Mesoscopic simulation studies the structure, dynamics and properties of large molecular ensembles with millions of atoms: Its basic interacting units (beads) are no longer the nuclei and electrons of quantum chemical ab-initio calculations or the atom types of molecular mechanics but molecular fragments, molecules or even larger molecular entities. For its simulation setup and output a mesoscopic simulation kernel software uses abstract matrix (array) representations for bead topology and connectivity. Therefore a pure kernel-based mesoscopic simulation task is a tedious, time-consuming and error-prone venture that limits its practical use and application. A consequent cheminformatics approach tackles these problems and provides solutions for a considerably enhanced accessibility. This study aims at outlining a complete cheminformatics roadmap that frames a mesoscopic Molecular Fragment Dynamics (MFD) simulation kernel to allow its efficient use and practical application.fSMILES) with connected molecular fragments to represent a molecular structure. The fSMILES notation and the following concepts and methods for building blocks 2-4 are outlined with examples and practical usage scenarios. It is shown that the requirements of the roadmap may be partly covered by already existing open-source cheminformatics software.The molecular fragment cheminformatics roadmap consists of four consecutive building blocks: An adequate fragment structure representation (1), defined operations on these fragment structures (2), the description of compartments with defined compositions and structural alignments (3), and the graphical setup and analysis of a whole simulation box (4). The basis of the cheminformatics approach (i.e. building block 1) is a SMILES-like line notation  contains supplementary material, which is available to authorized users. Molecular modelling and simulation aims at (at least) theoretically explaining and (at best) predicting the structures, properties and dynamics of molecules and molecular ensembles. Whereas the fundamental laws of nature are known in principle for nearly a century , their pDissipative particle dynamics (DPD) in particular is a well-established mesoscopic simulation technique to study the structure, dynamics and properties of very large molecular ensembles which may represent millions of atoms. Its basic coarse-grained interacting units (beads) are no longer the nuclei and electrons of quantum chemical ab-initio calculations or the fine-grained atom types of molecular mechanics but appropriate larger molecular shapes which may not necessarily be distinct chemical compounds at all -11]. Th. Th11]. A MFD kernel software for mesoscopic simulation has a simple architecture in principle: It comprises a main loop for a defined number of successive iteration steps to approximately solve the equations of motion. The positions and velocities of all beads are stored in appropriate array structures which represent the corresponding mathematical vectors. All topological information, e.g. the mutual connections between the beads of a specific molecule, is coded with speed- or memory-optimized data structures that represent the corresponding mathematical connection matrices. A simulation input consists of a set of initial bead positions for simulation start, the complete molecular connectivity information and numerous additional parameters that guide the simulation process like the number of simulation steps. The simulation output contains sets of bead positions for different simulation steps as well as corresponding calculated properties. All input or output of a simulation engine is usually provided or generated in form of adequate ASCII files which often comprise hundred thousands of lines. Thus performing a simulation task with a pure kernel software requires a manual setup and interpretation of these ASCII files - a tedious, time-consuming and above all error-prone venture that considerably limits practical usage and application.A virtue of Cheminformatics is to develop concepts, definitions, data structures, algorithms and toolbox software that allow an efficient and comfortable treatment of chemical entities and ensembles at the man-machine interface -23]. Th. Th23]. An MFC roadmap consists of four consecutive building blocks above the MFD kernel yOH, abbreviated as CxEy, may be approximated by x linearly connected methane fragments followed by y connected dimethyl ether fragments and a terminal methanol fragment . Th. ThfSMILfSMILES becomes available.Peptides and proteins consist of distinct sequences of amino acids which fold into secondary and tertiary 3D structures. With an adequate fragment representation for each amino acid and all possible dipeptides, the automated conversion of peptides and proteins into fSMILES:Figure The fragments and the fragmentation scheme for the used amino acids are depicted in Figure For proteins the amino acid sequences can be obtained from Protein Data Bank (PDB) files . These ffSMILES definition (see above and Appendix A). An additional polymer builder tool could realize tasks like the definition of different statistical monomer distributions within a polymer chain or the creation of specific  copolymers (including star/brush/comb structures) by adequate textual construction and manipulation of fSMILES strings.The simulation of polymers and polymer mixtures is a common application of mesoscopic simulation in general and of MFD in particular. Monomer definition features are already included into the fSMILES fragment structure representation productive, a basic function library is necessary that provides useful operations on these structures.In order to make the sketched fSMILES string to ensure its formal correctness. This comprises a left-to-right fSMILES string evaluation with separation and translation of every semantic unit  into an array of corresponding tokens (where possible forbidden characters or substrings are detected). Then, general checks (like the match of numbers of opening and closing brackets or the pairwise occurrence of ring closures) are performed and finally the token sequence is analysed in a consecutive manner for its syntactic validity.At first, syntax parser functions are mandatory which allow a detailed syntax check of a provisional fSMILES into a distinct fragment-connection table/matrix: This function performs the primary man-machine interaction in MFD where the human-comprehensible fSMILES string is converted to the machine-readable connection matrix. Since a mesoscopic MFD simulation may comprise thousands to millions of molecules, distinct functions for the initial mapping of their fragments to their spatial box coordinates are necessary to obtain the start configuration of the simulation. Figure fSMILES converter and the spatial fragment mapping functions for C 10E4 surfactant molecules with fSMILES string 9Methane-4DME-MeOH9Methane-4DME-MeOH in form of an ASCII input file for the MFD kernel that defines the start geometry and connectivity of the first two of 40884 C 10E4 molecules in the simulation box. Each C 10E4 molecule contains 14 fragments where the position and connectivity of each fragment is described in a single line: The “Index” column attributes an array position to each fragment, followed by the “Fragment” column defining the fragment, a “Potential-Index” column for additional fragment-fragment potentials (e.g. necessary for defining the stiffness of peptide and protein backbones), the “x y z” simulation box coordinates columns and finally relative “Bond-Offsets” for fragment-fragment connections by harmonic springs (where “-1” indicates a connection to the fragment in the previous line and “1” a connection to the fragment in the next line).For communication with the MFD simulation kernel, a converter function is necessary that transforms a valid fSMILES string, the maximum-distant fragment (denoted A) is evaluated by a first DFS run. In a second step, another DFS run is performed to find the maximum-distant fragment from fragment A (denoted B). Finally the fragment chain between fragments A and B is chosen for the spatial tube representation. The sketched method leads to true longest chains for acyclic fSMILES but not necessarily for cyclic fragment structures. In the latter case, the determined linear fragment chain is a heuristic result only but still sufficient for all practical purposes. If a “[START]” and an “[END]” fragment are defined (see Appendix A), then the longest linear chain between these tagged fragments is used. If specific additional intramolecular potentials between fragments are defined in order to influence the backbone stiffness of e.g. polymers, peptides or proteins the longest linear chain tube representation may also be regarded as an adequate start configuration for their structural unfolding. For specific large macromolecules like proteins, a known spatial fragment backbone may of course be directly mapped onto the corresponding box coordinates to speed up the simulation , different fragments may overlap and even penetrate each other. As a consequence, the problem of possible atomic entanglements or caging effects due to inadequate molecular start geometries is negligible . NonethefSMILES-related property calculations like fragment frequencies, monomer-fragment expansion or fragment expansion from frequency counts (as well as their corresponding collapsing methods) and stoichiometric or concentration calculation methods.Last but not least, the function library ought to contain support methods for Mesoscopic simulation targets large molecular ensembles which represent up to millions of molecules. The start geometry of the simulation box may be a pure random bulk mixture with molecules represented by spatial tubes (see above). But in many cases, this global “random soup” is unfavourable for a specific simulation. To allow for a more detailed spatial setting of the start configuration, the definition of simulation box compartments is mandatory. Common compartment types are spheres, layers or bricks which may be freely positioned in the simulation box with or without overlap. The size of the compartments determines the number of fragments it may contain. Each compartment may possess a specific molecular composition as well as specific orientation and alignment of its molecules/fragments. Figure The graphical visualization of the simulation box, i.e. the fragments at their box positions, is a central feature of every molecular simulation method. Mesoscopic MFD simulation box visualization in particular is challenged by its large size with up to millions of fragments. On the other hand the simulation box visualization is confined to adequate 3D spherical fragment rendering only since fragment connections are usually omitted without any loss of display quality. A simulation box visualization should allow arbitrary box rotations, specific fragment/molecule size scaling up to their exclusions, arbitrary re-colouring of fragments, zoom in/out functions as well as length measurement options. Last but not least the creation of simulation box movies that comprise reasonable simulation steps should be supported.One approach is the use of established open-source atom-based molecular visualization tools like Jmol  which caFor minimum memory consumption and very fast rendering, a simulation box slicer approach may be followed alternatively: The simulation box is cut into slices along an axis of interest and the slice graphics are rendered one by one which leads to a graphical illustration with parallel projection that alleviates through-space length comparisons and measurements. For fragment rendering a fast 2D radial-gradient paint may be used to create a 3D sphere illusion. The depth impression of the simulation box may be adjusted by a fog option. Figure fSMILES), defined operations on these fragment structures, the description of compartments with defined compositions and structural alignments and the setup and analysis of a whole simulation box may considerably alleviate and broaden the use of mesoscopic simulation techniques like MFD. In addition the MFC roadmap realization can draw an already existing Cheminformatics open-source solutions like CDK, BioJava or Jmol. Thus, MFC may be regarded as a crucial accelerator to propagate MFD and similar mesoscopic simulation techniques in the molecular sciences. The MFC layer itself may then be utilized by a graphical user interface (GUI) approach to finally build a rich simulation client for convenient practical application even in industrial environments. Last but not least, MFC creates new cheminformatics challenges like a computer-assisted/automated fragmentation support to split a chemical compound of interest into adequate molecular fragments.A consequent MFC roadmap with an adequate fragment structure representation (fSMILES): Fragment names with a maximum of 10 characters  and an optionally prefixed frequency number.Connection character ‘-’ for bonding of the main chain.Round brackets ’(’ and ’)’ indicating branches. They may be nested for arbitrary levels of branches.Square brackets ’[’ and ’]’ with an enclosed number which follow a fragment indicating a ring closure.Curly brackets ’{’ and ’}’ with an enclosed monomer definition. Monomers are defined as molecular structures but must contain at least 1 fragment with a [HEAD] and [TAIL] attribute: Structure elements that precede the monomer connect to the HEAD fragment, structure elements that follow the monomer connect to the TAIL fragment. Monomers are not allowed to be nested.Monomer labels that start with a ‘#’ character followed by a sequence of characters (first non-digit).Monomer labels may be preceded by a frequency number.A fragment of a molecular structure (but not of a monomer) may optionally contain a [START] or an [END] tag which may be used for orientation purposes. There is only one [START]/[END] pair allowed per structure.A molecule may consist of multiple independent parts . Each part must be framed by angle brackets ‘<’ and ‘>’. Parts are not allowed to be nested.The following rules allow the definition of a monomer or molecular fragment structure line notation (fSMILES definitions sketched in Appendix A:The following examples demonstrate the usage of the A-B-CA-B-C defines a connection of fragment A with fragment B and fragment B with fragment C.A-2B(E-F)-DA-2B(E-F)-D is identical to A-B-B(E-F)-DA-B-B(E-F)-D.3A-B3A-B is a shortcut notation for A-A-A-BA-A-A-B.A-B(D-E)-FA-B(D-E)-F defines a main chain A-B-FA-B-F with a side chain D-ED-E where fragment D is connected to fragment B.A-B[1]-C-C-C-D-E[1]A-B[1]-C-C-C-D-E[1] defines a ring closure between fragments B and E.A-B(D-E(G-H[1])-F)-I-A-K[1]-BA-B(D-E(G-H[1])-F)-I-A-K[1]-B defines a main chain A-B-I-A-K-BA-B-I-A-K-B with a side chain D-E-FD-E-F (connected to fragment B of the main chain) and another side chain G-HG-H (connected to fragment E of the first side chain). In addition there is a ring closure between fragment H of the second side chain and fragment K of the main chain.3A[1]-B-B-C[1]3A[1]-B-B-C[1] is a shortcut for A-A-A[1]-B-B-C[1]A-A-A[1]-B-B-C[1].3A(B)-D3A(B)-D is a shortcut for A-A-A(B)-DA-A-A(B)-D.Multiple ring closures at one fragment are marked by successive use of ring-closure brackets, e.g. fragment B in A-B[1][2]-4C-D[1]-4C-E[2]A-B[1][2]-4C-D[1]-4C-E[2] is connected to fragments D and E.The simplest structure of a monomer consists of a single fragment A with attributes [HEAD] and [TAIL], i.e. {A[HEAD][TAIL]}{A[HEAD][TAIL]}.D-#MyMonomer-FD-#MyMonomer-F with #MyMonomer equal to {A[HEAD]-B-B[TAIL]-C}{A[HEAD]-B-B[TAIL]-C} defines a structure where fragment D is connected to fragment A (the head) of the monomer and Fragment B (the tail) of the monomer is connected to fragment F. This definition is equivalent to D-A-B-B(C)-FD-A-B-B(C)-F.2{A[HEAD]-B-B[TAIL]-C}2{A[HEAD]-B-B[TAIL]-C} defines a structure of 2 monomers where fragment A (the head) of the second monomer is connected to Fragment B (the tail) of the preceding first monomer. This definition is equivalent to A-B-B(C)-A-B-B-CA-B-B(C)-A-B-B-C.Definitions like {A[HEAD]-{A[HEAD]-B-B[TAIL]-C}-B[TAIL]-C}{A[HEAD]-{A[HEAD]-B-B[TAIL]-C}-B[TAIL]-C} with nested monomers are forbidden.A[START]-B-C[END]A[START]-B-C[END] defines orientation information.A[START][END]-B-CA[START][END]-B-C is syntactically correct but makes no sense.A[START]-B[START]-C[END]A[START]-B[START]-C[END] is forbidden: There is only one [START]/[END] pair allowed per structure.3A[START]-B-C[END]3A[START]-B-C[END] is identical to A-A-A[START]-B-C[END]A-A-A[START]-B-C[END].<A-B-C><A-D><A-B-C><A-D> defines a molecule which consists of two independent parts A-B-CA-B-C and A-DA-D.<A-B[1]-C><A-D[1]><A-B[1]-C><A-D[1]> is forbidden since parts are not allowed to be connected in any way. The correct definition in this case would be (A-B[1]-C)(A-D[1])(A-B[1]-C)(A-D[1]) or A-B(C)-D-AA-B(C)-D-A.


