OPTN, HNRNPA1 or TARDBP mutations. ALS-FUS and ALS-SOD1 showed less cortical proteinopathy in relation to spinal cord pathology than ALS-TDP, where pathology was more evenly spread across the motor cortex-spinal cord axis. Neuronal pTDP-43 aggregates were rare in GAD67+ and Parvalbumin+ inhibitory interneurons, consistent with predominant accumulation in excitatory neurons. Finally, we show that cortical microglia, but not astrocytes, contain pTDP-43. Our findings suggest divergent quantitative, genotype-specific vulnerability of the ALS primary motor cortex to proteinopathies, which may have implications for our understanding of disease pathogenesis and the development of genotype-specific therapies.Degeneration of the primary motor cortex is a defining feature of amyotrophic lateral sclerosis (ALS), which is associated with the accumulation of microscopic protein aggregates in neurons and glia. However, little is known about the quantitative burden and pattern of motor cortex proteinopathies across ALS genotypes. We combined quantitative digital image analysis with multi-level generalized linear modelling in an independent cohort of 82 ALS cases to explore the relationship between genotype, total proteinopathy load and cellular vulnerability to aggregate formation. Primary motor cortex phosphorylated (p)TDP-43 burden and microglial activation were more severe in sporadic ALS-TDP disease than C9-ALS. Oligodendroglial pTDP-43 pathology was a defining feature of ALS-TDP in sporadic ALS, C9-ALS and ALS with  C9ORF72 (C9-ALS)  = 0.53, p = 0.03] = 0.62, p = 0.004], however this relationship was not present in C9-ALS  = 0.62, p = 0.0037] and FUS cases , but not other genotypes (p > 0.2) is the main inhibitory neurotransmitter in the brain. It is produced by glutamic acid decarboxylases (GADs) GAD65 and GAD67, of which GAD67 is generally constitutively active and produces > 90% of basal GABA levels . GAD67 pn = 7, control n = 3; see The main role of oligodendroglia is the support of neurons, either directly as cortical satellite glia or via the production of insulating myelin. As pTDP-43 accumulation is present within oligodendrocytes and their dysfunction has been implicated in pathogenesis of ALS , we sougn = 10) and C9-ALS cases (n = 10)(Olig2 is a transcription factor highly expressed in immature oligodendrocyte precursor cells (OPC)Fig. a while T (n = 10). Both th (n = 10)g and h. Microglia may phagocytose inclusions or cells that have been suitably marked for destruction as part of the innate immune system. As microglial activation correlates closely with the extent of pTDP-43 deposition Fig. , we nextBetz cells are giant pyramidal neurons unique to the primary motor cortex that reside within layer Vb, and can be distinguished histologically by their large size, accumulation of intracellular lipofuscin, and circumferential somatodendritic architecture. Similar to a previous study , we obseTogether, these results highlight cortical pTDP-43 oligodendrocyte pathology as a defining feature of ALS-TDP across all studied genotypes, and suggest a clear neuronal/glial subtype specific vulnerability to proteoaggregation in the ALS primary motor cortex.We report, to our knowledge, the first large-scale, digital microscopy-guided neuropathological analysis of the burden and pattern of proteinopathies of the primary motor cortex in ALS since the discovery of the main disease defining genotypes. A strength of our study is the combined use of image-analysis algorithms with statistical modelling to allow a comprehensive, fully quantitative approach to pathological assessment. We consider these quantitative traits to be more proximally related to genetic variables and pathobiology than clinical data (e.g. UMN vs. LMN predominance), which were limited, not quantitative and unlikely to be reflective of neuraxis involvement at the end stage of the disease. Importantly, the genetic stratification of our cohort allowed us to begin to assess ALS neuropathological endophenotypes, the utility of which has been demonstrated in other settings, such as frontotemporal lobar degeneration and hippocampal sclerosis , 27, 53.Our dataset stems from an independent, previously unpublished cohort of individuals with ALS stratified by genotype. It allows us to contribute several important findings to the field: First, in the group of ALS associated with phosphorylated (p)TDP-43 proteinopathy, sporadic disease was characterised by a higher pTDP burden in the motor cortex than C9-ALS, and all genotypes in this group demonstrated oligodendroglial pTDP-43 inclusion body pathology. Second, overall motor cortex proteinopathy burden of ALS-FUS and ALS-SOD1 is less severe than that of ALS-TDP. Third, inhibitory interneurons appear to be less prone to accumulation of microscopic pTDP-43 aggregates than excitatory neurons.TARDBP mutation case assessed ; however, we observed a trend towards higher oligodendroglial pTDP-43 burden in C9-ALS.A key point in discussions concerning selective vulnerability is the distinction between cellular vulnerability to proteinopathy, and vulnerability to degeneration itself. The toxicity of TDP-43 has been demonstrated in vitro through a range of mechanisms including its effects on endocytosis, autophagy and stress-granule formation , 37, 45.sed Fig. , however in mice , and clig et al. , using iSOD1G93A mouse model [We detected a differential vulnerability of specific cell types to the aggregation of pTDP-43. This can be summarised as follows: excitatory neurons within the motor cortex are more prone to form pTDP-43 aggregates than inhibitory interneurons, and oligodendroglial, but not astroglial, pTDP-43 pathology should be considered a defining feature of ALS-TDP in cases with and without proven genetic aetiology. We found evidence of oligodendrocyte pathology in every ALS-TDP case assessed in this study, demonstrating cortical oligodendroglial proteinopathy as a defining feature of the disease. However, the mechanisms by which oligodendroglial aggregations potentially cause dysfunction are unclear. While the cellular origin of TDP-43 pathology in humans is not known, studies have suggested that TDP-43 can be transmitted between cells both in culture and in vivo , 63. It se model , 61. WheC9ORF72 is highly expressed in myeloid cells [OPTN results in dysmyelination and axonopathy [SOD1 and FUS cases, which exhibited a relatively low extent of cortical proteinopathy, while the highest microglial response was seen in sporadic and C9ORF72 cases, which on average demonstrated a higher burden of concomitant pTDP-43. This suggests that TDP-43 proteoaggregation specifically determines the extent of the microglial response, rather than the expression of a mutant protein within microglia themselves. We found several examples of pTDP-43 aggregates within microglia  and Alzheimer’s disease , but sucIn summary, we used a quantitative immunohistochemical approach to comprehensively analyse pathology within the primary motor cortex in a large cohort of genetically-defined ALS cases. Our results demonstrate a clear genotype-specific vulnerability to ALS proteinopathy, which may provide targets for the design of future therapeutics through genotype-specific amelioration of cortical pathology.Additional file 1 Supp Fig. 1: Additional staining of pathology in the motor cortex and spinal cord. Staining for OPTN was highly positive in all neurons in controls (A), but absent in an OPTN mutation case (D), reflecting the truncation of the protein upstream of the antibody epitope. Asterisks in (B) mark the presence of Betz cells. Arrows in (E) mark mislocalized and aggregated FUS inclusions. Arrows in (F) mark the presence of aggregated SOD1 protein in a case with the SOD1 D101G mutation (case 31). As discussed in the text, antibody SOD1 SPC-206 did highlight solid compact and skein aggregates but had strong background staining (C), making it unsuitable for quantitative automated image analysis. P62 was used instead as a marker for compact SOD-1 associated protein aggregates; granular aggregation of misfolded wild-type SOD1, which has been suggested to be present in all genotypes of ALS, was not revealed by p62 immunohistochemistry [Supp Fig. 2: Significant variation in the extent of pTDP-43 pathology between TARDBP and OPTN ALS mutation cases. pTDP-43 pathology in the TARDBP case was sparse, and consisted almost exclusively of compact NCI . In contrast, pathology was severe and widespread in the homozygous ALS-OPTN case , with NCI, oligo inclusions and dystrophic neurites in all layers, including the subcortical white matter (e). Supp Fig. 3: CD68 staining between the primary motor cortex (a) and lumbar spinal cord (b) white and grey matter is positively correlated in all the genotypes tested; Pearson r, results as on figure. Best fit lines are manually added for illustrative purposes. Supp Fig. 4: Assessment of anterior horn neuron size. Anterior horn degeneration and shrinkage was most prominent in FUS (b) and SOD1 cases, and noticeably less severe in the single ALS-OPTN case (c), however there was significant intraindividual differences within genotypes (d). pTDP-43 aggregation in the anterior horn did not correlate with anterior horn neuron shrinkage/loss in either sporadic (blue dots) or C9ORF72 disease (red squares) (e). Supp Fig. 5: Calculated predominance ratios for single-IHC cohort using CD68 as a surrogate marker of neurodegeneration. Ratios were calculated by dividing the log expression of motor cortex CD68/mm2 by the log expression of anterior horn CD68/mm2. Lower ratios therefore represent a higher LMN burden of activated microglia. Supp Fig. 6: Betz cells occasionally display pTDP-43 aggregation but also occasionally nodular microgliosis and neuronophagia. MxIF analysis did not reveal significant evidence of pTDP-43 within Betz cells , but it can occasionally be seen in some cases . However we did also find evidence of nodular microgliosis surrounding large layer V neurons in some cases (e). UL = upper layers, DL = deeper layers. Scale bars where not indicated (μm): a = 40, b,e = 50. Supp Table : Numerical results for Olig and TPPP/p25 quantification.hemistry . Scale bion case 1. As disAdditional file 2.


