NANO-ELECTROSPRAY IONIZATION OF THE NEGATIVE IONS CAUSED BY ADDING ESTERS, ALDEHYDES AND KETONES IN A MOBILE PHASE FLOW

Nastajanje negativnih iona elektroraspršenjem za cijelu klasu biomakromolekula (peptidi, proteini, DNA, RNA, ugljikohidrati) je energetski nepovoljnije od nastajanja pozitivnih iona u ionskom izvoru spektrometra masa. Jedan od razloga je vezan uz nemogućnost postizanja stabilnog elektroraspršenja u negativnom načinu rada spektrometra masa. Nastajanje i analiza negativnih iona peptida i proteina ima izraziti značaj u području proteomike, gdje bi se takvom primjenom pospješila uspješnost analize ukupnog aminokiselinskog slijeda proteina i njegovih posttranslacijskih modifikacija. Prijašnja istraživanja pokazala su pozitivan utjecaj formaldehida na formiranje negativnih iona selektivnih modulatora androgenih receptora. Smatra se da bi sličan utjecaj mogli imati i drugi aldehidi, ketoni i esteri jer podliježu sličnoj reakciji u kiselom mediju kao i formaldehid. Proučavana je ionizacija negativnih iona peptida i proteina nano-elektroraspršenjem na hibridnom spektrometru masa koji se sastoji od T-vala, kvadrupola i analizatora vremena leta u neprekinutom slijedu (T-val Q-TOF MS). Tijekom predselekcije poboljšivača ionizacije (aldehida, ketona i estera) pokazalo se da su najveći utjecaj na nastajanje negativnih iona imali 2,2-dimetilpropanal, etil-metanoat i 2-fenil-2-oksoetanal. Da bi se pospješila ionizacija, odabrani poboljšivači ionizacije negativnih iona su umiješani u tok pokretne faze pri protoku od 0,4 L min-1 uz pomoć poslijekolonskog injekcijskog sustava. Rezultati su pokazali da se umiješavanjem mikromolarnih količina navedenih spojeva u matičnicu pokretne faze postiže ionizacija negativnih iona peptida i proteina u ESI-MS-u.For a wide variety of biological compounds (peptides, proteins, DNA, RNA, carbohydrates),negative ion formation by electrospray ionization is energetically less favorable than positive ion formation in mass spectrometer ion source. One of the reasons is inability to maintain stable electrospray in negative ion mode. Formation and analysis of negative ions of peptides and proteins is of great importance in proteomics research, as they would significantly improve amino acid sequence analysis of proteins and analysis of their posttranslational modifications. Previous research showed favorable effects of formaldehyde on the negative-ion formation of selective androgen receptor modulators. It is assumed that other aldehydes, ketones and esters would have similar effect because their reaction mechanism in acidic medium is similar to the reaction mechanism of formaldehyde. Hybrid T-wave quadrupole time-of-flight mass spectrometer (T-wave Q-TOF MS) was used for the study of nano electrospray formation of negative ions of peptides and proteins. Preselection of the negative-ion enhancers (aldehydes, ketones and esters) showed that the greatest effect on negative-ion formation had 2,2-dimethylpropanal, ethyl methanoate and 2-oxo-2-phenylethanal. The selected negative-ion enhancers were added postcolumn at a flow rate of 0,4 L min-1 to a mobile phase flow to enhance ionization. Results showed that formation of negativeions of peptides and proteins in ESI-MS was achieved by addition of micromolar quantities of the mentioned negative-ion enhancers to the mobile phase flow

A young researcher’s guide to NME/Nm23/NDP Kinase

Nucleoside diphosphate kinases (NDPKs) catalyze the exchange of the terminal phosphate from trinucleotides to dinucleotides through a high-energy phosphohistidine intermedier. They are encoded by NME genes and have been found, with a few exceptions, in all living beings. Besides their well-known function as key regulators of the cellular nucleotide homeostasis, they have been appointed numerous additional biochemical and biological functions. The discovery of NME1/NDPK A as the first metastasis suppressor opened new avenues in cancer research. Although the precise role of NME genes/proteins in cancer dissemination is not yet revealed, it seems that further intensive research in this field may lead to new advances in cancer diagnosis and prognosis, as well as encourage new therapeutic strategies.</p

Učinci turističkih transnacionalnih korporacija na zemlje u razvoju i manje razvijene zemlje

U današnjim globalizacijskim uvjetima kao glavni nositelji, odnosno akteri globalnog poslovanja nameću se transnacionalne korporacije koje su, što se tiče sektora turizma, prepoznatljive u obliku mega hotelskih, agencijskih i sličnih lanaca. Inozemni su hotelski lanci danas određeni standard koji svaka ozbiljnija turistička destinacija mora zadovoljiti ukoliko želi biti konkurentna na globalnom turističkom tržištu. S obzirom na značaj koji se pridaje izravnim inozemnim ulaganjima u turizam i očekivanja hrvatske Vlade od ulaska inozemnog kapitala u turistički sektor, svrha je rada razmotriti ulogu i značaj transnacionalnih korporacija u turizmu. Cilj je rada sintetizirati najvažnije učinke transnacionalnih turističkih korporacija na zemlje u razvoju i manje razvijene zemlje

Guidance of adjuvant instillation in intermediate-risk non-muscle invasive bladder cancer by drug screens in patient derived organoids: a single center, open-label, phase II trial.