The molecular basis for the increased resistance of astrocytes to a non-neuropathogenic strain of West Nile virus (WNV), WNV-MAD78, compared with the neuropathogenic strain WNV-NY remains unclear. Here, we demonstrated that the reduced susceptibility of astrocytes to WNV-MAD78 is due to a combination of both cellular activities as well as viral determinants. Analyses of the viral particle indicated that astrocyte-derived WNV-MAD78 particles were less infectious than those of WNV-NY. Additionally, inhibition of cellular furin-like proteases increased WNV-MAD78 infectious particle production in astrocytes, suggesting that high levels of furin-like protease activity within these cells acted in a cell- and strain-specific manner to inhibit WNV-MAD78 replication. Moreover, analysis of recombinant viruses indicated that the structural proteins of WNV-MAD78 were responsible for decreased particle infectivity and the corresponding reduction in infectious particle production compared with WNV-NY. Thus, the composition of the WNV virion was also a major determinant for viral fitness within astrocytes and may contribute to WNV propagation within the central nervous system. Whether the WNV-MAD78 structural genes reduce virus replication and particle infectivity through the same mechanism as the cellular furin-like protease activity or whether these two determinants function through distinct pathways remains to be determined. Flavivirus that has emerged recently as a significant threat to human health. Historically, most WNV infections have been asymptomatic or associated with a mild febrile illness known as West Nile fever. However, recent outbreaks in the Western hemisphere have been characterized by a marked increase in the percentage of neurological infections. Since 1999, infections have resulted in >17 000 cases of severe neurological disease in the USA alone (http://www.cdc.gov/westnile/statsMaps/), making WNV the leading cause of arboviral encephalitis in North America. The viral factors responsible for the increased incidence of neurological disease associated with these recent outbreaks remain poorly understood.West Nile virus (WNV) is a neurotropic member of the genus Vβ3  protein and integrin αer cells . ExposurN-linked glycans from the virion, abrogating attachment to the cell via C-type lectins and reducing infectivity on cells expressing DC-SIGN or DC-SIGNR  by crossing the blood–brain barrier (BBB) . InteracIn astrocytes, replication of an avirulent lineage 2 African isolate, WNV-MAD78, is delayed and reduced compared with that of a highly virulent lineage 1 isolate, WNV-NY  nor removal of the glycosylation site from WNV-NY (WNV-NYS156P) altered viral replication rates or peak viral levels  and WNV-NYMAD78(5′UTR→prM)] (MAD78(5′UTR→prM) and WNV-NYMAD78(E) were significantly lower than those of WT WNV-NY . Therefore, the WNV-MAD78 C and/or prM proteins were responsible for WNV-NYMAD78(5′UTR→prM)’s differential replication. The decreased level of infectious particle production of WNV-NYMAD78(5′UTR→prM) and WNV-NYMAD78(E) likewise corresponded with lower infectivity of the viral particles, as observed previously for the recombinant virus containing all the WNV-MAD78 structural genes (MAD78(E) exhibited a significantly higher particle/p.f.u. ratio than either WT WNV-NY or WNV-NYMAD78(5′UTR→prM) at 24 h post-infection, suggesting that the E protein has a greater impact than C or prM on the infectivity of WNV particles at early times during infection ] . Peak viT WNV-NY . Althougal genes . WNV-NYMnfection . NeverthThe results of this study suggest that the impairment of WNV-MAD78 replication in astrocytes is due in part to the increased production of defective particles compared with WNV-NY. Moreover, both viral and cellular factors contribute to this lower infectivity. Characterization of chimeras of WNV-MAD78 and WNV-NY indicated that the infectivity of astrocyte-derived WNV particles corresponds to the cognate structural proteins. Examination of the cellular factors involved in the astrocyte-specific restriction to WNV-MAD78 replication indicated that high levels of furin-like protease inhibited WNV-MAD78, but not WNV-NY, infectious particle production. In contrast, the classical innate antiviral pathways did not contribute to the reduced level of WNV-MAD78 replication.in vivo , 2 mM l-glutamine, 1 mM sodium pyruvate and 150 μg endothelial growth supplement ml−1 as described previously (Primary HBCAs (ABRI371) derived from normal human tissue were purchased from CellSystems and maintained according to the manufacturer’s instructions. Prior to use in experiments, HBCAs were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % FBS, antibiotic/antimycotic and non-essential amino acids (complete DMEM). All experiments with HBCAs were performed on cells passaged ≤14 times. HBMECs, obtained from K. S. Kim , were grown in RPMI 1640 supplemented with 10 % FBS, antibiotic/antimycotic, non-essential amino acids, MEM vitamins, 5 U heparin mlWorking stocks of WNV-NY strain 3356 were generated from infectious clone pFL-WNV  and NYMAD78(E) viruses were generated using the plasmids pA-NY, pA-MAD, pI-WNV-MAD(MluI-BamHI), pWNV-MADIC and pFL-WNV. The plasmids pA-NY and pFL-WNV were as described previously (MluI-BamHI) encoded the T7 promoter immediately preceding nt 1–3836 of WNV-MAD78 and pWNV-MADIC encoded the T7 promoter immediately preceding the entire genome of WNV-MAD78 . Plasmid pA-MAD, which encoded the structural genes of WNV-MAD, was generated by replacing the MluI/BglII fragment of MAD-AB (IC.Constructs encoding the recombinant portions of the NYMAD78(5′UTR→prM) construct, a cassette encoding WNV-MAD78 nt 500–966, WNV-NY nt 967–2495 and sequence corresponding to the vector backbone of pA-MAD (5′-CGGCAAGAACTCCGCTGTGGGAGTGGAGTCTAGAAATATTGAAAAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGC-3′) was synthesized by GenScript and inserted into pUC57. The cassette was subcloned, using Gibson Assembly Master Mix (New England Biolabs) as per the manufacturer’s directions, into the pA-MAD vector backbone (NsiI and NgoMIV digested).To generate the pA-NYMAD78(E) construct. A cassette consisting of WNV-NY nt 540–966, WNV-MAD78 nt 967–2468, WNV-NY nt 2470–2495 and sequence corresponding to the vector backbone of pA-NY (5′-CGGCTCTAGAGTCGAATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTG-3′) was synthesized by GenScript and inserted into pUC57. The cassette was subcloned, using Gibson Assembly Master Mix, into the pA-NY vector backbone (NsiI and NgoMIV digested).A similar strategy was used to generate the pA-NYS156P, pFL-WNV was used as template for PCR to amplify WNV-NY nt 1–1441 and 1421–4087. The NY 1–1441 fragment was amplified using a sense primer encoding a BamHI restriction site followed by the T7 promoter immediately preceding the 5′ end of the genome and an antisense primer encoding two point mutations (A1431G and G1433A). These mutations changed residue 156 of the E protein from Ser to Pro, removing the glycosylation site. The NY 1421–4087 fragment was amplified using a sense primer containing the corresponding mutation at positions 1431 and 1433 (T1431C and C1433T), and antisense primer annealing at position 4087 (DpnI to remove any template DNA lacking the desired mutations. The resultant PCR products were assembled with the vector backbone of pFL-WNV (BamHI and SphI digested) using Gibson Assembly Master Mix.To generate pA-NYion 4087 . The reaP156S. pWNV-MADIC was used as template for PCR to amplify WNV-MAD78 nt 1102–1440 and 1420–3836. The 1102–1440 fragment was amplified using a sense primer annealing at position 1102 and an antisense primer encoding two point mutations (G1430A and A1432G). These mutations changed residue 156 of the E protein from Pro to Ser, introducing a glycosylation site. The MAD 1420–3836 fragment was amplified using a sense primer containing the corresponding mutation at position 1430 (T1430C) and 1432 (C1432T), and antisense primer annealing at position 3836 (DpnI to remove any template DNA lacking the desired mutation. The resultant PCR products were assembled with the vector backbone of pI-WNV-MAD(MluI-BamHI) (SphI and BamHI digested) using Gibson Assembly Master Mix to generate pI-MAD78 WNV-MAD78P156S intermediate.A similar strategy was used to generate pA-MAD78ion 3836 . The rea(IC) was used as template for PCR to amplify WNV-MAD78 nt 9461–10962 using primers encoding a T9473C point mutation (XbaI restriction site at this location. MAD-CG (NgoMIV and XbaI digested) , a strain that has a low mutation rate, and sequenced by GeneWiz to confirm identity.All plasmids were maintained in IC plasmids were digested as indicated in in vitro transcription were generated by ligating the corresponding plasmid fragments as indicated in IC products were combined at a 1 : 1 molar ratio, precipitated, resuspended in water and ligated overnight. The ligated DNA was treated with 0.8 µg Proteinase K for 1 h at 37 °C, purified by phenol/chloroform extraction, precipitated and resuspended in 4–6 µl RNase-free water. The purified ligation reactions (1 µg) served as templates for in vitro transcription using the AmpliCap-Max T7 High Yield Message Maker kit (Cell Script). In vitro transcribed RNA was purified by phenol/chloroform extraction, precipitated with 5 M ammonium acetate and resuspended in water. For transfection into Vero cells, 12 µg RNA was transfected into 1×106 cells using the Neon transfection system (Invitrogen) with the following settings: 1150 V, 20 ms and two pulses. Culture supernatants were collected 7 days after transfection or when cytopathic effects were visible and clarified by centrifugation at 1500 g for 5 min. Total RNA was extracted from monolayers using TRIzol Reagent (Invitrogen) as per the manufacturer’s directions. The extracted RNA was used as template for reverse transcription PCR to amplify the relevant regions of the WNV genome. The resulting PCR fragments were sequenced to confirm the presence of the inserted mutations and proper gene arrangement. The recovered viruses were amplified once in Vero cells to generate the corresponding viral stock.The pA, pB and WNV-MAD2 incubator for 1 h at 37 °C with rocking, the inocula removed and a 0.9 % agarose/complete DMEM overlay was added. Cell monolayers were incubated for 48 h and a second overlay of agarose/complete DMEM containing 0.003 % neutral red  was added. Plaques were counted as follows based on the appearance of plaques: WNV-NY, WNV-MAD78/NY, WNV-NYMAD78(5′UTR→prM), WNV-NYMAD78(E) and WNV-NYS156P on days 3 and 4; WNV-NY/MAD78 on days 4 and 5; and WNV-MAD78 and WNV-MAD78P156S on days 6 and 7. All titres were performed in duplicate.Monolayers of Vero cells in six-well plates were washed once with Dulbecco’s PBS  followed by the addition of serial dilutions of viral samples. The cells were incubated in a 5 % COg for 5 min. Viral titres were determined by plaque assay on Vero cells.Cell monolayers were infected with WNV-NY or WNV-MAD78 at the indicated m.o.i. The amount of virus added to cultures to achieve the indicated m.o.i. was calculated using the titre of the viral stock on the respective cell type (HBCAs or HBMECs). After 1 h at 37 °C, the inoculum was replaced with complete DMEM. Culture supernatants were recovered at the indicated times and clarified by centrifugation at 1500 Supernatants were recovered from mock- or WNV-infected (m.o.i. 0.01) HBCAs at 48 h post-infection and