D-lactic acid) (PDLA) with different polyethylene glycol (PEG) segment synthesized PDLA-PEG-PDLA triblock copolymer through the ring-opening reaction of D-LA and PEG will be used as a toughening modifier. The microstructure, crystal structures and crystallization behaviors of this triblock copolymer were investigated by Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, X-ray diffraction (XRD), differential scanning calorimetry (DSC) and polarized optical microscopy (POM). The triblock copolymer is synthesized from the appearance of CH2 stretching vibration peak at 2910 cm−1 and C-O stretching vibration peak at 1200 cm−1 from PEG in FTIR spectra. Moreover, the chemical shift that is about 3.6 ppm in 1H NMR and 68.8ppm in 13C NMR proves this matter. The results of XRD and DSC reveal that PDLA and PEG are crystallized separately, and are not fully compatible, and microphase separation has occurred in this triblock copolymer. PEG can induce the triblock copolymer to accelerate the rate of crystallization, allowing it to crystallize more completely in the same amount of time. When the molecular weight of PEG is 6000 or the ratio of D-LA/PEG is 1/1, the crystallizability of PDLA-PEG-PDLA triblock copolymer is the best.Poly( The biggest and most prominent advantage of PLA is that it can be recycled in nature4. Its raw materials are renewable biological resources, so it is no longer dependent on petroleum and other resources. Its wastes are completely transformed into carbon dioxide and water through hydrolysis and a series of biological metabolism, which is harmless to human body and non-toxic, and has no pollution to the environment6.The rapid development of industry has brought great convenience to people’s lives, but the environmental pollution caused by the rapid development is also gradually exposed, coupled with the oil crisis caused by the extensive development and utilization of petroleum resources, the necessity and urgency of the research and development of degradable polymers are gradually highlighted. Among the various degradable polymers, polylactic acid (PLA) is the most promising as a green polymer materialL-lactic acid) (PLLA), poly (D-lactic acid) (PDLA) and poly  (PDLLA)7. However, compared with polyesters with higher rigidity, PLA is a semi-crystalline polymer, which usually has a slower crystallization rate without external force and produces some amorphous products during processing9. And its heat resistance is poor, which limits its application requirements in bioengineering, food packaging and other industries12.According to the chiral property of the structural unit, PLA can be divided into poly  and polymerizing with other substances . Of course, there are also some investigations on the crystallization behavior of PLA. For example, Zhang et al.19 adds lactide-caprolactone copolymer (LACL) into polylactide/poly (ε-caprolactone) (PLA/PCL) blends. PCL reduces the spherulite size of crystallization of PLA, and the nuclear density PLA/PCL/LACL blends is far higher than that of PLA and PLA/PCL. The results indicate that LACL has a capacity-enhancing effect on immiscible PLA/PCL blends, thus promoting the nucleation of PLA. The crystallization half-life of PLA/PCL/LACL and PLA/PCL is also lower than that of pure PLA, and the crystallization rate of the blends increases. Similarly, PLA, cellulose nanofibers (CNFs) and glycidyl methacrylate (GMA) grafted poly (lactic acid) (PLA-g-GMA) are mixed to fabricate PLA/CNFs and PLA/PLA-g-GMA/CNFs composites20. When 0.1 wt% CNFs are added, the crystallization rate of the composites is improved markedly. What’s more, the combination of plasticizer and nucleating agent (dioctyl adipate and ethylene bis stearic amide) can effectively improve the crystallization rate of PLA21.Currently, in order to improve its physical or thermodynamic properties, PLA is modified by adding other substances to form composites , a water-soluble polyether achieved by the progressive addition of ethylene oxide to water or ethylene glycol, has good molecular chain flexibility, non-toxicity and good biocompatibilityD-LA/PEG was prepared by ion exchange method. PEG, as the second component, was involved in the polymerization reaction, which changed the microstructure of the copolymer. The effect of different PEG molecular weight and the ratio of D-LA/PEG on the microstructure, crystal structure and crystallization behaviors of copolymer was studied by Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance spectroscopy (NMR), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and polarized optical microscopy (POM).In this work, PDLA-PEG-PDLA triblock copolymer with different PEG molecular weight and the ratio of D-lactide  was acquired from Shanghai Macklin Biochemical Co. Ltd., China. Stannous octoate (Sn(Oct)2, 95%) was obtained from Shanghai Aladdin Biochemical Technology Co. Ltd., China. Dichloromethane (CH2Cl2) (AR), Petroleum ether (AR) and xylene (AR) was supplied by Chengdu Kelong Chemical Reagent Company (China).Polyethylene glycol (PEG) (AR) was purchased from Chengdu Kelong Chemical Reagent Company (China), with molecular weights of 2000, 4000, 6000, and 8000. D-lactide using PEG as an initiator and stannous octoate as a catalyst26. The synthetic route is shown in Fig. D-LA and PEG with different molecular weight were dried at 50 °C in an electric blast drying oven for 2 h. The required D-LA and PEG with different molecular weights were accurately weighed by an electronic balance, then the medicine was placed in a dry three-necked flask, and nitrogen was passed through. After 5 minutes, the air in the flask was completely removed and the three-necked flask was placed in an oil bath pot and heated at a constant temperature. The temperature of the oil bath was 140 °C and the magnetic stirring was 15 minutes until the drug was completely dissolved. Sn(Oct)2/xylene was injected into the mixed solution. Dosage of Sn(Oct)2 was about 0.6 wt% of D-LA. The reaction was stirred at 160 °C for 4 h under nitrogen atmosphere. After the reaction, the crude product was dissolved into CH2Cl2 to wash the unreacted drug. And then petroleum ether was used as precipitant to further purify the product and obtain pure PDLA-PEG-PDLA triblock copolymer after filtration. The obtained precipitates were dried under vacuum at room temperature for 24 h. When the ratio of D-LA/PEG was constant, a series of block copolymers were prepared by changing the molecular weight of PEG . And when PEG molecular weight was fixed, a series of block copolymers were obtained by changing the ratio of D-LA/PEG .The PDLA-PEG-PDLA triblock copolymers were synthesized by ring-opening polymerization (ROP) of −1. A small amount of fully dried sample to be tested and potassium bromide were ground evenly in a mortar, then placed on a tablet press and pressed into translucent flakes at a pressure of 10 Mpa. The flakes were tested in a Fourier infrared spectrometer.A Thermo Nicolet 560 FTIR spectrometer (USA) was applied to monitor the FTIR spectra of the samples in the range of 4000–400 cm3 at 25 °C.The NMR resonance spectroscopy was carried out on a Swiss Bruker model AVANCE400 with tetramethylsilane as the internal standard in CDClλ = 0.15418) at 40 kV and 40 mA. The diffraction patterns were collected at 2θ between 5–60° at a scanning rate of 5°/min.The crystal structure was measured by X-ray diffractometer  with Cu-Kα radiation . About 8–10 mg specimens were heated from room temperature to 200 °C at the rate of 10 °C/min, maintained at 200 °C for 5 min to erase the thermal history. The samples after premelting were cooled to room temperature at a cooling rate of 10 °C/min, and then subsequently reheated to 200 °C at a rate of 10 °C/min, and the DSC curves were recorded. All the tests were conducted under nitrogen atmosphere.The POM technology was employed to investigate the crystal morphology on a CX40P polarizing microscope . The samples were placed on a hot table at 170 °C. After the samples were completely melted, they were pressed into thin sheets with cover glass, and then put into an incubator with a set temperature. After crystallization for a certain period of time, the samples were taken out for observation.−1, corresponding to the alcoholic hydroxyl group at the end of the block copolymer27. The two absorption peaks at 2990 and 1600 cm−1 is due to the stretching vibration of CH3 and the antisymmetric bending vibration, respectively, which prove the existence of CH3 functional group28. 2910 and 2890 cm−1 can be observed in this figure, the two closely spaced absorption peaks represent the absorption peaks of CH and CH2, respectively. And in the fingerprint region, there is a 1460 cm−1 bending vibration peak of CH2 and in the range of 1350 to 1310 cm−1 of CH bending vibration peaks. These absorption peaks prove the introduction of PEG structure in PDLA, and the presence of methyl and hypomethyl groups from PDLA and methylene groups from PEG in molecular chains, indicating that PDLA successfully copolymerized with PEG. The stretching vibration peak of C=O appears at 1760 cm−1, and the intensity of the peak is strong. Since it is connected with oxygen atoms, the electron absorption induction effect is greater than the conjugate effect of electron donor, which makes the absorption peak of the whole carbonyl group shift to the region of high wavenumber. And this is a big proof of the existence of ester groups. The peaks in the range from 1300 to 1200 cm−1 are strong, corresponding to the stretching vibration peak of C-O, and the absorption peaks close to 1120 cm−1, 1100 cm−1 and 1000 cm−1 is the anti-symmetric stretching vibration peak of C-O-C. From the above analysis, the conclusion that PDLA-PEG-PDLA block copolymer was synthesized successfully can be obtained preliminarily.In order to understand whether PDLA-PEG-PDLA triblock copolymer is successfully synthesized, the block copolymer samples synthesized with different segments of PEG were characterized by FTIR, as shown in Fig. 1H NMR) and 4 (13C NMR). Nuclear magnetic resonance (NMR) spectroscopy, which studies the absorption of radiofrequency radiation by atomic nuclei, is one of the most powerful tools for qualitative analysis of the composition and structure of a variety of organic and inorganic things29. In the 1H NMR, there are three groups of peaks, indicating that H atom in this copolymer has three chemical environments, and each group of peaks of different copolymers is roughly in the same position in both Fig. 3) protons in PDLA is observed around 5.15–5.20 ppm (peak c) and 1.60 ppm (peak a), respectively. The methylene protons in (CH2) group of PEG are around 3.63 ppm (peak b). When D-LA: PEG is 1/1, the chemical displacement of absorption peak moves in the direction of decreasing in Fig. The composition of the synthesized PDLA-PEG-PDLA triblock copolymer was determined by NMR spectroscopy, as shown in Fig. 13C NMR of PDLA-PEG-PDLA triblock copolymer. Among the isotopes of C, only 13C has spin phenomenon, and nuclear magnetic resonance absorption. The principle is the same as that of the hydrogen spectrum, but the chemical displacement range of the carbon spectrum is very wide, generally 0~250 