LAB on CHIP: capacitive stimulation of cells

The main target of the LAB on CHIP phD project, funded by Fondazione Cariparo, was to develop a device to allow the handy production of CHO cell clones in order to built recombinant proteins for therapeutic targets. In particular, a major aim is to reduce time and costs associated with clones manufacturing. Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications, because of their capacity for proper protein folding, assembly and post-translational modification. The quality and efficacy of a protein can be superior when expressed in mammalian cells versus other hosts such as bacteria, plants and yeast. Today more than 60 % of all recombinant protein pharmaceuticals are produced in mammalian cells. Expression vectors for recombinant cell line generation generally use a strong viral or cellular promoter/enhancer to drive the expression of the recombinant gene. But non-viral gene transfer remains the preferred approach to generate stable cell lines for manufacturing purposes: calcium phosphate transfection, electroporation, lipofection. Transfection is a complex process, and in order to be successful all steps involved must work efficiently. The device developed is based on the physical phenomenon called electroporation, that is the formation of temporary pores in the plasmatic membrane upon application of electric fields. As the biological world is intrinsically variable, common approaches for improving electrotransfection rely on time consuming empirical attempts. It is therefore important to develop new methods enabling a fine control of all critical parameters involved to identify causes of failure and to improve efficiency. On-chip electroporation through capacitive currents can be such a method. To directly assess the formation of pores in the cell membrane, we performed patch-clamp experiments during on-chip electroporation. Thus, most promising protocols were selected and assessed for their electrotransfection efficiency. Moreover, patch clamp experiments allowed to study the dynamics of pores formation and resealing. Regarding the develop of the device, the biocompatibility of titanium dioxide, selected as dielectric material, was tested. The inertness against cellular environment and the state of cell culture were considered. Cell cultures showed healthy state and normal development, good adhesion and normal replication time. No chemical reactions that can damage the culture were observed. The chemical inertness was considered in the reverse direction too. Metabolic products of cell culture did not lead to chemical corrosion of the dielectric surface. So, the patch-clamp on-chip electroporation recordings allowed to select the promising protocol that was tested on CHO cultures. The prototype proposed demonstrated electrotransfection through capacitive coupling between cell and chip. The electroporation efficiency obtained is around 30%. Moreover, the selectivity of the device was demonstrated, and its applicability both in electrotransfection and electroporation for staining application. Collateral results were obtained concerning the formation of pore on attached and free membrane and the possibility of study pore dynamic

Circulating CRP Levels Are Associated with Epicardial and Visceral Fat Depots in Women with Metabolic Syndrome Criteria

Sexual dimorphism accounts for significant differences in adipose tissue mass and distribution. However, how the crosstalk between visceral and ectopic fat depots occurs and which are the determinants of ectopic fat expansion and dysfunction remains unknown. Here, we focused on the impact of gender in the crosstalk between visceral and epicardial fat depots and the role of adipocytokines and high-sensitivity C-reactive protein (hs-CRP). A total of 141 outward patients (both men and women) with one or more defining criteria for metabolic syndrome (MetS) were consecutively enrolled. For all patients, demographic and clinical data were collected and ultrasound assessment of visceral adipose tissue (VFth) and epicardial fat (EFth) thickness was performed. Hs-CRP and adipocytokine levels were assessed by enzyme-linked immunosorbent assay (ELISA). Men were characterized by increased VFth and EFth (p-value < 0.001 and 0.014, respectively), whereas women showed higher levels of adiponectin and leptin (p-value < 0.001 for both). However, only in women VFth and EFth significantly correlated between them (p = 0.013) and also with leptin (p < 0.001 for both) and hs-CRP (p = 0.005 and p = 0.028, respectively). Linear regression confirmed an independent association of both leptin and hs-CRP with VFth in women, also after adjustment for age and MetS (p = 0.012 and 0.007, respectively). In conclusion, men and women present differences in epicardial fat deposition and systemic inflammation. An intriguing association between visceral/epicardial fat depots and chronic low-grade inflammation also emerged. In women Although a further validation in larger studies is needed, these findings suggest a critical role of sex in stratification of obese/dysmetabolic patients