inactivated by exposure to UV (254 nm) for 10 min at room temperature in a Stratalinker XL-1000 (Spectronics). Complete inactivation of infectious particles was confirmed by titrating on Vero cells. UV-inactivated supernatants were diluted 1 : 1 in 2× DMEM supplemented with 20 % FBS and treated with isotype control antisera, antisera to IFN-α and IFN-β or left untreated. Fresh HBCA monolayers were exposed to UV-inactivated supernatants for 6 h at 37 °C prior to infection with WNV-NY or WNV-MAD78 (m.o.i. 0.01). After 1 h at 37 °C, the inoculum was replaced with fresh supernatant treated as described above. Culture supernatants were collected 24 h after infection, clarified by low-speed centrifugation and subjected to plaque assays on Vero cells. The amount of antisera necessary to neutralize the IFN present in supernatants recovered from WNV-infected HBCAs was determined as described previously (HBCA monolayers infected with WNV-NY or WNV-MAD78 (m.o.i. 0.01) were removed from plates by trypsinization, washed twice with DPBS and fixed with 3 % paraformaldehyde. Cells were permeabilized with 0.2 % Triton X-100, blocked with 0.5 % heat-inactivated FBS and probed with WNV hyperimmune ascitic fluid  followed by Dylight 633 nm-conjugated goat anti-mouse IgG. For analysis, 100 000 single-cell events were collected using a FACS Canto (BD Biosciences).2). Equivalent amounts (100 µM) of the fluorogenic substrate pERTKR-amino-methylcoumarin (R&D Systems) was added to cellular lysates and fluorescence  was read every 20 s for 10 min using a Spectromax M5 microplate reader (Molecular Devices). As a control, 0.2 µg recombinant furin ml−1 (R&D Systems) was assayed. The concentration of furin inhibitor (Dec-RVKR-CMK) needed to eliminate all detectable furin activity in HBCA lysates was determined by treating HBCAs for 48 h with serial dilutions (5–50 µM) of Dec-RVKR-CMK and assaying cell lysates as described above. For inhibition experiments, HBCAs were inoculated with WNV-NY or WNV-MAD78 (m.o.i. 0.01). After 1 h at 37 °C, the inoculum was replaced with complete DMEM in the presence or absence of Dec-RVKR-CMK at a final concentration of 50 µM. Cultures were treated with a second dose of Dec-RVKR-CMK  at 24 h post-infection. Cell supernatants were collected at 48 h post-infection and clarified by centrifugation at 1500 g for 5 min. Viral titres were determined by plaque assays on Vero cells.Confluent monolayers of HBCAs or HBMECs were washed twice with DPBS and lysed in 180 µl assay buffer  as per the manufacturer’s instructions or quantitative real-time (qRT)-PCR. For the virus counter, samples were diluted 1 : 25 in Sample dilution buffer (ViroCyt) to a total volume of 100 µl and incubated in the dark for 30 min with 50 µl Combo dye, which stains nucleic acid and protein. Two-channel fluorescence was used to detect co-localization of nucleic acid and protein. Events with simultaneous detection within both channels were defined as virus particles by ViroCyt software. For qRT-PCR, RNA was extracted from supernatants (Qiagen) of HBCAs or HBMECS infected at an m.o.i. 0.01 and 0.1, respectively, with WNV-MAD78 or WNV-NY. Total viral RNA levels were determined by qRT-PCR analysis on a Roche LC480 using Veriquest One-Step SYBR Green Master Mix (Affymetrix Biosystems) with 50 ng RNA. The total number of genomic equivalents was determined by comparison with a standard curve made from known copies of WNV. Primers utilized were: WNV-NY E (fwd) 5′-GGACCTTGTAAAGTTCCTATCTCG-3′, WNV-NY E (rev) 5′-AGGGTTGACAGTGACCAATC-3′, WNV-MAD78 E (fwd) 5′-CTGTAAGGTGCCCATTTCC-3′ and WNV-MAD78 E (rev) 5′-CCTCTTCCCACCACAATGTAG-3′. The efficiency of each primer was determined prior to standard curve generation.HBCAs were infected as described above. At the indicated times, culture supernatants were collected, clarified by centrifugation at 5000 3, 1 % sodium deoxycholate, 1 % Triton X-100, 0.1 % SDS and 1× protease inhibitors; Sigma). Lysates were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Nitrocellulose membranes were blocked in 5 % non-fat dry milk diluted in Western wash buffer (PBS+0.1 % Triton X-100) and incubated with anti-WNV E  or anti-actin , followed by HRP-conjugated anti-rabbit antibodies. HRP signal was visualized using electrochemiluminescence .Cell monolayers were washed twice with modified DPBS (HyClone) and lysed with RIPA buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 0.02 % NaNSupernatants from HBCAs and HBMECs infected with WNV-NY or WNV-MAD78 were incubated at 37 °C. Aliquots were removed at the indicated times and titrated on Vero cells.se and significance were determined using two-tailed unpaired Student t-tests comparing WNV strains and chimeras.GraphPad Prism 5 was used to generate all statistical analyses. The


Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed. Gap junctions connect neighboring cells via intercellular channels that allow direct electrical communication and propagation of ions and small molecules between coupled cells . The chaper se is not the main driver of glutamate-dependent neuronal death following injury. Rather, the key determinant for expansion of cell death is the presence and extent of Cx36 gap junctions in the injured CNS regions. In the absence of neuronal gap junctions, NMDAR-mediated neuronal death is restricted to a limited number of neurons. However, in the presence of gap junctions, the expanse of cell death extends to nearly all coupled neurons. In addition, the increase in neuronal gap junction coupling that results from activation of group II mGluRs, is a significant, underlying driver of expansive neuronal death. We proposed that this is a general mechanism for neuronal death during different types of neuronal injury [It has been suggested previously , 21 thatl injury , 23. HowThe use of animal subjects in these experiments was approved by the University of Kansas Medical Center Animal Care and Use Committee (protocol number 2013–2126). The experiments were conducted in accordance with the National Institutes of Health guidelines. We used two groups of mice: wild-type  and Cx36 knockout (C57bl/6 background strain). The Cx36 knockout was originally created by Dr. David Paul  . Mice wein vitro 2 (DIV2). The Neurobasal medium also was supplemented with B-27 , 0.5 mM L-glutamine and 5 mg/100 ml gentamicin. The medium in cultures was changed twice a week. Lentiviral transduction (see below) was done in neuronal cultures on DIV3-6 (in Cx36 knockout mouse cultures) or DIV10 (in WT mouse cultures). DIV10 WT neurons were chosen for transduction since subsequent neuronal death experiments were conducted at DIV13, i.e., at the time when endogenous neuronal gap junction coupling is maximal in developing neurons [2; 10 mM glucose) to OGD conditions  before returning cells to normal culture conditions. For both NMDA and OGD injuries, the controls included substitution of the incubating medium with the same medium for 30 min.Neuronal cultures were prepared as reported previously  from the neurons , 26. For neurons , by addiHeLa cells, obtained from the American Type Culture Collection  were cultured in Dulbecco’s Modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 10 units/ml penicillin/streptomycin .In the present study, HeLa cells were used in scrape loading and other experiments in order to characterize the signal permeability and cell membrane localization of virally-transduced wild-type and mutant gap junction channels (see below). These cells were chosen because the cell lines (including HeLa) have been used effectively in these types of experiments , 28. In 5’-TTTGAATTCGCCTCCGGATGCACAGCGATG-3’; Cx36_R, 5’-TAAGCGGCCGCTTCACACATAGGCAGAGTC-3’; Cx43_F, 5’-TTTGAATTCaaagagaggtgcccagacatg-3’; Cx43_R, 5’-ATAGCGGCCGCTTTAAATCTCCAGGTCATC-3’; Cx31_F, 5’-ATCGAATTCACCATGGACTGGAAGAA-3’; Cx31_R, 5’-AGCGGCCGCTCTCTAGCACCTGCTTC-3’. cDNAs for Cx36, Cx43 and Cx31 were obtained from Thermo Scientific  corresponding to clone ID’s 5364942, 3673723 and 4983869, respectively, and used as templates in polymerase chain reaction. Amplified products were subcloned into pCMV-SPORT6  before final placement into pCDH-MSCV-MCS-EF1-Puro for lentiviral production . A plasmid for production of lentivirus expressing Cypridina luciferase (pCDH-LUC) was constructed previously [g and 15°C for 2 hours and pellets then were resuspended in a small volume of Neurobasal medium. Lentiviral titers were determined by seeding U-937 cells  in 24-well plates at 5 x 104 cells per well on the day of transduction with serial dilutions of the concentrated stock virus. U-937 cells then were incubated for 2 days. Cells expressing GFP were identified on a BD LSR II flow cytometer  with titers ranging from 5x107 to 2x108 infectious units per mL.Polymerase chain reaction products of mouse Cx36, connexin 43 (Cx43) and connexin 31 (Cx31) cDNA were created using Q5 High Fidelity DNA Polymerase  and the primers listed below: Cx36_F, eviously , and waseviously  for 4 hoThe three WT connexin expressing lentiviruses  and the control lentivirus (derived from pCDH-LUC) were singly transduced into HeLa cells or neuronal cortical cultures (obtained from Cx36 knockout mice), where their expression was characterized see . For tra5'-ACCATCTTGGAGAGGGCCCTGGAAGCCGCGGTG-3'; Cx36_L10A-R, 5'-CACCGCGGCTTCCAGGGCCCTCTCCAAGATGGT-3'; Cx36_Q16A-F, 5'-CTGGAAGCCGCGGTGGCCCAGCACTCCACTATGA-3'; Cx36_Q16A-R, 5'-TCATAGTGGAGTGCTGGGCCACCGCGGCTTCCAG-3'; Cx36_L10P-F, 5'-CCATCTTGGAGAGGCCTCTGGAAGCCGCGGTG-3'; Cx36_L10P-R, 5'-CACCGCGGCTTCCAGAGGCCTCTCCAAGATGG-3'; Cx36_Q16P-F, 5'-TGGAAGCCGCGGTGCCTCAGCACTCCACTATG-3'; Cx36_Q16P-R, 5'-CATAGTGGAGTGCTGAGGCACCGCGGCTTCCA-3'; Cx36_I22R-F, 5'-GGTGCAGCAGCACTCCACTATGAGGGGGAGGATCCTG-3'; Cx36_I22R-R, 5'-CAGGATCCTCCCCCTCATAGTGGAGTGCTGCTGCACC-3'.Using the WT mouse Cx36 lentiviral construct that is described above, three Cx36 mutants were created using a QuikChange Lightning Site-Directed Mutagenesis kit . The following primers were used to render two double substitution mutants of either L10A/Q15A or L10P/Q15P in addition to an I22R substitution mutant.: Cx36_L10A-F, 5'-CTACTCCACGGCCAGAGGGAAGGTGTG-3'; Cx43_G21R-R, 5'-CACACCTTCCCTCTGGCCGTGGAGTAG-3'.It has been shown earlier , that a The four mutant connexin lentiviral vectors, pCDH-Cx36(AA) (L10A/Q15A mutation), pCDH-Cx36(PP) (L10P/Q15P mutation), pCDH-Cx36(I22R) and pCDH-Cx43(G21R), were used to generate lentivirus that was singly transduced into HeLa cells  and into Cx36 knockout mouse neuronal cortical cultures (MOI = 20), where their expression was then characterized as described in Results.5’-GATCCGGAGACGGTGTACGATGATGACTTCCTGTCAGATCATCATCGTACACCGTCTCCTTTTTG-3’; shRNA1-R, 5’-AATTCAAAAAGGAGACGGTGTACGATGATGATCTGACAGGAAGTCATCATCGTACACCGTCTCCG-3’; shRNA2-F, 5’-GATCCGCATTTGTGTGGTGCTCAATCCTTCCTGTCAGAGATTGAGCACCACACAAATGCTTTTTG-3’; shRNA2-R, 5’-AATTCAAAAAGCATTTGTGTGGTGCTCAATCTCTGACAGGAAGGATTGAGCACCACACAAATGCG-3’.To study the effects of down-regulation of endogenous Cx36 protein, two shRNAs were designed using Invitrogen’s shRNA designer and blasted against mouse Reference Sequence (RefSeq) database. The shRNAs were cloned into pGreenPuro  per the manufacture’s recommendations and using the following oligonucleotides: shRNA1-F, Cypridina luciferase (pGreenPuro-shLuc) was constructed previously [shRNA1-Cx36 and shRNA2-Cx36 targeted the Cx36 cDNA positions at 611 and 1270, respectively. A plasmid that enables expression of a shRNA directed against eviously , and wasStaining