Photobacterium sp. A5-7 isolated from marine sediment was characterized. P57 could hydrolyze casein, gelatin and collagen. It showed the highest activity at 40°C and pH 8.0. P57 is a new subtilase, with 63% sequence identity to the closest characterized protease. Mature P57 contains a catalytic domain and an inserted PA domain. The recombinant PA domain from P57 was shown to have collagen-binding ability, and Phe349 and Tyr432 were revealed to be key residues for collagen binding in the PA domain. This study first shows direct evidence that the PA domain of a subtilase can bind substrate, which provides a better understanding of the function of the PA domain of subtilases and bacterial extracellular proteases from marine sediment.Bacterial extracellular proteases are important for bacterial nutrition and marine sedimentary organic nitrogen degradation. However, only a few proteases from marine sedimentary bacteria have been characterized. Some subtilases have a protease-associated (PA) domain inserted in the catalytic domain. Although structural analysis and deletion mutation suggests that the PA domain in subtilases is involved in substrate binding, direct evidence to support this function is still absent. Here, a protease, P57, secreted by  Organic nitrogen degradation is an important part of marine nitrogen cycle . ParticuVibrio metschnikovii RH530 , Bacillus (25.8%), Vibrio (19.7%) and Shewanella (7.6%) were the major groups and Photobacterium strains were distributed in all sediment samples. Among the strains, Photobacterium sp. A5–7 had the highest protease activity, which was isolated from the sediment sample from the A5 station site where the depth, temperature, pH and carbon/nitrogen ratio were 5.9 m, 24.7°C, 8.11 and 7.0, respectively . Using the genomic DNA as template, the 16S rRNA gene of strain A5–7 was amplified by PCR with primers 27F and 1492R  and sequ1  database1  to deter1  in the M1 . The ali1  and maxi1  methods 1 . Bootstrg and 4°C for 15 min. The cell-free supernatant was concentrated against polyethylene glycol (PEG) 20,000, and then dialyzed against 20 mM phosphate buffer (pH 7.0). The sample was loaded onto a DEAE-Sepharose Fast Flow column  equilibrated with the same buffer. Bound proteins were eluted with a linear gradient of 0-1 M NaCl in 20 mM phosphate buffer (pH 7.0). The protease activity of every fraction (4 mL) was measured with casein as substrate. Fractions having protease activity were further subjected to 12.5% SDS-PAGE. Then, fractions having protease activity and displaying a single band in SDS-PAGE gel were collected for further use. The purified protease was named P57.Strain A5–7 was cultivated at 15°C for 72 h in a marine LB medium supplemented with 0.3% (w/v) casein and 0.5% (w/v) gelatin in a rotatory shaker at 180 rpm. The culture was centrifuged at 12,000 The purified P57 was transferred from SDS-PAGE gel to a Sequi-Blot polyvinylidene difluoride membrane . Its N-terminal amino acid sequence was determined by the Edman degradation method with a PROCISE491 sequencer  at Peking University, China. The obtained N-terminal sequence of P57 was aligned with those of proteases in GenBank using BLASTP to determine the protease type.2HPO4-citric acid (pH 4.0–6.0), Na2HPO4-NaH2PO4 (pH 6.0–8.0), Tris-HCl (pH 8.0–10.0), and Na2CO3-NaHCO3 (pH 9.0–11.0). To evaluate the effect of NaCl on enzyme activity, the assay was carried out at 40°C and pH 8.0 with different salt concentrations from 0 to 3 M. The effects of enzyme inhibitors (PMSF (phenylmethylsulfonyl fluoride), EDTA (ethylene diamine tetraacetic acid), EGTA (ethylene glycol tetraacetic acid), o-P (o-phenanthroline), and IA (iodoacetic acid)) and metal ions  on the activity of P57 were evaluated by measuring the enzyme activity at 40°C and pH 8.0 after the enzyme was pre-incubated with each inhibitor or metal ion for 1 h at 4°C.The activity of P57 was measured as described by p-nitroaniline in 1 min (N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide (AAPL), N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF), N-succinyl-Phe-Ala-Ala-Phe-p-nitroanilide (FAAF), N-succinyl-Ala-Ala-Pro-Arg-p-nitroanilide (AAPR), N-succinyl-Ala-Ala-Pro-Lys-p-nitroanilide (AAPK) and N-succinyl-Ala-Ala-Val-Ala-p-nitroanilide (AAVA). Protein concentrations were determined by the Bradford method , elastin and synthetic peptides. The activities of protease P57 toward collagen and gelatin were measured by the method as described by Worthington Biochemical Corp. . For colin 1 min . The synd method  with bovTable 1). With the degenerated primers and the genomic DNA of strain A5–7 as template, a part of the gene encoding P57 was amplified by PCR and sequenced at Shanghai Biosune Biotechnology Corp. (China). By using specific primers and general primers (Table 1), the neighboring sequences of the obtained gene fragment were amplified by thermal asymmetric interlaced PCR (TAIL-PCR) (Table 1) according to this ORF, and the full gene encoding P57 was amplified by PCR from the genomic DNA of strain A5–7, and verified by sequencing. The gene sequence of P57 was deposited in GenBank database under the accession number KT923662.Based on the N-terminal amino acid sequence of P57 and the conserved sequence of the catalytic center of serine proteases , two degAIL-PCR) . Through2  of NCBI2 . Homolog2 (3  with defTable 1). The DNA fragment encoding EGFP with the same overlapping sequence was also amplified by PCR using the vector pEGFP-N1  as the template and the primers PA-GFP2 and PA-GFP(C) (Table 1). The two fragments were concatenated by the overlapping extension PCR . The recombinant PA-EGFP was expressed as a C-terminal His6-tagged protein. The transformant cells were cultivated in a 100 mL LB medium supplemented with 100 μg/mg ampicillin at 37°C and 180 rpm, until the absorbance at 600 nm reached 1.5. Then, isopropyl β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM. After induction for 24 h at 15°C and 150 rpm, cells were harvested by centrifugation. Cell pellets were suspended in a 35 mL lysis buffer (50 mM Tris-HCl pH 9.0) and disrupted by sonication. After centrifugation at 4°C and 12,000 g for 30 min, the supernatant was collected and the recombinant PA-EGFP protein was purified with a HisBind metal chelating column.The DNA fragment encoding the PA domain of protease P57 with an overlapping sequence of EGFP was amplified by PCR using the genomic DNA of strain A5–7 as the template and two primers  (sion PCR . The chiPA-EGFP as template. Mutated sites were introduced by the primers with single-point mutations. The mutated genes were sub-cloned into pET-22b (+) and transformed into E. coli BL21 (DE3). All mutations were confirmed by enzyme digestion and nucleotide sequencing. The expression and purification of the mutants were performed under the same conditions as those of PA-EGFP.Alignment of the PA domains from P57 and other reported S8 subtilases were performed by using Clustal W. The conserved aromatic residues and positively charged residues were chosen for mutagensis. Site-directed mutagenesis on the PA domain was carried out by overlapping extension PCR  using thg and 4°C for 10 min. The free fluorescence intensity in the supernatant before and after incubation was measured on a FP-6500 spectrofluorometer . Fraction of fusion protein bound (%) = (B-A)/B*100%, B and A represent the free fluorescence intensity in the solution before and after a fusion protein binds to collagen, respectively.To analyze the binding ability of the PA domain and its mutants toward collagen, 5 mg collagen fibers were mixed with 500 μL PA-EGFP, EGFP or the mutants (0.5 mg/mL) in 50 mM Tris-HCl buffer (pH 8.0). The mixture was incubated at 37°C for 2 h, and then centrifuged at 12,000 2HPO4-NaH2PO4 (pH 7.0) containing 10 mM Fe3+ was incubated at 4°C for 1 h to inhibit the enzyme activity. Then collagen fibers  was added and the mixture was incubated at 37°C for 2 h with stirring. After incubation, the mixture was centrifuged for 10 min at 12,000 g and 4°C. The supernatant was analyzed by 12.5% SDS-PAGE. BSA as a negative control was treated as P57.The collagen-binding ability of wild P57 was assayed as described by Circular dichroism (CD) spectra of the purified PA-EGFP and its mutants F349A-EGFP and Y432A-EGFP in 50 mM Tris-HCl buffer (0.1 mg/mL) were measured on a Jasco J810 spectropolarimeter (Japan) according to the method described by Photobacterium species, suggesting an affiliation with the genus Photobacterium. In the neighbor-joining and maximum-likelihood trees  based on the 16S rRNA gene sequences, strain A5–7 fell within the clade of the genus Photobacterium and formed a distinct intra-branch with type strain of Photobacterium aplysiae supported by high bootstrap values (>85%), indicating its close phylogenetic relationship to the latter. However, considering that strain A5–7 has only 96.8% 16S rRNA gene sequence identity to the type strain of P. aplysiae, strain A5–7 may represent a potential new species of the genus Photobacterium, which merits further study.Strain A5–7 was isolated from marine sediment collected from A5 station in Jiaozhou Bay, China, and the nearly complete 16S rRNA gene sequence (1536 bp) of the strain was