Stability to thermal treatment of dipeptidyl peptidase IV (DPP-IV) inhibitory activity of a boarfish (Capros aper) protein hydrolysate when incorporated into tomato-based products

Biofunctional peptide ingredients should retain their stability following standard processing operations in food‐based delivery vehicles. A boarfish protein hydrolysate, exhibiting anti‐diabetic activity was subjected to a range of thermal treatments following incorporation into tomato‐based soup and juice products. The dipeptidyl peptidase‐IV (DPP‐IV) inhibitory activity and peptide profile of the hydrolysate within the products were assessed before and after thermal treatment. The treatments applied had no effect on the DPP‐IV inhibitory activity or peptide profile of the protein hydrolysate. The heat‐treated (90°C x 1 min and 121°C x 42 s) juice‐fortified beverage had microbial counts within the acceptable limits for consumption when stored at 4°C for 30 days. Furthermore, the hydrolysate within the beverage products was resistant to simulated gastrointestinal digestion (SGID) regardless of whether it was heat or non‐heat treated, or stored for 30 days at 4°C. Therefore, tomato‐based beverages are suitable delivery vehicles for biofunctional peptide ingredients

Arrays of MicroLEDs and Astrocytes: Biological Amplifiers to Optogenetically Modulate Neuronal Networks Reducing Light Requirement

<div><p>In the modern view of synaptic transmission, astrocytes are no longer confined to the role of merely supportive cells. Although they do not generate action potentials, they nonetheless exhibit electrical activity and can influence surrounding neurons through gliotransmitter release. In this work, we explored whether optogenetic activation of glial cells could act as an amplification mechanism to optical neural stimulation via gliotransmission to the neural network. We studied the modulation of gliotransmission by selective photo-activation of channelrhodopsin-2 (ChR2) and by means of a matrix of individually addressable super-bright microLEDs (μLEDs) with an excitation peak at 470 nm. We combined Ca²⁺ imaging techniques and concurrent patch-clamp electrophysiology to obtain subsequent glia/neural activity. First, we tested the μLEDs efficacy in stimulating ChR2-transfected astrocyte. ChR2-induced astrocytic current did not desensitize overtime, and was linearly increased and prolonged by increasing μLED irradiance in terms of intensity and surface illumination. Subsequently, ChR2 astrocytic stimulation by broad-field LED illumination with the same spectral profile, increased both glial cells and neuronal calcium transient frequency and sEPSCs suggesting that few ChR2-transfected astrocytes were able to excite surrounding not-ChR2-transfected astrocytes and neurons. Finally, by using the μLEDs array to selectively light stimulate ChR2 positive astrocytes we were able to increase the synaptic activity of single neurons surrounding it. In conclusion, ChR2-transfected astrocytes and μLEDs system were shown to be an amplifier of synaptic activity in mixed corticalneuronal and glial cells culture.</p></div

Identification and characterisation of peptides from a boarfish (Capros aper) protein hydrolysate displaying in vitro dipeptidyl peptidase-IV (DPP-IV) inhibitory and insulinotropic activity

Twenty-two novel dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides (with IC50 values <200 µM) and fifteen novel insulinotropic peptides were identified in a boarfish protein hydrolysate generated at semi-pilot scale using Alcalase 2.4L and Flavourzyme 500L. This was achieved by bioassay-driven semi preparative reverse phase-high performance liquid chromatography fractionation, liquid chromatography-mass spectrometry and confirmatory studies with synthetic peptides. The most potent DPP-IV inhibitory peptide (IPVDM) had a DPP-IV half maximal inhibitory concentration (IC50) values of 21.72 ± 1.08 µM in a conventional in vitro and 44.26 ± 0.65 µM in an in situ cell-based (Caco 2) DPP-IV inhibition assay. Furthermore, this peptide stimulated potent insulin secretory activity (1.6 fold increase compared to control) from pancreatic BRIN-BD11 cells grown in culture. The tripeptide IPV exhibited potent DPP-IV inhibitory activity (IC50: 5.61 ± 0.20 µM) comparable to that reported for the known DPP-IV inhibitor IPI (IC50: 5.61 µM). Boarfish proteins contain peptide sequences with potential to play a role in glycaemic management in vivo

Stimulation ofChR2 positive astrocytesincreases glial cells calcium transients frequency.

