Frozen Cord Blood Hematopoietic Stem Cells Differentiate into Higher Numbers of Functional Natural Killer Cells In Vitro than Mobilized Hematopoietic Stem Cells or Freshly Isolated Cord Blood Hematopoietic Stem Cells

Adoptive natural killer (NK) cell therapy relies on the acquisition of large numbers of NK cells that are cytotoxic but not exhausted. NK cell differentiation from hematopoietic stem cells (HSC) has become an alluring option for NK cell therapy, with umbilical cord blood (UCB) and mobilized peripheral blood (PBCD34⁺) being the most accessible HSC sources as collection procedures are less invasive. In this study we compared the capacity of frozen or freshly isolated UCB hematopoietic stem cells (CBCD34⁺) and frozen PBCD34⁺ to generate NK cells in vitro. By modifying a previously published protocol, we showed that frozen CBCD34⁺ cultures generated higher NK cell numbers without loss of function compared to fresh CBCD34⁺ cultures. NK cells generated from CBCD34⁺ and PBCD34⁺ expressed low levels of killer-cell immunoglobulin-like receptors but high levels of activating receptors and of the myeloid marker CD33. However, blocking studies showed that CD33 expression did not impact on the functions of the generated cells. CBCD34⁺-NK cells exhibited increased capacity to secrete IFN-γ and kill K562 in vitro and in vivo as compared to PBCD34⁺-NK cells. Moreover, K562 killing by the generated NK cells could be further enhanced by IL-12 stimulation. Our data indicate that the use of frozen CBCD34⁺ for the production of NK cells in vitro results in higher cell numbers than PBCD34⁺, without jeopardizing their functionality, rendering them suitable for NK cell immunotherapy. The results presented here provide an optimal strategy to generate NK cells in vitro for immunotherapy that exhibit enhanced effector function when compared to alternate sources of HSC.     

In vitro and in vivo anti-inflammatory properties of imine resveratrol analogues

Resveratrol is a natural polyphenol found mainly on red grapes and in red wine, pointed as an important anti-inflammatory/immunomodulatory molecule. However, its bioavailability problems have limited its use encouraging the search for new alternatives agents. Thus, in this study, we synthetize 12 resveratrol analogues (6 imines, 1 thioimine and 5 hydrazones) and investigated its cytotoxicity, antioxidant activity and in vitro anti-inflammatory/immunomodulatory properties. The most promising compounds were also evaluated in vivo. The results showed that imines presented less cytotoxicity, were more effective than resveratrol on DPPH scavenger and exhibited an anti-inflammatory profile. Among them, the imines with a radical in the para position, on the ring B, not engaged in an intramolecular hydrogen-interaction, showed more prominent anti-inflammatory activity modulating, in vivo, the edema formation, the inflammatory infiltration and cytokine levels. An immunomodulatory activity also was observed in these molecules. Thus, our results suggest that imines with these characteristics presents potential to control inflammatory disorders.     

Citrate analysis using capillary electrophoresis and complexation with Eu<Superscript>3+</Superscript>-tetracycline

A sensitive assay for citrate was developed. Citrate was incubated with 50 μM Eu³⁺-tetracycline and the complex separated using capillary electrophoresis utilizing post-column laser-induced luminescence detection in a sheath flow cuvette. Signal was linear with citrate concentration from 10 μM to 200 nM. Injection volumes were 320 pL. For the 200 nM sample, this corresponds to the injection of 64 amoles of citrate. Separation time was?<?90 s with a total run time of 5 min. As an application the method was used to analyze citrate in agricultural and medicinal products. The method was also used to develop an assay for the enzyme citrate synthase.     

Efficacy of different interaction devices using non-immersive virtual tasks in individuals with Amyotrophic Lateral Sclerosis: a cross-sectional randomized trial

Background

Amyotrophic Lateral Sclerosis (ALS) is a rapid progressive neurodegenerative disease, characterized by a selective loss of motor neurons, brain stem and spinal cord which leads to deterioration of motor abilities. Devices that promote interaction with tasks on computers can enhance performance and lead to greater independence and utilization of technology.