determined. Sequence comparison showed that strain A5–7 shared the highest 16S rRNA gene sequence identities with known Figure 2A). This protease was named P57 in this study.A protease was purified from the culture of strain A5–7 by anion exchange chromatography with a yield of 15.9%. SDS-PAGE analysis showed that the purified protease had high purity, with an apparent molecular mass of approximately 45 kDa . P57 was stable at 30°C for at least 1 h, but unstable at temperatures higher than 30°C. The half time of its activity at 40°C and 45°C was 40 min and 7 min, respectively . P57 was active in a wide range of pH from 5.0 to 11.0, with the maximum activity at pH 8.0 . In the buffers containing 0–3 M NaCl, the activity of P57 peaked at 0.25 M NaCl, then declined with the increase of NaCl concentration, but still retained 50% of the maximum activity at 1.5 M NaCl .With casein as substrate, the optimum temperature of P57 was 40°C, and 10% of the maximum activity was retained at 0°C . P57 showed no activity toward elastin. Among the synthetic peptides, P57 had higher activities toward AAPL and AAPF and lower activities toward FAAF, AAPR, AAPK and AAVA (Table 3).Substrate specificity analysis showed that P57 could hydrolyze casein, gelatin and collagen, with the highest activity toward gelatin, the degenerated form of collagen  from sp. Ac10 , Apa1 . SignalP 3.0 . Multiple sequence alignment suggests that the catalytic triad of P57 is composed of Asp153, His188 and Ser492 . The highest sequence identity of P57 with reported S8 serine proteases is 63% according to BLASTP analysis against NCBI non-redundant protein database.The ORF of database , the prenalP 3.0  predictiE. coli BL21 (DE3), and purified by HisBind metal chelating column with a yield of 51.7%. Because P57 could hydrolyze insoluble collagen, we investigated the binding ability of the recombinant PA domain to insoluble collagen by fluorescence analysis. After PA-EGFP was mixed with insoluble collagen fibers for 2 h at 37°C, the precipitated collagen fibers displayed a bright green color, whereas the collagen fibers mixed with EGFP still displayed its own white color . This result suggests that the PA domain of P57 has collagen-binding ability. To further confirm this, we quantified the fluorescence intensity changes of the supernatant with different amounts of collagen in the mixture. As shown in Figure 4B, the fluorescence intensity in the supernatant decreased from 500 to 360 with the increase of collagen amount from 0 to 10 mg in the mixture, which indicated that the amount of PA-EGFP bound to collagen fibers increased with the increase of collagen amount in the mixture, thereby leading to a successive decrease of PA-EGFP amount in the solution. Taken together, these results indicate that the PA domain of P57 has collagen-binding ability, implying that the PA domain in P57 may be involved in substrate binding in the catalysis of P57 toward collagen.To study the function of the PA domain in P57 catalysis, the PA domain fused with EGFP (PA-EGFP) was expressed in 3+. As shown in Figure 4C, after P57 was incubated with collagen at 37°C for 2 h in the presence of 10 mM Fe3+, the amount of P57 protein in the supernatant significantly decreased, whereas the amount of BSA in the supernatant showed little change after incubation with collagen . This result indicated that P57 can bind to collagen at 37°C. The PA domains probably play an important role in the interaction of P57 with collagen because it has collagen-binding ability .In addition, to estimate the contribution of the PA domain to the interaction of P57 with collagen, the collagen-binding ability of P57 was assayed after the enzyme activity was inhibited by 10 mM FeFigure 3B), conserved aromatic residues  and conserved positively charged residues  in the PA domain of P57 were mutated to Ala, and all the mutants were expressed as EGFP-fused proteins. The mutants R403A-EGFP and F444A-EGFP could not be expressed as soluble proteins, probably because these two residues are important for the correct folding of the proteins. The binding abilities of the mutants to collagen were measured and compared with that of PA-EGFP. While the collagen-binding abilities of mutants K397A-EGFP, Y412A-EGFP, K419A-EGFP and Y452A-EGFP were partly reduced, the collagen-binding abilities of mutants F349A-EGFP and Y432A-EGFP were severely destroyed, implying that Phe349 and Tyr432 may be essential for the PA domain to bind collagen . In addition, CD spectra of PA-EGFP, F349A-EGFP and Y432A-EGFP were collected to analyze the structural changes that residue substitution mutation may cause in the mutants. There were few differences in the spectra of PA-EGFP, F349A-EGFP and Y432A-EGFP , suggesting that the loss of the collagen-binding ability of F349A-EGFP and Y432A-EGFP should be caused by amino acid substitution. Altogether, these data indicate that Phe349 and Tyr432 are likely two key residues for collagen binding in the PA domain of P57. The data also suggest that hydrophobic interactions likely play an important role in the binding of the PA domain to collagen because Phe349 and Tyr432 are both aromatic amino acids.To determine the key amino acid residues in the PA domain of P57 for collagen binding, site-directed mutagenesis was performed. It has been reported that aromatic residues and charged residues usually play a key role in the binding of binding domains to insoluble substrates . Based oPhotobacterium sp. A5–7 isolated from the sea sediment of Jiaozhou Bay in China was characterized. P57 is a serine protease of the S8 family. Among reported S8 serine proteases, P57 has the highest identity (63%) to VapT from V. metschnikovii RH530 (P-proprotein domain, mature P57 only contains the catalytic domain and the PA domain that is inserted in the catalytic domain. The N-terminal pre-sequence, which is required for protease folding and secretion (P-proprotein domain, which is also important in enzyme secretion through the cell membrane (In this study, the extracellular protease P57 from ii RH530 , which secretion , and themembrane , are allS. agalactiae and S. pyogenes show that the PA domain is located near the active-site cleft of the catalytic domain (Lactococcus lactis without the PA domain (Our results show that P57 is a subtilase with an inserted PA domain in its catalytic domain. Some subtilases with an inserted PA domain in the catalytic domain have been reported from plants and bacteria, and structural and deletion mutational analyses have suggested that the PA domains in some subtilases are involved in substrate binding. For example, the crystal structures of C5a peptidases from c domain . Three oc domain . CleavagA domain . The actA domain . DespitePseudoalteromonas sp. SM9913 (Geobacillus collagenovorans MO-1 (Alkalimonas collagenimarina AC40 (Myroides profundi D25 (2+ could increase the enzyme activity or the thermal stability of some subtilases (2+ only had a little effect on P57 activity. Previous reports showed that Mn2+ did not affect the enzyme activity of subtilase significantly (2+ significantly increased P57 activity. EDTA and EGTA are ion chelators, which usually inactivate subtilase that contains metal ion and/or lower their stability by depriving the metal ion (While most subtilases have no collagenolytic activity, some subtilases have been reported to be serine collagenolytic proteases that can degrade insoluble collagen, such as MCP-01 from . SM9913 , the theans MO-1 , AcpII fina AC40 , and myrundi D25 . P57 hasundi D25 , VapT (Kundi D25  and AcpIundi D25 , usuallybtilases . Howeverficantly  or slighficantly . Our resetal ion . The actPseudoalteromonas sp. SM9913 (Pseudoalteromonas sp. CF6-2 (Photobacterium sp. A5–7 was isolated from a marine sediment sample from the A5 station site in Jiaozhou Bay, China, where the depth, temperature, pH and C/N ration were 5.9 m, 24.7°C, 8.11 and 7.0, respectively (Extracellular proteases from marine sedimentary bacteria usually have some environment-adapted characters, including cold-adaptation, salt tolerance/activation, and an optimal pH equal or near that of seawater, such as proteases MCP-01 and MCP-03 from . SM9913  and pseup. CF6-2 . Consistectively . P57 hadPhotobacterium sp. A5–7 was characterized in this study. P57 is a new S8 subtilase that can degrade casein, collagen and gelatin. P57 has a PA domain inserted in its catalytic domain. The PA domain from P57 has collagen-binding ability, which represents the first direct evidence that the PA domain of a subtilase has substrate-binding ability. Phe349 and Tyr432 were further revealed to be key residues responsible for collagen binding in the PA domain of P57 by site-directed mutational analysis. Our results provide more insight into the function of PA domain of subtilases and shed light on marine sedimentary bacterial proteases and PON degradation.In summary, protease P57 secreted by marine sedimentary H-JL, B-LT, and X-XH performed the biochemical experiments. XS, B-XL, and X-YZ helped in protein purification. X-LC designed and directed the research. H-JL and X-LC wrote the manuscript. P-YL and X-YZ helped in data analysis and manuscript editing.The 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.