procedures were as we described previously , 19. CovFor the analysis of co-expression of GFP and NeuN (a neuron-specific nuclear protein) in neuronal cultures, fluorescent signals were visualized on a Nikon Eclipse 80i using FITC and Texas Red filters, respectively. Images were captured on a Photometrics ES2 digital camera using OpenLab software . For the analysis of expression of Cx36, Cx43 or Cx31 in stably-transduced HeLa cells, laser scanning confocal Nikon Eclipse C1 Plus microscope, Nikon 60X oil objective and Nikon EZ-C1 software were used. In each group, the analysis was done in three coverslips obtained from independent cell culture preparations or HeLa cell lines. The antibodies that were used for analysis of expression of WT connexins also recognized mutant proteins see . NegativExperiments were performed as reported in detail , 26. Brin = 6) 500 μM glutamate was added to the cell culture medium and was present in the medium for 24 hours prior to conducting MTT assay. This killed all neurons, but did not affect glial cell survival. The absorbance in these purified glial cultures was measured, averaged, and the result was subtracted from the individual absorbance data in neuronal culture groups so that the final result would represent only neuronal death/survival. Finally, the absorbance results in experimental groups were normalized to control groups.Neuronal viability in cultures was quantitatively evaluated by MTT assay. Experiments were performed as reported in detail , 19. Cul5 cells per well in an 8-well chamber slide. Before the test, the incubating medium was replaced with the medium containing Neurobiotin  and dextran Alexa Fluor 594 . Cells were scrape-loaded using a sterile scalpel by drawing 2 cuts from corner to corner to form an “X” across the well. The cells were incubated in the medium for 5 minutes at room temperature to allow the dyes to infiltrate the cut cells. The cells then were washed  and fixed in 4% paraformaldehyde for 30 minutes in the dark. The cells were washed again (3 times), permeabilized with PBS + 1% triton-X for 30 minutes, washed (3 times) and the nonspecific binding was blocked with PBS + 0.1% Tween-20 + 10% fetal bovine serum for another 30 minutes. After washing (3 times with PBS), a probe for Neurobiotin, AMCA Avidin-D , was added at 1:500 in PBS + 0.1% Tween-20 + 0.1% bovine serum albumin for 30 minutes. Cells were washed (5 times), the chamber slide was removed and the slide was mounted with Vectashield mounting medium. Fluorescent images of the scrape-loaded cells were captured with a Photometrics ES2 digital camera on a Nikon Eclipse 80i microscope using OpenLab software. A bright-field image and three fluorescent images were captured from the same microscope field using UV-2E/C, FITC, and Texas Red filters to measure DAPI, FITC, and Texas Red emission for AMCA Avidin-D, GFP and dextran Alexa Fluor 594, respectively. Each experiment was done in triplicate. The analysis of gap junction channel permeability was made based on the number of cells containing Neurobiotin , excluding the cells containing dextran Alexa Fluor 594 .Experiments were performed as reported . Tests wWe utilized ethidium bromide (EtdBr) uptake that was previously used to measure the activity of hemichannels in cultured neurons , 33. Expt-test  or ANOVA with post hoc Tukey and InStat software . Data are reported as mean ± SEM. The number of samples indicated represents the number of plate wells/coverslips with cells obtained at least from three independent cell culture preparations, i.e., in case of neuronal cultures—from three dams; in case of HeLa cells—from three seedings. At the end of all experiments, the data for each experimental group were combined. The analyses were performed blinded to the treatment conditions.Data were analyzed using the two-tailed Student's Cypridina luciferase gene (pCDH-LUC) or shRNA directed against Cypridina luciferase (shRNA-LUC) were used as controls.In the present study, we addressed whether: (1) changes in the amount of neuronal gap junctions directly affect neuronal death and survival and (2) neuronal death related to gap junctions depends upon their channel activity. We prepared three lentiviral plasmids  for each of the vectors the transduction efficiency in neurons was >90%, measured 48 hours post-transduction by expression of GFP (the transduction marker) and NeuN (a neuron-specific nuclear protein) . Though t-test; n = 3 in each group). This showed that the cells transduced with the control vector do not pass Neurobiotin, since they lack gap junctional coupling. However, the cells expressing any of the three WT connexin proteins  demonstrated intercellular dye transfer. In these cultures, Neurobiotin was observed both in cells immediately proximal to the scrape  and in some of their close neighbors. None of the cultures showed Neurobiotin labeling on the periphery of the cell monolayer. These data support the presence of functional gap junction channels, assembled from the ectopically expressed connexin proteins.To determine if the transgene-expressed connexins formed functional gap junctional complexes, scrape-loading dye transfer assays were performed in stably-transduced HeLa cell lines Fig  and 2E. t-test; n = 3 in each group). The data indicate that the mutant gap junction channels are impermeable to Neurobiotin and therefore do not support gap junctional communication between cells.Next we characterized the cells expressing Cx36  or Cx43 (G21R) containing amino acid substitutions. As with the WT proteins, each of the four mutant connexin expressing lentiviruses effectively transduced neurons in Cx36 knockout neuronal cortical cultures . The transductions induced only moderate reduction in neuronal survival over a three day period , and botA critical role for Cx36-containing gap junctions in NMDAR-dependent excitotoxicity and injury-mediated neuronal death was suggested previously , 23. HowEssentially identical results were obtained in cultures subjected to ischemic injury with a 30-minute OGD  via propagation of gap junction-permeable death signals between the coupled cells , 35, 36 Cypridina luciferase (control). As demonstrated in control tests , pCDH-Cx36(PP), pCDH-Cx36(I22R) and pCDH-Cx43(G21R), that induce dye-impermeable  gap junctions despite the fact they are present in the cell membrane Fig  and 4D. It has been reported that open hemichannels may play a role in mediating neuronal cell death. For example, inflammation-like conditions open Cx43 hemichannels in astrocytes that activates neuronal hemichannels and promotes NMDAR-mediated excitotoxicity in neurons . In addiin vivo and in vitro, prior to the gradual decrease over next several hours [We used neuronal somatosensory cortical cultures prepared from WT mice. Experiments were performed on DIV10, at the time when developing neurons abundantly express endogenous Cx36 and gap junctions . Moreoveper se, but rather the expression of neuronal gap junctions. In the present study, we provide data supporting the critical role of gap junctions in neuronal death. Using WT neuronal cortical cultures, we show that increases and decreases in the level of expression of Cx36-containing gap junctions augment and reduce, respectively, NMDAR-mediated excitotoxicity and ischemic neuronal death.Our previous studies showed that genetic or pharmacological blockade of neuronal (Cx36-containing) gap junctions prevents NMDAR-mediated excitotoxicity , 18, 19 We also observed that cultures that lack functional gap junctions between neurons  are not susceptible to NMDAR-mediated excitotoxicity, though they are vulnerable to ischemic neuronal death. The latter presumably is due to direct ischemic injury in neurons .Based upon existing knowledge of the structure and function of gap junctions, there are two competing  models by which gap junctions may contribute to cell death: via channel-dependent or channel-independent mechanisms. The first model (the “bystander cell death model”) has been proposed based on experiments in non-neuronal cells and posits that gap junctions contribute to cell death by propagation of a gap junction-permeable death signal(s) from dying cells to healthy cells , 35, 36.2+ [As described above, the simplest mechanistic explanation for the contribution of gap junction channels to cell death is that they allow propagation of pro-death signals between the coupled cells. Some of those death signals have been suggested based on studies with non-neuronal gap junctions. These include inositol-1,4,5-trisphosphate, reactive oxygen and nitrogen species and presumably Ca2+ , 37. SucIt is interesting to note that unitary conductance of Cx36 gap junction channels is about ten times lower than that of Cx43 channels: 10–15 pS and 130 pS, respectively  data fo and 7C. It should be mentioned, however, that there are several possibilities for the role of gap junctions in neuronal death other than outlined above. The first possibility is that the introduced connexin mutations disrupt some channel-independent, protein-dependent intracellular signaling to death pathways. Given the location of the amino acid substitutions, which were chosen based upon biochemical and structural studies, this explanation is highly unlikely. Employing three different mutations in Cx36  and a previously characterized Cx43 mutation  with idThe second possibility is that the genetic manipulations of Cx36 have an impact on the expression of other genes, which then indirectly influence neuronal death/survival. As discussed above, we show in the present study that up-regulation of Cx36 increases, and down-regulation decreases, neuronal death. Further, over-expression of Cx36  rescues the death phenotype in Cx36-deficient neurons. These findings agree with the previous observations on neuroprotective effects of Cx36 knockout in various models of neuronal injury –18. Impoin vivo and in vitro [in vitro [Although Cx36 knockout induces substantial (>95%) loss of neuronal gap junction coupling in the mouse brain , 45, thein vitro , 18  and may play a role in injury-mediated neuronal death –50. HoweIn conclusion, this study provides insight into the mechanisms of contribution of neuronal gap junctions in NMDAR-dependent excitotoxicity and ischemic neuronal death. Because clinical trials for NMDAR antagonists as neuroprotective agents largely failed , our stu


