Our passive cooling approach, realized using commonly available low-cost materials, could improve the performance of existing cooling systems and enable next-generation thermal management and refrigeration solutions.Demonstrations of passive daytime radiative cooling have primarily relied on complex and costly spectrally selective nanophotonic structures with high emissivity in the transparent atmospheric spectral window and high reflectivity in the solar spectrum. Here, we show a directional approach to passive radiative cooling that exploits the angular confinement of solar irradiation in the sky to achieve sub-ambient cooling during the day regardless of the emitter properties in the solar spectrum. We experimentally demonstrate this approach using a setup comprising a polished aluminum disk that reflects direct solar irradiation and a white infrared-transparent polyethylene convection cover that minimizes diffuse solar irradiation. Measurements performed around solar noon show a minimum temperature of 6 °C below ambient temperature and maximum cooling power of 45 W m Passive daytime radiative cooling presents a promising low-cost refrigeration solution but has thus far relied on specialized nanophotonic structures. Here Bhatia et al. show a directional approach that decouples solar reflectance and infrared emission to achieve superior cooling performance. Existing cooling processes primarily rely on vapor compression and fluid-cooled systems despite their complexity and high cost. Passive cooling approaches such as atmospheric radiative cooling, relying on the high transparency of earth’s atmosphere at mid-infrared (mid-IR) wavelengths, can lead to simple and low-cost refrigeration and cooling strategies that can augment existing thermal management solutions7. This radiative access to the cold upper atmosphere through the atmospheric window has been exploited since ancient times to achieve cooling below ambient temperature during the night13. However, during the day, radiative cooling solutions have to mitigate solar irradiation (on the order of 1000 W m−2), which is an order of magnitude greater than the radiative cooling potential (on the order of 100 W m−2) and can impede any cooling. Several recent studies have investigated approaches that rely on spectrally selective surfaces that minimize absorption in the solar spectrum while maximizing emission in the mid-IR wavelengths22. However, this tightly constrained problem that requires negligible absorption in the solar spectrum and maximum emission in the mid-IR necessitates specialized photonic structures that are expensive and may not be easily accessible. Furthermore, previous work on passive atmospheric radiative cooling has primarily focused on spectral selectivity to enhance cooling performance without much regard to the possibility of angular radiative control. A few studies have investigated the advantages of directional control to radiative cooling26 and have recently proposed angle-selective photonic structures28 to enhance cooling performance. While the benefit of blocking direct-solar radiation was well understood and fixed solar reflectors were used in several daytime radiative cooling experiments29, no previous demonstration has utilized the directional nature of incident solar irradiation to achieve sub-ambient radiative cooling.Passive atmospheric radiative cooling approaches take advantage of the spectral overlap of the radiative emission of terrestrial objects near ambient temperature and the transparent “atmospheric window” in the wavelength range from 8 to 13 µm−2. Using a proof-of-concept setup, we measured temperatures as low as 6 °C below ambient and maximum cooling powers of 45 W m−2 for a solar-white emitter and 30 W m−2 for a solar-black emitter around solar noon. The experimental setup fabricated using low-cost readily available materials—polished aluminum, white polyethylene sheet, and commercially available paint—exhibits the simplicity and ease of implementation of the approach.This work describes a directional approach to achieve sub-ambient passive atmospheric cooling during the day. The method takes advantage of the angular confinement of the solar flux in the sky—completely blocking radiative exchange in the narrow direct-solar direction while allowing energy transfer in other directions. We demonstrate, theoretically and experimentally, that significant cooling below ambient temperatures is possible for emitters that are reflective (white) or absorptive (black) in the solar spectrum, despite the large incident solar flux. Energy balance modeling predicts that this approach has the potential to achieve temperatures as low as 20 °C below ambient and cooling powers as high as 89 W m30. Previous studies primarily relied on spectrally engineered surfaces that maximize radiative emission in the atmospheric window, while reflecting the incident solar radiation33. However, achieving such spectral selectivity is challenging, particularly due to the large solar flux that needs to be rejected almost perfectly to prevent heating.Passive terrestrial daytime radiative cooling relies upon the spectral separation between the high atmospheric transmission at mid-IR wavelengths, coinciding with blackbody emission at ambient temperature, and solar irradiation. Figure 35. The plot also shows the angular atmospheric transmittance at a representative wavelength of 10.5 µm estimated using τatm = τ0(λ)θ1/cos10, where θ represents the zenith angle and τ0(λ) represents the atmospheric transmittance in the zenith direction. In comparison with radiance due to the sun, which is concentrated around the solar disk, atmospheric transmittance is nearly constant across all angles other than near the horizon. This angular restriction of the solar irradiation in the sky relative to the broad angular range of high atmospheric transparency in the mid-IR provides an opportunity to selectively emit to the part of sky away from the sun and achieve passive cooling.The angular confinement of solar irradiation in the sky enables a complementary approach to passive daytime radiative cooling. Figure Pcooling at a temperature T can be estimated by accounting for all contributions to the energy balance:Figure Prad, represents the power radiated by the emitter. The second term, Patm, represents the radiation emitted by the surrounding atmosphere at an ambient temperature Tamb that is absorbed by the emitter. These contributions can be evaluated by integrating the spectral directional radiance leaving or absorbed by the emitter over all wavelengths and solid angles (Ω) over the atmospheric hemisphere and excluding the solid angle subtended by the reflector (Ωrefl) for Patm, as shown in Eqs. The first term in Eq. IBB represents the spectral radiance of a blackbody, ε represents the spectral directional emittance of the emitter, εatm = 1 − τatm represents the spectral directional emittance of the atmosphere, and τcover represents the spectral directional transmittance of the cover.Here, −5 steradians (about 0.5° planar angle), and isotropic diffuse-solar radiation36. For the proposed configuration , as shown in Eq. Isolar-diffuse(λ) estimation are shown in Supplementary Note The incident solar irradiation comprises direct beam and circumsolar radiation emanating from the solar disk, equivalent to a solid angle of 6.87 × 10ion Fig. , the dirPrefl, represented by Eq. εrefl and temperature Trefl . Thus the effect of the reflector can be minimal for a highly reflective surface or if the solid angle subtended by the reflector at the emitter is small.The direct-solar reflector also emits radiation toward the emitter reducing its cooling power. The radiative contribution from the reflector toward the emitter cooling power Pcond-conv can be lumped together and quantified using an effective conductive-convective heat transfer coefficient hcond-conv as shown in Eq. In addition to the radiative contributions, conduction and convection from any support structure and surrounding air also reduces emitter cooling. These non-radiative parasitic losses λ < 2.5 μm: ε = 0) and solar-black (λ < 2.5 μm: ε = 1) emitters with perfect emission in the infrared (λ ≥ 2.5 μm: ε = 1) coupled with ideal direct-solar reflectors. The model assumes an easily available diffuse-solar cover with a typical solar reflectance of 0.8 and infrared transmittance of 0.937, and no parasitic heat gain . At the 25 °C ambient temperature, Prad = 335.9 W m−2 and Patm = 246.7 W m−2 for both the solar-white and solar-black emitters, giving a total cooling potential of 89.2 W m−2. The solar contribution depends on the magnitude of diffuse-solar radiation and emitter absorptance in the solar spectrum. Thus, for the presented case where the total Isolar-diffuse = 76 W m−2, Psolar-diffuse = 0.5 W m−2 for the solar-white emitter and Psolar-diffuse = 15.6 W m−2 for the solar-black emitter. Overall, the model shows that a solar-white emitter can have a maximum cooling power of 88.7 W m−2 and minimum temperature of 20 °C below ambient, while a solar-black emitter shows a maximum cooling power of 73.6 W m−2 and minimum temperature of 16 °C below ambient. Even higher cooling powers and lower sub-ambient temperatures are possible using a diffuse-solar cover with a higher solar reflectance and infrared transmittance. Thus we show that sub-ambient cooling is possible for a range of emitter properties using the proposed directional radiative cooling approach.The potential cooling performance of the proposed approach is predicted using an idealized model based on the radiative contributions described above. Figure We designed a proof-of-concept demonstration that obstructed direct-solar irradiation, diminished diffuse-solar irradiation, maximized emission in the atmospheric window, reduced infrared absorption, and minimized heat gain due to conduction and convection. The device Fig.  compriseThe design of the experimental setup and spectral properties of the reflector and cover allowed decoupling the solar irradiation and mid-IR emission from the emitter, enabling passive daytime cooling. Figure We performed outdoor measurements simultaneously on two devices placed next to each other, each comprising a polished aluminum direct-solar reflector, nanoporous polyethylene convection cover and painted copper emitter as described in the previous section. One device included an emitter coated with a solar-white paint while the emitter of the other device was coated with solar-black paint.  