Further, high mechanical stability was achieved by the use of organic/inorganic stacking of dielectric and channel layers. Thus, the IGZO transistor endured unprecedented bending strain up to 3.33% at a bending radius of 1.5 mm with no significant degradation in transistor performances along with a superior reliability up to 1000 cycles.Flexible In-Ga-ZnO (IGZO) thin film transistor (TFT) on a polyimide substrate is produced by employing a thermally stable SA7 organic material as the multi-functional barrier and dielectric layers. The IGZO channel layer was sputtered at Ar:O For decades, transparent and flexible thin film transistors (TFTs) have drawn much attention and have become the main focus of research in large-area electronics because they can be used in modern electronic applications, such as wearable computers, sensors, photodetectors and rollable displays123452/Vs), high optical transparency and environmental stability78910Cherenack et al. used an IGZO channel material for a flexible TFT and demonstrated a good device performance down to a bending radius of 10 mmPark et al. achieved a reliable bending performance up to 5000 cycles in the IGZO TFTs at a 5 mm bending radius (1.25% strain) without any performance degradationPark et al. demonstrated the flexibility down to 2 mm (corresponding bending strain is 2.5%) with a coplanar IGZO TFTs by adjusting the device structural design (island configuration) as well as its location at the neutral axis plane while being bent2, ZAO, SiNX, and Al2O312131517To achieve the flexibility, transistors have to sustain harsh bending strain. Researchers have employed semiconducting polymers as channel materials in flexible organic TFTs (OTFTs) because the weak intermolecular interactions or van der Waals forces between the polymer chains within the organic materials enhance the strain-bearing capability. However, the mobility of the OTFTs is still a challenging issue2/Vs) compared with those obtained by sputtering20212) gas flow rate ratio during sputtering418Generally, the IGZO thin film on an organic dielectric layer was fabricated either by solution processing or sputtering192021B. U. Hwang et al. deposited an ultrathin Al2O3 layer on the poly-4vinylphenol (PVP) dielectric layer prior to sputtering the IGZO channel layer2O3 has to be replaced by thermally stable organic dielectric materials which can resist the plasma damage during the IGZO sputtering. In addition, the interface quality between channel layer and dielectric layer plays a critical role in the device performance. In case of the inorganic dielectric layer, there have been several reports that explain the interface quality using positive bias stability (PBS) test1326To protect the surface of the organic gate dielectric layer against sputtering damage, 2 gas flow ratio while sputtering. Two different dielectric polymers, PMMA and commercially available poly acrylate based organic material (hereafter referred to as SA7) were employed as a gate dielectric layer. The SA7 polymer was previously used as a buffer layer to achieve a smooth surface and simultaneously to reduce the permeability1328In this work, we proposed a low-temperature fabrication process of flexible IGZO TFTs on a polyimide (PI) substrate and optimized the transfer characteristics by controlling the Ar:O2 gas flow of 100:1 sccm on different gate dielectric layers of commonly used PMMA as a reference and SA7 to study the morphology of the IGZO channel layer (el layer . The optg) as shown in g of around 115 °C but, the SA7 has no endothermic peak in the operating temperature range. At the Tg, the entangled polymer chains have multiple degrees of freedom and become movable in response to the applied thermal energy. Hence, the IGZO film deposited on the PMMA layer underwent strain (deformation) during the subsequent annealing process at 120 °C, resulting in unwanted cracks within the IGZO film  and differential scanning calorimetry (DSC) were utilized. GZO film . MeanwhiGZO film .−1 (CH3 twisting) and 1725 cm−1 (C=O of acrylate carboxyl group) were drastically decreased, and slight peak shift and intensity increase were observed at the 600~800 cm−1 wavelengths. Those variations are mainly ascribed to the interaction of IGZO atoms with the underlying polymer matrix313233−1 wavelengths were very small, and there were no observable changes in intensity at the 1150 cm−1 and 1725 cm−1. Based on above results, the SA7 was selected as a dielectric layer in the following highly bendable IGZO transistor fabrication.To analyze the interaction of IGZO layer with the underlying organic dielectric layer, FTIR were measured for PMMA, SA7, IGZO/PMMA and IGZO/SA7 . After d2 gas was also introduced during the sputtering to reduce the oxygen vacancies, which can reduce the subsequent annealing temperature. Seven samples were fabricated at different oxygen partial pressures , which ranged from OPP1 to OPP16, and their transfer characterizations are given in ds) of 30 V. Their electrical parameters extracted from the transfer curves are compared in −2 A across the entire bias range, and transistor characteristics were not observed. In the other extreme case of OPP16, the IGZO film became an insulator layer and transistor characteristics were also not observed. The on-current (Ion) value of the IGZO TFT decreases with an increase in the oxygen partial pressure during sputtering.Generally, the IGZO film deposited under only Ar gas flow experiences high oxygen vacancies, which results in a high current level, so that the transistor characteristics cannot be achievedon value is subsequently reduced. Further, a Hall measurement was performed to directly measure the carrier concentration and resistivity of the IGZO films deposited at different OPP values. It revealed that by increasing oxygen gas flow, the carrier concentration decreased, and the resistivity abruptly increased at OPP3  decrease, and the I at OPP3 . TherefoTo analyze the interface quality between the IGZO channel and the SA7 dielectric layer, a positive bias stress (PBS) measurement was performed. With increasing the PBS time, more major charge carriers are likely to be trapped at the channel/dielectric interface, resulting in a higher threshold voltage shift2627Lo et al. demonstrated the effect of the gas (Ar:O2) flow ratio on the trap density at the interface and reported a lower trap density for lower oxygen flow rates2/Vs, a threshold voltage of 6.5 V, and a Ion/off of 4.5 × 105. Hereafter, the TFTs fabricated at OPP1 condition were utilized for further bending studies. Considering the plasma-induced thermal damage on the organic dielectric layer, our hysteresis value is much lower than that (~10 V) from the IZO/ PVA-co-PMMA bottom-gated transistorsThe threshold voltage shift at OPP1 is +6.5 V, which is smaller than those of OPP2 (+8.8 V) and OPP3 (+9.7 V), indicating that the IGZO TFT fabricated at OPP1 has a lower defect trap density compared to the other devices. k is Boltzmann coefficient, T is temperature in Kelvin, q is electron charge, Cit = qNss is the effective capacitance of trap state at the dielectric/channel interface, Nss is the trap density at dielectric/channel interface and COX is gate oxide capacitance)102 = 3.9, Al2O3 ≈ 9.5). However, the SS value of SA7-based TFT is lower than that of PMMA-based TFT  than that (2.7 × 1012 cm−2ev−1) of PMMA-based TFT owing to the smoother surface and better interface interaction between the IGZO film and the SA7 layer.The subthreshold swing (SS) of our organic dielectric layer-based TFT is high (~3 V/dec) in comparison with the conventional IGZO TFT (~0.5 V/dec) with an inorganic dielectric layerlculated  and reveon value decreased as the bending radius was decreasing further (1.5 mm).Two IGZO TFTs fabricated at OPP1 were subjected to bending; one device was bent along the channel width direction inset of , and theThe transfer characteristics were also measured while being bent along the channel length or channel width direction . Both saon current. The mobility was also seriously reduced to 3.83 cm2/Vs after the first bending, but it remained constant for the following bending cycles, as shown in The surfaces of TFTs at pristine state, and while being bent at 3 mm and 1.5 mm radius were observed using an optical microscope . There aoff current decreased with the bending cycles along both bending directions. Interestingly, the transistor bent along the channel length direction exhibited a higher on/off current ratio after 50 cycles as a result of the resultant lower Ioff value, and it was then maintained for the following 500 to 1000 bending cycles. The optical images of the transistor channel obtained during the 1000 bending cycles along the channel length direction at a 1.5 mm bending radius showed no noticeable cracks  at a bending radius of 3 mm and 1.5 mm are shown in e cracks .off value. To clarify the effect of moisture on the Ioff current during the bending cycles, the sample, which was already subjected to 1000 bending cycles along the channel length direction in ambient condition, was placed in a glove box (nitrogen atmosphere), and measured the transfer characteristics at different time intervals for a week  without any performance degradation and demonstrated stable transistor characteristics even after 1000 bending cycles, thus showing the unprecedented flexibility of these inorganic-based transistors.In summary, we fabricated flexible IGZO TFTs on PI substrates with the help of SA7 organic material as dielectric/barrier layers. The oxygen vacancies  within the IGZO channel were optimized by supplying oxygen gas while sputtering the IGZO active film, and thus, subsequent annealing of the IGZO film to reduce the oxygen vacancies could be performed even at 120 °C, a suitable temperature that the underlying polymer substrate could withstand. The device fabricated at an Ar:O−6 mbar during the IGZO sputtering. To achieve a