Methods: The fluidity was altered by chemically crosslinking the polymer networks. The crosslinked P(CL-co-DLLA) substrate showed a solid-like property with a stiffness of 261 kPa, while the non-crosslinked P(CL-co-DLLA) substrate of 100 units (high fluidity) and 500 units (low fluidity) existed in a quasi-liquid state with loss modulus of 33 kPa and 30.8 kPa, respectively, and storage modulus of 10.8 kPa and 20.1 kPa, respectively. Results: We observed that MCF-7 cells on low fluidic substrates decreased the expression of E-cadherin, an epithelial marker, and increased expression of vimentin, a mesenchymal marker. This showed that the cells lose their epithelial phenotype and gain a mesenchymal property. On the other hand, MCF-7 cells on high fluidic substrates maintained their epithelial phenotype, suggesting that the cells did not undergo EMT. Conclusion: Considering these results as the fundamental information for material fluidity induced EMT, our system could be used to regulate the degree of EMT by turning the fluidity of the material.Background: We propose the potential studies on material fluidity to induce epithelial to mesenchymal transition (EMT) in MCF-7 cells. In this study, we examined for the first time the effect of material fluidity on EMT using poly(ε-caprolactone- The epithelial cell layer is a membranous tissue that forms the covering of the internal and external surfaces of organs to protect them from their environment. It also acts as a fundamental cell layer to segregate different compartments of the organism, transport materials through the compartments, and produce extracellular components . The intEpithelial to mesenchymal transition (EMT) is one of such events regulated partly by ECM, in which polarized epithelial cells undergo several biochemical changes and develop a mesenchymal phenotype . EMT is In our previous report, we showed that the material fluidity induces senescent type cell death in cancer cells which halt cancer metastasis . In thisco-D,L-lactide) (P(CL-co-DLLA)). We observed that the epithelial cells underwent EMT in non-crosslinked P(CL-co-DLLA) 500 substrate by a gradual decrease in the expression of E-cadherin, an epithelial marker, and an increase in the expression of vimentin. The expression of transforming growth factor-β (TGF-β) is also increased with an increase in the duration of cell culture on non-crosslinked P(CL-co-DLLA) 500 substrates. Our results suggest that material fluidity is a crucial factor to determine the EMT process. Moreover, the induction of EMT depends on the duration of cell culture on P(CL-co-DLLA) substrates as well as the fluidity or viscosity. Our system could be considered as an ideal substrate to induce EMT phenomena and to study the mechanism of regulation of EMT. Further, it is possible to regulate the degree of EMT by altering the fluidity of the material.Here, we prepared a fluidic cell culture platform using poly(ε-caprolactone-Tm) of poly(ε-caprolactone) (PCL) by tailoring the number of branched chains [Tm was adjusted below human body temperature by copolymerization of different ratios of CL with DLLA [Previously we had reported novel techniques to control the melting temperature  substrate were 34.7 and 36.8 kPa, respectively, and were calculated from the formula √ (G’)2+ (G”)2. The tensile test showed the crosslinked P(CL-co-DLLA) substrate had a stiffness of 261 kPa. However, the water contact angle (co-DLLA) substrates remained unchanged.The molecular weight (Mw) and molecular number (Mn) of copolymers of 100 units were 59,600 and 40,900 (Mw/Mn = 1.46) and copolymers of 500 units were 119,142 and 50,261, respectively (Mw/Mn = 2.37) as determined by gel permeation chromatography (GPC). The CL and DLLA contents of the copolymers were determined by  40 mol% . The vis 40 mol% . The shect angle a,b and sct angle c of crosco-DLLA) substrates on cell culture, the cover glasses were spin-coated with different concentrations (0.1 w/v% to 100 w/v%) of non-crosslinked P(CL-co-DLLA) 100 or 500 substrates to obtain a different thickness of materials on the cover glass. To determine the thickness of the substrates on the cover glass, the substrates were mixed with the dye nile red. co-DLLA) substrates at different concentrations. The confocal microscopic image of MCF-7 cells on crosslinked P(CL-co-DLLA) substrate showed a well-spread cell morphology (co-DLLA) 100 substrates. MCF-7 cells showed a well-spread morphology in 1 w/v% and completely rounded morphology in 50 w/v% or higher on non-crosslinked P(CL-co-DLLA) 100 substrates (w/v%) of non-crosslinked P(CL-co-DLLA) 500 substrates (To optimize the thickness of spin-coated non-crosslinked P(CL-rphology a. Intereco-DLLA) 100 or 500 substrates for three days and monitored the presence or absence of epithelial and mesenchymal proteins such as E-cadherin and vimentin, respectively.The effects of materials stiffness on the transformation of epithelial cells to mesenchymal phenotype have been studied well . On the co-DLLA) and non-crosslinked P(CL-co-DLLA) 500 substrates. About 10% and 40% of MCF-7 epithelial cells were transformed to a mesenchymal phenotype in crosslinked and non-crosslinked P(CL-co-DLLA) 500 substrates, respectively, at 24 h which was confirmed by the expression of the epithelial and mesenchymal markers such as E-cadherin and vimentin, respectively, in those cells (co-DLLA) substrate, about 65% and 75% of MCF-7 epithelial cells were transformed to mesenchymal cells at 48 h (co-DLLA) 500 substrate, 48 h, about 85% of MCF-7 cells and at 72 h (co-DLLA) 500 substrates. On the other hand, MCF-7 cells on non-crosslinked P(CL-co-DLLA) 100 substrates maintained their epithelial phenotype throughout the culture period  and non-crosslinked P(CL-co-DLLA) 100 and 500 substrates, we analyzed the expression of E-cadherin and vimentin mRNA across five days. On day two, MCF-7 cells on non-crosslinked P(CL-co-DLLA) 500 substrates showed a significant reduction in the expression of E-cadherin mRNA (co-DLLA) substrate. After four days, the expression of E-cadherin and vimentin mRNA reached the same level in both crosslinked P(CL-co-DLLA) and non-crosslinked P(CL-co-DLLA) 500 substrates. After four days MCF-7 cells in both crosslinked P(CL-co-DLLA) and non-crosslinked P(CL-co-DLLA) 500 substrates converted to mesenchymal cells.To confirm the EMT process in crosslinked P(CL-rin mRNA e and an rin mRNA f compareco-DLLA) and non-crosslinked P(CL-co-DLLA) 500 substrates  substrates. For that, we cultured and incubated the cells for at least four hours on the substrates coated with or without fibronectin. After 4 h we checked what percentage of cells adhered to the substrates. About 83% of cells adhered to crosslinked P(CL-co-DLLA) and non-crosslinked P(CL-co-DLLA) 500 substrates. However, only 63% of the cells adhered to non-crosslinked P(CL-co-DLLA) 100 substrates (co-DLLA) and non-crosslinked P(CL-co-DLLA) 100 and 500 substrates, respectively  substrates (co-DLLA) 500 substrates (co-DLLA) 100 substrate did not show the expression of visible focal adhesion protein (co-DLLA) substrates.The expression of focal adhesion is one of the crucial factors associated with the ability of cells to move during the EMT process . Thus, wbstrates . A visibco-DLLA) substrates, we cultured MCF-7 cells on those substrates for five days. The cell proliferation increased by increasing the culture period on non-crosslinked P(CL-co-DLLA) 500 substrates. In contrast, on the non-crosslinked P(CL-co-DLLA) 100 substrate, the proliferation initially increased until two days. From day 3 onward, proliferation decreased. This shows that non-crosslinked P(CL-co-DLLA) 500 substrates favored the proliferation behavior of the cells, however, non-crosslinked P(CL-co-DLLA) 100 declined  500 substrates show visible expression of focal adhesion proteins and promote the proliferation of cells.Cell-ECM adhesions are key mediators of cellular mechanotransduction . EMT is Material stiffness plays a vital role in the regulation of EMT or mesenchymal to epithelial transition (MET) and vice-versa. The stiffness-based effect on polyelectrolyte-based materials promotes endothelial to mesenchymal transition (EndMT) for vascular implant . MDA-MB-co-DLLA) (solid-like) and non-crosslinked P(CL-co-DLLA) (liquid-like)100 and 500 substrates. Our findings showed that the cells which grew on non-crosslinked P(CL-co-DLLA) 500 substrates had an increased expression of E-cadherin and a decreased expression of vimentin. This suggests that the cells on non-crosslinked P(CL-co-DLLA) 500 substrates underwent the EMT phenomena. This could also be confirmed by the expression of E-cadherin and vimentin mRNA. In contrast, cells on non-crosslinked P(CL-co-DLLA) 100 substrates maintained their epithelial phenotype.Furthermore, the diameter of the fiber materials used for the preparation of the cell culture scaffold influenced the EMT phenomena. MDCK cells grew on PCL fiber scaffolds of 0.5 μm cells to form colonies with an epithelial phenotype. On the other hand, cells that grew on 5 μm scaffolds appeared more fibroblastic with a phenotype reminiscent of EMT . It is kco-DLLA) and non-crosslinked P(CL-co-DLLA) 500 substrates. Although a better understanding of