and solar reflection (by the direct-solar reflector and diffuse-solar reflecting cover), we relax the optimization constraints that can lead to significantly improved cooling performance.39, made using readily available low-cost materials.We believe this proof-of-concept demonstration is a significant first step that validates the concept of directional passive daytime radiative cooling and opens possibilities for improved device design and performance. One inherent constraint with the proposed directional approach is the need for sun position tracking. While the need for solar tracking prohibits infinite scaling of this concept, it is not necessarily limiting. Supplementary Note 28. In addition, this demonstration also proves the viability of future angular-selective photonic devices for passive daytime radiative cooling42.  attached on the back of the thin copper disk (near the center) using thermally conducting silver paste. All thermocouples were calibrated prior to application using a precise immersion-style resistance temperature detector (RTD) sensor (Omega P-M-A-1/4-3-1/2-PS-12) and a chiller (Thermo Scientific A25). The RTD sensor and thermocouples were inserted into holes drilled in an isothermal copper block, which was immersed in the chiller water bath. The RTD temperature was read using a multimeter (Keithley 2000) and the thermocouples were read using a DAQ module (Measurement Computing USB-TC) with on-board cold junction compensation sensors enclosed in an aluminum box—similar to the configuration used for outdoor measurements. The calibration result for each thermocouple was used to correct the offset error and the slope error was propagated to calculate the measurement uncertainty (approximately ±0.2 °C), represented by the x-axis error bars shown in Fig. Emitter temperatures were measured using y-axis error bars shown in Fig. 21 or at different emitter temperatures by varying the fixed heater power and allowing the emitter temperature to respond based on thermal time constant of the device18. We measured the cooling power at different emitter temperatures using PID control, which allowed us to span the range of cooling powers at different operating conditions and perform measurements in a short time span (5 min per emitter temperature) when the weather conditions stayed relatively uniform.Cooling power was determined by measuring the electrical power input into Kapton® insulated flexible heaters (Omega KHR-2/2-P) attached to the back of the copper emitters. Each heater was connected to a sourcemeter (Keithley 2425) using a four-wire configuration and the input power was regulated by PID control implemented using LabVIEW. The sourcemeter accuracy and fluctuation in measured heater power  were used to calculate the cooling power uncertainty represented by the 46 of the solar position algorithm presented by Meeus47. The solar-reflector track path was then calculated from the computed sun position, for a fixed vertical distance from the emitter, so as to block the line of sight between the emitter and the sun during the time of the experiment. A reasonable vertical distance was chosen that would ensure a sufficiently small view factor between the emitter and the solar reflector (Supplementary Note We computed the sun position (zenith and azimuth angle) relative to our experimental setup at the time and date of our experiment using an adapted versionTM, Pike Technologies).We measured the direct-hemispherical reflectance of the reflector, polyethylene cover, and absorbers using a UV-Vis-NIR spectrophotometer  with an integrating sphere  and an FTIR spectrometer  with an integrating sphere  and the global tilted irradiance (GTI) were measured by a pyrheliometer  and a pyranometer , respectively. Both sensors were mounted on a two-axis tracker (EKO STR-32G) and aligned to point to the sun during tracking. The pointing accuracy of the tracker was <0.01°. The diffuse-solar irradiance was calculated as the difference between GTI and DNI.Supplementary InformationPeer Review File


Full-dimensional excited-state dynamics simulations including explicit solvation show an unprecedented two-step intersystem crossing mechanism with electronic- and nuclear-driven components in [Re(CO)3(imidazole)(phenanthroline)]+. 3(im)(phen)]+  using extensive full-dimensional excited-state dynamics simulations in explicit aqueous solution. Contrary to what has been observed in other transition metal complexes, the transition from the singlet to triplet states occurs via a two-step process, with clearly separable electronic and nuclear-driven components with two different time scales. The initially excited electronic wave function is a “molecular spin–orbit wave packet” that evolves almost instantaneously, with an 8 fs time constant, into an approximate 25 : 75 singlet-to-triplet equilibrium. Surprisingly, this ISC process is an order of magnitude faster than it was previously documented for this and other rhenium(i) carbonyl diimine complexes from emission spectra. Simulations including explicit laser field interactions evidence that few-cycle UV laser pulses are required to follow the creation and evolution of such molecular spin–orbit wave packets. The analysis of the dynamics also reveals a retarded ISC component, with a time constant of 420 fs, which can be explained invoking intramolecular vibrational energy redistribution. The emission spectrum is shown to be characterized by ISC convoluted with internal conversion and vibrational relaxation. These results provide fundamental understanding of ultrafast intersystem crossing in transition metal complexes.Changes of molecular spin are ubiquitous in chemistry and biology. Among spin flip processes, one of the fastest is intersystem crossing (ISC) in transition metal complexes. Here, we investigate the spin relaxation dynamics and emission spectrum of [Re(CO) The fact that Re has a larger spin–orbit coupling than Ru and Fe, and nevertheless Re(i) complexes displays a slower ISC rate than related Ru and Fe ones, is intriguing. Herein, we show that the process of ISC in [Re(CO)3(im)(phen)]+ is more complicated than anticipated and assigned experimentally.In this paper, we report the first full-dimensional nonadiabatic simulation of ISC dynamics in explicit aqueous solution of the rhenium carbonyl diimine complex [Re(CO)2The nonadiabatic dynamical simulations are performed with the SHARC (surface hopping including arbitrary couplings) approachDetails of the employed computational methods are given below as well as in Sections S1–S3 and Fig. S1–S4 in the ESI.2.13(im)(phen)]+ is solvated in a 12 Å truncated octahedron box of water plus a chloride ion. In the QM/MM procedure, only the Re complex is described quantum-mechanically by time-dependent density functional theory (TD-DFT), while the water molecules and chloride ion are in the MM region. We employ the Tamm–Dancoff approximation (TDA),Initial conditions to carry out dynamical simulations were generated from 500 snapshots of classical molecular dynamics (MD) simulations described elsewhere,1 (28%), 53 in S2 (35%), 25 in S3 (17%), 25 in S4 (17%), and 5 in S5 (3%). Out of these, 100 were simulated, and 94 were analyzed (6 trajectories were corrupted by network errors); from these, 29 started in S1 (31%), 32 in S2 (34%), 17 in S3 (18%), 14 in S4 (15%), and 2 in S5 (2%).The electronic states that will be initially excited were selected stochastically based on the excitation energies and oscillator strengths in the excitation window of 2.8 eV to 3.2 eV, which corresponds approximately to the experimental excitation wave length of 400 nm.3(im)(phen)]+ were rescaled, but not the velocities of MM atoms. Similarly, in the energy-based decoherence correction3(im)(phen)]+ atoms.The excited-state dynamics simulations were carried out with the SHARC2.0 suite.Frozen-nuclei dynamics simulations were also performed using the same settings, except that nuclear motion was set to zero, and the quantum-chemical data from the first time step of each trajectory was reused in all time steps. In this case, 94 trajectories were propagated until 50 fs.2.2, with the spin-free–spin-mixed transformation matrix U. The populations were fitted with the function f(t) = Rfastet/τfast– + (1 – Rfast)et/τslow– and errors were computed by bootstrapping. The densities of state in 0 and the singlet, triplet, or active states. The simulated emission spectrum is a two-dimensional convolution of the active–ground state energy differences and the oscillator strengths, with 0.25 eV × 100 fs Gaussians. The character of the involved electronic excited states was analysed with a charge transfer analysis carried out with TheoDORE,To ease interpretation, the spin-mixed coefficients from the SHARC simulations were transformed into the spin-free populations from 2.32 envelope. The maximum field strengths were chosen to transfer about 75% of the ground state population to the excited states. Only the transition dipole moments between S0 and the excited singlet states were considered.Simulations using explicit laser fields were carried out for one frozen-nuclei trajectory. The laser pulses used a randomly chosen polarization vector, a phase of 0, a central energy of 3.3397 eV (corresponding to the energy of a bright spin-mixed state for that geometry), and a sin31 to S4 singlet states, with no population in the triplet states. This is due to the fact that before excitation the electronic wave function is a pure singlet ground state , and the instantaneous vertical excitation  does not change the spin expectation value. However, after vertical excitation the electronic wave function is not anymore an eigenstate of the total Hamiltonian; due to the strong spin–orbit couplings it can be best described as a linear combination of spin–orbit eigenstates. Such a linear combination—that might be called a “molecular spin–orbit wave packet” (see Section S5 ≈ 0.002), and the instantaneous vertical excitation  does not change the spin expectation value. However, after vertical excitation the electronic wave function is not anymore an eigenstate of the total Hamiltonian; due to the strong spin–orbit couplings it can be best described as a linear combination of spin–orbit eigenstates. Such a linear combination—that might be called a “molecular spin–orbit wave packet” (phen)]+ contradicts (phen)]+, ISC does not primarily (<25%) occur from the lowest S1 state, similar to what we found3]2+ and has been postulated in other complexes.46The different population behavior before and after 30 fs hints at a two-step process. Indeed, the total singlet and triplet populations are best fitted with a biexponential model from which two time constants are obtained, see prompt ISC is present in these simulations, despite the nuclear