Određivanje aminokiselinske sekvencije farmaceutskih peptida MALDI-TOF tandemnom spektrometrijom masa

Although the peptide ion mechanisms of pre-dissociation, dissociation and post-dissociation have been well-studied over the past fifteen years, their practical application still has to be implemented into modern mass spectrometry-driven proteomics and bioanalysis. Unambiguous peptide sequence determination by mass spectrometry relies on the idea that only one continuous series of ions in mass spectrum can assure full sequence determination and meets the requirements of peptide analysis quality. This set of rules defined for the peptide analysis by tandem mass spectrometry would generally improve an overall reliability and data accuracy. Based on the process mechanisms of gas-phase peptide bond dissociation, a relatively small and large model peptides are unambiguously analyzed (bivalirudin and exenatide) showing that derivatization concepts of the C- or N-terminus derivatization (SPITC and Lys-tag) can be avoided.Iako su mehanizmi preddisocijacije, disocijacije i postdisocijacije iona izučavani dugi niz godina, postoji još cijeli niz mehanizama raspada iona koji se mogu korisno upotrijebiti u modernoj spektrometriji masa, proteomici i bioanalizama. Nedvojbena aminokiselinska analiza spektrometrijom masa počiva na ideji da samo jedan i kontinuirani slijed iona u tandemnom spektru masa može potpuno potvrditi cjelovitu aminokiselinsku sekvenciju i na taj način zadovoljiti zahtjeve nedvojbene analize peptida. Niz pravila kojima se definira cjelovita i nedvojbena analiza peptida tandemnom spektrometrijom masa u konačnici može unaprijediti iščitavanje rezultata analize i pouzdanost dobivenih podataka. Na teorijskim zasadama disocijacije iona u plinskoj fazi u ovom je radu prikazana cjelovita analiza peptida. Na modelnim farmaceutskim peptidima bivalirudinu i eksenatidu pokazano je kako se i bez raširene upotrebe derivatizacijskih tehnika (SPITC i Lys-tag) može ostvariti nedvojbena analiza, iako se modifikacije cisteina akrilacijom, metilacijom ili karboksimetilacijom pri tome ne mogu izbjeći

Hyphenated techniques liquid chromatography-mass spectrometry: basic methodology and applicati ons

Tekućinska kromatografi ja (LC) osnovna je višenamjenska separacijska tehnika koja se primjenjuje u modernim biološkim znanosti ma i srodnim područjima molekularne biologije kao što su analiti čka ili preparati vna kemija. Za razliku od mnogih drugih separacijskih tehnika koje nisu pogodne za razdvajanje termički nestabilnih molekula (npr. plinska kromatografija, GC) ili onih koje se ne mogu direktno spregnuti (npr. izoelektrično fokusiranje, IEF), tekućinska kromatografija može uspješno poslužiti za razdvajanje širokog raspona molekula kao što su to polimeri, male molekule farmaceutika ili njihovih metabolita, kao i pepti da i proteina. Odabirom takve metode tekućinske kromatografi je koja ne šteti analizi spektrometrijom masa (hlapljivi puferi, stabilan i nizak protok, upotreba polarnih organskih otapala), tekućinski kromatograf može se jednostavno spregnuti sa spektrometrom masa. Izvor iona, analizator (ili kombinacija više analizatora u istom instrumentu, tzv. tandemska spektrometrija masa) i detektor iona odabiru se u ovisnosti o vrstama analiza, a koje pak mogu biti jednostavne analize određenog iona ili kvalitati vno substrukturalne i kvanti tati vne analize kompleksnih smjesa. Da bi se olakšalo određivanje strukture ili kvanti tati vna analiza, u proteklom desetljeću su razvijene različite programske aplikacije i pretraživači baza podataka.Liquid chromatography (LC) is a basic versati le separati on technique widely used in modern life sciences especially in the fi elds closely related to molecular biology, namely in the analytical or preparative chemistry. Unlike many other separati on techniques, which are unsuitable for thermally degradable molecules (e.g. gas chromatography, GC), or which are not feasible for on-line coupling (e.g. isoelectric focusing, IEF), liquid chromatography can efficiently separate a very wide range of large molecules like polymers, peptides and proteins as well as small molecules like drug or drug metabolites. Choosing the proper LC method that uti lize mass spectrometry (MS) friendly conditi ons (volati le buff ers, stable and low fl ow rate, polar organic solvents etc.), liquid chromatography system can be easily hyphenated to mass spectrometer. Ionizati on source, analyzer (or combinati on of more analyzers in one instrument, the so called tandem mass spectrometry) and ion detector are selected according to the analysis requirements that might range from simple one ion qualitative analysis to substructural qualitati ve, and quanti tati ve analysis of complex mixtures. To facilitate elucidati on of structure or quanti tati ve analysis different soft ware applicati ons and data bases search engines were developed during the last decade