Wireless sensor networks (WSNs) have been widely applied in agriculture field; meanwhile, the advent of multi-domain software-defined networks (SDNs) have improved the wireless resource utilization rate and strengthened network management. In recent times, multi-domain SDNs have been applied to agricultural sensor networks, namely multi-domain software-defined wireless sensor networks (SDWSNs). However, when the SDNs controlling agriculture networks suddenly become unavailable, whether intra-domain or inter-domain, sensor network communication is abnormal because of the loss of control. Moreover, there are controller and switch info-updating problems even if the controller becomes available again. To resolve these problems, this paper proposes a new approach based on an Open vSwitch extension for multi-domain SDWSNs, which can enhance agriculture network survivability and stability. We achieved this by designing a connection-state mechanism, a communication mechanism on both L2 and L3, and an info-updating mechanism based on Open vSwitch. The experimental results show that, whether it is agricultural inter-domain or intra-domain during the controller failure period, the sensor switches can enter failure recovery mode as soon as possible so that the sensor network keeps a stable throughput, a short failure recovery time below 300 ms, and low packet loss. Further, the domain can smoothly control the domain network again once the controller becomes available. This approach based on an Open vSwitch extension can enhance the survivability and stability of multi-domain SDWSNs in precision agriculture. With the popularity of the Internet of Things (IoT), wireless sensor networks (WSNs) are widely researched and change our life considerably . EspeciaIn recent years, software-defined networks (SDNs) has been a hot subject of research, especially multi-domain SDNs ,11 that However, there is a problem as to how the reliability of multi-domain software-defined WSNs (SDWSNs) can be enhanced. In bad agricultural weather environments, wireless sensor networks are affected so seriously that the sensor link becomes unstable. Agricultural vehicle sensor networks include numerous dynamic mobile sensor nodes carried by vehicles, which means that mobile sensor nodes will frequently join and disjoin the network. Therefore, once the wireless link between the controller and switches is disrupted, or any domain controller leaving its network becomes unavailable, both intra-domain and inter-domain network communications become abnormal due to the loss of control , which wTo resolve the reliability problems of multi-domain SDWSNs in precision agriculture mentioned above, many attempts have already been carried out, such as cluster controllers , local aBased on the above analysis, this paper proposes a new approach based on the Open vSwitch extension for multi-domain SDWSNs in agriculture. This new approach can enhance intra-domain and inter-domain network communication performance and info-updating when the controller connection is lost. In addition, the scalability and flexibility of SDNs can be maintained.We developed a connection-state mechanism, a communication reliability mechanism on network L2 and L3, and an info-updating mechanism based on Open vSwitch. Through experiments, we show that, when a domain controller suddenly disconnects, the failure recovery time is short enough to guarantee an uninterrupted communication service in the agricultural intra-domain and inter-domain network, and the domain controller is smoothly recovered.This new approach focuses on intra-domain as well as inter-domain networks. It uses a proposed communication reliability mechanism that detects controller unavailability as soon as possible and keeps intra-domain and inter-domain communication normal after the controller becomes unavailable, which improves the reliability of multi-domain SDWSNs.The controller can access the network again and maintain both the info-updating of topology information in the controller and the rules in sensor switches.Specifically, the main contributions of this paper are as follows:The rest of the paper is organized as follows. This section provides the design details of the proposed communication reliability mechanism. In multi-domain SDWSNs, each agricultural network domain including numerous sensor switches is controlled by its own domain controller. These domain controllers work cooperatively or independently to ensure normal communication of the intra-domain and the inter-domain in the network data plane. As shown in The connection-state detection mechanism: The connection state is the link state between the controller and each switch; therefore, this mechanism means, when a controller is suddenly unavailable, the data plane mainly depends on the connection-state processing mechanism to enter recovery mode quickly and smoothly. This connection-state detection mechanism can detect the real-time state by probes messages, such as the OpenFlow Echo message . FailureThe stateful match mechanism: The connection states mainly consist of two states: connection-state and disconnection-state. Connection-state means that the domain controller informs the packet how to forward in switches and gateways. In contrast, disconnection-state means that the domain controller is unavailable and switches will enter failure recovery mode in sensor networks. To apply this connection state into both the controller and the switches, this paper extends a match field named connection-state in OpenFlow protocol. The OpenFlow entries can contain a connection state into their match fields. Therefore, as shown in The self-learning mechanism: When switches enter disaster recovery mode, it needs a self-learning mechanism to keep the agricultural intra-domain network normal. This self-learning mechanism is extended as a new OpenFlow protocol action, so the controller can use OpenFlow API to install an entry that consists of a flexibly self-learning action. The new self-learning mechanism is similar to the traditional switch L2-mechanism but can be invoked and inserted in the flow-table by a controller application, which maintains the flexibility of SDN. After a packet matches this entry consisting of a self-learning action successfully, then it will implement this self-learing action. This action process entails that the switch can parse the ingress port and mac address of the packet’s header. Then, a new entry will be generated and updated by this parsing information, which can forward a similar packet from the opposite direction; therefore, this new self-learning mechanism can ensure basic L2 communication in an intra-domain network when the controller becomes unavailable.The L3 mechanism: When the network enters disaster recovery mode, inter-domain communication still needs to rely on domain gateways. However, after the domain gateways lose control of the controller, gateways do not know how to route the packets to another domain, seriously restricting the inter-domain communication. We designed the L3 mechanism in the data plane for two types of packets in case of controller failure. The first type of packet is that which will transfer from this domain to another domain network. This is designed to insert a stateful entry into gateways, which can forward this type of packet to a neighboring domain when the loss of controller happens. Then, the controller of the neighboring domain can correctly direct the packet. The second type of packet is that which will enter this domain network. For these packets, the gateway switch will implement an ARP mechanism to obtain the next hop mac address so that it can finish packet routing in case of controller failure. This paper proposed this simple but effective method to ensure normal inter-domain network communication when domain gateways lose control.The domain sensor network loses control due to controller crashes or unstable wireless links in severe agricultural and weather conditions. However, when the SDN controller recovers from failure again or a new controller hands over the network, the controller will control the domain switches and gateways again in the agricultural sensor network. However, there is old staling information in the controller and switches. At this time, it becomes important to ensure the info-updating in both the domain controller and the switches because of the staling information when domain controllers control the network again.When the unavailable controller recovers and manages the domain switches again, the controller will send switches some request messages to retrieve the latest information such as flow tables, entries, and topology. This information represents the latest network state, but the domain controller’s database still stores the past network information. Therefore, the controller database information, especially entries and topology, should be updated according to the latest network information regarding the issuing of request messages. Besides the above information updated in the controller database, the info-updating mechanism still needs to be used in updating and deleting stale entries in some sensor switch nodes because of the changing topology during controller failure. As shown in To implement the proposed mechanism of According to the description of the communication reliability mechanism, the communication reliability module should consist of three functions: connection-state processing, intra-domain reliability, and inter-domain reliability. To implement them, the communication reliability modules should include three modules, which are described below.This module mainly extends a stateful match field of the OpenFlow protocol and the OpenFlow Switch so that the sensor packets can match different stateful entries according to the states of the controller. The connection-state module is implemented by a finite state machine, which can clearly realize the state-jump function. As shown in As shown in The intra-domain module mainly depends on a self-learning mechanism. We implemented the self-learning by extending the OpenFlow and OpenFlow Switch API. To ensure intra-domain reliability, the domain controller can proactively insert a disconnection entry with the self-learning action into each switch with, for example, “match: state = disconnection-state, action: self-learning” (see also the first entry in This reliability module consists of two parts. For the packet out of the domain, it relies on a stateful entry in the gateway. When the SDN controller is available in an agricultural sensor network, the inter-domain module in the controller will insert a stateful entry in the gateway, which can forward the packet to a neighboring domain gateway for further processing. When the controller becomes unavailable, the entry will be valid and start to work. When the packet missing match in the gateway matches this entry and is forwarded to the neighboring domain, the controller of the neighboring domain will correctly direct the packet. For the packets arriving to this domain and entering this domain network, the destination mac of the packet will be modified before the packet is forwarded to the destination node by existing entries. If not matching any entry to modify the header of the packet, this gateway can implement an ARP module in the gateway so that the gateway can actively trigger an ARP request to retrieve the destination mac. The processing of the ARP module is shown in After the sensor network and the domain SDN controller recover from failure, or new controllers hand over as primary controllers to control the network again, the controller should reconnect the switches and gateways and manage the domain network again in agriculture. However, the domain network states may have changed during the disaster period, such as in the topology, which leads to stale information in switches and the controller. Therefore, this paper proposes the design of an info-updating reliability mechanism module that can take charge of the updating of the information in switches as well as the controller’s database once the controller controls the domain network again.There are two main types of information to be updated in the