the molecular mechanism accounting for the activation and regulation of EMT is essential, the fluidity of the material is considered to be a crucial factor in determining cellular fate.The principal signaling pathways which induce EMT are TGF-β, Wnt and Notch. Many EMT pathways control the transcription factors of the Snail family and the ZEB family . TGF-β s1H NMR spectroscopy , respectively. The viscoelastic properties of non-crosslinked P(CL-co-DLLA) substrates were tested using a rheometer  and a viscoelastic spectrum which measured storage modulus (G’) and loss modulus (G’’).The synthesis of four-branched copolymers of ε-caprolactone and D,L, lactide (DLLA) of 60:40 ratio of 100 or 500 unit length was performed as described in our earlier reports ,22. The co-DLLA) 100 or 500 units were dissolved in toluene at different concentration ranges from 1 w/v% to 100 w/v%. The solutions were then mixed with a small proportion of nile red (TCI America) and spin coated on 15 mm glass coverslip at 15,000 rpm for 60 sec. For cell culture, four-branched P(CL-co-DLLA) 100 or 500 units were dissolved in toluene at 80 w/v% and 50 w/v% respectively. The prepared P(CL-co-DLLA) substrates were then spin-coated on glass coverslips of 24-well inserts (15 mm) or 6-well inserts (35 mm) as described above and sterilized using a low-pressure hydrogen peroxide gas plasma system CH-160C .Four-branched P(CL-co-DLLA) substrates were prepared according to previous reports [co-DLLA) substrates was characterized by a tensile test . The water contact angle was measured using a contact angle meter . Scanning electron microscope (SEM) images of crosslinked and non-crosslinked P(CL-co-DLLA) substrates were examined with SU-8000 . For cell culture, the substrates were cut into the size of 24-well insets (15mm) or 6-well inserts (35mm).The crosslinked P (crosslinked and non-crosslinked) substrates were coated with 10 µg/mL of fibronectin (Sigma-Aldrich) for 1 h and excess fibronectin was washed by phosphate buffered saline .Human breast epithelial adenocarcinoma cells  were cultured in minimum essential medium eagle’s (MEM) supplemented with 10% fetal bovine serum , 0.1 mM non-essential amino acid (NEAA), 1 mM sodium pyruvate, and 1% antibiotic- antimycotic . The cells were maintained in a humidified atmosphere of 5% COco-DLLA) 100 or 500 unit substrates of different concentrations (1 w/v% to 100 w/v%) were visualized under Leica SP5 confocal laser scanning microscope  and the 3D images were scanned.The spin coated non-crosslinked P(CL-4 cells/cm2 on a glass coverslip or P(CL-co-DLLA) (crosslinked and non-crosslinked) substrates and incubated at different time periods for up to three days. After the preferred incubation period, the cells were fixed by 4% paraformaldehyde  and blocked by 1% bovine serum albumin  (in PBS) for 30 min. The cells were stained independently with monoclonal anti-vinculin , anti-E-cadherin  and anti-vimentin  antibodies specific to vinculin, E-cadherin, and vimentin, respectively, followed by staining with Alexa Fluor® 488 antimouse secondary antibody . F-actin and nuclei were counterstained by tetramethylrhodamine B isothiocyanate-conjugated phalloidin (Sigma-Aldrich) and 4’,6-diamidino-2-phenylindole  respectively. The images were taken with a Leica SP5 confocal laser scanning microscope. The areas of the cell were measured by ImageJ .For immunostaining, MCF-7 cells were seeded at a density of 1 × 10co-DLLA) (crosslinked and non-crosslinked) substrates of 24- well plate inserts at a density of 1*104 cells/cm2. The cells were incubated at different time periods from 1 to 5 days. A cell proliferation assay was performed using cell counting kit-8  according to the manufacturer’s instructions. The cell adhesion assay was performed for 4 h and the relative cell adhesion was determined from the initial cell seeding density.MCF-7 cells were seeded on P(CL-5 cells/mL of MCF-7 cells were cultured on crosslinked and non-crosslinked P(CL-co-DLLA) substrates. The total cellular RNA was extracted using the RNeasy Kit (Qiagen) and extracted RNA was treated with DNaseI (Takara) to remove DNA contamination. Fifty ng of total RNA was reverse- transcribed into cDNA using the PrimeScriptTM 1st strand cDNA synthesis kit (Takara) according to the manufacturer’s protocol. The qRT-PCR was performed using Light Cycler 1.5 System (Roche). The reaction mixture was composed of FastStart DNA Masterplus SYBER GREEN I, 20 µM of forward primer, 20 µM of reverse primer, and 1 µL of cDNA which made up a total volume of 15 μL which was treated with DEPC water. The thermocycling conditions were 95 °C for 10 s, followed by 45 cycles of 95 °C for 10 s, 60 °C for 7 s, and 70 °C for 10 s. The relative change in gene expression was quantified by threshold cycle (Ct), 2−ΔΔCt method. Data were normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control in the same reaction as the gene of interest. The primers used in this study are in For the quantitative determination of mRNA expression, the qRT-PCR analysis was performed. 5 × 10p < 0.05 is considered statistically significant when comparing non-crosslinked P(CL-co-DLLA) to crosslinked P(CL-co-DLLA) substrates.All results were represented as mean ± standard deviation (SD) obtained from three independent experiments. The degree of significance of each data was performed by Student’s t-test, where co-DLLA) 100 and 500 units to understand the effect of materials fluidity on the EMT process. Initially, we checked the adhesion behavior and the expression of the focal adhesion protein of MCF-7 cells on these substrates. The adhesion of MCF-7 cells was enhanced by coating the surface of the substrates with fibronectin. The cells on non-crosslinked P(CL-co-DLLA) 500 substrates expressed a visible focal adhesion protein. However, cells on non-crosslinked P(CL-co-DLLA) 100 substrates did not show a visible focal adhesion. This could correlate with the proliferation behavior and EMT phenomenon of the cells on non-crosslinked P(CL-co-DLLA) 100 and 500 substrates. Cell proliferation increased with an increasing culture period on non-crosslinked P(CL-co-DLLA) 500 substrates. On the other hand, the proliferation decreased by increasing the culture period on non-crosslinked P(CL-co-DLLA) 100 substrates. EMT was observed with a decrease in the epithelial marker and an increase in the mesenchymal marker. We found the cells underwent EMT in non-crosslinked P(CL-co-DLLA) 500 substrates by a gradual increase in the expression of vimentin. At 72 h, about 100% of the cells underwent EMT. The principal signaling pathway which induced EMT was TGF-β. The expression of TGF-β also increased with increased time on non-crosslinked P(CL-co-DLLA) 500 substrates. This also suggests that the induction EMT process is associated with TGF-β. In contrast, the cells maintained their epithelial phenotype on non-crosslinked P(CL-co-DLLA) 100 substrates by maintaining the expression of E-cadherin. Our findings show that material fluidity is a crucial factor to determine the EMT process. Further, it is possible to regulate the degree of EMT by altering the fluidity of the material.EMT is a series of biochemical events in which polarized epithelial cells are transformed to a mesenchymal phenotype. In this study, we have prepared non-crosslinked (fluidic or viscous) material made of P(CL-


When fE = 0.8 eV, the relative bandwidth of the proposed MMA, which represents the frequency region of absorption beyond 90%, can reaches its maximal value of 72.1%. Simulated electric field distributions reveal that the broadband absorption mainly originates from the excitation of surface plasmon polaritons (SPPs) on the CGS graphene sheet. Furthermore, the proposed MMA is polarization-insensitive and has wide angles for both transverse-electric (TE) and transverse-magnetic (TM) waves in the broadband frequency range. The broadband absorption capacity of the designed MMA can be effectively adjusted by varying the Fermi energy level of graphene. Lastly, the absorbance of the MMA can be adjusted from 42% to 99.1% by changing the fE from 0 eV to 0.8 eV, which is in agreement with the theoretical calculation by using the interference 41theory. Due to its simple structure and flexible tunability, the proposed MMA has potential application prospects in tunable filtering, modulators, sensing, and other multispectral devices.We present a simple design of a broadband tunable metamaterial absorber (MMA) in the terahertz (THz) region, which consists of a single layer complementary gammadion-shaped (CGS) graphene sheet and a polydimethylsiloxane (PDMS) dielectric substrate placed on a continuous metal film. The Fermi energy level ( Terahertz wave metamaterial absorbers (MMAs) have recently attracted great attention and become one of the widespread research hotspots due to their promising applications in communication, imaging, detection and sensing, and stealth ,2,3,4,5.In the last few years, MMAs based on graphene have received increasing interest and have achieved tremendous progress. Various MMAs based on patterned graphene structures, such as disks , ribbonsfE = 0.8 eV. Both simulation and calculation results reveal that the MMA maintains excellent absorption