ppm30. Even if the chemical environment difference of C is very small, the peak can be separated in the carbon spectrum. The positions of absorption peaks in Fig. 3 from PDLA; the chemical shift at 68.8 ppm (peak b) is the carbon atom of CH2 from PEG; the chemical shift peak at 70.2 ppm (peak c) is the carbon atom of CH from PDLA and the chemical shift at 169.7 ppm (peak d) is the carbon atom from PDLA. The absorption peak of tritium chloroform was 76–77 ppm. So far, combined with the FTIR which has been analyzed before, it is a strong demonstration that the PDLA-PEG-PDLA block copolymer has been successfully synthesized.Figure 31. Diffraction peaks appear at 19.3° and 23.5°, corresponding to (120) and (032) crystal plane diffraction of PEG, respectively32. PDLA also has diffraction peaks at 19.3° is (203) crystal plane. There are also some weak diffraction peaks at 13.9° and 15.1° belonging to PDLA and PEG, respectively. The positions of the respective diffraction peaks are substantially the same, and as the molecular weight of PEG from 2000 to 6000 is increased, the intensity of the diffraction peak is also increased, but when the molecular weight is too large, reaching 8000, the strength is weakened. It may be that the over-molecular weight of PEG limits the diffusion of the molecular chain of the block copolymer, thereby limiting crystallization. When the molecular weight of PEG is 6000, the intensity of the diffraction peak of PDLA reaches the maximum, however, the position of the peak remains unchanged. The change of the diffraction peak of PEG is not obvious. The results reveal that the effect of different molecular weight of PEG on the crystallization of block copolymer is not significant when the content of PEG is fixed, and the crystal form does not change. It may be that there is little difference in the molecular chain size of PEG with different molecular weights. There is no significant difference in the molecular chain movement during crystallization, and the structure formed is the same. Only the changes in the diffraction peak intensity are shown in the XRD pattern. However, when the content of PEG in block copolymer is different, the structure of sample is obviously different.XRD analysis can effectively provide the crystal structure information of polymer. Figure D-LA: PEG ratio. When the content of PDLA is low (1/1), the diffraction peak at 13.9° does not appear, and the diffraction peak of PEG is dominant. With more of PDLA, the characteristic peak (16.8°) strength of PDLA gradually increases, while the diffraction peak of PEG gradually weakens. This further demonstrates that PEG induces the crystallization of the copolymer. At the same time, PEG and PDLA exhibit respective crystal diffraction peaks, meaning that the two blocks are not completely compatible in the copolymer and present a state of microphase separation.In Fig. D-LA/PEG, and the corresponding DSC detailed data are listed in Tables Figures 33. It is clear that with the increase of PEG molecular weight, copolymer molecular chains become softer, crystallize faster and have larger spherulites.Through POM, the effects of different holding times on the crystallization behavior of PDLA-PEG-PDLA triblock copolymers with different PEG segments can be visually observed. Figure D-LA: PEG at 100 °C during the isothermal crystallization process for different time . The crystallization of PEG is very rapid, and the spherulite size is large. But the spherulite density is low. We find only one spherulite, filling the field of vision, and even the part near the nucleus can be seen at 100 °C for 0.5 h. As the holding time is prolonged, more crystals grow and the density of spherulites increases. As for copolymers, in general, the larger the PEG content, the larger the spherulite size at the same time. Moreover, the change in the block ratio is more pronounced than the change in the molecular weight of the PEG on the crystallization behavior of the copolymer. This also fully demonstrates that PEG increases the mobility of molecular chains of block copolymers and induces their crystallization to some extent.Figure 2 and C-O, chemical displacement of protons and carbon atoms, respectively, confirming that the copolymerization has already taken place, and PEG segment is introduced into PDLA successfully. In the XRD pattern, PDLA and PEG have their own diffraction peaks. When the molecular weight of PEG is changed, there is no large change in the position and intensity of the diffraction peak. But when the PEG content is large (1/1), the diffraction peak of PDLA is weak. The crystallization peak and melting peak of PDLA and PEG also appear separately in the crystallization and melting curves of DSC. This is similar to the results of XRD, indicating that PDLA and PEG are not completely compatible and present a state of microphase separation. The POM results reveal that the addition of PEG allows the copolymer to form crystals faster and with a larger grain size. This may be PEG as a soft segment, which enhances the flexibility and fluidity of the molecular chain and promotes the crystallization of PDLA. In addition, compared with the change of PEG molecular weight, the difference of PEG content has a greater effect on crystallization behavior.PDLA-PEG-PDLA triblock copolymers are successfully synthesized by solution method. The FTIR and NMR results exhibit the characteristic peaks of CH


The electrical control and readout of molecular spin states are key for high-density storage. Expectations are that electrically-driven spin and vibrational excitations in a molecule should give rise to new conductance features in the presence of magnetic anisotropy, offering alternative routes to study and, ultimately, manipulate molecular magnetism. Here, we use inelastic electron tunneling spectroscopy to promote and detect the excited spin states of a prototypical molecule with magnetic anisotropy. We demonstrate the existence of a vibron-assisted spin excitation that can exceed in energy and in amplitude a simple excitation among spin states. This excitation, which can be quenched by structural changes in the magnetic molecule, is explained using first-principles calculations that include dynamical electronic correlations. Electrically manipulating molecular magnetism is a challenge to overcome for applications in high-density storage. Here, the authors use inelastic electron tunneling spectroscopy to show that a vibron-assisted spin excitation in a nickel-nickelocene complex exceeds a pure spin excitation in energy and amplitude. In these molecules, the reduced symmetry of the metal center aligns the magnetic moment of the molecule along certain favorable directions and spin excitations can be produced in the absence of a magnetic field owing to this magnetic anisotropy12. Along with spin excitations, electrons may also trigger molecular vibrational modes, or vibrons, that modify18 or even suppress20 molecular conductance. Even if the presence of both excitations could be evidenced in a molecular system22, experimental observations regarding their interplay have remained surprisingly elusive.Electrically-driven excitations among spin states27. These resonances are ascribed to tunneling electrons that have their spin flipped when elastically scattering off the molecular spin, but with sufficient energy to activate a vibrational mode in the molecule28. In principle, a similar scattering mechanism can be expected for tunneling electrons losing energy to both the molecular spin states and a vibrational mode.Individual molecular spins are also prone to the Kondo screening by the host electrons of the metal electrode. In this case, the electron–vibron interaction can produce resonances in the molecular conductance at the bias of the vibron’s excitation energy5H5)2, see Fig. With this in mind, here we use scanning tunneling microscopy (STM) to study a molecular complex with magnetic anisotropy, which includes a nickelocene molecule [Ni, as sketched in the inset of Fig. 30 and results in two possible molecular configurations, known as paired  surface (see Methods section), well-ordered molecular assemblies on the surface were found , ∣ϵ2∣ = 33.9 ± 0.5 meV (noted 2) and ∣ϵ3∣ = 38.1 ± 0.6 meV (noted 3). The data exhibited a negligible tip dependence.Figure 34, we assign 1 to a spin excitation. To confirm this assignment, we carried out DFT calculations (see Methods section). Using the relaxed structure of NiNc determined above, we find that the dxz and dyz frontier orbitals of Nc in the complex are spread out in a range of  ±1 eV around the Fermi level  and Nc (+1.6 μB) with a charge transfer of 0.1 electrons from the substrate. The DFT calculations point therefore to an effective spin of S = 1. The strong resemblance with the spin excitation of single Nc molecules35 further allows us to identify the spin spectrum with the one originating from a S = 1 system. Consequently, we model 1 via a spin Hamiltonian that includes axial magnetic anisotropyz-axis is chosen along the molecular axis running through the center of the Cp rings. Within this viewpoint, 1 is assigned to a spin excitation occurring between the ground state ϵ1∣ corresponding then to D. The fit to the line shape based on Eq.  associated to 2 as high as 1.5 meV  all the spectra recorded so far showed that the energy onset of 3 is the sum of the energy onsets of 1 and of 2, ∣ϵ3∣ = ∣ϵ1∣ + ∣ϵ2∣  a red shift is observed for 3 when approaching the tip towards NiNc as seen for excitation 2 (σ1/σ0)  Cu(100) surface. A molecular flux of 2.5 × 10−2 monolayer/min was used in order to obtain well-ordered molecular assemblies along with isolated Nc molecules. To build NiNc complexes, we then exposed the surface to a small amount of Ni atoms (0.05 monolayers). The single-nickel atoms were deposited from a Ni wire source (99.99% purity) onto the cold surface (≈10 K) through an opening in the cryostat shields.The measurements were performed in an ultra-high vacuum STM operating at 2.4 K. The Cu(100) surface was cleaned in vacuo by sputter/anneal cycles, while a sputter-cleaned etched tungsten tip was employed for tunneling. The tip was further prepared by controlled tip-surface contacts to ensure a monoatomically sharp copper apex. All the spectra were recorded with a lock-in amplifier (200 μV rms and 716 Hz) using a tip that was verified to have a negligible electronic structure in the bias range investigated. The d35 treating the adsorption, chemical and physical properties of adsorbed Nc molecules on Cu(100). In the present case, a Ni atom was added to the substrate and the adsorption of Nc on this nucleation center was studied. Electron transmission calculations were also performed at the density functional theory (DFT) level. In this way, two implementations of DFT were used: (i) VASP52 for the adsorption, (ii) TRANSIESTA for the transport calculations53. The molecular geometry was optimized using DFT at the spin-polarized generalized gradient approximation (GGA-PBE) level, as implemented in VASP52. In order to introduce long-range dispersion corrections, we employed the so-called DFT-D2 approach proposed by Grimme54. We used a plane wave basis set and the projected augmented wave (PAW) method with an energy cutoff of 400 eV. The two surfaces representing substrate and tip were modeled using a slab geometry with a 4 × 4 surface unit cell and six layers for the surface holding the tip-apex and the molecule and five layers for the approaching surface electrode. The k-point sampling was converged at 3 × 3, although the sampling was 11 × 11 for the transmission calculations. For these last calculations, The valence-electron wave functions were double-ζ plus polarization (DZP) basis sets for Nc and diffuse orbitals were used to improve the surface electronic description and a single-ζ plus polarization (SZP) basis set for the copper electrodes. The use of a DZP basis set to describe the adsorbate states is mandatory in order to yield correct transmission functions56. The vibrational modes of NiNc were calculated by diagonalizing the dynamical matrix obtained from VASP while only taking the degrees of freedom of the Nc molecule into account.We have continued the work of previous publicationsI/dV spectrum, we then write an Anderson Hamiltonian for a S = 1 molecular system with a single molecular orbital in the presence of a molecular vibration of frequency ω and axial magnetic anisotropy, Dk represents the quantum numbers for the metal states, assumed to be one-electron  in the implementation by Korytár and LorenteSupplementary InformationPeer Review File