High-risk human papillomaviruses (hrHPVs) infect keratinocytes and successfully evade host immunity despite the fact that keratinocytes are well equipped to respond to innate and adaptive immune signals. Using non-infected and freshly established or persistent hrHPV-infected keratinocytes we show that hrHPV impairs the acetylation of NFκB/RelA K310 in keratinocytes. As a consequence, keratinocytes display a decreased pro-inflammatory cytokine production and immune cell attraction in response to stimuli of the innate or adaptive immune pathways. HPV accomplishes this by augmenting the expression of interferon-related developmental regulator 1 (IFRD1) in an EGFR-dependent manner. Restoration of NFκB/RelA acetylation by IFRD1 shRNA, cetuximab treatment or the HDAC1/3 inhibitor entinostat increases basal and induced cytokine expression. Similar observations are made in IFRD1-overexpressing HPV-induced cancer cells. Thus, our study reveals an EGFR–IFRD1-mediated viral immune evasion mechanism, which can also be exploited by cancer cells. Human papillomavirus employs immune evasion strategies to establish a long-term infection. Here the authors show that the virus in the EGFR-dependent manner induces IFRD1, which blocks NFκB activating acetylation, and that this process can be suppressed by the EGFR inhibitor cetuximab. High-risk human papillomaviruses (hrHPVs) are absolutely species-specific small double-stranded DNA viruses that primarily target undifferentiated keratinocytes (KCs) of squamous epithelia via micro-wounds and abrasions. HrHPV infections can last up to 2 years despite viral activity in infected KCs, the expression of viral antigens and the presence of KC-expressed pattern recognition receptors (PRRs)123STAT1 and lowered STAT1 protein levels, explaining the impaired expression of IFN-stimulated genes131415Upon infection, hrHPV alters the immune-related response of KCs to various innate and adaptive immune stimuli, resulting in impaired expression of IFN-stimulated genes, interferon regulatory transcription factor-induced genes and NFκB-induced genes37891011In this study, we analyse NFκB activation and subsequent cytokine/chemokine production following IFN-γ and TNF-α stimulation in uninfected and HPV-infected primary KCs. Our study reveals that RelA acetylation, needed for NFκB transcriptional activityin vivo as documented by genome amplification, late gene expression and virus production during the differentiation-dependent life cycle of HPV1819To evaluate whether the KCs immune response following the exposure to IFN-γ and/or TNF-α is attenuated by hrHPV, we utilized a system that resembles the natural infection with hrHPV as closely as possible. Primary KCs stably maintaining the hrHPV genome as episomes (hrHPV+KCs) display similar growth properties as non-transfected KCs, and upon culture in organotypic raft cultures, mimic HPV infection The presence of HPV type 16 (HPV16) was clearly associated with an impaired capacity to respond to IFN-γ and to TNF-α, as shown by the lower messenger RNA (mRNA) expression and production of the IFN-γ and/or TNF-α-induced pro-inflammatory cytokines CCL2, RANTES (CCL5), interleukin (IL)-8 and the chemokines CXCL9, 10 and 11 by KCs . Not onlThese data suggest that hrHPV, besides impairing the innate immune response of KCsThe hrHPV-mediated deregulated expression of STAT1 refs , 15, 16 HDAC1 to 11) or sirtuin (SIRT1 to 7) expression (CREBBP (KAT3A), confirming previous observations stating that HPV upregulates CREBBP to enhance expression from episomal DNA21IFRD1  PCAF (KAT2B), CREBBP (KAT3A), p300 (KAT3B) and TIP60 (KAT5), as well as the HDACs 1 and 3 ref. . Since opression . The onlA21IFRD1 , which pA21IFRD1  and HDACA21IFRD1 . We hypopression , left anpression , left wepression , IFRD1 mRD1 mRNA , middle RD1 mRNA , middle,RD1 mRNA , right aRD1 mRNA , right l1 mRNA 25.IFRD1 was used to lower IFRD1 protein expression, a concomitant increase in the steady-state levels of acetylated RelA K310 in HPV16+KCs was seen when compared with control knockdown HPV16+KCs   stimulation, previously shown by us to be impaired in hrHPV-infected KCsoly(I:C) .IFRD1 soon after infection, thereby effectively decreasing the basal levels of transcriptionally active RelA and as a consequence the levels of pro-inflammatory cytokines induced via various innate and adaptive immune-mediated NFκB stimulatory pathways.Thus, hrHPV upregulates the expression of Tis family genes, which also includes IFRD1, in rat neocortical astrocytes and chromaffin cell line PC12, mouse C243 and IEC-18, and mammary epithelial cellsEGFR mRNA expression , MEK1 (PD98059) and RAF (GW5074), but not PI3K (LY94002) or JNK (SP60025), resulted in decreased expression of IFRD1 . Thus EGCCL2, IL-8 and CXCL9, although the expression of RANTES was abrogated -associated HPV early antigen-specific T cells in the protection against progressive disease46All together, our data indicate that HPV upregulates EGFR to drive IFRD1 expression as a tool to decrease basal and adaptive-immune system-driven cytokine expression. This may allow hrHPV to evade the host’s immune response. It is highly likely that this mechanism plays a role in other viral infections too and even extends to tumours.The use of discarded human foreskin, cervical and vaginal KC tissues to develop cell lines for these studies was approved by the institutional review board at the Pennsylvania State University College of Medicine and by the institutional review board at Pinnacle Health Hospitals. The Medical Ethical Committee of the Leiden University Medical Center approved the human tissue sections  used for staining. All sections and cell lines were derived from discarded tissues and de-identified, therefore no informed consent was necessary.Primary cultures of human epithelial KCs were established from foreskin, vaginal, vulva and cervical tissues, as previously described19Primary basal layer human foreskin KCs were seeded at 75,000 cells per well in 24-well plates and allowed to attach for 48 h. Cells received fresh medium (mock infected) or medium containing native HPV16 isolated from raft cultures at multiplicity of infection 100 for 24 h. Cells were washed and harvested for either RT–qPCR or western blotting analysis.−1 and 10% glycerol) in Escherichia coli for propagation and downstream purification of the shRNA clones. pLKO.1 contains the puromycin selection marker for transient or stable transfection. The construct against IFRD1 (NM_001550) was TRCN0000156194: 5′-CCGGCAGTTCTGAAACAGTTTCTTTCTCGAGAAAGAAACTGTTTCAGAACTGTTTTT-3′, RelA (NM_021975) was TRCN0000014687: 5′-CCGGCCTGAGGCTATAACTCGCCTACTCGAGTAGGCGAGTTATAGCCTCAGGTTTTT-3′ and the control was: SHC004 (MISSION TRC2-pLKO puro TurboGFP shRNA control vector): 5′-CCGGCGTGATCTTCACCGACAAGATCTCGAGATCTTGTCGGTGAAGATCACGTTTTT-3′. HPV16-positive KCs at ~60% confluence were transduced with lentivirus at multiplicity of infection 5–10 overnight, after which the medium was replaced. At least 72 h post transduction, cells were stimulated as indicated and target gene expression was assayed by RT–qPCR or western blotting.shRNAs were obtained from the MISSION TRC-library of Sigma-Aldrich. The MISSION shRNA clones are sequence-verified shRNA lentiviral plasmids (pLKO.1-puro) provided as frozen bacterial glycerol stocks  was designed using siRNA Target Finder software . Blast search revealed that the designed E2 siRNA does not match with the known human transcriptome. E2 and negative control #2 siRNA (sequence not provided by the manufacturer) were purchased from Ambion. HPV16+KCs were transfected with 50 nM siRNA E2 or negative control #2 using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. Forty-eight hours post transfection, cells were harvested or stimulated as indicated and target gene expression was assayed by RT–qPCR or western blotting.Non-infected primary KCs were seeded at 50,000 cells per well in 24-well plates and allowed to attach overnight. Cells were transfected with 500 ng DNA using Lipofectamine (Invitrogen), according to the manufacturer’s instructions. Cells were maintained in E-medium. Seventy-two hours post transfection, cells were harvested and target gene expression was assayed by RT–qPCR.−1; Merck serono), rituximab , rapamycin , PD98059 , GW5074 , LY94002  or SP60025 . Medium was changed every 2–3 days. After at least 72 h, cells were stimulated as indicated and target gene expression was assayed by RT–qPCR or western blotting.Subconfluent cells were cultured in respective complete growth medium in the presence of cetuximab , vorinostat , trichostatin A  or sodium butyrate  in the respective complete growth medium overnight. Medium was changed for the respective complete growth medium, cells were stimulated as indicated and target gene expression was assayed by RT–qPCR or western blotting. Since treatment with 10 μM entinostat showed a good increase in RelA K310 acetylation without signs of toxicity, subsequent experiments were performed using this dose.(HPV-positive) KCs were stimulated as indicated for 24 h. Cleared (HPV-positive) KC supernatants were added to the lower compartment of a transwell plate (Corning). The upper compartment was filled with PBMCs isolated from buffy coats (Sanquin). PBMCs were allowed to migrate for 16 h, after which the cells in the lower compartment were counted by flow cytometry in the presence of counting beads (Invitrogen), according to the manufacturer’s instructions. Myeloid cells and lymphocytes were differentiated by their respective size in the Forward Scatter (FSC)/Side Scatter (SSC) plot (data not shown).http://r2.amc.nl).The microarray dataTotal RNA was isolated using the NucleoSpin RNA II kit  according to the manufacturer’s instructions. Total RNA (0.5–1.0 μg) was reverse transcribed using the SuperScript III First Strand synthesis system from Invitrogen. TaqMan PCR was performed using the TaqMan Universal PCR Master Mix and pre-designed, pre-optimized primers and probe mix for CCL2, RANTES (CCL5), IL-8 (CXCL8), CXCL9 and GAPDH . Threshold cycle numbers (Ct) were determined using the CFX PCR System , and the relative quantities of complementary DNA per sample were calculated using the ΔΔCt method using GAPDH as the calibrator gene.Enzyme-linked immunosorbent assays (ELISAs) for CCL2, RANTES, IL-8 and CXCL9 were performed according to the manufacturer’s instruction .Expression of EGFR on KCs was analysed by flow cytometry using phycoerythrin (PE)-coupled mouse-anti-human EGFR . Per live gate, 50,000 cells were recorded using the BD FACS Calibur with Cellquest software (BD Bioscience) and data were analysed using Flowjo .For western blotting, polypeptides were resolved by SDS–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad). Immunodetection was achieved with anti-p65 , anti-phospho-p65 ), anti-acetyl-p65 , anti-IFRD1 , β-actin  primary antibodies, and horseradish peroxidase-coupled anti-mouse  and horseradish peroxidase-coupled anti-rabbit  secondary antibodies. Chemoluminescence reagent (Bio-Rad) was used as a substrate and the signal was scanned using the Chemidoc and accompanying software (Bio-Rad) to quantify the intensity of the bands as a measure of the amount of protein of interest in the blot. The relative amount was determined by calculating the ratio of each protein over that of the density measured for the housekeeping protein β-actin. Images have been cropped for presentation. Full-size images are presented in 2O2/MeOH for 20 min. Antigen retrieval was performed in boiling EDTA buffer (pH 9.0) for 12 min. After 2 h of cooling down to room temperature, slides were washed twice in distilled water and twice in PBS. Subsequently, incubation was performed overnight at room temperature with the primary IFRD1 antibody ; p16  and E2 (1:50) (provided by Dr F. Thierry). Second, sections were incubated with BrightVision polyhorseradish peroxidase anti-mouse/rabbit/rat immunoglobulin-G  for 30 min at room temperature. Washing between incubations was performed three times for 5 min in PBS. Immune complexes were visualized by applying a 0.05-M tris-HCl buffer (pH 7.6) containing 0.05% of 3,3′-diamino-benzidine-tetrahydrochloride and 0.0018% of H2O2. After 10 min, the reaction was stopped by rinsing with demineralized water. Finally, the tissue sections were counterstained with Mayer’s haematoxylin before addition of a coverslip.Four μm formalin-fixed, paraffin-embedded tissue sections from two random vulvar intraepithelial neoplasia cases were deparaffinized and rehydrated using graded concentrations of ethanol to distilled water. Endogenous peroxidise activity was blocked with 0.03% HP values were determined using Welch-corrected unpaired t-tests. *P<0.05, **P<0.01 and ***P<0.001.Statistical analysis was performed using GraphPad InStat version 3.00. B.T., R.G., E.S.J., C.J.M.M., J.M.B. and S.H.v.d.B. designed the experiments. B.T., R.G., L.P.L.P. and E.S.J. performed the experiments. L.P.L.P., E.M.G.M.v.E. and C.M. made viruses and cells. B.T. and S.H.v.d.B. wrote the paper. C.J.M.M., J.M.B. and S.H.v.d.B. supervised the project. All authors discussed the data.How to cite this article: Tummers, B. et al. The interferon-related developmental regulator 1 is used by human papillomavirus to suppress NFκB activation. Nat. Commun. 6:6537 doi: 10.1038/ncomms7537 (2015).Supplementary Figures 1-6