controller database—topology information and flow table information. First, when the sensor network recovers from network disaster, the topology may have changed during the disaster period; therefore, the controller will use LLDP protocol to obtain topology information. By sending LLDP packets requests to all switches, the controller can collect the current topology information such as the switch’s connection map relationship. Moreover, the controller will send request messages to switches to obtain the configuration information of the switches, such as port state. Then, this new topology information will update the old and stale information in the controller database so that applications such as the shortest-path-application can be used correctly based on these updated information. Second, it will update the flow table information in the controller database, such as entries and counters. In failure recovery mode, the switches adapt to the new topology and generate new entries in flow tables; therefore, the module of info-updating will send an OpenFlow status message to obtain the flow table information and replace this information in the controller database.When the controller controls the domain sensor network again, the topology may have changed so much that part of the entries in the switches have become stale and should be updated or deleted. The info-updating module can also update and delete these stale entries in the switches to achieve data plane connectivity and an auto-following strategy. The module mainly compares the latest topology information with the former topology. Then, it will find all of the switch nodes whose connection to other nodes has changed. Because the packets sent to these changing nodes will match the stale entries, these changing nodes will be unreachable. The module will update and delete these stale entries in the relevant switches directly. An example of info-updating reliability that clearly describes the entire process is given below. Let us suppose that the controller crashes due to bad agricultural weather and cannot control the sensor network. The controller then recovers from failure and manages the network again. In this section, we describe our experiment evaluation. We extended the RYU controller  and OpenIf the links between the domain controller and switches are suddenly interrupted, the proposed mechanism should take more time to detect the failure because of continuous detection; therefore, this controller failure type will result in more complexity and damage than other failure types. To be more compelling, we selected this type of link failure as our experimental agricultural scenario.There is a fail-secure mode in OVS that depends on a “NORMAL Port” action to simulate a traditional switch when the controller is not available . To compThe first experiment was to evaluate failure recovery time. We triggered the link failure between the domain controller and switches several times with different throughput. As shown in The failure recovery cumulative probability distribution can show how much time the network requires from failure to normal communication with different mechanisms. The distribution was in excess of 1000 ms in OVS’s own mechanism in both intra-domain and inter-domain tests. However, AL2 and AL3, compared with than BL2 and BL3, yielded a shorter recovery time. The 90th percentiles of the intra-domain communication in AL2 were only about 100 ms, maintaining a low recovery time when controller failure occurred. For inter-domain sensor network communication, we used communication reliability mechanisms, such as ARP and stateful entries in the gateway. Because it required more time to obtain mac and change the packet header, the AL3 yielded about 300 ms. Therefore, the short intra-domain and inter-domain failure recovery time indicates that our proposed mechanism can make network communication normal as soon as possible when the domain controller becomes unavailable in agricultural sensor networks.In comparison with our proposed mechanism, BL2 and BL3 led to relatively severe packet losses. The high packet loss severely impacts sensor communication quality. In contrast, whether it is intra-domain AL2 or inter-domain AL3 in our mechanism, the number of packet losses is below 1000, which means that the packet loss rate is below 13%. We designed the controller connection-state tracking module to achieve a real-time state in a short period, so it can decrease the effect of domain controller failure.To show the throughput stability over time, we triggered the controller failure at the 5th and 12th seconds, as shown in More importantly, Finally, we considered the relationship between topology scale and failure recovery performance when the controller becomes unavailable with sensor switches. Failure recovery time is measured as the number of area switches increases. The experiment result is shown in The small increase in failure recovery time is mainly affected by the number of switch nodes, the packet’s header parse, and the modification of switches. Whether in intra-domain switches or modified in the inter-domain gateway, the data packet will be parsed or modified to match the rules and forward, which inevitably leads to a small increase in failure recovery time. However, in general, the results show that our design does not cause a significant increase in failure recovery time, even though the topology scale increases.Recently, as the SDNs has stirred considerable attention, e.g., for control traffic balancing  and for However, it is easy for the data plane to lose control of the controllers when controller failure occurs. For example, the link between controller and switches link become unstable when the agriculture sensor network is faced with a bad natural environment. Moreover, the controller and switch info-updating also become a problem when the controller manages the network again. The attempts to resolve the problem are as follows: Controller cluster: The backup controller takes over the rule of the primary controller when the primary controller crashes or becomes unavailable. Optimizing the election mechanism  and the Data plane: In one study , a localIn multi-domain SDWSNs, each SDN domain controller manages its own domain sensor switches and sensor gateways in agriculture. Once the domain controller suddenly becomes unavailable, the data plane, including sensor switches and gateways, will lose control of the domain controller such that it leads to agricultural sensor network reliability problems in both inter-domain communication and intra-domain communication. For the above problems, this paper proposes connection-state tracking and a stateful match mechanism, an ARP mechanism, and an info-updating mechanism for agricultural multi-domain SDWSNs. Via these mechanisms, the experimental results show that, when the domain controller becomes unavailable, whether it is inter-domain or intra-domain, sensor switches can enter failure recovery mode as soon as possible so that there is stable throughput and a short failure recovery time below 300 ms. During the failure period, the packet loss is very low. Moreover, the domain can smoothly control the domain network when the controller becomes available again. Therefore, our proposed approach can enhance the survivability and stability of multi-domain SDWSNs in precision agriculture.More importantly, besides the agricultural scenario, it is worth considering that the proposed reliability mechanism can be applied in other network scenarios to improve the reliability of SDNs. Therefore, more work and evaluation of the proposed design is needed.


The product template (PT) domains, specifically in fungal non-reducing polyketide synthases (NR-PKSs), mediate the regioselective cyclization of polyketides dominating the final structures. However, up to date, the systematic knowledge about PT domains has been insufficient. In present study, the relationships between sequences, structures and functions of the PT domains were analyzed with 661 NR-PKS sequences. Based on the phylogenetic analysis, the PT domains were classified into prominent eight groups (I–VIII) corresponding with the representative compounds and cyclization regioselectivity . Most of the cavity lining residue (CLR) sites in all groups were common, while the regional CLR site mutations resulted in the appearance of finger-like regions with different orientation. The cavity volumes and shapes, even the catalytic dyad positions of PT domains in different groups were corresponding with characteristic cyclization regioselectivity and compound sizes. The conservative residues in PT sequences were responsible for the cyclization functions and the evolution of the key residues resulted in the differentiations of cyclization functions. The above findings may help to better understand the cyclization mechanisms of PT domains and even predict the structural types of the aromatic polyketide products. One hallmark of fungi is their capacity to synthesize diverse biological polyketide natural products with structural variation during synthesis by polyketide synthase (PKSs)121The fungal PKSs can be divided into three major classes according to the function and phylogeny, i.e., the non-reducing (NR), the highly-reducing (HR), and the partial-reducing (PR) PKSs17According to an earlier phylogenetic analysis of β-ketoacyl synthase (KS) domains, NR-PKSs were divided into three basal subclades (subclades I–III), each of them characterized a typical domain architectureAspergillus parasiticus PksA, which participates in aflatoxin B1 biosynthesisvia a PT-specific sequence insertion. The residues important for substrate binding and catalysis were revealed, and an internal pocket was identified, which can be visually divided into three regions, phosphopantetheine (PPT)-binding region, cyclization chamber, and hydrophobic hexyl-binding region. The crystal structure of PT domain in PksA provided a vital model for the structural study on other PT domains of which structures were still unknown.Up to date, only one PT domain (group IV) crystal structure has been studied from So far, the systematic knowledge about PT domains has been insufficient. The relationships between sequences, structures and functions of the PT domains have been unclear. In this study, the NR-PKS phylogenetic tree and PT phylogenetic tree were established based on all known fungal NR-PKSs protein sequences. The three-dimensional structures of different PT domains were modeled, and the relationships between catalytic pocket shapes and sizes, regioselective cyclization, and compound sizes were analyzed. The influence of the PT domain sequence variations on the differentiations of structures and functions was also discussed.All of the known fungal NR-PKSs amino acid sequences were searched and screened from the NCBI database to establish phylogenetic trees . The colAspergillus, Bipolaris, Colletotrichum and Talaromyces were found in six groups in the phylogenetic tree.The NR-PKS phylogenetic tree was constructed on the basis of the phylogeny and domain architectures. The resulting NR-PKS phylogenetic tree clearly classified the collected NR-PKS sequences into eight major groups (groups I–VIII) except a few sequences . CompareAll of the NR-PKSs contain SAT, KS, AT, PT and ACP domains with the PT domains locating between AT and ACP domains. Moderate differences of domain architectures were observed in eight groups. The C-terminus TE/CLC domains responsible for releasing products in all members in group V were absent. Most members in groups VI and VII were characterized by a CMeT domain located after the ACP domain, which can produce aromatics with methyl branching on their benzene rings. Most NR-PKSs  showed an additional ACP domain. A previous study revealed that the product-releasing domains including TE/CLC in each group could be further distinguished by different functionsThe phylogenetic tree of PT domains from 661 NR-PKS sequences was also constructed. The PT sequence boundaries were defined by computation tools according to the NR-PKSs differentiation and the sequence length is about 300 ~ 400 amino acids. The established PT domain phylogenetic tree