Alkaloidit Rhazya stricta in vitro -viljelmissä

Rhazya stricta Decne. (Apocynaceae) is a traditional medicinal plant in the Middle East and South Asia. It produces a large number of terpenoid indole alkaloids(TIAs), some of which possess important pharmacological properties. This study focused on the establishment of biotechnological production tools of R. stricta, namely undifferentiated cell cultures, and an Agrobacterium rhizogenes-mediated transformation method to obtain hairy roots expressing heterologous genes from the early TIA pathway. As Rhazya alkaloids comprise a wide range of structures and polarities it was necessary first to develop different analytical methods to determine the alkaloid contents and changes in their profiles in transgenic cultures and after various treatments. Targeted and non-targeted analyses from cell andorgan cultures were carried out using gas chromatography-mass spectrometry(GC-MS), high performance liquid chromatography (HPLC), ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Callus cultures were successfully initiated from five different explants onmodified B5 medium containing phytohormones. The phenotypes of the calli varied, but as was expected the callus cultures accumulated lower levels of alkaloids than wild type hairy roots and adventitious roots. Surprisingly, calli derived from stems had elevated levels of strictosidine lactam compared to other cultures. Transformation experiments revealed that only leaves but not cotyledons, hypocotyls or stem segments were susceptible to Agrobacterium infection and subsequent root induction. The transformation efficiency varied from 22% to 83% depending on the gene. Wild type and gus hairy root clones contained twofold higher amounts of alkaloids than adventitious roots. A total of 17 TIAs, including glycosylated alkaloids, were identified from hairy root extracts by UPLC-MS. GC-MS analysis allowed the separation of the most volatile and non-polar alkaloids in a single run. The composition of typical non-polar alkaloids indicated the occurrence of 20 TIAs belonging to nine different groups. The quantities of these alkaloids varied between clones in the order eburenine, vincanine, vallesiachotamine and yohimbine isomer II. The occurrence of pleiocarpamine, fluorocarpamine, vincamine, ajmalicine, and yohimbine isomers, analysed by GC-MS, and serpentine and its isomer, tetrahydrosecodinol as well as tabersonine, analysed by UPLC-MS, is reported here for the first time from R. stricta. Methyl jasmonate, a well-known elicitor, caused a significant increase in the total alkaloid content of wild type hairy roots as determined by NMR analyses. Detailed targeted analyses by GC-MS showed that the contents of eight out of ten studied alkaloids increased compared to non-elicited cultures. Another studied elicitor, chitosan, did not have any effect on individual alkaloid contents. Transgenic hairy root clones did not exhibit phenotype differences. Multivariate analysis from NMR data showed a clear discrimination between transformed and wild type/gus cultures. This was most probably due to differences in primary metabolites, as the total alkaloid content did not vary between different hairy roots and controls. In general, the production of individual TIAs, analysed by HPLC, was repressed in hairy roots transformed with geraniol synthase(ges) and geraniol 8-oxidase (g8o) genes compared to the wild types. Overexpression of the strictosidine synthase (str) gene resulted in a higher accumulation of serpentine, whereas the production of strictosidine lactam was decreased. There were no significant differences in the contents of other alkaloids compared to the wild type hairy roots. In conclusion, a simple and efficient gene transfer method is reported for R. stricta for the first time. New analytical methods were established which enabled comprehensive investigation of the alkaloids. These data might serve as a basis for further utilization of biotechnological methods for R. stricta and its further metabolic engineering.Rhazya stricta Decne. (Apocynaceae) on Lähi-idästä ja Kaakkois-Aasiasta kotoisin oleva perinteinen lääkekasvi. Se tuottaa lukuisia