P3 in cellular membranes. PI3K is a heterodimeric protein consisting of a regulatory and a catalytic subunit (p85 and p110 respectively). Heterologous expression of p110α in Saccharomyces cerevisiae leads to toxicity by conversion of essential PtdInsP2 into futile PtdInsP3, providing a humanized yeast model for functional studies on this pathway. Here, we report expression and functional characterization in yeast of all regulatory and catalytic human PI3K isoforms, and exploitation of the most suitable setting to functionally assay panels of tumor- and germ line-associated PI3K mutations, with indications to the limits of the system. The activity of p110α in yeast was not compromised by truncation of its N-terminal adaptor-binding domain (ABD) or inactivation of the Ras-binding domain (RBD). In contrast, a cluster of positively charged residues at the C2 domain was essential. Expression of a membrane-driven p65α oncogenic-truncated version of p85α, but not the full-length protein, led to enhanced activity of α, β, and δ p110 isoforms. Mutations impairing the inhibitory regulation exerted by the p85α iSH2 domain on the C2 domain of p110α yielded the latter non-responsive to negative regulation, thus reproducing this oncogenic mechanism in yeast. However, p85α germ line mutations associated with short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome did not increase PI3K activity in this model, supporting the idea that SHORT syndrome-associated p85α mutations operate through mechanisms different from the canonical disruption of inhibitory p85–p110 interactions typical of cancer.In higher eukaryotes, cell proliferation is regulated by class I phosphatidylinositol 3-kinase (PI3K), which transduces stimuli received from neighboring receptors by local generation of PtdIns P3 by phosphatidylinositol 3-kinase (PI3K) physiologically occurs in cellular membranes in response to the stimulation of growth factor receptors and other stimuli. PtdInsP3-responsive downstream effectors of PI3K include, sequentially, phosphoinositide-dependent kinase (PDK), Akt, and mTor protein kinases, involved in multiple functions related to transcriptional regulation and protein synthesis [P3-dependent signaling map in the phosphatase and tensin homolog deleted on chromosome ten (PTEN) phosphoinositide 3-phosphatase, a prominent tumor suppressor [PIK3R1  and PIK3CA , are commonly found in many tumor types [Synthesis of PtdIns3,4,5)ynthesis –3. Activ,4,5yntheppressor . Somaticor types –12. Suchor types ). Archetor types , which lor types , which ror types . AlthougSaccharomyces cerevisiae is an easily manipulatable and genetically tractable model organism for molecular studies on heterologously expressed proteins. We have previously developed a ‘humanized yeast’ model by heterologous expression of the mammalian PI3K catalytic subunit (p110α) artificially driven to yeast membranes by a C-terminal prenylation box [P3-dependent pathways, but expression of the mammalian class I PI3K severely depletes its substrate PtdInsP2 from the plasma membrane. The latter phosphoinositide is essential for polarized secretion and endocytosis in yeast, so its elimination by in vivo PI3K activity is reflected as growth inhibition [The application of the latest analytical technologies to cancer genomics, such as single cell whole exome sequencing performed on tumors by The Cancer Genome Atlas (TCGA) project and others, is revealing a plethora of mutations in these oncogenic pathways –19. Addrtion box , and thotion box –25. The hibition ,24.Here, we explore the applicability of this yeast setting to perform functional studies on PI3K by co-expression of its regulatory and catalytic subunits. We used a set of mutants to evaluate the aspects and define the limits of PI3K regulation that can be assayed in this heterologous model. We found that the key features related to oncogenesis, namely PI3K gain-of-function by p85-dependent recruitment to the plasma membrane, and disruption of inhibitory p85–p110 interactions can be traced in yeast. Moreover, p85α mutations associated with the non-oncological short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome –28 behavGAL1 promoter , p110α and β isoforms led to severe and mild, respectively, growth inhibition in yeast. This required their expression as membrane-directed proteins by attachment of an H-Ras C-terminal prenylation box (p110α–CAAX and p110β–CAAX), and was dependent on p110 catalytic activity  was involved in its interaction with the plasma membrane through electrostatic interactions . Therefoin vivo.In mammalian cells, interaction of p110 with the Ras oncoprotein via its RBD domain reinforces its recruitment to the plasma membrane thus contributing to PI3K activation . Yeast pin vivo. Hence, we generated a series of N-terminal-truncated versions of both p110α and β isoforms, as shown in The interaction between the regulatory subunit p85 iSH2 domain and the N-terminal adaptor-binding domain (ABD) of p110 facilitates interaction with the plasma membrane , triggerTo reconstruct in yeast a model that resembles mammalian PI3K activity and regulation, we extended our system by expressing PI3K regulatory subunits. For this purpose, we expressed HA-tagged versions of the p85α and β isoforms, in addition to a p65α oncogenic C-terminal truncation comprising amino acids 1–571 , its corS. cerevisiae lacks tyrosine phosphorylation as a prominent signaling event. Thus, it was expected that activation of p110 by p85-dependent recruitment was not naturally reproduced in yeast, imposing some limits to our analysis. Still, these results underscore the requirement of the recruitment of p110α and p110β to plasma membrane for its function. Since C-terminal prenylation is probably too strong as a membrane-directing signal for p110α to actually detect fine-tuning interactions with p85, we tested N-terminal myristoylation as an alternative p110α membrane-targeting strategy. As shown in Unlike higher cells, Finally, we introduced a CAAX prenylation signal on p85α and p65α, thus mimicking their membrane attachment by activated receptor tyrosine kinases (RTKs) in higher cells. We evaluated their effects on both plasma membrane-anchored p110 isoforms  and their corresponding soluble forms . Remarkably, p65α–CAAX enhanced the activity of all the class IA catalytic subunits tested  but not that of class IB (p110γ) D. In conThe above data show that the p65α–CAAX + p110α combination could be an appropriate setting to reproduce the positive effect of membrane recruitment of p110 by p85 in the absence of the inhibitory interaction imposed by full-length p85. Toxicity in yeast of the p65α–CAAX/p110α pair was dependent on its catalytic PI3K activity since (i) growth inhibition was counteracted by co-expression of PTEN A; (ii) iWe then combined p65α–CAAX with mutants in different p110α domains. Co-expression with the p110α ∆1-107–truncated mutant showed that p65α–CAAX requires the presence of the p110α ABD domain to exert activating interaction C. Also, Next, we used the p85α–CAAX to verify whether the negative regulation exerted by p85 on p110 in yeast involved inhibitory interaction between these two proteins. Indeed, the introduction of the oncogenic mutation E545K at the helical domain of p110α, which inverts the charge involved in electrostatic interactions with the nSH2 domain of p85α , releaseFinally, we tested whether the p110α H1047R mutant was sensitive to regulation by membrane-driven p85α. Interestingly, both p65α–CAAX and p85α–CAAX enhanced the activity of p110α H1047R D, suggesPIK3R1 (encoding p85α) mutations to be expressed as p85α–CAAX, including N564D and a series of selected p85α point mutations isolated in tumors, some previously assayed in vitro or in vivo in mammalian cells, but most of them with a yet unclear mechanism [The results above are suggestive that p85–p110 functional interactions can be traced in the yeast heterologous system. Thus, we chose a set of echanism ,12,36–38PIK3R1 mutations linked to the genetically inherited SHORT syndrome [PIK3R1 coding region mutations currently identified, as well as PIK3R1 mutations associated with colorectal cancer risk and with insulin resistance (HGMDR Human Gene Mutation Database)  B and D. atabase) B and D, atabase) B and D. atabase) C. OveralPIK3CA and PIK3R1 genes, coding for the p110α and p85α proteins, are prevalent in numerous tumor