Objective

To evaluate performance on a computer task in individuals with ALS using three different commonly used non-immersive devices.

Method

Thirty individuals with ALS (18 men and 12 women, mean age 59?years, range 44–74?years) with a mean score of 26, (minimum score of 14 and maximum 41) on the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) and 30 healthy controls matched for age and gender, participated. All participants were randomly divided into three groups, each using a different device system (motion tracking, finger motion control or touchscreen) to perform three task phases (acquisition, retention and transfer).

Results

Both the ALS and control group (CG) showed better performance on the computer task when using the touchscreen device, but there was limited transfer of performance onto the task performed on the Finger Motion control or motion tracking. However, we found that using the motion tracking device led to transfer of performance to the touchscreen.

Conclusion

This study presents novel and important findings when selecting interaction devices for individuals with ALS to access technology by demonstrating immediate performance benefits of using a touchscreen device, such as improvement of motor skills. There were possible transferable skills obtained when using virtual systems which may allow flexibility and enable individuals to maintain performance overtime.

Trial registration

Registration name: Virtual Task in Amyotrophic Lateral Sclerosis; Registration number: NCT03113630; retrospectively registered on 04/13/2017. Date of enrolment of the first participant to the trial: 02/02/2016.

     

In vitro cell-based assays for evaluation of antioxidant potential of plant-derived products

Several plant-derived compounds have been screened by antioxidant assays, but many of these results are questionable, since they do not evaluate the pharmacologic parameters. In fact, the development of better antioxidants stills a great challenge. In vitro cell-based assays have been employed to assess the antioxidant effect of various compounds at subcellular level. Cell-based assays can also reveal compounds able to enhance the antioxidant pathways, but without direct radical scavenging action (which could not be detected by traditional assays). These methodologies are general of easy implementation and reproducible making them suitable for the early stages of drug discovery. Hydrogen peroxide, a nonradical derivative of oxygen, can be employed as an oxidative agent in these assays due its biochemical properties (presence of all biological systems, solubility) and capacity to induce cell death. Truthfully, if their limitations are understood (such as difference on cell metabolism when in in vitro conditions), these cell-based assays can provide useful information about the pathways involved in the protective effects of phytochemicals against cell death induced by oxidative stress, which can be exploited to develop new therapeutic approaches.     

The Human Tau Interactome: Binding to the Ribonucleoproteome,and Impaired Binding of the Proline-to-Leucine Mutant at Position 301 (P301L) to Chaperones and the Proteasome