Behçet’s disease (BD), a multi-organ inflammatory disorder, is associated with the presence of the human leukocyte antigen (HLA) HLA-B*51 allele in many ethnic groups. The possible antigen involvement of the major histocompatibility complex class I chain related gene A transmembrane (MICA-TM) nonapeptide (AAAAAIFVI) has been reported in BD symptomatic patients. This peptide has also been detected in HLA-A*26:01 positive patients. To investigate the link of BD with these two specific HLA alleles, molecular dynamics (MD) simulations were applied on the MICA-TM nonapeptide binding to the two BD-associated HLA alleles in comparison with the two non-BD-associated HLA alleles (B*35:01 and A*11:01). The MD simulations were applied on the four HLA/MICA-TM peptide complexes in aqueous solution. As a result, stabilization for the incoming MICA-TM was found to be predominantly contributed from van der Waals interactions. The P2/P3 residue close to the N-terminal and the P9 residue at the C-terminal of the MICA-TM nonapeptide served as the anchor for the peptide accommodated at the binding groove of the BD associated HLAs. The MM/PBSA free energy calculation predicted a stronger binding of the HLA/peptide complexes for the BD-associated HLA alleles than for the non-BD-associated ones, with a ranked binding strength of B*51:01 > B*35:01 and A*26:01 > A*11:01. Thus, the HLAs associated with BD pathogenesis expose the binding efficiency with the MICA-TM nonapeptide tighter than the non-associated HLA alleles. In addition, the residues 70, 73, 99, 146, 147 and 159 of the two BD-associated HLAs provided the conserved interaction for the MICA-TM peptide binding. Behçet’s disease (BD) has caused recurrent inflammation of symptom complex in genital and oral ulceration, inflammatory lesion of central nervous systems, gastrointestinal tract and eyes leading to blindness . This diα and IL-8 might play a role in the pathogenesis of this disease [The exact etiology of this disease has not been clarified yet, but it is probably mediated by a combination of excessive immune reactions, genetics factors , immune  disease . In addi disease , 11. Rec disease .Streptococcus, Mycoplasma and Helicobacter pylori) and viruses  [The notion of an environmental trigger of BD in patients with genetic susceptibilities has long been advocated. Several infectious agents have been investigated, especially bacteria (rus B19) . The autrus B19) .On the other hand, the genetic association of the major histocompatibility complex (MHC) class I chain related to gene A (MICA) with BD has been reported in various ethnic groups , 16, andß-strands and α1 and α2 helixes, while α3 and ß2-microglobulin (ß2m) are the floor and non-covalent supported units, respectively, as shown in The HLA molecules are human equivalents of the MHC found in most vertebrates . They arThe association of BD with the HLA-B*51:01 allele in various ethnic groups in Japan and Korea has been frequently observed , while tThe starting structures of three of the HLA class I alleles were taken from the Protein Data Bank, being entries 1E27  for B*51+ counterions . Afterwards, the individual complex was solvated by TIP3P [3. The periodic boundary condition with the NPT ensemble and a simulation time step of 2 fs was used. All energy minimizations and MD simulations were performed using the SANDER module of AMBER 10. All bonds and angles concerned to hydrogen atoms were constrained by algorithm of SHAKE [-1∙Å2 for 500 ps in each stage accordingly. These subsequent steps could allow the peptide to adapt its geometry and orientation from the initial model to fit better within the peptide binding groove. Then, the constraints were completely removed and fully unrestrained MD simulations were performed until 50 ns. The convergences of energies, temperature, and global root mean-square displacement (RMSD) were used to verify the stability of the systems. The MD trajectories were collected every 0.2 ps from the production phase for further analysis.The four studied HLA/MICA-TM complexes were investigated by molecular dynamics (MD) simulations using the AMBER 10 software package  with theby TIP3P  water moof SHAKE . The lonof SHAKE . All MD of SHAKE . Each syß2-microglobulin) and MICA-TM peptide, were plotted in To study the structural stability of the four MD simulations, the RMSDs for all atoms of the four HLA alleles  complexed with the MICA-TM peptide compared with those of the starting structures were monitored along 50 ns of simulation time using the PTRAJ module implemented in AMBER10 package . The RMSvice versa in blue. Among the four systems, the HLA-A*11:01 allele showed the highest degree of protein flexibility in the region of peptide binding groove and in particular at the S7–S9 sub-sites (defined in α1 or α2 domains and the intermolecular hydrogen bonds (H-bonds) between the C-terminal of the MICA-TM peptide and the binding residues of the three HLAs , which is discussed in a later section.The backbone flexibility of the HLA-B*51:01, B*35:01, A*26:01 and A*11:01 complexes with MICA-TM were investigated by B-factor calculation over the last 25 ns trajectories . Note thvice versa was calculated over 100 snapshots of the production phase. The obtained DC energies of the HLA and MICA-peptide residues are plotted in Figs α1 and α2 helixes with additionally some residues of β-strands (The decomposition (DC) energies were calculated and used to scan for potentially important residues for binding . In orde-strands . Using t-strands , respectVan der Waal (vdW) interactions were found to play an important role in the complex, where the magnitude of the MICA-TM peptide binding with the BD-associated HLA alleles (B*51:01 and A*26:01) was greater than that for the corresponding matched non-BD-associated HLA alleles (B*35:01 and A*11:01). For the nonapeptide, as expected the non-polar residues interacted with HLA through vdW interactions . Based o…A > 120°, as previously reported [To investigate the intermolecular H-bond interactions between the HLA protein and the incoming short MICA-TM nonapeptide, the number of H-bonds was evaluated using the two acceptance criteria of (i) a distance between the proton donor (D) and acceptor (A) atoms of D−A ≤ 3.5 Å; and (ii) an angle of D−Hreported , 51. Theα1 helix (I80 in HLA-B*51:01 and N80 in HLA-B*35:01). Through the strong H-bond (70% occupancy) with the amide group of N80 , whilst this end of the peptide had no interaction with any HLA-B*51:01 residues but instead the P3 residue was stabilized by two α1-helix residues, Y9 and N70, with ∼90 and ∼50% H-bond occupations, respectively  and the anchor P5 residue weakly interacted with the α1 helix residue T73 (~40%). The Y99 residue on the β-sheet of HLA-A*26:01 somewhat stabilized the P3 residue of MICA-TM (~40%), while the α1 helix residue N66 in HLA-A*11:01 supported the P2 and P3 residues (~40–50%). It is worthy to note that the high protein flexibility of HLA-A*11:01  in biomolecular systems [Gbind of the HLA alleles with the peptide bound at the binding groove was calculated from a summation of the total MM energy (ΔEMM), the solvation free energy (ΔGsol) and the entropic term (TΔS). The last term was estimated from the normal mode analysis [EMM components suggested that the short peptide was better stabilized by the non-BD-associated HLAs in the gas phase, the large destabilization from the solvation effect on the HLA/peptide complex led to a lower total ΔGbind. Rather, within each paired HLA-I gene (HLA-A or HLA-B), the MICA-TM peptide interaction was stronger with the BD-associated allele, where B*51:01  > B*35:01  and A*26:01  > A*11:01  in To determine the MICA-TM peptide binding strength towards the four studied HLA alleles, the molecular mechanic/Poisson-Boltzmann surface area (MM/PBSA) approach was applied on the same set of 100 snapshots taken from the production phase. The MM/PBSA approach has been extensively used to predict the overall Gibbs free energy  are given in G value < -2 kcal/mol in either HLA-B*51:01 or HLA-A*26:01  are most frequently detected in BD patients. Herein, we aimed to search for the selective correlation between the two BD-associated HLA alleles (HLA-A*26:01 and HLA-B*51:01) and the MICA-TM peptide in comparison with two class matched non-BD-associated HLA alleles (HLA-A*11:01 and HLA-B*35:01) by MD simulations. From the simulations, more contact residues at the binding groove of the BD-associated HLA alleles stabilized the incoming MICA-TM peptide than at the non-associated alleles of the same class . The vdW force was found to be the main protein-protein interaction, but in addition strong H-bonds (>70% occupation) were likely formed with the backbone of the nonpolar peptide, and these were stronger in the BD-associated HLA alleles. The P2/P3 and P9 residues  acted as the anchor for the peptide accommodation at the binding groove of the BD-associated HLAs. The total binding free energy of the HLA/peptide complex suggested a significantly stronger binding strength of the MICA-TM peptide with the BD-associated HLA alleles in the same class (B*51:01 > B*35:01 and A*26:01 > A*11:01). The residues 70, 73, 99, 146, 147 and 159 provided the conserved interaction of the two BD-associated HLAs with the MICA-TM peptide. All the structural, dynamics and energetics information somewhat explain the recognition and selective binding of the MICA-TM peptide towards the specific HLAs related to the BD pathogenesis.S1 Figß2-microglobulin and MICA-TM peptide in the (A) B*51:01, (B) B*35:01, (C) A*26:01 and (D) A*11:01 HLA alleles bound to the MICA-TM peptide.Root-mean square displacements (RMSDs) of all atoms relative to those of the initial structure for the HLA/MICA-TM complex, peptide binding groove, (TIF)Click here for additional data file.S2 FigThe MICA-TM peptide and HLA residues at the binding groove are shown in green and white sticks.