Bladder cancer (BC) is the second most common urologic malignancy and the ninth most common malignancy worldwide. Surgical resection is the mainstay of treatment for patients with early-stage disease, whereas therapeutic options are limited for patients with advanced-stage or residual BC. Programmed cell death ligand-1 (PD-L1) is an important target for immunotherapy. It is known that PD-L1 is overexpressed in BC; a clinical trial involving PD-L1 immune checkpoint inhibitors in advanced BC is ongoing. In the present study, we used Western blot and quantitative real-time PCR (qPCR) to define the expression level of PD-L1 after cisplatin treatment in BC-derived cell lines. The signal activation was also evaluated by Western blot in BC-derived cell lines. We found that chemotherapeutic drug cisplatin can induce PD-L1 but not PD-L2 expression in BC-derived cell lines. Furthermore, the expression level of PD-L1 was increased in a dose- and time-dependent manner after cisplatin treatment. The cisplatin-induced PD-L1 expression is mainly mediated by ERK1/2 but not Akt/mTOR signal pathway. Moreover, we found that cisplatin activates transcription factor activator protein-1 (AP-1) to regulate PD-L1 expression. The chemotherapy drug such as cisplatin may trigger resistance of BC through PD-L1 up-regulation. The present study suggests that PD-L1 antibody should be used concomitantly with chemotherapy in the setting of advanced and metastatic BC. Bladder cancer (BC) is a commonly diagnosed urological malignancy with a very high recurrence rate. The standard treatment for managing BC is a complete transurethral resection of the bladder tumor (TURBT). Intravesical chemotherapy including mitomycin C, epirubicin, and doxorubicin or Bacillus Calmette-Guérin (BCG) for non-muscle-invasive BC is generally used as adjuvant therapy after complete endoscopic resection . For musEvasion of the immune system is a hallmark of cancer. Cancer cells can express many immune inhibitory signaling proteins that cause immune cell dysfunction and apoptosis . One of Avelumab, an immune checkpoint inhibitor of the PD-L1/PD-1 axis, has shown anti-tumor activity in patients with platinum-refractory metastatic urothelial carcinoma . However2448; cat. no. 2971), p-Akt , p-ERK1/2 , p-p38 , p-JNK , p-c-jun , Akt (cat. no. 9272), p38 (cat. no. 9212), c-jun (cat. no. 9165), mTOR (cat. no. 2983), PD-L1 (cat. no. 13684) were purchased from Cell Signaling Technology . ERK1/2 (cat. no. sc-94), JNK (cat. no. sc-7345), and β-Actin (cat. no. sc-47778) were purchased from Santa Cruz Biotechnology . Cisplatin (cat. no. 232120), Akt inhibitor (cat. no. 124005), PD98059 (cat. no. 19-143), U0126 (cat. no. 19-147), SP600125 (cat. no. S5567), SB203580 (cat. no. S8307) and Tanshinone IIA (cat. no. T4952) and all other chemicals were obtained from Sigma–Aldrich .Anti-mouse or anti-rabbit IgG–conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for p-mTOR  or 10-cm dishes for cell viability assays and collecting RNA or protein samples.Human BC cell lines (5637 and T24) were obtained from Bioresource Collection and Research Center  and maintained at 37°C under 5% COThe cellular lysates were prepared and proteins were then resolved on SDS/PAGE and transferred to Immobilon polyvinyldifluoride (PVDF) membranes. The blots were blocked with 4% BSA for 1 h at room temperature and then probed with rabbit primary antibodies against human proteins (1:1000) for 1 h at room temperature. After three washes, the blots were subsequently incubated with a donkey anti-rabbit peroxidase–conjugated secondary antibody (1:1000) for 1 h at room temperature. After three washes with TBST, the blots were then detected with Amersham ECL Western Blotting Detection Reagents  and photographed by using a ChemiDoc-It® Imaging Systems . Quantification of Western blot results was performed using ImageJ software1.49v . β-actin was used as internal control and densitometry values of each detected proteins were normalized to β-actin. Results are expressed as the mean ± standard deviation (SD) of three independent experiments.T).The quantitative real-time PCR (qPCR) analysis was performed using Taqman® one-step PCR Master Mix . A total of 100 ng of total cDNA was added per 25-µl reaction with sequence-specific primers and Taqman® probes. Sequences for all target gene primers and probes were purchased commercially from Applied Biosystems ; β-actin was used as the internal control. Quantitative RT-PCR assays were carried out in triplicate on a StepOnePlus sequence detection system. The cycling conditions were 10 min of polymerase activation at 95°C followed by 40 cycles at 95°C for 15 s and at 60°C for 60 s. The threshold was set above the non-template control background and within the linear phase of target gene amplification, to calculate the cycle number at which the transcript was detected  for 1 h at room temperature, washed again, and incubated with FITC–conjugated secondary antibody (1:100) for 1 h. Finally, cells were washed, mounted with DAPI containing solution, and photographed with a Nikon Ti2 microscopy System.t test for statistical significance and expressed as the means ± standard deviation (S.D.). A P<0.05 was considered statistically significant. The data containing more than two groups were performed using one-way analysis of variance (ANOVA) with Bonferroni’s post-hoc test. The difference was considered to be significant if the P-value was <0.05.All experiments were performed at least three times, each time in triplicate. Statistical comparisons between two samples were analyzed by Student’s Since PD-1/PD-L1 expression is the main indication for these immune checkpoint inhibitors, and the expression of these immune checkpoint proteins is up-regulated with the progression of BC, it is reasonable to hypothesize that PD-L1 overexpression may be involved in the progression of BC by providing an escape route for tumor cells to evade immune detection. Suppression of these proteins by immune checkpoint inhibitors or other strategies may effectively treat BC. Our results found that cisplatin dose-dependently promoted PD-L1 mRNA expression but not that of PD-L2 (another ligand for PD-1), in BC-derived cell lines A,B. The Multiple mechanisms can contribute to intrinsic tumor PD-L1 expression. Previous research indicates that activation of the Akt/mTOR pathway promotes immune escape by driving PD-L1 expression in lung cancer . TherefoThe AP-1 transcription factor is a dimeric transcription factor that is composed of c-Jun, c-Fos, activating transcription factor (ATF). The main AP-1 proteins in mammalian cells are c-Jun and c-Fos. When the cellular counterparts of the viral oncoproteins were discovered, the up-regulation of AP-1 proteins by overexpression or by oncogenic RAS was found to correlate with a positive effect on cell transformation . MAPK faThe anti-PD-L1 antibody MPDL3280A, a systemic cancer immunotherapy, has proven efficacy in the treatment of metastatic BC; tumors expressing high levels of PD-L1 had particularly high response rates . Here, wNumerous reports have suggested that intrinsic cellular changes are associated with carcinogenesis-induced PD-L1 expression. T-cell lymphoma cells carrying the oncogenic nucleophosmin/anaplastic lymphoma kinase (NPM/ALK), which is involved in malignant transformation, induce high levels of PD-L1 expression via STAT3 and ERK activation ,19. MoleAP-1 protein is a well-established pro-oncogenic transcription factor. The activation of AP-1 is rapidly induced by growth factors, cytokines and oncoproteins, which are implicated in the proliferation, survival, differentiation, and transformation of cells . A mouseThe MAPK cell signaling pathways play important roles in the regulation of cell growth, proliferation, and survival. Mutations within these pathways are frequently implicated in the pathogenesis of solid tumors . Agents In the present study, we demonstrate that cisplatin promotes PD-L1 expression through the ERK1/2 signaling pathway. This work provides evidence in support of the development of combination therapy using a conventional chemotherapy drug and a PD-L1 antibody for the treatment of BC.


