Activation or suppression of single PV neurons modified visual responses of postsynaptic Pyr cells in 6 of 7 pairs whereas that of single SOM neurons showed no significant modification in 8 of 11 pairs, suggesting that PV neurons can act solo whereas most of SOM neurons may act in chorus on Pyr cells.Neural circuits in the cerebral cortex consist primarily of excitatory pyramidal (Pyr) cells and inhibitory interneurons. Interneurons are divided into several subtypes, in which the two major groups are those expressing parvalbumin (PV) or somatostatin (SOM). These subtypes of interneurons are reported to play distinct roles in tuning and/or gain of visual response of pyramidal cells in the visual cortex. It remains unclear whether there is any quantitative and functional difference between the PV → Pyr and SOM → Pyr connections. We compared unitary inhibitory postsynaptic currents (uIPSCs) evoked by electrophysiological activation of single presynaptic interneurons with population IPSCs evoked by photo-activation of a mass of interneurons  GABAergic interneurons are divided into several subtypes, in which the two major groups in the rodent neocortex are those expressing parvalbumin (PV) or somatostatin (SOM)16. Recent studies in the mouse visual cortex reported notable differences in function between these subtypes of interneurons, such as scaling visual responses through divisive inhibition or sharpening response tuning through subtractive inhibition19, although there is some inconsistency among these reports20. Also it is suggested that PV-expressing and SOM-expressing interneurons control response reliability in a different way21. It is not fully clear, however, why their functions are so different and what mechanisms in cortical circuits underlie the different functions.In the cerebral neocortex GABAergic/inhibitory interneurons control tuning and/or gain of response of pyramidal (Pyr) cells to sensory stimuli24. By combining the methods of optogenetic, massive cell activation with those of electrophysiological single cell activation, we addressed these questions and found that about 14 PV neurons at least made strong connections with a postsynaptic Pyr cell while a much larger number of SOM neurons made weak connections. The activation/inactivation of single PV neurons modified visual responses of postsynaptic Pyr cells in 6 of the 7 pairs whereas that of single SOM neurons did not induce such a modification in 8 of the 11 pairs, suggesting that the operation mode of the two major subtypes of interneurons is different.To understand mechanisms underlying the different functional roles of these interneurons in cortical circuits, the quantitative information on functional connectivity with target Pyr cells is crucial. However, there has been no solid information about how many PV or SOM interneurons make functional connections with a postsynaptic Pyr cell, although some quantitative analysis was made previouslyin vivo in layer 2/3 of the visual cortex of mice in which each subtype of interneurons expressed channelrhodopsin-2 (ChR2). To activate single PV or SOM neurons we injected depolarizing currents into target interneurons through recording electrodes so as to generate action potentials which induced unitary IPSCs (uIPSCs) in postsynaptic Pyr cells. The intensity of injected currents was adjusted to generate single action potentials. We found that there were notable differences in uIPSCs between PV → Pyr and SOM → Pyr cell connections. In a pair of PV and Pyr cells uIPSCs had relatively high amplitudes and fast rising slopes  of Pyr cells evoked by action potentials of presynaptic PV or SOM neurons pes Fig. . The peapes Fig. . The meapes Fig. .Figure 12 to find parameters for generation of single action potentials. We found that the optimal intensity was 18 mW/mm2 for 1 ms width both for PV and SOM neurons, and there was no detectable difference in sensitivity to blue laser between PV and SOM neurons. In part of the experiments we increased the intensity to 20 mW/mm2 that did not induce multiple action potentials. We also observed that none of the 8 PV- or 7 SOM-negative neurons that were presumed to be interneurons on the basis of their soma shape generated action potentials in response to blue laser. Also we confirmed that none of the 11 GAD-positive but ChR2-negative cells showed action potentials to photoactivation. These results suggested that the photoactivation selectively generated action potentials of PV or SOM interneurons.Then we activated a population of PV or SOM neurons which make functional connections with a target Pyr cell under observation by emission of blue laser to the cortex through the microscope. An application of laser pulse of 1 ms width faithfully generated single action potentials of interneurons which in turn evoked IPSCs in postsynaptic Pyr cells  longer than that of the PV → Pyr connections (35.9 ± 2.7 ms)  larger than that of the PV → Pyr connections (30.9 ± 2.3 pC)  Fig. . Also thpC) Fig. . To conf− was assumed to take place in postsynaptic Pyr cells. This might reduce the charge currents of pIPSCs, in particular in the late phase, as reported previously25 and thus lead to an underestimate of the number of interneurons. Also there is another possibility that the same group of interneurons that have mutual connections (a kind of disinhibitory connection)29 might also be activated, and consequently the number of interneurons to a postsynaptic Pyr cell may be underestimated in the present study. These points will be discussed later.Because the total charge of pIPSC (pIPSQ) is assumed to be an integral of uIPSQ of each connection in case of linear summation, we calculated the ratio, pIPSQ/uIPSQ, for each pyramidal cell to approximately assess the number of interneurons of the same subtype that functionally connects with a postsynaptic Pyr cell. The values of the 8 PV → Pyr connections were densely distributed near the mean of 14.2 ± 1.8, while those of the 8 SOM → Pyr connections were distributed in a scattered manner from 29 to 122 with the mean of 63.8 ± 11.4 -expressing or Vasoactive Intestinal Polypeptide (VIP)-expressing neuronsin vivo condition blue laser emitted through the two-photon microscope system was localized to the volume of about 200 × 200 × 400 μm of the cortex so that possible connections from interneurons outside this area might elude activation. To overcome this possible problem we activated PV or SOM interneurons with diffuse blue light which covered the volume of about 5,500 × 5,500 × 300 μm of the sliced cortex. Initially we recorded uIPSCs of Pyr cells evoked by single action potentials of PV neurons or SOM neurons in the same way as in the in vivo experiments. As demonstrated in the in vivo cortex, uIPSCs of PV → Pyr cell connections were relatively large while those of SOM → Pyr cell connections were small  recorded from VGAT-ChR2-YFP mice in the PV neuron group, because FS cells in cortical layer 2/3 were reported to be PV neuronsell Fig. . The monell Fig. . In the 41, activation or suppression of a single interneuron by current injections might affect neighboring interneurons so that the observed effects might not be simply ascribable to manipulation of single cell activities. To test this possibility we recorded membrane potentials of a pair of PV cells which were located closely. Currents were injected into one of the pair to hyperpolarize membrane potentials. No detectable change was observed in the neighboring PV cells during injection of currents . For example, we reliably measured uIPSCs as small as 10 pA amplitude from SOM-Pyr cell connections. This value was still larger than the noise level of our electrophysiological system for voltage-clamp recording (around 2–3 pA). Thus it is unlikely that we missed very small IPSCs. Nevertheless we cannot completely exclude the possibility that inhibitory synaptic connections which were located far from the soma or had the low release probability of GABA might be overlooked. In excitatory connections in the hippocampus it was reported that proximal and distal glutamatergic synaptic events had the similar impact on neuronal outputs, independent of their spatial location47, although in the neocortex more distal excitatory inputs to pyramidal cells generated the lower amplitude of somatic EPSPs48. The distance-dependent scaling of synaptic currents along dendrites of hippocampal pyramidal neurons was shown to have a structural basis such as synapse number and AMPA receptor expression in their dendrites49. In some GABAergic connections, however, the distance-dependent scaling was not seen in inhibitory synaptic currents in hippocampal pyramidal cells51. In the neocortex there has been no report on the presence or absence of the distance-dependent scaling of IPSCs to the best of our knowledge. Thus we cannot exclude the possibility of the regional difference of GABAergic synapses as a reason for the difference in uIPSCs between the two types of connections. It is also possible, however, that SOM neurons effectively affect somatic action potential generation of Pyr cells in case a certain number of SOM neurons become synchronously active. In fact, we observed that the mean peak amplitude and rising slope of pIPSCs evoked by photoactivation of a mass of SOM neurons were not significantly different from those by the mass activation of PV neurons. If the distance from the soma solely determines the magnitude of IPSCs, the former currents should have been smaller and slower-rising. However, this was not the case. Thus, one should consider the number of active connections in addition to the cable decay of IPSCs to fully understand the function of PV → Pyr and SOM → Pyr cell connections.It is well known that axons of PV neurons mostly make synaptic contacts with the soma or proximal dendrites of Pyr cells while those of SOM neurons mainly target distal dendrites52. In the somatosensory cortex of adult mice it was reported that 14 of the 23 pairs were electrically coupled, but their coupling coefficient was only 1.43%41. Therefore, the possibility that our activation or suppression of single presynaptic PV cells with current injection affected activities of neighboring PV cells seems low, although cannot completely be excluded in the present study. Also there is a possibility that the manipulation of a single SOM neuron affected activities of neighboring PV cells directly through gap junctions. This possibility seems low, because electrical coupling in the neocortex was detected almost exclusively between GABAergic neurons belonging to the same class41.As mentioned in the Results section there is a possibility that activation or suppression of a single cell by current injections might affect neighboring cells so that the observed effects might not simply be ascribable to manipulation of single cell activities. However, we did not observe a detectable change in the neighboring interneurons during injection of hyperpolarizing or depolarizing currents. In our previous study only 3 of the 30 FS-FS cell pairs in layer 2/3 of the mouse visual cortex showed weak electrical couplingin vivo preparations. In the slice preparations the recording was carried out at the low temperature . Thus in the physiological condition the decay of SOM-induced pIPSCs might be longer than that of PV-induced pIPSCs, which makes the charge of currents of the former pIPSCs larger. This might reflect filtering property of distantly located synapses. This possibility is unlikely, however, because the peak amplitude and rising slope was not different from those of PV-induced currents. The slow decay of SOM-induced pIPSCs might be ascribable to the convergence of large number of connections.In spite of the notable differences in the four parameters of uIPSCs between PV → Pyr and SOM → Pyr cell connections, optically induced pIPSCs were not significantly different from each other in the slice preparations and slightly different only in their decay tau and total charge of currents in the in vivo condition the activation of ChR2-expressed interneurons by blue laser was limited to the volume of about 200 × 200 × 400 μm of the cortex. In the in vitro condition, on the other hand, the activation by diffuse blue light covered the volume of about 5,500 × 5,500 × 300 μm of the sliced cortex. Nevertheless it is possible that connections from interneurons outside this area might be missed from activation. In particular, some long range connections were sectioned in slice preparations. It was reported, however, that every potential presynaptic inhibitory input to Pyr cells was detected in slice preparations of the mouse frontal and somatosensory cortices23. In the present in vivo experiments it is possible that PV or SOM neurons whose soma were located outside of the irradiated area were also activated by blue laser because their dendrites also expressed ChR2  might suggest such a shunting effect in the massive activation of interneurons. Therefore, the present calculation may give an estimate of the minimum number of interneurons that converge to postsynaptic Pyr cells. Although we cannot exclude such an underestimate of the number of interneurons, the large difference in uIPSCs albeit the similar size of pIPSCs between the PV → Pyr and SOM → Pyr connections suggests that the number of each subtype of interneurons functionally connecting to postsynaptic Pyr cells is notably different.The present assessment of the number of presynaptic interneurons to postsynaptic Pyr cells is based on the assumption that the charge currents are linearly summated, as mentioned in the Results section. However, there is a possibility that the activation of many interneurons will affect the summation of the inhibitory events in Pyr cells. For example, a massive activation of multiple GABAergic synapses results in intracellular Cl59 crossed with Ai14 reporter line displayed electrophysiological fingerprint of FS interneurons60, suggesting that a proportion of PV neurons might be included in interneurons classified as SOM neurons in SOM-Cre mice in the present study. In the present study we analyzed the cortical tissue with immunohistochemistry, and demonstrated that 96–97% of ChR2-YFP expressing interneurons were immunoreactive for SOM, as shown in Supplementary Fig. Recently it was reported that 18% of reporter fluorescence protein-expressing interneurons in SOM-Cre miceIn sum, the present findings suggest that there is a much greater functional convergence of SOM cells than PV cells to postsynaptic Pyr cells and a small number of PV cells or even a single PV cell affects visual responses of target Pyr cells whereas a larger number of SOM cells do so when they work together in a mass.Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze/J, 2. SOM-ChR2-YFP mice, STOCK Sst < Tm2.1(cre)Zjh > J × B6;129S-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze/J, 3. VGAT-ChR2-YFP mice, B6.Cg-Tg(Slc32a1-COP4*H134R/EYFP)8Gfng/J. The animals were anesthetized with urethane (1.5–1.9 mg/g body weight), supplemented with additional doses of urethane when necessary. The level of anesthesia was continuously monitored by observing heart and respiration rates, which were not changed in response to a strong touch of the tail. A small custom-made head chamber to stabilize the animal’s head under a two-photon microscope was attached over the occipital region of the left hemisphere. The part of the skull and dura mater over the primary visual cortex were removed, and the exposed cortex was covered with agarose (1.5–2.0% in Ringer’s solution).All experimental procedures were approved by the Animal Experiment Committee of RIKEN Brain Science Institute and the experiments were carried out in accordance with the guidelines of the Committee. The following three types of mice in which PV, SOM or whole GABAergic interneurons express ChR2, of either sex, were used at postnatal days 45–70 (P45-70). 1. PV-ChR2-YFP mice, B6;129P-Pvalbtml (cre) Arbr/J × B6;129S-In vitro experiments PV-ChR2-YFP or SOM-ChR2-YFP mice of either sex were used at P25-35. For slice preparations, animals were anesthetized with Isoflurane  and then decapitated. The brains were rapidly removed and placed in the cold oxygenated artificial cerebrospinal fluid (ACSF). Coronal slices of the visual cortex (300 µm thick) were obtained using a tissue slicer (Vibratome 3000). Slices were placed in an incubating chamber filled with oxygenated ACSF at 31 °C for 30 min and then stored at room temperature (23–25 °C) until transferred to a submerged recording chamber. The recording was carried out at room temperature. The ACSF had the following composition (in mM): NaCl, 124; KCl, 3.0; CaCl2, 2.0; MgCl2, 1.0; NaH2PO4, 1.25; NaHCO3, 26.0; and glucose, 10.0, at pH 7.4. The ACSF was bubbled continuously with 95% O2–5% CO2. The flow rate of the ACSF was about 2.5 ml/min. An antagonist for NMDA receptors, DL-2-amino-5-phosphonovaleric acid  at 100 µM and an antagonist for AMPA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione  at 20 µM were added to the ACSF. In part of the experiments tetrodotoxin  at 1 µM was added to the ACSF.Images of cells were visualized with an upright microscope  equipped with a water immersion objective lens  and the laser scanning microscope system , which was coupled with a mode-locked Ti:sapphire laser . The mode-locked lasers were set at the wavelength of 800 and 930 nm for excitation of Alexa 594 which was injected into target neurons and Venus/YFP which were expressed in a given type of interneurons, respectively. Emitted fluorescence was divided into long wavelength (>570 nm) and short wavelength light with a dichroic mirror , and short-wavelength light was further filtered through a bandpass filter . Both wavelengths of emitted fluorescence were detected simultaneously using two photomultiplier tube detectors (PMTs). The microscope objective was shielded from possible stray light by covering the space over the animal’s head with lightproof cloth and clay.4, 130; CsCl, 3; HEPES, 10; MgATP, 4; Na3GTP, 0.3; EGTA, 1 and Na-Phosphocreatine, 10. The resistance of the electrode filled with this solution was 3–6 MΏ. The composition of the internal solution for current clamp recordings was as follows (in mM): K-Gluconate, 130; CaCl2, 0.1; HEPES, 10; MgATP, 4, Na3GTP, 0.3; MgCl2, 2; Na-Phosphocreatine, 10 and EGTA, 1. The resistance of the electrodes filled with this solution was 6–8 MΏ. For recording IPSCs from Pyr cells the former CsMeSO4-based solution was used while the latter K-Gluconate-based solution was used for inducing action potentials of inhibitory interneurons and recording visual stimulation-induced action potentials of Pyr cells. For analyzing the morphology of recorded cells Alexa 594 was added to the internal solution at 0.025 mM. The osmolarity of the solution was 280–290 mOsm, and pH was adjusted to 7.2–7.4 with CsOH or KOH.Whole-cell current-clamp recordings of membrane potentials were performed from YFP-positive interneurons at a depth of 110–230 μm from the cortical surface. Whole-cell voltage-clamp or current-clamp recordings of membrane currents/potentials were simultaneously performed from nearby pyramidal cell-like neurons. The mean distances between the PV → and SOM → Pyr cell pairs were 28 ± 5.0 and 36 ± 6.0 μm, respectively. These recordings were performed with a pair of multi-clamp amplifier (Multiclamp 700B). Recording electrodes were pulled from borosilicate glass capillary with filaments . The composition of the internal solution for voltage clamp recordings was as follows (in mM): CsMeSOWhole-cell recordings from pyramidal cells were targeted to YFP-negative neurons with large soma of pyramidal shape and large apical dendrites which were visualized by filling the extracellular space with Alexa flour during shadow patching. They were confirmed to be pyramidal cells by identifying spines along dendrites visualized with intracellularly injected Alexa and regular spiking pattern evoked by depolarizing current injection. The mean resting membrane potential of Pyr cells was 70.2 ± 1.8 mV (n = 16). Action potentials were generated in YFP-positive interneurons by injection of depolarizing currents of ˂250 pA for 5 ms duration. Evoked IPSCs were recorded from pyramidal cells at the holding potential of 0 mV. Membrane currents were filtered at 2–5 kHz and sampled at 20 kHz using custom made LabView software, and fed into a computer with an NI-DAQ board . Data analysis was performed using the MATLAB program. Series resistance was monitored before and after each recording session by applying small voltage steps (5 mV of 250 ms duration). The recording was excluded from further analysis if the series resistance was higher than 25 MΩ and/or changed >20% during recording.in vivo experiments. The membrane potentials were recorded with a multi-clamp amplifier (700B), filtered at 2–5 kHz and digitized at 10 kHz, and fed into a Pentium 4 personal computer with a digitizer computer interface . The analysis was made using Igor 4.01 program. Cells were not used for analysis if the resting membrane potential was more positive than −50 mV, series resistance was >25 MΩ, or if any of these parameters changed by 20% during data acquisition. The mean resting membrane potentials of Pyr cells were 66.6 ± 1.3 mV (n = 28).Dual whole-cell recordings were made from a pair of interneurons and Pyr cells in layer 2/3 of the visual cortex under infrared differential interference contrast optics. PV or SOM interneurons in cortical slices were visualized with an epifluorescence microscope . The composition of the internal solution was the same as in vivo preparations  or an LED source of blue light through the objective lens of the Olympus upright microscope in the slice preparations .To activate a particular type of interneurons we employed the two methods: Electrical stimulation of single cells under observation by current injection through recording pipettes and optical stimulation of a population of interneurons which expressed ChR2 through blue light. This optical stimulation was performed using 473 nm laser through the objective lens of the two-photon microscope in the Dual whole-cell recordings were performed from a pair of Pyr cell and interneuron to record uIPSCs evoked by single action potentials of PV, SOM or FS-GABA neurons in layer 2/3 of the visual cortex. The following parameters of uIPSCs were measured in traces obtained by averaging 15–30 consecutive responses: Peak amplitude (from the baseline to the peak of uIPSC); Rising slope (slope of a fitting line from 10–90% after onset of uIPSC); Total charge of currents as the integral of uIPSCs until the trace returned completely to the baseline; and Decay τ which was measured by fitting a single exponential to the decay phase of the uIPSCs.Visual stimuli were presented on an LCD monitor  30 cm from the eye of the animal, covering 80 × 50 degrees of the visual field. Square-wave gratings  at 100% contrast were moved on the monitor in 12 directions at 30 degree steps (from 0 to 330 degrees). The visual stimulation procedure was as follows: 2 s before, 2 s during and 2 s after the presentation of visual stimulus. In the experiments in which presynaptic single cells were activated or suppressed these 12 patterns of 2 s visual stimuli were presented 4–6 times in a randomly shuffled order before, during and after activation or suppression of the presynaptic interneurons.The number of action potentials during visual stimulation was counted before, during and after activation or suppression of presynaptic interneurons. If postsynaptic neurons did not generate action potentials in response to visual stimulation, the membrane depolarization during the presentation of visual stimuli moving in the same direction was averaged over trials . These visually evoked depolarizations were computed as areas under the trace of depolarization above the mean baseline level of membrane potentials in the pre-visual stimulation period. Cells were defined as visually responsive when one of the averaged signals was larger than 2 SDs of the baseline signals during the pre-stimulation period.29. Briefly, after fixation with 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer (PB), the brain blocks were cut into 40 µm-thick coronal sections on a freezing microtome. The brain sections were incubated overnight with a mixture of chicken anti-GFP antibody  and rabbit anti-PV  or rabbit anti-SOM . The sections were further incubated overnight with a mixture of AlexaFluor488-conjugated goat antibody against chicken IgY  and Cy3-conjugated donkey antibody against rabbit IgG , and counterstained by NeuroTrace 435/455 blue fluorescent Nissl stain  to identify the cytoarchitecture of the primary visual cortex.The following procedure was carried out as previously reportedThe sections were observed under a Leica TCS SP8 confocal laser-scanning microscope (Leica) equipped with a 25 × water-immersion objective lens  and HyD detectors. We acquired images at 5.0 Airy disk unit and 2048 × 2048 pixels. NeuroTrace 435/455 blue, AlexaFluor488 and Cy3 were sequentially excited with 405, 488 and 552 nm laser beam, and observed through 410–470, 495–550 and 560–610 nm emission prism windows, respectively. After taking images, we counted the number of neurons across cortical layers.t-test. The t-test was used in case the values plotted in representative neurons showed the normal distribution. Statistical evaluation of the normal distribution was made using the Kolmogorov–Smirnov test. In case the distribution of values was not normal, the Mann–Whitney U test was used.In the present study, values are given as the mean ± SEM, unless otherwise mentioned. For statistical analysis, values between two different groups of cells were compared with unpaired The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Information