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Synechocystis sp. PCC 6803 lacks the ability to catabolize wood sugars as an energy source. Here, we generated four Synechocystis strains that heterologously expressed XylAB enzymes, which mediate xylose catabolism, either in combination with or without one of three xylose transporters, namely XylE, GalP, or Glf. Except for glf, which is derived from the bacterium Zymomonas mobilis ZM4, the heterologous genes were sourced from Escherichia coli K-12. All of the recombinant strains were able to utilize xylose in the absence of catabolite repression. When xylose was the lone source of organic carbon, strains possessing the XylE and Glf transporters were most efficient in terms of dry biomass production and xylose consumption and the strain lacking a heterologous transporter was the least efficient. However, in the presence of a xylose-glucose mixed sugar source, the strains exhibited similar levels of growth and xylose consumption. This study demonstrates that various bacterial xylose transporters can boost xylose catabolism in transgenic Synechocystis strains, and paves the way for the sustainable production of bio-compounds and green fuels from lignocellulosic biomass.Wood sugars such as xylose can be used as an inexpensive carbon source for biotechnological applications. The model cyanobacterium  D-xylose begins with its transport into the cell by means of specific transporter proteins. Once inside the cell, xylose is isomerized by D-xylose isomerase (XylA) to yield D-xylulose, which in turn is irreversibly phosphorylated by the action of xylulokinase (XylB) to yield D-xylulose 5-phosphate, a pentose phosphate pathway (PPP) intermediate   .Few microorganisms are able to catabolize pentose sugars . Even in2 limit cyanobacterial growth and biosynthesis  strains heterologously expressing xylose-specific catabolic genes xylAB without or with one of three known xylose transporters . XylE (proton symporter), GalP (proton symporter), and Glf (uniporter) are members of the Major Facilitator Superfamily (hereafter MFS) class of transporter proteins  and TOP10 (Thermo Fisher Scientific) were used for DNA cloning and plasmid construction. The strains were grown at 37°C on solid LB medium or in liquid LB medium with shaking (220 rpm) in the presence of an appropriate antibiotic, if needed. Antibiotic concentrations used for selection were as follows: ampicillin (Fisher Scientific), 100 μg/ml for liquid and solid media; kanamycin (Fisher Scientific), 25 μg/ml for liquid and solid media; and spectinomycin (Fisher Scientific), 25 μg/ml for liquid medium and 50 μg/ml for solid medium.Zymomonas mobilis ZM4 was used as a source organism for amplification of xylose transporter-specific gene. The strain was grown at 28°C on solid YP medium or in liquid YP medium  with shaking (220 rpm).Synechocystis sp. PCC 6803, the kanamycin resistance cassette was spliced out from plasmid pUC4K (BamHI and inserted into pBluescript II SK+ (Stratagene) digested with BamHI. The neutral site sequence  (Synechocystis as upstream and downstream regions using the following primers:To construct the plasmids designed to insert xylose transporter genes into id pUC4K  using Ba site 1)  was amplKpnI-XhoI sites and SpeI-SacII sites of the modified pBluescript SK+ plasmid, respectively. Next, a ∼0.4-kb region encompassing the psbA2 promoter (hereafter psbA2 promoter) was amplified from Synechocystis genomic DNA using the following primers:5′-ACTCGGTACCGGCAATGCAATTAATTAAAAATGG-3′ (forward primer) and 5′-ACTCCTCGAGTCTATTGTTGGAAGGTTGCTG-3′ (reverse primer) for the upstream region; and 5′-ACTCACTAGTGTGAAAAAATATTGACATTAAGATATC-3′ (forward primer) and 5′-ACTCCCGCGGGGAACCAGATTTTTAGGATG-3′ (reverse primer) for the downstream region. The upstream and downstream fragments were inserted between the SalI-EcoRI sites of the plasmid. Then, the ∼0.5-kb 5ST1T2 double terminator region was amplified from the pBTac-1 plasmid (Boehringer Mannheim) using the following primers:5′-ACTCGTCGACGGTATATGGATCATAATTGTATGC-3′ (forward primer) and 5′-ACTCGAATTCTTGGTTATAATTCCTTATGTATTTGTC-3′ (reverse primer). The amplified fragment was inserted between the PstI-BamHI sites of the modified pBluescript II SK+ plasmid. Finally, the xylose transporter genes, xylE, galP, and glf, were amplified from genomic DNA isolated from E. coli K-12  or the Z. mobilis ZM4 strain (glf). The primers used to amplify the transporter genes were as follows:5′-ACTCCTGCAGCCAAGCTTGGCTGTTTTGG-3′ (forward primer) and 5′-ACTCGGATCCATTGAAGCATTTATCAGGGTTATTG-3′ (reverse primer). The fragment was inserted between the xylE; 5′-ACTCGAATTCATGCCTGACGCTAA-3′ (forward primer) and 5′-ACTCCTGCAGTTAATCGTGAGCG-3′ (reverse primer) for galP; and 5′-ACTCGAATTCATGAGTTCTGAAAGTAGT-3′ (forward primer) and 5′-ACTCCTGCAGCTACTTCTGGGAG-3′ (reverse primer) for glf.5′-ACTCGAATTCATGAATACCCAGTATAATTC-3′ (forward primer) and 5′-ACTCCTGCAGTTACAGCGTAGCAG-3′ (reverse primer) for EcoRI-PstI sites of the plasmid to generate three individual plasmids.The amplicons were inserted between the BamHI and inserted into the pBluescript II SK+ plasmid at the BamHI site. The neutral site sequence    was amplKpnI-SalI sites and SpeI-SacII sites of the plasmid, respectively. The psbA2 promoter and 5ST1T2 double terminator were amplified and cloned into the plasmid as described above. In the last step, the xylose catabolic genes xylAB were amplified from genomic DNA isolated from the E. coli K-12 strain using the following primers:5′-ACTCGGTACCATGACTATTCAATACACCC-3′ (forward primer) and 5′-ACTCGTCGACCACCTGCACCAGACCA-3′ (reverse primer) for the upstream region; and 5′-ACTCACTAGTTTGGGGCTGGCGGATT-3′ (forward primer) and 5′-ACTCCCGCGGCTAAGTCAGCGTAAATCTG-3′ (reverse primer) for the downstream region. The upstream and downstream fragments were inserted between the xylAB amplicon was inserted between the EcoRI-PstI sites of the plasmid.5′-ACTCGAATTCATGCAAGCCTATTTTGACCA-3′ (forward primer) and 5′-ACTCCCTGCAGGTTACGCCATTAATGG-3′ (reverse primer). The xylAB, only the region from the start codon of xylA to the end codon of xylB was amplified from the source organism; no other regulatory elements from the xylAB operon were included.Note that, in the case of Synechocystis strains were grown on solid or in liquid BG-11 medium at 30°C under 50 μE m-2s-1 light intensity with shaking (200 rpm). When OD730 reached ∼0.8, transformations were carried out as described , which was used as a positive control, was amplified using the following primers:Total RNA was isolated from 80-ml cultures of escribed . A TurboxylAB genes, the SuperScript One-Step RT-PCR system for long templates (Thermo Fisher Scientific) was used as per manufacturer’s instructions. PCR products were analyzed on a 0.8% agarose gel.5′-ACTCGAATTCATGAGAAACCCTGATACTTTGGGGCTGTGGACGAAAAC-3′ (forward primer) and 5′-ACTCCTGCAGCTAGAAATTAAGTTCGGCAGCTTGAACTTTTTCAATCTG-3′ (reverse primer). For a longer template, i.e., the Synechocystis strains (wild-type and strains possessing the xylAB genes) were grown under the conditions described above, until OD730 reached 0.6–0.8. Whole cell extracts were obtained and 20 μg protein samples were electrophoresed as described (escribed . After eSynechocystis strains (wild-type and strains harboring the xylAB genes) were grown as described above, until the OD730 reached ∼0.8. Then, 10 ml of the cells were washed thrice with BG-11 medium and OD730 was adjusted to 0.6. Next, 1 ml of the cultures was grown in the presence of 400 nM 14C-labelled xylose [D-(1-14C)]  in 10 ml snap-capped glass tubes under similar growth conditions for 1 h. Nine milliliters of BG-11 medium was added to the cultures and they were filtered through a 0.45 μm pore-size nitrocellulose membrane (Bio-Rad). The membranes were washed thrice with 10 ml BG-11 medium, air dried, and suspended in 10 ml scintillation fluid (PerkinElmer) for 24 h. Radioactivity was measured using an LS 6500 scintillation counter (Beckman).Synechocystis biomass was measured in terms of DW as previously described (Synechocystis strains were initiated in 100 ml BG-11 liquid medium at 30°C with shaking (200 rpm) and an initial OD730 of 0.05. The cultures were provided with: for autotrophic growth, 50 μE m-2 s-1 light; for mixotrophic growth, 5 mM (750 mg/L) xylose and/or 5 mM glucose (900.8 mg/L) along with 50 μE m-2 s-1 light; for light-activated heterotrophic growth (hereafter LAHG) conditions  to obtain cell-free media.D-Xylose Assay Kit (Megazyme) as per manufacturer’s instructions. The sequential action of xylose mutarotase and β-xylose dehydrogenase generates NADH molecules. The amount of NADH is stoichiometric with that of D-xylose and is calculated from the difference in OD340 values before and after β-xylose dehydrogenase action.The amount of xylose present in the filtered medium fractions was measured using a D-glucose and was calculated from OD510 values obtained for the samples relative to the OD510 value obtained for a standard.The amount of glucose present in the filtered medium fractions was measured using the D-Glucose Assay Kit-GOPOD Format (Megazyme) as per manufacturer’s instructions. Sequential action of glucose oxidase and peroxidase generates quinoneimine. The amount of dye formed is stoichiometric with that of Synechocystis strains during each time interval, using a previously described mathematical formula . The construction process involved two rounds of transformation. In the first round, one of the three xylose transporter genes, xylE or galP from E. coli K-12 or glf from Z. mobilis ZM4, was introduced into neutral site 1 . The plasmid constructs designed for integration of the transporter genes included the psbA2 promoter of Synechocystis origin, 5ST1T2 double terminator of E. coli origin, kanamycin resistance cassette, neutral site 1 divided into ∼600 bp upstream and ∼600 bp downstream regions, and one of the three aforementioned transporter genes .l site 1  in WT SyxylAB genes, which are responsible for funneling xylose into the PPP, were introduced into neutral site 2 . The plasmid construct designed to integrate the xylose catabolic genes included the same promoter and transcriptional terminator as used in the transporter gene-specific plasmid constructs, the spectinomycin resistance cassette, neutral site 2 divided into ∼970 bp upstream and ∼970 bp downstream regions, and xylAB genes . The xylAB genes consist of two individual genes, xylA (encoding D-xylose isomerase) and xylB (encoding D-xylulokinase), along with the ribosomal binding site (hereafter RBS) present between them.In the second round of transformation, the l site 2  in the tpsbA2 promoter to drive expression of both the transporter genes and the catabolic genes. psbA2 is a strong, light-sensitive promoter that enhances gene expression under high light conditions , which generated products of approximately 1.48, 1.40, and 1.42 kb in length, respectively . To verify segregation, the forward primer used to amplify the upstream region of neutral site 1 and the reverse primer used to amplify the downstream region of neutral site 1 were employed , and extension times in the PCR cycles were set specifically to amplify the wild-type (uninterrupted) neutral site 1. PCR amplifications yielded a ∼1.20-kb band representing neutral site 1 in the WT strain, but failed to generate any product for the transformants, indicating the absence of uninterrupted neutral site 1 and hence complete segregation of the strains .For the PCR analyses represented in xylAB in the strains obtained after the second round of transformation, i.e., X-Ut1, X-Ut2, X-Ut3, and X-Ut4, the same set of primers used to amplify the genes from genomic DNA of the source organism was employed , which generated products of approximately 2.85 kb in length . To verify the segregation with respect to neutral site 2, the forward primer used for amplification of the upstream region of neutral site 2 and the reverse primer used for amplification of the downstream region of neutral site 2 were employed , and extension times in the PCR were set specifically to amplify wild-type neutral site 2. PCR amplifications yielded a ∼1.93-kb band