Alternative mobile phase additives for the characterization of protein biopharmaceuticals in liquid chromatography – Mass spectrometry

When analyzing large complex protein biopharmaceuticals, ion-pairing agents imparting low pH are widely used as mobile phase additives to improve the chromatographic performance. However, one of the most effective additives in RPLC and HILIC, trifluoroacetic acid (TFA), is known as a strong suppressor of the MS signal and limits its use in hyphenated techniques. In this study, we evaluated a wide range of acidic additives to find alternatives to TFA that provided comparable chromatographic performance and improved MS sensitivity. It was observed that stronger acidic additives were required for intact level analysis compared to subunit level analysis and that the additive nature had a larger impact on the chromatographic performance in HILIC mode compared to RPLC. Therefore, four additives were identified as valuable alternatives to TFA in RPLC mode, namely, difluoroacetic acid (DFA), dichloroacetic acid (DClAA), trichloroacetic acid (TClAA), and methanesulfonic acid (MSA). Only one of these additives provided acceptable performance in HILIC mode, namely, TClAA.After evaluation of the MS performance, TClAA was discarded due to the apparent loss of intensity in both RPLC-MS and HILIC-MS mode. Together, these results demonstrate that for HILIC-MS analysis TFA remains the gold standard additive. However, DFA was found as promising alternative to TFA for RPLC-MS analysis and could play an important role in the development of methods for the characterization of the increasingly complex protein biopharmaceuticals

Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules

Background: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement

Phase I dose-escalation and pharmacokinetic study of temozolomide (SCH 52365) for refractory or relapsing malignancies

Temozolomide, an oral cytotoxic agent with approximately 100% bioavailability after one administration, has demonstrated schedule-dependent clinical activity against highly resistant cancers. Thirty patients with minimal prior chemotherapy were enrolled in this phase I trial to characterize the drug's safety, pharmacokinetics and anti-tumour activity, as well as to assess how food affects oral bioavailability. To determine dose-limiting toxicities (DLT) and the maximum tolerated dose (MTD), temozolomide 100–250 mg m−2 was administered once daily for 5 days every 28 days. The DLT was thrombocytopenia, and the MTD was 200 mg m−2 day−1. Subsequently, patients received the MTD to study how food affects the oral bioavailability of temozolomide. When given orally once daily for 5 days, temozolomide was well tolerated and produced a non-cumulative, transient myelosuppression. The most common non-haematological toxicities were mild to moderate nausea and vomiting. Clinical activity was observed against several advanced cancers, including malignant glioma and metastatic melanoma. Temozolomide demonstrated linear and reproducible pharmacokinetics and was rapidly absorbed (mean Tmax ~1 h) and eliminated (mean t1/2 = 1.8 h). Food produced a slight reduction (9%) in absorption of temozolomide. Temozolomide 200 mg m−2 day−1 for 5 days, every 28 days, is recommended for phase II studies. © 1999 Cancer Research Campaig

Temozolomide induces senescence but not apoptosis in human melanoma cells

Temozolomide (TMZ), a DNA alkylating agent used in the treatment of melanoma, is believed to mediate its effect by addition of a methyl group to the O6 position of guanine in DNA. Resistance to the agent may be in part due to the activity of O6-methylguanine-DNA methyl transferase (MGMT). In the present study, we show that sensitivity of melanoma cells to TMZ was dependent on their p53 status and levels of MGMT. Analysis of the mechanisms underlying reduced viability showed no evidence for induction of apoptosis even though marked levels of apoptosis was seen in TK6 lymphoma cells. Sensitivity of melanoma cells was associated with p53-dependent G2/M cell cycle arrest and induction of senescence. To verify the role of p53, the assays were repeated in presence of pifithrin-α, an inhibitor of p53. This resulted in increased viability of melanoma cells with wild-type p53 and reversed G2/M cell cycle arrest. Paradoxically, apoptosis was increased in melanoma but decreased as expected in TK6 lymphoma cells. These results are consistent with the view that TMZ is relatively ineffective against melanoma due to defective apoptotic signalling resulting from activation of p53. The nature of the defects in apoptotic signalling remains to be explored