<p>Cortical glial culture were co-incubated in fura-2-AM (<b>A</b>) and fluo-3-AM (<b>B</b>) and Ca²⁺ transients were monitored during UV [excitation (ex)380±20 nm] and blue light [excitation (ex) 470±20 nm] stimulation (200 ms light pulse @ 0.5 Hz; 10 min UV→10 min blue→10 min UV). The star (<b>*</b>) indicates the ChR-2 positive astrocyte. <b>C</b>, Time course of ChR-2 negative astrocyte during UV (left panel) and blue (right panel) illumination. Fura-2 downward peak indicates [Ca²⁺]_i increase, fluo-3 upward peak indicates [Ca²⁺]_i increase. <b>D</b>, Stimulation of the ChR-2 positive astrocyte with 470 nm light (blue column)increased calcium waves frequency to 566.7%±124.2% (UV vs Blue, paired t test p = 0.0002 – Blue vs UV, paired t test p = 0.0048). <b>E</b>,The increased Ca²⁺ waves frequency mediated by stimulation of ChR2 positive astrocyte was significantly reduced by APV 50 µM (UV vs Blue, paired t test p<0.0001 – Blue vs Blue+APV, paired t test p = 0.0019). Values are means ±SEM.</p

ChR2+ asctrocytic stimulation modulates neuronal calcium waves frequency.

<p><b>A</b>, Bottom, snapshots from Ca²⁺ experiments during stimulation with 380 nm (<b>i</b>) and 470 nm (<b>ii</b>) light. Green circles indicate neurons, one of which (blue circle) was co-localizated with the ChR-2 positive astrocyte (star). Top, time course of one of the not colocalized neurons (circled in green). <b>B</b>,Time course of all circled neurons mean relative fluorescence and (inset)single cell measurement of calcium wave frequency (paired t test p<0.0001). <b>C</b>, Concurrent patch clamp and Ca²⁺imaging time course of the neuron circled in blue in <b>A</b>(<b>iii</b>). The red arrow shows the first wave (top) syncronised with the first sEPSCs burst and the red arrows show following sEPSCs bursts concomitant to internal calcium concentration increase.</p

μLEDs finely modulate in time and space inward current in ChR2-transfected astrocytes.

<p><b>A</b>,The ChR2+ astrocyte was stimulated with the whole matrix (blue box) or variable number of μLEDs (black and red boxes, 9 and 2 μLEDs, respectively) while recording the elicited inward currents in voltage clamp mode. Fine targeting and pulsing of the μLEDs on the cell was achieved overlaying in real time the fluorescent image to the μLEDs using a specific designed software. <b>B</b>, ChR-2 inward currents of different amplitude were recorded pulsing the whole matrix (blue box in <b>A</b>, pulse duration 20 ms) at different voltages (grey traces represent μLED stimulation pattern). Inset, mean inward current vs power density from different cells. <b>C</b>, μLEDs (blue box in <b>A</b>) can be finely modulated in time with submillisecond precision producing proportionally longer and larger ChR-2 currents (grey traces represent μLED stimulation pattern). <b>D</b>, Inward currents produced when 2 μLEDs (<b>A</b>, red box) or 9 μLEDs (<b>A</b>, black box) were pulsed 5 times at 33 Hz at different time on different locations (grey traces represent μLED stimulation pattern). <b>E</b>, The μLEDs irradiance is stable over time. When long term optogenetic light stimulation (central trace indicated by the black arrow, 200 ms pulse at 0.5 Hz, full led) is performed onChR-2 positive astrocyte the μLEDs produced stable current transients (Top trace) and peak inward currents (filled circles).</p

MicroLEDs-inducedChR2 positive astrocytes stimulation increases EPSCs frequency and is glutamate mediated.

<p><b>A</b>, One of the ChR2 positive astrocyte in the field of view is light stimulated using 18 μLEDs (top left inset) while patch clamping from a nearby ChR2 negative neuron. Bottom right inset, a close-up of the ChR2-negative neuron showing that it is not illuminated by the μLEDs. <b>B</b>,Representative gap free patch clamp recording (black trace)performed on one of the 13 neurons that were modulated by the glial stimulationandstimulation pattern(blue trace)of the ChR2 positive astrocyte showing increase of synapticactivity following ChR2+ astrocytic light stimulation. <b>C</b>, Mean event frequency time course of the 13 neurons stimulated with the protocol as in <b>B</b> (blue trace) that showed a significant sEPSCs frequency increase over the baseline (black dashed line). <b>D</b>, The stimulation protocol was performed in 22 neurons, 13 of which showed a nearly 4-fold increase in the sEPSCs frequency. 9 out of the 22 neurons tested showed no significant sEPSCs frequency increase. Application of AMPA and NMDA receptor blockers after a significant increase of the sEPSCs frequency was established, reduced the latter to levels below the baseline level (all means paired t test vs control. No effect, p = 0.2635; Excitation, p = 0.0068; APV, p = 0.0371; NBQX, p = 0.0001; NBQX + APV, p = 0.0001.(Values are the means ± SEM).</p