ATP) channels. We have shown that treatment with the KATP channel agonist pinacidil increases survival of bees while decreasing viral replication following infection with FHV, whereas treatment with the KATP channel antagonist tolbutamide decreases survival and increases viral replication. Our results suggest that KATP channels provide a significant link between cellular metabolism and the antiviral immune response in bees.Honey bees are economically important pollinators of a wide variety of crops that have attracted the attention of both researchers and the public alike due to unusual declines in the numbers of managed colonies in some parts of the world. Viral infections are thought to be a significant factor contributing to these declines, but viruses have proven a challenging pathogen to study in a bee model and interactions between viruses and the bee antiviral immune response remain poorly understood. In the work described here, we have demonstrated the use of flock house virus (FHV) as a model system for virus infection in bees and revealed an important role for the regulation of the bee antiviral immune response by ATP-sensitive inwardly rectifying potassium (K Apis mellifera) play an economically vital role in global agriculture as pollinators of a wide variety of crops, in addition to being valued for the honey and other natural products that they provide. Declines in the numbers of both managed and wild pollinators have served to increase public awareness of bee health issues and prompted researchers to increase efforts to understand the factors driving these declines. While there exist a variety of factors that negatively impact the health and survival of both managed and wild bee populations, there is a growing consensus that the prevalence of parasites and pathogens, especially viruses, are among the most significant threats to pollinator health3. Viral infections have been found to be closely associated with weakened or dying bee colonies, as well as with colonies believed to be affected by Colony Collapse Disorder (CCD)7. The viruses that most commonly infect bees are nonenveloped, positive sense, single-stranded RNA viruses belonging to either the family Dicistroviridae or Iflaviridae in the order Picornavirales8. Bee colonies are commonly infected with multiple viruses concurrently11, and those infections may range from asymptomatic to producing overt deformities, paralysis, and even death. There exist multiple routes of transmission for bee viruses12, the most damaging being the ectoparasitic mite Varroa destructor, which can suppress overall immune responsiveness and cause seemingly harmless chronic infections to become acute and devastating14. Evidence suggests that this complex interplay between host, pathogen, and parasite significantly alters the critical balance that exists between viral replication strategies and the host immune defenses and is thought to be a significant factor driving colony loss13.Honey bees , Toll, and Immune deficiency (Imd) innate immune response pathways in response to viral infections, though the importance of these seems to vary19. The bee genome has been demonstrated to encode the major proteins associated with these immune pathways20, and bees have been shown to utilize an antiviral RNAi response24. Interestingly, however, bees have been found to possess only about one third as many genes associated with insect immunity as fruit flies and mosquitoes25, possibly suggesting a reduced reliance on the immune response of the individual. This discrepancy may be explained by the effectiveness of social immune barriers to infection that function at the colony level or it is possible that bees also rely on homeostatic mechanisms to control damage at the level of the cell or tissue to simply tolerate infection26. Studies using the fruit fly Drosophila melanogaster, for example, have shown that Jak/STAT deficient flies are still able to resist infection with high titers of virus, despite a reduced transcriptional response to infection and an increased viral load27. Conversely, RNAi deficient flies have been observed to experience rapid mortality in response to viral infections, despite experiencing viral loads no greater than those observed in wild type controls28. Similar findings have demonstrated the importance of the heart in maintaining homeostasis during the innate immune response of both mammals and insects during a viral infection and suggest that ATP-sensitive inwardly rectifying potassium (KATP) channels play an evolutionarily conserved role in mediating this interaction between the host and the virus29.One of the primary invertebrate antiviral defense mechanisms is an evolutionarily-conserved, post-transcriptional gene silencing mechanism known as RNA interference (RNAi), which recognizes the presence of double-stranded RNA to initiate targeted RNA degradationATP channels belong to the family of inwardly rectifying potassium (Kir) channels, which are a class of potassium (K+) selective ion channels that conduct larger inward currents at membrane potentials negative to the equilibrium potential of K+ than outward currents at potentials positive to it under physiological conditions30. Kir channels mediate K+ transport across the membrane and are responsible for stabilizing the resting membrane potential of the cell and for regulating action potential duration in electrically excitable cells such as cardiac muscle30. KATP channels are heteromeric octamers composed of four pore-forming Kir channel subunits and four regulatory sulfonylurea receptor (SUR) subunits31 that are regulated by the relative level of ATP present in the cytosol and consequently provide an important link between the metabolic state of the cell and its membrane potential30. In mammals, roles for KATP channels have been described in a variety of different tissues, including heart, skeletal muscle, vascular smooth muscle, pancreas, and brain32. While much of the research investigating KATP channels focuses on their role in the sarcolemma and the cytoplasmic membrane, it is worth noting that KATP channels have also been identified in the mitochondrial membrane33. Evidence suggests that mammalian KATP channels facilitate cardiovascular tolerance to endotoxic shock by linking vasoreactivity to metabolic demand, allowing coronary smooth muscle cells to adapt to the systemic stress caused by the innate immune response to infections34. Relative to mammals, considerably less is known about the physiological role of insect Kir channels and KATP channels. The majority of research focused on insect Kir channels has been conducted using fruit flies35 and mosquitoes36, though a recent study has added bedbugs to the list37. Much of this research has focused on the role of Kir channels in Malpighian tubule function and their potential for exploitation as a novel insecticide target in Aedes and Anopheles mosquitoes40. Research related to the insect innate immune response has found that KATP channels appear to play a role in mediating the survival of Drosophila during viral infections similar to that observed in mammals29. This finding has led to the further observation that insect KATP channels have a function in modulating antiviral RNAi, presumably by facilitating tissue-specific regulation of innate immune response mechanisms by the cellular environment of the heart41.Kir channels in the honey bee or any other hymenopteran. Recently published findings have shown that KATP channel modulators can significantly alter heart rate in the honey bee42, but there is no information available about the relationship between KATP channels and the antiviral immune response of bees. Unfortunately, investigations of bee antiviral immune responses must contend with several major obstacles. One of those obstacles is that bee colonies are often covertly infected with one or more viruses at any given time, posing a challenge for researchers focused on the outcome of infection with a single virus. Another is that infectious clones for honey bee viruses have not been developed, despite the fact that numerous bee viruses have been sequenced49. Some research has been conducted using semi-purified virus preparations8, but complete removal of contaminating viruses is often impossible, making it difficult to accurately characterize infection dynamics. Recent work has proposed and tested Sindbis virus expressing enhanced green fluorescent protein as an experimental model of honey bee virus infection, using it to show that nonspecific double-stranded RNA triggers an antiviral response that controls viral infection in bees50. In this study, we demonstrate the use of a different virus, flock house virus (FHV), as another experimental model for the study of viral infections in bees. FHV belongs to the family Nodaviridae, whose members are nonenveloped, icosahedral viruses with genomes composed of two single-stranded, positive sense RNAs51. FHV has been well characterized structurally and genetically and has served as a model system for the study of host-virus interactions in a variety of arthropod species52. Here we report that FHV infects adult bees, causing rapid onset of mortality and accumulation of viral RNA. Furthermore, infection-mediated mortality can be altered by pre-exposure to KATP channel modulators. Pharmacological activation of KATP channels reduced the onset of mortality and the level of viral RNA production following FHV infection, whereas inhibition of KATP channels increased both the rate of mortality and viral replication.At this time, very little is known about the role of K7 plaque-forming units (pfu) of FHV/bee, experienced 70% mortality within the first 24 h and 100% mortality by 72 h post injection. Bees injected with 1.5 × 106 pfu of FHV/bee experienced 30% mortality within the first 24 h and 100% mortality was not observed until 144 h post injection. Bees injected with 1.5 × 105 pfu of FHV/bee or 1.5 × 104 pfu of FHV/bee were indistinguishable from vehicle controls at 24 h post injection and 100% mortality was not observed within the time period monitored during this study. Log-rank tests of the Kaplan-Meier survival curves indicated a significant difference in survival between bees challenged with 1.5 × 105 pfu of FHV/bee or more and bees challenged with vehicle . Increasing amounts of FHV RNA1 were detected in infected bees using quantitative real-time PCR (qPCR)  Fig. . These dATP channel agonist pinacidil increased survival of honey bees following infection with 1.5 × 106 pfu of FHV/bee, whereas treatment with the KATP channel antagonist tolbutamide decreased survival, relative to a untreated control  and bees treated with tolbutamide . When infected with a higher titer of virus (1.5 × 107 pfu of FHV/bee), survival in pinacidil-treated bees was somewhat less pronounced, but was still significantly increased relative to untreated bees  , mortality in tolbutamide-treated bees became more pronounced and survival was significantly decreased relative to untreated bees   Figure . When in) Figure .Figure 3ATP channel modulators are affecting viral replication, relative changes in the amount of FHV RNA1 were assessed using qPCR following treatment with pinacidil or tolbutamide  were used for all experiments. Bee colonies were maintained as previously described63 according to standard beekeeping practices using commercial hives that were housed in an apiary located at the Virginia Tech Price’s Fork Research Facility . For all laboratory experiments, frames of emerging worker brood were removed from the hive and housed in an incubator at 32 °C with a relative humidity of 50–80%. Newly emerged bees were collected from these frames over the course of 48 h and sorted into cages in groups of approximately 25 per cage. All cages were also provided with ¼ portions of a queen mandibular pheromone (QMP)-impregnated strip (Mann Lake Ltd.) to reduce stress by simulating the presence of an egg-laying queen. Bees remained housed in incubators under these conditions for the duration of all experiments.European honey bees , while untreated control groups received sucrose solution supplemented only with 1% DMSO. Preliminary testing demonstrated no significant effects of DMSO on either bee mortality or viral replication. For all experiments, bees were provided access to the same sucrose solution for the duration of the test. For all experiments lasting more than 48 h, bees were also provided ad libitum access to honey starting at 72 h.Stock solutions of tolbutamide and pinacidil (Sigma Aldrich) were initially prepared in dimethyl sulfoxide (DMSO). Cages of newly emerged bees were provided 64. Viral stocks were prepared in 10 mM Tris-HCl, pH 7.5. Infections were performed by injection  of 50.6 nl of a viral suspension into the thorax of each bee. Injection of the same volume of 10 mM Tris-HCl, pH 7.5, was used as an injection control. For all experiments involving drug treatments, bees were injected following 24 h of exposure to drug-supplemented sucrose solution as the only source of food and water.All experiments involving viral infections utilized FHV, kindly provided by Dr. Anette Schneemann , that was purified as previously described7 pfu of FHV/bee , 1.5 × 106 pfu of FHV/bee, 1.5 × 105 pfu of FHV/bee, or 1.5 × 104 pfu of FHV/bee and survival was compared