Tight junctions and the modulation of barrier function in disease

Tight junctions create a paracellular barrier in epithelial and endothelial cells protecting them from the external environment. Two different classes of integral membrane proteins constitute the tight junction strands in epithelial cells and endothelial cells, occludin and members of the claudin protein family. In addition, cytoplasmic scaffolding molecules associated with these junctions regulate diverse physiological processes like proliferation, cell polarity and regulated diffusion. In many diseases, disruption of this regulated barrier occurs. This review will briefly describe the molecular composition of the tight junctions and then present evidence of the link between tight junction dysfunction and disease

The Contributions of Alessandro D'Atri to Organization and Information Systems Studies

This book is dedicated to the memory of Professor Alessandro (Sandro) D'Atri, who passed away in April 2011. Professor D'Atri started his career as a brilliant scholar interested in theoretical computer science, databases and, more generally information processing systems. He journeyed far in various applications, such as human-computer interaction, human factors, ultimately arriving at business information systems and business organisation after more than 20 years of researc hbased on "problem solving". Professor D'Atri pursued the development of an interdisciplinary culture in which social sciences, systems design and human sciences are mutually integrated. Rather than retrospection, this book is aimed to advance in these directions and to stimulate a debate about the potential of design research in the field of information systems and organisation studies with an interdisciplinary approach. Each chapter has been selected by the Editorial Board following a double blind peer review process. The general criteria of privileging the variety of topics and the design science orientation and/or empirical works in which a design research approach is adopted to solve various field problems in the management area. In addition several chapters contribute to the meta-discourse on design science research.This book is dedicated to the memory of Professor Alessandro (Sandro) D'Atri, who passed away in April 2011. Professor D'Atri started his career as a brilliant scholar interested in theoretical computer science, databases and, more generally information processing systems. He journeyed far in various applications, such as human-computer interaction, human factors, ultimately arriving at business information systems and business organisation after more than 20 years of researc hbased on "problem solving". Professor D'Atri pursued the development of an interdisciplinary culture in which social sciences, systems design and human sciences are mutually integrated. Rather than retrospection, this book is aimed to advance in these directions and to stimulate a debate about the potential of design research in the field of information systems and organisation studies with an interdisciplinary approach. Each chapter has been selected by the Editorial Board following a double blind peer review process. The general criteria of privileging the variety of topics and the design science orientation and/or empirical works in which a design research approach is adopted to solve various field problems in the management area. In addition several chapters contribute to the meta-discourse on design science research.Monograph's chapter

Effects of fronto-temporal 5 Hz-tACS on subjective and objective sleepiness

Recent findings showed that oscillating transcranial Direct Current Stimulation (osc-tDCS) and transcranial Alternating Current Stimulation (tACS) applied during wake and sleep can interact with the ongoing brain activity in a frequency-specific manner. Here, we evaluated the efficacy of a bilateral 5Hz-tACS on fronto-temporal areas in modulating physiological sleepiness during wakefulness, by inducing synchronization in cortical and subcortical structures involved in the sleep onset. Twenty-six healthy volunteers (18-35 years) participated in two within-subject sessions (Active and Sham), one week apart and in counterbalanced order. Each session involved: 5-min pre-stimulation EEG; 10-min tACS stimulation; 5-min post-stimulation EEG. The self-reported levels of sleepiness were collected with the Karolinska Sleepiness Scale (KSS) and Visual Analogue Scale (VAS) at the beginning and at the end of the two experimental sessions. A sinusoidal AC was applied (current intensity: 0.6 mA; max current density=0.531 mA/cm2) by two electrodes, located on FT7 and FT8. Power spectra of the 28 EEG derivations were computed by a fast Fourier transform routine in 2 s epochs across the EEG bands. Results show that 5Hz-tACS on fronto-temporal area induces an increase (10-15%) of EEG power in lower frequency bands (delta and theta) but fails to induce any effect on subjective sleepiness. The specific topographic pattern of changes involving frontal increase in the delta band and occipital increase in the theta range seems to simulate the dynamics characterizing an early sleep onset. However, no causal relation can be traced on the basis of the current results between these rhythms and changes on sleepiness