smooth surface and a considerable reduction in the permeability, 2 μm thick SA7  was spin-coated as a barrier layer on the PI substrate and annealed at 120 °C for 2 hrs2O3:Ga2O3:ZnO = 1:1:1 atomic ratio) target at a pressure of 3 × 10−6 mbar at different oxygen partial pressures (OPP), which was defined by OPP (%) = PO2/(PO2 + PAr) × 1002 flow rate was varied from 1.01 sccm to 19 sccm with an Ar flow rate at 100 sccm under a constant chamber pressure of 5 mTorr.Bottom-gated IGZO TFTs were fabricated on a 100 μm thick PI substrate. To avoid undesirable crack formation during the fabrication, the thin flexible PI was attached to a rigid glass substrate using an elastic poly-dimethyl siloxane  interlayer. Owing to the adhesive nature of PDMS, the PI substrate can be attached to the PDMS/glass substrate tightly at an annealing temperature of 120 °C and at a vacuum pressure of 3 × 10To see the effect of the OPP on the device performance, we deposited IGZO film at seven different OPP conditions that varied from 0%, 1%, 2%, 3%, 4%, 8% and 16%; we named them as OPP0 , OPP1, OPP2, OPP3, OPP4, OPP8 and OPP16, respectively. After the deposition of the active layer, source and drain (S/D) electrodes (Ti/Au: 5/50 nm) were deposited using a shadow mask with a channel length and width of 100 μm and 2 mm, respectively. Prior to the detachment of the TFTs from the PDMS/glass supporting substrate, the whole setup was annealed at 120 °C. The TFT easily peeled off at the interface between the PI and PDMS with a small external force. The IGZO channel region, including the source/drain electrodes, was observed using an optical microscope before and after the separation from the PDMS/glass substrate .−1. The surface morphology and roughness of the SA7 organic dielectric and the IGZO active layer were analyzed using an atomic force microscope . A scanning electron microscope  was used to analyze the cross-sectional view of the fabricated TFT. A Hall measurement was used to measure the carrier concentration of the IGZO channel. All electrical characterizations were performed using a semi-conductor parameter analyzer (Keithley 4500 C) inside a glove box at room temperature.Thermal properties of the PMMA and SA7 were measured using TGA  and DSC . FTIR spectra were recorded using a FTIR Spectrometer  in the range 600~2250 cmHow to cite this article: Kumaresan, Y. et al. Highly Bendable In-Ga-ZnO Thin Film Transistors by Using a Thermally Stable Organic Dielectric Layer. Sci. Rep.6, 37764; doi: 10.1038/srep37764 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Methylmercury selectively damages the central nervous system (CNS). The tumor necrosis factor (TNF) superfamily includes representative cytokines that participate in the inflammatory response as well as cell survival, and apoptosis. In this study, we found that administration of methylmercury selectively induced TNF-α expression in the brain of mice. Although the accumulated mercury concentration in the liver and kidneys was greater than in the brain, TNF-α expression was induced to a greater extent in brain. Thus, it is possible that there may exist a selective mechanism by which methylmercury induces TNF-α expression in the brain. We also found that TNF-α expression was induced by methylmercury in C17.2 cells  and NF-κB may participate as a transcription factor in that induction. Further, we showed that the addition of TNF-α antagonist (WP9QY) reduced the toxicity of methylmercury to C17.2 cells. In contrast, the addition of recombinant TNF-α to the culture medium decreased the cell viability. We suggest that TNF-α may play a part in the selective damage of the CNS by methylmercury. Furthermore, our results indicate that the higher TNF-α expression induced by methylmercury maybe the cause of cell death, as TNF-α binds to its receptor after being released extracellularly. Methylmercury is an environmental pollutant known as the cause of Minamata disease, and severe central nervous system damage12456Microglia and astrocytes, types of brain cells, regulate the neuroprotective immune response that participates in maintaining brain homeostasis. However, there are cases in which they have instead been shown to be the cause of damage to neurons. In recent years, glial contributions have been noted in the progression and exacerbation of neurodegenerative disorders including Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease8910et al.et al. investigated the effect of adding recombinant CCL2 to culture media on the methylmercury sensitivity of primary rat neurons. Instead of CCL2 reducing methylmercury toxicity, they found it amplified toxicity at high concentrationset al. reported that methylmercury induces IL-6 expression in astrocytes, and that IL-6 exhibits a defensive effect against the neuronal damage caused by methylmercuryActivated microglia and astrocytes produce inflammation mediators , inflammatory cytokines and other products. The production of inflammatory cytokines is known to damage neurons through the invasion of inflammatory cells and cytotoxic T cells1214The TNF superfamily (TNFSF) are representative inflammatory cytokines that participate in processes such as cell survival, induction of apoptosis, the inflammatory response, and cell differentiation. Approximately 20 molecular species have been identified, including cytokines that participate in the induction of apoptosis, such as: TNF-α, lymphotoxin α, the Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL), as well as the RANK ligand (RANKL) and the CD30 ligand (CD30L)1920Male C57BL/6 mice were injected subcutaneously with methylmercuric chloride . Seven days after methylmercury administration, the cerebrum and cerebellum were dissected and any changes in the expression of 18 TNFSF genes were analyzed using quantitative PCR. The expression of TNF-α (TNFSF2) and CD30L (TNFSF8) were found to have increased markedly in both the cerebrum and cerebellum after methylmercury administration . AdditioNext, methylmercury was administered to mice under the same conditions as the experiment depicted in CD30L expression in the cerebrum and cerebellum was significantly elevated on the seventh day; however, by contrast to TNF-α, CD30L expression also rose on the fifth day in the kidneys at approximately the same rate as in the cerebrum and cerebellum . The expWe found a concentration- and time-dependent induction of TNF-α expression resulting from the treatment of C17.2 cells, which are mouse neural stem cells, with methylmercury . The TNF222425As discussed above, TNF-α levels were found to increase significantly in culture medium as a result of methylmercury treatment . TNF-α t27+-dependent transporters39In this study, methylmercury was shown to specifically induce TNF-α expression in brain tissue, and significantly induce TNF-α expression in cultured neural stem cells. TNF-α is an inflammatory cytokine produced by monocytes, macrophages, and fibroblasts. In the central nervous system, astrocytes and microglia are involved in TNF-α production, and in particular, activated microglia produce TNF-α continuously293031333536In addition, although the methylmercury administered in our study to the mice accumulated at a higher concentration in the liver and kidneys than in the brain, the induction of TNF-α expression was found to be most pronounced in brain tissue . This su414322Cytokines are known to regulate the expression of other cytokines, which results in a chain reaction among the cytokines46C57BL/6 male mice (8 weeks old) were purchased from Japan SLC Inc. . Mice were kept in plastic cages  under pathogen-free conditions in a room at 22 ± 2 °C and 55 ± 20% relative humidity, with a light/dark cycle of 12 hr. Mice had free access to commercial food that had been sterilized by radiation , and filtered water ad libitum. The Tohoku University Committee for Animal Experiments approved all animal experiments, and the experiments were performed in accordance with the Regulations for Animal Experiments and Related Activities at Tohoku University and Guidelines for Proper Conduct of Animal Experiments by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. After an adaptation period, mice were randomly divided into control (n = 5) and methylmercury-treated groups (n = 5). Methylmercuric chloride, dissolved in physiological saline, was administered via a single subcutaneous injection at a dose of 25 mg/kg. Control mice received injections of saline only. The cerebrums, cerebellums, kidneys, and livers of the mice were dissected under isoflurane anesthesia. Tissue samples were immediately preserved in Isogen II . Aliquots of the samples were taken for analysis of their total mercury content.® RT reagent kit  according to the manufacturer’s instructions. qPCR was undertaken using SYBR Premix EX Taq (Takara) and a Thermal Cycler Dice® (Takara) with the oligonucleotides shown in Total RNA was isolated from mouse tissue and C17.2 cells using an Isogen II kit (Nippon Gene) according to the manufacturer’s protocol. Reverse transcription was performed using 1 μg of total RNA as the template and a PrimeScript+, Lumex Ltd., Russia) based on the atomic absorption spectrometry. This instrument can real-time detect extremely low mercury concentration in biological samples (detection limit was 1 ug/kg). The accuracy of mercury measurement was ensured by using reference NIES CRM No. 13 .The total mercury concentrations in the various tissues of the mice (approximately 10 mg for each tissue) were measured by Zeeman Mercury Spectrometer (RA-9152 atmosphere at 37 °C. C17.2 cells (4 × 105 cells) were plated in 6-well plates and cultured for 24 hr, followed by treatment with methylmercuric chloride.The C17.2 cell line, which is composed of neural stem cells that were derived from the external germinal layer of mouse cerebellum, were cultured in Dulbecco’s modified eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/mL of penicillin, and 100 mg/mL of streptomycin in a humidified 5% COTNF-α secreted into culture medium was quantified using a Quantikine ELISA kit  according to the manufacturer’s protocol.2, 1 mM dithiothreitol [DTT] and 10% Nonidet P-40) containing protease inhibitor  for 10 min on ice, and centrifuged at 15,000 × g at 4 °C. The supernatant (post-nuclear fraction) was removed, and the pellet was resuspended in nuclear lysis buffer  containing protease inhibitor (Roche). The resulting suspension was sonicated and centrifuged at 15,000 × g at 4 °C to obtain the nuclear fraction. The protein concentration of each fraction was determined using a DC protein assay kit . Proteins in nuclear and post-nuclear fractions were separated by SDS-polyacrylamide gel electrophoresis, transferred to an Immobilon-P membrane , and visualized using primary antibodies against p65 , lamin A/C , GAPDH (Santa Cruz Biotechnology), and horseradish peroxidase-coupled secondary antibodies .C17.2 cells were washed twice with cold PBS and lysed with a hypotonic buffer .