terpeeni-indolialkaloideja (TIA), joista useilla on tärkeitä farmakologisia ominaisuuksia. Tämä tutkimus keskittyi bioteknologisten menetelmien kehittämiseen R. stricta -kasville. Näihin menetelmiin lukeutuivat erilaistumattomien solukkoviljelmien perustaminen sekä Agrobacterium rhizogenes -bakteerin avulla aikaansaadut siirtogeeniset karva-juuret, jotka ilmensivät varhaisen TIA-synteesireitin geenejä. Rhazya-alkaloidit muodostavat laajan kirjon kemiallisia, erilaisen polaarisuuden omaavia rakenteita. Tästä syystä oli tärkeää kehittää uusia analyysimenetelmiä alkaloidi-pitoisuuksien määrittämiseen ja alkaloidiprofiilien muutosten seuraamiseen eri siirtogeenisten linjojen ja käsittelyiden välillä. Kohdennetut ja kohdentamattomat analyysit solu- ja solukkoviljelmistä suoritettiin kaasukromatografia-massaspektrometrialla (GM-MS), korkean erotuskyvyn neste-kromatografialla (HPLC), erittäin korkean suorituskyvyn nestekroma-tografialla (UPLC-MS) ja ydinmagneettisella resonanssispektroskopialla (NMR). Kallusviljelmät perustettiin viidestä eri kasvinosasta modifioidulle, kasvutekijöitä sisältävälle B5-kasvatusalustalle. Kallusten fenotyypit vaihtelivat, mutta kuten oletettua, ne tuottivat pienempiä määriä alkaloideja kuin villityypin juuret tai karvajuuret. Yllättäen varren soluista perustetut kallusviljelmät tuottivat suurempia määriä striktosidiinilaktaamia kuin muut soluviljelmät. Vain lehdet, toisin kuin sirkkalehdet, alkeisvarret tai varren osat, olivat alttiita agrobakteeri-infektiolle ja siten karvajuurten muodostumiselle. Transformaatio-tehokkuus vaihteli 22 ja 83 %:n välillä riippuen kohdegeenistä. Villityypin sekä gus-geeniä ilmentävät karvajuurilinjat tuottivat kaksinkertaisia määriä alkaloideja verrattuna tavallisiin juuriin. UPLC-MS -menetelmällä karvajuurista tunnistettiin kaikkiaan 17 terpeeni-indolialkaloidia, mukaan lukien glykosyloidut alkaloidit. GC-MS -analyysi mahdollisti myös kaikkein helpoimmin haihtuvien, poolittomien alkaloidien erottelun samalla kertaa. Tyypillisten poolittomien alkaloidien koostumus viittasi 20 TIA:n esiintymiseen, jotka voitiin edelleen jakaa yhdeksään ryhmään. Suurimmat vaihtelut alkaloidien pitoisuuksissa eri solulinjojen välillä havaittiin ebureniinin, vinkaniinin, vallesiakotamiinin ja johimbiinin isomeeri II:n kohdalla, tässä järjestyksessä. GC-MS:lla analysoitua pleiokarpamiinia, fluorokarpamiinia, vinkamiinia, ajmalisiinia ja johimbiini-isomeereja sekä UPLC-MS:lla analysoitua serpentiiniä ja sen isomeereja, tetrahydrosekodinolia ja tabersoniinia ei ole tätä ennen ole raportoitu R. stricta -kasvista. NMR-analyysi osoitti metyylijasmonaatin, joka on yleinen elisiittori, lisäävän merkittävästi alkaloidien kokonaismäärää villityypin karvajuurissa. Kohdennettu GC-MS -analyysi osoitti, että kahdeksan alkaloidin pitoisuus kymmenestä lisääntyi verrattuna käsittelemättömiin karvajuuriin. Toisaalta kitosaanilla, toisella elisiittorilla, ei havaittu olevan vaikutusta yhdenkään yksittäisen alkaloidin pitoisuuteen. Siirtogeeniset karvajuuret eivät poikenneet fenotyypiltään villityypin karvajuurista. NMR-datan monivarianssianalyysi osoitti kuitenkin selkeän eron siirtogeenisten ja villityypin tai gus-karvajuurien välillä. Tämä johtui todennäköisimmin eroista primäärimetaboliittien pitoisuuksissa, koska alkaloidien kokonaispitoisuudessa ei ollut eroa eri karvajuurilinjojen ja kontrollien välillä. Yksittäisten HPLC:lla analysoitujen terpeeni-indolialkaloidien tuotto oli yleisesti vähentynyt geraniolisyntaasi- (ges) sekä geranioli-8-oksidaasi-geenejä (g8o) ilmentävissä karvajuurilinjoissa verrattuna villityypin karvajuuriin. Striktosidiinisyntaasi-geenin (str) yli-ilmeneminen johti kohonneeseen serpentiini pitoisuuteen ja pienentyneeseen striktosidiinilaktaamin tuottoon. Muiden alkaloidien kohdalla ei havaittu merkittävää eroa villityypin karvajuuriin. Tässä työssä esitetään ensimmäistä kertaa yksinkertainen ja tehokas geenin-siirtomenetelmä R. stricta -kasville. Lisäksi kehitettiin joukko uusia analyysi-menetelmiä alkaloidien kokonaisvaltaiseen analysoimiseen. Nämä tulokset luovat pohjan bioteknologisten menetelmien laajemmalle käytölle R. stricta -kasvin kohdalla ja sen metabolian muokkaamiselle