performance when the incident angle is below 50° for both transverse electric (TE) and transverse magnetic (TM) polarized terahertz waves. By controlling the graphene Fermi energy from 0 eV to 0.8 eV, the absorbance of the MMA can be easily tuned from 42% to 99.1%. Hence, the proposed tunable MMA based on graphene is believed to have promising applications in terahertz sensing, detection, imaging, and cloaking.In this work, we propose a novel MMA based on a periodic patterned graphene structure, which can achieve polarization-insensitive and wide-angle broadband terahertz absorption with flexibly active tunability. The proposed tunable MMA is composed of a single-layer periodical complementary gammadion-shaped (CGS) graphene array placed over a polydimethylsiloxane (PDMS) dielectric substrate deposited on a gold film. First, the structure design of the proposed terahertz MMA is demonstrated. Then, the absorption mechanism of the proposed MMA is clarified by analyzing the field distributions and multireflection interference theory. Next, the absorption characteristics of the proposed MMA are analyzed by varying the oblique incident angles of terahertz waves and the Fermi energy level of the graphene as well. The calculated results based on the multireflection interference theory model are in agreement with simulated results. Numerical results indicate that the relative bandwidth, which presents the frequency range of absorption beyond 90%, can reach its maximum value of 72.1% when 4) rotational symmetry, and the graphene conductivity of the MMA can be controlled by applying a DC voltage Vg. The design scheme of the proposed terahertz tunable MMA is illustrated in tg, whose relative permittivity can be calculated by εg = 1 + iσg/(ε0ωtg) [σg is the graphene surface conductivity, ε0 is the vacuum permittivity constant, and ω is the angular frequency of the incident terahertz wave. In this study, we assume an effective medium with a thickness of tg = 1 nm to replace the three-layer graphene sheets [σg of graphene can be modelled as a thin surface sheet and characterized by the Kubo formula [σg = σintra + σinter, where σintra and σinter denote intra-band and inter-band conductivities, respectively. In view of the Pauli exclusion principle, the inter-band contribution of graphene conductivity can be neglected in low terahertz and far-infrared regions [σg is only determined by intra-band contribution σintra, which can be simplified and approximately described as [ω, fE, τ, and T are the radian angular frequency of the incident terahertz wave, chemical potential (Fermi energy), relaxation time, and environmental temperature, respectively. The relaxation time τ characterizes the plasmon decay on account of impurities, which is fixed to 0.1 ps, and T is the kelvin temperature, which is fixed to 300 K in this study. In addition, e represents the charge of an electron, ℏ is the reduced Planck’s constant, and Bk is the universal constant representing the Boltzmann constant. The approximate estimated theoretical relation between Ef and Vg can be expressed as [fv is the Fermi velocity, Vg is the extra bias voltage, and ε0 and st are the effective relative permittivity and thickness of the middle dielectric layer, respectively. According to the equation above, it can be found that the absorption property of the proposed MMA can be controlled via bias voltage between the CGS graphene layer and the continuous metallic film. The continuous metallic film of the MMA is formed with gold, with the relative permittivity obtained from the Drude model [xp = yp = 25 μm, l = 24 μm, w = 2.5μm, st = 14 μm. The unit cell structure of the proposed MMA was set to be periodical along the x- and y-axis with periods of 25 μm to avoid diffraction at the normal incidence rate for frequencies up to 12 THz. In this configuration, the surface plasmon polaritons (SPRs) of the single-layered CGS graphene can be excited, therefore it makes sense to utilize the resonant absorption characteristics of graphene surface plasmon polaritons (SPPs) to enhance the absorption of incident terahertz wave [The graphene is numerically modelled by an effective medium with a certain thickness /(ε0ωtg) ,38, where sheets , since te sheets ,41,42. T formula ,43,44: σ regions . Thus, iribed as :(1)σg(ω)essed as :(2)Ef=ℏvde model . Since tde model . The finrtz wave ,48.x- and y-axis directions (x-y plane) are used, and incident terahertz waves are set to propagate along the z-direction, as shown in k is set to be in the xoz or yoz plane and the electric field is in the x-axis direction (TE) or in the y-axis direction (TM). The absorbance of the proposed MMA can be calculated by the formula A(ω) = 1 − T(ω) − R(ω) = 1 − |S21(ω)|2 − |S11(ω)|2, where T(ω) = |S21(ω)|2 and R(ω) = |S11(ω)|2 are transmittance and reflectance, respectively. In our design, the transmittance is zero (S21(ω) = 0), owing to the thickness of the bottom continuous gold film, which is greater than the skin depth across the whole terahertz region. Thus, the absorbance can be simplified as A(ω) = 1 − |S11(ω)|2. To verify the absorption efficiency of the proposed MMA, three-dimensional full wave numerical simulations were performed in CST Microwave Studio based on the finite element method (FEM). In simulation, the periodic boundaries in both T = 300 K, relaxation time of graphene τ = 0.1 ps, and the initial Fermi energy fE = 0.8 eV. As shown in Firstly, the absorbance performances of MMAs with and without the CGS graphene sheet under normal incident terahertz waves were investigated, and the simulated results are depicted in To better understand the operation principle of the proposed MMA structure, we calculated the wave impedances of the MMA with and without the CGS graphene sheet from the simulated reflection coefficients according to the S-parameter retrieval method . On averzE) of the electric field distributions on the y-z plane of the unit cell structure at absorption peak frequencies of 2.7 THz and 4.4 THz, respectively, are illustrated. It can be found that the z-component (zE) of the electric field is mainly concentrated near the interface between the CGS graphene sheet and the PDMS substrate at 2.7 THz and 4.4 THz, respectively. The tight field confinement around the CGS graphene sheet on the top layer can be observed clearly, which means that all the electric fields are trapped and subsequently dissipated on the graphene layer. The strong terahertz wave absorption supposedly caused by excitation of different resonance modes. In To reveal the physical mechanism of the proposed MMA based on CGS graphene, an intuitive and straightforward method is to investigate the electric field distributions in resonance. As shown in rε and ts are the relative permittivity and thickness of PDMS substrate, respectively; k0 is the free space wave number; and iθ. In addition, fE = 0.8 eV, which is in good agreement with the simulated result. This result indicates that the interference theory based on the Fabry-Pérot-like resonance cavity can be used to legitimately explain the physics mechanism of the proposed MMA.To further reveal physical insights from the proposed graphene-based MMA, quantitative analysis based on interference theory was employed in this work. Interference theory based on Fabry-Pérot-like resonance cavity can provide a profound understanding of the underlying physics for the proposed MMA. According to interference theory, the MMA can be considered as a Fabry-Pérot-like resonance cavity, which would induce multiple EM interference effects in multireflection in the incident terahertz waves ,13,14,534) rotational symmetry of the unit cell structure (not show). Here, we consider the absorption performance of the proposed MMA (fE = 0.8 eV) under different oblique incident angles for both TE and TM mode polarization waves. Both the simulated and calculated absorbance under different oblique incidence angles for TE and TM waves are depicted in For practical application, the absorption performance of the proposed MMA should be robust to the different polarization angles and oblique incident angles for both TE and TM waves. Obviously, the proposed terahertz MMA is polarization-independent for both TE and TM waves due to the uniaxial four-fold (CAs shown in fE) of the CGS graphene sheet placed on the top layer of the MMA structure is adjusted dynamically by changing the external bias voltage Vg, as shown in fE) of the CGS graphene sheet was continuously tuned from 0 to 0.8 eV through external bias voltage. fE) under normal incident TE waves. It is obvious that the absorption level is enhanced gradually with the increasing fE, while the operating frequency range is nearly unchanged. Moreover, the simulated average absorbance increases from 42% to 99.1% gradually with the increment of fE from 0 to 0.8 eV, while the operating frequency range remains nearly unchanged, which is ell consistent with the calculation result based on interference theory. Thus, it can be concluded that the proposed MMA with flexible tunability could be used as a tunable broadband spatial amplitude modulator or attenuator in terahertz regimes. In addition, as distinguished from the previous MMAs consisting of isolated graphene