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Our results show that in a diverse plankton system comprised of ~464 operational taxonomic units (OTUs) with at least 97% 18 S identity, as few as two or three key OTUs, i.e. large diatoms, Phaeocystis, and mixotrophic/phagotrophic dinoflagellates, can explain a large majority of the spatial variability in the carbon export potential (76–92%). Moreover, we find based on a community co-occurrence network analysis that ecosystems with lower export potential have more tightly coupled communities. Our results indicate that defining plankton communities at a deeper taxonomic resolution than by functional groups and accounting for the differences in size and coupling between groups can substantially improve organic carbon flux predictions.Despite our current realization of the tremendous diversity that exists in plankton communities, we have little understanding of how this biodiversity influences the biological carbon pump other than broad paradigms such as diatoms contributing disproportionally to carbon export. Here we combine high-resolution underway O This is generally predicted and modeled to be a function of Plankton Functional Types (PFTs). PFTs refer to collections of organisms based on explicit biogeochemical roles or requirements. Conventional PFTs include picoautotrophs, diazotrophs (N2-fixers), calcifiers , and silicifiers 7. These PFTs are expected to contribute differently to carbon export production and carbon export efficiency. For example, most diatoms are believed to be efficient carbon exporters to depth because of their large size and heavy silica shells, which make them sink faster8.The biological carbon pump, fueled by nutrient delivery to the sunlit layers of the ocean, is believed to be a function of multiple processes, including net community production (NCP), where NCP is equal to gross primary production minus community respiration. Plankton communities regulate NCP through their influence on the fate of organic matter production, which can be either recycled or exported to depth depending on phytoplankton size and density10 with in-depth molecular taxonomic measurements13 to explore the relationship between the carbon export potential and plankton community assemblages at the Western Antarctic Peninsula (WAP). High-throughput sequencing has recently been shown to be a powerful tool to explore the influence of plankton community structure on carbon export in the oligotrophic oceans14. We apply similar molecular tools at the WAP, where large spatial variability in iron and light availability drives steep gradients in plankton diversity and NCP16. Furthermore, the WAP may represent the “canary in a coal mine” of the Southern Ocean, a region that has a disproportionate influence on global climate17. The WAP region has experienced drastic changes19 over the last 50 years warming at an unprecedented rate20. Community production at the bottom of the food chain, and local and remote upper trophic levels  are likely to be affected by this rapid warming and associated sea ice loss21.In this study, we combine high-frequency NCP estimates22. Cassar et al. (2015)10 hypothesized based on theoretical considerations that the POC-normalized NCP may, under some conditions, provide insight into the apparent sinking rate and turn over time of the organic carbon inventory in the mixed layer. We found that the relative abundance of diatoms was positively correlated with NCP  but that there was no significant correlation with NCP/POC. Diatoms were further divided into centrics and pennates. Near the coast, on the continental shelf, and in the open ocean, the relative abundance (%) of centrics averaged 36.8 ± 16.2, 22.9 ± 26.5, and 5.6 ± 4.7; whereas pennates averaged 14.2 ± 8.2, 23.9 ± 15.5, and 22.2 ± 11.9, respectively. While there were no significant offshore-onshore trends in the relative abundance of pennate diatoms, the relative abundance of centric diatoms was significantly higher near the coast compared to the open ocean . A positive correlation was observed between the relative abundance of centric diatoms and NCP , as well as centric diatoms and NCP/POC . Surprisingly, the relative abundance of pennate diatoms was not significantly correlated with NCP or NCP/POC, which may be explained by the difference in size between centrics and pennates in the region (see discussion below). However, should this relation hold in other regions of the Southern Ocean, a correlation between HPLC pigment estimates of total diatom abundance and NCP may not be observed. This could explain why earlier studies found no clear correlation between HPLC diatom relative abundance and NCP in the WAP region15 and in the Subantarctic Zone south of Australia10.Using DNA sequencing, we first characterized community structure at a taxonomic level comparable to PFTs and High Performance Liquid Chromatography (HPLC) pigment-based definitions Fig. . At this11. Based on the relative abundance of these 20 OTUs, a stochastic search variable selection (SSVS) analysis identified which OTUs were the best predictors of NCP and NCP/POC  or NCP/POC . Many Fragilariopsis species are ice algae and the high abundance in some areas may be due to sea ice melt seeding rather than new cell growth. Our results suggest that PFTs may not be good predictors of NCP in some regions and that the intra-PFT variability may be greater than inter-PFT in terms of NCP.Little is known about the carbon export potential of particular eukaryotic plankton species. The vast array of plankton attributes  and the associated food web dynamics  are likely to be first-order controls on the carbon export potential. In order to gain insight into how NCP relates to the presence of specific eukaryotic plankton, we further characterized plankton assemblages at the OTU level  based on a 97% DNA similarity threshold Fig. . In totaStellarima (OTU_8) in a north bloom near Palmer Station and Proboscia (OTU_6) dominating the south ice edge bloom  was not correlated with NCP, but positively correlated with NCP/POC. Phaeocystis accounted for only 9.0% of the total 18 S reads on average, but co-dominated (34.4%) with diatoms (36.1%) at station (600.040) that had the highest NCP/POC  and NCP/POC (OTU_318) , a group of amoeboid heterotrophic protists with members known to feed on diatoms as well as other protists and bacteria36. Foraminifera, the protists known to carry autotrophic endosymbionts, are also within this supergroup. The ecology and physiology of Rhizaria are not well understood and are not detected by traditional HPLC analyses due to their lack of pigments. However, the recent discovery of their significant contribution to plankton diversity and abundance worldwide37 suggests that they must serve an important ecological function.At the higher taxonomic level (supergroup), NCP and NCP/POC were also negatively correlated with Rhizaria  than Proboscia alata and Actinocyclus actinochilus (a strain closely related to Stellarima), which belong to or are closely related to the identified key diatom OTUs that are best predictors of NCP. Under varying Fe and light availability growth conditions , they are significantly different in size. The biovolume per cell for P. alata and A. actinochilus are more than two orders of magnitude larger than that of F. cylindrus  on two equally divided subsets of 18 S OTUs data based on their NCP/POC values , measures the number of neighbors a node is connected with; the network centralization describes to which extent certain nodes are highly connected to others ; the clustering coefficient measures the degree of clustering between nodes, calculated as ratio of observed vs. maximum number of edges given the number of neighbors; and the characteristic path length is the average shortest path length (number of steps) to connect two nodes. The detailed properties of the low and high NCP/POC networks are summarized in Fig. Other than the effects of biomass, size and phytoplankton growth rates, species interactions and particularly grazing are considered regulators of carbon export. To investigate the influence of interactions on the carbon export potential, we performed a co-occurrence network analysis using SparCC  they do not necessarily reflect direct causal ecological relationships, 2) they may result from niche differentiation and interactions other than predation, including mutualism, competition, and parasitism, and 3) could in some cases be fortuitous or symptomatic of other abiotic and biotic factors not captured by our analyses, such as higher trophic level activity , aggregation, and bacterial activity. For example, the low NCP/POC regions are mostly in the stable open ocean environments that are more homogeneous, resulting in less niche differentiation and more correlations between community members44. In contrast, more niche partitioning and fewer correlations