was similar to that of NR-PKSs, also classified into eight groups .Until now, 55 NR-PKSs among the 661 NR-PKS sequences have been investigated for their regioselective cyclization modes . Successvia C2-C7 aldol condensation. The main NR-PKSs in this group are involved in the biosynthesis of aromatic portions in the resorcylic acid lactones, exemplified by hypothemycin Hpm3Aspergillus nidulansAspergillus terreus forms the first rings via C3-C8 rather than C2-C7The phylogenetic analysis suggested that each group of PT domains associates with specific regioselective cyclization and compound size. In group I, most PT domains catalyze the first-ring cyclization via C2-C7 with pentaketide backbones. The TE/CLC domains in this group are in charge of cyclizing the second rings.In group II, the NR-PKSs include the most known THN synthases from a variety of genera responsible for the synthesis of 1,3,6,8-tetrahydroxynaphthalene (THN) analoguesvia C2-C7 cyclization, such as naphthopyrone heptaketide synthase Alb1A. nidulans also generates pentaketide THN with shorter polyketide chains11The NR-PKSs in group III synthesize longer polyketide chains via C4-C9 regiospecificity. The only one crystal structure of PT domain in PksA from A. parasiticus23All the PT domains in group IV cyclize the first ring via C6-C11 register. It seems that these NR-PKS members produce multiple aromatic polyketides with the most different polyketide backbones by regioselectivity cyclization modes. Group V is composed of the NR-PKSs catalyzing the synthesis of different polyketide backbones, such as griseofulvin heptaketide synthase GsfA from Penicillium aethiopicumA. terreusA. nidulansAspergillus. nigerAll of the NR-PKSs in group V lack TE/CLC domains and they mainly form the first ring Penicillium brevicompactumAll of the NR-PKSs in groups VI and VII associate with C2-C7 specific cyclization, and most of them have a CMeT domain. The compounds derived from group VI are single aromatic ring natural products such as 5-methylorsellinic acid from Armillaria mellea with C2-C7 cyclizationGroup VIII is a newly separate group according to our phylogenetic analysis. Different from other groups, this group contains not only the NR-PKS members from ascomycetes and but also from basidiomycetes. The only studied polyketide synthase in group VIII is orsellinic acid synthase ArmB from The above phylogenetic analysis for PT domains indicated that the cyclization modes of C2-C7, C4-C9, C6-C11, C1-C6, and C3-C8 were found in eight groups. C2-C7 cyclization is the most common mode that spreads over seven groups except group IV. And the C4-C9 and C6-C11 cyclization modes only appear in groups IV and V, respectively.A. parasiticus PksA responsible for C4-C9 cyclization has been the only PT crystal structure reported to date. The internal pocket of this PT domain extends 30 Å from the surface to the bottom  of PT domain in aflatoxin synthase PksA can perfectly adapt to accept the substrate hexanoyl starter unitIn order to analyze the relationship between the cavity volumes of PT pockets and compound sizes, the cavity volumes of PT catalytic pockets were calculated by using CASTp . It was The C2-C7 cyclization modes cover the groups I–III, and VI–VIII. The NR-PKSs in groups I, VI and VIII generate the tetraketides as the common metabolites. Interestingly, the compounds generated by groups I, II and VI, such as orsellinic acid17112342P. aethiopicum GsfA and A. nidulans PkgA in group V have been found to catalyze C1-C6 and C2-C7 heptaketide cyclizations, respectively. It is speculated that the cavity structures of group V cannot be suitable for the C6-C11 heptaketide cyclization, leading to the emergence of special C1-C6 and C2-C7 heptaketide cyclizations  sites of each PT sequence were identified from the catalytic pockets selected by CASTpIt was indicated that the catalytic pockets of each PT group are composed of 31–45 CLR sites . The CLRA. parasiticus PksA) are highly conserved.The sequence differentiation of PT domains were also analyzed. The conservation analyses of all amino acid residues were performed with the evolutionary conservation scores calculated by ConSurfA. parasiticus PksA prevail) belonging to the 27 common CLR sites have been validated to be important residues for activity by mutationsFurthermore, most of the conserved residues in 661 PT sequences locate at the catalytic pockets . There aA. parasiticus PksA prevail), are involved in the formation of finger-like region. In the corresponding positions of other groups, these CLRs are replaced by the bulkier residues , preventing the formation of finger-like region. The previous experimental data showed that the specific CLR G1491 was mutated to Leu, resulting in no apparent activity of the PT domainA. nidulans MdpG prevail) substitute the bulkier residues in other groups, including Phe and Ala, to generate finger-like region. Similarily, M1378 and G1423 in group V take the place of the conserved Val and Pro in other groups, respectively, resulting in the increase of the pocket volumes.In groups IV and V, however, besides the 27 common CLR sites, 11 specific CLR sites and 8 specific CLR sites were found to only appear in the catalytic pockets of these two groups, respectively . These rFurthermore, nearby the catalytic dyad, Thr (1354) in group IV and Thr (1385) in group V, are replaced by Pro in all C2-C7 groups. The spatial structure of Pro may influence the ring architecture and prevent the transfering of the first ring with C2-C7 regio-specific cyclization.The above analysis suggested that the CLRs related with the catalysis are highly conserved in the main catalytic cavity of the pockets. The volume diversification of PT pockets affects the different cyclization regiospecificity and even compound formations. Residue mutation is the main reason to change the sizes and shapes of the PT pockets.In present study, the relationships between sequences, structures and functions of PT domains in fungal polyketide synthases were analyzed and elucidated. In the evolutionary process, the PT domains diverge into eight groups with functional distribution to synthesize diverse polyketides with variety of substrates by different cyclization modes. The functional differentiation is attributed to the changes of the cavity shapes and sizes with CLR mutations arised from the sequence variations of PT domains. The above findings may help to better understand the cyclization mechanisms of PT domains and even predict the structural types of the products especially with aromatic polyketide backbones from NR-PKS protein sequences. It could be prospected to manipulate the fungal PKS genes and further regulate the biosynthetic pathway to obtain the target metabolites.The amino acid sequences of 55 fungal NR-PKSs with known cyclization modes were collected from NCBI database (Genbank)The NR-PKS sequences and PT sequences were aligned with MAFFTThe three-dimensional models of PT domains were constructed using comparative protein modeling methods . The strThe structural mapping and pocket architecture visualization were displayed using VMDThe evolutionary conservation of amino acid positions in the PT sequences was estimated by using ConSurf algorithmHow to cite this article: Liu, L. et al. Bioinformatical Analysis of the Sequences, Structures and Functions of Fungal Polyketide Synthase Product Template Domains. Sci. Rep.5, 10463; doi: 10.1038/srep10463 (2015).Supplementary Figures 1-6


RGCs extend their axons during embryonic life, forming the optic nerve, and organize in a retino-collicular map in the SC during the first postnatal week. Around post-natal day 6, while retino-collicular mapping is underway, axons from layer V neurons in V1 reach the SC, forming the cortico-collicular map, which aligns with the retino-collicular map in the SGS, so that corresponding sources of visual inputs are mapped topographically and adjusted with respect to the visual field: retinal nasal-temporal and V1 medial-lateral axes project onto the anterior -posterior  axis in the SC , a layered structure in the midbrain involved in multisensory processing, controlling visual orientation and attention May, . The supC Figure  and dorsC Figure . EvidencC Figure . HoweverPotential candidates are the Eph tyrosine kinase receptors and their membrane-bound ligands, the Efns (ephrins), key players in the formation of visual maps  and because the graded expression of EphAs was quantitatively altered, from a smooth to an oscillating gradient (change in periodicity). This approach, and subsequent work using the Isl2-EphA3KI model, yielded crucial findings about the mechanisms of retino-collicular and cortico-collicular mapping which have been impacting both experimental , which presents a quantitative perturbation of the EphA receptor gradients in the retina . The analysis of the retino-collicular mapping in these animals showed no targeting perturbation, suggesting that Efna3 is not directly involved in retino-collicular mapping  carries molecular cues  allowing subsequent sensory map alignment (cortico-collicular map) Movie . In suppES and MR wrote and edited the manuscript and designed the figure and the movie.The 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.


Domain motion is intrinsically linked to the functionality of the enzyme, and we can define the position of the conformational equilibrium for individual steps in the catalytic cycle.NADPH-cytochrome P450 reductase is a multi-domain redox enzyme which is a key component of the P450 mono-oxygenase drug-metabolizing system. We report studies of the conformational equilibrium of this enzyme using small-angle neutron scattering, under conditions where we are able to control the redox state of the enzyme precisely. Different redox states have a profound effect on domain orientation in the enzyme and we analyse the data in terms of a two-state equilibrium between compact and extended conformations. The effects of ionic strength show that the presence of a greater proportion of the extended form leads to an enhanced ability to transfer electrons to cytochrome  Internal motions in proteins range from bond vibrations through local loop movements to large-scale domain motion and occur across an extremely wide range of time scales (femtoseconds to seconds). The choreography of local loop motions in the catalytic cycle has been studied in some enzymes8, but in other instances much larger-scale movements of whole domains are important11. This is particularly true in electron transfer pathways; electron transfer (ET) is generally carried out by proteins associated in large dynamic complexes, and in such systems domain motion can be required to provide access for the protein partner(s) to the redox centre(s)15.The concept of an energy landscape for a folded protein requires that proteins exist as an equilibrium population of conformational states. Interconversions between these states are of fundamental importance to biological function16, which includes cytochrome P450 reductase 21, the cancer-related novel reductase 122, and methionine synthase reductase24, as well as the bacterial proteins sulfite reductase25 and CYP BM326. These enzymes (or their reductase components) have three domains: an FMN-binding domain, related to flavodoxins, an FAD- and NADPH-binding domain, related to ferredoxin/flavodoxin reductases, and a ‘linker’ domain, which may serve to position the other two domains. The FMN domain is connected to the linker and FAD domains through a highly flexible ‘hinge’. In all the members of this family the ET pathway involves the sequence NADPH → FAD → FMN → acceptor, and there is good evidence from mutagenesis and kinetic experiments for motion of the FMN-binding domain to allow it to accept electrons from the FAD and deliver them to the acceptor29.An important family of ET proteins which depends on domain movement in this way is that of the diflavin reductases18, and in the biosynthesis of secondary metabolites in plants30. In man, polymorphisms or mutations in CPR can lead to changes in drug metabolism32, and to disordered steroidogenesis and skeletal malformations34. The conformation of truncated soluble CPR seen by X-ray crystallography19 