disks, ribbons, and multi-resonators, the proposed MMA, which possesses a net CGS graphene layer with inherent continuous electrical connection, may greatly simplify the previously redundant gating structures to just one voltage gate and realize the flexible tunability of the absorption properties. The design strategy of this tunable broadband terahertz MMA can be easily scaled to infrared or visible regimes if the dimensions are reduced to nano- or even lower scales.Furthermore, the electrically tunable properties of the broadband terahertz MMA were also investigated numerically. Graphene, as one kind of tunable photoelectric material, is often applied to regulate the absorption amplitude and frequency range of a MMA. The surface conductivity of a graphene sheet relates primarily to its Fermi energy, which can be easily controlled by applying bias voltage or electrostatic chemical doping ,34,35. HfE = 0.8eV. The simulated electric field distribution of one unit cell structure reveals that the stronger broadband absorption of the MMA mainly originates from the continuous excitation of the fundamental and second order graphene SPP resonances. Additionally, multiple-reflection interference theory was used to quantitatively analyze the absorption performance of the designed MMA with wide-range incidence angles and different Fermi levels (fE). The theoretical calculations and full-wave simulations are in excellent agreement with each other. The MMA has been proven to be insensitive to the polarization states of incident terahertz waves, and the absorbance remains at 80% with incident angles up to 45° for TE waves and up to 65° for TM waves. Compared to conventional MMAs consisting of isolated graphene disks, ribbons, multi-resonators, or multi-layered structures, the continuous net CGS graphene structure greatly simplifies the electrostatic gating structure to achieve flexible tunability. By controlling the Fermi level (fE) via external bias voltage of the graphene sheet, the absorbance can be tuned continuously from 42% to 99.1%. This work offers a new perspective on the simple design of graphene-based tunable terahertz broadband MMAs. Benefitting from these promising properties, including the simple structure, absolute polarization insensitivity, wide incident angle, and the flexible tunability of the broadband absorption, the proposed MMA could be engineered for various applications in broadband terahertz spatial amplitude modulators, attenuators, sensors, and other optoelectronic devices.In conclusion, we have demonstrated a new efficient route for achieving a broadband terahertz tunable MMA using a net CGS graphene sheet deposited on a PDMS dielectric spacer supported by a metallic reflecting plate. Simulation results indicate that the relative bandwidth of the proposed MMA, which represents a frequency region of absorption beyond 90%, reaches its maximal value of 72.1% when


Phyllitis scolopendrium, thriving in a forested area along a radiation gradient at the entrance of a cave, we examined selected biochemical, morphological, and physiological frond traits of the ferns from three different habitats. Sampling was performed two times during the vegetation season, in April and June. We also measured frond optical properties to point out the differences in leaf/light interactions between different plant samples. According to frond size, the middle habitat, receiving 125 µmol m−2s−1 of photosynthetically active radiation at both sampling times, appeared to be the most favourable. The production of UV-absorbing substances was highest in the habitat with the lowest radiation level. At the beginning of the season, the level of photosynthetic pigments in this habitat was the same as in the other habitats, while it was significantly lower in June when the tree canopy was closed. Frond reflectance was similar when comparing habitats and different sampling times. The most significant differences were obtained in the UV-A and near-infrared regions. The reflectance spectra depended mainly on frond biochemical properties, which altogether explained 54% (p ≤ 0.05) of the spectra variability. Frond transmittance depended on both, morphological parameters, explaining 51% (p ≤ 0.05), and frond biochemistry, explaining 73% (p ≤ 0.05) of the spectra variability. P. scolopendrium was revealed to be highly plastic regarding light conditions. The shapes of the frond reflectance and transmittance optical curves were similar to those typical of leaves of seed plants. The fronds exhibited high morphological plasticity when comparing different habitats. However, their biochemical and optical traits differed more between the two sampling times than between the habitats.Ferns display an elevated degree of phenotypic plasticity to changes in irradiance levels; however, only a few reports deal with their response to different light conditions. To get an insight into the extent of phenotypic plasticity of the fern  Radiation environment may vary in time and space . ChangesThe interactions between solar radiation and plants are very complex, depending on the species’ biochemical and structural leaf traits. These are a consequence of the species’ genetic potential and specific environmental conditions in the habitat of a plant . All thePhyllitis scolopendrium is a fern species that thrives at moist sites, such as wooded limestone ravines, steep north-facing slopes, and cave entrances, where it can colonise sites along a gradient of environmental conditions, including light [P. scolopendrium, thriving in a forested area along a radiation gradient at the entrance of a cave, and measured their optical properties in order to point out the differences among plant specimens from the different habitats, sampled at different times during the vegetation season.ng light ,27, as ang light . An impong light . This abng light . In spitng light . In the Phyllitis (syn. Asplenium) scolopendrium (L.) Newm., is a perennial, rhizomatous, evergreen fern belonging to the Aspleniaceae family [A. scolopendrium L. var. scolopendrium, which is distributed broadly throughout Europe and Asia, and a rare tetraploid taxon A. scolopendrium L. var. americanum (Fern.) Kartesz and Gandhi that can be found in the eastern United States, Mexico, and Canada [Hart’s-tongue fern, e family . It is ae family . It has d Canada . Cold wid Canada .Experimental plants were sampled under the Little Natural Bridge near the entrance of the Zelške jame cave system in the Rakov Škocjan valley . Rakov Škocjan is a karst valley located at the northern foot of the Javorniki hills.Omphalodo-Fagetum sylvaticae forest, (2) the middle habitat with somewhat lower light levels, and (3) the lower habitat with poor light conditions  the upper habitat, which is the farthest from the cave system in the nditions  in the vAt the time when the tree canopy was closed, average mid-day temperature and relative humidity at the plant level were rather uniform, namely, 20.5 ± 0.3 °C and 76 ± 4% for the upper habitat, 21.3 ± 0.4 °C and 71 ± 4% for the middle habitat, and 19.6 ± 0.5 °C and 76 ± 3% for the lower habitat.Photosynthetically active radiation (PAR) was measured with a data logger  and a quantum sensor . The radiation spectra were measured on the day of sampling in April and June using a portable spectrometer . A white reference panel  was used for calibration of the spectrometer to 100% reflectance prior to measurement. Ten measurements of PAR and ten measurements of solar radiation spectra from 200 to 1100 nm were performed at the plant level for each of the three habitats.a, chlorophyll b, and carotenoid contents were determined as described by Lichtenthaler and Buschmann [−2). Anthocyanin contents were determined according to Drumm and Mohr [−2). We also measured the contents of the total methanol-soluble UV-B- and UV-A-absorbing substances, as proposed by Caldwell [All of the biochemical analyses were conducted on vital, fully developed fronds. Chlorophyll uschmann ,36. Extrand Mohr , with exCaldwell . The absMorphological traits were studied on transverse frond sections, which were analysed under a light microscope  equipped with a digital camera  and CellSens software . 