Effetti della stimolazione transcranica a corrente alternata (tACS) nella modulazione dei livelli di sonnolenza e vigilanza

Rationale e obiettivi Le tecniche di stimolazione elettrica transcranica rappresentano metodiche non invasive in grado di interferire con determinate configurazioni elettrofisiologiche mediante l’induzione di deboli correnti elettriche attraverso lo scalpo. In particolare, la stimolazione transcranica in corrente alternata (tACS) agisce modulando l’attività ritmica cerebrale mediante la sincronizzazione (entrainment) tra frequenze di stimolazione e frequenze endogene. Nello specifico, l’obiettivo del presente studio è stato facilitare la manifestazione di attività EEG rapida e/o diminuire quella lenta, in direzione di un incremento dei livelli fisiologici e soggettivi di vigilanza e/o riduzione di sonnolenza. Materiali e metodi Lo studio è stato condotto su un campione di 10 soggetti adulti di sesso maschile, secondo un disegno procedurale within- subject. Il protocollo ha previsto due distinte sessioni di tACS (Attiva o Sham), ciascuna con registrazioni EEG di veglia pre e post-stimolazione. In aggiunta, questionari di misurazione dei livelli di sonnolenza autopercepita (Karolinska Sleepiness Scale -KSS, Visual Analogue Scale -VAS) sono stati somministrati nelle fasi immediatamente precedenti e successive al protocollo sperimentale. In accordo con gli obiettivi, una tACS con frequenza di 30 Hz (beta range) e intensità compresa tra 0 e 0.6 mA è stata applicata bilateralmente in aree frontali per una durata totale di 15 minuti. Variabili quantitative (potenze spettrali delle canoniche bande di frequenza EEG) e qualitative (punteggio globale ai questionari di sonnolenza soggettiva) sono state considerate nelle successive analisi. Risultati Le analisi sulle variazioni delle potenze spettrali EEG pre e post-stimolazione mostrano l’assenza di effetti principali per i fattori “Banda” e “Stimolazione” e la presenza di un lieve effetto di interazione in aree corticali fronto-centrali. Scomponendo tali effetti attraverso successivi confronti post hoc, si evidenziano due principali fenomeni relativi all’incremento e decremento di potenza spettrale rispettivamente delle bande EEG delta e theta. Analizzando dettagliatamente le singole frequenze incluse in ciascuna banda, è stato possibile osservare che, mentre il fenomeno di riduzione del theta in seguito a stimolazione attiva (coerente con le ipotesi di ricerca), si manifesta diffusamente in un ampio spettro di bin di frequenza, l’incremento del delta (contrario alle ipotesi di ricerca) caratterizza selettivamente un unico bin di frequenza (3 Hz). Connessa alla breve durata degli effetti, l’analisi del time course ha rivelato la massima espressione dei fenomeni osservati nei primi minuti successivi alla stimolazione, seguita da una graduale riduzione nelle finestre temporali successive. Le stime dei livelli di sonnolenza autopercepita, pur descrivendo una riduzione di sonnolenza nella fase post-stimolazione associata alla condizione di stimolazione Attiva, non rilevano variazioni significative dovute specificatamente agli effetti della stimolazione. Conclusioni Dai risultati emerge che la tACS, sebbene in grado di produrre variazioni dei pattern EEG associati a determinate condizioni di sonnolenza e vigilanza, richiede la considerazione di numerose variabili nella determinazione complessiva di direzione e pervasività degli effetti. Da un punto di vista clinico- applicativo, l’individuazione dei parametri procedurali e metodologici ottimali in grado di garantire il raggiungimento degli effetti desiderati potrebbe aprire affascinanti prospettive nel trattamento non- farmacologico di patologie caratterizzate da Eccessiva Sonnolenza Diurna