The detection of chemical messenger molecules, such as neurotransmitters in nervous systems, demands high sensitivity to measure small variations, selectivity to eliminate interferences from analogues, and compliant devices to be minimally invasive to soft tissue. Here, an organic electrochemical transistor (OECT) embedded in a flexible polyimide substrate is utilized as transducer to realize a highly sensitive dopamine aptasensor. A split aptamer is tethered to a gold gate electrode and the analyte binding can be detected optionally either via an amperometric or a potentiometric transducer principle. The amperometric sensor can detect dopamine with a limit of detection of 1 μM, while the novel flexible OECT-based biosensor exhibits an ultralow detection limit down to the concentration of 0.5 fM, which is lower than all previously reported electrochemical sensors for dopamine detection. The low detection limit can be attributed to the intrinsic amplification properties of OECTs. Furthermore, a significant response to dopamine inputs among interfering analogues hallmarks the selective detection capabilities of this sensor. The high sensitivity and selectivity, as well as the flexible properties of the OECT-based aptasensor, are promising features for their integration in neuronal probes for the in vitro or in vivo detection of neurochemical signals. The ability to detect small-molecule neurotransmitters is crucial for understanding neuronal information processing, related neurochemical processes, and brain functions in general . DopaminOrganic electrochemical transistors (OECTs) have emerged as robust alternatives to state-of-the-art sensors, since they were introduced by White et al. in 1984 , due to In the present work, we embedded interdigitated OECTs (iOECTs) in a flexible polyimide substrate and utilized aptamer-modified gold electrodes as gate electrodes. Split aptamers were employed to get rid of the electrochemical response of the blank sample, in which one receptor fragment (aptamer1) is covalently attached to the surface of a gold electrode, as described in our previous work , and theThe same gold electrode can be also utilized as a gate in the potentiometric iOECT transducer system and therefore shares the same recognition process as the amperometric transducer principle. However, the iOECT transducer relies on a change in the gate potential caused by the binding events between aptamer2/target and aptamer1 . We founAptamer 1: TTC GCA GGT GTG GAG TGA CGT CG-(CH2)6-SHAptamer 2: MB-(CH2)6-CGA CGC CAG TTT GAA GGT TCGwere purchased from FRIZ Biochem . Ten micromolar TE buffer (pH 8.0) was used to prepare the stock solutions of both DNA probes, whose concentrations were separately determined by using UV–vis spectroscopy to obtain the average absorbances value at 260 nm.Dopaminehydrochloride (DA) and its analogues, as well as the chromium etchant, were purchased from Sigma . Epoxy  and polydimethylsiloxane  were used for the encapsulation of the flexible chip, which was described in detail in our previous work ,26. Two v/v) dimethyl sulfoxide and 1% (v/v) 3-glycidoxypropyltrimethoxysilane with a channel area  of 30 μm × 22 μm were utilized, which had similar dimensions as individual cardiomyocyte-like HL-1 cells and could record action potentials from electrogenic cells [Flexible OECTs were fabricated according to the previously reported methods , includiic cells .2SO4 solution over the potential range of −0.15 V to 1.55 V with a scan rate of 1 V/s and a step width of 0.01 V for final electrochemical annealing and determining its surface area (a scan rate of 0.1 V/s) [An Au macroelectrode (ME), which was used as the gate electrode for the iOECTs, was firstly annealed with a hydrogen flame for ~10 s to remove the organic contaminations from the surface, followed by immersing it into ethanol and Milli-Q water for further cleaning. Oxidation and reduction scans were performed in a 50 mM H0.1 V/s) ,34. PrioA conventional three-electrode setup was used for the electrochemical characterization of the amperometric sensors, including a platinum wire coil as a counter electrode, a micro Ag/AgCl electrode saturated  as a reference electrode, and the modified gold electrodes described above as working electrodes. Cyclic voltammetry and square wave voltammetry (SWV) were recorded by an Autolab potentiostat PGSTAT302  in a 0.5 μM aptamer2 solution dissolved in 10 mM Tris buffer. The potential for SWV measurement varied from −0.6 V to 0.2 V (vs. Ag/AgCl) at a selected AC frequency . Dopamine hydrochloride powder  was firstly dissolved in 10 mM Tris buffer and then added to the 0.5 μM aptamer2 solution to obtain different concentrations of dopamine. Subsequently, the aptamer1-modified gold electrode was incubated in the electrolyte for 10 min, which showed the highest current response to 10 μM dopamine , followeThe measurements of the output characteristics of the flexible iOECTs and the transfer characteristics for the detection of dopamine targets were performed using a Keithley 4200 semiconductor analyzer  . FurtherHigh background signals are a common problem associated with conventional amperometric aptamer sensors where a redox tag is attached to the distal end of the aptamer. To this end, an electrochemical sandwich assay, obtained by splitting a full aptamer into two fragments, is regarded as an available platform to suppress the background signal and to enhance the potential change on the gate electrode induced by analyte binding for potentiometric transducer ,35. In tFurthermore, the selectivity of the amperometric SAESA was evaluated by comparing the current response of the aptasensor for dopamine relative to its analogues, which are in part also important neurotransmitters or interfering sample species with redox characteristics similar to dopamine a,b. A diVds) varying from −1.0 V to 0.2 V, and a gate–source bias (Vgs) in the range of −0.5 V and 1.0 V, were measured by using a standard Ag/AgCl pellet as the gate electrode (Ids) increased with Vds following Ohm’s law [Vgs, especially for a Vgs higher 0 V. This observation can be attributed to the depletion mode characteristics of PEDOT:PSS-based OECTs. Once a positive gate bias is applied at the gate bias, anions from electrolytes accumulated around the gate electrode and cations inversely penetrated into the PEDOT:PSS channel, which compensated the pendant sulfonate anions on the PSS, resulting in a de-doping of PEDOT. As a consequence, the hole density in the channel decreases, accompanied by the decline of the drain–source current. The transconductance, which represents the capability to convert changes in the gate potential into variations of the source–drain channel current [Vds was 8 mS/V , the gate potential exhibited weak dependence on the target concentration. The rise in the target concentration from 5 fM to 1 nM resulted in an almost linear increase in the potential shift. This regime was followed by the third region for high analyte concentration (Cdopamine > 1 nM), where the potential change saturated. The detection limits and the detection range of the flexible iOECT sensor are summarized in Vgs by the immobilization of ssDNA molecules on the gate electrode, were observed [KD) of the split aptamer sequences with dopamine targets for the amperometric and potentiometric transducers using a Langmuir equation [To permit a comparison of both transducers, the calibration curve was determined as well, by plotting the shift of the gate potential versus target concentration . A typicobserved . Additioequation  were 5 µFurthermore, the novel iOECT-based transducer exhibited an extremely wide detection range for dopamine concentrations, varying from 5 fM to 1 nM. Since both amperometric and potentiometric transducers are using the same gold electrode, they can be operated together to broaden the detection range of the combined sensor (5 fM to 1 nM potentiometric + 5 μM to 50 μM amperometric), and thus extend its field of applications for the detection of dopamine in plasma, cells, and urine. In addition to the sensitivity and detection range, the selectivity was evaluated as a crucial parameter of the aptasensor performance. The response of the transfer characteristics of the iOECTs to selected interfering substances such as ascorbic acid (AA), uric acid (UA), glutamate (Glu), and gamma-aminobutyric acid (GABA) was characterized for this purpose . Five inIn this work, flexible interdigitated organic electrochemical transistors (OECTs) were utilized for detecting the small molecule neurotransmitter dopamine. The background signal of the amperometric sensor significantly decreased by introducing split ssDNA aptamers with a resultant detection limit of 1 μM. This sandwich assay, formed by two aptamer fragments plus the analyte, exhibited high selectivity for the detection of dopamine among other neurotransmitters and excellent reusability. In comparison, the iOECT transducer exhibited a significantly improved sensitivity for dopamine detection with an ultralow detection limit, which was nine orders of magnitude lower than the corresponding amperometric transducer and lower than all other previously reported electrochemical sensors. The high sensitivity of the flexible iOECT-based aptasensor can be attributed to its intrinsic amplification capability. Furthermore, the high selectivity of the aptamer receptor could be conserved for the flexible iOECT transducer, where dopamine was distinctly distinguished from other important neurotransmitters. The high sensitivity, selectivity, regeneration capability, and conformability of the flexible OECT-based aptasensor pave the way for medical applications where excellent sensor performance is required without limitations from motions or the shape of the target object.