motion being frozen, evidences that this is a purely electronic effect.To disentangle the different contributions to ISC, we first investigate the time evolution of the electronic wave function decoupled from nuclear motion, using SHARC dynamics simulations with the full initial distribution of geometries but freezing nuclear motion. The resulting, specific and summed-up, electronic populations of the frozen dynamics are shown in prompt ISC, the retarded ISC component with τ2 = 420+350–200 fs can only be understood by considering nuclear relaxation. t = 0 fs, the singlet states S1 to S6 are primarily distributed between 2.8 eV and 3.7 eV and the triplet states T1 to T8 between 2.5 eV and 3.6 eV. According to the chosen excitation window, the active state is located between 2.8 eV and 3.2 eV within the singlet manifold. In this energy range, the triplet density is approximately three times as large as the singlet one, due to the three MS components of the triplet. At around t = 10 fs, nuclear relaxation has begun to shift all electronic states to lower energies and activate nonadiabatic transitions (IC) that move the active state density down. On the same time scale, the extremely efficient prompt ISC quickly establishes a statistical distribution with an approximately 25 : 75 (singlet : triplet) ratio. This basically reflects that ISC is so fast that the system rapidly finds a dynamic equilibrium between singlet and triplet states, where the point of equilibrium is controlled by the densities of states. At later times, as the system continues vibrationally relaxing to lower energies, eventually there is less and less singlet state density available, forcing the overall spin density to become more and more triplet-like. It is precisely this process that is responsible for the retarded temporal ISC component visible in In contrast to the purely electronic et al.prompt ISC. The fact that both simulations find this ultrafast initial decay is encouraging, showing that very distinct treatments of nuclear motion  complexes from the last decade.i) carbonyl complexes was anomalously slower than in Ru or Fe complexes and do not scale with the spin–orbit coupling of the metal center.Intriguingly, neither the 3(im)(phen)]+ (144 fs)i.e., we calculated a time-resolved emission spectrum from our trajectory data, temporally convoluted with a 100 fs Gaussian corresponding to the experiments.τem1 = 7 fs and τem2 = 154 fs. While τem1 is below the experimental time resolution, τem2 is in excellent agreement with the experimental value of 144 fs,Puzzled by the discrepancy between the experimentally assigned time constant for ISC in + rivals the ISC time scales of [Ru(bpy)3]2+ or [Fe(bpy)3]2+ complexes,One important conclusion of this finding is to realize that the simulated emission time i) carbonyl diimine complexes have predominant MLCT character—or more precisely a mixture of MLCT and LLCT, which we denote as “Re(CO)3 → phen”. As t = 0 with minor contributions coming from IL states (phen → phen), LLCT (im → phen) states, and additional small contributions from molecular orbitals that are delocalized over all ligands. One issue intensely discussed in the literature is the role of the IL states.3IL states were assigned to be populated by 11–20% and to decay to the lower 3MLCT states within 1–3 ps.3IL time evolutions gives a rise time of about 26 fs (slightly slower than prompt ISC), a maximum population of 17%, and a decay time constant of 1370 fs.Another important gain of our spin relaxation dynamics study is the elucidation of the nature of the electronic states involved. Decomposing the total electronic wave function in terms of charge transfer contributions,At this point it is fair to wonder how the dynamics simulations, which assume a δ-pulse excitation, correlate with experiments with finite-duration laser pulses. To answer this question, we have carried out three simulations with frozen nuclei including explicit laser pulses of different durations, from a width of 0.1 fs (an approximate δ-pulse) to 85 fs (phen)]+ evidence a two-step ISC process—resulting from the interaction of extremely efficient, electronically-driven ISC and nuclear relaxation—with time constants of 8 fs and 420 fs, respectively. The ultrafast ISC component quickly establishes a dynamical equilibrium between singlet and triplet states in a 25 : 75 ratio, whereas nuclear relaxation shifts the singlet–triplet ratio towards a pure triplet state on a slower time scale. These results will allow for new interpretations of spin relaxation phenomena in transition metal complexes—for example in terms of spin–orbit wave packets—which are key to design photonic materials of current interest. To observe the presented ultrafast few-fs ISC experimentally, very short—few-cycle—ultraviolet laser pulses are necessary, as the longer pulses typically employed currently (phen)]+) are longer than the equilibration between singlet and triplet states takes, making singlet–triplet transitions unobservable. This work also showcases the potential of current computational technologies to unravel spin relaxation and vibrational coupled dynamics in systems with strong relativistic effects.Contrary to the conception that nuclear relaxation is needed to drive ISC, the herein presented simulations on [Re(CO) ≈ 85 fs  in [Re(CThere are no conflicts to declare.SM carried out all parts of the simulations and data analysis, and wrote the initial manuscript draft. LG and SM conceptualized the research, interpreted the results, and wrote the final manuscript.Supplementary informationClick here for additional data file.


In this study, a surface diffraction two-dimensional (2D) grating structure was placed on the topmost layer of distributed Bragg reflectors (DBRs) for biosensing. Bloch surface wave (BSW) resonance was realized by coupling a 2D subwavelength hole-array grating and could be excited at different locations: the surface of 2D-grating layer or the inter-face between the DBR and bio-solution. Material losses in the multilayer dielectric were measured to test the robustness of this scheme. Both the surface diffraction-grating BSW (DG-BSW) and the alternative guided grating-coupled BSW (GC-BSW) configuration showed markedly enhanced angular sensitivity compared to conventional prism-coupled schematics. Exciting these modes using a grating-coupling technique appears to yield different extreme sensitivity modes with a maximum of 1190°/RIU for DG-BSW and 2255°/RIU for GC-BSW. Refractive index sensors with a high figure of merit may be realized via such compact configurations. Specially designed photonic devices represent the possibility of real-time, label-free selective detection of various chemical and biological species for a variety of medical research and environmental monitoring applications and particularly for the optical detection of miniscule quantities of molecules in highly diluted solutions –3. Optic−1) [The most popular surface wave resonance-based sensing technology is the surface plasmon resonance (SPR) method , 16 whic−1) , 18.−1 [p-polarized light and 930 nm/RIU for s-polarized light. Most 1D photonic crystal (1DPC)-based sensors utilize complicated Kretschmann prism-coupled structures to excite BSW. Few researchers have explored grating-coupled-based BSW sensors or other new designs to reduce the complexity of bulk optical components. Vijay et al. [BSW is a promising alternative to SPPs. BSW technology based on the low optical loss all-dielectric structure has higher sensitivity and adjustable field enhancement than other surface waves and can be combined with different chemical surface modification methods and optical detection mechanisms –21. Many−1 , 25. Rec−1  demonstry et al.  did repoTwo-dimensional (2D) grating devices –30 have θ (angle between the incident beam and Z-axis) and the azimuth angle φ (angle between the negative X-axis and the incident beam projection in the x–y plane) are used to describe the propagation direction of the incident light. In the numerical calculations, we used a five-period DBR (LH)5 where the L dielectrics have RI of 1.46 (SiO2 at the working wavelength of λ0 = 657 nm) and the H layers are made of TiO2 with the RI of 2.57. The RIs of both TiO2 and SiO2 over the range of 0.43 to 0.8 μm are expressed as [A schematic diagram of the surface diffraction-grating BSW configuration is shown in Fig. essed as :1\documdL = 100 nm and dH = 70 nm.The imaginary parts of the refractive indices refer to the losses in the dielectric layers. These losses include the intrinsic material absorption and the scattering losses in the incident light (3N4) was deposited on top of the DBR with an air-hole pattern [2) was inserted between the Bragg mirror and the sub-wavelength hole-array grating. The grating layer is designed to couple the propagating illuminations to the BSW mode. As described above, grating is essentially a 2D periodic array of structural features made from air holes. In the numerical simulations described below, only the physical dimensions of the grating  were adjusted to excite BSW under different illumination conditions and to optimize the reflection profiles.To fabricate the surface diffraction-grating BSW sensor, a 116-nm-thick layer of silicon nitride  layer, where the thickness of the region to be probed is 2 μm. We did not need to precisely control the thickness of the sensing layer in this case, because the outer surface of the probed region does not significantly affect BSW mode excitation. Resonance is formed as s-polarized light falls incident on the DBR through the grating at a certain angle, and multiple reflections occur at the bottom defect layer formed by the solution to be tested. The surface defect state structure alters the electromagnetic field distribution on the bottom of the DBR due to surface wave resonance, and multiple reflections in the defect layer form coherent interference. The electromagnetic field is locally enhanced and can fully act on the sample molecules to be tested.We placed a bio-solution layer with RI near 1.333 (pure water) adjacent to the outmost high refractive index : s-polarized light in both cases. Reflectivity curves of these modes as a function of angle of incidence and wavelength are shown in Fig. θ = 4.3° by incident angle interrogation. In the GC-BSW device, the resonant angle θ = 7° corresponds to an incident wavelength of 633 nm. We found that although a resonant peak with higher quality factor Q (>103) value can be obtained by optimizing the device parameters, the wavelength sensitivity and angle sensitivity of the BSW sensor reached only about 100 nm/RIU and 280°/RIU