100× magnification was used to measure thicknesses of the fronds, mesophyll, epidermis, and cuticle, while 400× magnification was used to determine stomata length and density. The latter two parameters were studied only for the lower frond surface. All of the measurements were performed on the central parts of vital and fully developed fronds.A portable chlorophyll fluorometer  was used for measurements of the potential and effective photochemical efficiencies of the photosystem (PS) II, which were evaluated according to Schreiber et al. . Prior tThe optical traits of the fronds, namely, reflectance and transmittance, were measured from 300 to 800 nm on vital and fully developed fronds on the day they were collected. Measurements were performed using a Jaz Modular Optical Sensing Suite portable spectrometer  that was fitted with an ISP-30-6-R integrating sphere  and a QP600-1-SR-BX optical fibre .Total adaxial reflectance spectra were recorded during illumination of fronds with a UV–VIS/near-infrared (NIR) light source . Before measurement, the spectrometer was calibrated to 100% reflectance using a white reference panel .To measure the transmittance spectra, we first calibrated the spectrometer to 100% transmittance with a light beam that passed directly into the integrating sphere. Afterward, the integrating sphere was placed at the abaxial frond surface, while the UV–VIS/NIR light source illuminated the adaxial frond surface.p ≤ 0.05. The figures for mean relative reflectance and transmittance spectra of the fronds from the three studied habitats, sampled at two different times during the vegetation season, were drawn in Microsoft Excel 2016 .Normal distributions of the data were evaluated using Shapiro–Wilk tests. Homogeneity of variance from the means was analysed using Levene’s tests. One-way analysis of variance (ANOVA) according to Duncan’s posthoc multiple range tests was used to assess differences between the six considered groups for each measured parameter. To investigate the relationships between the selected frond traits, we performed Pearson’s correlation analysis. IBM SPSS statistics 22.0  was used for these statistical calculations, with significance accepted at Detrended correspondence analysis was used for the exploratory data analysis using the CANOCO for Windows 4.5 programme package. Due to the gradient lengths obtained (< 3 SD) , we usedp ≤ 0.05) between the lower habitat and the remaining two habitats  throughout the whole spectrum, except for the NIR region (p ≤ 0.05), explaining 9% of the reflectance spectra variability. The RDA plot showing the strength of the associations between frond biochemical parameters and the regions of the reflectance spectra (p = 0.01) and 13% (p = 0.01), respectively, chlorophyll a explained 12% (p = 0.02), and UV-A-absorbing substances explained additional 6%. April samples are positioned in the left part of the plot, while June samples are mainly located in the right part of the plot.The RDA plot showing the strength of the associations between frond morphological parameters and frond reflectance spectra revealed the relatively low importance of frond morphology in explaining the reflectance spectra. Only frond thickness was significant , while upper and lower epidermis, mesophyll, and lower cuticle thicknesses explained 5% (p ≤ 0.05) or 6% (p ≤ 0.05) each. The RDA plot showing the strength of the associations between frond biochemical parameters and the regions of the transmittance spectra revealed that UV-B- and UV-A-absorbing substances explained 30% (p = 0.001) of the spectra variability each, while carotenoids and chlorophyll a explained an additional 11% (p = 0.001) and 2% (p = 0.018), respectively. Altogether, the biochemical parameters explained 73% of the transmittance spectra variability  value, which is characteristic of high radiation environments [Platycerium bifurcatum and determined a higher accumulation of carotenoids and anthocyanins in the fronds of plants grown under lower R/FR ratios. However, this was not the case in our study. Frond pigment contents of the P. scolopendrium samples from April were comparable for the three habitats; an exception was seen for the contents of UV-absorbing substances, which were significantly higher for the lower habitat. For the second sampling, the contents of photosynthetic pigments increased, with the exception of the lower habitat, where we observed a decrease. The increase in photosynthetic pigments was more pronounced for chlorophyll b, which resulted in a lower chlorophyll a to b ratio. This change in the chlorophyll a to b ratio is acclimation to an altered radiation regime [P. scolopendrium. Flavonoids are very important substances for plants as they are involved in the interactions of plants with other organisms and mitigate different environmental stresses, including protection against high radiation levels, which results from their antioxidative potential [P. scolopendrium [Plant environment is very complex since it is affected by a variety of abiotic and biotic factors, among which light, as a primary source of energy, presents a crucial factor. Light conditions for experimental plants differed among habitats and regarding the time of the season, with the exception of the middle habitat, where the level of PAR was 125 µmol mon layer . At the ronments . In our ronments  analysedn regime . On the n regime ,44. In sn regime . Howeverotential . Shade potential , as is apendrium ,49. Kaempendrium . In spitpendrium . TherefoP. scolopendrium from the upper and middle habitats, effective photochemical efficiency (yield) was negatively affected in spring prior to the unfolding of leaves in the tree canopy. On the other hand, potential photochemical efficiency (Fv/Fm) was relatively high [−2, which ranged from 0.7 to 0.8 [P. scolopendrium consist of guard cells that contain more chloroplasts than the guard cells of angiosperms, and they can open widely even under conditions of limited photosynthetic carbon dioxide fixation [Asplenium. Namely, A. ceterach seems to be more efficient under full sunlight conditions in comparison to A. trichomanes, where a decrease in maximum photochemical efficiency and slower energy flow through PSII were observed [The chlorophyll fluorescence measurements in our study may indicate the presence of stress in plants ,52. For ely high . The Fv/7 to 0.8 . Therefo7 to 0.8 . The stu7 to 0.8  showed t7 to 0.8 . The stofixation . In addiobserved .Changes in frond optical properties during the season depend on changes in pigment contents, anatomy, and senescence processes . The refAccording to frond size, the middle habitat revealed to be the most favourable. The production of UV-A-absorbing substances was higher in the overwintered fronds in comparison to the fronds of the current season. However, when comparing different habitats, it was highest in the habitat with the lowest radiation level. An increase in photosynthetic pigments and a decrease in UV-absorbing substances during the season in the upper and middle habitats, and just the opposite response in the lower habitat, were likely a consequence of the trade-off between input in plant assimilation and plant protection. Frond spectral signatures (reflectance) differed less between the habitats than between the two different sampling times and were mostly related to frond biochemical properties, which should be taken into account when monitoring this species with the remote sensing technique. On the other hand, frond transmittance depended on both, frond morphology and biochemistry.P. scolopendrium exhibits a highly plastic response regarding the examined physiological, biochemical, and morphological traits, which enables plants to use the advantage of specific environmental conditions. The higher production of UV-A-absorbing substances in this species, which were produced under less favourable conditions, is likely in the function of protection against pathogens that could develop under such conditions in winter and in a low radiation environment. This deserves further investigation, since it presents an important aspect of the future success of this fern species.We can conclude that


In particular, the suggested graphene/ZnO NRs/graphene hybrid-nanostructure-based devices reveal comparable photocurrents at a bidirectional bias, which can be a promising platform to integrate 1D and 2D nanomaterials without complex patterning process for UV device applications.High crystalline ZnO nanorods (NRs) on Zn pre-deposited graphene/Cu sheet without graphene transfer process have been fabricated by self-catalyzed vapor-phase transport synthesis. Here, the pre-deposited Zn metal on graphene not only serves as a seed to grow the ZnO NRs, but also passivates the graphene underneath. The temperature-dependent photoluminescence spectra of the fabricated ZnO NRs reveal a dominant peak of 3.88 eV at 10 K associated with the neutral-donor bound exciton, while the redshifted peak by bandgap shrinkage with temperature and electron-lattice interactions leads a strong emission at 382 nm at room temperature. The optical absorption of the ZnO NRs/graphene hetero-nanostructure at this ultraviolet (UV) emission is then theoretically analyzed to quantify the absorption amount depending on the ZnO NR distribution. By simply covering the ZnO NR/graphene/Cu structure with the graphene/glass as a top electrode, it is observed that the current-voltage characteristic of the ZnO NR/graphene hetero-nanojunction device exhibits a photocurrent of 1.03 mA at 3 V under a light illumination of 100 μW/cm Especially, its self-organized growth as single-crystalline nanostructures leads to quantum confinement and small size effects, which reveals novel properties in optical and electrical aspects [Zinc oxide (ZnO) has been widely used in photodetector, solar cell, and display applications ,2,3 due  aspects ,5,6 rela aspects , photoco aspects , biosens aspects , and pie aspects . Graphene, a single layer of carbon atoms, has also superb physical properties such as high electrical conductivity, ultrahigh mobility, and high transparency . It can 2 ambient was reported. The broken C-C bonding of graphene by heating at high temperature of 600 °C above in O2 ambient results in etch-pit