An Unusually High Blood Alcohol Level in a Burnt Child Homicide Victim

Background: Alcohol detection is one of the most prevalent requests in forensic toxicology laboratories for medico-legal investigation. Interpretation of analytical toxicology results is important in deciding the cause and manner of death. The aim of the present case report is to introduce an unusually high blood alcohol level in a homicide victim and interpretation of analytical toxicology results considering crime scene investigation. Case Presentation: In the present study, we report a case of a burnt body on which autopsy examination and forensic toxicology analyses were carried out. Autopsy examination showed several stab wounds in the neck and thorax regions. Forensic toxicology analysis results showed 1055 mg/dL ethanol and 71 mg/dL methanol in right heart blood using headspace gas chromatography. The leading cause of death was stab wounds. Burning seemed to be a tool for covering up the homicide. Use of alcoholic fire accelerant and contamination of the corpse with alcohol was the main reason to detect high blood alcohol levels.Conclusion: It is essential to determine ethanol in different sampling sites to reach a reliable and scientific interpretation of analytical toxicology results. Also, some important factors such as crime scene investigation should be considered in the interpretations of unusual forensic toxicology results

Occupational Exposure to Paving Asphalt Fumes, Impact on Liver and Kidney Function Test Parameters

Background: Asphalt is a mixture of aliphatic and aromatic hydrocarbons. It also contains different kinds of heavy metals. Hot mix asphalt emits hazardous volatile substances. Asphalt is used in many industries, causing serious adverse health effects for workers. Therefore, investigation of the health status of workers is of crucial importance. The present study aimed to assess the impact of asphalt vapor on hepatic and renal function tests parameters and the clinical parameters in road paving workers compared to healthy subjects.Methods: In a cross-sectional study, 41 road asphalt paving workers and 120 healthy non-exposed subjects were selected. The serum enzymes of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin, triglyceride, urea, and creatinine levels were assessed using diagnostic kits. A medical practitioner examined all subjects for any possible medical condition.Results: Liver and kidney function test parameters were significantly higher in road asphalt paving workers compared to their healthy counterparts (P<0.05 for all parameters). About 63% of workers exposed to asphalt fumes for more than 10 years showed cardiac, pulmonary, and metabolic complications. Conclusion: Significant elevations in liver and kidney function test parameters suggest that exposure to asphalt fumes is associated with hepatic and renal dysfunction in asphalt-exposed workers

