at all used doses, did not influence the motor coordination measured via the rotarod test. The results from this study show that KSO effectively attenuated acute and repeated effects of ethanol and morphine. Thus, KSO possesses NPFF-like anti-opioid activity in these behavioral studies.

Glycosylation Changes in Serum Proteins Identify Patients with Pancreatic Cancer.

After more than a decade of biomarker discovery using advanced proteomic and genomic approaches, very few biomarkers have been involved in clinical diagnostics. Most candidate biomarkers are focused on the protein component. Targeting post-translational modifications (PTMs) in combination with protein sequences will provide superior diagnostic information with regards to sensitivity and specificity. Glycosylation is one of the most common and functionally important PTMs. It plays a central role in many biological processes, including protein folding, host-pathogen interactions, immune response, and inflammation. Cancer-associated aberrant glycosylation has been identified in various types of cancer. Expression of cancer-specific glycan epitopes represents an excellent opportunity for diagnostics and potentially specific detection of tumors. Here, we report four proteins (LIFR, CE350, VP13A, HPT) found in sera from pancreatic cancer patients carrying aberrant glycan structures as compared to those of controls.