to injection controls. In order to assess the effect of tolbutamide and pinacidil treatment on survival following infection with FHV, bees were treated with either drug-supplemented or unsupplemented sucrose solution and then injected with either virus or a vehicle control. Pinacidil was tested against 1.5 × 106 pfu of FHV/bee and 1.5 × 107 pfu of FHV/bee. Tolbutamide was tested against a viral challenge of 1.5 × 106 pfu of FHV/bee and 1.5 × 105 pfu of FHV/bee.Preliminary testing compared survival of bees following vehicle control injections and sham injections, in which the bee thorax was punctured by the needle without delivery of fluid, and found that neither group experienced significantly greater mortality than uninjected bees over a 7 d period. For all survival experiments, six replicates of approximately 25 bees were used for each treatment group. Survival was observed 12 h following injection and then every 24 h for 7 d. In order to assess the effect of infection with increasing titers of FHV on bee mortality, groups of bees were injected with approximately 1.5 × 106 pfu of FHV/bee. In order to assess the effect of tolbutamide and pinacidil treatment on FHV RNA expression following infection, groups of bees were fed either unsupplemented or drug-supplemented sucrose solution, as described for survival studies, and then injected with 1.5 × 106 pfu of FHV/bee. For all treatments, 6 bees were collected at each time point, dissected, and then frozen together in liquid nitrogen for pooled analysis of RNA expression. Frozen samples were maintained at −80 °C until RNA isolation was performed. The head and abdomen were removed from each insect, as PCR inhibitors have been noted in the eye65 and preliminary experiments found that inclusion of the gut also inhibited PCR. The first time point for each treatment (0 h) was collected immediately following injection to be used as a calibrator, or reference sample, and subsequent time points were collected at 6, 12, and 24 h following infection.In order to assess the level of viral replication in bees, a group of otherwise untreated bees was injected with 1.5 × 107 pfu of FHV/bee and after 24 h, surviving bees were homogenized in groups of approximately 10 individuals in 10 mM Tris-HCl, pH 7.5. Virus particles were collected from the supernatant and concentrated using a polyethylene glycol (PEG) virus precipitation kit (Biovision) according to manufacturer instructions. Groups of bees were then injected with virus isolate as described for infections with purified virus. At 0, 24, and 48 h post-injection, 6 bees were collected for pooled analysis of RNA expression, as described for viral RNA expression.In order to demonstrate infectivity, groups of otherwise untreated bees were injected with approximately 1.5 × 1066: forward 5′-AGGAATGGAAGCTTGCGGTA-3′, reverse 5′-AATTTTCATGGTGGATGGTGC-3′, and probe FAM-5′-ATGCCAACACTGTCCTTTCTGGAGGTA-3′-TAMRA. Primers and probe used for the detection of DWV RNA have also been previously described66: forward 5′-ATCAGCGCTTAGTGGAGGAA-3′, reverse 5′-TCGACAATTTTCGGACATCA-3′, and probe FAM-5′-CGCATGAACAAGTTCGGCGTT-3′-6-TAMRA.The following primers and probe were designed for the detection of FHV RNA1: forward 5′-GGACCGAAGTGCGGTGATG-3′, reverse 5′-CAGTTTTGCGGGTGGGGGG-3′, and probe FAM-5′-TGCCGCAATGAAGGATGTCT-3′-TAMRA. Primers and probe used for the detection of β-actin have been previously describedTotal RNA was isolated from each sample using TRI Reagent RT  and then 1 µg was reverse transcribed using a high capacity cDNA reverse transcription kit (Applied Biosystems). The thermal cycling parameters used for reverse transcription were 25 °C for 10 min, 37 °C for 120 min, and then 85 °C for 5 min. Gene expression was determined using qPCR on a StepOne Real-Time PCR System (Applied Biosystems). Each PCR reaction (20 µl) contained TaqMan Fast Advanced Master Mix (Applied Biosystems), 100 ng cDNA template, 1 µM forward and reverse primers, and 250 nM TaqMan probe. The thermal cycling parameters used for qPCR were 95 °C for 20 sec for enzyme activation followed by 40 cycles of denaturation at 95 °C for 1 s and annealing/extension at 60 °C for 20 s.T (ΔΔCT) method, which determines the quantity of target in a sample relative to the quantity of target in a reference sample68, was used to assess the change in FHV RNA1 expression at each time point relative to the 0 hr sample. Measurements of target gene expression were normalized using Apis mellifera β-actin as the endogenous control. Three technical replicates were analyzed for untreated bees injected with 1.5 × 106 pfu of FHV/bee and four technical replicates were analyzed for all other samples. Serial dilutions of different inputs of target and reference genes were performed to verify that efficiencies of target and reference are approximately equal. Viral RNA expression is reported as the mean fold change, measured in relative quantity units (RQ) ± RQmax/RQmin, relative to the calibrator sample for each treatment. Because the relative quantitation is exponentially related to the threshold value (CT), calculation of the statistical error results in an asymmetric distribution of the upper and lower limits, precluding direct statistical comparisons between expression levels in different treatment groups. Data analysis and calculation of RQ, RQmax, and RQmin were performed using StepOne Software v2.3 (Applied Biosystems).The comparative CAll data analysis and graph construction was performed using GraphPad Prism 7 . Survival experiment results are reported as Kaplan-Meier survival curves, displaying mean values ± standard error, with significant differences between the survival curves determined by the log-rank (Mantel-Cox) test.Supplemental Figures


Anterior cervical diskectomy and fusion (ACDF) is a well-established surgical treatment. Several types of intervertebral spacers can be used, but there is increasing evidence that PEEK cages yield insufficient fusion and thus less clinical improvement. The study aim was to assess the outcomes of single-level ACDF with an empty PEEK cage partially coated with titanium.post hoc comparison was made to closely matched patients from another similar trial treated with identically designed, empty, uncoated PEEK cages.This prospective multicenter single-arm clinical study collected follow-up data at 6, 12, and 18 months. A n = 49) seemed to have somewhat better fusion scores and significantly better pain relief at 6 M than the matched controls (n = 49), but these differences did not persist at 12 M or 18 M. Patients (with either implant) who achieved complete bony fusion had significantly better improvement of pain at 6 M and disability at 6 M and 12 M than patients that remained unfused.There were 49 of 50 patients (98%) who met the MCID of 3+ points of improvement on VAS pain or had an 18-month VAS ≤ 1. Yet even by 18 months post-op, only 40 of 50 (80%) PEEK + Ti patients achieved complete bony fusion. The PEEK + Ti group  contains supplementary material, which is available to authorized users. Anterior cervical diskectomy and fusion (ACDF) is a widely used, effective treatment for radiculopathy –3 and myBut the problem with PEEK is that bony fusion of the vertebrae occurs too slowly and remains incomplete too often when ACDF is performed with empty PEEK cages without additional instrumentation , 17–20. post hoc comparison of these patients to matched controls implanted with an uncoated but otherwise identical PEEK cage in an earlier similar trial. The present report completely replaces an earlier interim analysis on a subset of the patients reported previously in conference abstracts [The aim of this study was to investigate the radiographic and clinical outcomes of a clinical trial performing single-level ACDF using empty PEEK cages coated with titanium on the top and bottom surfaces. This paper also reports a bstracts , 26.The PIERCE-PEEK study  was approved by the Ethics commission of the Charité University Hospital on May 22, 2006. The follow-up study for the PEEK + Ti group with an identical study design was approved by the local Institutional Review Board of DRK Kliniken Berlin. Patients were permitted to join the study only if they provided written informed consent and were considered competent to do so.post hoc comparison to another group of patients, for whom similar prospective multicenter data had already been collected during the couple years prior to this study using an identically designed implant without any titanium coating, as described further below.The research was designed as a prospective, multicenter, single-arm clinical study. Enrollment took place from July 2009 until August 2012 in two hospitals in a major European city. We also decided to make a Patients were eligible for enrollment if they were age 18+, had a degenerative condition between C3 and T1 with clinical signs of radiculopathy or myelopathy, and were otherwise indicated for a single-level ACDF. Exclusion criteria were typical and have been described previously . All patAll patients received a standard single-level anterior cervical diskectomy and fusion (ACDF) with implantation of a cage (described in detail below) by one of three surgeons at our hospitals. The cages were not filled with any kind of material. Additional instrumentation was not used. Post-operative bracing was not prescribed. Further details can be found elsewhere , 28.All patients received a PEEK cage with a thin layer of titanium on the top and bottom surface where the cage makes contact with the vertebral bodies  Fig. . The titAll clinical data was collected at pre-op and at 6 months, 12 months, and 18 months post-op.X-rays were taken at every study timepoint and digitalized: anterior/posterior perspective of the neck in a neutral position and lateral perspective of the neck in flexion, neutral, and extension positions. The x-rays were evaluated by two independent clinicians: 1) one of two radiologists and 2) one of three neurosurgeons. In the few rare instances when the first two evaluators did not agree, the opinion of the others was sought, which always led to agreement.Drawing on previous guidance from the US FDA and the Spinal Interbody Research Group –31, the Pain was measured with a visual analogue scale (VAS), yielding a score of 0–10, as detailed previously .Patient functioning was assessed with the Neck Disability Index (NDI), yielding a score of 0–100, as detailed previously .The improvement on each of these measures at each study follow-up was calculated by subtracting the follow-up score from the pre-op score. The minimum clinically important differences (MCID) were considered to be at least 2.9 points of improvement for VAS and 20 points for NDI, as explained previously .The overall outcome at 18 months post-op was assessed by the surgeon using Odom’s criteria .post hoc comparison of these patients to a group of similar patients who had received a plain PEEK implant, in a separate but nearly identical clinical study (the “PIERCE-PEEK” study) [After enrollment was entirely completed, the decision was taken to make a ” study) . Except p test and a transformed Wilson score interval for the 95% CI, according to the recent recommendations and formulas from Fagerland and colleagues [Descriptive statistics were used to characterize the patient sample. Summary statistics of radiographic and clinical outcomes were graphed. The PEEK + Ti group was compared to the PEEK only comparison group, in regards to the fusion score, the change of VAS, and the change of NDI at each of the three follow-up timepoints, as well as the Odom criteria, using Mann–Whitney rank sum tests. The odds ratio of achieving complete fusion at each follow-up timepoint, depending on which implant was used, was calculated using the McNemar mid-lleagues , 34. Corp-value was assigned the meaning of a cutoff point between “statistically significant” versus “statistically non-significant” values. Instead, we leave it to the readers to interpret the results themselves, keeping in mind that a p-value of 0.05 (just for example) would basically mean that there is a 5% chance that the corresponding results could have been found by random chance of the study sampling and do not represent the real results that would be found in the complete population from which the study sample was drawn. Although we did always view results with p ≥ 0.15 as non-significant, we do not imply that results with p < 0.15 are necessarily significant. We recommend that readers carefully consider the meaning of each result reported, the meaning of each associated p-value, and the corresponding risks of making type I and type II errors .No particular The software used for statistical analysis is reported in Additional file The study sample included 27 males and 23 females. The median (range) age was 56 (22–76). The operated level was C3/C4 for 3 patients, C4/C5 for 12, C5/C6 for 19, C6/C7 for 14, and C7/T1 for 2. There was absolutely no missing data.Although the vast majority of patients achieved complete fusion by 12 and 18 months, a noteworthy