types as verified by deep sequencing analyses in the frame of projects such as TCGA [In physiological conditions in higher eukaryotic cells, receptors activated by tyrosine phosphorylation recruit the p85–p110 PI3K heterodimer to the plasma membrane. The interaction between pYXXM domains in receptor/adaptor proteins and p85 SH2 domains releases inhibitory interactions between such domains and the C2/helical domains in the p110 catalytic subunit. This allosteric regulation allows a conformational switch in the PI3K heterodimer, leading to full activation of the kinase in the proximity of its phosphoinositide substrate ,36,40,41 as TCGA –19. Hots as TCGA ,40,42,43S. cerevisiae crucial aspects of PI3K regulation is that it provides an environment free of all the complex layers of regulation present in higher cells, but yet in an in vivo context. Thus, it permits studies of structure–function relationship properties inherent to the expressed proteins in the absence of additional inputs. However, limits to the use of the yeast system include the apparent lack of sensitivity to interaction with Ras, as well as its dependence on protein production and stabilization. When assaying catalytic p110 subunits in yeast in this and previous studies [Here, we show that the key mechanistic events involved in pathologic PI3K hyperactivation, i.e. p85-driven plasma membrane recruitment and disruption of p85-imposed inhibition of p110, can be reproduced and traced in yeast. The key feature of reproducing in  studies ,21, we f studies ,30. HoweAmong all class I PI3K catalytic isoforms individually tested, the p110α isoform appeared more active than the p110β isoform in terms of yeast growth inhibition, while p110γ and p110δ did not interfere with yeast growth when expressed alone. This could be related to a lower basal activity of the latter isoforms, as well as to a more limited ability to efficiently attach to membranes in the absence of the regulatory subunit, because when the oncogenic p85 truncation p65α–CAAX was co-expressed, all compatible class IA catalytic subunits  were equally and strongly activated.By assaying p110 N-terminal truncations, we found that removal of the whole ABD domain does not eliminate intrinsic p110α and p110β PI3K activity, as long as the enzymes were artificially directed to the membrane. However, as expected, the ABD was necessary for p85-dependent activation, as p110α Δ1-107 did not respond to activation by p65α–CAAX. Nevertheless, these experiments are hampered by the difficulty in detecting by immunoblot the p110 N-terminal truncations, probably by compromised stabilization.The artificial introduction of a C-terminal prenylation CAAX box in PI3K regulatory subunits mimicked in yeast its recruitment by tyrosine-phosphorylated RTKs in mammalian cells, causing an up-regulation of p110 activity by p65α–CAAX, or its down-regulation in the case of p85α–CAAX on membrane-driven Myr–p110α or p110β–CAAX. These results favor the notion that the regulatory interplay between p85 and p110 subunits can be achieved in yeast. Accordingly, mutations affecting the negatively charged p110α loop spanning residues 541–546, which interact with the positively charged p85 nSH2 domain , such asInteresting mechanistic observations can also be inferred from the yeast model about performance of the most common p110α missense point mutation in clinical oncology, namely H1047R, mapping at the kinase domain ,42. FirsPIK3R1 found in tumors, is thought to disrupt the inhibitory interaction of the iSH2 domain in p85 with the C2 domain in p110 [PIK3R1, which preferably target the cSH2 p85α domain, displayed in our yeast system an ability to modulate p110α indistinguishable from that of the wild-type p85α protein, even when large C-terminal truncations eliminating most of the cSH2 were assayed. This supports the idea that these mutations may cause pathology by different molecular mechanisms than those involved in cancer. The mutation R649W, the most common p85α variant associated with SHORT syndrome, causes impaired PI3K signaling, probably by defective association with tyrosine-phosphorylated protein effectors, such as IRS-1 [PIK3R1.To validate the usefulness of our system on assessing the mechanism of putative pathogenic mutations in p85α, we chose a series of mutants described in the clinics. Among the mutations linked to cancer, two of them reproduced unequivocally the behavior pattern of p65, suggesting that they fully disrupted n/iSH2–helical/C2 inhibitory interactions. These were K379E, which introduce a negative charge at a key residue of the nSH2 domain ,49, as d in p110 ,30,45. T in p110  and Jais in p110  respecti in p110  or R574T in p110  did not as IRS-1 ,28,50. OS. cerevisiae YPH499 strain (MATa ade2-101 trp1-63 leu2-1 ura3-52 his3-Δ200 lys2-801). Growth conditions as well as serial dilution spot growth assays were described previously [MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YDR007w::kanMX4) by using the 15e compound (PI3Kα inhibitor II Echelon®) as described previously [All agar growth assays were performed with the eviously .E. coli and yeast and other basic molecular biology methods were carried out using standard methods. Plasmids pYES2 and pYES3/CT (Invitrogene), pYES2–GFP–Akt1, pYES2–GFP–Akt1 (K179M), pYES2–PTEN, pYES2–PTEN (C124S), YCpLG, YCpLG–p110α–CAAX, YCpLG–p110α (K802R)–CAAX, YCpLG–p110α, YCpLG–p110α (H1047R), YCpLG–p110α (E545K), YCpLG–p110β, and YCpLG–p110β–CAAX were already described [PIK3CA nucleotides from plasmids RCAS–Myr–p110α, RCAS–MM–p110α, and RCAS–FLAG–p110α [BamHI/XbaI sites at 5′ tails and then subcloned into plasmid YCpLG–p110α [PfuI DNA polymerase (Stratagene) and subsequent digestion with DpnI according to the QuikChangeTM (Stratagene) recommendations.Transformation of escribed ,21. YCpLAG–p110α  with priXbaI/SalI  or BamHI/SalI (p110α truncation plasmids) YCpLG linker sites the appropriate cDNAs, obtained from the corresponding pRK5 plasmids. The pRK5–Myc–p110 catalytic subunit and pRK5–Myc–p110 truncation plasmids will be described elsewhere, and were generated by cloning into pRK5 or pRK5–Myc the appropriate cDNAs, amplified by PCR using specific oligonucleotides and the corresponding plasmid templates: pCMVSPORT6 p110δ (mouse sequence) or pCMVSPORT6 p110γ (human sequence) , or YCpLG–p110α or YCpLG–p110β  [EcoRI/XhoI pYES3 linker sites the appropriate cDNAs with EcoRI/SalI flanking sites, obtained from the corresponding pRK5 plasmids. The pRK5–HA–PI3K-regulatory subunit plasmids will be described elsewhere, and were generated by cloning into pRK5 the appropriate cDNAs, amplified by PCR using specific oligonucleotides and the following plasmid templates: pCG–HA–p85α , or pOTB7–p85β (human sequence), pCMVSPORT6–p55α (mouse sequence), pCMVSPORT6–p55γ (human sequence), and pCMVSPORT6-p101 (human sequence) . Site-directed mutagenesis with the QuikChangeTM kit on pYES3–p85α–CAAX was performed in order to obtain the different p85α mutations. All the oligonucleotide sequences used in the present study are available upon request. When analyzing the sequences to verify the point mutations, we observed that the pCG–HA–p85α template used corresponded to the deposited sequence GenBank AAA39886.1, which bears a missense mutation as compared with the canonical NM_001077495.2 entry, involving a change of Glu510 to glycine (E510G). To verify that the presence of this amino acid change did not affect the validity of our results, we changed this residue back to glutamic acid by site-directed mutagenesis in our yeast expression vectors for HA–p65α and HA–p85α  and tested them for their influence on yeast growth in co-expression with different p110 constructs used in the present study. The results obtained were totally unaffected by the E510G substitution in any case (results not shown).Plasmids YCpLG–p110γ, YCpLG–p110γ–CAAX, YCpLG–p110δ, YCpLG–p110δ–CAAX, YCpLG–p110α(∆1-186), YCpLG–p110α(∆1-186)–CAAX, YCpLG–p110α(∆1-154)–CAAX, YCpLG–p110α(∆1-107), YCpLG–p110α(∆1-107)–CAAX, YCpLG–p110β(∆1-190), YCpLG–p110β(∆1-190-CAAX), YCpLG–p110β(∆1-117), and YCpLG–p110β(∆1-117–CAAX)  were constructed by subcloning into the myc-tag) . PlasmidWestern blotting assays were carried out by standard techniques. Anti-G6PDH antibody  was used as loading control. Monoclonal anti-HA antibodies  were used as primary antibodies to detect the expression of the HA-tagged regulatory PI3K subunits.