LAB on CHIP: capacitive stimulation of cells

The main target of the LAB on CHIP phD project, funded by Fondazione Cariparo, was to develop a device to allow the handy production of CHO cell clones in order to built recombinant proteins for therapeutic targets. In particular, a major aim is to reduce time and costs associated with clones manufacturing. Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications, because of their capacity for proper protein folding, assembly and post-translational modification. The quality and efficacy of a protein can be superior when expressed in mammalian cells versus other hosts such as bacteria, plants and yeast. Today more than 60 % of all recombinant protein pharmaceuticals are produced in mammalian cells. Expression vectors for recombinant cell line generation generally use a strong viral or cellular promoter/enhancer to drive the expression of the recombinant gene. But non-viral gene transfer remains the preferred approach to generate stable cell lines for manufacturing purposes: calcium phosphate transfection, electroporation, lipofection. Transfection is a complex process, and in order to be successful all steps involved must work efficiently. The device developed is based on the physical phenomenon called electroporation, that is the formation of temporary pores in the plasmatic membrane upon application of electric fields. As the biological world is intrinsically variable, common approaches for improving electrotransfection rely on time consuming empirical attempts. It is therefore important to develop new methods enabling a fine control of all critical parameters involved to identify causes of failure and to improve efficiency. On-chip electroporation through capacitive currents can be such a method. To directly assess the formation of pores in the cell membrane, we performed patch-clamp experiments during on-chip electroporation. Thus, most promising protocols were selected and assessed for their electrotransfection efficiency. Moreover, patch clamp experiments allowed to study the dynamics of pores formation and resealing. Regarding the develop of the device, the biocompatibility of titanium dioxide, selected as dielectric material, was tested. The inertness against cellular environment and the state of cell culture were considered. Cell cultures showed healthy state and normal development, good adhesion and normal replication time. No chemical reactions that can damage the culture were observed. The chemical inertness was considered in the reverse direction too. Metabolic products of cell culture did not lead to chemical corrosion of the dielectric surface. So, the patch-clamp on-chip electroporation recordings allowed to select the promising protocol that was tested on CHO cultures. The prototype proposed demonstrated electrotransfection through capacitive coupling between cell and chip. The electroporation efficiency obtained is around 30%. Moreover, the selectivity of the device was demonstrated, and its applicability both in electrotransfection and electroporation for staining application. Collateral results were obtained concerning the formation of pore on attached and free membrane and the possibility of study pore dynamicsScopo principale del progetto di Dottorato "LAB on CHIP" finanziato dalla Fondazione Cariparo è stato lo sviluppo di un dispositivo che agevoli la creazione di cloni di cellule CHO per la produzione di proteine a scopo terapeutico. In particolare il fine ultimo è quello di ridurne tempi e costi associati alla produzione. Le cellule di mammifero in coltura sono ormai il sistema più diffuso per la produzione di proteine per applicazioni cliniche. La qualità e l'efficacia di una proteina possono essere superiore se essa è espressa in cellule di mammifero rispetto ad altri organismi, quali batteri, piante e lieviti. Ad oggi più del 60 % di tutte le proteine ricombinanti per applicazioni farmaceutiche è prodotto in cellule di mammifero. Vettori di espressione per la creazione di linee cellulari stabili da DNA ricombinante utilizzano vettori virali per indurre l'espressione del gene. Ma la transfezione senza l'ausilio di virus rimane l' approccio prediletto per la generazione di linee stabili per questi scopi. La transfezione è un processo complesso e, affinchè avvenga con successo, tutti i sottoprocessi coinvolti devono svolgersi efficientemente. Il dispositivo proposto si basa sul fenomeno fisico chiamato elettroporazione, che non è altro che la formazione di pori temporanei nella membrana plasmatica a seguito dell'applicazione di opportuni campi elettrici. I comuni approcci utilizzati per migliorare la transfezione tramite elettroporazione richiedono tempi lunghi e possono essere inefficaci. E' importante poter sviluppare metodi nuovi che permettano un controllo di tutti i parametri critici coinvolti in modo da poterne identificare le cause in caso di fallimento e dunque migliorare l'efficienza. L'elettroporazione su chip utilizzzando correnti capacitive può essere un valido approccio. Per poter rilevare la formazione di pori, sono stati fatti esperimenti di patch-clamp su chip durante l'elettroporazione. In tal modo sono stati selezionati i protocolli più promettenti. Per quanto riguarda lo sviluppo del dispositivo, ne è stata verificata la biocompatibilità. Si è valutato lo stato delle colture cellulari che hanno mostrato normali sviluppo, adesione e tempo di replicazione. Non sono state rilevate reazioni chimiche tra il mezzo di coltura e il diossido di titanio. Non si sono inoltre rilevati problemi di corrosione o danneggiamento dell'ossido a causa di prodotti metabolici della cellula. Gli esperimenti di patch-clamp hanno permesso di selezionare un protocollo che è stato poi testato sulle cellule in coltura. Il prototipo sviluppato ha dimostrato l'elettroporazione di cellule CHO in coltura, ottenendo un'efficienza media del 30 %. E' stata inoltre dimostrata la selettività di tale dispositivo e la sua applicabilità sia per la transfezione che per l'introduzione nella cellula di marcatori. Risultati "collaterali" ottenuti riguardano la dimostrazione della formazione di pori temporanei sia sulla membrana adesa che su quella libera e la possibilità di studiare la dinamica dei por