minority of patients remained distributed among the other grades of radiographic fusion at all follow-ups Fig. .Fig. 3HiMost patients had moderate pain at pre-op Fig. . The majSimilarly, most patients had moderate disability at pre-op Fig. . The majThe overall surgeon-rated outcome according to Odom’s criteria was “excellent” for 13 patients, “good” for 31, “fair” for 6, and “poor” for none.There were 49 of 50 patients (98%) who either met the MCID of VAS pain improvement ≥2.9 or had an 18-month pain score ≤1. There were 41 of 50 patients (82%) who either met the MCID of NDI improvement ≥20 or had an 18-month NDI ≤10. There were 44 patients (88%) with an “excellent” or “good” Odom’s criteria. There was only one patient (2%) who did not meet any of these three aspects of success. There were 38 patients (76%) who met all three aspects.There were no revisions or further cervical operations for any of the 50 patients within the 18 month follow-up period.The comparison patients were closely matched to the cases, as described in more detail in Additional file The fusion scores appeared somewhat better in the PEEK + Ti group than in the comparison group at 6 months, but this possible difference did not persist at 12 months and was entirely gone at 18 months  but not at 12 months  or 18 months . Patients who achieved complete fusion had greater improvement of pain scores at the 6-month follow-up, but this difference disappeared at later follow-ups , at 12 months , and 18 months . Patients who achieved complete fusion had greater improvement of disability scores at the 6-month and 12-month follow-ups, but non-fused patients caught up by 18 months . The distribution of overall surgeon-rated outcomes according to Odom’s criteria was better in the patients who achieved complete fusion by 18 months than those that did not (p = 0.015).The fusion score at 18 months correlated with the surgeon-rated overall outcome according to Odom’s criteria  yields very good clinical outcomes. Almost every patient obtained clinically important relief of neck pain, and about 3 in 4 patients achieved all three clinical criteria of success. However, the radiographic outcomes for this cage remain less than desirable, implying that some patients did not obtain as much benefit from ACDF with this cage as perhaps they could have.The comparison of these two cohorts suggests that although titanium is not irrelevant, coating only the top and bottom surface remains inadequate. The PEEK + Ti group was about 2.6 times as likely to achieve fusion at 6 months than the plain PEEK group, but this difference disappeared at later timepoints. Correspondingly, the PEEK + Ti group had more improvement of pain at 6 months but not later.This lack of lasting advantage in the PEEK + Ti group is not surprising, considering that the lateral walls of the cage – both internally and externally – remain uncoated, and thus no different from the plain PEEK cage. Perhaps partial coating of the implant endplates improves the bony ongrowth at those junctures, . That might explain the better fusion rate at 6 months: bony ongrowth at the endplates might reduce motion on dynamic x-rays sooner. But measurable radiographic fusion requires bridging bony growth between the two vertebrae, across the implant, both around its external lateral surfaces and through its interior hole. If those lateral and internal surfaces remain uncoated, then the fusion across the cage will be no better than for a plain PEEK cage. So, obviously enough, the implants should be entirely coated in titanium.More broadly and importantly, this study supports the strong findings of our previous study  that acIn conclusion, ACDF is an effective treatment for radiculopathy and myelopathy. Achieving bony fusion of the vertebrae is important for maximizing the clinical improvement. The speed and frequency of achieving bony fusion is suboptimal with PEEK cages. Titanium coating appears useful, but coating only the top and bottom surfaces is inadequate. A large prospective clinical trial of PEEK cages coated entirely with titanium would be an important next step toward improving treatment.Additional file 1:Methods: Protocol for Selecting Matching Comparison Patients. (DOC 26 kb)Additional file 2:Methods: Software Used for the Statistical Analysis. (DOC 22 kb)Additional file 3:Results: Outcome of Matching Comparison Patients. (DOC 22 kb)


An efficient synthesis of dihydro-isoquinolines via a Pd–catalyzed double C–H bond activation/annulation featuring a short reaction time, high atom economy and the formation of a sterically less favoured tertiary C–N bond. via a Pd–catalyzed double C–H bond [a C(sp2)–H and a C(sp3)–H bond] activation/annulation (CHAA) reaction is presented. This methodology features a short reaction time, high atom economy (loss of H2O only) and the formation of a sterically less favoured tertiary C–N bond. This fast (30 min) and environmentally benign radical C–H activation approach has demonstrated the potential direction for the future design/development of fast and efficient C–H direct functionalization processes.An efficient synthesis of dihydro-isoquinolines  Pioneers like Yu, Gaunt, Kündig, Baudoin, Shi and Rao et al. in this area have mainly focused on two typical strategies: (1) Pd-induced oxidative addition onto an aryl halide followed by intramolecular C(sp3)–H bond activation to give cyclized product 2, see 3)–H bond activation of alkane 3 followed by the oxidative addition of an aryl or alkyl halide and reductive elimination for the introduction of alkyl or aryl substituents into the system –H bond. Herein, we report our latest discovery of the first direct double activation of C(sp2)–H and C(sp3)–H bonds for the synthesis of dihydro-isoquinolines under radical processes  or Pd(iv)-catalyzed radical processes.iii) intermediate 9 and Pd(iv) species 10 may be formed during the processes –C(sp3) bond construction via a sequential C(sp2)–H and C(sp3)–H bond activation. As described in 2)–H activation to give a Pd(ii) intermediate 12, upon the radical addition, one or two aliphatic chain(s) (depends on the oxidation state of the Pd centre) could be introduced for the further C(sp3)–H bond activation, analogous to the initial step under CMD processes. After the formation of the bicyclic system the highly reactive Pd(iii/iv) species undergoes a reductive elimination to give an intermediate Pd ring followed by further reductive elimination or work up to give the final product 16.Inspired by the proposed reactive intermediates, we envisaged a new pathway for the direct C in a combination of radical initiators , in all cases, either unreacted starting material was recovered or hydrolysis was observed for the reactions with benzamides. Even under the reaction conditions we have previously reported for the synthesis of hydroxyisoindolones, we did not observe any of the proposed products (either the directly coupled product or the CHAA product). Interestingly, however, when N-methoxyl benzamide 17a2 (10 mol%) and 10.0 equivalents of TFA in DCE at 100 °C, our proposed product 18a was isolated in 5% yield  was an excellent source for the C–H activations. TFA seemed to be crucial for this type of transformation in order to make the C(sp3)–H activation proceed while other acid or base additives did not provide any of our desired product 18b during our studies. The reaction condition screening focused on the stoichiometry of the acid as well as the screening of the solvents. We found that the acid additive is crucial for this transformation, as demonstrated in ESI Table S1.18b in 69% and 75%, respectively , our desired product 18b was isolated in 55% yield together with benzoic acid (27%) and methyl benzoate (18%). Surprisingly, no hydroxyl isodolone was detected from the 1H NMR of the crude reaction mixture which indicates that our desired reaction is more favored than the hydroxyl isoindolone formation processes. Benzamides bearing electron deficient substituents at the para-position are good substrates and the corresponding dihydro-isoquinolines 18c–18f were synthesized in good yields. Electron rich benzamides are also tolerated; the products 18g and 18a were also facilitated in good yields. Either electron rich or electron deficient substituents at the ortho-position would provide dihydro-isoquinolines, such as 18h–18j, synthesised in 54–94% yields. Reaction of a meta-substituted benzamide gave rise to the corresponding product 18k exclusively in 43% yield which consistently agreed with the selectivity that we have reported previously.N-isopropoxy benzamide was also examined and isoquinoline 18l was obtained in 75% yield. The reaction of N-methyl benzamide also works and the corresponding product 18m was successfully synthesized, albeit with a low yield (10%). The low yielding reaction is due to the low reaction conversion as most of the starting material was left without having been converted into the dihydro-isoquinoline. Unfortunately, the attempts of the synthesis of N-phenyl dihydro-isoquinoline 18n failed to provide the desired product , compared to the corresponding reaction with DTBP. The reaction with N-methyl benzamide also worked as the desired N-methyl dihydro-isoquinoline 18m was isolated in 10% yield, together with 88% unreacted starting material. Unsymmetrical tertiary alcohols are also tolerated in the reaction, while the reaction of 1,1-dimethyl propanol with benzamide 17b gave the two regio-isomers 18o and 18o′ in 63% and 8%, respectively . These results may indicate that the later C–H activation of an alcohol is more likely to proceed at the more substituted carbon centre. Unfortunately, alcohols 6a–6c failed to give the corresponding dihydro-isoquinolines, only the background radical hydrolysis products benzoic acid and/or methyl benzoate were formed.Peroxides are not the only efficient coupling partners; we were pleased to find that when the inorganic salt Kectively . However3)–H bonds. An intramolecular cyclization reaction of alkenes for the formation of five membered-lactams has been described.18p was observed, instead only unreacted starting material was recovered in 94% yield after column chromatography  totally shut down the reactivity. After heating the reaction mixture under standard conditions for one hour, no desired product was observed and only 95% of the unreacted starting material was recovered. Unfortunately, the trapping reaction using TEMPO gave us a complicated reaction mixture with 92% of the starting material recovered .19 was also successful, which may indicate that this reaction may undergo C–H activation of the directed arene first followed by a second C–H activation of the alkyl group after the addition of the radical to the Pd centre –H activation processes may be involved in the selectivity determining step.To have a better understanding of the reaction mechanism, we set up competitive KIE studies. A 1 : 1 mixture of benzamide tert-butanol-D6 were also carried out. A much higher KIE value of 4.7 : 1 compared to the KIE value 1.9 : 1 for ortho-C(sp2)–H activation (3)–H activation processes may be involved in the rate determining step.3)–H activation is comparable with the KIE value of 5.8 : 1 obtained by Baudoin3)–H activation reactions –Pd(iv) cycle for the reaction pathway. The reaction of palladacycle 19 with 1.0 equivalent of tertiary butoxide radical (0.5 equiv. of DTBP) did not provide our desired product in greater than 50% yield (ii) species ultimately into Pd(iv), which may be involved in the key reaction mechanism instead of Pd(iii) intermediate.According to our initial proposal, as well as the evidences we have collected during the mechanistic studies, we have proposed a plausible reaction mechanism based on the Pd–H activation occurred first under acidic conditions to give a palladacycle A, (iv) species B may form, which would give the active precursor C after ligand exchange of the tert-butanol with trifluoroacetate. The further reaction is involved in a CMD process for the activation of a C(sp3)–H bond to form a bicyclic intermediate, D. The final product was then formed after a reductive elimination as well as a further elimination of PdO, which had also been proposed by Daves during their studies of syn-elimination of PdO.ii) species for the next catalytic cycle.As we have shown in cycle A, . Under rvia sequential C(sp2)–H and C(sp3)–H bond activation of benzamides reacting with tertiary alcohols. These novel radical processes featured (1) high reactivity – two intermolecular unactivated C–H bonds were directly functionalized, together with a challengeable tertiary amide bond formation; (2) high efficiency – reactions were generally fast, in most of the cases reactions were completed within 30 minutes; (3) modular synthesis – the heterocyclic ring systems were constructed in a clickable fashion. Detailed mechanistic studies are still in progress.In conclusion, we have reported a novel approach for the synthesis of a series of useful dihydro-isoquinolines Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file.