Azadirachta indica A. Juss secondary metabolites (AISM) formulated soap and cream for management of infectious and oxidative stress-related diseases (OSD).Infectious diseases, particularly those due to multi-drug resistant bacterial strains are almost impossible to cure globally. In this study we investigated the inhibitory effects of The antibacterial, radical scavenging and cytotoxic effects of the neem cosmetics were examined by serial dilution, spectrophotometric and hemolytic techniques respectively, while the AISM in the essential oils (EOs) were elucidated by Gas chromatography-mass spectrometry (GC-MS) and retention index.Staphylococcus aureus, Listeria ivanovii, Enterobacter cloacae, Mycobacterium smegmatis, and Streptococcus uberis) and two confirmed multi-drug resistant bacterial strains  at 0.80 mg/mL. Conversely, at less than 0.50 mg/mL the neem soap produced with AISM demonstrated bactericidal effects against most of these test pathogens linked to infectious diseases. The neem soap containing AISMs displayed noteworthy effects in scavenging radicals associated with OSD at < 1.76 mg/mL. The cosmetics were not toxic to human red blood cells below 0.70 mg/ mL. To our known, the AISM predominantly caryophyllene (30.02%), phytol (14.12%), elemene (13.40%) and linoleic acid (10.5%) exceptional inhibitory effects in neem cosmetics are reported here for the first time.The neem cosmetics without AISM exhibited bacteriostatic effects against five reference bacterial strains  contains supplementary material, which is available to authorized users. Escherichia coli, vancomycin-resistant Enterococcus faecalis, Vibrio Spp., and methicillin-resistant Staphylococcus aureus (MRSA) are known public threats in hospitals and have become more resistant because of natural selection processes, and over-prescription and misuse of antibiotics  as shown in Table The inhibitory effects (supporting document 1) of the neem cosmetics produced with and without AISM on test pathogens are as presented in Tables E. cloacae, the four creams exhibited a similar effect, while NMLSBC displayed a superior MIC value (0.400 mg/mL). The NMLSBC displayed higher inhibitory effects than other neem cream products with lower MBC values against the five test pathogens, however, its effect was lower than that of positive control drug  had better effects against the pathogens Table . However value 0.00 mg/mL.p < 0.05), while the effect of NMSBS against LP •, and NO • were similar (s). At a higher dose (0.20 mg/ mL) all test neem soap samples scavenged the three radicals differently , creams  as well as neem cosmetics (NMS and NMC) produced devoid of AISM were examined on three different radicals . The radical scavenging effects of neem soaps and positive control (ascorbic acid and rutin) on the radicals were concentration dependent Figs. , 2 and 350 values for the different soaps and creams, regression equations from the standard curves generated for each radical was used while t-test analysis was employed for significantly different of % radical scavenging effects against the concentrations . Interestingly, the antioxidant capacities of NMLSBS and NMLSBC were remarkably better than the two positive control drugs  of the investigated substance because of formation of a neutral molecule of DPPH-H upon donation of H atom from the substance . The strns Figs. , 2 and 3p < 0.05) than that reported for neem seed oil (0.939) by Akpan, [The physicochemical properties (PCP) of the crude neem seed oil (NSO) and essential oils (EOs) extracted by mechanical cold press and hydrodistillation from the aerial parts respectively are as presented in Table y Akpan,  and highy Akpan, . The RD 2O) for the neem soap (NMS) produced without AISM. Nevertheless, the total fatty matter (77.30–80.14%) for the four neem soaps and other physicochemical parameters were better than the specifications of the SON for toilet soap and similar to neem soap produced elsewhere [The physicochemical properties of neem soaps produced with and without the AISM are as presented in Table lsewhere .Table 8PThe cytotoxicity effects on neem cosmetics are as presented in Table A. indica leaf and stem-bark incorporated unto the recipes of neem cosmetics are as presented in Table The chemical profiles of secondary metabolites (SM) isolated from A. indica SMs are reported here for the first time in the genus Azadirachta. The neem cosmetics produced with AISM were more active against Gram-positive bacterial than the test Gram-negative bacterial strains. This may be linked to net repulsion of the complex outer membrane in Gram-negative bacterial, reported in previous studies to contain hydrophilic lipopolysaccharide which exhibits higher tolerance toward hydrophobic terpenes and oxygenated terpenes [Biochemical investigations of inhibitory effects on pathogens and radicals of two different neem extracts (neem seed oil and EO) revealed higher bioactive and physicochemical properties of NC formulated with SM from the EO. Characterization of SM in the leaf and stem EOs integrated into the recipe of NC shown more terpenoids including phytol 18, one sesquiterpenoid ledol 14), a monoterpene (camphene 2) and sesquiterpene (curcumene 8) in the stem-bark (SBEO). Moreover, the content of bioactive linoleic acid (17), hexadecenoic acid (15), caryophyllene oxide (12), linalool oxide (6) and terpinene (5) were more in SBEO. Accordingly, the NC made incorporating SMs from SBEO and LEO had the highest activity followed by NC produced with the SM from the SBEO, while NC produced with only crude neem seed oil (NSO) displayed the least activity. To the best of our knowledge, this enhanced bioactivity of neem cosmetics formulated from the leaves and stem bark of , a monotterpenes . Anotherterpenes .A. indica is known to inhibit low-density lipoprotein oxidation even in the early phase [Phytol one of the major SM in the SBEO was reported  to demonly phase . Other bly phase , pinene ly phase  and linoly phase  may haveA. indica, especially in the stem-bark  indicates that the stem-bark EO will be feasible as a fresh potent source in the hunt for principal agent for the management of oxidative stress-related ailments such as skin cancers , scabiesA. indica leaves and stem-bark in tropical and subtropical countries.None of the neem cosmetics displayed hemolytic effects on human red blood cells (HRBC) below 0.68 mg/ mL. Mild cytotoxic effect was observed in neem soap formulated with combined stem-bark and leaf SMs at > 0.69 mg/ mL. This may be attributed to the frail nature of HRBC since there has never been any report on human toxicity after using a decoction of This present study indicates that besides the known uses neem tree, the neem cosmetics integrated with the stem-bark and leaf essential oil has robust bioactive secondary metabolites; therefore, the EO could be a novel applicant as new antimicrobial agent in this current era of rising multidrug-resistant bacterial strains, likewise as an alternative to synthetic antioxidant and effective bioactive chemical in pharmaceutical and cosmetics industries.Additional file 1:Screening of the products against 7 Bacterial strains at different concentrations. (DOCX 4399 kb)


Strong light-graphene interaction is essential for the integration of graphene to nanophotonic and optoelectronic devices. The plasmonic response of graphene in terahertz and mid-infrared regions enhances this interaction, and other resonance mechanisms can be adopted in near-infrared and visible ranges to achieve perfect light absorption. However, obtaining near-absolute polarization insensitivity with ultra-narrow absorption bandwidth in the visible and near-infrared regimes remains a challenge. In this regard, we numerically propose a graphene perfect absorber, utilizing the excitation of guided-modes of a dielectric slab waveguide by a novel sub-wavelength dielectric grating structure. When the guided-mode resonance is critically coupled to the graphene, we obtain perfect absorption with an ultra-narrow bandwidth  of 0.8 nm. The proposed design not only preserves the spectral position of the resonance, but also maintains >98% absorption at all polarization angles. The spectral position of the resonance can be tuned as much as 400 nm in visible and near-infrared regimes by tailoring geometrical parameters. The proposed device has great potential in efficient, tunable, ultra-sensitive, compact and easy-to-fabricate advanced photodetectors and color filters. The ultra-wideband spectral response and the huge carrier mobilities make graphene a strong candidate as a backbone of novel nanophotonic and optoelectronic devices. Indeed, graphene-based solar cells8, saturable absorbers for the mode-locked ultra-fast lasers11, photodetectors13, transparent electrodes16, optical modulators18 and third harmonic generation20 are demonstrated. Further incorporation of graphene into high-performance optical devices makes strong light-graphene interaction compulsory. Localized surface plasmons and surface plasmon polaritons supported by graphene in the terahertz (THz) and mid-infrared (MIR) regimes can enhance this interaction and perfect absorption can be achieved25. In contrast, in the visible and near-infrared range (Vis-NIR), graphene’s optical response is dominated by interband transitions, giving a real conductivity with no plasmonic response and graphene effectively mimics a dielectric with considerable loss26. Unpatterned and suspended graphene absorbs A = πα = 2.3% of the normally incident light without spectral selectivity, defined by the fine structure constant α = e2/ℏc. A = 2.3% is actually a significant value for a one-atom thick material. Nevertheless, the absolute strength of the absorption is low and it limits graphene’s further application in optoelectronics and photovoltaics in the visible spectrum, an example related to our work being low responsivity in photodetectors28.Ever since the experimental isolation of graphene in 200430. This method can confine large optical fields and significantly enhance the absorption in graphene. However, these designs require deposition of a large number of dielectric layers  on top of graphene. This results in not only fabrication difficulty, but also bulky devices. Another option is placing graphene near plasmonic nanostructures34 and relying on the near-field enhancement effect of surface plasmons or surface plasmon polaritons. This method suffers from absorption peak broadening because of the background absorption coming from the metals, which is detrimental to color sensitivity. Moreover, significant Joule losses decrease device efficiencies35.Since graphene plasmons cannot be excited outside the THz and MIR ranges, graphene has to be coupled to other resonant mechanisms to improve the light absorption in the Vis-NIR ranges. Enormous research efforts have been devoted to enhancing the local optical field around graphene and increase light-graphene interaction in the Vis-NIR ranges. The methods to achieve these can be classified into four categories. First, graphene can be placed inside a Fabry-Pérot (FP) cavity42. The difference between two methods is the structures where GMRs are excited. The third method is to couple graphene to the GMRs of photonic crystal slabs (PhCs)43 or 1-dimensional photonic crystals (1DPCs), excited by sub-wavelength grating (SWG) couplers45. The guided-mode leaks out evanescent waves to graphene. Critical coupling of the guided-modes to graphene then produces perfect absorption. Notably, the latter can achieve an ultra-narrow absorption bandwidth of 0.03 nm due to placing graphene far away from 1DPC45. Although promising in theory, this method enforces extreme fabrication accuracy on not only the SWG, but also on the 1DPCs to achieve the target wavelength and store very high energy in 1DPC to achieve critical coupling. Similar to the methods that adopt FP cavity, this method also results in bulky components. The final method is to excite the GMRs of either unmodulated slab-waveguide structures beneath the grating50, or the grating itself  51, which acts as a waveguide whose core is refractive-index modulated bulk38. These GMRs then interact with graphene to increase light-graphene interaction. Compared to the third method, this method offers more compact designs and a more facile fabrication process. This is because it does not require 1DPC, but only a SWG, two or three dielectric films with only one being patterned, and possibly a perfect back metal mirror or Bragg reflector. However, the downside is the larger bandwidth because low-Q resonances can be coupled to graphene, due to the smaller distance between GMR and graphene. Thus, there is a fundamental trade-off between the two methods in terms of the fabrication complexity and Q-factor of resonances. While the previous studies for the guided-mode resonances focused mainly on the absorption enhancement, polarization insensitivity remained an issue, because of incorporating 1-dimensional SWGs. Although polarization-insensitivity between s and p-polarizations is achieved by the square lattice in ref.43, the bandwidth, measured as full-width at half-maximum (FWHM), 14 nm is quite high because of the circular shape of the holes. The sharp features of gratings allow for enhanced field localizations and high-Q resonances. Hence, an ideal unit-cell would use a rectangular grating structure with rotational symmetry to preserve both strong light absorption and achieve polarization insensitivity.The third and fourth methods can be classified together as coupling of graphene to guided-mode resonances (GMRs) that define the phase-matched coupling between the free-space radiation and the supported (guided) modes with a leaky nature due to their complex wavenumberIn the present paper, we employ the finite-difference-time-domain (FDTD) method to numerically propose a novel design that uses a 2-dimensional sub-wavelength grating structure to excite the GMRs of an unmodulated slab waveguide. The critical coupling of the GMRs to graphene yields 99.2% light absorption with 0.8 nm FWHM at 678 nm, under p-polarized (TM) light. The proposed device preserves perfect absorption >98% at all source polarizations as well as the spectral position of the