It was found that the contact was deformed via a plastic flow mechanism, which differs from the slip on lattice planes frequently observed in metals, and that the crystallinity became disordered. The various irregular relaxed structures formed during the deformation process affected the conductance.Various types of nanometer-sized structures have been applied to advanced functional and structural devices. Inherent structures, thermal stability, and properties of such nanostructures are emphasized when their size is decreased to several nanometers, especially, to several atoms. In this study, we observed the atomistic tensile deformation process of zirconium nanocontacts, which are typical nanostructures used in connection of nanometer-sized wires, transistors, and diodes, memory devices, and sensors, by  As the size of metals decreases to the nanometer scale, dislocation motion is suppressed, or even absent, while applying external forces. When the grain size of polycrystalline metals is decreased to 10–100 nm, grain boundary sliding and grain rotation govern deformation instead of dislocation-mediated slip1234568910111011915161718192021222324250  and 1.8G0. These peaks do not correspond to the positions of integral multiples of G0. Thus, the features of the conductance quantization are not observed272829303132330 variation in the duration of one imaging frame (66.7 ms). Since the total number of observed plateaus is 90, the ratio of the 0.5G0 plateaus accounts for 19%.0 plateaus. 0 plateaus. The number of 0.5G0 plateaus is 11 and the duration ranges from 70 to 2430 ms. Most of the plateau lengths of metallic NCs measured using mechanically controllable break junction methods and scanning tunneling microscopy are, at most, approximately 45 μs17272829353637We next focused on the contact structures corresponding to the 0.5G0 against the minimum cross-sectional width. The width ranges from one to five atom(s). The observation frequency decreases with the contact width. In particular, note that the ratio of one-atom-wide contacts accounts for 49% of all contacts.For the NCs with fcc structure, e.g., gold (Au), Ag, and palladium (Pd), and that with an hcp structure, e.g., Zn, the tensile deformation occurs via slip along primary slip systems, i.e., {111}– < 110 > and (0001)–[2The deformation region observed in this study was smaller than several atom width and as a result, plastic flow deformation, which is different from well-known deformation observed in bulk states, such as slip, shear band in glass, and phase transformation1234567911in situ transmission electron microscopy combined with piezomanipulation of specimens was developed394041915162021222325The atomistic behavior of deformation in metallic NCs have been investigated intensively by theoretical and computational methods in decades915161718192021222324252632464748In studies of NCs, the deformation including the variation in cross-sectional area has been discussed on the basis of the conductance measurements350 between times g and i. The conductance of the two peaks observed in the present study (0.5G0 and 1.8G0) are lower than this limit. Generally, peaks in conductance histograms imply the formation of stable structures. If deformation occurs via slip, the minimum cross-sectional area varies stepwise in a regular manner, which may lead to the emergence of periodic peaks in conductance histograms, e.g., features of the quantization of conductance that are observed in NCs of fcc metalsThe one- to five-atom-wide contacts before fracture were formed by plastic flow deformation. In 0 peak was the highest, as observed in 0 peak were observed, the deformation via slip was suppressed. The duration time of the 0.5G0 structures was recorded in a wide range (70–2430 ms)(The 0.5G–2430 ms) and the –2430 ms). Thus, wMetals that show the quantization of conductance are limited: s-electron metals, i.e., some of fcc structure noble metals, Au17202729313637385354554755565758596027283033556264−2/s, which is 102–103 times larger than those in the deformation tests of nanometer-grained polycrystalline metals2925The average strain rate in this experiment was 1.4 × 10It is reported that when homogeneous slip occurs in metallic NCs, the yield stress increases to several gigapascals, corresponding to the theoretical strength of metals10111041in situ TEM and investigated the relationship between their structures and conductance. We found that Zr contacts deformed in a plastic flow manner, which differed from slip mechanisms, when the contact size decreased to less than several atoms. Irregular variation in the minimum cross-sectional area and atomic configuration of the contact region was observed during plastic flow deformation. We inferred that plastic flow deformation contributed to the emergence of aperiodic broad peaks at 0.5G0 and 1.8G0 in the conductance histogram.We observed the tensile deformation process of Zr NCs by in situ TEM combined with a conductance measurement system using piezo-manipulation of two nanotips–5 Pa vacuum. We observed the structural variation using high-resolution TEM lattice imaging with a charge-coupled-device camera and measured the conductance using a two-terminal method. High-resolution imaging frames were obtained at intervals of 66.7 ms.We used How to cite this article: Yamada, K. and Kizuka, T. Transformation from slip to plastic flow deformation mechanism during tensile deformation of zirconium nanocontacts. Sci. Rep.7, 42901; doi: 10.1038/srep42901 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In this work, plastic deformation was locally introduced at room temperature by nanoindentation on a γ-TiAl-based alloy. Comprehensive analyses of microstructures were performed before and after deformation. In particular, the Burgers vectors, the line directions, and the mechanical twinning systems were studied via accurate electron channeling contrast imaging. Accommodation of the deformation are reported and a scenario is proposed. All features help to explain the poor ductility of the TiAl-based alloys at room temperature. Titanium aluminide alloys have attracted considerable attention due to their unique combination of properties, such as high specific strength and stiffness, good creep properties, and resistance against oxidation and corrosion ,2, whichOne of the main weaknesses of TiAl alloys is that they are brittle at Room Temperature (RT)—i.e., below their brittle-to-ductile transition temperature, which lies between 800 and 1000 °C . Despite2 as a minor phase. The α2 phase is hexagonal  observations on an electron transparent lamella ,13. The The fully dense Ti–46.8Al–1.7Cr–1.8Nb (at %) sample was obtained in the form of investment cast-bars  from Howmet. The as-received bars were hot isostatically pressed at 1250 °C and 125 MPa for 4 h, then subjected to a homogenization treatment in a furnace under vacuum at 1270 °C for 24 h . Then, tBecause deformation occurs mainly in the γ-phase , plasticDetailed characterizations of microstructures before and after deformation were performed by aECCI using a Zeiss Auriga Scanning Electron Microscope  operating at 10 kV. aECCI is a non-destructive method offering the ability to provide—inside a SEM—TEM-like diffraction contrast imaging of sub-surface defects (at a depth of about one hundred of nanometers) on centimetric bulk specimens. Defects such as dislocations can be characterized by applying the TEM extinction criteria ,16. BecaEBSD experiments were carried out on a Zeiss Supra 40 SEM  operating at 20 