Uloga laparoskopije u liječenju raka jajnika

Laparoscopy is usually undertaken in young women with adnexal disease that is believed to be benign, but found to be malignant at surgery. Some surgeons use laparascopy for staging, but mainly for presumed stage I or II ovarian cancer. Also, laparoscopy can be useful for selecting women who can be optimally debulked at primary surgery. There are some concerns about using laparoscopic approach in women with ovarian cancer: port-site metastasis, possibility of intraoperative rupture of tumor and eff ect of pneumoperitoneum. Studies showed that rate of port-site metastasis after laparoscopic surgery in women with ovarian cancer is low and usually occurs when there is peritoneal carcinomatosis or distant metastatic disease. Also, it seems that laparoscopy with CO(2) pneumoperitoneum does not reduce the overall survival in women with intraabdominal metastases. Preoperative rupture, surface capsular invasion and positive peritoneal cytology are more relevant in terms of prognosis than intraoperative rupture, but further research is needed. Conventional laparoscopy and robotic-assisted laparoscopy are comparable in both early and advanced disease. These approaches are not inferior to laparotomy and they are acceptable access in selected patients.Laparoskopija se najčešće koristi kod žena za koje se mislilo da imaju benignu bolest, ali se tijekom operacije ispostavilo da se radi o malignoj bolesti. Neki kirurzi koriste laparoskopski pristup za stupnjevanje bolesti i to najčešće za pretpostavljeni I ili II stadij. Također, laparoskopija može biti korisna za odabir žena kojima se može primarno napraviti „debulking“. Postoji nekoliko briga prilikom korištenja laparoskopskog pristupa: pojava metastaza na mjestu ulaska troakara, mogućnost intraoperacijske rupture tumora i učinak pneumoperitoneuma. Studije su pokazale da je udio metastaza na mjestu ulaska troakara nizak i da se najčešće javlja kod žena koje imaju karcinomatozu peritoneuma i udaljene metastaze. Također, čini se da laparoskopija s CO2 pneumoperitoneumom ne smanjuje ukupno preživljenje žena s intraabdominalnim metastazama. Preoperacijska ruptura, površinska invazija kapsule i pozitivan peritonealni ispirak su važniji za prognozu od intraoperacijske rupture, no daljnja istraživanja su potrebna. Konvencionalna i laparoskopija pomoću robota su usporedivei u ranoj i u proširenoj bolesti.Ovi pristupi nisu inferiorni laparotomiji i prihvatljivi su kod odabranih pacijenata