are anticipated in high NCP/POC regions associated with the dynamic and heterogeneous conditions on the continental shelf and at the ice edge. Finally, a greater proportion of net community carbon production could be in the form of DOC in tightly coupled communities (in lower NCP/POC ecosystems). Overall, it will be important to further investigate these results and validate them with estimates of in situ growth rates, photophysiology, microzooplankon growth and grazing rates, and relations to other biotic and abiotic components of the ecosystem.There are several caveats to our study. A lack of steady-state in the mixed-layer carbon budget may blur some of the relationships. Normalizing NCP to POC provides us with a qualitative approach to evaluate how productive the ecosystem is for a given amount of biomass at the ocean surface. However, POC reflects an amalgam of detrital and living organic matter. Our normalization only partly accounts for the multitude of factors that are believed to influence the carbon export potential, including the proportion of detrital matter, aggregation, physiological status, plankton migration and many other processes. These factors vary as a function of the stage of the plankton ecosystem over time (e.g. blooming vs. senescence), which is not taken into account with our normalization. Uncertainty in the ratios of net community O39. In our study, the most dominant group OTU_4  was found not to be a good predictor of net community production. Future studies of interannual variability in this and other regions of the Southern Ocean will be critical to assess the generality of our observed patterns. However, if our results are an accurate reflection of other regions of the Southern Ocean, functional group level studies should be interpreted with caution as the relation of organic carbon fluxes to taxonomy is more nuanced than currently depicted.This study explored the rich genomic complexity of polar ecosystems in the context of net community production, further untangling the intricate interplay of plankton community characteristics critical to carbon fluxes at the ocean surface by selecting through statistical inferences candidate key species and interactions. Within the diverse plankton communities of the WAP, as few as 2 to 3 key OTUs can explain a significant proportion of the spatial variability in carbon export potential during our observational period. Microzooplankton  also arose as important negative predictors of carbon export potential, likely due to enhanced organic matter remineralization at the ocean surface. Using specific OTUs, as opposed to broad functional groups, substantially improved our ability to explain NCP variance from 35% to 92%, and NCP/POC variance from 16% to 76%. These results suggest that the PFT currency needs to be adjusted based on the biogeochemical process or element under study. As an example, diatoms require silica, and their biogeography is strongly delineated by Si availability. However, this functional grouping based on Si requirements does not necessarily translate into a cohesive group when pertaining to carbon export potential2) to the inert gas argon (Ar). The biological O2 supersaturation can be estimated from O2/Ar because O2 and Ar have similar solubility properties. The O2/Ar method is suitable to explore the relation of NCP to plankton assemblages because the O2 residence time in this study is comparable to plankton community turnover time. The residence time of O2 at the ocean surface is calculated as the MLD divided by the piston velocity. For a mixed layer depth ranging from 11 to 22 m (mean 13 m) in sampled stations, with the gas exchange piston velocity calculated at each station using the weighted 60-day wind speed49, the O2 residence time in the mixed layer of 8 to 19 days (average of 11 days)  as described in9. Assuming steady state and neglecting vertical mixing across the mixed layer boundary, NCP in units of mmol O2 m−2 day−1 was calculated as:ρ is mixed layer density (kg m−1), and k is the gas transfer velocity for O2 (m day−1) estimated from NCEP/NCAR daily reanalysis winds (Supplementary Table 51 and a gas exchange weighting as described in49. Mixed layer depth was determined using a density threshold ∆σθ = 0.03 kg m-3 from CTD profiles following52 (Supplementary Table 2 was then converted to carbon (mmol C m−2 day−1) using O2/C = 1.453. NCP is averaged over 10 min at the time point of water collection from the ship’s flow-through seawater line.The biological O(O2/Ar) measured in this region is highly correlated (r2 = 0.83) with the NCP calculated from the seasonal DIC drawdown54.A recent study has shown that the NCP56. In order to assess whether the assumption of steady-state is valid in our study region, we estimated the change in POC inventory over the days prior to ship sampling. To that end, we first estimated the time-derivative of chlorophyll between the time of sampling and 8 days earlier using MODIS ocean color data at 8 days 9 km resolution. We then converted Chl to POC using an empirical relationship derived from in situ Chl and POC (bottle measurements) data measured at the Palmer LTER  in the mixed layer is negligible compared to NCP. In the Southern Ocean, DOC production is a small fraction of NCP59 to avoid mismatch with haptophytes. Amplicons from each sample were tagged using dual index fusion primers60 and a “heterogeneity spacer”61 was used to improve sequencing quality. The “heterogeneity spacer” is a 0–5 bp of spacer added to the index sequence in order to allow different samples be sequenced out of phase and thus improve the amplicon library sequencing quality. Each PCR reaction (25 µl) consisted of 1U of Platinum Taq DNA Polymerase High Fidelity (Invitrogen), 1 × High Fidelity Buffer, 200 µM dNTPs, 2 mM MgSO4, 0.2 µM each primer, and 5–30 ng extracted environmental DNA template. PCR was conducted with an initial activation step at 94 °C for 3 min, followed by 30 three-step cycles consisting of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 1 min, and a final extension step of 72 °C at 10 min. PCR products in triplicates for each sample were then pooled and purified using QIAquick PCR Purification kit, and the concentration of amplicon DNA was quantified using a Qubit dsDNA assay. Amplicons from different locations were pooled in equimolar amounts  and submitted to Duke Institute for Genomic Sciences and Policy (IGSP) for sequencing using Illumina MiSeq. 300PE platform. The averaged read count per sample was 105,253 after demultiplexing.In the lab, from each sampled station one filter was first split in two halves, one for DNA extraction and the other for later RNA studies. 500 µl of the RNAlater from the original tube was loaded onto an Amicon 10 k column and the column was spun for 15 minutes at 14,000 g to get rid of most of the RNAlater and concentrate cells suspended in RNAlater. The resulting solution (~50 µl) was then added back to the one half filter. The cells on filter were lysed by bead-beating using 0.2 g of zirconium beads in 400 µl of buffer AP1 (Qiagen). DNA was extracted and purified using Qiagen DNeasy Plant Mini kit following the manufacture’s instruction. 18 S (eukaryotic) rDNA gene fragments were amplified by PCR using V4 primer sets: 18 S forward (5′- CCAGCASCYGCGGTAATTCC-3′) and reverse (5′-ACTTTCGTTCTTGAT-3′), modified from62. Assembled reads (mean length 424 bp) were further processed including quality filtering and chimera checking using USEARCH, and clustered into 97% OTUs following the UPARSE pipeline63. Representative sequences were aligned and assigned taxonomic identity based on the SILVA rRNA database64 using the software package QIIME65.Paired-end reads were assembled using PANDAseq66. The predictor variables were mean-centered and variance-scaled (i.e. divided by standard deviation). If the dependent variable yi (NCP or NCP/POC) is assumed to be a linear function of potential predictors xi (relative abundance of OTUs), the SSVS can be set up as follow:I is an p × p identity matrix, xi is a 1 × p vector, β = T is a p × 1 vector, and yi, α and σ2 are scalars. i and p represent sample index and number of variables, respectively. α, β and σ2 are considered unknown, with βj=0 indicating that variable xi,p is the nonselected predictor. Unknown β has prior distribution of δ0 denotes Dirac delta function that places all its mass at zero. Other parameters have prior distributions of π ~ Beta, σ−2~Ga, and a, b, aj, bj, μ0, βj is defined as B denotes burn-in. OTUs were ranked by p(βj) (Supplementary Table p(βj) after the selected OTUs.We searched for a subset of predictors, i.e. relative abundance of the top 20 OTUs, best fitting NCP or NCP/POC using Stochastic Search Variable Selection (SSVS)67 and are as follows: 18AF 5′-AACCTGGTTGATCCTGCCAGT-3′ and 18BR 5′- TGATCCTTCTGCAGGTTCACCTAC-3′. PCR products were purified using either QIAquick PCR Purification Kit (Qiagen) or ExoSAP-IT (Affymetrix) and sequenced by Sanger DNA sequencing (Genewiz). Sequences were edited using Geneious Pro 5.6.4 software and BLASTn sequence homology searches were performed against the NCBI nucleotide non-redundant (nr) database to determine species with a cutoff identity of 98%.Diatoms were isolated from the Western Antarctic Peninsula along the PalmerLTER sampling grid in 2013 and 2014. Diatom species were identified by morphological characterization and 18 S rRNA gene (rDNA) sequencing. DNA was extracted with the DNeasy Plant Mini Kit according to the manufacturer’s protocols (Qiagen). Amplification of the nuclear 18 S rDNA region was achieved with standard PCR protocols using eukaryotic-specific, universal 18 S forward and reverse primers. Primer sequences were obtained from−2 s−1 (low light) or 90 μmol photons m−2 s−1 (growth saturating light). Light intensities were chosen based on previous culture-based experiments70. Cultures were grown in the synthetic seawater