kV. In order to discriminate the different order variants of γ-TiAl, fine EBSD analyses were performed at a step of 75 nm with Channel 5 as the indexation software.Experimentally, the twin nature (true or pseudo-twin) was determined using high-resolution spot mode EBSD. Patterns were collected by manually pointing the electron beam at both sides of the Twin Boundary (TB). The corresponding EBSD patterns c,d clearThe evolution of ROI1—before and after deformation—is presented in g were accessible by tilting and rotating the specimen: g1 is shown in EBSD gave n system ,21. AfteROI2 is composed by two twinned grains A (left) and B (right) with their surface plane as vely see a. The co grain A a were anUnfortunately, good channeling conditions were not reachable in the right l alloys ,22.In addition, an NT contrast .At the location where B2 intercepts the TB, a stress concentration appeared. It resulted in a local distortion of the boundary. Therefore, the TB seems to be a strong obstacle to the propagation of the deformation, and at higher load it may cause microcracking at its vicinity, as observed in References ,25,26.From this knowledge, and taking into account our results, we propose the following scenario see c:Under Furthermore, we can suggest that the low load used (500 µN) was just high enough to generate a complex and non-uniaxial stress field at the tip of the indent. This led to the activation of the main deformation mechanism , but it was too low for dislocation glide. For higher loads, both mechanisms were activated subsequently, and led to the formation of a buckling area, according to the previous scenario. At RT, twinning was observed to be the main deformation mechanism.Twinning was accommodated by ordinary dislocation mechanism, leading to the canalization of the deformation.TB could play the role of obstacle to the propagation of deformation to neighbor grains, leading to a stress concentration at the vicinity of the boundary. Therefore, the true twin seems to be one of the weak links explaining the poor ductility of γ-TiAl at RT.In summary, RT nanoindentation tests combined with aECCI observations before and after deformation brought novel insights into the γ-TiAl deformation mechanisms:


Snake venom metalloproteases, in addition to their contribution to the digestion of the prey, affect various physiological functions by cleaving specific proteins. They exhibit their activities through activation of zymogens of coagulation factors, and precursors of integrins or receptors. Based on their structure–function relationships and mechanism of action, we have defined classification and nomenclature of functional sites of proteases. These metalloproteases are useful as research tools and in diagnosis and treatment of various thrombotic and hemostatic conditions. They also contribute to our understanding of molecular details in the activation of specific factors involved in coagulation, platelet aggregation and matrix biology. This review provides a ready reference for metalloproteases that interfere in blood coagulation, fibrinolysis and platelet aggregation. Snake venoms are cocktails of pharmacologically active proteins and peptides. They are used as offensive weapons in immobilizing, killing and digesting the preys ,2. Some 2+-dependent, endoproteolytic enzymes that are classified into three different classes: P-I, P-II and P-III [Snake venom metalloproteases (SVMPs) are Znnd P-III ,4. They nd P-III . SVMPs sBlood coagulation factors circulate as zymogens and they get activated through limited proteolytic cleavage during the breach of the blood vessel in a sequential manner leading to formation of fibrin clot that stops the blood leakage. All procoagulants from snake venoms characterized to date are proteases; they activate a zymogen of specific coagulation factors in the coagulation cascade and hasten clot formation. Unlike snake venom serine proteases, which activate various zymogens in the coagulation cascade , SVMPDaboia russelli) venom (RVV-X) is the well-characterized protein . Theyils, see ). As witils, see ,17. Bothing FXaν .N-terminal M domain interacting with C-terminal C domain  in addition to a solvent water molecule, which in turn is bound to the conserved Glu . The identity of fourth ligand as water is ascertained by quantum mechanical and molecular mechanical simulations [s) and the “arm” (Da)  at the N-terminal, and the “hand” (Ch) towards the C-terminal. The Cw subdomain extends from Ds and Da subdomains to form the C-shaped arm structure while the Ch subdomain forms a separated core of made of a unique α/β -fold structure  can be identified and may function in specific protein–protein interactions [Structural studies of RVV-X and other related P-III enzymes ,21,22,23C domain A. One ofD domain ,21,22,23D domain ,32,33,34D domain  enzymes ulations . The D drm” (Da) . The bouelements . The Da integrin  with somtructure . Within ractions .2+ [h/light chain portion may act as a scaffold to accommodate the elongated FX molecule. Ca2+ is likely to induce conformational changes in the Gla domain of FX, which might be necessary for the RVV-X recognition [P-IIId SVMPs is a subgroup that has additional subunits forming larger complexes. For example, RVV-X is a P-IIId complex  consisti2+ . A dockiognition , consistognition . RVV-X iA large number of snake species contain prothrombin activators in their venoms , true vipers (Bitis and Cerastes) and colubrids, Dispholidus typus . Altews, see ,60,63. Tews, see ,68,69,70ews, see ,72.A. rhodostoma venom or by plasmin do not show antiplatelet effects comparable to the protease [Naja nigricollis venom, showed that the degradation products of fibrinogen formed by this protease failed to inhibit platelet aggregation [Some α-fibrinogenases, described above, inhibit platelet aggregation ,74. Becaprotease . Thus, tregation . This SVregation . We specAgkistrodon rhodostoma  venom that inhibited platelet aggregation [2+ rise, completely blocked formation of thromboxane B2 and inositol phosphates in platelets stimulated by 0.1 U/mL of thrombin. In contrast, it inhibited significantly thromboxane but not inositol phosphates formation of platelets stimulated by a high concentration of thrombin (1 U/mL). They showed that incubation of platelets with kistomin resulted in a selective cleavage of platelet membrane glycoprotein Ib (GPIb) [2β1 and αIIbβ3. The release of 25- and 35-kDa fragments from GPVI suggests that kistomin cleaved GPVI near the mucin-like region. Hsu et al. identified that kistomin cleaves Glu205-Ala206 and Val218-Phe219 peptide bonds using synthetic peptides [Crotalus atrox that also cleaves vWF and GPIbα [In 1992, Huang et al. purified a P-I SVMP from regation . It inhiregation . This enb (GPIb) . These rb (GPIb) . Kistomib (GPIb) . Kistomib (GPIb) . Kistomib (GPIb) . It cleapeptides . Thus, Pnd GPIbα .Naja mocambique mocambique (=Naja mossabica) cleaves GPIbα [1-Glu282 was useful in identifying the thrombin-binding site; the sulfated tyrosine/anionic segment Y276DYYPEE282 are important for the binding of thrombin and the botrocetin-dependent binding of vWF [N. kaouthia (N. siamensis), N. nivea (N. flava), N. nigricollis crawshawii, N. nigricollis pallida, N. nigricollis nigricollis, N. atra, N. haje, N. naja, N. melanoleuca and N. oxiana, but not in N. sputatrix venoms [2+-agarose column and purified Nk from Naja kaouthia venom that cleaves GPIbα [2+-agarose column and NGF is co-purified with SVMPs [Mocarhagin, a 55-kDa SVMP from es GPIbα . The GPIg of vWF . Interesg of vWF . In bothx venoms . They ales GPIbα . During th SVMPs . They shNaja naja venom [Naja SVMPs, fails to inhibit ristocetin-induced platelet aggregation. Instead, it inhibits collagen-induced aggregation of washed platelets [2β1 mAb 6F1 suggested that NN-PF3 binds to α2β1 integrin in a sequence-dependent manner only but does not cleave α2β1 integrin. However, there is a drastic reduction in several intracellular signaling [Naja SVMPs.Interestingly, another distinct P-III SVMP, NN-PF3, that inhibits platelet aggregation was purified and characterized from ja venom . NN-PF3,latelets . Westernignaling . FurtherBothrops jararaca (Brazilian pit-viper) venom is a P-III SVMP with MDC domains [241TRKKHDNAQ249C* (*Cys residues form the disulfide bond) binds to α2I domain and interferes with the interaction between α2I domain and collagen. Using Ala scanning method, they identified the importance of RKK tripeptide sequence for this interaction [50 of 1.3 mM. These peptides bind near the metal ion-dependent adhesion site of the human integrin α2I-domain [2I but does not induce a large scale conformational rearrangement of the I domain [Jararhagin from  domains . The RGD domains . The cle domains . Jararha domains . The cle domains . Ivaska  domains . The peperaction . FinallyI-domain . The pepI domain .2β1 integrin, but not GPVI receptor [2β1 integrin on the platelet surface [2I domain [2β1 integrin [1 subunit of α2β1 by jararhagin results in the loss of pp72syk phosphorylation and thus β1 subunit appears to be critically involved in collagen-induced platelet signaling [2β1 integrin occurs by two independent motifs, which are located on D and C domains, respectively [In contrast, the inhibition of collagen-induced aggregation is driven by interference with the αreceptor . However surface ,97. The integrin ,98. The ignaling . Using rectively . The rolCrotalus atrox venom [Crotalus atrox venom [Crotalus viridis venom [Bothrops alternatus venom [Agkistrodon acutus venom [Gloydius halys venom [Naja kaouthia venom [2β1. Interestingly, acurhagin (87% identity with jararhagin) selectively inhibits platelet aggregation induced by collagen and suppresses tyrosine phosphorylation of several signaling proteins in convulxin-stimulated platelets [2β1, or cleaving platelet membrane glycoproteins [Bothrops barnetti venom that inhibits platelet aggregation induced by vWF plus ristocetin and collagen was characterized [2A domain of α2β1 integrin and thus, inhibits collagen-induced platelet aggregation [In addition to jararhagin (described above), several other P-III SVMPS, such as atrolysin A from ox venom , catrocoox venom , crovidiis venom , alternaus venom , acurhagus venom , halydinys venom  and kaouia venom  inhibit latelets . Thus, aproteins . Recentlcterized . It presregation . DespiteTrimeresurus albolabris venom activates platelets through a mechanism involving GPVI [Crotalus horridus horridus venom induce platelet aggregation [A small number of SVMPs have been shown to induce platelet aggregation. Alborhagin, a P-III SVMP isolated from ing GPVI . It induing GPVI  to convuing GPVI ,110,111.regation . They inregation .2+-dependent proteolytic activity leads to either destruction of the receptor or release of new ligands. Thus, M domain plays critical role in most of the pharmacological activities exhibited by SVMPs. However, in a significant number of instances, SVMPs exhibit their functions by non-enzymatic mechanisms through selective binding to key proteins. In such cases, non-enzymatic domains, such as D and C domains, as well as non-enzymatic subunits, such as snaclecs, play important roles. At times, these domains are proteolytically “processed” and exhibit independent pharmacological effects [In snake venoms, three distinct classes of SVMPs, P-I, P-II and P-III, are produced ,4. These effects ,4,113. IMacrovipera labetina transmediterranea venom inhibits platelet aggregation induced by thrombin and arachidonic acid with IC50 of 40 and 50 nM, respectively [vβ3 and, to a lesser extent, with αvβ6 and αvβ1 integrins. It does not bind to α2β1 integrin. These studies support the importance of DC domains in the inhibition of platelet aggregation through non-enzymatic mechanisms. Structure–function relationships of these DC domains will help in determining the integrin selectivity and binding.As mentioned above, precursor of SVMPs are “processed” into various proteolytic products ,114. Accectively . It inhiIIbβ3 [IIbβ3, αvβ3 and α5β1 integrins [IIbβ3 integrin with a high degree of selectivity [As with DC domains, “processed” D domains were also isolated as disintegrins from crotalid and viperid venoms. Disintegrins are among the potent inhibitors of platelet aggregation peptides ,123,124.IIbβ3 ,128. TheIIbβ3 . Disintentegrins , while Kectivity . This RGectivity  and refeectivity ,136,137.In significant number of P-III SVMPs, RGD sequence is replaced by various tripeptide sequences  and at 2β1 integrin on the platelet surface. Using overlapping peptides from C domains of atrolysin A and jararhagin, Kamiguti et al. identified two peptides each corresponding to identical segments [2β1 integrin and not through GPVI. VKC-jararaca, but not VKC-atrox, induced a rapidly reversible weak aggregation [365PCAPEDVKCG374 and 372KCGRLYCK379 in C domain of jararhagin which could bind to vWF [Thus far, polypeptides containing only C domain have not been isolated from snake venoms. Therefore, C domains are recombinantly expressed to evaluate their role in platelet functions. C domain of atrolysin A potently inhibits collagen- but not ADP-stimulated platelet aggregation . These ssegments . These pregation . Pinto ed to vWF . They rud to vWF . The intd to vWF ,146. Simd to vWF . These s2+-dependent manner and provide excellent selectivity. Thus, these non-enzymatic subunits impart to distinct properties. Correctly modified and folded Gla domain is important for optimal activity. It defines the Ca2+-dependence, as Ca2+ ions are required for proper folding of Gla domain. Carinactivase-1 and multactivase fail to activate prethrombin-1 and descarboxyprothrombin in which Ca2+-binding has been perturbed. On the other hand, Ecarin, which does not have this subunit, activates prothrombin, prethrombin-1 and descarboxyprothrombin with equal efficiency. This functional difference helps in measuring normal prothrombin versus descarboxyprothrombin in the plasma of warfarin-treated individuals [Thus far, only snaclecs are found to be associated with P-III SVMPs ,12,47,58ividuals . Thus, tProteases recognize and interact with specific substrates by binding them through various functional residues distributed among different sites. Each of these sites plays a specific role in the overall function of the enzyme. Our understanding of the chemical and biophysical interactions of various substrates with their respective enzymes has helped us to define these sites. Based on the interactions of SVMPs with various substrates, receptors and integrins (discussed above), we would like to propose new definitions of additional functional sites. We will also provide distinguishing features of these new sites in comparison with established functional sites.2+ coordinated by three conserved His side chains and a water anchored to a conserved Glu [It is the region where substrate molecules bind (binding site) and undergo a chemical reaction . Binding site correctly orients the substrate for catalysis, while residues in the catalytic site play mechanistic role in lowering the activation