resonance. To the best of our knowledge, this is the first study that illustrates an FWHM below 1 nm with near-absolute polarization independence. Furthermore, the presence of a waveguide structure, instead of 1DPCs or FP cavities, relaxes the device fabrication. The spectral position of the resonance is easily tunable by changing the key geometrical parameters of the device. We first show the enhancement of graphene absorption in a simple optimized grating-core-substrate structure and scrutinize the effect of geometrical parameters on the device characteristics. In the second part, we add a metal back mirror and optimize the geometry accordingly to achieve critical coupling, and hence perfect absorption with the ultra-narrow bandwidth. We discuss the design considerations to maximize absorption strength and minimize absorption bandwidth, for each case.2O5), unmodulated core dielectric region by a unique 2-dimensional sub-wavelength polymethyl-methacrylate (PMMA) dielectric grating. The structure is supported by glass (SiO2) substrate. We call this two-port structure Device I. Figure 52. Another equivalent unit-cell for the structure is presented in Fig. PMMA.Our proposed device in Fig. ϕ = 0, p-polarization). In the x and y directions, we use periodic boundary conditions, while in the z-direction perfectly matched layers (PML) are adopted and they are placed 5 wavelengths away from the structure. We use two monitors that are at an adequate distance away from the structure to find the reflected (R) and transmitted (T) power. Absorption is then found by using A = 1 − R − T formula. The dispersion effect of the dielectric materials are retrieved by ellipsometric measurements, and they are in good agreement with the models based on Sellmeier equation53. In the material fits, the only non-zero extinction coefficient comes from Ta2O5, and is equal to 10−5. It is then reasonable to regard the device as lossless without graphene. Graphene is modeled as a 2-dimensional material with infinitesimal thickness and a complex surface conductivity that includes the chemical potential, scattering rate and temperature effects, based on Kubo formulation54. The surface conductivity is divided into intraband and interband contributions, as given in Eqns j is the imaginary unit, e is the elemental charge, w is the angular frequency, T is the temperature, ℏ = h/2π is the reduced Planck’s constant, Γ is the scattering rate, and μc is the chemical potential. kb is Boltzmann’s constant.To investigate the spectral response of Device I, we utilize finite-difference-time-domain simulations. In the numerical simulations, we illuminated the unit-cell with a broadband plane-wave at normal incidence whose electric-field is polarized in x-direction  and 220 nm, respectively. We simulated Device I without graphene to gain insight about the resonance excitation and to confirm that the device is almost lossless. The simulation result in Fig. 2O5. It is then safe to associate the absorption in subsequent simulations to graphene. In Fig. 36. Another condition for the occurrence of the resonance is the phase-matching condition, i.e. the real part of the complex wavevector component in the travelling direction of the guided-mode is equal to the components coming from the reciprocal lattice vector of grating and the incident wave48. Eq. β is the propagation wavenumber along the guiding direction, k0 is the free-space wavenumber, neff is the effective refractive index of the mode inside the core, m is the diffraction order, λ0 is the free-space wavelength and Λ is the grating period. Writing an analytical expression for the phase-matching condition in Device I is cumbersome because our grating diffracts incoming waves in not only the x-direction but also in the y-direction, as opposed to the explicitly used devices with 1D grating51 does. Therefore, the guided-mode’s propagation direction is not simply the x-direction as in Device 0. This makes numerical methods necessary to fully discern the resonances. Another interesting feature of GMRs is their Fano lineshape. It arises due to the interaction between a sharp resonance channel and the broad radiation continuum55. Critically, the zeroth order diffracted wave is a propagating wave, while all other diffracted orders are evanescent due to the sub-wavelength nature of the grating. Therefore, we have evanescent coupling of the incident wave to the guided-mode.First of all, we set the design parameters 2O5 to increase the light-graphene interaction, thanks to the guided-wave confined in the waveguide28. The spectral response of the configuration is shown in Fig. 47 also found that simple 1D grating and unmodulated slab waveguide structure has a theoretical maximum absorption of 60% without the addition on back-mirror. We, therefore, attribute both the near-stable spectral position of resonance and slightly enhanced absorption to our novel grating design.We then sandwiched monolayer graphene between the grating and Ta47. While Fig. Figure 2 is enhanced two orders of magnitude with respect to the incident light, |H|2 is enhanced one order of magnitude. The enhanced optical field on graphene is directly responsible for the absorption enhancement, in accordance with Eq. 457,ε0 is the vacuum permittivity, λ and I(λ) are the wavelength and the intensity of the light, respectively, n″ and n′ denote the imaginary part (extinction coefficient) and real part of the complex refractive index, respectively. The enhancement of fields is slightly higher than the designs incorporating slab waveguide as the guiding layer50. We also observe that the standing wave pattern is created in both the z and x-directions. While the former is because of the waveguiding effect, the latter is due to the refractive index modulation in the x-direction45. The electric field profile in Fig. 59.To get further comprehension about the improvement in the light-graphene interaction, we extracted the electric and magnetic field profiles for both off-resonant and on-resonant cases. A comparison of the fields in the off and on-resonant cases, shown in Fig. 60. In addition, when the guided-mode travels along the periodically segmented waveguide61, the periodicity of the grating defines Bloch modes. Different effective refractive indices of the Bloch modes in the grating region42 consequently define different effective mode refractive indices for TE and TM modes of the waveguide. The excitation condition of TE and TM modes are different on top of this62. Thus, the devices utilizing 1D gratings show polarization dependency42 as they excite the GMRs at different wavelengths. Moreover, dissimilar field distributions result in different absorption strengths.Because it is the most important aspect of our design, we devoted this subsection to the further dissection of polarization insensitivity in Device I, by analyzing what causes the devices with 1D grating, such as Device 0, to be polarization sensitive in the first place. Firstly, different boundary conditions imposed on different polarization states, which results in form birefringence63.f is the fill-factor of grating, n1 and n2 are the refractive indices of the grating material, and its surrounding, respectively. n⊥ are the refractive indices of the effective medium for the s-polarized , and p-polarized waves (E-field perpendicular to grating grooves), respectively. Any arbitrary incident polarization can then be decomposed into a superposition of s and p-polarizations to find the effective refractive index as a weighted sum of n⊥.Without considering the waveguiding effects and the complicated properties of Bloch modes, we adopted Rytov’s formulas given in Eqns 52. To compare Device I with the devices using 1D grating, we kept the polarization angle at ϕ = 0 degrees, and rotated the unit-cells of each for 0, 30, 60, and 90 degrees. The result of this comparison is shown in Fig. From Eqns 2O5-PMMA (core-PMMA) and Ta2O5-SiO2 (core-substrate) boundaries are highly related to the effective mode index, neff, of the guided-mode if we regard the structure as an asymmetric dielectric slab waveguide. This is done by considering the SWG as a medium with an effective dielectric permittivity58. Therefore, we start our discussion here by briefly reviewing the fundamental results of asymmetric dielectric slab waveguide theory64.Results of the previous subsection demonstrate that GMRs can be excited when the phase-matching condition is satisfied, and the evanescent wave from the diffraction grating can be coupled to one of the guided waveguide modes. Although the waveguiding effects on the Bloch modes of the periodically patterned waveguide are complicated, we can still sufficiently understand how the spectral response of Device I depends on the structural parameters, by considering the optical field around graphene. Optical fields at the Ta2 regions, respectively. nSWG is the effective refractive index of the SWG, different for TE and TM modes. Eqs eff is determined primarily by the thickness of the core to the wavelength ratio  are satisfied, whereupon the interference and dissipation leads to perfect absorptionA = 1 − R formula, and maximizing it requires minimizing the reflection, which is due to the externally leaked energy from the system that is not harnessed on graphene. This can occur due to either undercoupling or overcoupling of the GMR to graphene43. In both cases, the external leakage rate is not equal to the intrinsic absorption in graphene. The fact that critical coupling depends on the external leakage rate makes SiO2 thickness a critical parameter, as it controls the FP cavity modes in it46 and consequently reflection losses25.To achieve perfect absorption and retain the sub-micron thickness of Device I, we added a thick gold layer beneath Device I, to arrive at Device II, shown in Fig. PMMA, and To achieve critical coupling, we set the geometrical parameters to their optimized values, i.e. 45. Thus, the bandwidth is narrower in devices where graphene is placed far away from the optical field confined by GMR. Although this strategy results in a narrower bandwidth, the necessity of creating a very high-Q GMR puts vigorous stress on the fabrication process. To increase the Q-factor of resonances in Device II, it is better to confine the mode to the core, i.e. increase neff. The narrower resonances at larger core thicknesses and smaller unit-cell periods in Fig. eff increases the energy storage inside the core region and decreases the leakage to the graphene58. The critical coupling condition is then satisfied by the very high-Q GMR, and the resultant bandwidth is low. This is the reason why PMMA and w are tailored accordingly to meet critical coupling condition.We now want to emphasize the key design strategy in achieving the ultra-narrow bandwidth. To achieve critical coupling in a system where the external leakage rate is low, a very high energy storage, or equivalently, a very high-Q GMR in the resonant structure is needed2 thickness in the absorption spectrum, we varied it over a wide range from 100 nm to 500 nm, and the result is shown in Fig. 46 and it is because of the phase-shift introduced by the SiO2 layer and the FP modes defined by it46. Another reason may be that the system evolves from critical coupling to overcoupling or undercoupling, as altering glass thickness changes external leakage rate43, while the intrinsic loss rate remains almost the same. An interesting observation is that glass thickness starts having an effect on resonance wavelength below a certain value. Besides this, when To examine the effect of the SiO68 means that although the grating has 60° rotational symmetry, overall structure will not have this feature.We inspected the polarization-independence of Device II, and the result is shown in Fig. eff increases. 69.The sensitivity of GMR to geometrical parameters gives Device II a great tunability in visible and NIR ranges. As a general guideline, the resonance can be red-shifted by an increase in We now accentuate the fundamental difference in the design methodologies of Device I and Device II. In Device I, the effective mode refractive index had to be kept at an intermediate value because too high or too weak confinement of the mode to the core was detrimental to absorption. However, in Device II we could be able to confine the mode as much as we can to the core, without considering transmitted losses, and critical coupling gives both perfect absorption and ultra-narrow bandwidth. The same design strategy in Device I would result in absorption and transmission approaching each other, and to 50%.66. Other 2D materials, such as transition-metal dichalcogenides show stronger light-matter interaction than graphene, in the order of 10% in the visible range70. Compared to the intrinsic 2.3% absorption in graphene, larger intrinsic absorption in TMDs would mean satisfying the critical coupling condition requires a larger external leakage rate for matching. So, the absorption spectrum associated with them are inherently broader. From the viewpoint of light sensing or biosensing applications, using graphene is advantageous due to narrower bandwidth. However, the broader resonances associated with TMDs would mean a larger average absorption in the visible spectrum and the presence of a direct bandgap combined with it makes monolayer TMDs a great candidate for photodetection and photovoltaics72.Up to now, this paper dealt with achieving perfect absorption with monolayer graphene. However, the concept of critical coupling is general and can be applied to other 2D materials, such as transition-metal dichalcogenides (TMDs), and multilayer graphene to yield perfect absorption with polarization insensitivity. To explain the effect of multilayer graphene, we increased the number of graphene layers in the simulations, and the result is demonstrated in Fig. 2 and Ta2O5 can all be coated by Physical Vapor Deposition (PVD) methods such as sputtering. Chemical Vapor Deposition (CVD) grown graphene can be transferred and PMMA grating structure can be realized by electron-beam lithography. The numerical findings of this paper propose that the core thickness and the unit-cell periodicity are the most important factors in determining the resonance position and bandwidth, as well as the absorption strength. This makes the precise deposition of Ta2O5 and electron-beam lithography of the 2D grating structure the most important steps. The fabricated samples can be characterized by Fourier Transform Infrared Spectroscopy (FTIR) technique for the resonances in NIR, whereas confocal microscopy can be utilized for resonances in visible range. We believe that the deviations of experimental findings from theoretical results can be restricted to a controlled region by the physical laws and explanations presented in this paper.The experimental verification of this work starts with the fabrication flow of choosing a Silicon or quartz substrate. Choice of substrate does not affect Device II as the thick gold layer means that there will be insignificant amount of light reaching there. Thus, a thin (5–10 nm) Titanium layer can be coated beneath gold for better adhesion, without affecting the optical properties. After that gold, SiOIn conclusion, the enhancement of light-graphene interaction is numerically analyzed in this paper and the near-perfect polarization insensitivity of proposed design to incident light is verified. Our novel sub-wavelength dielectric grating excites the guided modes of an asymmetric dielectric slab waveguide. The critical coupling of the guided mode resonances to graphene resulted in perfect light absorption that is tunable by structural parameters in the visible and NIR ranges. The bandwidth of the resonance, 0.8 nm resulted in a very high Q-factor of 850. To the best of our knowledge, this bandwidth is smaller than all of the relevant device proposals with sub-micron thickness. Overall, our simple yet robust, and easy-to-fabricate device holds great promise in ultra-sensitive color detection and design of advanced photodetectors. Our future work will focus on the fabrication of the device and extending the tunability range of the guided-mode resonance.Supplementary Information