medium, AQUIL, enriched with filter sterilized vitamin and trace metal ion buffer containing 100 μmol L−1 EDTA. The growth media also contained 300 μmol L−1 nitrate, 200 μmol L−1 silicic acid and 20 μmol L−1 phosphate. Premixed Fe-EDTA (1:1) was added separately for total iron concentrations of either 1370 nmol L−1 (pFe19) or 3.1 nmol L−1 (pFe21.7) to achieve high iron and low iron media, respectively. All media preparation and subsampling were performed under a positive-pressure, trace metal clean laminar flow hood. Cultures were grown in acid-washed 28 mL polycarbonate centrifuge tubes  at least in triplicates (n ≥ 3) and maintained in exponential phase by dilution. Specific growth rates of successive transfers were calculated from the linear regression of the natural log of in vivo chlorophyll a fluorescence using a Turner 10-AU fluorometer during 3–7 days at exponential phase. The error bars of specific growth rates . Diatom biovolumes were calculated according to71 where pennate diatoms were modeled after elliptic prisms and centric diatoms were modeled after cylinders.To estimate biovolumes of each diatom species, frustules were viewed using an Olympus BX61 Upright Wide Field Microscope with the differential interference contrast imaging mode and a 60X/1.42 Oil PlanApo N objective lens. Valve apical length (AL), transapical width (TW), and pervalvar height (PH) dimensions were estimated with Scion Image software , betweenness centrality, and closeness centrality.All OTUs were used in the initial dataset. The rare taxa, i.e. OTUs appearing less than three times in at least 20% of the samples, were removed from the dataset. The OTU abundances were then standardized to the median sequencing depth. The dataset was then divided into two equally sized subsets based on their NCP/POC values ≥ or ≤ the median NCP/POC. For each subset the correlations between OTUs and the p-values were computed using the SparCC method2 of the model (≤0.05).Forward stepwise regression was performed in JMP statistical discovery software from SAS. To reduce the number of regressors in the stepwise regression, which increase the possibility of collinearity and over-fitting, only the top 20 most dominant OTUs were included in the analysis. The best model was selected based on Akaike’s information criterion correction (AICc). AICc is suitable for small sample size (here n = 21), and it reduces over-fitting by greater penalty for increasing number of parameters included in the model. Even though significant in stepwise regression, some OTUs  using function “varpart” in the “vegan” package. Briefly, the unique (non-overlapping) or shared (overlapping) fractions of NCP or NCP/POC variance explained by OTUs were calculated by partial regressions and then decomposing the adjusted RSequences and associated metadata have been deposited in the NCBI Sequence Read Archive under the accession number SRP119815. Other datasets are available from the corresponding author on reasonable request.Supplementary Figures and TablesSupplementary Table S6


The olfactory bulb transforms not only the information content of the primary sensory representation, but also its underlying coding metric. High-variance, slow-timescale primary odor representations are transformed by bulbar circuitry into secondary representations based on principal neuron spike patterns that are tightly regulated in time. This emergent fast timescale for signaling is reflected in gamma-band local field potentials, presumably serving to efficiently integrate olfactory sensory information into the temporally regulated information networks of the central nervous system. To understand this transformation and its integration with interareal coordination mechanisms requires that we understand its fundamental dynamical principles. Using a biophysically explicit, multiscale model of olfactory bulb circuitry, we here demonstrate that an inhibition-coupled intrinsic oscillator framework, pyramidal resonance interneuron network gamma (PRING), best captures the diversity of physiological properties exhibited by the olfactory bulb. Most importantly, these properties include global zero-phase synchronization in the gamma band, the phase-restriction of informative spikes in principal neurons with respect to this common clock, and the robustness of this synchronous oscillatory regime to multiple challenging conditions observed in the biological system. These conditions include substantial heterogeneities in afferent activation levels and excitatory synaptic weights, high levels of uncorrelated background activity among principal neurons, and spike frequencies in both principal neurons and interneurons that are irregular in time and much lower than the gamma frequency. This coupled cellular oscillator architecture permits stable and replicable ensemble responses to diverse sensory stimuli under various external conditions as well as to changes in network parameters arising from learning-dependent synaptic plasticity. The mammalian olfactory bulb responds to odor stimulation by generating fast oscillations in its electrical field potential. Such oscillations are indications that a substantial number of principal neurons in the olfactory bulb are coordinating their activities in time, which often means that their action potentials are synchronized, or partly synchronized, such that the pattern of small differences in their spike times contains olfactory sensory information. We are interested in the mechanisms by which olfactory bulb circuitry can transform sensory information from the temporally unsophisticated spike rates of primary sensory neurons into this sophisticated cortical format. We present a biophysically explicit, multiscale dynamical model of the olfactory bulb network that generates these oscillations. The elements of this model are designed to adhere to experimental findings from individual neurons, membrane currents, and synapses as well as the functional network. Together, these elements generate gamma oscillations exhibiting the full range of properties of those in the biological system. We show that these dynamics arise from an inhibition-coupled oscillator framework, a type of dynamical system with some established mathematical properties. This finding enables us to understand how the olfactory system translates sensory information for distribution in the central nervous system, and how different areas of the brain can mechanistically coordinate with one another so as to regulate the flow of sensory information to appropriate target structures. The mammalian main olfactory bulb (OB) plays a central role in processing and relaying olfactory information from the primary sensory epithelium to subcortical and cortical areas . This prIt is generally accepted that OB gamma oscillations are intrinsic, and mediated by a fast negative feedback loop formed between principal output neurons  and a class of inhibitory GABAergic interneurons , interacting via dendrodendritic synapses in the external plexiform layer (EPL) of the OB  and sharply heterogeneous afferent activation levels. The explicit, multiscale nature of this dynamical model further enables the elaboration, explanation, and experimental testing of the underlying mechanistic details that may underlie these observed physiological phenomena.To address this question, we developed a conductance-based, dynamically detailed biophysical model of the OB network. The present model is based on our earlier two-layer model of cholinergic neuromodulation in the OB , but embin vitro recordings from olfactory bulb [j] denoted the MC in the ith column and the jth row). In some figures, model neurons were denoted by a single index to enable their distribution along a single axis . In such cases, that single index z was related to the two indices i and j as follows: z = N * i + j + 1, where N was 5 for MCs and PGCs and 10 for GCs.The “default” OB network model contained 25 mitral cells (MCs), 25 periglomerular cells (PGCs) cells and 100 granule cells  and Esyn is the reversal potential . The function B(V) implemented the Mg2+ block for the NMDA current, and was defined as B(V) = (1 + [Mg2+]exp(−0.062V)/3.57)−1A currents, B(V) = 1. The gating variable s represented the fraction of open synaptic ion channels and obeyed first-order kinetics [F(Vpre) was an instantaneous sigmoidal function of the presynaptic membrane potential, F(Vpre) = 1/(1 + exp(−(Vpre –θsyn)/σ)). The half-activation potential (θsyn) of the synapse was set to 0 mV for AMPA/NMDA receptor synapses and -40 mV for GABAA synapses; the parameter σ was set to 0.2 for AMPA/NMDA currents and 2.0 for GABAA currents [α and β) were expressed as α = 1/τα and β = 1/τβ, where τα and τβ were the synaptic rise and decay time constants respectively. For AMPA receptor currents, τα = 1 ms, τβ = 5.5 ms; for NMDA receptor currents, τα = 52 ms, τβ = 343 ms; and for GABAA receptor currents, τα = 1.25 ms, τβ = 18 ms [The MC→PGC and MC→GC synapses were mediated by both AMPA and NMDA receptors, whereas the PGC→MC and GC→MC synapses were mediated by GABAed as in :Isyn=Wgurrents; ). The masynapses . W denotkinetics , 85:dsdcurrents . Consequ = 18 ms , 23, 86;u0 was the pre-odor value (simulated pure air input) and us the steady-state value after odor excitation. The parameter r determined the transition rate from u0 to us (set to 100) and torn the time of odor onset. Different MCs  received different levels of afferent activation; the corresponding values of u0 and us were drawn from uniform distributions u0 ∈  and us ∈ . Additionally, all cells in the network received random excitatory inputs representing intrinsic and extrinsic sources of uncorrelated background noise. These nonspecific inputs were modeled as uncorrelated Poisson spike trains mediated exclusively by AMPA receptors; specifically, they comprised instantaneous steps followed by exponential decays with a time constant of 5.5 ms [Odor stimulation was modeled as in . A sigmof 5.5 ms . When plA simulated local field potential (sLFP) was constructed by filtering the mean (somatic) membrane potentials across all MCs . Filterii was the phase of each MC spike in the network relative to the sLFP peak. This synchronization index (SI) measures the degree of phase locking between MC spikes and sLFP oscillations rather than the absolute synchrony of MC spikes in time. Nevertheless, when substantial numbers of MC spikes are evoked, the SI also is a good measure of absolute spike synchrony. When all MC spikes have identical phases, the index achieves its maximal value of unity. The oscillation index (OI) corresponded to the peak of the frequency power spectrum of the sLFP, which was normalized to the largest peak value generated from ten sets of simulations with different random seeds. The oscillation frequency was determined from the position of the spectral peak in the power spectrum [The synchronization (or phase-locking) index was calculated as follows :κ=1/N[∑spectrum .The model was implemented in the neuronal simulator package NEURON, version 7.3 , using t