Uloga laparoskopije u liječenju raka jajnika

Laparoscopy is usually undertaken in young women with adnexal disease that is believed to be benign, but found to be malignant at surgery. Some surgeons use laparascopy for staging, but mainly for presumed stage I or II ovarian cancer. Also, laparoscopy can be useful for selecting women who can be optimally debulked at primary surgery. There are some concerns about using laparoscopic approach in women with ovarian cancer: port-site metastasis, possibility of intraoperative rupture of tumor and eff ect of pneumoperitoneum. Studies showed that rate of port-site metastasis after laparoscopic surgery in women with ovarian cancer is low and usually occurs when there is peritoneal carcinomatosis or distant metastatic disease. Also, it seems that laparoscopy with CO(2) pneumoperitoneum does not reduce the overall survival in women with intraabdominal metastases. Preoperative rupture, surface capsular invasion and positive peritoneal cytology are more relevant in terms of prognosis than intraoperative rupture, but further research is needed. Conventional laparoscopy and robotic-assisted laparoscopy are comparable in both early and advanced disease. These approaches are not inferior to laparotomy and they are acceptable access in selected patients.Laparoskopija se najčešće koristi kod žena za koje se mislilo da imaju benignu bolest, ali se tijekom operacije ispostavilo da se radi o malignoj bolesti. Neki kirurzi koriste laparoskopski pristup za stupnjevanje bolesti i to najčešće za pretpostavljeni I ili II stadij. Također, laparoskopija može biti korisna za odabir žena kojima se može primarno napraviti „debulking“. Postoji nekoliko briga prilikom korištenja laparoskopskog pristupa: pojava metastaza na mjestu ulaska troakara, mogućnost intraoperacijske rupture tumora i učinak pneumoperitoneuma. Studije su pokazale da je udio metastaza na mjestu ulaska troakara nizak i da se najčešće javlja kod žena koje imaju karcinomatozu peritoneuma i udaljene metastaze. Također, čini se da laparoskopija s CO2 pneumoperitoneumom ne smanjuje ukupno preživljenje žena s intraabdominalnim metastazama. Preoperacijska ruptura, površinska invazija kapsule i pozitivan peritonealni ispirak su važniji za prognozu od intraoperacijske rupture, no daljnja istraživanja su potrebna. Konvencionalna i laparoskopija pomoću robota su usporedivei u ranoj i u proširenoj bolesti.Ovi pristupi nisu inferiorni laparotomiji i prihvatljivi su kod odabranih pacijenata

Diferencijacija Pseudomonas i Stenotrophomonas vrsta izolovanih iz riba primenom molekularnih metoda i MALDI-TOF metode