formation [Compared with the solution process that has been widely adopted for implementing graphene-ZnO hybrid-structures ,15, the ormation . Then, tIn order to use the ZnO nanostructures in ultraviolet (UV) device applications, it is necessary to develop highly crystalline ZnO NRs without the defects induced by metal catalysts or the defects formed on graphene during the fabrication process to utilize its superb optoelectronic properties. It is known that carbon materials including amorphous carbon, graphite flakes, and photoresists can be used as preferential nucleation sites for growing ZnO nanostructures . Here, Z2 scale). Then, the fabricated graphene/ZnO NRs/graphene hybrid-nanojunction devices were characterized by measuring the current-voltage (I-V) curves under dark and illumination conditions.In this respect, we firstly demonstrate the fabrication of vertically aligned ZnO NR on graphene/Cu sheet by using self-catalyzed vapor-phase transport (VPT) synthesis. The growth of ZnO NRs on Zn pre-deposited graphene/Cu sheet was performed at 700 °C using a thermal-vapor-deposition technique. The pre-deposited Zn-metal on graphene has a role of a seed to grow the ZnO NR as well as of passivating the graphene underneath. Then, the fabricated ZnO NRs on graphene/Cu sheet were investigated by the low temperature-dependent photoluminescence (PL) spectra ranging from 10 to 140 K. To elucidate the optical absorption depending on the ZnO NR distribution, we carried out the optical simulation for the emission peak (382 nm) determined by the PL measurement at room temperature. By simply covering the ZnO NRs/graphene/Cu sheet with the graphene/glass as an electrode, we fabricated hetero-nanojunction devices in a large area (~cm−4 Torr). Here, the Cu foil was vertically placed on a substrate holder in the central isothermal growth zone of the reactor during the growth process. The temperature of the chamber was ramped up to 1060 °C with a mixture of Ar  and H2  at the processing pressure of 470 mTorr. The Cu foil was typically annealed for 2 h. The furnace temperature was then decreased to the process temperature of 1020 °C with 30 sccm of H2 gas at 570 mTorr. When the furnace temperature reached 1020 °C, graphene layers started to grow on the Cu foil by the mixed gases of CH4  and H2  while flowing 2000 sccm of Ar as a carrier gas to the reaction chamber at 600 mTorr for 30 min. The reaction chamber was then cooled down to room temperature with an average rate of 14 °C/min under the same flow rates of Ar and H2 gases without methane. Graphene was synthesized on Cu sheet by the CVD process with a mixture of methane and hydrogen as described in detail elsewhere ,26,27. T−3 mbar. To prevent the oxidation of graphene, 500 sccm of H2 gas was injected while the temperature was rising at a heating rate of 20 °C/min, and the inside of the reactor was flushed until the set point was reached. When the reactor reaches the source temperature of 610 °C and the growth zone temperature reaches 700 °C, the vaporized Zn-metal was transferred into the growth zone by flowing 1000 sccm of N2 (99.9999%) as a carrier gas for 60 s. Here, the VPT equipment consisting of 3 source zones and 3 growth zones accurately controls the temperature of each zone with a temperature controller , and the substrate and Zn material were located in the center of the source and growth zone. Then, the Zn-metal layer (~20 nm) covering the graphene plays a role of a seed to grow the ZnO NR and passivates the graphene underneath. After shutting off the supply of H2 gas, 30 sccm of O2 (99.999%) gas was injected into the growth zone in the reactor, where the mixed gases of O2 and N2 flow at the rates of 1.7 and 2 standard liters per minute, respectively. The gas pressure inside the reactor was maintained at 2.4 mbar during 30 min of growth. After the reaction was complete, it was cooled to room temperature. After loading the graphene/Cu sheet in the growth zone, Zn metal  contained in an alumina boat was placed in the center of source zone of the reactor quartz tube. Initially, the reactor was kept in vacuum at a pressure of ~102 intensity  were measured using a B1500A semi-conductor device parameter analyzer  by applying a series of voltages ranging from −3 V to 3 V for dark and illumination conditions.The morphological and structural properties of the ZnO NRs grown on the graphene/Cu sheet were examined using field emission-scanning electron microscopy (FE-SEM)  with magnification of 1.2k~12k at an acceleration voltage of 10 kV and X-ray diffraction (XRD)  with an X-ray source of Cu Kα radiation (λ = 1.5406 Å), respectively. The emission spectra of the temperature-dependent PL intensity were measured using Spectra Physics 404, Cryostat-HS-4, He-Cd laser-IK3202R-D, and Monochromator-SPEX 1072 in a temperature range from 10 to 140 K. The signal was amplified with analyzer-EG&G-5206. The Raman analysis was carried out using the laser excitation of 514 nm (~1 mW) polarized by a GX-AN360  filter. Each spectrum was obtained by 10 acquisitions in 10 s of accumulation time. The I-V characteristics of the glass/graphene-ZnO NRs-graphene/Cu heterostructure under dark and illumination condition of 100 μW/cmn, k) of Cu, ZnO, and graphene were taken from the References [The full-wave electromagnetic simulation using Lumerical’s finite-difference time-domain (FDTD) software  was performed to analyze the optical absorption of the ZnO NRs depending on the NR distributions. The complex refractive indices  larger than 1. Then, the morphological properties of the ZnO NRs grown on the graphene/Cu sheet in comparison with the ZnO NRs on Cu sheet were examined by cross-sectional FE-SEM images as shown in 2 gas was injected to prevent the oxidation of graphene while the growth zone temperature reaches 700 °C, and then the vaporized Zn-metal was transferred to cover the graphene/Cu sheet. According to the previous research [To characterize the graphene on Cu sheet, Raman analysis was performed by applying a background subtraction and normalization procedure on the measured Raman spectrum since there is a high background signal due to the emission of Cu sheet. Then, it is clearly observed the main G-peak and 2D-peak of graphene at ~1584 and ~2676 cmresearch , the Zn research ,34. HereLow temperature-dependent PL spectra of the ZnO NRs at a temperature range from 10 to 140 K have been plotted on an arbitrary scale, as illustrated in Generally, optical absorption in ZnO occurs only in the UV region. Since the morphology of ZnO NRs has a high surface-to-volume ratio, there can be defects on the surface of the ZnO NRs ,22. ThenTo quantify the amount of optical absorption of the ZnO NRs depending on the NR distributions, we carried out the full-wave electromagnetic simulation using the FDTD method. The simulation structure presented in eff = nZnO∙x + nair∙(1 − x), where nair = 1, nZnO = 1.72 at a 382 nm wavelength and x is the volume fraction of the ZnO NR array with respect to the thin-film counterpart. Then, the estimated values of neff are 1.48, 1.36, 1.28 and 1.24 for the interspacing among the ZnO NRs of 100, 200, 300, and 400 nm, respectively. Hence, the ZnO NRs with the neff less than 1.3  without any patterning process by simply covering the ZnO NRs/graphene/Cu sheet with the graphene/glass as a top electrode as illustrated in 2 intensities of light illumination. The inset of 2 by a pressure, while the ZnO NRs on a graphene/Cu sheet were synthesized through the VPT method. In the ZnO NRs on the graphene/Cu sheet, the graphene has an intrinsic property because the process proceeds in a high vacuum chamber in an in situ state. Meanwhile, the electrons of the graphene in the graphene/glass structure move into the oxygen atoms contained in the glass substrate (SiO2) [To characterize the electrical properties of the graphene/n-type ZnO NR/graphene hybrid-structure, we fabricated the device in a large scale (~cme (SiO2) , resultiWhen the light illuminates this hybrid nanostructure, photocurrents  arising from the ZnO NR/graphene/Cu sheet are slightly higher than that  from ZnO NR/graphene/glass as shown in The vertically aligned ZnO NRs were grown on graphene/Cu sheet using the catalyst-free VPT synthesis. The pre-deposited Zn-metal on graphene has a role of a seed to grow the highly crystalline ZnO NR without defects as well as passivating the graphene underneath. Using the graphene/Cu as a substrate also eliminates the need for a graphene transfer process, simplifying the process and avoiding physical defects of graphene caused by the transfer process. The fabricated ZnO NRs with a length of ~20 μm and a diameter of 150–200 nm shows one strong UV emission at 382 nm at room temperature. The optical absorption at UV emission of this hybrid-structure is much enhanced as compared to that of a thin-film counterpart, and the amount of absorption can be optimized according to the diameter and interspacing among the ZnO NRs. The developed graphene/ZnO NR/graphene heterojunction devices exhibit comparable photocurrents at a bidirectional voltage bias, which provides a promising way to integrate 1D and 2D nanomaterials with a cost-effective and continuous fabrication process over a large area.