In plants, the subunits of these thylakoid membrane protein complexes are both chloroplast and nuclear encoded. How the chloroplast-encoded subunits of multi-spanning cytochrome b6 are targeted and inserted into the thylakoid membrane is not fully understood. Experimental approaches to evaluate the cytochrome b6 import mechanism in vivo have been limited to bacterial membranes and were not a part of the chloroplast environment. To evaluate the mechanism governing cytochrome b6 integration in vivo, we performed a comparative analysis of both native and synthetic cytochrome b6 insertion into purified thylakoids. Using biophysical and biochemical methods, we show that cytochrome b6 insertion into the thylakoid membrane is a non-spontaneous co-translational process that involves ALB3 insertase. Furthermore, we provided evidence that CSP41 (chloroplast stem–loop-binding protein of 41 kDa) interacts with RNC-cytochrome b6 complexes, and may be involved in cytochrome b6(petB) transcript stabilization or processing.The cytochrome  The 220-kDa cytochrome b6 f complex dimer occupies an electrochemically central position in the electron-transport chainb6 f provides an electronic connection between the two photosynthetic reaction centers of PS I and PS II, allowing linear electron transfer from the H2O electron donor to the NADP (nicotinamide-adenine dinucleotide phosphate) acceptorb6 f and PS I complex participate in cyclic electron transfer, which generates an electrochemical proton gradient across the thylakoid membrane without net production of reducing equivalentsb6 f requires numerous regulatory factors for coordinated transport, insertion and assembly of these complexes subunits from both chloroplast and nuclear originPhotosynthetic electron transport is accomplished by three hetero-oligomeric integral oxygenic photosynthetic membrane protein complexes: Photosystems I (PS I) and II (PS II), and the cytochrome trans-thylakoid pH gradient as its sole energy source7. All of these are found in the soluble stromal fraction. However, the “spontaneous” pathway does not seem to require any soluble factors or energy source for protein insertion into membraneHowever, for the import into the thylakoid membrane of proteins from both nuclear and chloroplast origin, four independent precursor-specific transport pathways had been proposed Commonly, nuclear-encoded multi-spanning proteins are targeted to the thylakoid membrane by hydrophobic amino acid sequences from either their transmembrane segments or from a cleavable signal sequence (ss)11b6 binds a one covalently c-type haem as well as two non-covalently b-type haems and consists of four transmembrane helices, while the signal for this integral protein integration with thylakoid membrane remains unknown13b6 f complex, the first step involves the transcriptional activation of the chloroplast petBD operon (encoding cytochrome b6 and subunit IV)petB and petD mRNAs are translated into the polypeptides that undergo insertion into the membrane and form the polytopic monomeric core of the cytochrome b6 f complex. In the next step the monomers form a dimer (CS) which is stabilized by lipids, and simultaneously a Rieske ISP-cytochrome f sub-complex (RF) is formed. This sub-complex then interacts with the CS to form a cytochrome b6-subunit IV-ISP-cytochrome f sub-complex (CSRF). Regardless of the formation of the CSRF complex, small subunits  form an additional sub-complex which may interact with the RFb6 f complex is formed.The chloroplast-encoded cytochrome b6 f complex assembly process requires a complex coordination between transcription, translation, chloroplast membrane transport, membrane insertion and sub-complexes assembly.Hence, cytochrome b6 import mechanism in vivo were limited to bacterial membrane and therefore did not involve the chloroplast environment16171819To date, experimental approaches to evaluate the cytochrome b6 integration into the thylakoid membrane. Our comparative analysis of both native and synthetic cytochrome b6 revealed that an unfolded cytochrome b6 can be anchored into the thylakoid membrane by hydrophobic interactions that can be removed by chaotropic action. Hence, we excluded the spontaneous pathway for insertion of cytochrome b6 into the thylakoid membrane in vivo. Furthermore, our results indicate that the proper integration of cytochrome b6 is co-translationally mediated by other proteins. Indeed, we determined ALB3 insertase is a crucial protein for cytochrome b6 insertion into thylakoid membrane. Furthermore, we identified cpFtsY and CPS41 (chloroplast stem–loop-binding protein of 41 kDa) as other proteins involved in this process. These data are not limited to cytochrome b6, but also provide a new insight into the mechanisms involved in the insertion of integral membrane proteins integration into the thylakoid membrane.Hence, the objective of the present study was to examine the mechanism governing cytochrome b6 inserts into the thylakoid membrane. For these experiments, we used import assays for the insertion of different variants of cytochrome b6 proteins into purified Pea thylakoids. Our main criterion for correct insertion was that the cytochrome b6 has to be integrated with the membrane and thus cannot be extracted along with extrinsic, non-inserted proteinsb6 has to be properly oriented in the membrane, with the C- terminus and N-terminus at the stromal side of the thylakoid membrane.The primary aim of this work was to analyse the mechanism by which cytochrome b6 integration with the Pea thylakoid membrane, the protein was purified from Synechocystis sp. PCC 6803 as described in ref. b6 protein sequences from Pea and Synechocystis sp. PCC 6803 was above 87% and positions with identical amino acid residues were above 79%. The circular dichroism (CD) spectra of isolated native cytochrome b6 indicated a high proportion of the α-helical structure characterised by negative maxima at 208 and 222 nm  was almost entirely chaotropic-extractable by a mild concentration of urea (4 M) and not detectable in the membrane fraction . The GuHCl treatment led to a relatively flat spectrum indicating a substantial loss of secondary structure . Furthermore, both denaturized and refolded spinach apocytochrome b6 was sensitive to 4 M urea extraction , since this protein inserts into the thylakoid membrane by an apparently spontaneous pathwayb6 into the thylakoid membrane occurs spontaneously, the spontaneous insertion of mature PsbW into the thylakoid membrane observed in the same experimental model validates our methodological approach.In order to validate our cytochrome In order to probe the structure of the soluble form of the synthetic PsbW protein, biophysical analyses by CD and MS (mass spectroscopy) were performed. The CD spectra of denatured and DDM refolded PsbW differed significantly in secondary structure, indicating that the PsbW protein forms a transmembrane α-helix in hydrophobic environments of DDM . The obtin vitro experiments verified that synthetic PsbW is indeed spontaneously inserted into the isolated thylakoid membrane. The thylakoid import assays showed that the PsbW inserted into the thylakoid membranes and sorted efficiently also in an absence of a stromal fraction  and in the presence of apyrase  process termed co-translational translocation, chloroplast import experiments were performed using cell free in vitro system.The results of the import assays questioned both spontaneous and Sec-dependent mechanisms for cytochrome b6 expression was performed in the linked system and transcription and translation reactions were separated. Translations were carried out in the presence of thylakoid membrane or thylakoid membrane with a stroma fraction. As shown in b6 into membrane due to degradation of thylakoid and stromal translocation proteins. Furthermore, in 2627b6 is Sec-independent. Following the import assay of native cytochrome b6, protein integration with thylakoid membrane was tested with the use of a chaotropic agent  after immunoprecipitation of cross-linked proteins with an antibody against cytochrome b6. These proteins were identified with MS using Mascot Distiller software as shown in b6 without crosslinker was used as a control of the specificity of the cross-linking  or for untranslated plastid mRNA stabilization (CSP41). The cytochrome b6 complexes at the membrane. Furthermore, we have demonstrated that GTP hydrolysis is required for cytochrome b6 insertion. As presented in b6 integration, suggesting that cpSRP–cpFtsY complex occupies functional ALB3 integration sites.The MS analysis of cross-linking products shown that cpSRP, cpFtsY and ALB3 form a complex with RNC-cytochrome b6 import into the thylakoid membrane was by the Sec/ALB3 insertion site or by ALB3 itself. To test this hypothesis, cell-free