36, suggesting that the interflavin ET is ‘gated’. Furthermore, in the CPR conformation seen in the crystal it is difficult to see how cytochrome P450  could approach close enough to the FMN for ET to occur18.CPR is located on the endoplasmic reticulum where it is a key component of the P450 mono-oxygenase system which plays a central role in drug metabolism38, but it remains to be established precisely where in the reaction cycle it occurs. To address this question we examine the conformational equilibrium of CPR in solution using small-angle neutron scattering (SANS) and transient kinetics. The power of solution scattering in studying domain organisation in proteins is well established; by using SANS rather than SAXS we are able to control the redox state of the enzyme precisely without the problems associated with reduction of the flavins by X-ray-induced photo-electrons37. These experiments allow us to relate the domain movement to individual steps in the catalytic cycle of the enzyme.There is thus evidence for the existence of domain motion in CPR and for its importance in catalysisr), derived from the neutron scattering (SANS) curves, for each state of the enzyme studied, while Table g, and the maximum dimensions, Dmax, for each state. , which was based on NMR and SAXS data on wild-type CPR, or the crystal structure of the ΔTGEE mutant of CPR, which has a deletion in the flexible hinge42 (PDB 3ES9); in molecule A of the crystal structure of the mutant the FMN domain has rotated away from the linker and FAD domains so as to expose the FMN to the solvent . Analysis using either of these models for the extended state, while allowing the ratio of compact to extended states to vary, gave reasonable fits to the scattering curves for all the samples studied  oxidised human CPRied Fig. ; Table 119; this single conformation fits the scattering curve with a χ2 value of 1.87, and the experimental estimate of Rg is close to that calculated from the crystal structure: 24.7 (±0.1) vs. 24.88 Å. Introduction of a second conformation produces only a marginal increase in the goodness-of-fit; using the specific structural models for the extended state described above leads to a fit with 90% of the compact state and 10% of the extended state, giving χ2 values of 1.75 and 1.64  with cyt c leads to a burst of cyt c reduction by those CPR molecules which are in a reactive (‘open’ or ‘extended’) state, followed by a slower reduction of cyt c by those CPR molecules that exist in a cyt c unreactive (‘closed’ or ‘compact’) conformation and which need to change to the ‘open’ conformation in order in interact with cyt c. At low salt 21% reduction of cyt c takes place within the 2ms dead-time 37 seen in the compact conformation are abolished show an increased population of the extended conformation51, since in both models these pairs of residues are distant from one another. Scrutton’s laboratory has used FRET measurements between dyes attached to the naturally occurring cysteine residues in the FMN and FAD domains to study domain movement38. The results were discussed in terms of a model in which the compact (‘closed’) form of the enzyme corresponds to the crystal structure and the extended (‘open’) form to the structure of the ΔTGEE mutant42; the positions of the cysteines are such that the transition to this extended form would lead to an increase in the FRET signal38. Hedison et al. propose that the oxidised form of the enzyme is predominantly in the extended conformation and that coenzyme binding and ET lead to successively greater proportions of the compact form38. This is not consistent with the SANS results presented here, which show clearly that the oxidised enzyme is almost wholly in the compact state; the same conclusion has been reached by NMR50 and mass spectrometry51. However, these FRET experiments are entirely consistent with the structural experiments if the model for the extended state is taken to be that of Huang et al.37, since in this model the FRET signal between the dye-labelled cysteine residues would be expected to decrease as the proportion of the extended form increased. Recently, Kovrigina et al.52 have studied the domain movement by measuring FRET between dyes attached to two specifically engineered cysteines; consistent with our present results, they concluded that oxidised CPR is in a compact conformation. The NMR and FRET results thus lead to a slight preference for the model of Huang et al.37 for the extended state. However, it should be emphasised that the model based on the ΔTGEE mutant fits the SANS data slightly better, and this mutant forms a stable complex with heme oxygenase53.Two structural models of the extended statein vitro reaction starting with the fully oxidised enzyme is shown in Fig. −-reduced species. Analysis of equilibrium redox titration data54 led to an approximate estimate of [FAD-FMNH2]/[FAD•-FMN•]~11, with only a small amount of FADH2-FMN. Intermolecular ET to cyt c takes place from FMNH218, and then probably from FMN•55. This represents a 0-2-1-0 cycle of redox states (in terms of numbers of electrons). It has been suggested that in vivo the ‘resting state’ of CPR is a 1e−-reduced (FAD-FMN•) state, reduction by NADPH leading to a 3e−-reduced state and ET to cytochrome P450 taking place only from FMNH2 – that is a 1-3-2-1 redox cycle18. An unambiguous choice between these two cycles cannot yet be made.The catalytic cycle of CPR for an eq ~9; Fig. − - NADP+ species, the product of the initial hydride transfer, the proportion of the extended state is ~30%. The retention of a significant population of the compact state in this species is consistent with the obvious requirement for a conformation with the FAD and FMN in close proximity for interflavin electron transfer. Indeed, relaxation kinetics shows that the rate of interflavin electron transfer increases on binding NADP+ 28. On the subsequent dissociation of NADP+, the population of the extended state increases to ~40%, facilitating electron transfer to cytochrome P450 (or cyt c).SANS allows us to relate our results on domain movements to the catalytic cycle secure in the knowledge that the redox state of CPR is well defined in our experiments. The oxidised enzyme is essentially completely in the compact state  plasmid construct66. Cells were grown to the mid-log phase in TB medium at 37 °C prior to induction by reducing the growth temperature to 15 °C to exploit the cold-shock promoter. CPR was purified as described previously66, with modifications. The purification involved use of a 2′5′-ADP affinity column; the pure protein was eluted using a 20% glycerol solution rather than 2′-AMP in order to avoid undesired persistent binding of the 2′-AMP. A final stage of purification included the use of size exclusion liquid chromatography in order to isolate the purely monomeric form of the protein, essential in small angle scattering experiments. The protein concentration was calculated using a molar extinction coefficient of ε450 = 22,000 M−1cm−1.The gene for human fibroblast CPR lacking the N-terminal membrane-anchoring region  was expressed in c reduction assays following absorbance change at 550 nm were carried out in 100 mM BES , pH 7.0, with 50 μM cytochrome c and 50 μM NADPH at 25 °C. Burst-phase kinetics of the reduction of cytochrome c by fully reduced CPR was studied by stopped-flow under anaerobic conditions at 10 °C. The stopped-flow apparatus  was placed inside a glovebox  in an atmosphere with an oxygen content of 5 ppm or less. All transient kinetics studies were carried out in 100 mM BES pH 7.0 buffer. A solution containing 10 μM CPR and 200 μM NADPH was incubated for 5 minutes in anaerobic conditions before starting any measurements. The reduced protein solution was rapidly mixed with an equal volume of 100 μM cytochrome c in the 2 μL flow cell and the change in absorbance at 550 nm after the 2 ms dead-time of the instrument was recorded. 2000 data points were measured over a time period of 1 s in order to ensure that the full burst phase, as well as the transition to the slow steady-state phase was observed. In order to provide an initial reading for A550 in the absence of reduction, the cytochrome c solution was also mixed with the buffer solution only.Steady-state cytochrome 315 = 8040 M−1 cm−1 and ε340 = 6220 M−1 cm−1 for sodium dithionite and NADPH respectively. The redox titrations were carried out as previously described67. A small aliquot of the reducing agent solution was added to the protein solution and the spectrum was recorded after a period of equilibration  in a nitrogen atmosphere with < 5ppm oxygen. All samples were in 100 mM BES, pH 7.0, buffer. All solutions were purged in a nitrogen atmosphere before introduction to the glovebox and then incubated for 4–6 hours on ice before continuing. The reducing agent titrants were prepared inside the glovebox using degassed buffer and their concentration determined using ε68. In this case the flow rate on the FPLC  was set to 0.3 mL/min and SANS measurements were taken throughout the whole protein elution time with a frame duration of 60 s in a custom made quartz flow-cell  with a 1 mm path length.SANS measurements were carried out on D22, the high-flux neutron diffractometer at the Institut Laue-Langevin, Grenoble, France. The data collection parameters are given in Supplementary Table q range from the Guinier region of the monomer to the solvent. The 2-dimensional 3He detector was positioned at different distances from the sample with an off-centered direct beam in order to provide a q-range of 0.01–0.6 Å−1, where q = 4π sin θ/λ and 2θ is the scattering angle at a wavelength of 6 Å ± 10%. The raw scattering data were reduced using the instrument specific software GRASP69, merged to produce the full scattering curves and buffer subtracted and normalised for concentration as appropriate using NIST SANS reduction macros in IGOR pro70. Where samples were found to show small amounts of unavoidable aggregation, the scattering curves were fit to the Beaucage model71 to ensure that their influence on the Guinier region was negligible.Data were recorded at two collimation lengths (5.6 m and 2.8 m) and respective sample-to-detector distances (5.6 m and 1.4 m) in order to provide a full 72. Determination of hydrodynamic parameters was performed using PRIMUS73, where Rg was determined using the Guinier approximation, and Dmax and P(r) were calculated using GNOM74. Model-independent ab initio molecular envelopes were generated using DAMMIF75. Fifteen independent DAMMIF runs were averaged using DAMAVER76 to obtain a typical molecular shape and filtered using DAMFILT to produce a refined model revealing only the most common structural features.Initial data processing and analysis were carried out using programs from the ATSAS suite77 and the ATSAS suite. Either a pool of specific structural models, as discussed in the text, or a pool of 10,000 conformational samples created using the RRT (rapidly exploring random tree) sampling tool provided with a PDB structure and a selection of flexible residues, were used. For the specific structural models, the compact state was represented by the crystal structure of soluble  oxidised human CPR19; the Rg value calculated from this structure using CRYSON41 is 24.88 Å. To represent the more extended state we used either the model we described earlier37 , which was based on NMR and SAXS data on wild-type CPR, or the crystal structure of the ΔTGEE mutant of CPR42 (PDB 3ES9). In the crystals of this mutant, which has a deletion in the flexible hinge42, there are three molecules in the asymmetric unit, in each of which the FMN domain has moved relative to the linker and FAD domains. In molecule A the position of the FMN domain is well-defined and can be seen to have rotated away from the linker and FAD domains in such a way as to expose the FMN to the solvent, with a distance between the two isoalloxazine rings of ~29 Å; we have used molecule A as a model for the extended state .Theoretical scattering curves were calculated for each of the sampled conformations using CRYSON41. A best fit model to the experimental data was determined using MultiFoXS40 in partial mode where precomputed scattering intensities were used. For most fits the maximum q-value was set at 0.2 Å−1 due to the levels of experimental noise present in the high-q data. The best-scoring models comprising one or more states from the pool provided were identified by using the χ2 value describing the goodness-of-fit to the experimental data.Rigid body modelling was carried out using software from the IMP (Integrative Modelling Platform) suiteSupplementary Information