Gastrointestinal sarcoidosis in the absence of pulmonary disease is rare. Likewise, cytomegalovirus (CMV) reactivation in the stomach is also rare. We present a 67-year-old woman with symptomatic CMV gastritis and gastroduodenal sarcoidosis who presented with epigastric pain, nausea, and vomiting. Initial gastric biopsies revealed CMV gastritis. Repeat assessment demonstrated worsening disease requiring antiviral treatment. After this, further investigations into ongoing epigastric pain demonstrated noncaseating granulomas on repeat gastrointestinal biopsies. A diagnosis of sarcoidosis was established and treated with prednisone to resolution. Management of sarcoidosis involves assessment for multiorgan disease including pulmonary and cardiac evaluation, with initial treatment involving corticosteroids.Helicobacter pylori infection, remote cholecystectomy, a remote 10 pack-year smoking history, and hyperlipidemia. Her symptoms had not improved with acid suppression or gastric emptying agents. She denied B symptoms, extraintestinal manifestations of inflammatory bowel disease, changes in diet, or recent travel. Medications at the time of presentation were metformin, domperidone, and pantoprazole. There was no contributing family history. Physical examination demonstrated mild epigastric pain without organomegaly or masses, with the remainder of the examination being unremarkable. No rashes or joint abnormalities were observed on examination.A 67-year-old woman of Middle Eastern origin presented with acute on chronic diffuse abdominal pain associated with nausea and vomiting. Her medical history was significant for cirrhosis (Child-Pugh Class A) initially suspected to be secondary to nonalcoholic steatohepatitis in the context of obesity, hyperlipidemia, and liver nodularity on computed tomography (CT), as well as mild pancytopenia, gastroesophageal reflux disease, previous 9/L, hemoglobin of 105 g/L, mean cell volume of 80.2 fL, and platelets of 88 × 109/L. Remaining blood work was as follows: aspartate aminotransferase of 45 U/L, alanine aminotransferase of 54 U/L, alkaline phosphatase of 150 U/L, γ-glutamyl transferase of 26 U/L, total bilirubin of 12 μmol/L, albumin of 24 g/L, and international normalized ratio of 1.2. Previous investigations for cirrhosis and autoimmune enteropathies included negative hepatitis C serology, antihepatitis A virus immunoglobulin M (IgM), antinuclear antibodies, antineutrophil cytoplasmic antibodies, antismooth muscle antibody, antienterocyte antibodies, antigoblet cell antibodies, antitransglutaminase antibodies, and antiparietal antibodies. Furthermore, human immunodeficiency virus serologies were negative. Antimitochondrial antibody was intermittently positive with low titers. There was diffuse polyclonal hypergammaglobulinemia on serum protein electrophoresis without abnormal bands. Abdominal ultrasound demonstrated mild ascites and no splanchnic vein thromboses.Complete blood count revealed mild pancytopenia: white blood count of 3.2 × 10H. pylori, treponemal organisms, acid-fast bacilli, dysplasia, or malignancy.Initial esophagogastroduodenoscopy (EGD) and endoscopic ultrasound revealed a polypoid, heterogeneous, friable, solid 0.8-cm nodule arising from the second layer of the gastroesophageal junction. Similar nodules were also visualized in the gastric body. Biopsies of the gastric lesions demonstrated active chronic gastritis with expansion of the lamina propria by a chronic inflammatory infiltrate without granulomas. CMV inclusions were detected by both routine and immunohistochemistry (IHC) Figure . A repeaThe patient was treated with oral valganciclovir for 2 weeks after the first EGD. This was followed by intravenous ganciclovir for 6 weeks for unrelenting epigastric discomfort after the second EGD; subsequent EGDs for persistent pain demonstrated persistently inflamed polypoid lesions in the antrum and duodenum Figure . AlthougEnsuing investigations including thoracic and abdominal CT, CT enterography, and colonoscopy did not reveal any evidence of inflammatory bowel disease. There were multiple subcentimeter lymph nodes noted throughout the mediastinal, precordial, and mesenteric chains, as well as persistent splenomegaly of 21 cm, all of which were stable from previous imaging. A core needle para-aortic lymph node biopsy showed focal granulomas with epithelioid-type histiocytes. No clonal B-cell population was seen with gene rearrangement studies. Bone marrow biopsy was not pursued in consultation with hematology, and review of remote bone marrow biopsy performed 10 years ago was unremarkable. Given the history of cirrhosis, core needle biopsies of the liver were pursued. These demonstrated granulomatous hepatitis with bridging fibrosis Figure . The pat10 Similarly, CMV reactivation in the intestinal tract is rare in immunocompetent individuals.2 Both CMV gastritis and gastric sarcoidosis present with nonspecific symptoms, such as abdominal pain, nausea, and vomiting. Clinical suspicion for CMV would be high in patients with immunosuppression (such as transplant patients or patients undergoing intense chemotherapy), whereas suspicion for sarcoidosis would be higher in patients with concurrent pulmonary symptoms and nongastrointestinal symptoms of sarcoidosis such as subcutaneous nodules.Gastric sarcoidosis and intestinal sarcoidosis are rare conditions with 2.5% and 3.4% of systemic sarcoid patients respectively affected with a lack of clear pulmonary involvement being uncommon.11 However, there has been only one reported case of CMV infection in sarcoidosis, where a female patient with pulmonary sarcoidosis was found to have fever, night sweats, left upper quadrant pain, and elevated liver enzymes with high CMV IgM titers and elevated CMV polymerase chain reaction viral load contributing to the diagnosis.12 Although pulmonary CMV disease was suspected in this case, viral inclusions on pathology was not confirmed.12 An alternative explanation may be that CMV infection may have exposed the presence of gastroduodenal sarcoidosis in our patient, with immunosuppression and inflammation induced by subclinical sarcoidosis allowing for CMV reactivation, possibly through mechanisms involving macrophage activation and tumor necrosis factor-α. In support of this hypothesis, tumor necrosis factor-α is involved in granuloma formation in sarcoidosis and is also an important cytokine for viral replication.14 Finally, it is possible, although less likely, that CMV infection unveiled a de novo presentation of sarcoidosis. Further studies are required to elucidate the association between these 2 diseases.The longstanding history of cirrhosis and lymphadenopathy suggests that this patient likely had pre-existing systemic sarcoidosis with initial subclinical pathology. With granulomatous changes seen on stomach biopsies, Crohn's disease, tuberculosis, autoimmune enteropathy or hepatopathy, sarcoidosis, syphilis, fungal infections such as coccidiosis and cryptococcus, neoplasm, and drug effect were all on the differential. It remains unclear if CMV gastritis preceded gastroduodoenal sarcoidosis or vice versa. Several possible explanations exist for this unique presentation. Immune dysregulation associated with systemic sarcoidosis or an unknown trigger could have led to CMV reactivation, followed by manifestation of sarcoidosis in the stomach and duodenum. In support of this hypothesis, sarcoidosis is believed to be associated with abnormal cell-mediated immunity involving CD4, CD8, and CD19 T-cells which may increase the risk of opportunistic infections.Author contributions: K. Leung and U. Khan wrote the manuscript. I. Teo provided the pathology images. P. James and J. McCurdy revised the manuscript. J. McCurdy is the article guarantor.Financial disclosure: None to report.Previous presentation: This case was presented at the Department of Medicine Research Day; June 15, 2017; Ottawa, ON, Canada.Informed consent was obtained for this case report.


Since the outbreak of the novel SARS-like coronavirus (SARS-CoV-2) in Wuhan, China, more than 4,000,000 cases have been reported globally. Recently, a large number of reports have been published to discuss the possible roles of environmental factors in transmission of the novel coronavirus. They have focused especially on the impact of two air parameters, temperature and humidity, on the spread of SARS-CoV-2.Various reports have drawn contradictory conclusions even when using the similar meteorological data and epidemic data collected in cities in China from January to April 2020. For example, Liu et al.  analyzedWhat is the reason behind these contradictory findings? One reason is the different types of data included in the analyses. Some researchers collected counts of confirmed cases or new cases or total cases, but other researchers calculated the cumulative incidence rate. Different places (cities) have different population sizes and population densities. Considering that the population size significantly regulates the spread rate of COVID-19 , and theAnother reason for the opposite conclusions drawn from different researchers may be the weak correlations between temperature and humidity and SARS-CoV-2 propagation. For example, correlation coefficients acquired from the COVID-19 pandemic in New York, USA, were as low as 0.25–0.4 . The corU-shaped curve at various RH at about 20°C , the spreading efficiency was as high as that at a high RH (60–80%), while the spreading efficiency was at the lowest at moderate-range RH . The inout 20°C . A 30-yeout 20°C .Given that the pressure of saturation vapors is correlated with the temperature exponentially, both water vapor levels and temperatures influence RH. Therefore, either temperature or RH has effects on evaporation, thus influencing size of the droplets . The surIt is suggested that, at a high humidity, such as humidity >70%, viruses survive under moist conditions in the droplets under physiological contents of salts; at moderate humidity (40–60%), salts are concentrated due to the evaporation that deactivates the viruses; and, at low humidity, humidity <30%, salts crystallize out of solution, which may keep the virus active . It is aContrasting with the high humidity areas, RHs of Iran, Iraq, India, the midwestern United States, and other arid areas in the middle and low latitudes are usually lower than 20%. Although the midday temperatures in these areas are usually >20°C (or even > 30°C), the night temperature may below 10°C , which may facilitate the spread of the virus. The influenza virus can survive for more than 24 h in the environment of 23–25% RH and 7–8°C . ConsideAlthough the viral transmission may be mainly determined by the humidity, the temperature may still impart a moderate influence on COVID-19 pandemic . Now, thMost of the current studies have been based on the correlation analysis between the meteorological data and the epidemic data. However, direct studies with SARS-CoV-2 under different environmental conditions are still lacking. The environment variables determining the viral persistence and spread of SARS-like coronaviruses through aerial habitats are still less understood. Besides the studies of epidemiology, biological sciences and medical sciences, the contributions of environmental research are urgently warranted for controlling the spread of SARS-CoV-2 globally.SY conceptualized the analysis and wrote the original draft. S-CJ and Z-LL reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.S-CJ was employed by the Chengdu KangHong Pharmaceutical Group Comp. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.