Fibromyalgia Syndrome: A Metabolic Approach Grounded in Biochemistry for the Remission of Symptoms

Fibromyalgia syndrome (FMS) is a chronic, complex, and heterogeneous disorder of still poorly understood etiopathophysiology associated with important musculoskeletal widespread pain, fatigue, non-restorative sleep, and mood disturbances. It is estimated to afflict 2–3% of the worldwide population, with clean prevalence among women. The objective of this paper is to propose a novel treatment for symptomatic remission of FMS, grounded in biochemistry and consisting in the withdrawal from the diet of molecules that can indirectly trigger the symptoms. The hypothesis develops from the evidence that low serotonin levels are involved in FMS. Serotonin is synthesized starting from the essential amino acid tryptophan. The presence of non-absorbed molecules in the gut, primarily fructose, reduces tryptophan absorption. Low tryptophan absorption leads to low serotonin synthesis that triggers FMS symptoms. Moreover not-absorbed sugars could also produce a microbiota deterioration activating a positive feedback loop: the increasing microbiota deterioration reduces the functionality of absorption both of fructose and tryptophan in the gut, entering a vicious circle. The therapeutic idea is to sustain serotonin synthesis allowing the proper tryptophan absorption. The core of the cure treatment is the exclusion from the diet of some carbohydrates and the marked reduction of some others. The main target is the limitation of total dietary fructose as marked as possible. It could be an effective strategy to get the remission of symptoms acting on the impaired biochemical pathways. The straying from the treatment is expected to cause the reappear of the symptoms

Fibromyalgia Syndrome: A Case Report on Controlled Remission of Symptoms by a Dietary Strategy

A 34-year-old woman suffered from significant chronic pain, depression, non-restorative sleep, chronic fatigue, severe morning stiffness, leg cramps, irritable bowel syndrome, hypersensitivity to cold, concentration difficulties, and forgetfulness. Blood tests were negative for rheumatic disorders. The patient was diagnosed with Fibromyalgia syndrome (FMS). Due to the lack of effectiveness of pharmacological therapies in FMS, she approached a novel metabolic proposal for the symptomatic remission. Its core idea is supporting serotonin synthesis by allowing a proper absorption of tryptophan assumed with food, while avoiding, or at least minimizing the presence of interfering non-absorbed molecules, such as fructose and sorbitol. Such a strategy resulted in a rapid improvement of symptoms after only few days on diet, up to the remission of most symptoms in 2 months. Depression, widespread chronic pain, chronic fatigue, non-restorative sleep, morning stiffness, and the majority of the comorbidities remitted. Energy and vitality were recovered by the patient as prior to the onset of the disease, reverting the occupational and social disabilities. The patient episodically challenged herself breaking the dietary protocol leading to its negative test and to the evaluation of its benefit. These breaks correlated with the recurrence of the symptoms, supporting the correctness of the biochemical hypothesis underlying the diet design toward remission of symptoms, but not as a final cure. We propose this as a low risk and accessible therapeutic protocol for the symptomatic remission in FMS with virtually no costs other than those related to vitamin and mineral salt supplements in case of deficiencies. A pilot study is required to further ground this metabolic approach, and to finally evaluate its inclusion in the guidelines for clinical management of FMS