The effect of strontium carbonate on cross-linking of pectin based free

Introduction: : Recently, considerable attention has been devoted to biopolymers because of their unique properties and high usage in different fields such as pharmaceutical industry, health promotion and cosmetic applications .Pectin is a high-molecular weight and anionic natural heteropolysaccharide extracted from cell walls of higher plants which is non-toxic and biocompatible.(1) A cross-link is a bond that links one polymer chain to another. Strontium (Sr+2) establish covalent bonds (chemical method) and physical interaction (physical method) between anionic pectin chains that makes polymer stronger. Methods and Results: : Firstly, specific amounts of pectin were dissolved in distilled water. Then, a fixed amount of triethyl citrate (1:6 ratio related to total polymer content) was added to solution as a plasticizer. After 24 hour, free film was cross-linked with 1% strontium carbonate solution for 1 minute .For swelling experiment, a piece of dried cross-linked and non cross-linked pectin based free film was weighted and immersed in flasks of dissolution test containing 250 ml of acidic media(PH=1.2) and phosphate buffer media(PH=6.8) at 37 ℃. sampling time was started from 2 minutes to 60 minutes. To quantify the swelling process, the swelling index, Is (%),was calculated(2). Thickness of both cross-linked and non cross linked pectin based free film was 0.16. Is for cross-linked free film in buffer media was 104% and in acidic media was 94%. Non cross-linked free film was ionized in buffer media while Is was 163% for non cross-linked free film in acidic media. Conclusions: Cross-linker greatly reduced the ionization of the free film in phosphate buffer media and resisted against excessive swelling of the free film in acidic media. Due to positive charge of acidic media and negative charge of pectin free film ,more swelling was expected in acidic media versus buffer media. The Cross-linking had no significant effect on thickness. According to investigate the effect of cross-linker on free film , more experiments needed to be done

Determination of Methadone and Tramadol in Vitreous Humor Specimens Using Dispersive LiquidLiquidMicroextractionandUltraHighPerformance Liquid Chromatography

Background: Drug abuse is spreading rapidly all over the world. Methadone and tramadol are among not only the most abused opioids but also important from the forensic point of view. Therefore, we need to devise a simple and sensitive method for the sample preparation and identification of abused drugs in postmortem specimens.Methods: A simple and rapid Dispersive Liquid-Liquid Microextraction (DLLME) technique coupled with Ultrahigh Performance Liquid Chromatography (UHPLC) was developed for the extraction and analysis of methadone and tramadol from postmortem vitreous humor samples. Different parameters affecting the extraction recovery, such as the type and volume of extraction and dispersion solvents, pH value, sensitivity, and specificity, were optimized and studied. Results: Under optimized conditions, the recovery ranges were 82.3%-89.6% and 85.4%-87.1% for methadone and tramadol, respectively. The linear range was 25-100 ng/mL for both methadone and tramadol with a correlation coefficient (R2) of more than 0.98. Limit of Detection (LoD) and Limit of Quantification (LoQ) were 3 and 8 ng/mL for methadone and 6 and 16 ng/mL for tramadol. The accuracy level of the methods for methadone and tramadol detection were 99.4%-100% and 99.7%-99.9%, respectively. The method was specific enough for the qualitative and quantitative determination of methadone and tramadol.Conclusion: The obtained results showed that DLLME combined with UHPLC is a fast and straightforward method for determining methadone and tramadol in postmortem vitreous humor specimens

Discrimination Between Drug Abuse and Medical Therapy : Case report of a tranylcypromine overdose-related fatality

Tranylcypromine is an effective antidepressant from the class of monoamine oxidase inhibitors and is structurally related to amphetamine. However, reports differ regarding the potential metabolism of tranylcypromine to amphetamine and methamphetamine within the human body. We report a 25-year-old woman with severe depression who died due to a fatal tranylcypromine overdose in 2016. She had been prescribed tranylcypromine one day previously and had no history of previous suicide attempts or substance abuse. The body was transferred to a forensic medicine department in Tehran, Iran for the autopsy. A urine sample was positive for tranylcypromine, amphetamine and methamphetamine using gas chromatography/mass spectrometry after derivatisation with heptafluorobutyric acid. As amphetamines were present in the urine sample, it was assumed that the tranylcypromine had been converted to amphetamines metabolically. As such, it is possible that the legitimate use of certain prescription drugs may complicate the interpretation of test results for illegal drugs