The tau protein is central to the etiology of several neurodegenerative diseases, including Alzheimer''s disease, a subset of frontotemporal dementias, progressive supranuclear palsy and dementia following traumatic brain injury, yet the proteins it interacts with have not been studied using a systematic discovery approach. Here we employed mild in vivo crosslinking, isobaric labeling, and tandem mass spectrometry to characterize molecular interactions of human tau in a neuroblastoma cell model. The study revealed a robust association of tau with the ribonucleoproteome, including major protein complexes involved in RNA processing and translation, and documented binding of tau to several heat shock proteins, the proteasome and microtubule-associated proteins. Follow-up experiments determined the relative contribution of cellular RNA to the tau interactome and mapped interactions to N- or C-terminal tau domains. We further document that expression of P301L mutant tau disrupts interactions of the C-terminal half of tau with heat shock proteins and the proteasome. The data are consistent with a model whereby a higher propensity of P301L mutant tau to aggregate may reflect a perturbation of its chaperone-assisted stabilization and proteasome-dependent degradation. Finally, using a global proteomics approach, we show that heterologous expression of a tau construct that lacks the C-terminal domain, including the microtubule binding domain, does not cause a discernible shift of the proteome except for a significant direct correlation of steady-state levels of tau and cystatin B.The tau protein is a member of the family of microtubule-associated proteins (MAPs)¹ that in humans is coded by the MAPT gene on chromosome 17q21.31 (1). Initially, described as a factor that binds to and stabilizes microtubules (MTs) (2), interest in the tau protein grew when it was shown to represent the main constituent of intracellular protein aggregates, termed neurofibrillary tangles (NFTs), observed in Alzheimer''s disease (3, 4). Similar tau aggregates have since been described in other, less common dementias, including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick''s disease and dementia pugilistica, a form of dementia observed in athletes who had been exposed to repeated traumatic brain injury (5).Despite its early recognition as a MT binding molecule, the physiological function of the tau protein is still being debated (6). At least, in part, this uncertainty is born from the observation that tau knockout mice are rather nonconspicuous in their phenotype (7, 8). Ongoing attempts to define additional roles for this protein have, over the years, generated several hypotheses, including that the tau protein modulates neurite outgrowth and axonogenesis (8, 9), bridges the microtubule and actin cytoskeletons (10), and acts as a scaffold for tethering the Src family tyrosine kinase Fyn to PSD-95/NMDA receptor complexes (11). The predominant expression of tau in neuronal axons suggests a role in brain function. Significantly, in all tauopathies, a group term used to describe dementias with pathological tau protein involvement, the tau protein is observed to detach from microtubules and to form aggregates. There also is compelling evidence from a body of work with transgenic models that the cellular toxicity observed in the aforementioned dementias relies on the presence of the tau protein (12). Consequently, it seems plausible that the cellular toxicity observed in AD and other dementias does not relate to a loss of function of the tau protein but represents a gain of toxic function the protein exhibits in its microtubule-detached form.The tau molecule can be crudely subdivided into an amino-terminal projection domain (PD), a microtubule-binding domain (MTB), and a carboxy-terminal domain (C'') (13). The protein has long been known to exhibit some remarkable biochemical characteristics, including an ability to withstand harsh acid and heat treatments that would cause a majority of other proteins to precipitate (2, 14). These characteristics have been attributed to tau being natively unfolded and possessing a highly dynamic character (15). The tau protein is also known to be a substrate for several post-translational modifications (PTMs), and the list of tau modifying enzymes that have been described is long. In particular, tau phosphorylation has been recognized to occur in vivo and in disease, and tau hyperphosphorylation at sites within the MTB domain and at nearby sites flanking the MTB has been shown to promote detachment of tau from microtubules (16). There further is broad agreement in the field that levels of several other tau PTMs are raised in tauopathies, including nitration (17), ubiquitination (18), sumoylation (19), and truncation (20, 21). Less agreement exists on the degree to which specific PTMs contribute to disease manifestation in individual tauopathies (22). Lacking also are insights into other physiological protein interactions the tau protein engages in and, surprisingly, to our knowledge, no systematic screen for tau binders has been reported. Thus, except for its well-established binding to microtubules (2), members of the Src family of protein kinases (23, 24), Hsp70 (25)/Hsp90 (26, 27), and reports on its interaction with F-actin (28), ApoE3 (29), a subset of peptidyl-prolyl cis-trans isomerases (30, 31), α-synuclein (32), PACSIN1 (33), and negatively charged polymers, including nucleic acids (34, 35), relatively little is known about other nonenzymatic interactions the protein engages in.In an attempt to address this unmet need, we set out to define molecular interactors of the tau protein in the human neuroblastoma SH-SY5Y cell model. The study made use of advanced instrumentation and workflows that included comparative mass spectrometry based on isobaric tags. We observed that the tau interactome is dramatically enriched in cellular components involved in the regulation and execution of RNA-processing and translation. We document that the previously known ability of the tau protein to bind to nucleic acids is partially responsible but not sufficient by itself to explain this binding propensity of the tau protein. We narrowed down the binding preference of individual binders to N- and C-terminal domains within tau and document that several interactors, including 14-3-3 proteins, heat shock proteins, and the proteasome, exhibit a strong binding preference for the C terminus of tau. When comparing the interactomes of wild-type and mutant tau (P301L) linked to frontotemporal dementia, we observed that interactions with the aforementioned C-terminal tau binding partners are diminished for mutant tau. Despite the strong binding of tau to the ribonucleoproteome, its overexpression does not seem to affect the global steady-state levels of cellular proteins, and only the levels of cystatin B, a natural inhibitor of cysteine proteases, were modified and correlated directly with the levels of heterologously expressed tau.