Unit cell of the array consists of three vertically stacked gold micro rings separated from each other by 30 µm thick SU-8 layers. The proposed array also exhibits a filtering response in its transmittance spectrum. The characteristics are explained by plasmon hybridization method. The transmission, reflection and absorption spectra of the structure are measured and they show a good agreement with corresponding simulated results.In this article we present a three-dimensional loop Yagi-Uda array for efficient, polarization independent and directional absorption of THz radiation over a narrow frequency range (f Recently in Tera-Hertz (THz) regime (0.1–10 THz) metamaterial absorbers have attracted attention for their compactness and perfect absorption. They have potential applications in the field of non-invasive sensing, thermal and biomedical imaging, food safety, anti-reflection coatings and spectroscopy10. In recent years different narrowband13, multiband16 and broadband20 THz metamaterial absorbers have been reported. Different physical mechanisms to explain the working principle of these absorbers are, trapping waves in isotropic Mie resonators21, combination of out of phase currents and non-zero order Bragg modes22, standing wave resonances in metal-insulator-metal structure23, destructive interference of multiple reflections from stacked resonators and Perfect Electrical Conductor (PEC) background24. A THz metamaterial based on free space impedance matching through independent tuning of its electrical and magnetic resonances was reported by Hu Tao et al.25. Different microwave receiving antennae are also designed to match their effective impedance with free space impedance for efficient absorption of the radiation at a desired frequency.Metamaterials are the periodic array of subwavelength composite engineered structures that possess different exotic electromagnetic properties like negative refractive index27 and printed circuit form31. Towards higher frequency range like Infrared (IR) and optical regime, design of Yagi-Uda has been adopted from their radio frequency counterpart. Kosako et al. revisited the iconic design of Yagi Uda in nanometer scale to operate in visible frequency range32. In Near- Infrared (NIR) frequency range 3D Yagi-Uda antenna was reported by Daniel Dregely et al.33, which consists of three parallel linear dipole elements embedded in polymeric matrix. It was demonstrated that just like its microwave counterpart, 3-D Yagi-Uda nanostructure can be used as an efficient absorber albeit polarization dependent when illuminated by a NIR source. In THz regime, Ding et al. reported the simulation studies of polarization independent metamaterial broadband stop filter using a stack of identical resonators34.Yagi Uda antenna is a well established candidate to radiate directional beam when operated in transmitting mode and can also be used as a directional absorber while in receiving mode. It has been extensively studied in the frequency ranges covering microwave and millimeter waves, both in metallic wires35, which is analogous to the hybridization of wavefunctions in molecular orbital theory. This intuitive approach has been successfully applied to understand the optical properties of various complex nanostructures in IR and visible frequency range41.In this work, we present antenna comprised of a stack of three circular ring elements of decreasing radii as shown in Fig. d, Rf and Rr) of these three ring elements are chosen to be 43, 47 and 54 µm, respectively. The width (w) of each ring element is considered to be 10 µm. The complex permittivity of gold in THz regime is described by Drude model with plasma frequency ωp = 1.366e16 rad/sec and collision frequency ωc = 5.648e13 rad/sec42. The THz permittivity and loss tangent of SU-8 are taken as 2.92 and 0.055, respectively43. The periodicity of the array (Px and Py) is chosen to be 130 µm in both x and y- directions . Figure THz far field properties of the Yagi-Uda array were numerically simulated followed by the experimental verifications of simulated results. All simulations were performed using CST Microwave studio. A normal incident x-polarized plane wave is considered throughout the numerical studies  for both front and back side illumination in Fig. 7 THz forTo elucidate the far-field optical properties of the proposed Yagi-Uda structure a detailed study of plasmon hybridization scheme is presented in this section. We start our discussion with single-layer ring array. The unit cell (top view) is shown in Fig. 45. Simulated transmittance and reflectance spectra of the circular ring array for three different ring radii are shown in Fig. The lowest energy plasmon mode in a circular ring is same as that in an infinitely long cylindrical rod where the guided wavelength of the plasmon is equal to the circumference of the ring rf and Gfd in Fig. After single ring study, we studied an array of double stacked rings. The structure is fully embedded in SU-8. The vertical gap  and 1 THz (II’), respectively, as marked in Fig. 35.Higher energy intermediate plasmon The Z-components of electric field distribution at bottom surfaces of three rings at different resonant minima are shown in Fig. 46. So the Yagi-Uda array can be used as a band stop filter to suppress the transmission over a broad frequency range.As observed in the transmittance spectrum of Yagi-Uda structure  close to the frequency of 0.657 THz as shown in Fig. In summary, we have demonstrated a loop Yagi-Uda absorber in THz, which is consisting of three stacked micro rings of decreasing radii from bottom to top. We have explained its optical property using plasmon-hybridization method. It exhibits resonant directional absorbance property with absorption amplitude higher than 90%. The measured transmssion, reflection and absorption spectra are found to be well matched with the corresponding simulated results. The proposed structure can be used as a band reject filter to suppress the transmission from 0.2 to 1.1 THz.A square array of Yagi-Uda structure of area 5 mm by 5 mm was fabricated on a 1 mm thick THz-transparent fused-silica substrate. A 30 µm thick SU 8–2025 layer was spin coated on the substrate and was first soft baked at 65 °C and then at 95 °C for 3 and 5 minutes, respectively (Pre-exposure bake). Then soft baked SU8 was exposed to 365 nm wavelength UV light through a 5 mm × 5 mm window of an iron oxide mask followed by a post exposure bake at 65 °C and 95 °C for 1 and 6 minutes, respectively. Pre and post-exposure baking was performed to dry the film and to reduce internal stress in it, respectively. Sample was then immersed in SU8 developer, followed by rinsing in IPA and then dry cleaned by pressurized dry nitrogen gas. A final hard baking at 150 °C for 15 minutes was carried out to make the SU8 extremely hard. A single layer PPR (S-1813) was spin coated onto the hard baked SU8 layer and solvent was evaporated by baking it at 90 °C for 4 minutes. Image of micro-ring structures of the reflector layer are then transferred from the iron oxide mask to PPR layer by standard optical lithography technique and subsequent development of UV exposed PPR by MF-319 developer, followed by rinsing in DI water and then dry cleaned by pressurized pure nitrogen gas. After that Ti (5 nm)-Au (190 nm) was sputtered and subsequently lift-off was performed to remove the remaining resist. The whole procedure was repeated two more times to create subsequent driver and director array as shown in Fig. Transmittance and reflectance of the fabricated sample was measured using THz-Time Domain Spectroscopy (THz-TDS). THz-pump pulse was generated by dipole photoconductive antenna fabricated on a Low temperature Gallium Arsenide (LT-GaAs) substrate  by a 10 femtosecond Ti: sapphire laser beam with 80 MHz repetition rate. A Quartz beam splitter was used to simultaneously measure both transmittance and reflectance in the same set up.The transmitted and reflected THz pulse was detected by electro-optic sampling process. In this process, 800 nm optical probe pulse and transmitted (or reflected) THz signal were together impinged on a Zn-Te (Zinc-Telluride) crystal followed onto a quarter-wave plate and a Wollaston prism. After that, the probe pulse was sent to a balanced photodiode with rise time of 100 nanosecond whose output is recorded by a lock in amplifier (SR-865). The measurement was performed at room temperature. A 1 mm fused silica substrate was used as the reference for transmittance and a 0.2 µm thick gold mirror was used as the reference for reflectance. The recorded time domain signal was amplitude against the time delay between pump and probe pulses. We repeated the experiment three times and performed FFT (Fast Fourier Transform) on them. The averaged Fourier transform signal is normalized with respect to the corresponding reference samples.Supplementary Information


Malignant glioma is the most fatal of the astrocytic lineage tumors despite therapeutic advances. Men have a higher glioma incidence than women, indicating that estrogen level differences between men and women may influence glioma pathogenesis. However, the mechanism underlying the anticancer effects of estrogen has not been fully clarified and is complicated by the presence of several distinct estrogen receptor types and the identification of a growing number of estrogen receptor splice variants. Specifically, it is generally accepted that estrogen receptor alpha (ERα) functions as a tumor promoter, while estrogen receptor beta (ERβ) functions as a tumor suppressor, and the role and therapeutic significance of ERβ signaling in gliomas remains elusive. Thus, a deeper analysis of ERβ could elucidate the role of estrogens in gender-related cancer incidence. ERβ has been found to be involved in complex interactions with malignant gliomas. In addition, the prognostic value of ERβ expression in glioma patients should not be ignored when considering translating experimental findings to clinical practice. More importantly, several potential drugs consisting of selective ERβ agonists have exhibited anti-glioma activities and could further extend the therapeutic potential of ERβ-selective agonists. Here, we review the literature to clarify the anti-glioma effect of ERβ. To clarify ERβ-mediated treatment effects in malignant gliomas, this review focuses on the potential mechanisms mediated by ERβ in the intracellular signaling events in glioma cells, the prognostic value of ERβ expression in glioma patients, and various ERβ agonists that could be potential drugs with anti-glioma activities. Glioma is the most common primary malignant brain tumor and has a poor prognosis. Currently more efforts should be directed toward developing more efficacious and targeted therapeutic paradigms. A known gender bias exists in tumor development: women have a lower incidence than men, indicating protective effects of estrogen. Evidence from