native spinach cytochrome b6 translations were carried out in the presence of thylakoid membrane with the stroma fraction in a presence of ALB3 antibody. We did not observe this effect in the presence of antibody against cytochrome f (negative control) or against C-terminus of cytochrome b6  that the protein should be integrated with the membrane and hence resistant to chaotropic extraction, (ii) and that it should be properly oriented within the membrane.The mechanisms responsible for targeting and insertion into the thylakoid membrane of both nuclear and chloroplast-encoded proteins are still not well understood. The presence of signalling sequence, protein structure and origin, are crucial for the choice of transport pathway involved in import processin vitro translation products suggested that this protein may be inherently stable in aqueous phases and hence a good model system for studying membrane proteins in generalAs a control for our experimental model, we tested if subunit W (PsbW) of the photosystem II is spontaneously inserted properly into the thylakoid membrane. PsbW is a single-span thylakoid membrane protein that is synthesized with a cleavable hydrophobic signal peptide and integrated into the thylakoid membrane by an apparently spontaneous mechanismb6 can spontaneously insert into the bacterial cytoplasmic membraneb6 protein is largely unstructured in aqueous solution, it acquires a highly α-helical structure upon interaction with certain types of lipid prior to full insertionRecently, it was reported that cytochrome b6 was induced by dialysis of solubilised cytochrome b6 to buffer with DDM. The disordered structure of the solubilised form of cytochrome b6 in the presence of DDM was converted into a folded structureIn our study, the folding of cytochrome b6 isolated from Synechocystis sp. PCC 6803 was observed nevertheless. Furthermore, completely denatured cytochrome b6 was only partially imported to the thylakoids and could also be easily removed by urea washes. The formation of α-helices is a key event during membrane protein insertion, but the precise timing of α-helix formation is unclear in most casesin vitro insertion is extremely difficult to address usually because of the insoluble nature of membrane proteins in aqueous buffer. Herein we show that DDM stabilizes native cytochrome b6. While soluble cytochrome b6 showed no detectable α-helix content, upon addition of DDM, there was a rapid increase in secondary structure formation. This indicates that the hydrophobic regions of cytochrome b6 are able to form α-helical structures in a hydrophobic environment, leading to protein insertion into the membraneb6 is not able to insert into the thylakoids by a spontaneous pathway.The formation of α-helices is a key event during spontaneous protein insertion into the membrane. Energetic considerations strongly suggest that these must form prior to full protein insertion39b6 into the thylakoid membrane has been also suggested17b6 and the opposite orientation in the cytoplasmic membrane after expression in E. coli suggests that other pathways are utilized. Indeed, previous studies in bacteria show that the fusion of cytochrome b6 to MBP  directs the cytochrome b6 onto the Sec-dependent pathwayb6 protein are recognised by the E. coli Sec translocon leading to integration of this protein into the bacterial inner membrane although in an opposite orientation as compared to that in the thylakoidThe Sec-dependent pathway for the integration of cytochrome b6 is not a typical passenger for unassisted integration since it lacks the cleavable N-terminal hydrophobic domain, which is missing in the prokaryotic or plastid-encoded counterparts. Consequently, a “helical hairpin”-type loop insertion mechanism is not feasible for cytochrome b6. Two variants of an SRP-dependent pathway exist, the posttranslational and the co-translational variantCytochrome b6 is a chloroplast protein targeted in a GTP-dependent process termed co-translational translocation, we performed in vitro chloroplast import experiments. Although the cytochrome b6 protein was not synthesized in the wheat germ extracts but in the human in vitro protein system, the protein was efficiently transported into the thylakoid membranes. Mass spectrometry analysis of the cytochrome b6 crosslinked proteins allowed identification of cpSRP54, cpFtsY (both GTPase), ALB3 and CSP41 as potentially involved in targeting and insertion of cytochrome b6 protein into the thylakoid membrane. As reported previously, cytochrome b6 elongating nascent chain interacts with the cpSRP54 but not with cpSecYb6 insertion could be co-translational and mediated by cpFtsY and TMH (trans membrane helices) in order to enter the ALB3. cpFtsY would interact with the thylakoid membrane to support cpSRP-dependent targeting. Furthermore, a conserved amphipathic helix located at the N-terminus of cpFtsY that is both necessary and sufficient for interaction with the thylakoid membraneIn order to confirm directly that the cytochrome b6 import into the thylakoid membrane. These results are consistent with earlier studies showing that nuclease pretreatment did not remove the cytochrome b6-RNC complexes from the membrane44et al.0IIIWe also show that impairment of ALB3 limits cytochrome However, so far, it has not been shown that these proteins or any other membrane protein strictly requires both ALB3 and cpSecY for insertion into the thylakoid membrane of plants. Moreover, it was also shown that the D1 nascent peptide may also have the ability to localize to the membrane independently of cpFtsY and cpSecY via ALB3. However, the subsequent integration events may require the use of cpSecY for lateral movement or assembly of the D1 protein from the ALB3 translocon into the lipid bilayerb6 integration. However, antibodies against cpSecY prevent cpSecA-dependent protein translocation2627Herein, we show that thylakoid membrane treatment with anti-cpSecY antibody was not able to prevent the cytochrome b6 or RNC-cytochrome b6 complexes with cpSecYb6 into the thylakoid membrane as depicted in Neither this nor previous studies have shown interaction of cytochrome b6in vitro translation mixtures provided a first evidence that CSP41 interacts with RNC-cytochrome b6 complexes, and maybe be involved in petB transcript stabilization or in a process of monocistronic petB mRNA synthesis. CSP41 proteins are highly abundant chloroplast proteinsin vitro to the 3′ end of the petD mRNArbcL), PSI , and PSII  core proteins, and 16S and 23S rRNAspetD mRNA, encoding subunit IV from the cytochrome b6 f complex, and is transcribed as a part of the polycistronic cluster psbB-psbH-petB-petD, which is subsequently processed to monocistronic mRNAspetD mRNA of the cytochrome b6 f complex was 20% reduced comparing to WTb6 insertion into thylakoid membranes requires further studies.Our mass spectrometry analysis of cytochrome b6 protein into the thylakoid membrane. Our data demonstrated that the formation of a complex between cpSRP–cpFtsY and ALB3 is a necessary step in the integration of cytochrome b6 into thylakoid membranes. However, future studies that combine more exact membrane fractionation approaches with RNC profiling are required to understand the interplay between cytochrome b6 and ALB3 insertase and/or ALB3/SecYEG translocon as well as the role of CSP41.Herein, we provided evidence for the role of SRP54, FtsY, and ALB3 in targeting and insertion of cytochrome Pisum Sativum, cv Calvedon) were germinated, planted in plastic trays and grown hydroponically as described in ref. Seeds of Pea (−1 CHL) were incubated (5 min on ice) with micrococcal nuclease (8 units μL−1) in the presence of 10 mM CaCl2, protease inhibitor cocktail  and 1 mM DTT (dithiothreitol). The digestion was stopped with 20 mM EGTA. Finally, the isolated thylakoid membranes were washed twice and resuspended in HMS buffer  apocytochrome b6 as a fusion protein (ss-cytochrome b6) to the C-proximal part of the signal pre-sequence of the Oxygen Evolving Complex .The PsbW protein (GenBank: CAA59409.1) was obtained from GenScript at purity above 85%. Cytochrome Ala-X-Ala consensus sequence that is recognised by a thylakoid processing peptidase (TPP) and that cleaves off this transit sequence was changed to protect fusion protein from being cleaved (SLQSDFKELAHKCEASKIAGFALVTSALVASGRSA). An additional Lys was incorporated into C-terminus in order to allow for biotin labelling. The molecular weight of synthetic protein was assessed with the MALDI and ESI-MS techniques , using the Mascot database search engine The http://www.matrixscience.com/help/scoring_help.html)For identification, the proteins were analysed in electrophoresis, stained with Coomassie solution and blotted. Furthermore, protein bands were cut out and subjected to in-gel digestion. After reduction of cysteine residues with DTT and alkylation with iodoacetamide, proteins were digested with trypsin and the resulting peptide mixtures analyzed by liquid chromatography/MS. After preprocessing of the raw data using Mascot Distiller software, output lists of precursor and product ions were compared for identification to the NCBI database using the Mascot database search engine −1 final) for 2 h on ice. The digestion was terminated with 4 mM PMSF (phenylmethylsulfonyl fluoride) and the thylakoid membranes collected . The proteins  were incubated with the thylakoid membranes for approximately 1 h. Import incubation mixtures contained the excess of thylakoid membrane (50 μg CHL), stromal extract (30 μg protein) and 10 mM ATP (adenosine 5′-triphosphates). All experiments were carried out at 25 °C under a green safe light, and a microstirrer/heater thermocouple with the reaction medium was used to ensure that the proteins were in a homogeneous distribution and there was a proper temperature within the subphase below the thylakoid membrane. If present, the stromal extract was equivalent