under non-azimuthal illuminations. Our 3D RCWA simulations are consistent with the literature [φ, accordingly.We designed optimized BSW structures under the two sets of sensing conditions as shown Figs. terature . We tookθ = 7° and φ = 10° is shown in Fig. θ = 7.6° and φ = 12°. Considering the difficulty of small angle monitoring, we chose a relatively large angle to couple the BSW. The resonance peak is insensitive to polar angle changes but very sensitive to azimuthal angle changes. We calculated the electric field distribution of the sample point  to recognize the resonance  of the resonant dip and the dip peak height. As shown in Fig. θ increased. The resonance peak height increased as resonance peak FWHM decreased. At a larger polar angle, the BSW resonance shifted toward a larger azimuthal angle due to the wave vector matching effect.Figure H only slightly affects the resonance characteristics; they introduced an extinction κL = 10−4 to the low index layers calculated via transfer matrix method (TMM). Lossy materials are necessary to observe a dip in the reflectance spectrum [Lossless materials  are assumed in most numerical simulations , 25, 30.spectrum .2 materials can excite the BSW dip peak in the reflection spectrum. Losses in the DBR degrade the BSW line shape obtained in the lossless case. We analyzed the perturbation effect induced by non-zero values for κ on the resonances. In the DG-BSW case, the FWHM of the resonances first decreased and then increased as the extinction coefficient increased from 0 to 10−3, while the resonance depth did the opposite. We achieved the optimal BSW resonance line shape when the extinction coefficient κ reached 10−4. The resonance quickly dropped off as the coefficients further increased (κH = 10−2). In the GC-BSW configuration, the line width increased slowly as κH increased as did the BSW resonance peak value. The resonance dip grew wider as energy losses within the biosensor increased.To study the influence of loss, we assessed the azimuthal reflection spectra from DG-BSW and GC-BSW structures Figs.  and 3 wi2 materials yield optimal BSW resonance. When considering loss, an imaginary part as large as 10−3 can suppress the amplitude of the reflection and the Q of the resonance without affecting the position of the peak. Our simulations also showed that extinction coefficient values play a vital role in determining the optimum compromise between the depth and width  of BSW resonance.Our results suggest that lossless TiOΔφ is the change in azimuthal angle and Δnbio is the change in sensing layer refractive index. The reflectivity curves as a function of azimuthal angle for different biomolecule values are shown in Fig. λ0) and incident angle (θ) are fixed at 657 nm and 4.3° respectively  causes the azimuthal angular shift between the resonance peaks to grow larger at small azimuthal angles.The primary goal of this study was to establish a design schematic for label-free sensing platforms based on a 2D grating to excite BSWs, so we continue to explore sensing locations in order to optimize and enhance its performance as a RI sensor. RI biosensors are generally designed to detect small refractive index modulations caused by variations in biomolecule concentration ratios. We thus consider the azimuthal sensitivity  and FWHM (red bar) as shown in Fig. Figure of merit (FOM)  is anothIt is worth noting that the sensitivity of both of the BSW configurations we tested is one order of magnitude higher than that of the conventional prism-based scheme (see Table N = 5) to realize high-sensitivity BSW resonances with low side-bands. A surface DG-BSW configuration and alternative guided GC-BSW schematic were designed based on RCWA methodology. A theoretical sensitivity of 2255°/RIU was achieved with a small polar angle of the illumination (< 10°) and azimuthal angle sweeps around the same values. Angular sensitivity was one order higher than that of sensors based on prism-coupled polar illuminations . The optimized GC-BSW sensor showed a particularly large increase in sensitivity (twofold) and narrower BSW resonance compared to the DG-BSW biosensor. Both 2D-grating-coupled sensor platforms tested in this study show low quality factor compared to traditional BSW RI sensors, but they may be enhanced by tuning the period (Λ), hole radius (r), and thickness (h).In this study, we explored surface diffraction 2D grating configurations and sensing applications. We built a multilayer dielectric heterostructure from subwavelength hole-array grating and distributed Bragg reflection (DBR) with few periods (The proposed schemes for exciting Bloch surface waves, DG-BSW and GC-BSW, represent new class compact configurations for highly sensitive biosensing and may afford a valuable opportunity to engineer nanoscale “lab-on-chip” technologies in the future.


Cardiovascular disease is a leading cause of death in zoo-housed great apes, accounting for 41% of adult gorilla death in North American zoological institutions. Obtaining a timely and accurate diagnosis of cardiovascular disease in gorillas is challenging, relying on echocardiography which generally requires anesthetic medications that may confound findings and can cause severe side effects in cardiovascularly compromised animals. The measurement of brain natriuretic peptide (BNP) has emerged as a modality of interest in the diagnosis, prognosis and treatment of human patients with heart failure. This study evaluated records for 116 zoo-housed gorillas to determine relationships of BNP with cardiovascular disease. Elevations of BNP levels correlated with the presence of visible echocardiographic abnormalities, as well as reported clinical signs in affected gorillas. Levels of BNP greater 150 pb/mL should alert the clinician to the presence of myocardial strain and volume overload, warranting medical evaluation and intervention. Cardiovascular disease (CVD) is one of the leading causes of death in both humans and great apes –5. WhileGorilla gorilla gorilla) deaths in North American zoological institutions are due to fibrosing cardiomyopathy [Forty-one percent of captive adult western lowland gorilla  spreadsheets. Whole blood and plasma samples in EDTA taken at the time of evaluation were submitted to the Smithsonian’s National Zoological Park along with the standard data collection sheet. All samples were processed within 48 hours of sampling on the Triage BNP test machine  according to manufacturer recommendations. The amount of BNP present in the sample were displayed as a number in pg/mL. For the purpose of statistical analysis, readings that read as “< 5” were converted to the value of “4”, and readings that read as “> 5,000” were converted to the value of “5,000”.All descriptive statistics and analyses were run on SPSS V20 and MathCad 2013. Descriptive statistics were calculated for all variables over all gorillas and subgroups of sex and health. Exploratory analyses were examined by grouping but were not useful because subjective groupings based upon medical diagnoses proved more reliable. Assumptions were tested for each test of hypothesis and variance-stabilizing transformations were applied using natural logs and Box-Cox methodology accordingly. When correlations were computed they were Pearson’s for continuous measures and Spearman’s was used to test measures with two or more categories (dichotomous or polychotomous) measures. General linear models were developed using factors  as well as covariates (age) and terms for nonlinearity if necessary. Since age was highly correlated with health status, age was used as a covariate in the model to increase precision of the model design. This procedure statistically removes from the model that part of the variability that is predictable from age alone. Canonical discriminant analyses were modeled when required as a natural extension of regression analysis for predicting group membership. Post hoc classification tables were based upon Fisher’s classification functions.Records were analyzed for 116 zoo-housed gorillas 10 years of age and over, 51 females and 65 males. Gorillas were assigned into groups based upon health assessment: Group 1 (n = 85) consisted of gorillas that were apparently healthy; Group 2 (n = 9) contained gorillas demonstrating clinical signs consistent with cardiovascular decompensation including but not limited to coughing, lethargy, exercise intolerance, social withdrawal, dyspnea, or grabbing at the chest; and Group 3 (n = 22) contained gorillas that were currently asymptomatic, and were undergoing medical management for CVD based upon diagnosis via previous echocardiographic examination.Results of BNP testing from individual animals in these groups were examined through canonical discriminant functions . The meafemale = 1.489, SDmale = 1.642) was not significant (p = 0.473). A pooled variance t-test was used to examine the difference between male (3.632) and female (2.910) lnBNP means in each group and was significant (p = 0.016). Male BNP values showed significant positive correlation with age  and LVIDd , and negative correlation with EF  but not health status . Female BNP values showed no correlation with LVIDd  but significant negative correlation with EF  and positive correlation with age  and health status . When examined by health status the Group 1 BNP results did not correlate with LVIDd, EF or with age. Group 2 BNP results likewise had no correlates. Group 3 correlated BNP values with age  and LVPWd .Correlations of BNP and cardiac measurements were significant and positive for age  and LVIDd , while the correlation of EF and BNP was significant and negative . Levene’s test by sex  histogram plots with log transformation applied for a) all gorillas included in the study (n = 116); b) gorillas assigned a health status of “1” (n = 85); c) gorillas assigned a health status of “2” (n = 9); and d) gorillas assigned a health status of “3” (n = 22).(ZIP)Click here for additional data file.S2 FigHistogram plots with normal curves applied depicting for all gorillas: a) age (years); b) interventricular septal end diastole ; c) left ventricular internal diameter end diastole ; d) left ventricular posterior wall diastole ; and e) ejection fraction .(ZIP)Click here for additional data file.S3 FigHistogram plots with normal curves applied depicting gorillas assigned a health status of “1”: a) age (years); b) interventricular septal end diastole ; c) left ventricular internal diameter end diastole ; d) left ventricular posterior wall diastole ; and e) ejection fraction .(ZIP)Click here for additional data file.S4 FigHistogram plots with normal curves applied depicting gorillas assigned a health status of “2”: a) age (years); b) interventricular septal end diastole–IVSd (cm); c) left ventricular internal diameter end diastole–LVIDd (cm); d) left ventricular posterior wall diastole–LVPWd (cm); and e) ejection fraction–EF (%).(ZIP)Click here for additional data file.S5 FigHistogram plots with normal curves applied depicting gorillas assigned a health status of “3”: a) age (years); b) interventricular septal end diastole–IVSd (cm); c) left ventricular internal diameter end diastole–LVIDd (cm); d) left ventricular posterior wall diastole–LVPWd (cm); and e) ejection fraction–EF (%).(ZIP)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