The general objective of this study was to reexamine two views of creativity, one positing that there is a general creative capacity or talent and the other that creativity is domain-specific. These two views were compared by (a) testing correlations among measures of domain-general and domain-specific creativity and (b) examining how the general and the specific measures was each related to indices of knowledge, motivation, and personality. Participants were 147 college students enrolled in a foreign language course. Data were collected on participants’ domain knowledge, motivation, and creative personality, as well as four measures representing “General or Domain-Specific Creative Ideation” or “Creative Performance and Activity”. Results indicated that the four measures of creativity were correlated with one another, except for “General Performance and Activity” and “Domain-Specific Ideation.” A canonical correlation indicated that knowledge, motivation, and personality were significantly correlated with the four creativity measures . Multiple regressions uncovered particular relationships consistent with the view that creativity has both general and domain-specific contributions. Limitations, such as the focus on one domain, and future directions are discussed. There is a debate whether creativity is domain-general or domain-specific e.g., . Pluckerr = .18) among three different types of artwork in a group of 4th, 5th, and 6th grade students. The domain-specificity view holds that creativity emerges within fields. The critical evidence for this position is based on low correlations among different domain-derived artifacts created by the same person. For example, One important difference between the two views of creativity involves contributions of knowledge and personality. Theories of domain-specific creativity often emphasize knowledge, and in particular domain-specific knowledge e.g. . AlthougThe primary objective of the present research was to offer a fine-grained examination of how both general and domain-specific creativity are related to knowledge, motivation, and personality. This research is in fact unique in that it included measures of knowledge, motivational, and personality variables and examined each as predictor of both general and domain-specific creativity. Participants included a total of 147 undergraduates from a large university in the southeastern United States. Each student was enrolled in a Korean language course at the time of the investigation. The fact that participants were only Korean language students suggests a limitation but it also provides an opportunity to define domain-specific work very carefully. Seven classes at three different levels  participated in this study. Students completed a set of instruments as an extra credit activity in the classroom.Runco Ideational Behavior Scale . This is an important point because it clearly distinguishes the RIBS from the Performance and Activity measures described below.Creative Activities and Accomplishment Checklist , but this particular research project was focused on the language domain, and in particular the foreign language domain. Creative ideation within this domain was assessed using a scale adapted from the original RIBS . For theCreative performance and activity in the foreign language domain was assessed using a scale that was adapted from the CAAC . Ten iteM = 50, SD = 9.78).Here again, it was necessary to be realistic and chose an available index to estimate knowledge. There are a multitude of options, but the decision was made to obtain data from student records as estimates rather than extend the time necessary for data collection by adding an additional measure. Thus course grades in Korean classes were collected and used as the estimate of domain-specific knowledge in foreign language. The raw scores  were obtained from instructors, and the scores were converted to standard T-scores (Motivation in foreign language was measured using the Korean Motivation Questionnaire (KMQ). This was modified from the Science Motivation Questionnaire II SMQ; . The SMQCreative Personality Scale . Motivation was significantly correlated with General Creative Ideation , Domain-Specific Creative Ideation , and Domain-Specific Creative Performance and Activity . Motivation also significantly correlated with CPS creative personality scores . Finally, creative personality significantly correlated with General Creative Ideation  and General Creative Performance and Activity .As can be seen in r = .51, p < .01). General Creative Ideation was also significantly correlated with Domain-Specific Creative Ideation  and Domain-Specific Creative Performance and Activity . General Creative Performance and Activity was significantly correlated with Domain-Specific Creative Performance and Activity . However, the relationship between General Creative Performance and Activity and Domain-Specific Creative Ideation was not significant . Finally, Domain-Specific Creative Ideation correlated with Domain-Specific Creative Performance and Activity .The four measures of creativity  were related to one another, with the exception of General Creative Performance and Activity and Domain-Specific Ideation. The correlation between General Creative Ideation and General Performance and Activity was significant , 15% of General Creative Performance and Activity , 15% of Domain-Specific Creative Ideation , and 9% of Domain-Specific Creative Performance and Activity .The data were then analyzed using four multiple regression analyses. Each used one of the creativity measures as a criterion. The four models tested included domain-specific knowledge, motivation, and creative personality measures as predictors. Results are presented in p < .01) and creative personality  significantly predicted General Creative Ideation, and creative personality predicted General Creative Performance and Activity . Domain-specific knowledge was negatively associated with General Creative Performance and Activity . Motivation also predicted Domain-Specific Creative Ideation  and Domain-Specific Creative Performance and Activity .Rc = .49, p < .01). Thus, and quite importantly, 24.3% of variance in creativity criteria can be explained by domain knowledge, motivation and creative personality.A canonical correlation using domain-specific knowledge, motivation, and creative personality as predictors and the four creativity measures as criteria was significant  were largely inter-related. The exception was Domain-Specific Creative Ideation, which was not significantly correlated with General Creative Performance and Activity. The significant correlations certainly do not imply that the various measures are redundant with one another nor are interchangeable. Correlations were only moderate, which makes perfect sense. It would be a surprise if General Creative Ideation was completely independent of Domain-Specific Creative Ideation, or if General Creative Performance and Activity were not at all associated with Domain-Specific Creative Performance and Activity. It makes sense that Domain-Specific Creative Activity is associated with General Creative Ideation and Activity—after all, they are all about creativity, and general components of creativity could contribute to domain specific creativity, even if they are not redundant. Recall also R2 < 16%), but the contributions of the particular predictors did vary from criterion to criterion. Personality contributed to both General Creative Ideation and General Creative Performance and Activity, for instance, an outcome that replicates previous research , when the knowledge, motivation, and personality predictors were regressed, did not differ in any dramatic way  was negatively related to General Creative Performance and Activity. This finding is in line with the previous research e.g.,  showing Previous research compared general and domain-specific creativity by examining correlations among the measures of creativity. Support for general creativity was inferred from significant correlations among creativity measures in diverse domains e.g.,  and fromThe focus on foreign language represents a notable limitation in the present investigation. This was essentially a test of only one domain (language), and one culture for that matter. Other domains and samples would need to be tested in future research. As a measure of domain knowledge, we used students’ course grades. Although we used standardized scores for the grades, another limitation is that grading can vary from course to course, and in fact the assessment criteria may vary as well. This investigation also relied on particular measures of domain-specific knowledge, motivation, and personality as predictors of creativity. Measures that cover a wider variety of specific personality traits  might uncover relationships that differ from the present results. It would be most beneficial to include assessments other than self-reports in future research. Certainly future research is needed to examine both general and specific indices to better understand the multidimensional nature of creativity.


Given the critical importance of the plastic deformation in the martensite phase for the shape memory effect and superelasticity, we investigated here the plastic deformation behaviour of a single crystal of α″ (orthorhombic) martensite of Ti-27 mol%Nb shape memory alloy obtained by the stress-induced martensitic transformation of a single crystal of the parent β phase. Four operative plastic deformation modes were observed, including two dislocation slips and two twinnings. To the best of our knowledge, two of these plastic deformation modes (one dislocation slip and one twinning) were discovered for the first time in this study. The identified slip and twinning systems in the martensite phase have corresponding slip and twinning systems in the parent β phase with which they share many similarities. Therefore, we believe that the plastic deformation of the α″ martensite is inherited from that of the parent β phase. In particular, Ti base shape memory alloys (SMAs) composed of non-toxic elements have attracted considerable attention as new biomedical SMAs. The most widely applied SMA in the biomedical field is Ni-Ti alloy. However, the risk of triggering an allergic reaction to Ni by using a Ni-Ti SMA has been pointed out6. To address this issue, Ni-free Ti base SMAs have been developed in recent years. The shape memory effect and superelasticity in Ti base SMAs are due to the martensitic transformation between the β phase  and the α″ phase . The research on the material design  and fundamental aspects of martensitic transformation29 in Ti base alloys has rapidly grown. One of the biggest problem of Ti base SMAs from a practical viewpoint is a low critical stress for plastic deformation, which causes a small shape recovery strain9 and unstable superelasticity during the cyclic deformation32. While a lot of effort has been dedicated to increasing the critical stress for plastic deformation18, there have been only a few reports on the plastic deformation modes in Ti base SMAs. The plastic deformation of metastable β-Ti alloys in the β phase has been systematically investigated by many researchers38, in particular the effect of the loading orientation, test temperature and alloy composition on the main plastic deformation modes , are operative in the α″ martensite phase of Ti base alloys.Noble Ti alloys have been actively investigated in the past two decades owing to their high biocompatibility and unique mechanical properties, which make them a choice material for biomedical applications39. Otsuka and co-workers48 have succeeded in making and investigating the deformation behaviour of single-crystalline martensite of