For the purpose of precise antibiotic susceptibility testing it is necessary to clearly distinguish Pseudomonas and Stenotrophomonas genera, considering acquired resistance of Pseudomonas species, as well as the intrinsic resistance of Stenotrophomonas species. This is why in the identification of the 51 isolates originated from fish, the following methods were used: standard PCR, 16S rRNA gene sequencing, and MALDI-TOF. The results of the standard PCR test, 16S rRNA gene sequencing and MALDI-TOF analysis confirmed 35 strains to belong to the Pseudomonas genus. Standard PCR test and VITEK MS device confirmed that 10 strains belong to Stenotrophomonas maltophilia species. Three strains were positive in both standard PCR tests for Pseudomonas and Stenotrpohomonas. 16S rRNA gene sequencing identified these 3 strains to be 99% Pseudomonas sp. and 99% Stenotrophomonas sp. VITEK MS first identified these three strains as 99% Stenotrophomonas, and in the repeated identification it identified them as 99% Pseudomonas. MALDI TOF/TOF 4800 Plus device identified these strains as Stenotrophomonas. Three strains were negative in both standard PCR tests for Pseudomonas and Stenotrpohomonas. 16S rRNA gene sequencing identified these 3 strains to be 99% Pseudomonas sp. and 99% Stenotrophomonas sp. VITEK MS first identified these three strains as 99% Stenotrophomonas, and in the repeated identification it identified them as 99% Pseudomonas. MALDI TOF/TOF 4800 Plus device identified these strains as Stenotrophomonas. Although modern test methods that have very high specificity (PCR, 16S rRNA gene Pseudomonas and Stenotrophomonas species for 6 isolates could not be reached using the above mentioned methods.S obzirom na značaj stečene rezistencije Pseudomonas vrsta, kao i na intrinzičnu rezistenciju Stenotrophomonas vrsta, a u cilju preciznog ispitivanja osetljivosti na antibiotike, neophodna je jasna diferencijacija pripadnika ovih rodova bakterija. U tom cilju su u identifikaciji 51 izolata poreklom od riba korišćene metode: standardni PCR, 16S rRNA sekvenciranje gena, MALDI-TOF. Rezultati standardnog PCR testa, 16S rRNA sekvenciranja gena i MALDI-TOF analize su za 35 sojeva potvrdili pripadnost rodu Pseudomonas. Standardnim PCR testom i primenom aparata VITEK MS utvrđeno je da 10 sojeva pripada vrsti Stenotrophomonas maltophilia. U 16S rRNA sekvenciranju gena 3 soja koja su bila pozitivna u oba standardna PCR testa identifikovana su kao 99% Pseudomonas sp. i 99% Stenotrophomonas sp. VITEK MS je ova tri soja u prvoj identifikaciji identifikovao kao 99% Stenotrophomonas, a u ponovljenoj identifikaciji kao 99% Pseudomonas. Ti sojevi su na aparatu MALDI TOF/TOF 4800 Plus bili identifikovani kao Stenotrophomonas. U 16S rRNA sekvenciranju gena tri soja koja su bila negativna u oba standardna PCR testa su identifikovana kao 99% Pseudomonas sp. i 99% Stenotrophomonas sp. Aparat VITEK MS je ova tri soja identifikovao u jednoj identifikaciji kao 99% Stenotrophomonas, a u drugoj identifikaciji 99% kao Pseudomonas. Ti sojevi su na aparatu MALDI TOF/ TOF 4800 Plus bili identifikovani kao Stenotrophomonas. Iako su u ovom istraživanju korišćene savremene metode ispitivanja koje imaju vrlo visoku specifičnost (PCR, 16s rRNK sequencing, MALDI TOF) precizna diferencijacija Pseudomonas i Stenotrophomonas vrsta nije mogla biti postignuta

Characterization of a S-adenosyl-l-methionine (SAM)-accumulating strain of Scheffersomyces stipitis

S-adenosyl-l-methionine (SAM) is an important molecule in the cellular metabolism of mammals. In this study, we examined several of the physiological characteristics of a SAM-accumulating strain of the yeast Scheffersomyces stipitis (M12), including SAM production, ergosterol content, and ethanol tolerance. S. stipitis M12 accumulated up to 52.48 mg SAM/g dry cell weight. Proteome analyses showed that the disruption of C-24 methylation in ergosterol biosynthesis, a step mediated by C-24 sterol methyltransferase (Erg6p), results in SAM accumulation by S. stipitis M12 compared to the wild-type strain. A comparative proteome-wide analysis identified 25 proteins that were differentially expressed by S. stipitis M12. These proteins are involved in ribosome biogenesis, translation, the stress response, ubiquitin-dependent catabolic processes, the cell cycle, ethanol tolerance, posttranslational modification, peroxisomal membrane stability, epigenetic regulation, the actin cytoskeleton and cell morphology, iron and copper homeostasis, cell signaling, and energy metabolism. [Int Microbiol 2015; 18(2):117-125]Keywords: Scheffersomyces stipitis · S-adenosyl- l-methionine (SAM) · SAM accumulating yeast · C-24 sterol methyltransferase (Erg6p

Combination of cyclopamine and tamoxifen promotes survival and migration of MCF-7 breast cancer cells – interaction of Hedgehog-Gli and Estrogen receptor signaling pathways