various sources indicates that endogenous estrogens could have beneficial effects against glioma development . First, As a steroid hormone in humans, the biological function of estrogen is primarily mediated by binding to two classical receptors: estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), which were identified and cloned in 1986 and 1996, respectively . Our undAlthough ERα and ERβ are structurally similar, their ligand binding domains actually exist sufficient differences to allow them to be selective for different ligands . Recent Given these research findings regarding the role of ERβ in preventing glioma, the protective effects of estrogen signaling cannot be ignored. The status and exact role of estrogen and ERs in glioma requires further research, and here, we review the literature regarding the roles of ERβ in malignant gliomas Table . A thoroSignificant research efforts have been directed toward clarifying the relationship between malignant glioma and ERβ and investigating nuclear ERβ and the cytoplasmic part of ERβ, which have been shown to significantly influence glioma. Dominguez et al.  showed tSareddy et al.  have expThe findings of Sareddy et al.  suggesteThe Egr-1 gene is an immediate early response gene that encodes a transcription factor that plays a regulatory role in cell growth, differentiation, and apoptosis. Kim et al.  investigERs, including ERα and ERβ, are traditionally considered transcription factors that bind to EREs in the promoter region, resulting in the regulation of gene expression . However6-guanine of DNA) function, the PI3K/AKT/mTOR signaling pathway may help to promote the expression of a series of anti-apoptotic factors and activate survival signals, which rid the cells of the cytotoxic effects caused by TMZ treatment [Temozolomide (TMZ) is a common first-line postsurgical drug used for malignant gliomas. However, the efficacy of TMZ is still poor because genetic resistance could be often observed. As mentioned above, ERβ has become an important tumor suppressor, as well as a key regulator of signal transduction in glioma cells; however, little is known regarding the role of ERβ in the chemotherapeutic response to TMZ. It is well known that PI3K/AKT/mTOR signaling plays a critical role in regulating protein synthesis, proliferation and survival and has been implicated in multiple drug resistance (MDR) in a variety of cancer types. Sveral key components of this pathway, including PI3K, AKT, epidermal growth factor receptor (EGFR) and Ras, have been observed to be frequently mutated in most tumor cells and are significantly related to the hyperactivation of this pathway . Indepenreatment , 26.Using the novel highly selective ERβ agonist liquiritigenin and TMZ-resistant U138 glioma cells, Liu et al.  suggesteTMZ treatment in conjunction with liquiritigenin has been demonstrated to be a feasible therapeutic option with several benefits . First, In vitro analyses have revealed that each ERβ isoform has distinct transcriptional activity [ERβ has been found to be a potential tumor suppressor as mentioned above. Further identification of various ERβ isoforms further increases the complexity regarding the biomedical role of ERβ . At presactivity , 29. Theactivity , 31, 32.activity , 21. HowLi et al.  have evaBatistatou et al.  for the Interestingly, Batistatou et al.  further Furthermore, Kefalopoulou et al.  for the In summary, the expression of ERβ decreased with increased astrocytoma grade and could be a good independent prognostic factor of patient prognosis. Unveiling the complex mechanism of tumor initiation and progression is a necessary condition for optimizing tumor diagnosis and evaluation and to design new treatment strategies that are tailored and individualized. Further studies at the clinical and molecular levels are needed to confirm the current findings and to examine whether these findings can be translated to clinical practice.ERβ-selective agonists are considered to be potential therapeutic agents for a wide variety of cancers –43. Theiin vitro and in vivo xenograft models of gliomas and tested its therapeutic significance using recently developed selective ERβ modulators. Their findings indicated that ERβ agonists could promote both the expression and tumor-suppressive functions of ERβ. They demonstrated that liquiritigenin significantly reduced in vivo tumor growth in a xenograft model [Recently, a great number of selective ERβ agonists have been developed and are being investigated and considered for therapeutic use . Liquirift model . Signifift model , 9, althin vitro, on a glioma U87 cell line, and in vivo on a murine xenograft model of the same tumor.Paterni et al.  investigAs mentioned above, the plant-derived ERβ agonist liquiritigenin has been proven to suppress the growth of subcutaneous glioma xenograft tumors . In addiSeveral efforts have been directed towards the development of ER-selective ligands . In partThis review focuses on the therapeutic significance of the ERβ pathway in gliomas and suggests that functional activation of the ERβ pathway may be a potential therapeutic target for gliomas. Although ERβ has been shown to have tumor-suppressive potential, more efforts should be directed toward clarifying the exact roles of ERβ in the etiology and progression of malignant glioma. Likewise, limited information regarding which promoters drive classical ER transcription in glia or which ERβ splice variants are expressed in glia and glial tumors is currently available. Further identification of certain genes that are specifically regulated by ERβ warrants further research. Because ERβ agonists are currently being tested in clinical trials and have been well tolerated with few side effects, the identification of ERβ agonists as therapeutic agents could be readily extended to clinical use, and thus, ERβ agonists could represent a novel class of drugs for treating gliomas in the near future.


We reveal that the presence of a CoO/Pt interface enables efficient transport of electrons and higher catalytic activity for PtCo NPs. These results are consistent with theoretical calculations suggesting that lower activation energy and higher exothermicity are required for the reaction at the CoO/Pt interface.Despite numerous studies, the origin of the enhanced catalytic performance of bimetallic nanoparticles (NPs) remains elusive because of the ever-changing surface structures, compositions, and oxidation states of NPs under reaction conditions. An effective strategy for obtaining critical clues for the phenomenon is real-time quantitative detection of hot electrons induced by a chemical reaction on the catalysts. Here, we investigate hot electrons excited on PtCo bimetallic NPs during H 2 oxidation by measuring the chemicurrent on a catalytic nanodiode.The real-time quantitative detection of hot electrons provides critical clues to understand the origin of the enhanced catalytic performance of bimetallic nanoparticles (NPs). Here, the authors investigate hot electrons generated on bimetallic PtCo NPs during H Extensive experimental studies demonstrate that hot electrons are crucial for explaining the kinetics of catalytic surface reactions because the transport of hot electrons facilitates the formation of a transient state in the molecules6. However, the microscopic mechanisms of hot-electron-mediated chemistry are still unclear because of the extremely fast relaxation of hot electrons via electron–electron and electron–phonon interactions1.In the field of heterogeneous catalysis, there is much interest in understanding how hot electrons, which are associated with energy dissipation and conversion processes during surface reactions, affect catalytic activity and selectivity13. The architecture of these devices allows for thequick extraction of hot electrons across the metal–semiconductor interface before thermalization, thereby providing key evidence of non-adiabatic charge transfer during surface reactions. In a previous study on Pt nanoparticle (NP)/Au/TiO2 catalytic nanodiodes, we showed that the size-dependent catalytic activity of Pt NPs was quantitatively described by the chemicurrent, which is the flow of hot electrons generated on the Pt NPs during a chemical reaction 9. Furthermore, the kinetics of H2 oxidation following the Langmuir–Hinshelwood mechanism were investigated by measuring the magnitude of the number of hot electrons at different concentrations of hydrogen using Pt NP/graphene/TiO2 nanodiodes10.Recently, metal–semiconductor catalytic nanodiodes have been developed as a powerful tool for detecting and utilizing hot electrons generated on nanocatalysts under various surface reactions16. Despite considerable focus on various catalytic reaction studies 18, there are still questions about the underlying causes of improved performance because the structure, chemical composition, and oxidation state of bimetallic NPs can change under reaction conditions21. Recently, the presence of oxide–metal interfacial sites formed by surface segregation of bimetallic NPs were specifically suggested to be responsible for increased catalytic activity23. However, the physical nature and fundamental role of oxide–metal interfaces are still elusive because of a lack of definitive evidence.In contrast with conventional monometallic NPs, bimetallic NPs have opened a new pathway that could control the electronic structure and binding energy in catalysts, resulting in superior catalytic performance2 oxidation and clarify the origin of the synergistic catalytic activity of PtCo NPs with corresponding chemicurrent values. To investigate the dynamics of hot electrons on nanocatalysts, we use catalytic NP/Au/TiO2 nanodiodes composed of stoichiometric PtCo bimetallic NPs prepared via the co-reduction method using two metals. In both chemicurrent and turnover rate measurements, we observe that the catalytic activity of the bimetallic PtCo NPs is significantly enhanced compared with monometallic Co or Pt NPs. Through X-ray photoelectron spectroscopy (XPS) analysis, transmission electron microscopy (TEM), and density-functional theory (DFT) calculations, we confirm that this improvement is attributed to the presence of a CoO/Pt interface stabilized on the PtCo NP surface under reaction conditions. By estimating the chemicurrent yield, we conclude that the catalytic properties of the bimetallic NPs are strongly governed by the oxide–metal interface, which facilitates hot electron transfer on the NPs.Herein, we report the real-time detection of hot electrons generated on bimetallic PtCo NPs during exothermic H2 nanodiodes using the Langmuir–Blodgett technique. Here, the hot electrons generated on the NPs pass across the Au/TiO2 Schottky interface if they obtain sufficient energy (1–3 eV) from the chemical reaction to transport irreversibly through the interface. Furthermore, because the thickness of the metal layer is less than the electron mean free