During their follow-up period, patients with < 3 interventions had a significantly better renal function than those with ≥ 3 surgical interventions (p = 0.04). Additionally, linear regression analysis showed that eGFR was demonstrated to decline with increasing numbers of stone episodes (r2 = 0.169). It was also noted that patients who began early medical management remained stone free during follow-up compared to those who had medical management after ≥ 2 stone episodes, of whom all had a recurrent episode. Our long-term longitudinal study of cystine stone formers highlights that patients who are stone free and receive early metabolic stone screening and medical management after their initial presentation have the lowest recurrence rates and tend to preserve their renal function. Hence, prompt referral for metabolic assessment, and the stone and fragments entirely removed (SaFER) principles are key to preventing stone episodes and improving long-term function.There is a lack of studies looking at the longitudinal follow-up of patients with cystine stones. We wanted to assess the journey of cystinuric patients through our specialist metabolic stone clinic to improve the understanding of episodes, interventions and current outcomes in this patient cohort. After ethical approval, all patients who attended our metabolic stone clinic from 1994 to 2014 with at least one cystine stone episode were included in our study. Data were retrospectively analysed for patient demographics, stone episodes or intervention, clinical parameters and patient compliance. Over a period of 21 years, 16 patients with a median age of 15.5 years underwent a mean follow-up of 8.6 years (1–21 years). The mean number of surgical interventions was 3.1 (1–8/patient), but patients who were stone free after their first treatment had lower recurrences ( Despite constituting only 1% of adult and 6–10% of paediatric urinary tract calculi , patient0,+AT) of the transepithelial sodium-independent antiporter in the renal proximal tubules, causing impaired reuptake of the dibasic amino acids cystine, ornithine, lysine and arginine (COLA) [Ka of 8.3 [Cystine stones form as the result of defects in one or both of the subunits (rBAT and be (COLA) –8. Whilea of 8.3 , 8. This0,+AT and mutations in this gene follow an inheritance pattern on autosomal dominance with incomplete penetrance [Dysfunction of this transport mechanism has been attributed to mutations in solute carrier (SLC) genes SLC3A1 and SLC7A9. SLC3A1 at locus 2p16.3 codes for rBAT and mutations in this gene follow an inheritance pattern of autosomal recessive with high penetrance . To datenetrance . To datenetrance . PatientIn recent years, much of the published research around cystinuria has focused on the genetic diversity of this condition . Due to n = 16). Electronic patient records were accessed and both clinic letters and investigation results were reviewed.Following ethical approval  for long-term longitudinal follow-up of cystine stone patients, all patients between the ages of 5 and 90 years presenting to our University teaching hospital with at least one cystine stone episode over a period of 21 years between 1994 and 2014 were retrospectively identified , shock wave lithotripsy (SWL) or ureteroscopy (URS). A second spread sheet was created on the database to provide in-depth analysis of each patient consultation with our Consultant Chemical Pathologist in the Metabolic Stones Clinic; this consisted of a further 18 categories including body mass index (BMI), salt and fluid intakes, urinary pH, urinary cystine:creatinine ratio, estimated glomerular filtration rate (eGFR) and patient medications.Patients were stratified into a number of groups to analyse the full patient journey; this included those who were stone free after their first treatment versus those who required multiple treatments to be stone free, and those who were seen in the specialist Medical Stones Clinic after their first stone episode versus those who were seen after more than one stone episode . The stone-free rate (SFR) was ascertained on USS in 13 patients and CT scan in 3 patients.t test, Mann–Whitney U test and Pearson’s Chi-square test were used to check for statistical significance where appropriate.Analysis of the results was undertaken in both Microsoft Excel and IBM SPSS version 22. Descriptive statistics were calculated on Excel and confirmed on SPSS to ensure accuracy. The Shapiro–Wilk test for normality was used where necessary to check for parametric/non-parametric data. Independent samples Sixteen patients underwent mean follow-up of 8.6 years range 1–21 years). Female:male ratio was 1:1. The median age at diagnosis of cystinuria was 15.5 years   initially presented to their general practitioner, with just under half of those (4/9) being admitted to the emergency department the same day. Only one patient attended the Emergency Department as their initial presentation. The ratio of patients who lived closer than 5 miles from our centre to patients who lived further than 5 miles away was 1:1.Information on the presenting complaint was available for all patients Fig. ; four paThe surgical treatment in these patients included PCNL in nine patients of which four had PCNL as the solo procedure, URS in six patients of which t had URS as the solo procedure, with most patients having a combination of PCNL, SWL or URS procedure. While SWL was used as a sandwich therapy, it was not used as a solo treatment in any patient. The medical treatment was in form of D-Penicillamine (500 mg twice a day) in four patients, potassium citrate (10 ml three times a day) in five patients and a combination of them in seven patients.p = 0.091). The mean number of interventions in group I was also lower (2.75/patient compared to 4.00/patient in group II) (p = 0.272). One patient from the stone-free group underwent nephrectomy due to extremely poor renal function and a heavy stone burden at their initial presentation.The mean number of surgical interventions per patient was 3.1 (range 1–8/patient) Fig. ; group IPatients were also stratified into two groups according to recurrence status. Ten patients (62.5%) suffered recurrence during the follow-up period. All patients who had a recurrent episode were found to have at least one stone in the same kidney as the initial stone had been. There was no significant difference observed between the number of patients with diagnosed chronic kidney disease and their recurrence status in our cohort.2) compared to patients who underwent ≥ 3 surgical interventions (eGFR − 63.1 mL/min/1.73 m2) over the follow-up period (p = 0.04). Additionally, linear regression analysis showed that eGFR was demonstrated to decline with increasing numbers of stone episodes (r2 = 0.169)  were available for 12/16 patients due to some patient’s data being documented prior to the introduction of electronic patient records. The mean eGFR was significantly better in patients with < 3 interventions (eGFR − 87.6 mL/min/1.73 m69) Fig. .n = 10), six (60.0%) were categorised as overweight or obese. Nine patients had both BMI and urinary pH data on record; using the last recorded urinary pH and BMI measurements, those with a BMI > 25 were more likely to have a urinary pH of < 7 (p = 0.016). There was no correlation of note between a BMI in the overweight/obese categories and reduced renal function.Data on detailed metabolic evaluation of our cystinuric cohort were available for 12 patients. The mean number of appointments in the Metabolic Stones Clinic per patient per year was 2.02 (SD 0.71). Of those who had BMI data recorded in the clinic (Patient compliance was assessed from their urine pH measurements and fluid intake. While the mean urine pH increased from 6.6 (5.2–7.4) to 6.9 (5.4–7.8), the fluid intake was between 3 and 3.5 L in seven patients, 3.5–4 L in five patients, 4.1–4.5 L in three patients and over 4.6 L in one patient. Patients were divided into two groups based on when they started prophylactic treatment to prevent stone recurrence (group III and group IV); group III began appropriate prophylaxis after their first episode, whereas group IV were seen after ≥ 2 stone episodes  undergoing tradition stone treatment  compared to 17 patients treated with minimally invasive treatments , fewer patients in the modern era underwent nephrectomy or deterioration of renal function with these minimally invasive treatments [The strength of our study is the comprehensive medical stone database and the journey of the cystinuric patients, which confirm multiple interventions they need in their treatment. However, with an early diagnosis, medical management and better compliance these patients have reduced episodes of surgical intervention and preserve their renal function in the long term. However, our sample size is small in keeping with the disease prevalence and this has been recognised in other studies –17. As iThe effect on Quality of life (QoL) and the cost of treatment were not calculated in this study . A recenConsidering all of the studies on cystine stones are retrospective series from individual hospitals, perhaps there is merit in multicentre studies to get meaningful data. Similarly, as each study is from a different unit across the world, there is a lack of standardisation of data collection and reporting process. More must be done to assess patient compliance to medical therapy and ways to improve it . SimilarOur long-term longitudinal study of cystine stone formers highlights that patients who are stone free and receive early metabolic stone screening and medical management after their initial presentation have the lowest recurrence rates and tend to preserve their renal function. Hence, prompt referral for metabolic assessment, early multidisciplinary input and the stone and fragments entirely removed (SaFER) principles are keys to preventing stone episodes and improving long-term function in this cohort of patients.