BACKGROUND In intermediate-risk non-muscle invasive bladder cancer (NMIBC) clinical guidelines suggest an adjuvant instillation with a chemotherapeutic agent. However, the agent and regimen are not clearly defined. Worldwide, less than 15% of patients receive this adjuvant chemotherapeutic instillation. We recently developed a pipeline for the generation of patient derived organoids (PDO) in NMIBC. In this phase II trial, we aim to use our in vitro pipeline to select the most effective drug for chemotherapeutic instillation in NMIBC patients. METHODS Patients with first diagnosis of intermediate-risk NMIBC that are directed to transurethral resection of bladder tumor (TURBT) are enrolled. During TURBT, tumor is sampled, and specimens are directed to generate PDO. Once the PDO are formed, drug screens on them for Epirubicin, Mitomycin C, Gemcitabine and Docetaxel are performed. The drug with the highest antitumor activity in vitro will then be selected for 6 adjuvant intravesical instillations once weekly. Thereafter, patients are followed according to clinical guidelines by cystoscopy. DISCUSSION The aim of this trial is to use drug screens in PDO to precise treatment selection for adjuvant instillation therapies in patients with intermediate-risk NMIBC. The ultimate goal of this trial is to reduce the risk of cancer recurrence. In the future, we aim to conduct clinical multicenter trials with an increased sample size, a broader panel of compounds and a focus on the reduction of cancer recurrence by precision delivery of care. Trial registration NCT05024734

Validacija ICP-MS metode za određivanje metala u tragovima prema ICH Q3D smjernicama u fosamprenavir kalciju

Fosamprenavir calcium is an active pharmaceutical ingredient (API) in which trace elements must be monitored by ICH Q3D Guideline. It delivers appropriate method to ensure the quality of the product and its safe use. Elemental impurities in API are separated in three classes based on their toxicity and probability of their occurrence in the product. ICP-MS has proven to be a suitable technique with the appropriate sample preparation method. Due to the presence of oxidising agents in the sample preparation, determination of osmium is problematic. In the presence of oxidising nitric acid, a highly volatile and toxic osmium tetraoxide is formed. Due to its high volatility, the recovery of osmium has reached the value of 287 %, which exceeds recovery limit (70–150 %). However, osmium can be stabilised by complexation with thiourea in the hydrochloric acid solution (recovery = 87 %). In that way, the loss of osmium is reduced and good results are achieved in terms of usability, accuracy, and precision. A method with addition of thiourea has been successfully validated through main validation parameters: linearity, quantitation limit, selectivity, accuracy, precision (which included repeatability and intralaboratory reproducibility), and robustness. Each of these validation parameters met the acceptance criteria, and therefore it was concluded that the method is suitable for the determination of trace elements in fosamprenavir calcium by ICP-MS. This work is licensed under a Creative Commons Attribution 4.0 International License.Fosamprenavir kalcij je aktivna farmaceutska tvar (API) u kojoj se elementi u tragovima moraju nadzirati prema ICH Q3D smjernicama. ICH Q3D smjernice donose odgovarajuću metodu za osiguravanje kvalitete proizvoda i njegove sigurne uporabe. Elementarne nečistoće u API-ju razdvajaju se u tri razreda na temelju njihove toksičnosti i vjerojatnosti da se pojave u proizvodu. ICP-MS se pokazala kao prikladna tehnika ako se primijeni odgovarajuća metoda pripreme uzoraka. Zbog prisutnosti oksidirajućih sredstava u pripremi uzoraka problem se javlja kod određivanja osmija. Osmij u prisutnosti oksidirajuće dušične kiseline daje visoko hlapljiv i toksičan osmij tetraoksid. Zbog visoke hlapljivosti, iskorištenje osmija iznosi 287 %, što premašuje zadanu granicu iskorištenja (70 – 150 %). Međutim, osmij se može stabilizirati kompleksiranjem s tioureom u otopini klorovodične kiseline (povrat od 87 %). Na taj se način smanjuje njegov gubitak, a postižu se dobri rezultati u pogledu točnosti, preciznosti i robusnosti metode. Ovo djelo je dano na korištenje pod licencom Creative Commons Imenovanje 4.0 međunarodna