Despite the essential role of protein intake for health and development, very little is known about the impact of protein restriction on neurobiological functions, especially at different stages of the lifespan. The dopamine system is a central actor in the integration of food-related processes and is influenced by physiological state and food-related signals. Moreover, it is highly sensitive to dietary effects during early life periods such as adolescence due to its late maturation. In the present study, we investigated the impact of protein restriction either during adolescence or adulthood on the function of the mesolimbic (nucleus accumbens) and nigrostriatal  dopamine pathways using fast-scan cyclic voltammetry in rat brain slices. In the nucleus accumbens, protein restriction in adults increased dopamine release in response to low and high frequency trains of stimulation (1–20 Hz). By contrast, protein restriction during adolescence decreased nucleus accumbens dopamine release. In the dorsal striatum, protein restriction at adulthood has no impact on dopamine release but the same diet during adolescence induced a frequency-dependent increase in stimulated dopamine release. Taken together, our results highlight the sensitivity of the different dopamine pathways to the effect of protein restriction, as well as their vulnerability to deleterious diet effects at different life stages. The regulation of food intake in an ever-changing environment is a central survival process. Healthy diet requires a balanced intake of the three main macronutrients  . ProteinThe dopamine system plays a central role in food-seeking behaviors, food preference, and in the motivation to eat –19. ReceEarly life periods like childhood and adolescence are periods of particular vulnerability to the deleterious impact of various diets on corticolimbic circuits and reward-related processes –47. InteHere, we investigated the impact of protein restriction either during adolescence or adulthood on the function of the mesolimbic (NAc) and nigrostriatal  dopamine pathways using fast-scan cyclic voltammetry (FSCV) in rat brain slices. We found that protein restriction induced opposite effects on NAc dopamine release depending on age, with restriction increasing dopamine release in adults but decreasing it in adolescents. In the dorsal striatum, however, dopamine function following protein restriction was increased only in adolescents and not adults.n = 13) or at adulthood  for adult groups (n = 15). Rats were housed in groups of 2–3 in individually ventilated cages (46.2 × 40.3 × 40.4 cm), in a temperature (21 ± 2 °C) and humidity (40–50%) controlled environment with a 12 h light/dark cycle (lights on at 7:00 a.m.) and with food and water available ad libitum. All testing and tissue harvesting occurred in the light phase. Procedures were performed in accordance with the Animals (Scientific Procedures) Act 1986 and carried out under Project License PFACC16E2 granted by the UK Home Office after review from the local Animal and Welfare Ethical Review Board (AWERB) at the University of Leicester.Male Sprague Dawley rats (Charles River Laboratories) were received either at weaning  for adolescent groups  or were switched to a modified AIN-93G diet containing 5% protein from casein  containing 18% protein. One week after arrival rats either continued on standard laboratory chow diet . Slices were kept at room temperature (20–22 °C) in aCSF saturated with 95% O2 and 5% CO2 for at least 1 h before the start of recordings.Rats were deeply anesthetized with chloral hydrate , decapitated, and brains were removed and transferred to ice-cold artificial cerebrospinal fluid (aCSF) containing in mM: 126 NaCl, 10 glucose, 26 NaHCO2 and 5% CO2 at 30 °C. Slices were allowed to equilibrate for 30 min prior to recordings. A twisted stainless steel bipolar stimulating electrode  was placed at the surface of the slice within the NAc core or the dorsal striatum  were confirmed by examining current–voltage plots showing oxidation (~+0.6 V) and reduction (~−0.2 V) peaks using TarHeel software. Background (non-Faradaic) current was measured for 1 s between 4 and 5 s before the stimulation and subtracted from the signal. Dopamine currents (in nA) were then converted to dopamine concentration (in nM) using the calibration of each electrode against a known standard dopamine concentration. [DA]o peaks were measured following any stimulation artefacts as previously described o AUC and clearance times  were analyzed using two-way ANOVAs with Age (Adults and Adolescents) and Diet  as between-subject factors. [DA]o AUC in response to single pulses were plotted as cumulative probability and compared using Kolmogorov–Smirnov test. [DA]o peaks and AUC from frequency-response curves were analyzed using three-way and two-way repeated measures ANOVA using Age (Adults and Adolescents) and Diet  as between-subject factors and frequency  as within-subject factor. 5 p/1 p [DA]o ratios were calculated by dividing the average [DA]o peak value at 20 Hz by the average [DA]o peak value at 1 Hz for the same recording site, and were analyzed using two-way ANOVA with Age (Adults and Adolescents) and Diet  as between-subject factors. Sidakʼs and Dunnettʼs post hoc tests were performed when required.For single pulse stimulation, [DA]Statistical analyses were conducted using GraphPad Prism 8. All values were expressed as mean ± standard error of the mean (SEM). The alpha risk for the rejection of the null hypothesis was 0.05.Upon publication, all data analyzed in this paper will be available on Figshare (10.25392/leicester.data.c.5008904).F = 0.5, p = 0.5; Day, F = 85.5, p < 0.001; Diet × Day, F = 0.4, p = 1.0). In contrast, protein restriction during adolescence significantly decreased weight gain, relative to control diet  = 19.8, p < 0.001; Day, F = 478.7, p < 0.001; Diet × Day, F = 234.0, p < 0.001). Both NR and PR adult rats exhibited similar low weight gain . NR adolescent rats showed substantial weight increases (+118 ± 4 g), indicating a normal developmental growth whereas PR rats showed only a modest increase in their weight  = 23.14, p < 0.001; Age, F = 97.8, p < 0.001; Diet × Age, F = 70.3, p < 0.001; Sidak’s post hoc tests p = 0.4 for Adults and p < 0.001 for Adolescents), demonstrating that protein restriction in adolescence disrupted normal growth.We first investigated the impact of protein restriction during either adolescence or adulthood on weight and weight gain Fig. . As we pF = 229.8, p < 0.001; Fig. F = 35.1, p < 0.001; Diet × Age, F = 0.1, p = 0.7). A more detailed analysis of macronutrient breakdown showed that PR groups had an lower energy intake from protein but an increased intake from carbohydrate and fat  = 35.1, p < 0.001; Diet × Macronutrient, F = 394.7, p < 0.001; all Sidak’s post hoc tests p < 0.05; Fig. Analysis of the average daily food intake for each cage showed that adolescent rats have a higher energy intake than adults  = 0.1, p = 0.7; Diet, F =  0.7, p = 0.4; Diet × Age, F = 17.8, p < 0.001). Protein restriction at adulthood induced a significant increase (+167 ± 49%) in NAc dopamine release in response to single pulse stimulation compared to NR control rats (Sidak’s post hoc tests p < 0.01). In contrast, protein restriction during adolescence significantly decreased NAc dopamine release evoked by single pulse stimulation . Further analyses confirmed that protein restriction in adulthood significantly changed the distribution of [DA]o AUC values toward the right, demonstrating a greater proportion of large dopamine responses to single pulse, compared to control animals  = 0.7, p < 0.05). In adolescents, protein restriction significantly induced a left-shift of the distribution of [DA]o AUC, confirming a reduced dopamine response  = 0.8, p < 0.05). Importantly, analyses of [DA]o peaks evoked by single pulse confirmed the age-dependent differential effect of the diet  = 1.4, p = 0.2; Diet, F = 0.2, p = 0.7; Diet × Age, F = 7.6, p < 0.05), without revealing significant differences in either adults (Sidak’s post hoc test p = 0.1) or adolescent rats (Sidak’s post hoc test p = 0.09). To examine whether the diet-induced changes in dopamine release were mediated by differences in dopamine reuptake, we measured the T80 clearance time. T80 was significantly shorter in adolescent groups compared to adults  = 6.5, p < 0.05). However, protein restriction at adulthood or during adolescence did not significantly change dopamine clearance  = 0.1, p = 0.7; Diet × Age, F = 2.7, p = 0.1; see also Supplementary Fig. o peak amplitude or the time course of dopamine uptake, but likely a combination of both factors.In the NAc, protein restriction had a different impact on dopamine release evoked by single pulse stimulation depending on the life stage  to brief bursts of action potentials at high frequency  o peak amplitude  = 0.04, p = 0.9; Age, F = 0.08, p = 0.8, Diet × Age, F = 1.4, p = 0.2) or dopamine clearance  = 0.03, p = 0.9; Age, F = 0.9, p = 0.4, Diet × Age, F = 2.1, p = 0.2; see also Supplementary Fig. In the dorsal striatum, protein restriction had no effect on dopamine release evoked by single pulse stimulation whether rats were exposed to the diet during adulthood or adolescence  = 81.6, p < 0.001; Age, F = 5.8, p < 0.05; Frequency × Age, F = 16.9, p < 0.001; see also Supplementary Fig. F = 2.2, p = 0.1; Diet × Frequency, F = 6.7, p < 0.001; Diet × Age, F = 0.4, p = 0.5; Diet × Frequency × Age, F = 0.8, p = 0.5). Separate analyses for each age group confirmed that protein restriction has no significant effect on frequency-dependent striatal dopamine release in adults  = 0.2, p = 0.6; Frequency, F = 63.8, p < 0.001; Diet × Frequency, F = 4.2, p < 0.05; Sidak’s post hoc tests: all p > 0.7). In contrast, protein restriction in adolescent rats significantly increased stimulation-evoked striatal dopamine release  = 4.8, p < 0.05; Frequency, F = 19.2, p < 0.001; Diet × Frequency, F = 2.7, p = 0.05), especially in response to phasic-like stimulations . Moreover, both low and high stimulation frequencies increased dopamine release in the adolescent PR group , whereas this is only observed for high frequencies in the NR control group . Similar results were observed by measuring the AUC of dopamine evoked release  = 11.7, p < 0.01) but was not altered by protein restriction  = 1.8, p = 0.2; Age × Diet, F = 0.001, p = 1.0).Similar to what we observed in the NAc, the ‘phasic/tonic’ ratio of striatal dopamine release was lower in adolescent slices  and high frequency burst-like stimulation (5p at 20 Hz). Dopamine neurons exhibit an elevated firing rate during adolescence , 53, 54 –10 Hz anIn the dorsal striatum in adolescents, we observed an opposite pattern compared to the NAc. As such, evoked dopamine release was increased after adolescent protein restriction, especially at high stimulation frequencies. Such an effect partially rules out the hypothesis of a global amino acid deficiency. However, the nigrostriatal dopamine pathway matures earlier than other dopamine pathways  and may 0 peak amplitudes, this suggests that neither protein restriction during adolescence nor adulthood impacts dopamine transporter functioning , the European Commission [grant #GA 631404 to JEM], The Leverhulme Trust [grant #RPG-2017-417 to JEM] and the Tromsø Research Foundation [grant #19-SG-JMcC to JEM). The authors declare no competing interests.Supplemental Tables and Figures