to 1.3 times the CHL concentration. The import assays were terminated and unincorporated proteins removed by washing the thylakoid membrane three times in 2 mL of ice-cold 50 mM HEPES-KOH buffer, pH 8.0 with 330 mM sorbitol .In order to remove external endogenous proteins domains, thylakoid membranes were pre-incubated with proteinase K  and exposed to light (~300 μmol photons m−2 s−1) at 4 °C, in order to restore the ΔpH. Subsequently the thylakoid membranes were incubated with trypsin  units mg−1) for 10 min on ice. The digestion was stopped with trypsin inhibitor , and membranes were washed twice in HM enriched with 60 μg mL−1 trypsin inhibitor . The thylakoids were finally collected by centrifugation at 20,000 × g for 10 min at 4 °C and resuspended in stromal extract or in 15 μL of HM buffer containing 5 μg of trypsin inhibitor.The Michl, −1 protein) in 0.1 M Na+ citrate buffer, pH 6.5) was added for 1 h at 30 °C. Subsequently, after this treatment an equal aliquot of enzyme solution was added and the incubation continued for another 30 min. Controls received 0.1 M Na+ citrate buffer, pH 6.5, instead of the enzyme solution. The final pH of the samples was kept at 6.5–7.0. The proteolytic reaction was stopped by addition of the carboxypeptidase inhibitor .Thylakoid membranes prior to carboxypeptidase B  treatment were supplemented with procaine and Triton X-100 at final concentrations of 3 mM and 0.01%, respectfully. Then, carboxypeptidase B  were incubated for 15 to 30 min on ice in either 0.1 M NaHCO3-NaOH, pH 12.5 or 4 M urea in 50 mM HEPES-KOH pH 7.7, 50 mM potassium acetate, 100 mM mannitol, 5 mM magnesium acetate, 2 mM DTT and a protease inhibitor cocktail. After these incubations, the membranes were washed and collected in HMS buffer .The proteins associated with but not integrated into the thylakoid membranes are not resistant to chaotropic agentsb6 construct were performed with the Human in vitro Protein Expression Kit for DNA Templates (Thermo Scientific). For in vitro transcription of the full length cytochrome b6 plasmid pT7CFE1-b6 containing the 5′ UTR consisting of EMCV internal ribosome entry site (IRES) was used. pT7CFE1-b6 was isolated using a standard mini prep protocol . To avoid compromising protein expression yield, RNase A was used during the purification. Transcription with T7 polymerase was performed according to Human in vitro Protein Expression Kit for DNA. The reaction was performed at 32 °C for 90 min in a 20 μL reaction mixture and a total of 1 μg of DNA template was used. The mRNA (2 μg) generated from the transcription was added to each translation reaction mix containing all of the machinery for protein expression . Translation reactions were performed essentially according to manufacturer’s recommendations for a 50 μL translation reaction. However, some modifications were made for insertion of cytochrome b6 into thylakoid membrane. Radioactive [35S]methionine  and additional RNase and protease inhibitors  were used during the translation assays. Mannitol was added (100 mM) to stabilize thylakoid membrane. Cytochrome b6 was synthesized in vitro in the presence of thylakoid membrane (250 μg mL−1 of CHL) with or without the fresh prepared stromal fraction  for 2 h at 30 °C. To increase protein yield, 2 μL more of the transcription mixture (1.0 μg μL−1) and 1 μL of energy mix was added for each reaction being performed after 2 h of translation and each reaction mixture was incubated for another 2 hours. The total reaction volume was 100 μL. Each translational sample (25 μL) was analysed by SDS-PAGE and the autoradiography.Coupled transcription/translation reactions of spinach cytochrome −1 final) for 2 h on ice. The digestion was terminated with 4 mM PMSF and the thylakoid membranes collected . In order to remove endogenous RNA during translation reactions thylakoid membranes or stroma were pre-incubated with RNase A (5 ng RNase A) for 15 min at 37 °C. Prior to translation reaction ribonuclease inhibitor  was added (2 μl RNasin ribonuclease inhibitor (40 units/μL).In order to remove external domains of endogenous proteins, thylakoid membranes or stroma were pre-incubated with proteinase K (40 μg mLN-hydroxysuccinade ester bis[2-(succinimidyloxycarbonyloxy) ethyl] sulfone, Pierce) or DMA . Reactions were then quenched with a large molar excess of DTT in an equal volume of HMS buffer. The thylakoid membranes were than incubated with 1% DDM (30 min at 0 °C) and insoluble materials were removed by ultracentrifugation at 50,000 rpm (Beckman SW60 Ti rotor) at 4 °C for 1 h. The resultant supernatant was further incubated (30 min) with an excess (5 μg mL−1) of antibody against cytochrome b6 N-terminusTo isolate targeted RNCs, translation reactions were diluted with one volume of ice-cold 50 mM HEPES-KOH, pH 7.8, 1 M potassium acetate, 10 mM magnesium acetate buffer (HMS) and centrifuged for 1 min at 12,000 × g. The thylakoid membranes pellets were re-suspended to the original volume (100 μl) in HMS buffer and treated (for 30 min at 0 °C) with the membrane-permeable crosslinkers: BSOCOES ® (ab53468). After washing steps  the membranes were incubated with goat anti-rabbit IgG (H + L) secondary antibodies (BioRad) and detected using ECL . Densitometry was performed using Image Lab software v. 4.1 (BioRad). We use the same exposure time for all autoradiography.The protein concentration was determined with the BCA (bicinchoninic acid) reagent according to the manufacturer’s instructions (Pierce). Following the normalization of protein concentrations, samples were mixed with an equal volume of 2X Laemmli sample buffer and incubated for 5 min at 95 °C prior to separation by Tricine/Tris SDS-PAGE as described in ref. How to cite this article: Króliczewski, J. et al. ALB3 Insertase Mediates Cytochrome b6 Co-translational Import into the Thylakoid Membrane. Sci. Rep.6, 34557; doi: 10.1038/srep34557 (2016).


All the synthesized compounds were evaluated for their in vivo anti ulcerogenic activity using Albino rats (weighing 180–220 g). The interactions between the compounds and active site residues of H+/K+ ATPase were investigated by molecular docking studies using autodock vina 4.0. SCH28080 was used to validate the docking results. Also the drug likeliness of these compounds was predicted using Molinspiration server in light of Lipinski’s rule of five. All the six synthesized compounds exhibited higher anti-ulcer activity as compared to omeprazole. These novel hybrid compounds showed comparable anti-ulcer potential of 72–83% at dose level of 500 µg/kg, whereas omeprazole showed 83% anti-ulcer activity at dose level of 30 mg/kg. The results clearly indicate that these novel benzimidazole-pyrazole hybrids can present a new class of potential anti ulcer agents and can serve as new anti-ulcer drugs after further investigation.A series of six novel benzimidazole-pyrazole hybrid molecules was synthesized and characterized using elemental analysis (CHN) and spectroscopic methods ( To check the hypothesis docking studies were carried out and very good binding energies of our compound with the target comparable with omeprazole and SCH28080 were obtained. This suggests that H+/K+ATPase inhibition is a possible mechanism of action of these compounds, but involvement of other mechanisms cannot be ignored. Hypothesis can be validated through in vitro studies, which is part of our future plans.Recently some new derivatives of mercapto benzimidazole have been reported for their strong inhibitory potential against H 20379-4  5,7-dihy 20379-4 , 4- ≤10 that they have strong absorption.Drug likeliness of our synthesized compounds is given in Table All the compounds have TPSA within the range. The drug-likeness data of our compounds suggested that these compounds can be used as drugs because all of the above mentioned descriptors for these molecules are within the range.Docking scores of first three best docked posses are given in Table Docking scores of synthesized compounds is also better than SCH28080, omeprazole, its sulfenic acid derivative (SC) and sulfenamide derivative (SA) predicting that synthesized compounds will be more active. This is also observed in in vivo activity test as dose for omeprazole was very higher than the dose of compounds (5a–f) suggesting that these compounds are far more potent than omeprazole.In post docking analysis it was observed that compound 5b, 5c, and 5f are binding to the active site in almost same pose, whereas, 5a, 5d, and 5e are binding to the active site in totally different pose. According to docking score poses of compounds 5a, 5d and 5e are energetically better than the posses of compounds 5b, 5c and 5f as shown in Fig. SCH28080 covers the Cys 813 in the binding pocket. It can be observed that compound 5d covers the Cys 813 in the binding pocket in the same way as SCH28080 as expressed in Fig. All of the compounds (5a–f) are showing more interactions with the amino acid residues surrounding the active site as compared to other reference molecules. It can be seen in the Fig. The test compounds 5a, 5b, 5c, 5d and 5e did not causes any mortality up to dose of 100 mg/kg. 5f was found lethal at the dose of 100 mg/kg.+/K+ ATPase were established through docking studies. It was found that our compounds showed higher binding affinities as compare to omeprazole. This suggest that compounds (5a–f) might be acting through the same mechanism as omeprazole and other related compounds but involvement of other mechanism cannot be ignored. The study confirmed that these molecules can present a new class of lead molecules for drug discovery as H+/K+ ATPase inhibitors.Six novel benzimidazole-pyrazole hybrids were synthesized and evaluated for their anti-ulcer activity. All the compounds exhibited potent anti-ulcer activity at lower dose levels as compared to the standard, omeprazole. Further the molecular interaction of the these novel hybrids molecules with the target H