In mammals, fine-tuned regulation of gene expression leads to transcription initiation from diverse transcription start sites (TSSs) and multiple core promoters. Although polysome association is a critical step in translation, whether polysome selectively uses TSSs and core promoters and how this could impact translation remains elusive.In this study, we used CAGE followed by deep sequencing to globally profile the transcript 5′ isoforms in the translatome and transcriptome of human HEK293 cells at single-nucleotide resolution. By comparing the two profiles, we identified the 5′ isoforms preferentially used in translatome and revealed a widespread selective usage of TSSs (32.0%) and core promoters (48.7%) by polysome. We discovered the transcription initiation patterns and the sequence characteristics that were highly correlated with polysome selection. We further identified 5804 genes significantly enriched or depleted in translatome and showed that polysome selection was an important contributing factor to the abundance of related gene products. Moreover, after comparison with public transcriptome CAGE data from 180 human tissues and primary cells, we raised a question on whether it is a widely adopted mechanism to regulate translation efficiency by changing the transcription initiation sites on the transcription level in cells of different conditions.Using HEK293 cells as a model, we delineated an indirect selection toward TSSs and core promoters by the translation machinery. Our findings lend additional evidence for a much closer coordination between transcription and translation, warranting future translatome studies in more cell types and conditions to develop a more intricate regulatory model for gene expression.The online version of this article (10.1186/s12864-019-5650-0) contains supplementary material, which is available to authorized users. The flow of genetic information is tightly controlled at multiple levels to maintain proper phenotypes and achieve cellular fitness. The regulation of transcription, the first step in gene expression, has been extensively studied and its complexity has been elaborated mostly owing to highly efficient next-generation sequencing techniques –3. BesidThe noncoding part of mRNA, including the 5′ UTR (with the 5′ cap), the 3′ UTR and the poly(A) tail, is responsible for most translational regulation on gene expression. The 5′ UTR is of special importance to translational initiation where protein synthesis is principally regulated: during the initiation step in eukaryotes, eukaryotic initiation factors recruit the small ribosomal subunit 40S) to form a pre-initiation complex that scans the 5′ UTR to locate the start codon, after which the initiation factors are released and the large ribosomal subunit (60S) is recruited to form the elongation-competent 80S ribosome . Several0S to forCap Analysis of Gene Expression (CAGE) is a powerful method widely used to profile the 5′ ends in organisms like human, fly and yeast –3, 15–18In this study, we employed CAGE followed by deep sequencing to globally profile the transcript 5′ isoforms in the translatome of human HEK293 cells at single-nucleotide resolution. This allowed us to precisely annotate the 5′ ends, portray the 5′ end distributions, define the 5′ UTR and identify core promoters used by polysome. By comparing them with the counterparts from HEK293’s transcriptome, we revealed selective usage of the TSS-derived 5′ ends by polysome, thus delineating an indirect selection toward TSSs and core promoters by the translation machinery. In addition, quantitative measurement of transcript abundance with CAGE allowed us to investigate the transcript enrichment after polysome selection, enabling the identification of highly enriched or depleted gene products in translatome. All these differences between transcriptome and translatome highlight the important roles of polysome in regulating gene expression, the interplay between transcription and translation and the necessity of developing a more intricate model to explain the underlining mechanisms.A flowchart of our work before data analysis is shown in Fig. p-value < 0.001 by proportion test), suggesting that transcripts with canonical ORFs were more likely to be translated. We identified a considerable fraction of TCs from intron and the coding sequence (CDS) in transcriptome (similar to the findings in human . B. B31]. Bo groups . Furtherel motif . ImportaA could generate transcripts from n different core promoters (n is determined by GRCh37 annotations from Ensembl), we used {p1, p2,  … , pn} and {t1, t2,  … , tn} to denote the abundance of core promoter-derived TCs in translatome and transcriptome, respectively. We first defined gene A’s abundance in translatome (Ep) and transcriptome (Et) as follows:Suppose a gene Sfc) as follows to measure polysome preference toward genes:We then defined a fold-change score . We then looked into the top 50 translatome-enriched genes  ranked by the p-values (Table Sfc < 0.5) enriched in the RNA binding motif containing genes , included no histone or ribosomal genes. Considering that histones and ribosomal proteins are highly abundant in cells and polysome association is a prerequisite for translation ; (2) Sdu = 0 when gene A only uses 1 core promoter , or uses ≥2 core promoters  but the core promoter-derived TCs account for the same proportions between translatome and transcriptome ; (3) Sdu = 1 when gene A only uses TCs (in translatome) that are not detected in transcriptome by the Illumina sequencing. For simplicity, we only calculated Sdu for genes using at least 2 core promoters  in HEK293 cell line. Importantly, we found that, the correlation between Sdu and Sfc is very low (Pearson correlation R = -0.04), demonstrating that Sdu give additional information independent of Sfc. We identified 62 genes with Sdu > 0.5 and further analysis of them showed that they were enriched in the myocardin gene family . By contrast, the top 62 genes with smallest Sdu were only enriched in the gene families of histones and ribosomal proteins , suggesting that differential usage of core promoters was very rare for histone and ribosomal genes in HEK293 cell line. Here we listed > 4700 genes (including the aforementioned 62 × 2 genes) with their Sdu scores in Additional file This equation applies only when gene In this work, we use CAGE followed by deep sequencing to systematically compare the transcript 5′ ends between the translatome and transcriptome of human HEK293 cells. The revealed preferential usage of many 5′ ends by polysome shows that, after transcriptional selection of TSS and core promoters, the translation machinery again makes such selection. This comparison leads to the identification of highly selected TSSs, core promoters and gene products in translatome. It also gives rise to the transcription initiation patterns and the sequence characteristics highly correlated with polysome selection. These findings delineate an indirect selection toward TSSs and core promoters by the translation machinery, emphasizing closer than expected interplay between transcription and translation.2.HEK293 cells were cultured in Dulbecco’s Minimal Essential Medium  supplemented with 10% FBS (GIBCO #10099–141), 100 units/ml penicillin, 100μg/ml streptomycin (GIBCO #15140–122) and 2 mM L-glutamine (Sigma) at 37 °C and 5% COPolysome fraction is isolated by 10–50% sucrose gradient using the method from Bor et al. (2006) with minor modifications . In brieThe cells without cycloheximide treating were lysed with TRIzol reagent  and total RNA was extracted following TRIzol protocol. The polysome-associated RNA was also extracted using TRIzol with the same method.Two 27 bp-tagged deep sequencing libraries were prepared using the methods described in Valen et al. (2009) and Takahashi et al. (2012) , 36. In Raw sequencing data were first processed using fqtrim (version 0.94) to remove the 3′ sequencing adaptor, 5′ barcodes (CTTCAGCAG and GATCAGCAG for translatome and transcriptome RNA library respectively) and low-quality reads (with parameters -q 20 -m 1) , 27. Rean tags were observed in a tag set of (n-1) × 27 nt length follows a Poisson distribution with N is the human genome length and T is the number of uniquely mapped reads). Based on this λ, it is obvious that the probability of n ≥ 4 is less than 0.001, which corresponds to the p-value. Therefore, the above two steps guaranteed that the identified tag clusters had significant p-values and were not background noise.Tag clusters were identified with the following two steps. First, tags that overlapped on the same strand were grouped into a tag set. Second, any tag set with at least four tags were defined as a tag cluster. Suppose tags randomly distributed on the human genome were background noise, the probability Different genomic features could overlap and the positions of TCs could be situated within two or more features at the same time. When this happened, we used the method described in Ref.  to assigi) a TC was classified into SP class if the distance between the 25th and 75th percentile of its tag positions was < 4 nt, or the distance between the 15th and 85th percentile was < 6 nt; (ii) if a TC did not meet (i) but the ratio between its highest peak and second highest peak was > 2 and the highest peak accounted for > 20% of all tags in it, the TC was classified into DP class; (iii) If distance between any two consecutive peaks  was > 5 nt and the TC was in neither SP nor DP class, it was classified into MP class; (iv) if a TC did not meet (i), (ii) or (iii), the TC was classified into BP class.We classified TCs (with ≥100 tags) into four shape classes with a method similar to those from previous studies , 16. BriIn this study, the expressed transcripts were defined as those with at least one TC identified within the 5′ UTR or the 100 nt upstream region. The expressed genes were defined as those with at least one annotated transcript expressed in transcriptome. To make comparison between samples, RPM was used to normalize the read number of each cluster, which was defined as follows:http://www.r-project.org/). In the case of multiple hypothesis testing, we used BH method to correct p-values (unless otherwise specified)  [Statistical analysis (including the hypothesis testing) was performed with the R language (ecified) . Sequenc motifs] –33. Mult motifs] –43.http://fantom.gsc.riken.jp/5/datafiles/latest/), including the TCs and the representative TSSs for 180 human tissues and primary cells  [All CAGE data from the FANTOM5 project were downloaded from