path (<15 nm)9, direct detection of the hot electrons is possible before thermalization caused by electron–electron scattering and electron–phonon coupling. To study the dynamics of hot electrons on NPs, monolayer arrays of NPs deposited on Au/TiO2 nanodiodes were confirmed by scanning electron microscopy (SEM)  Fig. . After ft energy –3 eV froxCoy NPs with different compositions  using the co-reduction method with two precisely mixed metal precursors to study the dynamics of hot electrons on bimetallic NPs. The structures of the PtxCoy NPs were characterized by TEM, high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), and STEM-energy dispersive X-ray spectroscopy (STEM-EDS). As shown in the typical TEM images and size distribution histograms  show an average size range of 2.5–2.8 nm, which are slightly larger than those of the monometallic Pt1Co0 NPs (1.7 nm) and smaller than those of the monometallic Pt0Co1 NPs (5.1 nm). The measured d-spacings of the PtCo bimetallic NPs are less than the value of the Pt (111) plane (2.26 Å). The elemental distributions of the synthesized NPs were assessed using STEM-EDS mapping, which demonstrates that the Pt and Co atoms were randomly mixed within the lattice by forming alloys with different stoichiometric ratios of Pt to Co  in which the chemical compositions determined by both measurements were well matched with the targeted molar ratios of the Pt and Co precursors  exhibit typical (111), (200), and (220) diffraction peaks of the crystalline face-centered cubic (fcc) Pt phase, respectively, with no observable impurities of CoOx or phase-segregated metals16. The peak positions were also shifted slightly to a higher angle as more Co was incorporated into the PtCo NP, thus demonstrating the formation of bimetallic NPs with a disordered alloy phase, which agrees with the STEM-EDS mapping. The lattice parameters calculated from the (220) diffraction peak are 3.925 and 3.851 Å for Pt1Co0 and Pt3Co1, respectively, which are consistent with the reported lattice constants of Pt and Pt3Co125. However, the pure Co  NPs show the characteristic pattern indexed to the cubic Co3O4 structure26, indicating that the metallic Co NPs are easily oxidized—even at atmospheric conditions—to Co3O4, which is thermodynamically more stable than the CoO phase. The average crystallite sizes were calculated from XRD using the Debye–Scherrer equation; they are consistent with the NP sizes obtained from TEM , 00, and (2 + O2 → H2O) has been chosen for the reaction kinetic study because it is a simple and representative reaction and it is closely correlated to a variety of hydrogen-based energy systems. To investigate the flow of hot electrons induced by the exothermic reaction on the catalytic NPs, we carried out current measurements on the PtxCoy NP/Au/TiO2 catalytic nanodiodes in a H2 (15 Torr) + O2 (745 Torr) mixture as well as in pure O2 at elevated temperatures . We observed a definite deviation between the currents measured with and without catalytic H2 oxidation, indicating that the differences in magnitude of the currents were associated with hot electrons  generated on the surface of the PtxCoy NPs , exhibit a somewhat higher current than the smaller Pt1Co0 NPs (1.7 nm), even though the detection efficiency of hot electrons is higher in smaller NPs due to the shorter travel length for the hot electrons9. This implies that the enhanced chemicurrent obtained on the PtCo bimetallic NPs indeed originates from their improved catalytic properties and is beyond the size effect of the NPs.Figure xCoy NPs supported on SiO2 substrates at temperatures of 80–110 °C under identical reaction conditions  measured on the mono-layered Ptons Fig. . Here, tons Fig.  and 8. TNPs Fig. . These f3Co1, Pt1Co1, Pt1Co3), we conducted ex situ XPS and in situ TEM measurements. As shown in Fig. 3Co1 NPs consist of metallic Co and CoO and the amount of CoO species increased by 23% because of further oxidation during H2 oxidation. Unlike cobalt, regardless of the composition of the PtCo bimetallic NPs, platinum is rarely oxidized and maintains its metallic properties during the reaction , we found that additional Co atoms began to segregate after a few seconds (~14 s), grew atom-by-atom along the {111} surface of the Pt3Co1 NP (~54 s), and formed a monolayer of CoO {111}, which is identified by lattice spacing measurements  with a Fusion heating holder (Protochips inc.), enabling dynamic observation of the NP surface at atomic-scale resolution  calculations34. For our model system, the CoO island structure on a Pt (111) surface, denoted as CoO/Pt, was investigated and compared with the Pt (111) surface  and CoO/Pt interface sites  by 0.16 eV. This result can be understood by the Pt–O bond at the CoO/Pt interface not undergoing major atomic rearrangement when forming *OH, while the same Pt–O bond on the Pt (111) surface requires the transfer of *O from the hollow site to the top site to form *OH  is higher than that on the Pt (111) surface (0.79 eV), supporting the experimental results that hot electron generation is enhanced on the PtCo bimetallic NPs with a CoO/Pt interface. All these results thus point to the conclusion that local electronic perturbations caused by the presence of the CoO/Pt interface do indeed contribute to enhanced catalytic activity of the PtCo bimetallic NPs. Since it is possible that the reaction could also occur on the CoO cluster (island) itself instead of at the CoO/Pt interface, we also considered the same reactions occurring at the top sites of the island model of the cobalt monoxide. The high activation barrier (1.34 eV) indicates that H2 oxidation on the cobalt monoxide cluster itself will be unlikely  using electron beam evaporation with a titanium target and a patterned aluminum shadow mask (4 × 6 mm2). To modify the Fermi level of the TiO2 film, the wafer was annealed in air at 380 °C for 2 h, while the sheet resistance was monitored. The electrode, a 50 nm Ti layer and 150 nm Au layer, was then deposited using electron beam evaporation through a second aluminum shadow mask (5 × 5 mm2). The titanium layer is the ohmic contact with the TiO2 thin film and the Au layer comprises the nanodiode’s two ohmic electrodes. Finally, a 10 or 30 nm Au thin film was deposited through a third patterned mask (2 × 1 mm2). This produced Au/TiO2 nanodiodes with a 4 mm2 active area.The metal−semiconductor nanodiodes were fabricated as follows: First, we deposited a thin (250 nm) layer of Ti on a 500 nm SiOxCoy NPs  on the Au/TiO2 nanodiode, we used the Langmuir–Blodgett technique. The solution of PtxCoy NPs was first dispersed in water on a Langmuir–Blodgett trough  at room temperature. As the water-supported thin film layer of nanoparticles reached equilibrium, the layer was compressed by moving the mobile barrier at a rate of 15 cm2 min−1 while monitoring the surface pressure with a Wilhelmy plate. Finally, 2D arrays of PtxCoy NPs on the Au/TiO2 nanodiode were created by lifting the submerged substrate from the water. Before setting the diode into the water, the nonactive portion of the diode surface was covered with Teflon tape, and the PtxCoy NPs were left only on the active diode area  after removing the Teflon tape. The resulting monolayer arrays of NPs on the Au/TiO2 nanodiode and SiO2 substrate were confirmed by SEM, as shown in Fig. To assemble the 2D monolayer of Pt2 oxidation reaction was performed in an ultra-high vacuum batch reactor (1 L) with a base pressure of 5.0 × 10−8 Torr. The reaction chamber was evacuated and isolated with a gate valve before it was charged with 15 Torr of H2 and 745 Torr of O2 at room temperature. A catalyst sample was placed on a ceramic heater in the batch reactor, and the temperature was monitored by a thermocouple and fluctuated no more than 0.5 K. The reaction mixture was circulated continuously through the reaction line by a Metal Bellows recirculation pump at a rate of 2 L min−1. The H2 molecules were monitored as a function of reaction temperature (80−110 °C). After equilibrating for 1 h, the reaction mixture was continuously analyzed through an online gas chromatograph. The reaction mixture was separated for analysis using a DS iGC 7200 gas chromatograph equipped with a thermal conductivity detector and a 6 ft long, 1/8'' outer diameter stainless steel 80/100 mesh size column. H2O conversion was reported in terms of TOF and was calculated on the basis of product molecules of H2O produced per metal surface site per second of reaction time.The H–10 Torr. The TEM measurements were performed using a Tecnai TF30 ST at 300 kV and HRTEM, HAADF-STEM, and STEM-EDS mapping analyses were performed using a Titan cubed G2 60-300 (FEI) at 300 kV with a spherical aberration corrector (CEOS GmbH). EDS analysis was carried out along with four integrated silicon-drift EDS detectors (ChemiSTEMTM technology) at a collection solid angle of 0.7 srad. An aberration-corrected environmental TEM  operating at 300 kV with the Fusion heating holder (Protochips inc.) was used for the in situ TEM characterization. Field emission SEM was conducted using a Verios 460 SEM instrument. For electrical characterization of the nanodiodes, I–V curves and the short-circuit current were measured using a Keithley Instrumentation 2400 sourcemeter under various chemical reaction conditions. The reaction rates of the H2 oxidation reaction were measured using a gas chromatograph (DS iGC 7200) in a batch reactor system.XPS spectra were acquired using a Thermo VG Scientific Sigma Probe spectrometer equipped with an Al–Kα X-ray source (1486.3 eV) and an energy resolution of 0.5 eV full width at half maximum under ultra-high vacuum conditions of 1042. The D3-corrected43 revised Perdew–Burke–Ernzerhof exchange-correlation functional45 with the projector augmented wave method was used46, and the kinetic cutoff energy was set to 500 eV. The nudged elastic band method was employed to find a minimum energy pathway and to calculate the activation barriers48. Geometry optimization and nudged elastic band calculations were performed until the residual force on each atom was less than 0.05 eV Å−1 and 0.1 eV Å−1, respectively. K-points were sampled using a 2 × 2 × 1 Monkhorst–Pack mesh49. To represent the Pt (111) surface, 48 atoms in a (4 × 4) surface unit cell with three atomic layers was modeled with spacing more than 15 Å in the z-direction. To represent the CoO/Pt interface, we modeled periodic CoO islands on a three-layered Pt (111) slab (Supplementary Fig. 35.Spin-polarized density-functional theory calculations were performed to optimize the atomic structures and calculate the electronic energies using the Vienna Ab initio simulation package codeThe authors declare that all data supporting the findings of this study are available within the paper and its supplementary information files.Supplementary InformationDescriptions of Additional Supplementary FilesSupplementary MoviePeer Review File