The drug IBU was applied as a model drug to investigate the drug release behaviour by ultraviolet spectrophotometry measurements. The investigation results demonstrate that mesoporous silicate molecular sieves by the aerosol-assisted method are spherical with a core–shell structure. As the drug carrier, it has good structural stability and can achieve drug controlled release which is expected. It exhibits safety to a certain degree. Therefore, the aerosol-assisted synthesis method provides a new idea for the synthesis of sustained-release drug carriers.The mesoporous silicate molecular sieves were synthesized with polyether F127 as the template by the aerosol-assisted method for loading and release of ibuprofen (IBU). The synthesized samples were characterized by scanning electron microscopy, transmission electron microscopy, X-ray diffraction and N Drug-Mesoporous molecular sieve refers to a three-dimensional porous material with a pore size of 2–50 nm. It is mainly characterized by the use of surfactant molecular aggregates as a mesoporous template, and the formation of pore structure in the molecular sieve can be achieved through the interface assembly process of surfactant molecular aggregates and inorganic species. Its structure and performance are between the amorphous inorganic aluminosilicate porous material and the inorganic zeolite molecular sieve porous material with a crystalline structure. It not only has a regular pore structure and an amorphous pore wall, but also has some excellent properties which are not available in other porous materials . Since tThere are three main methods for the synthesis of mesoporous molecular sieves: the sol–gel method, the hydrothermal synthesis method and the phase transition method –27. HoweHerein, we synthesized the novel mesoporous silicate molecular sieves with polyether F127 as a template by the aerosol-assisted method. The morphology of most synthesized samples is spherical with a particle size of about 200 nm. Meanwhile, the well-developed spherical particles have a core–shell structure. The layer of annular pores is arranged in parallel on the outside of the spherical particles, and the three-dimensional short worm-like pore structure is arranged inside the spherical particles. The drug IBU was loaded in the sample as a drug model to study the loading and release of a drug. With the loading of the drug, although the ordering of the carrier is affected to some extent, it still maintains the original structure. At the same time, it can quickly reach the adsorption equilibrium and have a significant slow-release effect during the drug loading process.2.2.1.The process of aerosol synthesis of molecular sieves is shown in The IBU was added into an appropriate amount of ethanol. The mix was magnetically stirred until dissolved. An amount of molecular sieves were added and stirred continuously. After the loading process was completed, the sample was filtered by vacuum suction filtration, washed by ethanol and dried at room temperature to obtain a molecular sieve loaded with IBU.2.2.2.3.et al. [The transmission electron microscopy (TEM) images reveal the well-developed spherical particles with a core–shell structure . It can et al. . This st2.4.θ value, the larger the mesoporous structure pore diameter. As can be seen from the figure, the (100) characteristic peak of the IBU/F127 sample shifts significantly to the right, so the pore size of the carrier is reduced.X-ray diffraction (XRD) is one of the most important means to characterize the structure of molecular sieves. XRD spectra can be used to analyse the sample structure, channel order and other information. As 2.5.2 adsorption–desorption isotherms of the mesoporous silicate molecular sieves by the aerosol-assisted method and that supported IBU are shown in figure 5a. Both samples have the characteristics of type IV and type I isothermal curves, which indicates that the pore structure of the molecular sieve carrier has maintained the original appearance, and basically no pore blocking and collapse have occurred. From the changes of the adsorption amount of these samples, single-layer adsorption occurred in the low-specific pressure region P/P0 < 0.01, and the jump in the N2 adsorption amount is attributed to the filling of micropores, which indicates that the molecular sieve carrier prepared by the aerosol method has a small number of microporous structural units. The intensity of the isotherm of the IBU/F127 sample is very low, and the adsorption capacity is small, which indicates that the sample's pore structure is affected by the loading of the drug. These conclusions are also confirmed in figure 5b. Mesoporous pore size of samples decreased from 8.5 nm to 6.9 nm owing to the loading of drug. The specific surface area of the molecular sieve prepared by the aerosol method is 371.2277 m2 g−1, and the pore volume is 0.3186 cm3 g−1. With the loading of the drug, the specific surface area and pore volume of the sample gradually decrease. This is because after loading the drug, IBU is deposited in the pore channel, which reduces the pore size and pore volume. The specific surface area of IBU/F127 is decreased to 285.1357 m2 g−1, and the pore volume is decreased to 0.2351 cm3 g−1.The N2.6.−1. The residual drug concentration in the solution was determined by the ultraviolet method, and the loading of IBU on the molecular sieve was calculated by the subtraction method. The loading curve is shown in The molecular sieve prepared by the aerosol method was as the carrier, and the concentration of IBU was 35 mg g2.7.The amount of IBU released per unit molecular sieve was measured by the ultraviolet method. 2.8.−1, and over 80% cell viability could be observed in the HIEC-6 cells incubated with IBU or IBU/F127 for 48 h, exhibiting safety to a certain degree.The bio-safety of materials was measured by the Cell Counting Kit-8 (CCK-8) assay. As shown in 3.3.1.Tetraethyl orthosilicate (TEOS) was used as the silicon source, and polyvinyl ether-polypropylene ether-polyvinyl ether triblock copolymer (F127) was used as template agents, respectively. The raw material composition is TEOS: F127: ETOH: H2O: HCl = 1 : 0.005 : 18 : 24 : 0.075. The deionized water, ethanol (ETOH), template (F127) and concentrated hydrochloric acid were added to the conical flask and mixed well. The TEOS was added after the template was dissolved. Then the mix was hydrolyzed at room temperature for 4 h. The aerosol spray was generated by the mixed liquid through the aerosol generator and the dried at 300°C in a tube heating furnace. In this process, as the volatile solvent evaporated, the non-volatile matter was self-assembled into the mesoporous structure to obtain a white powder. The white powder was dried at 100°C overnight and baked at 540°C for 6 h to remove the template to obtain the final product. The product was labelled as F127.3.2.2 adsorption–desorption isotherm (N2-TPD) of the sample was measured using the American-based Quantachome company's Autosorb-1 physical adsorption instrument. The specific surface area was calculated using the modified Brunner Emmet Teller method. The adsorption pressure corresponding to the pore volume was P/P0 = 0.99. Calculation of pore size distribution of mesoporous molecular sieves was by the Barret Joyner Halenda method.XRD analysis was performed in a FEI Tecnai G2 F30 X-ray powder diffractometer. TEM images were obtained on the Tecnai G220 Stwin-type electron microscope from FEI. The acceleration voltage was 200 kV, and the sample was ultrasonically oscillated in absolute ethanol before the test. SEM images were recorded using a Helios with an acceleration voltage of 10 kV. The N3.3.−1). The mix was magnetically stirred until dissolved. An amount of molecular sieves were added. Samples were taken at regular intervals and diluted appropriately for ultraviolet testing. Two hundred and seventy-three nanometres was the wavelength of incident light. According to the IBU standard solution, the standard curve was measured as C = 0.613A-0.009 . The loading of IBU per unit mass molecular sieve was calculated by the differential subtraction method. The specific calculation formula was as follows. L was the loading of IBU in the molecular sieve, m0 was the initial mass of IBU in the solution, cn was the concentration of the residual drug solution, M0 was the initial mass of the solution, V was the volume of the sample solution (1 ml) and M was the mass added to the molecular sieve:Liquid ultraviolet spectrophotometry was used for quantitative analysis of drug molecules. The IBU was added into an appropriate amount of ethanol . IBU/F127 was pressed into tablets and put into the release solution. The sample was stirred at room temperature and taken at regular intervals. The same amount of fresh medium was replenished. The supernatant taken was diluted appropriately for ultraviolet testing. The amount of IBU released per unit molecular sieve was as follows. R was the amount of IBU released per drug-loaded molecular sieve, nc was the measured IBU concentration in the released V0 solution, V0 was the total volume of the release medium (500 ml), V is the sampling volume (5 ml) and M is the mass of the load tablet:The simulated intestinal fluid (pH = 7.4 phosphate-buffered solution) was used as the release system solvent. The IBU was added into an appropriate amount of phosphate-buffered solution. The mix was magnetically stirred. Samples were taken at regular intervals and diluted appropriately for ultraviolet testing. Derivative spectroscopy was used to process the data and the regression equation of the derivative spectrum of IBU in phosphate buffer was dl(A) = 0.762C-0.017  assay was used to determine the bio-safety of materials. Cell lines HIEC-6 were maintained in RPMI 1640 (Life Technologies) with 10% fetal bovine serum (Sigma) in a humidified atmosphere at 37°C with 5% CO4.In conclusion, we present a simple and efficient aerosol-assisted method to prepare novel mesoporous silicate molecular sieves. The investigation results demonstrate that mesoporous silicate molecular sieves by the aerosol-assisted method are spherical with a core–shell structure. The morphology of most synthesized samples is spherical with a particle size of about 200 nm. The layer of annular pores is arranged in parallel on the outside and the three-dimensional short worm-like pore structure is arranged inside the spherical particles. The drug IBU was applied as a model drug to investigate the drug release behaviour by ultraviolet spectrophotometry measurements. As a drug carrier, it has good structural stability and can achieve drug controlled release which is expected. It exhibits safety to a certain degree. Therefore, the aerosol-assisted synthesis method provides a new idea for the synthesis of sustained-release drug carriers.