the FANTOM website (es.org/) .Sfc > 2); (2) picked top 100 genes from the unchanged genes ; (3) picked the genes ranked from 101th to 200th from the translatome-depleted genes . This way, we obtained three groups consisting of 100 genes each with similar average RPM in transcriptome . We retrieved protein abundance data  in mouse fibroblasts (NIH3T3 cell line) from Ref. [We picked three groups of genes with the following criteria: (1) picked top 100 genes from the translatome-enriched genes  under accession number E-MTAB-7382.Raw sequencing data used in this work are available in the ArrayExpress database Additional file 2:Figure S2. Typical examples for the 4 TC shape classes. SP class (A) are characterized by a sharp peak that stands for the majority of tags in a TC. BP class (D) do not have any peak much stronger than the others in a TC. The 5’ end distributions in DP (B) and MP (C) classes are somewhere between SP and BP classes (refer to Methods for more details). The TC information  are placed on top of each example. (PNG 336 kb)Additional file 3:Table S1. TCs with changed 5’ end distributions between translatome and transcriptome. “Chr” stands for “chromosome”. “TC start” and “TC end” gives the genomic range of TCs on the human genome. “Gene Symbol” shows the genes where TCs are located. “Source” shows where TCs come from. P-values are calculated with KS test and adjusted with BH method. Any two TCs in sequential rows from Translatome and transcriptome correspond to the same core promoter and thus have the same p-value. (XLSX 268 kb)Additional file 4:Table S2. Length difference of the 5’ UTR between translatome and transcriptome. “Length Difference” shows the difference in length of the 5’ UTR. “Length Status” shows whether the length of the 5’UTR in translatome is the same as, or shorter than, or longer than that in transcriptome. The definitions of the other column names are the same as in Additional file Additional file 5:Table S3. Usage frequency of HEK293-derived representative TSSs in 180 human samples. All 3248 representative TSSs are from HEK293 translatome. In the table, “1” means the representative TSS (row name) is also used as representative TSS in the sample (column name). The name of each representative TSS contains the information of chromosome, strand and genomic position. (XLSX 1773 kb)Additional file 6:Table S4. TCs with significantly changed RPM between translatome and transcriptome. “z-score”, “p-value” and “q-value” are calculated with the R package of DEGseq. TCs are ranked by the q-values. (XLSX 2126 kb)Additional file 7:Calculation of TC fold change (FC) with polysome-free RNA instead of total RNA. (DOCX 13 kb)Additional file 8:Table S5. Comparison of genes’ abundance between translatome and transcriptome. “Ep”, “Et” and “Sfc” are all defined in the main text. “p-value” and “q-value” are calculated with the R package of DEGseq. Genes are ranked by the q-value. Genes with too few reads to calculated the p-values are removed from this table. (XLSX 925 kb)Additional file 9:Table S6. Differential usage of core promoters from the same gene by polysome. Under each gene name, there are two (or more) rows corresponding to two (or more) core promoters of the gene. Each gene has one Sdu score, which is defined in the main text. (XLSX 660 kb)


Enadenotucirev is a chimeric adenovirus with demonstrated preclinical tumor-selective cytotoxicity and a short half-life. Further clinical mechanism of action data showed that enadenotucirev can gain access to and replicate within different types of epithelial tumors. This phase 1 dose escalation study assessed intravenous (IV) dose escalation with enadenotucirev to establish the maximum tolerated dose (MTD) and subsequently identify a suitable schedule for repeated cycles.n = 22) and expansion (n = 9), delivery of enadenotucirev between 1 × 1010 and 1 × 1013 viral particles (vp) on days 1, 3, and 5 (single cycle) was used to determine an appropriate MTD. Subsequent treatment cohorts  examined the feasibility of repeated dosing cycles in either 3-weekly or weekly dosing regimens.Sixty-one patients with advanced epithelial tumors unresponsive to conventional therapy were enrolled and received enadenotucirev monotherapy as part of this study. During the phase 1a dose escalation  of grade 3 or higher were hypoxia, lymphopenia, and neutropenia. The frequency of all TEAEs (notably pyrexia and chills) was highest within 24 h of the first enadenotucirev infusion and decreased upon subsequent dosing. Additionally, delivery of three doses of enadenotucirev over 5 days optimized pharmacokinetic and chemokine profiles in the circulation over time.Enadenotucirev displayed a predictable and manageable safety profile at doses up to the MTD of 3 × 1012 vp.This study provides key clinical data in patients with solid epithelial tumors following treatment with IV enadenotucirev monotherapy and supports further investigation of enadenotucirev in combination with other therapeutic agents at doses up to the MTD of 3 × 10ClinicalTrials.gov Identifier: NCT02028442). Trial registration date: 07 January 2014 – Retrospectively registered.(The online version of this article (10.1186/s40425-019-0510-7) contains supplementary material, which is available to authorized users. The use of oncolytic viruses is rapidly emerging as a novel therapeutic approach against cancer –4. Such Systemic delivery is a major goal in the field of oncolytic viruses . IntraveEnadenotucirev is a group B Ad11p/Ad3 chimeric oncolytic adenovirus generated by directed evolution that has potent and tumor-selective cytotoxicity, inducing a non-apoptotic immunogenic death process , 13 and Enadenotucirev is being tested in several trials in an international clinical program. One of these studies (the Mechanism of Action [MOA] study) demonstrated that enadenotucirev can be successfully administered by IV infusion or IT to a range of epithelial tumor types with a predictable and manageable safety profile . This stWe now report the primary findings of the phase 1 EValuating OncoLytic Virus Efficacy (EVOLVE) study. EVOLVE was designed to assess the safety and tolerability of enadenotucirev dose escalation administered by IV infusion in patients with colorectal cancer (CRC) and other solid epithelial tumors to determine the maximum tolerated dose (MTD), as well as selecting a suitable schedule for repeat cycle IV infusion in patients with metastatic CRC (mCRC) or urothelial cell carcinoma (UCC).The EVOLVE study was a phase 1 (NCT02028442), multicenter, non-randomized, open-label study to investigate the administration of enadenotucirev monotherapy in patients with advanced or metastatic epithelial solid tumors. The EVOLVE study design is shown in Fig. The study consisted of single-cycle dose escalation and dose expansion cohorts and a repeat cycle cohort (phase 1a), as well as additional optimal schedule-finding repeat cycle cohorts (phase 1b). Key study decisions were taken by centralized study decision-making committees. Safety and tolerability data (as well as supporting laboratory data) were reviewed at each point of study treatment adjustment by a Clinical Events Committee (CEC). Safety data were reviewed by a Data and Safety Monitoring Committee (DSMC) at the end of phase 1a. Patients were followed up in phase 1a for 9 months and in phase 1b for overall survival.10 viral particles (vp) infused over 5 min, determined to allow a 40-fold safety margin below the no observed adverse effect level from the relevant Good Laboratory Practice toxicology study. Three patients were enrolled sequentially with at least a 14-day window and followed up during the dose-limiting toxicity (DLT) assessment period (28 days after the first enadenotucirev administration). If no DLTs were observed, the next cohort was enrolled at a 10-fold higher dose. Expansion of a cohort, from three to six patients, was required if one of the three patients experienced a DLT at a given dose. If two or more participants had a DLT at a given dose, then no further patients received this dose and the previous lower dose level was defined as the MTD. Adverse events (AEs) that met any of the following criteria were considered DLTs if they were treatment-related and confirmed to meet the DLT criteria by the CEC.self-limiting or medically controllable toxicities  and alopecia.Grade 3 or higher non-hematological toxicity lasting more than 3 days despite optimal supportive care, except for:Not all enadenotucirev doses were administered over the 5 days owing to AEs considered treatment-related (cycle 1 only).9/L concurrently with a temperature elevation of > 38.5 °C lasting more than 5 days).Febrile neutropenia  treatment cycles of enadenotucirev monotherapy. This cohort was initiated once six patients had been followed up for at least 28 days after the day 5 administration of enadenotucirev in the dose expansion stage. Phase 1b was initiated once six patients had been treated in the phase 1a repeat cycle stage to further assess repeated Q3W and weekly (Q1W) dosing schedules. A summary of the dosing regimens used during EVOLVE is shown in Additional file Dose escalation stage (in phase 1a): solid tumor of epithelial origin not responding to standard therapy or for which no standard treatment exists.Dose expansion and repeat cycles stages (phase 1a and phase 1b): mCRC not responding to standard therapy and no more than three previous lines of systemic therapy for advanced disease, or no more than four previous lines of systemic therapy for advanced disease if one of the four lines was an anti-epidermal growth factor receptor therapy given as a single agent or combined with a previously administered chemotherapy regimen. In phase 1b, patients with advanced or metastatic UCC who had received no more than one chemotherapy-containing regimen and one other systemic biologic agent were also included.Patients were eligible if they were aged 18 years or over with an Eastern Cooperative Oncology Group performance status of 0 or 1 and predicted life expectancy of 3 months or more with adequate renal, hepatic, and bone marrow function and coagulation test results. Eligibility also required patients to have a gap of at least 3 weeks since their last dose of IV systemic chemotherapy and 2 weeks since their last oral dose of capecitabine, with recovery to grade 1 from the effects  of any previous therapy. Patients had to meet one of the following specific inclusion criteria for the different parts of the study.Key exclusion criteria included any condition or illness that would compromise safety or interfere with