In the context of a large treatment gap for common mental disorders (CMDs) and shortage of mental health specialists in low- and middle-income countries, there is increasing evidence of the effectiveness of task sharing of counselling interventions to increase access to mental health care for CMDs at primary health care level. This study evaluated the relationship between levels of exposure to a task-shared counselling intervention and psychosocial outcomes  in chronic care service users with comorbid depression in South Africa guided by the Medical Research Council process evaluation framework. Implementation and participant-level factors that promote greater exposure were also investigated.The study design was a cohort study comprising of 173 participants referred by primary health care nurses for the task-shared counselling intervention. The study site comprised four primary health care facilities in a sub-district of the Dr. Kenneth Kaunda district in the North West Province of South Africa. The participants were assessed for psychosocial outcomes at three time points: baseline, 3 months and at 12 months. The number of counselling sessions each participant was exposed to was collected for each participant. Linear regression models were used to test the influence of counselling exposure on each of the psychosocial variables between baseline and endline. In-depth qualitative interviews were conducted on 29 randomly selected participants, stratified according to exposure to counselling sessions, and analysed using framework analysis.p = 0.064). The WHODAS scores decreased significantly more from baseline to endline among those who received 5–8 sessions  compared to those with 0 sessions . No significant differences as a function of levels of counselling exposure from baseline to endline was observed for OSS-3 scores. An improvement in ISMI scores from 1–4 sessions to 5–8 sessions was found . The qualitative process evaluation indicated that the service was acceptable and accessible; but that session attendance was hindered by women’s’ caregiving burden, poor counsellor attributes and poor referral processes.Findings from the cohort study indicated a significant reduction in depression severity at 12 months. Internalised stigma and functional disability improved from baseline to endline. Participants receiving 5–8 sessions have the greatest reduction in PHQ9 scores from baseline to endline  compared to those with 0 sessions  was found to optimize functional ability, reduce stigma, and potentially reduce depression symptoms. In order to enhance session attendance, lay counsellor delivered psychosocial interventions need to pay attention to (i) counsellor selection criteria, particularly person-centred care qualities; and (ii) strengthening referral processes in contexts where mental health literacy is low. Non communicable diseases (NCDs) have become the leading cause of global mortality, accounting for 68% (38 million) of deaths in 2012  I could not [cook or do laundry] before as I wanted to do nothing when I woke up in the morning all I wanted to do was to just sit. [I: Mmm] So now I have the strength to do it I wake up in the morning and do everything that I am supposed to do She […] gave me a paper and told me to go to [the lay counsellor] I did not know who [the counsellor] was and she took me to her […] Yes, she [explained the referral process]. I wouldn’t have gone anywhere if she didn’t.  reported being walked over to the counsellor’s room or getting introduced to the counsellor after being diagnosed/identified with depression.Participant 9; 1–4 sessions)I was very nervous, not knowing the outcome got me feeling scared. I am at fault because I did not come and I promised them (I: Mmm) I did not know what was going to happen (I: Mmm) That is the reason - I do not want to lie. Alright, the sessions really changed me a lot the first time when I attended the session I would shout (at) my children at home for nothing and I would feel hurt, now it has brought back my life because I would sleep the whole time and would not cook and eat, I would not wash the dishes living in unkempt conditions but now it was helped me a lot as I do not do those things anymore and I do not undermine myself […] .I would say the sessions made that possible, because it helped me to want to get up and do something with my life, like selling achar for income, and not just sitting waiting on my husband. […] right now, I’m making means to get extra money for the household, when my husband can’t buy some things I can use some of the money to supplement. I can now talk [about my condition] I have informed my children about my status […] when I came I told [the counsellor] I want to tell my children that I am HIV positive […] I was afraid of telling them but I ended up telling them and they accepted me […] I now don’t hide when I take my medication […]  to deal with poverty through improved budgeting and identifying income generating opportunities as ability to manage identified problems.Yes, it was easy for me to accept because as the scenarios were being read, I could relate them to my personal experiences. […] The depression counselling manual and sessions were designed and structured (using vignettes and protagonists) so as to help service users feel safe and not exposed while sharing their experiences. Identifying with the protagonist’s experience of the depression-stress-factor of the day allowed for the group to discuss the issue in a safe way. The vignettes presented depression factors in a way that was relatable for the service users.Participant 16; 1–4 sessions; emphasis added).[Thandi’s story] is the one that made us talk about life […] It had an impact because I had similarities with her I have to look after my husband as he uses a wheelchair and the baby so at times the time that’s set which is mostly 8am to 9am I don’t have time because I have to prepare for the people going to school first then I have to attend to my husband and I have a baby on the other side so it’s just lack of time for me I saw that going for counselling will help me in a lot of ways [compared to what] I had received before.  to continue with the counselling sessions with expectation for further improvement.Participant 18; 5–8 sessions)[[The counsellor] is good. She does her job with integrity without discrimination […] she is not the type of person that judges people; she just really knows how to talk to people, with everyone […] she [made telephone reminders], but then I already knew when to attend my sessions, if I had an issue with attending I’d phone to alert her.  framework for the evaluation of complex interventions, this study had two objectives: (i) to evaluate the relationship between levels of exposure to the task-shared counselling intervention component of the collaborative care model, and psychosocial outcomes  in chronic care service users with comorbid depression; and (ii) to understand implementation and participant-level mechanisms of impact that promoted greater exposure to the intervention guided by the MRC framework for process evaluation. The majority of the sample enrolled in the study were women, a demographic feature characteristic of primary health facilities providing chronic care in South Africa –48.The intervention led to a clinically significant reduction of depression symptoms at 12 months follow up. Service users who were exposed to 5–8 counselling sessions showed greater reduction in PHQ9 scores  compared to those who had no exposure to the intervention. This outcome corroborates international findings of similar studies with lay counsellor led interventions  and addsParticipants who received of 5–8 sessions had better PHQ 9 outcomes at 12 months compared to those having zero or 1–4 sessions, suggesting 5–8 sessions to be the optimal dosage. It is not clear why service users who did not take up the intervention had similar PHQ9 outcomes to those who received 1 to 4 counselling sessions. One explanation could be service users with 0 counselling sessions did not, in-fact, need the service while those with 1 to 4 counselling sessions needed to continue with the counselling sessions. More robust methods are needed to investigate this further. Qualitative interviews with participants who received 5–8 sessions suggests that more sessions helped to promote greater attainment of skills to help negotiate life circumstances better. This finding is supportive of the NICE guidelines based on evidence from mostly high-income countries that recommends 6 to 8 sessions for low-intensity psychosocial intervention as optimal .Evidence shows that functional disability is strongly associated with depression –55. WhilExposure to more sessions also produced better benefits for self-stigma. There was a significant difference in reduction in internalized stigma between participants who attended 1–4 sessions compared to those who attended 5–8 sessions at endline. The fact that the session on internalized stigma was generally delivered later in the programme could potentially explain this finding. It is not clear why service users who did not take up the intervention had better self-stigma outcomes than those who received 1 to 4 counselling sessions. As with the PHQ9 outcomes it could be service users with 0 counselling sessions did not need the service while those with 1 to 4 counselling sessions needed to continue with the counselling sessions. More robust methods are needed to further investigate this. Self-stigma has a negative impact on self-esteem and self-efficacy and is considered a risk factor for poor mental health , 59. LitIn relation to social support, the overall OSS-3 mean scores showed no significant improvement for perceived social support. This is similar to Petersen et al.’s  findingsIn relation to reasons for greater counselling attendance, the qualitative process evaluation interviews revealed that availability of services, awareness of helpfulness of counselling sessions and counsellor qualities helped with attending more counselling session. It appears having insight, the process of becoming increasingly aware of one’s mental state , promoteIn addition, the referral process emerged as important in promoting greater uptake and adherence to the counselling sessions. Service users who had received 5–8 sessions reported that the referring nurse accompanied them to the counselling room or introduced them to the counsellor following the referral. This group also reported meeting with the counsellor at set times. In contrast, participants who did not take up the service and those who attended 1–4 sessions reported unexplained referral process, contextual issues making it difficult to attend counselling sessions, inconsistent appointments and not meeting with the counsellor on the day the referral was made. Linking service users with service providers can help with improving access to health services . From thFeeling better, contextual issues including having a day job, caring for sick family members and helping with funeral arrangements were reasons reported for dropping out before completion of counselling sessions. This tended to affect female service users who played the role of primary care giver more. Not only were their services seen as expendable, they were not able to attend to their own health care needs, with the needs of other family members outweighing their own.The findings of this evaluation of a lay-counsellor delivered psychosocial counselling intervention for chronic service users found that it helped reduce depression symptoms, at 12 months follow-up; with 5–8 sessions found to be optimal in this regard. Participants receiving this optimal number of sessions displayed improved functionality and reduced self-stigma, reported to have been empowered to self-manage, be more self-reliant, and capacitated with skills to improve their quality of life. Factors found to optimize attendance of counselling sessions included counsellors with person-centred care qualities, referral processes that included being provided with an explanation as to how counselling could benefit the service user by the referring nurse, and being connected to the counsellor either by being walked to the counselling room or introduced to the counsellor.