Vibrio (V.) vulnificus infection is a rare disease whose death rates exceed 50% despite aggressive antibiotic treatment and surgical debridement. The aim of this study was to assess the ability of specific anti-V. vulnificus immunoglobulins Y (IgYs) for preventing and treating V. vulnificus infections. IgYs were produced by immunizing egg laying hens with inactivated whole cell bacteria. Peritoneal cytokines, blood's bacterial load, and survival curves were obtained from both prophylactic and therapeutic mouse models. The results showed that the specific IgYs (i) inhibited the growth of V. vulnificus in vitro, (ii) dramatically reduced the inflammatory response and blood's bacterial load, and (iii) improved the survival rate of V. vulnificus-infected mice. These results prove that anti-V. vulnificus IgYs can be markedly effective means for the prophylaxis and the therapy of V. vulnificus infections. Vibrio (V.) vulnificus is a halophilic Gram-negative pathogen that is naturally present in estuarine waters, contaminated oysters, and other shellfish [V. vulnificus infection may progress to severe skin lesions and septicemia in people with predisposing conditions, including liver diseases, hereditary hemochromatosis, and a compromised immune system [V. vulnificus infection-caused septicemia has a mortality rate equal to or higher than 50%. And with no antibiotic therapy, this figure could raise up to 100% [V. vulnificus infections has been increasing in parallel with global warming [V. vulnificus infections are still inadequate. Moreover, although V. vulnificus is usually vulnerable to most antibiotics, several studies have reported that it could resist multiple antibiotics because of the ongoing misuse of antibiotics in aquaculture farming [hellfish , 2. V. ve system . Infecti to 100% . During  warming . HoweverAntibody therapy is potentially one of the most important methods that can replace antibiotic treatment . CompareAs compared with IgGs, the noninvasive, low-cost, and simple collection method from egg yolk makes IgYs into favorable therapeutic agents capable of controlling infectious diseases . In factV. vulnificus to prevent and treat V. vulnificus infection in the respective mouse model.Therefore, the aim of the present study was to explore the capability of IgYs obtained via immunization against inactivated whole-cell V. vulnificus strain (No.182487342) we used for this work. The strain was isolated from the blood of an infected patient and cultured in thiosulfate citrate bile salt sucrose agar, blood agar plates, and 3% sodium chloride alkaline peptone broth .The Laboratory Department of the First Affiliated Hospital of Sun Yat-sen University kindly provided the Male CD1 mice, aged 6 to 8 weeks, were bought from Liaoning Changsheng Biotechnology Co., Ltd. . The 20-week-old, roman powder laying hens were purchased from Zhengda Egg Industry Co., Ltd. . All animal experiments were conducted according to protocols approved by the Animal Ethics Committee of Sun Yat-sen University. The design of all the mouse experiments aimed at minimizing the animals' numbers. All efforts were made to reduce to a minimum the animals suffering and distress.V. vulnificus bacteria were killed by incubating them with 0.4% formaldehyde at 37°C for 24 h, next washed with phosphate-buffered saline (PBS), and stored frozen until used. A 1 : 1 mixture of complete Freund's adjuvant with 1.8 ml of 1 × 109 CFU/ml bacteria was injected into the hens' breast muscle. Next, to strengthen immunization at weeks 4, 8, and 12, the hens were injected a 1 : 1 mixture of 1.8 ml of 1 × 109 bacterial CFUs with incomplete Freund's adjuvant.4. The separated egg yolks were mixed with distilled water (pH 5) at 4°C overnight. Next, the suspensions were centrifuged at 10,000 rpm for 10 min. The obtained supernatants were salted out by adding 30% ammonium sulfate. Following centrifugation, the crude extracts were dissolved in PBS and finally passed through a ultrafiltration cartridge tube type 100 KD UF to concentrate the proteins.Eggs were collected until the detected IgYs' titers were over 1 × 10V. vulnificus. Briefly, 100 μl of inactivated bacteria was incubated overnight at 4°C. The coated plates were washed three times with 0.05% Tween-PBS and blocked with 5% bovine serum albumin. Next, the plates were incubated with serial dilutions of IgYs and washed again with Tween-PBS. This was followed by an incubation with 100 μl of 1 : 10,000-diluted HRP-conjugated rabbit anti-IgY antibody. Finally, after adding the TMB substrate, color development was stopped with 2 M H2SO4. The absorbance was measured at 450 nm in a microplate reader .An enzyme-linked immunosorbent assay (ELISA) served to figure out the level of IgYs against whole-cell 5 CFU/ml in 3% sodium chloride alkaline peptone broth. Next, the specific and nonspecific IgYs were added to the bacterial broth, making the final concentrations of the specific IgYs equal to 2, 1, and 0.5 mg/ml and that of the nonspecific IgYs equal to 2 mg/ml. An alike volume of plain PBS was added to the control group. Finally, the optical density (OD) was read at 600 nm.To perform the growth inhibition assays, the bacterial density was adjusted to 10V. vulnificus IgYs, 8-week-old CD1 mice were intraperitoneally injected with 2 × 106 bacterial CFUs (~23.3 LD50) at a given time point before or after the administration of a single dose of IgYs. The mice from the experimental groups were given the specific IgYs produced by hens immunized with inactivated whole-cell V. vulnificus. The mice from the negative control group received nonspecific IgYs produced by hens with no earlier immunization, and the mice from the blank control group received plain PBS with no IgYs.To assess the prophylactic and therapeutic efficacy of the specific anti-V. vulnificus infection. TNF-α levels in lavage fluids were determined via ELISA. Meanwhile, whole blood samples were collected from the orbits of infected mice to assess the numbers of blood stream live bacteria via the plating method. Finally, the survival curves of V. vulnificus infected mice were evaluated.Peritoneal lavages with 0.5 ml saline were performed 5 hours after α levels, and blood bacterial load experiments were found by using a t-test for pairwise comparisons. Kaplan-Meier survival curves were analyzed using the log-rank test. All the results were expressed as means ± standard deviations (SDs). p values<0.05 were considered significant.All data were graphed and analyzed using GraphPad Prism version 8 . The levels of statistical significance for ELISA assays, bacterial growth inhibition assays, TNF-V. vulnificus IgY titers. The hens immunized with inactivated whole-cell V. vulnificus had significantly higher IgY titers than control hens had. Moreover, the IgY titers were correspondingly reduced by gradual increases in V. vulnificus antigen dilution. This showed that the antigens had successfully stimulated the IgY antibodies production in the hens  2 hours after the intraperitoneal administration of a single dose of IgYs (2 mg/ml in 0.5 ml) or of plain PBS (0.5 ml). The specific IgYs elicited a 100% protection against the death from V. vulnificus infection, as compared to the 10% survival of the control (no IgYs) group   .V. vulnificus IgY-injected mice was 100%. By contrast, only 10% of the control group mice survived   or PBS (0.5 ml) 1 h. The survival of the specific anti- 0.0001) . These rV. vulnificus exert excellent prophylactic and therapeutic effects on this bacterial infection by significantly mitigating the inflammatory response and microbial load in the blood, and by increasing the survival rate of the infected mice.To our knowledge, this is the first proof that the specific IgYs produced by laying hens immunized with inactivated whole V. vulnificus infection is one of the main causes of seafood-related deaths. The infection occurs through accidental skin cuts while handling contaminated aquatic products, or by eating raw seafoods, such as lobster [V. vulnificus infection is quick and it progresses fast. Notwithstanding an aggressive antibiotic therapy and surgical interventions, the fatality rate stays high [V. vulnificus strains isolated worldwide due to the use of antibiotics by the aquaculture farming [V. vulnificus infection gave excellent results [ lobster . The onsays high . Early p farming . Therefo farming , 10. Ear results , 13. How results .Helicobacter pylori infections. Although the administration of antibodies from other species may theoretically cause IgE-mediated allergic reactions or Arthus' serum sickness, no adverse clinical events were reported for IgYs administered via the respiratory or gastrointestinal tract [Unlike IgGs that are extracted from mammalian serum, IgYs are obtained from the egg yolk by a noninvasive procedure. Each immunized hen functions as a small manufacturing plant, producing a steady stream of targeted antibodies and transferring them into the egg yolk. The cumulative amounts of antibodies produced by each hen are much higher than those extracted from the serum of a mammal. This makes the large-scale production of IgYs workable at a cost that is much lower than that of IgGs , 9. Nowaal tract , 14. Andal tract , 14, 15.V. vulnificus bacteria to immunize laying hens. Our present results show that the specific IgYs thus produced exert significant prophylactic and therapeutic effects on V. vulnificus infection. They could reduce the inflammatory response and blood bacterial load and keep at 100% the survival rate of V. vulnificus-infected mice. No obvious adverse reactions occurred in the IgY-treated mice. Therefore, our findings constitute the cornerstone of future clinical trials. Interestingly, the nonspecific IgYs also exerted partial inhibitory effects in vitro and mitigated the inflammatory response and lowered the blood bacterial load in vivo in comparison with the control (No IgYs) group. This might be ascribed to the fact that both the nonspecific and the specific antibodies recognized and bound portions of the same epitopes. However, aside their partial prophylactic and therapeutic effects on V. vulnificus infection, the nonspecific antibodies could not improve the survival rate of V. vulnificus-infected mice.We used the inactivated whole V. vulnificus bacteria served as antigens for immunization. Earlier studies had shown that VvhA and RtxA1 were the main virulence factors of V. vulnificus infection as silencing the corresponding genes significantly reduced the virulence of V. vulnificus strains [V. vulnificus infections.In the present study, inactivated whole  strains , 13, 16.V. vulnificus infections. These antibodies could significantly mitigate the inflammatory response and lower the blood bacterial load while keeping at 100% the survival rate of infected mice. These preclinical findings constitute the foundations for future clinical trials confirming the effectiveness of specific IgYs in preventing and treating V. vulnificus infections.Our study showed that specific IgYs showed an excellent prophylactic and therapeutic efficacy against