representing neutral site 2 in the WT strain, but failed to generate any product for the transformants, indicating the absence of uninterrupted neutral site 2 and hence complete segregation of the strains .To test for the presence of the catabolic genes petA (sll1317), which encodes apocytochrome f, a core subunit of the cytochrome 6fb complex, was tested. As a negative control for each strain, we examined whether the corresponding heterologous genes could be amplified from DNase-treated RNA samples obtained from each strain by PCR, to confirm the absence of any leftover DNA in the RNA samples.Once the segregation of the strains obtained after the second round of transformation was confirmed, transcription of the heterologous genes for the transporters and the catabolic genes in the X-Ut1, X-Ut2, X-Ut3, and X-Ut4 strains was examined by RT-PCR. For the expression studies, both a positive and negative control were included. For the positive control, expression of Figures 4A,B). These results indicate the successful transcription of all the heterologous genes and corroborate the quality of the RNA samples.When cDNA samples from the aforementioned strains were subjected to PCR using the same primer sets used to confirm gene insertion, products of the expected size were amplified. A ∼0.98-kb band was present for all positive controls and heterologous gene-specific bands were absent in the negative controls for all strains  against XylA and XylB proteins overexpressed in E. coli. The antibodies were used to detect the presence of the proteins in the total cell extracts obtained from the X-Ut1, X-Ut2, X-Ut3, and X-Ut4 strains, and the WT strain was used as the negative control. XylA protein, with a molecular weight of ∼44 kD . Expression of XylB showed that the RBS present between xylA and xylB of E. coli origin functioned in Synechocystis. Antibodies probing both of these proteins showed no cross-reaction with proteins present in the WT cell extract.To study the expression of the xylose catabolic genes, f ∼44 kD , was pref ∼44 kD , was detSynechocystis, we tested the ability of the X-Ut1, X-Ut2, X-Ut3, and X-Ut4 strain and also of the WT strain to take up D-[14C] xylose. At the end of a 1-h incubation period, the strains showed various degrees of radioactivity. The levels of radioactivity in X-Ut1, X-Ut2, X-Ut3, and X-Ut4 were ∼653%, ∼136%, ∼77%, and ∼100% greater than those observed in the WT strain . Thus, the transformants were better able to take up and use xylose than was the WT.To probe the activity of xylose transporter proteins in To study and compare the growth patterns exhibited by the strains generated after the first and second transformation events and the WT strain, we cultured the strains under different conditions . Biomass, i.e., DW, measurements were taken at 24-h intervals for 7 days in the case of dark and light-activated heterotrophic growth (LAHG) conditions and at 6-h intervals for 3 days for autotrophy and mixotrophy. In the absence of xylose, biomass yields were similar for all the strains. None of the strains were able to grow in darkness (data not shown). Under LAHG conditions in the presence of 5 mM glucose (hereafter LAHG-glucose conditions), at the end of the seventh day, the strains showed biomass yields of 0.329 ± 0.010 g/L. Under autotrophy and mixotrophy in the presence of 5 mM glucose (hereafter mixotrophy-glucose conditions), at the end of third day, the biomass yields obtained for all strains were 0.244 ± 0.015 g/L and 0.993 ± 0.013 g/L, respectively.Figure 7A). Under mixotrophy conditions in the presence of 5 mM xylose (hereafter mixotrophy-xylose conditions), the WT, X-Tr1, X-Tr2, and X-Tr3 strains showed biomass yields of 0.331 ± 0.014 g/L, which were similar to those obtained when grown under autotrophy, indicating the inability of the strains to utilize xylose. However, the X-Ut1, X-Ut2, X-Ut3, and X-Ut4 strains showed greater biomass yields under mixotrophy-xylose conditions than under autotrophy . Biomass accumulation results demonstrate that all four of the transformants carrying the catabolic genes, namely X-Ut1, X-Ut2, X-Ut3, and X-Ut4, utilize xylose. After 7 days of culture under LAHG-xylose conditions, the X-Ut1, X-Ut2, and X-Ut3 strains showed ∼62, 28, and ∼60% greater biomass yields than did X-Ut4, respectively . Under mixotrophy-xylose conditions, the strains carrying heterologous transporters showed higher biomass values at each time point. Notably, at the end of the second day of culture, X-Ut1, X-Ut2, and X-Ut3 strains showed ∼92, ∼34, and ∼94% greater biomass values than did the X-Ut4 strain . For both LAHG-xylose and mixotrophy-xylose conditions, biomass measurement data for X-Ut1, X-Ut2, X-Ut3, and X-Ut4 strains at each time point are presented in Supplementary Figures Under LAHG conditions in the presence of 5 mM xylose (hereafter LAHG-xylose conditions), only X-Ut1, X-Ut2, X-Ut3, and X-Ut4 strains exhibited growth . The absence of two distinct growth phases and the simultaneous uptake of both the sugars did not indicate diauxie in these strains (data not shown).To examine the ability of the xylose consuming strains to grow in the presence of xylose and glucose, we cultured the strains under LAHG and mixotrophic conditions in medium supplemented with 5 mM each of xylose and glucose (hereafter LAHG-mixed sugar and mixotrophy-mixed sugar conditions). The xylose consuming strains showed similar biomass yields under both conditions, in contrast to the variable yields exhibited in the presence of xylose alone . On the other hand, the X-Ut1, X-Ut2, X-Ut3, and X-Ut4 strains that possessed the xylAB genes showed varying degrees of xylose consumption under both growth conditions . After 7 days of culture under LAHG-xylose conditions, xylose consumption was ∼122, 55, and ∼131% greater in the X-Ut1, X-Ut2, and X-Ut3 strains, respectively, than in X-Ut4 . Under the same conditions, the maximum xylose uptake rates exhibited by the X-Ut1, X-Ut2, and X-Ut3 strains were ∼78, ∼45, and ∼80% higher than that exhibited by X-Ut4 . Under mixotrophy-xylose conditions, the strains carrying heterologous transporters showed higher xylose uptake than did the strain relying only on endogenous transporter/s at each time point. Notably, at the end of the second day of culture, the X-Ut1, X-Ut2, and X-Ut3 strains showed ∼205, ∼80, and ∼209% higher levels of xylose uptake than did X-Ut4 . Under the same conditions, the maximum xylose uptake rates exhibited by the X-Ut1, X-Ut2, and X-Ut3 strains were ∼64, ∼26, and ∼65% higher than that exhibited by X-Ut4 . For both LAHG-xylose and mixotrophy-xylose conditions, the sugar uptake data at each time point are presented in Supplementary Figures To measure xylose uptake by the strains generated after each transformation event and by the WT strain, we first cultured the strains under LAHG-xylose and mixotrophy-xylose conditions. The WT, X-Tr1, X-Tr2, and X-Tr3 strains that lacked the catabolic genes Figure 9B), in accordance with the biomass accumulation data. Under LAHG-mixed sugar conditions, all of the glucose was consumed by the end of the sixth day, while under mixotrophy-mixed sugar conditions, all of the glucose was consumed by the end of 48 h (data not shown). In the presence of mixed sugars, the maximum sugar uptake rates exhibited by the xylose consuming strains were very similar, in contrast to the results obtained when xylose was the only organic carbon source. Also, a sharp reduction in the maximum xylose uptake rates was observed when glucose was present along with xylose compared to when xylose was the sole sugar, with the exception of the X-Ut4 strain, which exhibited similar maximum xylose uptake rates under LAHG-xylose and LAHG-mixed sugar conditions , indicating the involvement of endogenous sugar transporter/s. This also indicates that the xylose uptake observed in strains possessing non-native transporters was due to activities of both endogenous and heterologous transporters. GlcP (sll0771) is the only known MFS-type glucose transporter identified in Synechocystis , indicating that the E. coli RBS functions in Synechocystis. Heterologously expressed E. coli xylAB and xylFGH genes, which encode xylose catabolic enzymes and an ATP-binding cassette (ABC)-type transporter and contain unmodified RBSs, were recently shown to be involved in xylose transport and catabolism in engineered Synechocystis . This indicates that, the growth exhibited by the strains expressing xylAB genes was the result of utilization of xylose and not due to photosynthetic electron transport. Synechocystis strain expressing xylAB genes with another strain expressing xylAB and transporter coding xylFGH genes in the presence of four different concentrations of xylose, under modified LAHG conditions wherein the cultures received daily illumination of 50 μE m-2 s-1 for 1 h. At the end of ninth day under modified LAHG conditions, the latter strain reached higher optical density values only at xylose concentrations of 10 and 50 mM . Thus, among the heterologous transporters studied, GalP was the least efficient. However, we were unable to determine which of the other two transporters, XylE and Glf, was more active, since a bottleneck may exist that influences the catabolic activities of XylAB or enzymes of the PPP of Synechocystis. We did not find any significant difference in the biomass yields of X-Ut1 and X-Ut3 strains grown in the presence of 10 and 20 mM xylose under mixotrophic conditions at the end of third day (data not shown). During preliminary experiments performed in our laboratory, we observed that the WT, X-Tr1, X-Tr2, and X-Tr3 strains that lacked the xylAB genes and hence the ability to catabolize xylose showed no difference in growth under mixotrophy-xylose (up to 20 mM xylose) and autotrophy conditions (data not shown). These observations indicate the absence of osmotic stress and the presence of an efficient eﬄux system in Synechocystis.Our results either directly or indirectly show that we successfully expressed the xylose catabolic genes hocystis . In thise., LAHG . For LAHnd 50 mM . We sougnsporter , we founFigures 7A,B, 8A–C and 10A,B), it showed the lowest radioactivity among the xylose utilizing strains in the D-[14C] xylose uptake study . This can be explained by the bidirectional transport ability of Glf , indicating that a lag phase exists before xylose metabolism is fully established.Although X-Ut3 was one of the two most efficient strains in terms of dry biomass accumulation and xylose uptake  knockout enhanced the production of keto acids, and half of the carbon in the keto acids could be traced to catabolized xylose (Synechocystis strains, in addition to the aforementioned strategies, would increase the amount of carbon available for biotechnological conversion. The work reported here represents an important step toward engineering a Synechocystis strain that is able to harvest carbon from the second most abundant sugar source in nature and use it to biosynthesize a range of useful compounds.The strains generated in this work, especially X-Ut1 and X-Ut3, can be used directly or modified further. Introducing the d xylose . An exced xylose . EfficieSR and QH conceived the research. SR, YZ, MK, and WM designed and performed the experiments. SR analysed the data. SR and QH prepared the manuscript. All the authors have read and approved of the manuscript.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.