The role of PD-L1 as a prognostic and predictive biomarker is an area of great interest. However, there is a lack of consensus on how to deliver PD-L1 as a clinical biomarker. At the heart of this conundrum is the subjective scoring of PD-L1 IHC in most studies to date. Current standard scoring systems involve separation of epithelial and inflammatory cells and find clinical significance in different percentages of expression, e.g., above or below 1%. Clearly, an objective, reproducible and accurate approach to PD-L1 scoring would bring a degree of necessary consistency to this landscape. Using a systematic comparison of technologies and the application of QuPath, a digital pathology platform, we show that high PD-L1 expression is associated with improved clinical outcome in Triple Negative breast cancer in the context of standard of care (SoC) chemotherapy, consistent with previous findings. In addition, we demonstrate for the first time that high PD-L1 expression is also associated with better outcome in ER- disease as a whole including HER2+ breast cancer. We demonstrate the influence of antibody choice on quantification and clinical impact with the Ventana antibody (SP142) providing the most robust assay in our hands. Through sampling different regions of the tumour, we show that tumour rich regions display the greatest range of PD-L1 expression and this has the most clinical significance compared to stroma and lymphoid rich areas. Furthermore, we observe that both inflammatory and epithelial PD-L1 expression are associated with improved survival in the context of chemotherapy. Moreover, as seen with PD-L1 inhibitor studies, a low threshold of PD-L1 expression stratifies patient outcome. This emphasises the importance of using digital pathology and precise biomarker quantitation to achieve accurate and reproducible scores that can discriminate low PD-L1 expression. DNA-based high-throughput mutational analysis , 2 and hIt has long been thought that the ultimate cure for cancer would arise from harnessing the host immune system that, through its adaptive nature, could eradicate cancer cells even as they evolve. Indeed, in breast cancer, the presence of Tumour Infiltrating Lymphocytes (TILs) is a positive prognostic factor , 5 and aMost predictors of drug response also have a pure prognostic value. Indeed, PD-L1 expression has been studied in multiple cancer types and correlated with various clinicopathological parameters –19. In gAt the heart of this conundrum is the subjective scoring of PD-L1 IHC. Current standard scoring systems for PD-L1 as a companion diagnostic involve separation of epithelial and inflammatory cells and find clinical significance in percentages of expression above or below 1% , 35. CleWe conducted a retrospective case-control study involving a cohort of TNBC patients; ethical approval was obtained and tissue was acquired through the Northern Ireland Biobank (NIB ref: 15-0168). The dataset was compiled for a cohort of 109 patients and included a range of clinical and pathological parameters Supp . All of Original haematoxylin and eosin (H&E)-stained sections of all of the blocks were reviewed for tumour block selection. Following block selection, a new H&E section was cut and the slide was annotated for TMA construction. Where possible, from each selected donor block, nine representative areas were annotated for targeted coring – 3 tumour epithelial rich cores (T), 3 tumour cores from lymphoid rich areas (TL), and 3 stromal rich tumour cores (TS) in the 90-patient cohort. The TMA was constructed with 1-mm-diameter cores using a manual tissue arrayer  as described previously .The PD-L1 IHC Reference Standard consists of a genetically defined 11-spot custom cell line array (CLMA) with a range of negative, low, medium, and strong protein expressing cell lines developed using Horizon Discovery's gene editing technology. Parental cells were purchased from a commercial source and were characterised short tandem repeat (STR) analysis. Cell lines were engineered under the control of promoters of different strengths that resulted in a range of controlled PD-L1 protein expressing cell lines by IHC. Individual cell lines in the 11 spot CLMA were extensively characterized and verified using molecular assays, IHC, and quantitative digital pathology by Horizon Discovery.All IHC was performed in an integrated laboratory (Northern Ireland Molecular Pathology Laboratory) that has UK Clinical Pathology Accreditation . SectionTILs were assessed by three independent experienced immunohistochemists on H&E sections as recommended by the International TILs working group 2014 . TILs weDigital pathological analysis of the IHC stained TMA slides was performed using QuPath , 41, an Quantitation of PD-L1 was conducted as previously described . BrieflyManual chromogenic RNAScope for PD-L1  was performed on TMA sections as previously described . RNAscopLin's concordance analysis was used to examine the relationship between the PD-L1 scores using different antibodies with two-sided correlation coefficients reported. Results were interpreted using the cutoffs proposed by McBride . Fisher'In order to assess the reliability of PD-L1 analysis using the digital pathology platform, QuPath, we first utilised a genetically defined 11-spot custom cell line array PD-L1 IHC Reference Standard from Horizon Discovery . We compIn addition to IHC, PD-L1 may also be quantified using in situ hybridisation using RNAScope. In situ PD-L1 mRNA expression was not detectable in the null cell line standards (A1-2). As seen with the IHC experiment, more varied expression was observed for the low to medium expressing cell line standards . Increased expression was seen in the high cell line standards (C3-5) although there appears to be discordance between protein expression (measured by IHC) and mRNA (as measured by RNAScope) in 2 of the 3 high cell lines (C4-5) . This coBased on these results, we took forward the SP142 IHC on the Benchmark platform as well as the RNAScope assay for assessment in patient samples.In order to fully investigate the role of PD-L1 as a biomarker we initially focussed our assessment on TNBC as it demonstrates the greatest range of PD-L1 expression and most significant correlation with patient outcome. Therefore, we constructed a bespoke TMA that would allow us to compare not only the different technology platforms  but also different tumour areas. For each patient a total of nine cores were selected; three tumour rich cores (T), three tumour cores with high stroma (TS), and three tumour cores tumour from a lymphoid rich region (TL) .Microscopic IHC analysis with SP142 PD-L1 antibody on individual TMA cores demonstrated various expression patterns of PD-L1 in the different tissue compartments  PD-L1 expression across all 9 cores per patient. A threshold of 1% was used to stratify IHC-based PD-L1 expression as high (>1%) or low (<1%) in keeping with the clinically relevant thresholds reported in the literature with 44% of cases called PD-L1 positive using the SP142 antibody. Median expression was used to dichotomise the mRNA expression. While similar trends were observed with high PD-L1 expression associated with increased relapse free survival (RFS), only assessment of PD-L1 expression by IHC using the SP142 antibody was significant correlation (HR0.536 (95%CI 0.316-0.94) p=0.0294) . SimilarNext we wanted to compare the correlation with survival of PD-L1 expression in the different regions of the tumour . Galon and colleagues were the first to highlight the importance of the immune contexture showing that type, density, and location of immune cells within colon cancer predict clinical outcome , 50. TheFollowing on from this we wanted to further determine the importance of the cell context of PD-L1 expression. Therefore PD-L1 was categorised based on expression within the stroma (lymphoid cells) vs the tumour nest . Analysis was restricted to the tumour rich (T) cores given the significant correlation with clinical outcome. The range of expression was very similar and there was a highly significant correlation between stromal and tumour derived expression  . Further2 = 0.7456). Furthermore, as previously reported in the context of anthracycline based chemotherapy [2 = 0.151) and when analysed in the context of low (<1%) and high (>1%) PD-L1, the percentage of TILs observed was significantly higher in the high PD-L1 cases (p = <0.0001)  using the methodology recommended by the International TILs Working Group 2014  to reporotherapy , high TIotherapy . A weak <0.0001) . It is iIn order to further characterise the composition of the immune infiltrate in the context of PD-L1 expression, we utilised IHC and gene expression analysis that had been previously carried out on a subset 67/109) of the TNBC cohort  of the T and a siWe then wanted to extend the analysis of PD-L1 expression into other breast subtypes and this was achieved using an additional cohort accessed through the Northern Ireland Biobank (NIB). This was made up of 300 breast cancer patients, all of which were treated with FEC, creating a relatively uniform clinical cohort . Given tAs expected, high PD-L1 expression was associated with improved RFS and OS in TNBC patients (HR 0.355 (95%CI 0.148-0.84) p= 0.019 and HR 0.255 (95% CI 0.087-0.861) p=0.0269, respectively) . HoweverThis is an important finding for this poor outcome group of patients which represents 20-30% of all breast cancer cases and is associated with a disproportionately high number of deaths especially in the first five years following diagnosis.The role of PD-L1 as a prognostic and predictive biomarker is an area of great interest within oncology due to its association with the novel immune checkpoint inhibitors. It is also showing great promise in the treatment of a range of cancer types. However, inconsistent results