ObjectiveCoronary computed tomography angiography (CCTA) is a noninvasive diagnostic modality that remains underutilized compared to functional stress testing (ST) for investigating coronary artery disease (CAD). Several patients are misdiagnosed with noncardiac chest pain (CP) that eventually die from a cardiovascular event in subsequent years. We compared CCTA to ST to investigate CP.MethodsWe searched MEDLINE, PubMed, Cochrane Library, and Embase from January 1, 2007 to July 1, 2018 for randomized controlled trials (RCTs) comparing CCTA to ST in patients who presented with acute or stable CP. We used Review Manager (RevMan) [Computer program] Version 5.3  for review and analysis.Results2 = 0%). A significant reduction was seen in myocardial infarctions (MIs) after CCTA compared to ST: 115 vs. 156 . On subgroup analysis, the CCTA arm had fewer MIs vs. the ST with imaging subgroup  and stable CP subgroup . The CCTA arm showed significantly higher invasive coronary angiograms and revascularizations and significantly reduced follow-up testing and recurrent hospital visits. A trend towards increased unstable anginas was seen in the CCTA arm.We included 16 RCTs enrolling 21,210 patients; there were more patients with hyperlipidemia and older patients in the ST arm compared to the CCTA arm. There was no difference in mortality: 103 in the CCTA arm vs. 110 in the ST arm (risk ratio [RR] = 0.93, 95% confidence interval [CI] = 0.71-1.21, P = .58, and IConclusionsOur analysis showed a significant reduction in downstream MIs, hospital visits, and follow-up testing when CCTA is used to investigate CAD with no difference in mortality. Coronary heart disease is one of the leading causes of death, globally. Annually, more than 20 million patients undergo workup for angina . PatientWe conducted a systematic review and meta-analysis to compare CCTA to ST with subgroup analyses of ST (with and without imaging which has never been done before) and CP (acute chest pain [ACP] or stable chest pain [SCP]). Over the years, few meta-analyses comparing CCTA to ST have been published, and the outcomes are variable; these are summarized in Table Data sources and searchesWe completed a systematic review according to the Preferred Reporting Items for Systematic Review and Meta-analyses (PRISMA) guidelines . We searStudy selectionThree reviewers  reviewed the abstracts and selected 59 articles for a full review. A total of 16 RCTs met the predefined inclusion criteria for qualitative and quantitative analysis comparing CCTA to ST: myocardial perfusion imaging or scan, stress electrocardiogram (bicycle or treadmill), stress echocardiogram, pharmacologic nuclear scan, graded exercise testing, and pharmacologic ST  and odds ratio (OR), and inverse variance for the continuous data to estimate the standardized mean difference in Review Manager (RevMan) [Computer program] Version 5.3 . We reported results as forest plots. We used online GraphPad Online Version 8  to compare the baseline characteristics and to calculate the number needed to treat (NNT) to prevent one adverse event. A two-tailed P-value of < .05 was considered statistically significant. We assumed a 1:1 ratio in each arm except for the ACRIN/PA trial, which randomized patients in a 2:1 ratio where we used the same ratio for outcomes; this failed to uncover any event in both arms . BaselinHeterogeneity2 statistics to calculate the heterogeneity. I2 > 50% was considered substantial heterogeneity, as explained in the Cochrane Handbook for Systematic Reviews [We used I Reviews . We perfWe included 16 RCTs with 21,210 patients . Patients in the ST arm were older than those in the CCTA arm  and had more hyperlipidemia . There was no difference in baseline body mass index, hypertension, diabetes, smoking status, and baseline use of aspirin. Three studies used ST without imaging for a total of 1,110 patients (595 in the CCTA arm and 515 in the ST without imaging arm) ,22,26.Primary endpoints were all-cause mortality and new myocardial infarction (MI) during the follow-up period. Secondary endpoints included ICA after ST, true positive ICA, revascularizations, new unstable anginas, emergency room (ER) visits or hospital admissions during the follow-up period, follow-up tests, complications  attributed to CCTA compared to ST, direct discharges from ER, ER cost and total cost, and radiation dose. The results are summarized in Table Primary Endpoints2 = 0%). The subgroup analyses for ST with imaging , ST without imaging , ACP  and SCP  found no differences ; this was mainly noted as a reduction in MI in the SCP subgroup patients  compared to the ACP subgroup that showed no difference . The CCTA arm also had significantly reduced MIs compared to ST with imaging  with no difference compared to ST without imaging . Both the ACP  and SCP  subgroups had more ICA post-CCTA. ICA was common after CCTA compared to ST with imaging  and without imaging . This finding was consistent in both the ACP  and SCP  subgroups and in ST with imaging  and without imaging  compared to ST . Of note, I2 was reduced to 0% with exclusion of the SCOT-HEART trial and without affecting significance [2 = 60%), ST without imaging , ACP , and SCP  was significantly higher after CCTA . A similar trend was seen on subgroup analysis of ACP , SCP , ST with imaging  and ST without imaging, .New unstable angina: There was no difference in new unstable anginas in the CCTA group vs. ST group . I2 was reduced to 16% without the CAPPA trial, but the results became statistically insignificant. The subgroup analysis of ACP and SCP and ST with imaging revealed no difference between CCTA and ST, though there were significantly reduced ER visits or hospital admissions in the CCTA arm compared to ST without imaging ; sensitivity analysis did not reduce the heterogeneity. The ST with imaging subgroup  and ACP subgroup  showed no difference in follow-up testing. ST without imaging  and the SCP subgroup  had a significant reduction in follow-up testing after CCTA . Five studies reported direct ER discharges without admission to the hospital ; sensitivity analysis did not reduce heterogeneity.Direct discharge from the ER:Cost analysis: Eight studies reported cost, but only five studies were usable as these reported mean cost and standard deviation.Three studies reported the ER costs. There was a trend towards a decrease in ER costs in the CCTA arm . Sensitivity analysis, without the CT-COMPARE trial, reduced the heterogeneity to 0% and the results became statistically significant .Five studies reported the total cost. There was no difference between the two arms . Sensitivity analysis without the CT-COMPARE reduced the heterogeneity to 45%; however, the results remained statistically insignificant. The subgroup analysis for the cost in the United States and cost elsewhere also had significant heterogeneity with no difference between the subgroups .Radiation dose: Four studies reported the cumulative radiation exposure usable for our analysis. The CCTA arm had significantly higher radiation exposure . Sensitivity analysis failed to reduce the heterogeneity.Our meta-analysis of 21,210 patients comparing CCTA to ST demonstrated a significant reduction in the primary endpoint of MIs in the CCTA group without any difference in mortality. The reduction in MI was secondary to a significantly reduced number of events in the SCP group. The reduction in MIs is likely due to the early diagnosis of obstructive CAD and subsequent early initiation of aggressive medical management and revascularizations. Recently published five-year outcomes of the SCOT-HEART trial, which enrolled patients with SCP, also showed a significant reduction in MIs over five years . This diThis analysis also showed increased ICA and revascularizations, which also lead to significantly reduced MIs (NNT of nine to prevent one MI for each ICA). This early difference in ICA and revascularization may be lost after an extended follow-up as suggested by the five-year outcomes of the SCOT-HEART study . This inAfter the initial randomization and workup with either CCTA or ST, ER visits and rehospitalizations were significantly reduced in the CCTA arm; this differs from a previously published meta-analysis that showed no difference in ER visits and rehospitalizations -11. A liThe increased rates of angiographically confirmed CAD post-CCTA is another significant finding that suggests that CCTA has a better positive predictive value than ST (with or without imaging) to identify obstructive CAD at a time when current guidelines do not support the routine use of CCTA in intermediate-risk patients. Although our analysis showed an increasing trend towards unstable anginas in the CCTA arm, we hypothesize that this trend is likely the consequence of higher rates of revascularization in the CCTA group.The cost analysis had substantial heterogeneity for both ER visits and downstream costs. The trials included in our analysis were conducted in different countries with different healthcare systems and cost structures -28. In oLimitationsOur study had several significant limitations. First, a lack of long-term follow-up in the individual RCTs (≤25 months) that may not include events, hospitalizations, and revascularizations beyond 25 months would magnify the risks of ICA and revascularization and obscure potential long-term benefits. This may be true for ACP trials as short follow-up may have masked the advantage for either arm. Also, some outcomes were not reported by most studies, leading to substantial heterogeneity that persisted even after sensitivity analysis. In addition, we were unable to estimate radiation exposure from all studies between the two groups since they reported data in a variable form. Also, only three studies used ST without imaging, and the other studies used a combination of imaging and non-imaging ST; this leads to substantial overlap between the groups and has a risk to introduce bias in our results. Finally, these trials, although relatively modern, did not utilize high-sensitivity cardiac troponin tests. Their hypotheses must be tested again with the advent of these tests.Our analysis is the largest to date of 16 RCTs and found a significant reduction in post-CCTA MIs with increased ICA and revascularizations. In the future, more RCTs are needed utilizing scoring methods to identify more robust downstream investigations, cost analysis, and radiation exposure.