Hedgehog-Gli (Hh-Gli) signaling pathway is one of the new molecular targets found upregulated in breast tumors. Estrogen receptor alpha (ERα) signaling has a key role in the development of hormone-dependent breast cancer. We aimed to investigate the effects of inhibiting both pathways simultaneously on breast cancer cell survival and the potential interactions between these two signaling pathways. ER-positive MCF-7 cells show decreased viability after treatment with cyclopamine, a Hh-Gli pathway inhibitor, as well as after tamoxifen (an ERα inhibitor) treatment. Simultaneous treatment with cyclopamine and tamoxifen on the other hand, causes short-term survival of cells, and increased migration. We found upregulated Hh-Gli signaling under these conditions and protein profiling revealed increased expression of proteins involved in cell proliferation and migration. Therefore, even though Hh-Gli signaling seems to be a good potential target for breast cancer therapy, caution must be advised, especially when combining therapies. In addition, we also show a potential direct interaction between the Shh protein and ERα in MCF-7 cells. Our data suggest that the Shh protein is able to activate ERα independently of the canonical Hh-Gli signaling pathway. Therefore, this may present an additional boost for ER-positive cells that express Shh, even in the absence of estroge

Supplementary material for the article: Nišavić, M.; Janjić, G. V.; Hozić, A.; Petković, M.; Milčić, M. K.; Vujčić, Z.; Cindrić, M. Positive and Negative Nano-Electrospray Mass Spectrometry of Ruthenated Serum Albumin Supported by Docking Studies: An Integrated Approach towards Defining Metallodrug Binding Sites on Proteins. Metallomics 2018, 10 (4), 587–594. https://doi.org/10.1039/c7mt00330g

Supplementary material for: [https://doi.org/10.1039/c7mt00330g]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2134]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3243

Supplementary material for the article: Nišavić, M.; Janjić, G. V.; Hozić, A.; Petković, M.; Milčić, M. K.; Vujčić, Z.; Cindrić, M. Positive and Negative Nano-Electrospray Mass Spectrometry of Ruthenated Serum Albumin Supported by Docking Studies: An Integrated Approach towards Defining Metallodrug Binding Sites on Proteins. Metallomics 2018, 10 (4), 587–594. https://doi.org/10.1039/c7mt00330g

Supplementary material for: [https://doi.org/10.1039/c7mt00330g]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2134]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3243

Hiperspektralni senzori i primjena u šumarstvu

Od samoga početka razvoja daljinskih istraživanja ljudi su pokušavali stvoriti alat za proučavanje Zemljine površine i dinamičnih pojava na njoj. Napretkom tehnologije najprije za vojne, a potom i za istraživačke potrebe, razvijeni su senzori za prikupljanje podataka snimanjem elektromagnetskoga spektra (multispektralni i hiperspektralni). U trenutku kada su postali dostupni za uporabu u civilnoj zajednici, postale su očigledne potencijalne koristi uporabe hiperspektralnih podataka. Razvoj hiperspektralnih senzora slijedio je razvoj računalne tehnologije i napredak softvera za obradu velikoga broja prikupljenih podatka. Danas se hiperspektralni senzori sve više upotrebljavaju za razne namjene: proučavanje ekosustava, atmosfere, klime, hidrologije, iskorištavanja minerala, načina korištenja zemljišta, zemljišnoga pokrova i vegetacije. Zahvaljujući velikomu broju raznovrsnih informacija koje pružaju, hiperspektralni senzori imaju veliku mogućnost primjene u nadzoru i upravljanja okolišem te pomažu unapređenju upravljanja i donošenju boljih odluka pri ranom definiranju problema. Prva primjena hiperspektralnih senzora u šumarstvu u Hrvatskoj vezana je uz program Europske zajednice iz 2005. godine, kada su provedena prva snimanja linijskim hiperspektralnim senzorom. U sklopu provedenih snimanja izdvojene su prve spektralne krivulje obične jele i bijele imele, na osnovi kojih je prvi put izdvojena vrsta unutar vrste, odnosno pomoću hiperspektralnih snimaka razlučeni su grmovi poluparazitske vrste unutar krošanja domaćina te su definirane procedure za snimanja na velikim površinama