Cu-Al-Ni SMAs by this method.One of the reasons for the limited number of reports on the plastic deformation of α″ martensite is the difficulty in making single-crystalline martensite samples, which are nonetheless very helpful for analysing plastic deformation modes. Even when a single crystal of the parent β phase is cooled below the martensitic transformation temperature, an α″ martensite crystal with multi-variants forms. This type of structure is called a self-accommodated microstructure, i.e. without any apparent change in shape. Here, we focused on the stress-induced martensitic transformation that enables to selectively grow a specific variant of martensite from the parent phase. This variant selection by external stress is at the origin of the transformation strain and superelasticity in shape memory alloys49. Single-crystalline α″ martensite of Ti-27 mol%Nb was obtained here by a stress-induced martensitic transformation from a single crystal of the parent β phase, and its plastic deformation behaviour in terms of crystallographic orientation dependence and deformation modes was systematically investigated.In this study, the binary alloy Ti-27 mol%Nb was chosen because it is known to undergo a stress-induced martensitic transformation at room temperature49. The chemical composition analyses  between the uniaxial compression stress and the lattice deformation strain along the compression axis. The CV(s) with the largest negative value of U is (are) then the favourable variant(s)54. The favourable CV(s) expected to be formed in this study were determined from the calculated values of U for each compression axis  , e.g. the 20, while in none of the martensite plates could we observe internal twins in this study, indicating that the stress-induced martensite plate consisted of a single CV. This means that the stress-induced martensite plate in this study was single crystalline. In all the samples, the habit plane(s) of the plate-shaped martensite was determined by a two-face trace analysis. The results are shown in Table 57. As is shown in Supplementary Table b family, in good agreement with the experimental results shown in Table b habit plane, was observed, which corresponds to CV5  is shown in a standard stereographic triangle of the parent β phase in Fig. 49. However, in Samples #1, #6 and #8, the stress-induced α″ martensite could not reversely transform into the parent β phase by unloading  investigated in the next section.It has been reported that Ti-27 mol%Nb polycrystals exhibit superelasticity at room temperaturein-situ OM were introduced into the single crystalline α″ martensite. Figure σslip), i.e. the second yielding stress, of Samples #1–#6 was determined from the stress-strain curves  are shown with dashed lines in the stereogram in Fig. o slip direction significantly deviated from the MRSSP, whereas those for the [101]o slip direction were close to the MRSSP. The Schmid factor for the MRSSP is shown in Table τMRSSP) in each slip direction was calculated by the σslip and Schmid factor, and the results are given in Fig. τMRSSP of the [110]o slip showed the following orientation dependence: τMRSSP increased when the compression axis approached the [001]o direction.In all the samples, wavy trace lines, which correspond to plastic deformation in SMAs, were observed during compression after the second yielding. This means that the wavy trace lines observed by 001 Fig. . NumerouK1) was experimentally determined by a two-face trace analysis as follows: Twin A o; and Twin C K1 of Twin A and Twin C were close to {130}o, therefore we can consider that they correspond to the previously reported {130}o44. On the other hand, the K1 of Twin B was approximately (103)o, which has never been reported in orthorhombic crystals60 .In Sample #1, deformation bands were observed after the second yielding that exhibit features different from those of a dislocation slip: the interface between band and matrix was straight and sharp Fig. , and theη1, conjugate twinning plane K2, conjugate shear direction η2, and magnitude of shear s, of the newly discovered twin . Finally, the twinning elements of Twin B were determined as follows: K1 = (103)o, η1 = K2 = η2 = [101]o, and s = 0.3327.The other twinning elements, i.e. the shear direction in-situ OM observation was performed 38. Similarly to the <111>b slip in the parent β phase, the slip lines in the α″ martensite were wavy, and the slip planes were located parallel to the slip directions [110]o and [101]o. The crystal structure of α″ martensite was orthorhombic; however, it was close to that of the original parent β phase (bcc) owing to the small lattice deformation strain in this alloy9. In addition, the slip traces introduced in the α″ martensite did not disappear by the reverse transformation as shown in Supplementary Fig. Two slip systems with a slip direction parallel to either [110]o in α″ martensite has been observed in the tensile-deformed Ti-20 mol%Nb alloy41, there is no report on dislocation slips along [101]o in α″ martensite. The Burgers vectors  of <111>b slips in the parent β phase, and [110]o and [101]o slips in the α″ martensite phase are indicated in Fig. o slip is b = [101]o, and is thus perpendicular to the shuffling of the (001)o basal plane along the adjacent [010]o direction as a result of the martensitic transformation. In addition, in the case of a perfect dislocation , the magnitude of b for this [101]o slip is about twice as large as that of the [110]o and <111>b slips. We consider that this perfect dislocation can be dissociated into two partial dislocations and a planar fault. However, more work is needed to understand the dislocation dissociation, and systematic studies by transmission electron microscopy (TEM) are under way in our group.While the dislocation slip along [110]b b o b 44. Castany and co-workers44 insisted that the {332}b o b o et al. On the other hand, although the newly discovered {103}o b o et al.42 reported that a complicated ‘structure shuffling’ is necessary for the operation of a {130}o o η1 of this twin (o. Further observations of the twinning interface by high-resolution TEM are necessary to clarify the {103}o All of the observed deformation twins in this study correspond to the {332}o twinning (Twin A), 0.41 for (130)ooo and [101]o slips) were the predominant plastic deformation modes in this study. As shown in Fig. o slip, whereas it was small in the [101]o slip. This means that the slip plane of [101]o slip easily changed depending on the compression axis, and was controlled only by the resolved shear stress. A certain anisotropy of τMRSSP was found with the [110]o slip since τMRSSP increased when the compression axis approached [001]o. As mentioned above, the plastic deformation behaviour of the α″ martensite has many similarities with that of the parent β phase. There are some reports on a similar anisotropy of resolved shear stress in the parent β phase of Ti alloys38. In general, this anisotropy of resolved shear stress is well known as a twinning/anti-twinning sense asymmetry in bcc metals and alloys. In the case of compression, the resolved shear stress becomes lower when the loading axis is <001>b, while it becomes higher when the loading axis is <110>b. This feature is successfully explained by a non-planar core structure of screw dislocations in bcc metals and alloys62. The origin of the anisotropy of the τMRSSP observed in the α″ martensite is probably closely related to that in the parent β phase since the compression axis of Sample #2  before loading was close to [001]b, and that of Samples #3 (τMRSSP = 171 MPa), #4 (τMRSSP = 180 MPa) and #6 (τMRSSP = 181 MPa) to [110]b (or [111]b) as shown in Fig. Figure o are necessary. However, only the favourable variant of single-crystalline α″ martensite is obtained by stress-induced martensitic transformation of a single crystal of the parent β phase (i.e. only the CV5 is available in compression). This means that the compression axis in the α″ single crystal always approaches [100]o because the lattice deformation strain by the martensitic transformation along this direction has the largest negative value9. Moreover, even if a α″ martensite sample with a compression axis along [010]o was obtained, the compression axis of the sample would be readily converted to [100]o by the reorientation of the martensite variant. The lattice deformation strain along [010]o has the largest positive value, therefore the plastic deformation of the α″ martensite along [010]o is available in tension, not in compression.To completely understand the orientation dependence of the plastic deformation in α″ martensite, further experiments with a compression axis along [010]Dislocation slip discussed in this study corresponded to the macroscopic slip deformation which was observed as slip traces by OM and SEM. On the other hand, the microscopic slip deformation was possibly introduced to the parent phase and/or stress-induced martensite phase by the movement of habit plane during the stress-induced martensitic transformation. This microscopic slip deformation should play an important role in the stress-induced martensitic transformation, therefore, the further study for the microscopic slip deformation is necessary to obtain the stable superelasticity.o, (2) dislocation slip along [101]o, (3) {130}o ob slip and the {332}b o slip and (103)oIn summary, single-crystalline α″ martensite was successfully obtained by a stress-induced martensitic transformation from a single crystal of the parent β phase, and the plastic deformation behaviour of the different α″ martensite single crystals was systematically investigated. The operative plastic deformation modes in α″ martensite were as follows: (1) dislocation slip along [110]3) were cut by a precision diamond wheel cutter for the compression tests. Specimens were solution treated at 1173 K for 3.6 ks in an Ar atmosphere followed by water quenching, and the oxidised surface layer that formed by water quenching was removed by chemical etching at 333 K with a solution of HF:HNO3:H2O = 7:8:10 in volume. The final specimen size, after etching and before the compression tests, was approximately 2 × 2 × 5 mm3. The chemical composition of the samples after the solution treatment was determined by an inductively coupled plasma analysis  and inert gas fusion method . Cyclic loading-unloading compression tests were performed using an Instron-type mechanical testing machine  at room temperature with a nominal strain rate of 3.3 × 10−4 s−1. The sample surface was observed before and after the compression test by an optical microscope (OM) with a differential interference contrast (DIC) mode . In-situ OM observation with DIC mode during heating from room temperature to 373 K was performed for the specimen after compression. In-situ video OM observations of two adjacent surfaces parallel to the compression axis were also recorded during the compression tests by two high-resolution charge-coupled device (CCD) camera systems . The habit planes were determined by two-face trace analyses. The compression strain was precisely measured from the videos using a motion analyser software . The deformation microstructure was observed by a scanning electron microscope (SEM) equipped with a back-scattered electron (BSE) detector . The following lattice parameters of the parent β (ab) and α″ martensite  phases reported in a previous study9 were used for the calculation of the interaction energy (U), lattice deformation strain due to the stress-induced martensitic transformation, habit plane using the phenomenological theory of martensite crystallography (PTMC), and coordinate conversion between β and α″ phases: ab = 0.3290 nm, ao = 0.3225 nm, bo = 0.4770 nm and co = 0.4615 nm.A polycrystalline Ti-27 mol%Nb alloy rod was fabricated by Ar-arc melting using a high purity Ti (99.99%) and Nb (99.9%), and was used as mother alloy. Single crystals were prepared by an optical floating zone method with a grow rate of 5 mm/h under high-purity Ar flow. The single-crystal orientation was determined by X-ray back Laue diffraction. Rectangular specimens (3 × 3 × 6 mmSupplementary InformationSupplementary_Video_1Supplementary_Video_2