Molecular pathology is an essential part of pathology complementing conventional morphological tools to obtain a correct integrated diagnosis with appropriate assessment of prognosis and prediction of response to therapy, particularly in cancer. There is a concern about the situation of molecular pathology in some areas of Europe, namely, regarding the central role of pathologists in assessing somatic genomic alterations in cancer. In some countries, there are attempts that other laboratory medicine specialists perform the molecular analysis of somatic alterations in cancer, particularly now when next generation sequencing (NGS) is incorporated into clinical practice. In this scenario, pathologists may play just the role of “tissue providers,” and other specialists may take the lead in molecular analysis. Geneticists and laboratory medicine specialists have all background and skills to perform genetic analysis of germline alterations in hereditary disorders, including familial forms of cancers. However, interpretation of somatic alterations of cancer belongs to the specific scientific domain of pathology. Pathologists are necessary to guarantee the quality of the results, for several reasons: (1) The identified molecular alterations should be interpreted in the appropriate morphologic context, since most of them are context-specific; (2) pre-analytical issues must be taken into consideration; (3) it is crucial to check the proportion of tumor cells in the sample subjected to analysis and presence of inflammatory infiltrate and necrosis should be monitored; and 4) the role of pathologists is crucial to select the most appropriate methods and to control the turnaround time in which the molecular results are delivered in the context of an integrated diagnosis. Obviously, there is the possibility of having core facilities for NGS in a hospital to perform the sequence analysis that are open to other specialties , but also in this scenario, pathologists should have the lead in assessing somatic alterations of cancer. In this article, we emphasize the importance of interpreting somatic molecular alterations of the tumors in the context of morphology. In this Position Paper of the European Society of Pathology, we strongly support a central role of pathology departments in the process of analysis and interpretation of somatic molecular alterations in cancer. Cancer is a complex disease. Hereditary and environmental factors are important in its development. Cancer results from accumulation of genetic and epigenetic alterations in cells , under tEvery type of cancer has its own molecular profile. Even more, each individual cancer in a particular patient has its unique repertoire of molecular abnormalities . With thFor many years, cancer has been diagnosed on the basis of its microscopic appearance. Several decades ago, identification of proteins by immunohistochemistry became an important tool in cancer diagnosis . Later oPathology is the medical discipline responsible for the diagnosis of diseases based on the morphological appearance under the microscope of tissue or cytological samples. Cancer is one of the areas where the progress in pathology is most dynamic and significant. The role of pathologists in diagnosis of cancer, as well as assessment of prognostic and predictive factors, is crucial. The task for pathologists is not only the evaluation of the gross appearance of the lesion together with microscopic assessment of neoplastic tissue. To guarantee high reliability of pathological diagnosis, the so called pre-analytical conditions  must be controlled. Another crucial issue is the appropriate sampling of most relevant diagnostic areas, as well as selection of the most informative tumor areas for additional ancillary techniques , 6.Generally speaking, a patient does not have a proven cancer diagnosis, until a pathologist establishes such diagnosis in a histologic or cytologic specimen. Obviously, there are few exceptions to this statement, for some malignancies and disseminated tumors, in which obtaining a tissue sample may be too risky for the patient.Pathologists are not laboratory specialists who identify, quantify, and report molecules from blood or other biological samples. We are medical specialists interpreting the microscopic appearance of the tumor in the setting of the molecular, clinical, and imaging features. Occasionally, similar microscopic pictures can lead to different diagnoses, depending on age, tumor site, or clinical scenario.Interpretation by pathologists is also essential for correct interpretation of molecular research data resulting from the analysis of tissues and cells , 8. The The genetic alterations that are detected in cancer tissues can be germline or somatic. Cancer-related germline genetic abnormalities occur in the setting of clinical situations with an increased risk for the development of cancer. Familial cancer syndromes are the paradigmatic examples. Germline alterations are important for tumor development, and their identification may be important for prognosis or even for prediction of response to certain types of systemic anticancer treatment. Germline alterations are detected in tumor tissue but also in normal non-neoplastic tissues from cancer patients. Peripheral blood is the most frequently used biological material for identification of germline changes. Management of DNA and RNA from peripheral blood is less challenging than that obtained from tumor tissue, as the nucleic acids in the blood are less negatively influenced by pre-analytical conditions.Identification and interpretation of germline alterations in cancer do not usually require integration of the results in the context of morphological features of the tumors. It may be perfectly performed by geneticists, who have the appropriate expertise, including skills to classify the identified variations regarding their impact and distinguish between pathogenic changes from those that are not. Bioinformatic support is essential. Since the tumors occurring in the setting of familial cancer syndromes may sometimes have specific morphologic features, it is desirable to have pathologists as part of the multidisciplinary teams interpreting results of germline molecular testing and establishing screening or surveillance strategies. However, it does not seem necessary that pathologists have the lead in these clinical strategies.Occasionally, analysis of somatic mutations may lead to identification of a genetic change that may be suspicious to be in fact a germline alteration. Additional conventional germline mutation analysis  in close collaboration with clinical geneticist may be mandatory to confirm the germline nature of these genetic variants.BRCA1/2 mutations or microsatellite instability (Lynch syndrome) should be noted in the pathology report with a recommendation that clinical geneticists have to be involved to perform genetic counseling of the patient with this family.Evidence of BRAF, KIT) that have different significance depending on tumor type [The scenario of somatic genomic alterations of cancer is different from that of the germline ones. The clinical and pathologic contexts are important. Integration of molecular results with microscopic features is necessary. There are tissue-specific differences in tumorigenesis and the organization of individual oncogenic signaling pathways. There are many examples of somatic alterations  as well as bioinformaticians, who should be integrated as full-fledged staff members. This is currently the situation in a significant proportion of tertiary hospitals across Europe; however, in some areas, there are administrative difficulties for an efficient incorporation.Incorporation of NGS into clinical practice is having a tremendous impact in management of cancer patients . In someIn some centers, NGS equipment is based in pathology departments. In other centers, NGS facility is shared in central core facilities, available to several different specialties. In some of these centers, pathologists have access to NGS equipment and perform somatic molecular interpretation followed by delivery of an integrated pathologic report. In other centers, NGS is performed by other specialists, and pathologists are only requested to simply provide tissue samples, without too much interaction, and insufficient involvement in reporting the results. We are strongly convinced that this role of just “tissue providers” would be totally inappropriate, carrying important risks of incorrect interpretation of molecular findings outside of the tissue context, and might be thus potentially detrimental to patient safety.Sometimes, hospital administrators do not consider the abovementioned conditions and take unfortunate decisions allowing molecular testing and interpretation of cancer somatic alterations in facilities, outside pathology departments, without guaranteeing integration into the appropriate pathologic context. These wrong decisions are usually based on economical or organizational parameters, not supported by scientific evidence.Another important fact which must be taken into consideration is the issue of tumor heterogeneity. There is increasing evidence that tumors are composed of subpopulations of cells with distinct genetic alterations , 18. TumAs mentioned before, the clinical and microscopic context is essential to interpret appropriately the results. This is relevant not only for tumor morphology but also to interpret the role of non-cancer microenvironment in conjunction with molecular data. However, it is important to emphasize that any molecular test is just one small part of the entire complex pathologic diagnosis. Traditional pathological features, such as gross findings, tumor stage, histological type, and grade based on microscopic evaluation and molecular findings, should be therefore integrated in a combined pathologic report. Moreover, digital pathology, which is recently being implemented in many pathology departments, may become an additional useful tool to integrate molecular results in the appropriate pathological context . AdvanceIndications for molecular analysis in pathology have changed over the last decade. Molecular pathology was initially performed virtually exclusively for diagnostic purposes in hematologic disorders and solid tumors, and pathologists were the solely responsible for deciding when and which specific test should be performed in a particular case. Nowadays, however, a significant proportion of the molecular tests are performed for predictive purposes, to identify subsets of cancer patients who will have higher probability to respond to certain anticancer treatment. This has led to a change of roles, and the tests are now frequently requested by clinician. Under such circumstances, a close interaction between oncologists and pathologists is necessary, with efficient workflow and decision-making strategies, in which Molecular Tumor Boards are excellent scenarios. Nowadays, many pathology departments organize Molecular Tumor Boards, involving molecular biologists, bioinformaticians, and oncologists, to discuss the relevance of NGS findings for targeted therapies in different organ tumors  together. These Molecular Tumor Boards represent ideal platforms for a comprehensive discussion of all aspects of molecular diagnostics and the consequences for targeted therapies. It should be emphasized, however, that pathologist should play a role in the decision about the most appropriate molecular assay that must be performed, particularly for cancer diagnosis Fig. .Fig. 1WoThe number of molecular tests that are incorporated to regular testing of tissues is constantly growing, in concordance with development of novel targeted therapies requiring new predictive biomarkers . In thisCurrently, a new paradigm for cancer care is emerging, where treatments are identified based on the specific genetic profile of an individual’s tumor, regardless of where it first originated. Pathologists diagnose tumors based on a specific histological type and the organ of origin. However, organizations such as the American Food and Drug Administration (FDA) have recently shown a willingness to accept novel trial designs—such as basket studies, which enroll patients for treatments based on the biology of their tumor, not their tumor type. These “tumor-agnostic” agents target gene alterations thought to drive tumor growth regardless of cancer histology. “Tumor-agnostic” therapies are based on identification of microsatellite instability or high tumor mutational burden for immuno-oncological therapies . Other eMoreover, incorporation of somatic molecular testing integrated in pathology has a profound positive impact on attractiveness of pathology as a medical discipline in medical students who may decide to become pathologists. Furthermore, it has also important positive consequences in training and postgraduate education. The responsibility of incorporating molecular assays into routine pathology also requires deep understanding of the field by pathologists, not only for those directly involved in the workflow of the molecular tests.An international strategy should be considered to give support to countries where molecular pathology is performed so far only in minimal extent, in order to face the scenario of increasing technological complexity and hardware requirements .The issue of liquid biopsy (testing of circulating cell-free DNA released by the tumor—ctDNA) was not within the scope of this paper, which was exclusively focused on somatic molecular testing in tumor tissue. However, it is a very promising technique, regularly performed in many pathology departments across Europe. Although, at present, it is limited to a selected group of neoplasms such as lung cancer, its use will be probably extended also to other types of cancer in the near future.https://www.cen.eu), should be applied. These are then translated into ISO international standards after 1 or 2 years. Involvement of pathologists in the entire process of molecular analysis ensures optimization of pre-analytical variables, such as cold ischemia time and optimal fixation.Technical issues are important to obtain correct results –30. PathQuality control is a very important issue. Pathology departments should demonstrate to be capable to perform somatic molecular testing in optimal conditions, at all steps of the technical process at the pre-analytical, analytical, and post-analytical levels. External quality control by accredited agencies is required.Cold ischemia time is the period between tissue surgical removal and fixation. It is an important factor, since DNA, RNA, and proteins are altered during such period, with a clear impact on the results of testing using molecular techniques . Tissue As mentioned before, selection of the appropriate tissue block for molecular analysis is crucial in the light of tumor heterogeneity . It is iIn this article, the importance of interpreting somatic molecular alterations of tumors in the light of morphology has been emphasized. A central role of pathology departments is necessary in the process of analysis and interpretation of somatic genomic alterations in cancer, not just as tissue providers for their interpretation outside this context. There are many reasons from the scientific, technical, and logistics points of view. The issue is important in terms of patient safety, since patients deserve clinically relevant interpretation of somatic alterations in cancer, being performed and interpreted under optimal conditions, quality control of the whole procedure, and optimal turnaround time.