have been obtained in the analysis of the PD-L1 biomarker. We propose that lack of an objective, reproducible and accurate approach to PD-L1 quantification and scoring may, at least in part, underlie this. Therefore, the aim of this study was to perform a comprehensive study of PD-L1 as a biomarker in breast cancer using automated tumour recognition and quantitative IHC image analysis together with objective scoring systems on uniform patient tissue cohorts with extensive clinical annotation. We show that the use of different antibodies during IHC can have a significant impact on the detected PD-L1 expression and its association with clinical outcome. This was compared to mRNA expression as determined by RNAScope. We show here that high PD-L1 expression, as measured by IHC using the Ventana SP142 clone on a Ventana automated platform and quantified using the QuPath analysis platform, is significantly associated with better clinical outcome in TNBC treated with current standard of care chemotherapy. This observation was confirmed using a second TMA in which a large proportion of the TNBC cases were replicated. When immune topography was taken into account, tumour rich cores showed the greatest predictive power with analysis of PD-L1 expression regardless of cell type  demonstrating the most clinical relevance. Looking at breast cancer as a whole, there was also an association between high PD-L1 expression and improved clinical outcome. Moreover, we show for the first time that PD-L1 expression significantly predicts clinical outcome in all ER negative patients, including HER2 enriched disease.This study highlights the potential risk of obtaining varied results when investigating the role of PD-L1 as a biomarker and may explain some of the inconsistencies within the field. As previously mentioned, there is a range of commercially available antibodies for PD-L1 IHC studies. Mahoney et al. have shown that the region of PD-L1 against which the antibody is raised can have a significant impact on the staining patterns observed . They coCurrent standard scoring systems for PD-L1 as a companion diagnostic to immunotherapy find clinical significance in percentages of expression above or below 1% . We alsoThis sensitivity and specificity has allowed us to elucidate a role for PD-L1 in predicting response in patients within the ER negative HER2 enriched subgroup. To the best of our knowledge, this is the first time the utility of PD-L1 as a biomarker has been demonstrated to predict improved outcome in this subgroup. Along with TNBC, this is a subgroup of unmet clinical need. While use of HER2 targeted agents has had undeniable clinical benefit, response rates are low with best responses rates (~50%) observed in the neo-adjuvant setting with a combination of targeted agents , 60. FurOf interest, high levels of TILs are associated with better response to Trastuzumab in both the neo-adjuvant and adjuvant setting potentially associated with antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism of action of the drug . FurtherMuenst et al.  previousIn addition to highlighting the novel role of PD-L1 in predicting outcome in ER-negative breast cancer, this study has also shown that high PD-L1 expression is associated with improved outcome in breast cancer as a whole. This is in contrast to the overall findings from two recent meta-analyses showing high PD-L1 expression associated with poor outcome , 66. HowThe finding that PD-L1 expression, regardless of the cell type , is prognostic is keeping with the guidelines for the companion assay for Atezolizumab using SP142 . FurtherThis study highlights the need for high quality information at multiple levels  in order to accurately interpret the utility of a biomarker. We have shown the impact of high-resolution image analysis on PD-L1 interpretation from antibody choice to IHC versus mRNA-based quantification. This comprehensive comparison would not have been possible without the objective, reproducible and accurate PD-L1 quantification provided by our digital pathology platform QuPath. We believe this study provides vital information impacting on the clinical delivery and interpretation of PD-L1 as a biomarker and provides clarity to the role of PD-L1 in the prediction of response to standard of care in breast cancer.


Non-toxic liquid-metals offer a viable solution for flexible electrodes due to deformability and low bulk resistivity. However, fabrication processes utilizing liquid-metals suffer from high complexity, low throughput, and significant production cost. Our team utilized an inexpensive spray-on stencil technique to deposit liquid-metal Galinstan electrodes in top-gated graphene field-effect transistors (GFETs). The electrode stencils were patterned using an automated vinyl cutter and positioned directly onto chemical vapor deposition (CVD) graphene transferred to polyethylene terephthalate (PET) substrates. Our spray-on method exhibited a throughput of 28 transistors in under five minutes on the same graphene sample, with a 96% yield for all devices down to a channel length of 50 μm. The fabricated transistors possess hole and electron mobilities of 663.5 cm Since the 1960’s, integrated circuit electrodes have been fabricated from traditional copper, nickel, silver, and gold metals . As the 6 S/m, a desirable vapor pressure (<1 × 10−6 Pa at 500 °C) compared with mercury (0.1713 Pa at 20 °C), and a stable liquid state across a broad temperature range (−19 °C to 1300 °C) [Liquid-Metal (LM) Galinstan is a commercially available eutectic alloy comprising of 68% gallium, 22% indium, and 10% tin that exhibits a conductivity of 2.30 × 101300 °C) ,5.To deposit liquid-metal on electronic devices, mask deposition and microcontact printing have been adopted to improve manufacturing yield to over 95%, while simultaneously decreasing feature size down to 1 µm ,11,12. SIn this article, we demonstrate the use of a low-cost novel spray-on deposition technique for LM using off-the-shelf equipment that improves upon contemporary deposition techniques. We demonstrate the utility of the spray-on deposition of liquid-metal with a proven flexible material combination, consisting of Galinstan source and drain electrodes, an electrolytic gate comprised of honey, and a graphene channel that forms a flexible graphene field-effect transistor . GalinstIt is important to note the quality of monolayer graphene produced commercially has been an issue when fabricating graphene devices, and verification of quality is mandatory before experimentation . The RamTo complete the three-terminal graphene field-effect transistors (GFET) device, honey was loaded into a syringe and drop-casted between each electrode pair to act as an electrolytic gate dielectric, as seen in gV) sweep from ±5 V with a drain voltage (dV) of 10 mV and the charge transport characteristics were plotted in To demonstrate the utility of our spray-on LM technique, graphene charge transport characteristics for several GFET devices with varying channel lengths  were extracted from Current-Voltage (I-V) measurements taken via an Agilent 4155C Semiconductor Parameter Analyzer  and probe station. Each GFET device was subjected to a gate voltage , that can be actuated by altering the gate voltage [The ambipolar nature of all graphene field-effect transistors is made clear with the V-shape in dulation . Notice c oxygen . Therefoc oxygen . To our  voltage ,24,25.The high gate-leakage current density at the smaller channel lengths may be due to the reduced electrode separation distance between the graphene-dielectric interface. It is noted that in the aforementioned process, the PET stencil did not form an ideal, airtight contact with the graphene in all devices during fabrication. Therefore, there were stray microscale LM-spray residues deposited under the masked areas, and in some cases shorted the source and drain electrodes. In addition, for ease of fabrication, the honey dielectric was drop-casted by hand and applied unnecessary pressure to the LM electrodes causing the LM to move, hence the failure of our methods to produce repeatable working devices at the 50 μm channel length size. Due to these circumstances, there was additional stray conductance between the source/drain electrodes and gate electrode, and the additional gate-leakage current density at smaller channel lengths is to be expected. However, the readers are encouraged to optimize this spray-on process with more ideal stencil-mask materials that provide stronger adhesion to graphene, and produce minimal LM residue. Additionally, other liquid dielectrics, such as lower-conductivity ionic gels, can be utilized in place of honey. Lastly, traditional back-gated graphene field effect transistor topologies can be utilized to eliminate stray conductance produced from overlap of the top-gate dielectric.mg) of a 1 mm GFET, The transconductance (Transconductance:Electron and Hole Mobility:The extracted hole and electron mobilities measured for the GFET devices are shown in It has been demonstrated that our spray-on LM method is a viable technique to fabricate nanomaterial devices. We have shown that top-gated graphene field-effect transistor devices with feature sizes down to 100 μm can be fabricated and exhibit standard charge transport characteristics with minimal effort. All devices were produced in a time frame of 30 s per transistor with a reliability of 96%. Our methods can be improved with fully-automated spray and drop-casting techniques, or even with back-gated devices, to improve the speed and reliability of this method. Furthermore, a strong-adhesion stencil mask and higher-resolution stencil-cutter can be used to significantly improve the minimum channel length achieved in this first attempt. This technique, with further modification, can be used inexpensively to mass-produce nanomaterial devices.