Alginate Free Films: Cross-linking with Strontium Carbonate

Introduction: Alginates are non-toxic, hydrophilic, biocompatible, biodegradable and low cost polymers which make them suitable for many biomedical applications. Due to their good tissue compatibility, they have been widely used in enhancing the healing process. However, they have limitations in mechanical characteristics and drug release. Adding metal ions could add some features to these films in order to improve their properties. The purpose of this study is to compare alginate blend films cross-linked with different concentrations of strontium in order to submit one as the best in topical applications. Methods and Results:Sodium alginate (2 g/100 g) was first dissolved in 50 mL de-ionized water. Di butyl phthalate (16%w/w) was added as plasticizer. After dissolution, the solution was poured into Petri dishes and dried at 40°C for 24 h. Alginate films were chemically cross-linked by immersing in different concentrations of strontium carbonate (0.1-1-10% w/v) for 1 minute. The films were then dried at oven at 40 °C for 24 h. The swelling test was performed in acidic(HCl 0.1N) and phosphate buffer media (pH=6.8) for 90 minutes. Thickness of alginate film before and after the strontium crosslinking procedure was0.178mm and 0.26 mm, respectively. Swelling index (IS) in acid and buffer media was 280.70% and 263.64%, respectively. Conclusions:The swelling test of cross-linked films has demonstrated satisfying results by increasing in swelling properties, hence promising outcome for wound healing conditions. Also, the results showed pH-responsive swelling behaviors with surprisingly more swelling in acidic media compared to buffer

Playground v2.5: Three Insights towards Enhancing Aesthetic Quality in Text-to-Image Generation

In this work, we share three insights for achieving state-of-the-art aesthetic quality in text-to-image generative models. We focus on three critical aspects for model improvement: enhancing color and contrast, improving generation across multiple aspect ratios, and improving human-centric fine details. First, we delve into the significance of the noise schedule in training a diffusion model, demonstrating its profound impact on realism and visual fidelity. Second, we address the challenge of accommodating various aspect ratios in image generation, emphasizing the importance of preparing a balanced bucketed dataset. Lastly, we investigate the crucial role of aligning model outputs with human preferences, ensuring that generated images resonate with human perceptual expectations. Through extensive analysis and experiments, Playground v2.5 demonstrates state-of-the-art performance in terms of aesthetic quality under various conditions and aspect ratios, outperforming both widely-used open-source models like SDXL and Playground v2, and closed-source commercial systems such as DALLE 3 and Midjourney v5.2. Our model is open-source, and we hope the development of Playground v2.5 provides valuable guidelines for researchers aiming to elevate the aesthetic quality of diffusion-based image generation models.Comment: Model weights: https://huggingface.co/playgroundai/playground-v2.5-1024px-aestheti

Heroes and Demigods: Aristotle's Hypothetical "Defense" of True Nobles

Although the commentary on Aristotle’s problematic discussion of slavery is vast, his discussion of nobility receives little attention. The fragments of his dialogue On Noble Birth constitute his most extensive examination of nobility, and while their similarity to the παμβασιλεύς of the Politics has recently been recognized, their relevance to natural slavery has hitherto gone unnoticed. Yet by declaring that true nobles – particularly the god-like ἀρχηγός – preternaturally possess superhuman characteristics, Aristotle precludes their easy inclusion in the kind “human” in a manner inversely mirroring the preternatural subhumanity of natural slaves. Building on recent scholarship which argues that Aristotle’s “defense” of natural slaves is better understood as an indictment, On Noble Birth becomes most coherent if read as a hypothetical investigation into what would be required for “nobility” to name something true rather than equivocal, with the conclusion that “true nobility” is an empty set