energy to make the reaction proceed faster. Specific amino acid residues, cofactors and/or ions play critical roles in the catalytic mechanisms in protein enzymes. For example, each residue in the catalytic triad  plays a role in catalysis in serine proteases. The Acid–Base–Nucleophile triad generates a nucleophilic residue for covalent catalysis . The resrved Glu ,25. ThisSubstrate binding site can be quite elaborate and complex; higher the complexity better is the substrate selectivity. The substrate binding site is divided into several subsites—the regions, which are on the enzyme surface that interact with individual amino acid residues on either side of the substrate cleavage site. The subsites on the amino side of the cleavage site are labeled as S1, S2, S3, etc. (non-prime subsites), while those on the carboxyl side are labeled as S1′, S2′, S3′, etc. (prime subsites). Generally, these are discontinuous sites and thus, the residues forming these subsites are not contiguous in the protein sequence. P1 amino acid residue of the substrate associates with S1, P2 with S2, etc. Similarly, P1′ amino acid residue binds to S1′, P2′ with S2′, etc. P1-P1′ peptide bond of the substrate is proteolytically cleaved. Both non-prime and prime subsites could contribute to substrate selectivity and affinity. Paes Leme et al.  determin241TRKKHD246C* (as numbered in jararhagin) that interacts with human integrin α2I-domain [This is a secondary binding site, remote from the active site, on the enzyme. Exosites provide additional substrate (or inhibitor) selectivity. For example, thrombin (a serine protease) has two distinct electropositive surface regions, exosite I and exosite II, that contribute to the specificity of thrombin ,151. TheI-domain ,94,95. BI-domain ,157,158.Small regulatory molecules interact with this site on the enzyme to activate or inhibit  the specific enzyme. In general, the non-covalent and reversible interaction of the allosteric effector often results in a conformational change. In homotropic allosterism, the modulator molecule is the substrate as well as the regulatory molecule for the target enzyme. It is typically an activator of the enzyme. In contrast, in heterotropic allosterism, modulator is not the enzyme’s substrate. In this case, the modulator may be either an activator or an inhibitor. Although multimeric proteins  are considered to be prone to allosteric regulation, even monomeric proteins  exhibit heterotropic allosterism ,160,161.Both exosite and allosteric site are on the surface of the enzyme or receptor. In the case of exosite, one part of the substrate or inhibitor interacts with the exosite while the other part interacts with the active site. Thus, exosite typically must be occupied first for optimal activity. In contrast, a substrate molecule  or a ligand  interacts with the allosteric site and a second substrate molecule interacts with the active site. The binding at the allosteric site enhances or decreases the binding or catalysis at the active site. Thus far, no allosterism has been documented in SVMPs.241TRKKHD246C* exosite of SVMPs [Exosites and/or allosteric sites can be present in the same domain as the orthosteric site, such as active site (in enzymes) or agonist binding site (in receptors). These sites are thus closer to the orthosteric site and located on the enzymatic M domain and we name them as “p-exosite”  and “p-allosteric site”  A,B. The of SVMPs ,94,95. IEnzyme complexes, such as RVV-X, carinactivase-1 and multactivase ,47,58, aDuring our analyses of the literature, we found two interesting, somewhat unusual behaviors of SVMPs. We have highlighted these observations as they will be useful in future research strategies in the field of SVMPs as well as other proteases.125I-jararhagin to determine specific binding to platelets [125I-jararhagin to platelets. The inactive jararhagin showed excellent specific binding to platelets  time is one the earliest one-step clotting time . Russellthrombin . Since cthrombin ,180.IIbβ3), vitronectin receptors (αvβ3) and fibronectin receptor (α5β1). Disintegrins with MLD motif are heterodimeric disintegrins and bind to α4β1, α4β7 and α9β1 integrins. When their second subunit contains RGD, they bind to α5β1 integrin [1β1 integrin [2β1 integrin through its RSECD sequence located in the D domain [vβ3 integrin [1β1 integrin and integrin-linked kinase modulate angiotensin II effects in vascular smooth muscle cells and thus, are potential targets to the development of more effective therapeutic interventions in cardiovascular diseases [SVMPs and their domains have also significantly contributed as research tools and also in the development of therapeutic leads. Although classical snake venom D and DC domains are proteolytically released from PII and PIII SVMPs ,113,114,integrin . Disinteintegrin ,188,189.integrin ,190,191.D domain . Leberagintegrin . Howeverintegrin ,137,190 integrin , fibroblintegrin ,194, chointegrin , osteoblintegrin , and neuintegrin . Recent diseases ,199. ThuIIbβ3 integrin and are approved for the treatment of acute coronary ischemic disease and prevention of thrombotic complication in balloon angioplasty and stenting [Agkistrodon contortrix contortrix venom, exhibits potent antiangiogenic effects in in vitro and in vivo models [1β1/α2β1 integrins are being developed as potent antagonists of angiogenesis for the treatment of angiogenesis disorders and cancer [2β1 integrin are being developed as another class of inhibitors of platelet aggregation for antithrombotic therapy [The high specificity, selectivity and affinity of D domains have helped the scientific community to design potent therapeutic agents for various human diseases. For example, RGD-disintegrins resulted in the successful design of two therapeutic drugs that inhibit αstenting ,201. Intstenting  and echistenting , respecto models ,203,204.o models . A chimeo models . Based od cancer , whereas therapy . Thus, r therapy ,209,210.Agkistrodon contortrix contortrix venom, fibrolase and its recombinant analog, alfimeprase was developed as a clot-buster drug for myocardial infarction and stroke due to its thrombolytic properties [2 receptor antagonist) were shown to improve reperfusion after thrombolysis with alfimeprase but not tissue plasminogen activator [SVMPs and their catalytically active M domains are also important in the development of therapeutic agents. A direct fibrinolytic enzyme from operties ,212,213.operties ,215, butoperties . Despitectivator . A carefSVMPs, and their domains and complexes have evolved to bind to various integrins, receptors and extracellular matrix proteins. They activate or inactivate proteins through enzymatic or non-enzymatic mechanisms and interfere in blood coagulation and platelet aggregation, and contribute to venom toxicity, particularly to hemorrhagic activity and venom distribution in the prey or victim. The understanding of their structure–function relationships and mechanism of action has contributed significantly to basic sciences including protein chemistry, enzymology, hematology, angiogenesis and cancer biology, and also helped in the development of diagnostic and therapeutic agents. Further studies on this group of toxins will contribute to unlocking several complex physiological processes and pathological effects in blood coagulation, platelet aggregation, hemorrhage, matrix biology, angiogenesis and cancer biology. Their structure–function studies will enhance the potential in developing novel diagnostic and therapeutic agents.