The cytoplasmic accumulation of the nuclear TAR DNA-binding protein 43 (TDP-43) is a pathologic hallmark in amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and other neurological disorders. However, most transgenic TDP-43 rodent models show predominant nuclear distribution of TDP-43 in the brain. By expressing mutant TDP-43 (M337V) in the brains of rhesus monkeys and mice, we verified that mutant TDP-43 is distributed in the cytoplasm of the monkey brain and that the majority of mutant TDP-43 remains in the nuclei of the mouse brain. The primate-specific caspase-4, but not mouse homologue caspase-11, could remove the NLS-containing N-terminal domain and generate fragmented TDP-43 that accumulates in the cytoplasm. Moreover, increased expression of caspase-4 in the monkey brain promotes the cytoplasmic accumulation of endogenous TDP-43, and suppressing caspase-4 reduces the cytoplasmic distribution of endogenous TDP-43 in cultured human neural cells. Our findings suggest that primate-specific caspase-4-mediated cleavage of TDP-43 accounts for its cytoplasmic mislocalization in the primate brains and may serve as a potential therapeutic target.The online version of this article (10.1007/s00401-019-01979-0) contains supplementary material, which is available to authorized users. Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, characterized by muscle weakness due to the degeneration of large motor neurons. TAR DNA-binding protein of 43 kDa (TDP-43) accumulates and forms aggregates in the brains of patients with ALS and frontotemporal lobar degeneration (FTLD) , 7, 35. The cytoplasmic distribution of mutant TDP-43 appears to be species dependent. This is because most transgenic mouse models of ALS show the predominant nuclear localization of transgenic TDP-43 , 59 and 13 vg/ml for AAV-hTDP-43, 2.3 × 1013 vg/ml for AAV-GFP, and 1.8 × 1013 vg/ml for AAV-caspase-4. The following antibodies were used: mouse anti-human TDP-43  for the N-terminal TDP-43 (1-261 amino acids), rabbit anti-TDP-43  for a synthetic peptide corresponding to residues surrounding Gly400 of human TDP-43, rabbit anti-phospho-TDP-43 (Ser409/Ser410) , rabbit anti-LSD1 , rabbit anti-COX-IV , rabbit anti-PDI , rabbit anti-HSP90 , mouse anti-GFP , rabbit anti-NeuN , rabbit anti-GFAP , rabbit anti-caspase-4  and mouse anti-GST . All secondary antibodies were purchased from Jackson Immuno-Research Laboratories. Inhibitors of caspase family members included ZVAD-fmk (Selleckchem), DEVD-fmk (Tocris Bioscience), LEHD-fmk (Tocris Bioscience), and LEVD-fmk (BioVision).The human mutant TDP-43(M337V) cDNA , 59 was Monkeys were housed in accordance with Chinese National standards, which are consistent with the standard set forth in the eighth edition of the NRC Guide for the Care and Use of Laboratory at the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Peking Union Medical College, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), International. The animal use and experiments followed the protocol that was approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences. This study was conducted in strict compliance with the “Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Science (est. 2006)” and “The use of non-human primates in research of the Institute of Laboratory Animal Science (est. 2006)” to ensure the personnel safety and animal welfare.All mice (C57BL/6) were bred and maintained in the animal facility at Emory University under specific pathogen-free conditions in accordance with institutional guidelines of the Animal Care and Use Committee at Emory University. The studies followed the protocol approved by the Animal Care and Use Committee at Emory University.n = 12 each group, six males and six females per group) were anesthetized by i.p. injection of 2.5% Avertin, and their heads were placed in a Kopf stereotaxic frame (Model 1900) equipped with a digital manipulator, a UMP3-1 Ultra pump, a 10 μl Hamilton microsyringe. A 33G needle was inserted through a 1 mm drill hole on the scalp. Injections occurred at the following stereotaxic coordinates: 3.1 mm posterior to bregma, 1.0 mm lateral to the midline, 4.7 mm ventral to the dura, with bregma set at zero. The microinjections were carried out at a rate of 0.2 μl/min. The microsyringe was left in place for an additional 10 min before and after each injection. 0.5 μl AAV-TDP43, AAV-caspase-4 or AAV-GFP virus was stereotaxically injected into the right substantia nigra of mice. Viral injection of monkey brains was performed using the facilities at Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Peking Union Medical College.Adult wildtype C57BL/6 mice at 8 months of age (n = 6 each group). Each monkey was anesthetized by intraperitoneal injection of 0.3–0.5 mg of atropine, followed by 10–12 mg of ketamine, and 15–20 mg of pelltobarbitalumnatricum per kg body weight. The monkeys were stabilized on a stereotaxic instrument (David Kopf). The precise position of substantia nigra for stereotaxic injection was located by MRI before injection. Ten μl viruses were injected at five different locations in the right substantia nigra  in which the injection site was determined and the depth of needle insertion was calculated from the pre-operatively taken MRI. After the injection was given for 12 weeks, the monkeys with substantia nigra injections were subjected to behavioral analysis, and at the end of behavioral analysis, their brain tissues were isolated for immunohistochemical analysis. We also injected AAV-caspase-4 viruses (10 μl) into the prefrontal cortex of three monkeys. After 12 weeks, we isolated the injected brain tissues for studying the effect of caspase-4 on the cleavage of TDP-43.For AAV viral injection into the monkey brain, we injected AAV-GFP or AAV-TDP-43 into the substantia nigra of male monkeys at the age of 8–12 years . The balance beam apparatus consists of 1 m beams with a flat surface of 12 mm or 6 mm width resting 50 cm above the table top on two poles. A black box at the end of the beam is the finish point. Nesting material from home cages in the black box serves to attract the mouse to the finish point. A lamp serves as an aversive stimulus, shining light above the start point. The time required for a mouse to cross to the center (80 cm) is measured by two motion detectors: one at 0 cm that starts a timer and one at 80 cm that stops the timer. The video camera records the performance.All animal tests were performed in accordance with NIH guidelines for procedures and approved by the Institutional Animal Care and Use Committee of Emory University. Mouse behavior was assessed using a rotarod . Mice (C57BL/6) were trained for 10 min on three consecutive days with the rotarod speed at 5 rpm, and testing commenced after 3 days. The speed of the rod was set to 5 rpm and increased by 0.1 rpm/s. Each mouse went through three trials, and the average data of each group charted. The moribund mice were scored as “dead” and euthanized, and tissues were collected  who died at 67–74 years of age and were confirmed to have TDP-43 aggregates via postmortem histologic analysis. Non-ALS control tissues were obtained from neurologically unaffected patients  who died at 53–92 years of age.g for 3 min, and then placed for initial growth in a 50% glial-conditioned medium . The cells were cultured in neurobasal/B27 medium.Mouse neural crest-derived N2A cell line and human neuroblastoma SH-SY5Y cell line were purchased from ATCC and cultured in DMEM/F12 medium . Medium was changed every 2 days. For culturing mouse primary neuronal cells, cortical neurons were isolated from the cortex of postnatal day 1 mice. Dissected tissue was treated with 0.0625 mg/ml trypsin and 0.0625 mg/ml DNasein 1 × HBSS buffer without calcium and magnesium for 10 min at 37 °C. Cells were washed once with the tissue culture medium, centrifuged at 1500 × For analyzing TDP-43 expression and distribution in the monkey brain, we used three AAV-TDP-43 or AAV-GFP monkeys for Western blotting and another three AAV-TDP-43 or AAV-GFP monkeys for immunocytochemical studies. Animals were anesthetized and perfused with 10 ml 0.9% NaCl, and then with 20 ml of 4% paraformaldehyde in 0.1 M PBS through the left cardiac ventricle. Brains were removed and fixed overnight in the same solution and cryopreserved with 15% and 30% sucrose before sectioning into 10 µM sections with a cryostat (Leica CM1850) at − 20 °C. Sections from monkey or mouse brains or cultured cells were fixed in 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 in PBS for 30 min, blocked with 3% normal donkey serum in 3% BSA for 1 h, and incubated with primary antibodies in 3% BSA overnight at 4 °C. After several washes with PBS, the brain sections or fixed cells were incubated with secondary antibodies conjugated with either Alexa-488 or Alexa-594 (Invitrogen). 0.01 μg/ml DAPI was used to label the nuclei. Fluorescent images were taken with a Zeiss Axiovert 200 MOT microscope of the 40 ×/0.6 lens or 63 ×/0.75 lens, equipped with a digital camera  and Openlab software (Improvision). The immunostaining analysis of TDP-43 subcellular distribution in the injected monkey or mouse brains was performed completely blinded on standardized 40 mm sections. The monkey brain sections were prepared using a brain slicer including the injected regions . Each brain region was used to take at least six images (40 × magnification) that can clearly reveal the subcellular distribution of TDP-43. For the quantitative analysis of differential subcellular location of TDP-43 in the monkey and mouse brain, the numbers of cells showing the nuclear or cytoplasmic TDP-43 per image were presented as the mean ± SEM, and the quantitative data were obtained from three monkeys or six mice per group. Densitometry analyses of fluorescent intensities of aggregates were quantified by ImageJ software .3, 2 mM ATP, pH 8.0) containing protease inhibitor (Roche) and 100 μM PMSF. Ten percent lysates were stored as the total lysate sample. Nuclei and cellular debris were pelleted (P1) at 800 × g for 5 min. The supernatant (S1) was transferred to a new tube and centrifuged at 20,000 × g for 30 min at 4 °C to obtain the mitochondria-enriched pellet (P2). The supernatant (S2) was then used for the soluble cytoplasmic fraction. The S2 was centrifuged at 100,000 × g for 30 min at 4 °C to obtain the endoplasmic reticulum-enriched pellet (P3). Crude nuclear pellets were washed four times with ice-cold homogenization buffer to remove cytoplasmic contaminants. For