the evaluation of the safety of the study treatment, significant immunodeficiency, splenectomy, previous allogeneic or autologous bone marrow or organ transplantation, active infections or viral disease, use of anti-viral agents in the last 14 days, major surgery in the last 4 weeks or radiotherapy in the last 3 weeks, another primary malignancy in the last 3 years , and a central nervous system metastasis that was symptomatic and/or required treatment.Demographic and baseline data were collected at screening. The incidence, nature, and severity of AEs were characterized using the National Care Institute Common Terminology Criteria for Adverse Events (CTCAE) Version 4.03. Additionally, changes over time and maximum changes in laboratory data were analyzed.Although antitumor activity was not the primary outcome of the study, the best overall response to enadenotucirev was monitored using the Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1, as assessed both by the investigator and by an independent reviewer. Tumor imaging was performed every 8 weeks (phase 1a) or every 9 weeks (phase 1b) until disease progression.The concentration of enadenotucirev in the blood was measured using quantitative polymerase chain reaction (qPCR) to detect genomic viral DNA as described previously . Whole bThe concentration of shed enadenotucirev was quantified via qPCR as described previously . During Serum anti-enadenotucirev antibody response was assessed using a Meso Scale Discovery  enzyme-linked immunosorbent assay, as described previously . During Blood samples were collected at EOI on each dosing day. 100 μL of blood samples were added to monolayers of HT-29 cells along with negative and positive controls. The extent of infection was scored depending on viral staining and monolayer destruction. If no staining of cells or plaque formation was observed, the sample was scored as negative.Cytokine levels were assessed using a FlowCytomix assay as part of phase 1a as previously described , and a LHexon staining was assessed using immunohistochemistry with a pan hexon monoclonal antibody  as previously described .t-tests and multiple means were compared using Tukey-Kramer honest significant difference analyses. A post hoc analysis categorized treatment-emergent adverse events (TEAEs) and laboratory test results by dose level . For this post hoc analysis, coding of all AEs was updated to the latest version of the Medical Dictionary for Regulatory Activities, Version 19.1.Viral kinetic, pharmacodynamic, and safety data were analyzed using descriptive statistics, frequency counts, and percentages, and were also shown graphically, if appropriate. Individual means were tested using Student’s 13 vp). One patient withdrew consent during the dose expansion stage of phase 1a, with eight of nine patients (88.9%) completing the treatment period. All patients (6/6) completed the repeat dosing cycle treatment period in phase 1a.Patients were aged between 36 and 79 years, were primarily men (75.4%), and all were Caucasian. During phase 1a, 50 patients were screened for study eligibility between September 24, 2012 and March 28, 2014 . One patient developed hypoxia and dyspnea and a second acute lung injury (CTCAE grade 3); both DLTs were considered treatment-related and resulted in treatment discontinuation after the first dose. The dose investigated was subsequently de-escalated to 3 × 1012 vp (cohorts 5 and 6) and later re-escalated to 6 × 1012 vp (cohort 7). The infusion durations of cohorts 6 and 7 were increased to provide an infusion rate comparable to cohort 3, and the MTD was determined to be 6 × 1012 vp/40 min.Having increased the dose in 10-fold increments starting at 1 × 1012 vp/15 min owing to the overall frequency and severity of TEAEs (including severe hypoxia and raised hepatic transaminases) across the two initial phase 1b cohorts at the 6 × 1012 vp/40 min dose. At the end of the study, the investigators considered the MTD to be 3 × 1012 vp, irrespective of infusion time or dosing schedule.However, after initiation of phase 1b and following review of the additional safety data generated in this part of the study, the CEC and DSMC recommended reducing the dose for both dosing schedules (Q3W and Q1W) to 3 × 1012 vp) experienced grade 3 pyrexia, with none being considered a serious AE or leading to dose discontinuation  were among the most commonly reported, by 45 (73.8%), 41 (67.2%), and 13 (21.3%) patients, respectively. All chills and influenza-like illness events were grade 1 or 2, and only two patients , with an increase in frequency seen at doses of 1 × 1012 vp  at doses higher than 3 × 1012 vp, four patients (15.4%) receiving 3 × 1012 vp had proteinuria considered treatment-related.Assessment of renal function revealed that TEAEs of renal failure and acute renal injury considered treatment-related occurred at doses above 3 × 1012 vp. These were considered treatment-related in three patients (11.5%) receiving 3 × 1012 vp and 12 patients (41.4%) at doses above 3 × 1012 vp.Investigations relating to changes in hepatic function (increased transaminases) were seen more commonly at doses above 3 × 1012 vp but were reported in 14 (23%) and seven patients (11.5%) at doses of 3 × 1012 and above, which were considered treatment-related in 12 (19.7%) and six patients (9.8%), respectively. Anemia was observed across the dose range but most cases were not considered treatment-related. Falls in total white blood cell count were seen at doses of 1 × 1012 vp and above, and the proportion of patients with neutropenia increased with dose (two patients [7.7%] at doses of ≤ 3 × 1012 vp compared with five patients [17.2%] at doses > 3 × 1012 vp), and all were considered treatment-related. Likewise, decreased platelet counts increased with dose (six patients [18.8%] at doses of ≤ 3 × 1012 vp compared with 13 patients [44.8%]) at doses > 3 × 1012 vp). Falls in platelet count (reported as either thrombocytopenia or platelet count decreased) were observed in 19 patients (31.1%) and considered treatment-related in all patients , predominantly at doses of 3 × 1012 vp and above. These events were considered treatment-related in five patients.Intestinal obstruction  was reported in seven patients, all at doses of 3 × 10max) between days 1 and 5  of cycle 1. By day 61, genomic DNA levels had dropped to below the level of quantification (LOQ) in 26/28 (92.9%) blood samples , which showed extensive areas of cell necrosis and evidence of viral replication  was detected at all doses in all but six patients, and shedding frequency and concentration were generally related to infusion concentration Fig. a. Sheddi10 vp), and overall in 17 of the 31 patients  was related to dose level, detected in all but the lowest dosing cohort (1 × 10Urine shedding (phase 1a and phase 1b) could be detected in all samples; however, for phase 1a, this was only at low concentrations because no legitimate samples gave a concentration above the LOQ . Transient increases were observed after the first dose in most patients, and these were dose-dependent Fig. a–d. WhenEnadenotucirev dosing on subsequent days (i.e. day 3/5) was associated with a reduced frequency of TEAEs per dose during cycle 1  have correlated with signs of toxicity . Herein,max on day 5 relative to after the first dose. The increase was greater than can be explained by accumulation alone and is consistent with a response to the initial virus dose leading to suppression of virus clearance activity by cells of the macrophage lineage, such as Kupffer cells in the liver. However, no change in half-life was observed, which may be due to the sensitivity of the assay and sampling schedule used. This increase in circulating virus loading occurred in addition to the diminished induction of blood cytokine responses and side effect manifestation. These findings are consistent with the hypothesis that innate immune cells can be desensitized, allowing improved delivery and fewer side effects with subsequent doses.The EVOLVE study represents the first assessment of the pharmacokinetic behavior of a systemically administered group B adenovirus in humans. The use of a clustered administration regimen (three doses given in 5 days) resulted in a modest increase in CThe EVOLVE study provided further evidence of the advantages of enadenotucirev in the context of IV dosing with oncolytic viruses. Consistent with previous reports of group B adenoviruses in the general population , 19, a l12 vp) in combination with additional cancer therapeutics. Further investigations into the potential of enadenotucirev will take advantage of this unique platform to incorporate transgene add-ons and use systemic dosing to deliver therapeutic agents to the tumor microenvironment. Further modifications have utilized matrix-degrading enzymes to remove interstitial barriers and to improve the spread of enadenotucirev between tumor ‘islands’ [Taken together, these observations will inform the ongoing clinical program evaluating enadenotucirev . (DOCX 43 kb)Additional file 2:Figure S1. Comparison of viral kinetics following different doses and schedules. a Scatter plot of calculated half-life of enadenotucirev in blood in cycle 1, day 1 (phase 1a) by dose. The red box represents outlier box plot quantile analysis, and the green diamonds represent mean and 95% CI at each dose. b Scatter plot of viral clearance half-life by schedule in phase 1b. The horizontal line represents the mean of each schedule. c Scatter plot of viral clearance half-life for each patient on each visit, coloured by dose . Figure S2. Biopsy of skin metastasis after treatment with enadenotucirev. Skin biopsy taken after four cycles of enadenotucirev dosing , 107 days after first exposure . Figure S3. Mean urine viral shedding. As quantified by qPCR with bars representing the mean and error bars representing a the range observed by dose during phase 1a and b by schedule in phase 1b. Figure S4. Representative viral infectivity assay images. Images taken during the viral infectivity assay displaying a negative, b cells stained positive for virus, c plaques in monolayer, and d complete/partial monolayer destruction or quantifiable scoring. Figure S5. Cytokine levels in the blood by dosing schedule during phase 1b. As measured using a Luminex bead-based multiplex assay. a Mean concentration of IFN-γ by schedule. b Mean concentration of IL-6 by schedule. c Mean MCP-1 concentration by schedule. d Mean TNF-α concentration by schedule. e Total number of TEAEs of interest  occurring within 24 h of infusion across cycles. (DOCX 3593 kb)