Održavanje rituksimabom u uznapredovalom folikularnom limfomu: kontroverzije

Rituximab is a chimeric monoclonal CD20 antibody used in the treatment of CD20 positive non-Hodgkin lymphomas and has revolutionized treatment approach to these hematologic malignancies in the last decade. The main aim of this review is to present data on the use of rituximab in the treatment of follicular lymphoma (FL). We will focus on rituximab maintenance strategies in the first and second line treatment. Th is approach has improved the outcome in FL patients with better progression-free survival in all patients and better overall survival in relapsed setting. Regardless of good results, this strategy has generated controversies in medical community in the range from the lack of overall survival benefit in first line setting, adverse effects of possible overtreatment and toxicities to its unknown role in the era of novel agents. The existing data suggest that rituximab maintenance should be a rational therapeutic option for all patients with FL responding to first line therapy and transplant-ineligible patients responding to reinduction.Rituksimab je kimerično antiCD20 protutijelo koje se koristi u liječenju CD20 pozitivnih ne-Hodgkinovih limfoma te je promijenilo paradigmu liječenja ovih hematoloških neoplazma u prošlom desetljeću. Glavni cilj ovoga preglednog rada je predstaviti njegovu primjenu u folikularnom limfomu (FL) s naglaskom na terapiju održavanja. Ova strategija doprinijela je boljem preživljenju bez progresije bolesti u prvoj i drugoj liniji terapije, odnosno boljem ukupnom preživljenju u bolesnika s relapsom FL-a. No, usprkos dobrim rezultatima, održavanje rituksimabom je doprinijelo kontroverzi u medicinskoj zajednici. Navedene nedoumice potječu od nedostatka poboljšanja ukupnog preživljenja u prvoj liniji terapije, moguće toksičnosti do nepoznate uloge u eri novih lijekova za liječenje FL-a. Prema postojećim podacima zaključujemo da terapiju održavanja rituksimabom treba ponuditi bolesnicima s FL-om koji su odgovorili na prvu liniju terapije te bolesnicima s relapsom FL-a koji su odgovorili na reindukciju, a nisu kandidati za liječenje autolognom transplantacijom matičnih stanica

Characterization of Vemurafenib-Resistant Melanoma Cell Lines Reveals Novel Hallmarks of Targeted Therapy Resistance

Regardless of the significant improvements in treatment of melanoma, the majority of patients develop resistance whose mechanisms are still not completely understood. Hence, we generated and characterized two melanoma-derived cell lines, primary WM793B and metastatic A375M, with acquired resistance to the RAF inhibitor vemurafenib. The morphology of the resistant primary WM793B melanoma cells showed EMT-like features and exhibited a hybrid phenotype with both epithelial and mesenchymal characteristics. Surprisingly, the vemurafenib-resistant melanoma cells showed a decreased migration ability but also displayed a tendency to collective migration. Signaling pathway analysis revealed the reactivation of MAPK and the activation of the PI3K/AKT pathway depending on the vemurafenib-resistant cell line. The acquired resistance to vemurafenib caused resistance to chemotherapy in primary WM793B melanoma cells. Furthermore, the cell-cycle analysis and altered levels of cell-cycle regulators revealed that resistant cells likely transiently enter into cell cycle arrest at the G0/G1 phase and gain slow-cycling cell features. A decreased level of NME1 and NME2 metastasis suppressor proteins were found in WM793B-resistant primary melanoma, which is possibly the result of vemurafenib-acquired resistance and is one of the causes of increased PI3K/AKT signaling. Further studies are needed to reveal the vemurafenib-dependent negative regulators of NME proteins, their role in PI3K/AKT signaling, and their influence on vemurafenib-resistant melanoma cell characteristics

Altered Expression of Shorter p53 Family Isoforms Can Impact Melanoma Aggressiveness

Cutaneous melanoma is the most aggressive form of skin cancer. Despite the significant advances in the management of melanoma in recent decades, it still represents a challenge for clinicians. The TP53 gene, the guardian of the genome, which is altered in more than 50% of human cancers, is rarely mutated in melanoma. More recently, researchers started to appreciate the importance of shorter p53 isoforms as potential modifiers of the p53-dependent responses. We analyzed the expression of p53 and p73 isoforms both at the RNA and protein level in a panel of melanoma-derived cell lines with different TP53 and BRAF status, in normal conditions or upon treatment with common anti-cancer DNA damaging agents or targeted therapy. Using lentiviral vectors, we also generated stable clones of H1299 p53 null cells over-expressing the less characterized isoforms Δ160p53α, Δ160p53β, and Δ160p53γ. Further, we obtained two melanoma-derived cell lines resistant to BRAF inhibitor vemurafenib. We observed that melanoma cell lines expressed a wide array of p53 and p73 isoforms, with Δ160p53α as the most variable one. We demonstrated for the first time that Δ160p53α, and to a lesser extent Δ160p53β, can be recruited on chromatin, and that Δ160p53γ can localize in perinuclear foci ; moreover, all Δ160p53 isoforms can stimulate proliferation and in vitro migration. Lastly, vemurafenib-resistant melanoma cells showed an altered expression of p53 and p73 isoforms, namely an increased expression of potentially pro-oncogenic Δ40p53β and a decrease in tumor-suppressive TAp73β. We therefore propose that p53 family isoforms can play a role in melanoma cells’ aggressiveness