nuclear purification, the pellets were re-suspended in 374 μl of buffer  with 26 μl of 4.6 M NaCl to generate the final concentration at 300 mM NaCl, homogenized with 20 full strokes in Teflon homogenizer on ice, and sonicated for 10 s. The homogenized samples were kept on ice for 20 min and then centrifuged at 24,000 × g for 20 min at 4 °C.Monkey or mouse brain tissues were homogenized for 25 strokes with a dounce homogenizer ice-cold buffer . Equal amounts (10 μg) of the extracts were incubated with corresponding substrates in 100 μl caspase-4 activity assay buffer  for 30–60 min at 37 °C. Cold water (0.8 ml) was used to stop the reactions, and the reaction mixtures were iced for at least 10 min. The free AFC fluorescence was quantified using the CytoFluor multi-well plate reader  with excitation and emission wavelengths at 400 nm and 500 nm, respectively. All readings were standardized using the fluorescence intensity of an equal volume of free 7-amino-4-trifluoromethylcoumarin (AFC) solution (40 mM), normalized by the protein concentrations and expressed as nmol/min/mg protein.2, 100 µg/ml purified rabbit creatine kinase, 50 mM phosphocreatine, 1 mM ATP, pH 7.6). Monkey or mouse tissues form the brain striatum and cortex were homogenized at 1 g/ml in cold assay buffer using 20 strokes of a glass dounce hand homogenizer and were centrifuged at 500 × g for 5 min at 4 °C to pellet unbroken tissues and membranes. The supernatant was collected and stored on ice, while protein concentrations were determined using a BCA Protein Assay Kit (Thermo Scientific). The lysates (200 μl) at 0.5 mg protein/ml were incubated with GST-TDP-43 beads (200 μl) at 37 °C with 300 rpm shaking for 24 h. The beads were centrifuged and combined with the protein loading dye (0.2% SDS) for SDS-PAGE and Western blotting analysis to detect expression of the cleaved GST-TDP-43 using an anti-GST antibody. An anti-C-terminal TDP-43 antibody was used to detect the presence of C-terminal TDP-43 in the supernatant. Different caspase inhibitors at the concentrations (50–100 μM) were included in the lysates to inhibit caspase activity during the incubation process with GST-TDP-43 beads.Purified GST-mut-TDP-43 (M337V) in Sepharose beads were diluted in cold assay buffer  according to the manufacturer’s protocol. At 48 h following transfection, cells were harvested for immunofluorescent staining and Western blotting. For caspase-4 knockdown experiment, the cultured human neural SH-SY5Y cells were transiently transfected with caspase-4 siRNA (Gene Pham Co. sequence GUGUAGAUGUAGAAGAGAAtt or AAGUGGCCUCUUCACAGUCAUtt) or control siRNA (scrambled sequence) using RNAi Max transfection reagent (Invitrogen) according to the manufacturer’s protocol. At 48 h following transfection, cells were harvested for immunofluorescent staining and Western blotting.4 cells/100 µl/well) was dispensed in a 96-well plate. Each well of the plate was added with 10 µl CCK-8 solution, and incubated for 2 h at 37 °C. The absorbance values at the wavelength of 450 nm were spectrophotometrically measured using an CytoFluor multi-well plate reader . Each sample was tested in three replicates. SH-SY5Y cells without transfection and tunicamycin treatment were used as negative and blank controls. The values (mean ± SEM) of treated SH-SY5Y cells were calculated as  % of control cells.The cell viability assay was done using Cell Counting Kit-8 (CCK-8) , which determines the number of viable cells in proliferation and cytotoxicity. It utilizes tetrazolium-8-[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium] monosodium salt that produces a water-soluble formazan dye in living cells. The amount of this dye is directly proportional to the number of living cells, offering a sensitive assay to detect viability of many cell lines including SH-SY5Y cells , 56. Brit test for comparing two groups. When analyzing multiple groups, we used one-way ANOVA to determine statistical significance. For mice or monkey that was repeatedly subjected to behavioral tests, we analyzed the data using two-way ANOVA. Data are mean ± SEM. Calculations were performed with GraphPad Prism software.Statistical significance was assessed using the two-tailed Student’s Our previous studies demonstrated that stereotaxic injection of mutant TDP-43 in the striatum of mice led to abundant nuclear TDP-43 in the injected brain . Using aThree months after the injection, the AAV-GFP monkeys did not show any motor symptoms, but all the TDP-43-injected monkeys developed the obvious weakness of the left upper limb, whose movement is controlled by the contralateral right substantia nigra that was injected with AAV-TDP-43 Fig. b. These The neurological phenotypes of the TDP-43-injected mice and monkeys confirmed the neurotoxicity of overexpressed mutant TDP-43 and also allowed us to compare TDP-43’s expression and localization in these two different species. The brains of mice and monkeys were dissected after euthanization and prepared for Western blotting and immunocytochemistry. We used an antibody against a C-terminal peptide corresponding to residues surrounding Gly400 of human TDP-43 to detect the overexpressed exogenous TDP-43. Immunocytochemistry revealed that injection of the control AAV-GFP yielded diffuse green fluorescence throughout the entire cell . However, the injected mutant TDP-43 was diffusely distributed in the cytoplasm or adjacent to the nuclei in the monkey brain Fig. a. In conThe above findings have shown that different subcellular localization of TDP-43 is species-dependent even when mutant TDP-43 is overexpressed. To investigate whether there is any difference in TDP-43 cleavage, we performed Western blotting using anti-C-terminal TDP-43 antibody. Interestingly, truncated TDP-43 fragments 35- and 25-kD) were abundant in the monkey brain, whereas full-length TDP-43 is expressed at a comparable level as endogenous TDP-43 in the AAV-GFP-injected monkey. However, in the mouse brain, full-length TDP-43 was much more abundant than C-terminal truncated TDP-43, which is consistent with the previous findings from transgenic TDP-43 mice , 52, 55.5-kD wereThe above striking difference led us to examine the differential processing of TDP-43 in the monkey and mouse brains. TDP-43 consists of 414 amino acids with NLS in the N-terminal region and NES in the region close to the C-terminus  that was produced in bacteria and then purified Fig. b, c and In vitro experiments revealed that multiple caspases  cleave TDP-43 in cultured cells , 29, 42.Caspase-4 is an enzyme that cleaves proteins proteolytically at an aspartic acid residue and contributes to inflammatory activation in mono-myelocytic cells , 50. IntCaspase-4 is bound to the endoplasmic reticulum (ER) and can be activated under ER stress . PreviouA previous study reported the increased amounts of caspase-4 in the spinal cords of ALS patients . WesternIf caspase-4 is absent in the mouse brain, but is responsible for the cytoplasmic accumulation of TDP-43, transgenic expression of caspase-4 in the mouse brain should also lead to the cytoplasmic accumulation of TDP-43. We generated AAV vector-expressing human caspase-4 under the control of the ubiquitin promoter Fig. a and conTo provide direct evidence for the causative role of casapse-4 in the cleavage of TDP-43 in cells, we used siRNAs to knockdown the expression of endogenous caspase-4 in cultured human neural SH-SY5Y cells. The siRNA (siCAS4-1) had higher efficiency on silencing endogenous caspase-4 than siCAS4-2 . The siCAS4-1 was then transfected into SH-SY5Y cells for 48 h, and the transfected cells were treated with tunicamycin (1 μg/ml) for 12 h, which is a known ER stress activator for caspase-4 induction , 58. As If TDP-43 is cleaved by activated caspase-4, increased activity or expression of caspase-4 should promote the cytoplasmic distribution of endogenous TDP-43 in the monkey brain. To test this idea, we injected the same titer of AAV-caspase-4 or control AAV-GFP into the prefrontal cortex in 11-year-old wildtype monkeys. After 3 weeks, the injected brain prefrontal cortex was isolated for immunofluorescent staining with antibodies to caspase-4 and C-terminal TDP-43. Double immunofluorescent staining showed that endogenous TDP-43 in the normal monkey brain is predominantly distributed in the nucleus Fig. a. HoweveMislocalization of TDP-43 in the cytoplasm and loss of its nuclear distribution are the major pathological hallmarks in ALS and FTLD , 15, 35 TDP-43 is a major component of cytoplasmic aggregates in the brains and spinal cords of nearly all patients ~ 97%) with ALS and in ~ 45% of FTLD cases % with AL, 35, 40.Recent studies suggest that impaired nuclear–cytoplasmic transport contributes to ALS. The mutations in C9orf72 mRNA, which can also cause ALS by impairing the nuclear–cytoplasmic transport , 62, canIn the current studies using non-human primates, we identified four lines of evidence supporting caspase-4 as a critical contributor to the cytoplasmic accumulation of TDP-43 in the primate brains. First, caspase-4 is found in non-human primates and humans, but not in mice. Second, caspase-4 cleaves TDP-43, but its mouse homologue caspase-11 does not. Third, co-expression of caspase-4 with mutant TDP-43 in the mouse brain leads to the cytoplasmic redistribution of TDP-43. Lastly, overexpression of caspase-4 can increase the cytoplasmic distribution of endogenous TDP-43 in the monkey brain, whereas suppressing caspase-4 expression can reduce the distribution of endogenous TDP-43 in the cytoplasm in cultured human neural cells.In vitro studies demonstrated that TDP-43 was initially cleaved after Asp174 by caspase-4 , which cIt should be pointed out that overexpression of TDP-43 can only partially mimic ALS pathology that is caused by endogenous mutant TDP-43. Similarly, neurodegeneration in TDP-43 transgenic mice may not depend on TDP-43 cleavage and is mediated by different mechanisms. The critical role of nuclear TDP-43 in gene transcription and RNA processing is well documented , 39, 47.Our studies indicate that caspase-4 plays a critical role in the cytoplasmic accumulation of TDP-43. The relevance of this finding to other pathological conditions is also supported by the regulation of caspase-4 activity and expression. Caspase-4 is an endoplasmic reticulum (ER) membrane-bound enzyme and is activated under ER stress , which cSupplementary material 1 (PDF 1272 kb)Supplementary material 2 (MP4 489 kb)Below is the link to the electronic supplementary material.