Atomic layer deposited TiO2 for implantable brain-chip interfacing devices

In this paper we investigated atomic layer deposition (ALD) TiO2 thin films deposited on implantable neuro-chips based on electrolyte-oxide-semiconductor (EOS) junctions, implementing both efficient capacitive neuron-silicon coupling and biocompatibility for longterm implantable functionality. The ALD process was performed at 295 \ub0C using titanium tetraisopropoxide and ozone as precursors on needle-shaped silicon substrates. Engineering of the capacitance of the EOS junctions introducing a thin Al2O3 buffer layer between TiO2 and silicon resulted in a further increase of the specific capacitance. Biocompatibility for long-term implantable neuroprosthetic systems was checked upon in-vitro treatment

Hemodynamics and risk assessment 2 years after the initiation of upfront ambrisentan‒tadalafil in pulmonary arterial hypertension

BACKGROUND: Upfront combination therapy with ambrisentan and tadalafil has been reported to improve the condition of patients with pulmonary arterial hypertension (PAH) more than with either drug alone. However, little is known about the long-term associated changes in hemodynamics and risk assessment scores. METHODS: This was a multicenter, retrospective analysis of clinical data in 106 patients with newly diagnosed PAH. Clinical evaluations, including demographics, medical history, World Health Organization (WHO) functional class (FC) and 6-minute walk distance (6MWD), right heart catheterization, and Registry to Evaluate Early and Long-Term PAH Disease Management (REVEAL) risk score 2.0, were assessed over 48 months of ambrisentan‒tadalafil therapy. RESULTS: At baseline, 9 patients (9%) showed a low (8) REVEAL risk score. At a median follow-up of 2 years, 45 patients (43%) showed a low, 47 patients (44%) showed an intermediate, and 14 patients (13%) showed a high REVEAL score, along with improvements in WHO FC, 6MWD and a decrease in mean pulmonary artery pressure and N-terminal pro brain natriuretic peptide (all p 50% or a stroke volume within the limits of normal. CONCLUSIONS: Initial combination therapy with ambrisentan and tadalafil in PAH improves the REVEAL risk score in proportion to decreased PVR and preserved stroke volume but still insufficiently so in approximately 50% of the patients.SCOPUS: ar.jDecretOANoAutActifinfo:eu-repo/semantics/publishe

Lysosomal lipid switch sensitises to nutrient deprivation and mTOR targeting in pancreatic cancer

Objective: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with limited therapeutic options. However, metabolic adaptation to the harsh PDAC environment can expose liabilities useful for therapy. Targeting the key metabolic regulator mechanistic target of rapamycin complex 1 (mTORC1) and its downstream pathway shows efficacy only in subsets of patients but gene modifiers maximising response remain to be identified. Design: Three independent cohorts of PDAC patients were studied to correlate PI3K-C2γ protein abundance with disease outcome. Mechanisms were then studied in mouse (KPC mice) and cellular models of PDAC, in presence or absence of PI3K-C2γ (WT or KO). PI3K-C2γ-dependent metabolic rewiring and its impact on mTORC1 regulation were assessed in conditions of limiting glutamine availability. Finally, effects of a combination therapy targeting mTORC1 and glutamine metabolism were studied in WT and KO PDAC cells and preclinical models. Results: PI3K-C2γ expression was reduced in about 30% of PDAC cases and was associated with an aggressive phenotype. Similarly, loss of PI3K-C2γ in KPC mice enhanced tumour development and progression. The increased aggressiveness of tumours lacking PI3K-C2γ correlated with hyperactivation of mTORC1 pathway and glutamine metabolism rewiring to support lipid synthesis. PI3K-C2γ-KO tumours failed to adapt to metabolic stress induced by glutamine depletion, resulting in cell death. Conclusion: Loss of PI3K-C2γ prevents mTOR inactivation and triggers tumour vulnerability to RAD001 (mTOR inhibitor) and BPTES/CB-839 (glutaminase inhibitors). Therefore, these results might open the way to personalised treatments in PDAC with PI3K-C2γ loss