The structure of the putative membrane-binding tandem PX-PH domain module of the yeast protein Bem3 is reported. Saccharomyces cerevisiae (strain S288c) has been determined to a resolution of 2.2 Å . It shows that the domains adopt a relative orientation that enables them to simultaneously bind to a membrane and suggests possible cooperativity in membrane binding.The structure of the tandem lipid-binding PX and pleckstrin-homology (PH) domains of the Cdc42 GTPase-activating protein Bem3 from  However, only a minor fraction of these domains show high affinity and specificity for one PIP, whereas other PH domains either do not recognize PIPs or interact very weakly and the functional significance of their interactions is not clear  domain, which directly follows the PX domain within the primary sequence of Bem3 and consists of approximately 100 amino acids  and the primers listed in Table 1Tobacco etch virus (TEV) protease cleavage site. For phasing and unambiguous assignment of the amino-acid sequence in the structure, point mutations were introduced by site-directed mutagenesis at positions 680 (K680M) and 688 (L688M) to incorporate l-selenomethionine (SeMet).Bem3 constructs encompassing the regions containing amino acids 500–765 or 500–750 (comprising the PX and PH domains of Bem3) were amplified from yeast genomic DNA (strain S288c) using 2.2.Escherichia coli BL21-CodonPlus (DE3)-RIL cells were transformed with the corresponding plasmid and allowed to grow at 37°C until the OD600 reached approximately 0.60–0.65. Expression was induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM and growth was continued overnight at 20°C. The SeMet-labelled mutants were expressed using conditions described elsewhere  glycerol, 2 mM β-mercaptoethanol (β-ME), 1 mM PMSF and a protease-inhibitor cocktail including 50 µg ml−1 chymostatin, leupeptin, antipain and pepstatin (Sigma)] and disrupted in a fluidizer (Microfluidics). The lysate was incubated with 1% CHAPS for 30 min. Insoluble material was removed by centrifugation at 50 000g for 50 min at 10°C and the supernatant was applied onto an Ni2+–NTA HisTrap column pre-equilibrated with buffer A . After extensive washing of the column, His-tagged proteins were eluted with a linear gradient of buffer B (buffer A supplemented with 500 mM imidazole). The polyhistidine tag was cleaved overnight at 4°C using 0.06 mg TEV protease per milligram of recombinant protein during dialysis . After a second Ni2+–NTA affinity purification to remove uncleaved protein and TEV protease, final purification was achieved by size-exclusion chromatography  in the final storage buffer .The cells were harvested by centrifugation, resuspended in cell-lysis buffer  obtained from the initial screen was further improved by varying the pH and the concentration of PEG. The best diffracting crystals of wild-type Bem3 and the SeMet derivative grew in the form of hexagonal rods within 1 d. The final crystallization conditions are summarized in Table 2To determine the initial crystallization conditions, Bem3 was subjected to crystallization trials at 293 K by the sitting-drop vapour-diffusion method using the following commercially available protein crystallization screens: The PACT Suite, The PEGs Suite and The JCSG Core I–IV Suites . The crystallization drops were prepared by mixing 100 nl protein solution with 100 nl reservoir solution with the help of a TTP Labtech Mosquito LCP crystal liquid-handling robot and were incubated against 70 µl mother liquor in 96-well plates. The best condition [0.1 2.4.v/v) glycerol and flash-cooled in liquid nitrogen prior to data collection. The Bem3 crystals were maintained at 100 K and the collection of X-ray diffraction data was performed on beamline X10SA at the Swiss Light Source (SLS), Paul Scherrer Institute, Villigen, Switzerland. The data were indexed and integrated with XDS and scaled with XSCALE  with phenix.autosol  and was followed by automated model building in phenix.autobuild   and β6PH and β7PH (amino acids 709–715) within the PH domain were flexible and could not be resolved. Even though six amino acids are missing between the two domains, the presented structural assembly within the asymmetric unit represents the only possible arrangement, and a different connectivity between the PX and PH domains is excluded because the positions of the respective C- and N-termini are too remote. Using the native data set only, the sequence could not be unambiguously assigned within parts of the PH domain because of a lack of electron density for the side chains .The crystal structure of the tandem PX-PH domains shows the well characterized folds of both protein domains as reported previously  revealed that the corresponding Arg478 interacts with PIPs  showed that this abolished binding of the PH domain to PIP2, rendering the protein incapable of correctly localizing to the polar site at the cell membrane  and this part of the protein displays a large positively charged basic surface . The similar orientation thus suggests a possible synergy between the domains with regard to the recognition of PIPs within a membrane, both with respect to the affinity of binding and the possibility of the recognition of a distinct membrane or compartment within the cell by the relative abundance of PIPs.Interestingly, the putative membrane-binding sites of both domains (basic motifs I and II within the PX domain and the PIP-binding site within the PH domain) are oriented in a similar direction Fig. 1a and thce Fig. 2b. The s2 between the PX and PH domains , resulting in average B factors of 71 and 96 Å2 for the PX and PH domains, respectively. The elevated B factors of the PH domain can be understood by considering the different crystal contacts. Whereas the PX domain has large contact areas (∼1210 Å2) with symmetry-related molecules, the PH domain has only small contact areas , thus allowing the increased mobility. While we can only speculate on the possible role of this flexibility, it is conceivable that a less-than-rigid relative orientation of the two lipid-binding domains might allow different membrane curvatures in different specific situations.The interface of ∼620 Åin Fig. 2a, resul4.In this publication, we present the three-dimensional structure of the tandem PX-PH domains of the yeast protein Bem3. The structure shows that the putative lipid-binding domains adopt a relative orientation that enables them to simultaneously bind to a membrane. Therefore, future studies should address the possibility of cooperativity between the domains with regard to membrane interaction as well as the resulting specificity with respect to the relative abundances of PIPs in a target membrane.S. cerevisiae, 6fsftandem PX-PH domains of Bem3 from PDB reference:


Together, these results reveal a previously unrecognized key function for p35/CDK5 as a mediator of mesenchymal cell fibrotic responses. The results suggest a potential pathogenic role for elevated p35 expression and CDK5 activity in SSc, and raise the possibility that their selective pharmacological targeting might represent a novel treatment approach in fibrosis.The mechanisms underlying persistent fibroblast activation and myofibroblast phenoconversion in underlying multi-organ fibrosis in systemic sclerosis (SSc) remain incompletely understood, hindering effective therapies to slow or reverse the process. Cyclin-dependent kinase 5 (CDK5) is a pleiotropic member of the CDK family originally identified in neuronal cells. In contrast to other CDKs, CDK5 activity depends on its CDK5R1 subunit p35. Here we demonstrate that expression of p35 and CDK5 activity are induced by TGF-ß in fibroblasts and adipocytic cell types. Levels of p35 are markedly elevated in both SSc skin biopsies and explanted SSc fibroblasts, as well as in fibrotic skin in mice. Ectopic p35 and CDK5 suppressed adipogenic markers while stimulating collagen production and myofibroblast markers, whereas RNAi-mediated CDK5 knockdown abrogated TGF-β fibrotic responses in a Smad-independent manner. Pharmacological inhibitors of CDK5 likewise prevented and reversed TGF-β responses in fibroblast monolayers and in  Systemic sclerosis (SSc) is associated with multi-organ fibrosis . Skin fiIn vitro, TGF-ß treatment up-regulated p35 expression and CDK5 activity in human and mouse skin fibroblasts, mesenchymal progenitor cells and adipocytes. In adipocytes, ectopic expression of p35 and CDK5 caused suppression of adiponectin synthesis while stimulating collagen. In fibroblasts, ectopic p35 elicited fibrotic responses whereas p35/CDK5 silencing abrogated stimulation of these responses by TGF-β. Moreover, pharmacological inhibitors of CDK5 prevented, as well as reversed, stimulation of collagen synthesis and myofibroblast differentiation in fibroblast monolayer cultures and in ex vivo skin organ cultures, ameliorated collagen overproduction in SSc fibroblasts, and prevented and reversed skin fibrosis in distinct in vivo models of SSc. These results suggest, for the first time, that deregulated CDK5/p35 signaling may have a pathogenic role in SSc fibrosis, and identify the TGF-ß-CDK5 axis as a potential novel target for therapy.Cyclin-dependent kinase 5 (CDK5), a conserved member of the CDK family expressed principally in the central nervous system, is a pleiotropic non-receptor serine-threonine kinase implicated in angiogenesis, apoptosis, cell migration and senescence . In lighn = 6) and healthy controls (n = 3) were examined. Results of qPCR indicated that p35 mRNA levels were markedly elevated in SSc fibroblasts  also showed increased p35 protein levels and p35 mRNA expression compared to PBS-treated control mice (n = 3–4)  Figure .in situ  vectors  using Lison, WI) . pRL-TK ca). In the first protocol, six- to eight-week-old female C57BL/6J mice  were given s.c. bleomycin (10 mg/kg) daily for 14 consecutive days, and sacrificed on day 28. In the second protocol, replication-deficient type 5 adenovirus encoding TβRIca or LacZ (6.7 × 107 pfu/mouse) was injected i.c. on two occasions separated by four weeks, and mice were sacrificed four weeks following the second injection [Animal protocols were institutionally approved by the Animal Care and Use Committee of Northwestern University or the University of Erlangen-Nuremberg. Skin fibrosis was induced by two distinct approaches: subcutaneous (s.c.) bleomycin, or intracutaneous (i.c.) adenovirus expressing constitutively-active mutant Type I TGF-β receptor (TβRInjection . In bothnjection .® 594-conjugated secondary antibodies (Invitrogen) for 60 min. Nuclei were identified by 4›-6-diamidino-2-phenylindole (DAPI) staining. Non-immune IgG was used as a negative control in each experiment. Following stringent washing, slides were examined under a Zeiss UV Meta 510 confocal microscope . Each experiment was repeated at least three times with consistent results.Fibroblasts were pretreated with roscovitine (5 μM) for 15 min, followed by incubation in media with 10 ng/ml TGF-β2. Twenty-four h later, cells were fixed in 4% paraformaldehyde, washed in phosphate buffered saline (PBS), and incubated with primary antibodies against αSMA  or Type I collagen  for 120 min, followed by AlexaConsecutive 4-μm serial sections of paraffin-embedded mouse skin were stained with hematoxylin and eosin (H&E) or Trichrome . Dermal t-test. p < 0.05 was considered statistically significant. In the animal studies, differences between the groups were tested for their statistical significance by non-parametric Mann–Whitney U test.Data are presented as means ± SD. Significance of differences between experimental and control groups was determined by Student's


Wormcam imagery provided a vivid depiction of macrofauna functioning in an environment not previously observed, and affirmed the importance of fine-scale temporal observations of the benthic environment in situ. Observation of macrofauna presence and bioturbation during the summer contradicted previous studies that found this area to be azoic during methane activity and sulfide build-up. Sulfate concentrations decreased while sulfide and dissolved inorganic carbon concentrations increased during the summer. This coincided with changes in the depth and rates of bioturbation. Summer burrow depths (~0.8 cm) and rates (~0.4 cm h-1) were significantly less than spring burrow depths (~3.0 cm) and rates (~1.0 cm h-1). While sulfate reduction and OM degradation increased with temperature at a microscopic level, macroscopic OM degradation was reduced. As a result, reduced conditions dominated and a thin aerobic sediment layer, a few millimeters in thickness, was visible at the sediment surface. Decreases in macrofauna burrow depth and rates diminishes the area of influence of bioturbators, limiting bioturbation and subsequently the important ecosystem functions these organisms provide.Organic matter (OM) production and degradation is important in coastal estuaries, and OM fate is strongly influenced by the coupled interactions of bioturbation and biogeochemistry. From April to September 2013 sediment cores and a benthic observing system, Wormcam, were used to investigate the  Organic matter (OM) deposition and degradation is of critical importance in coastal estuaries given its role in biogeochemical cycling processes . The fatIn coastal estuaries bioturbation is largely driven by macrofauna  with ver-1 [Cape Lookout Bight is a semi-enclosed coastal lagoon that experiences high deposition of organic rich material from both autochthonous and allochthonous sources . Sedimen-1 , and mic-1 ,23. As a-1 . At extr-1 ,25. The -1 . Due to -1 ,28; this-1 .Based on previous studies of the biogeochemical profiles of Cape Lookout Bight it was assumed that macrofauna presence and influence is minimal to non-existent . AnotherAssessments of bioturbation in the bight have not been made since the early 1980s. Since that time methodological advances have improved the ability of scientists to assess macrobenthic function . Studiesin situ, a method of observation that could validate or challenge earlier assessments of the importance of macrofauna function in Cape Lookout Bight [in situ observation of the sediment-water interface, fine-scale (on the order of hours) temporal observations have been limited to only a few days or weeks [in situ study of benthic systems. In our study we used Wormcam to assess macrofauna presence and bioturbation in Cape Lookout Bight relative to sediment biogeochemistry. Presence was determined by active biogenic structures as no abundance data was collected. The specific objectives of our study were to 1) assess the temporal presence of macrofauna in Cape Lookout Bight, 2) quantify macrobenthic function through burrowing rates and the maximum depth of bioturbation, and 3) assess the relationship between macrobenthic function and sediment biogeochemistry through rates of sedimentation and optical geochemistry patterns.Traditional SPI has enhanced the understanding of sediment-organism interactions ,36, partut Bight ,29,30. Dor weeks ,32,39. Oor weeks . Using aor weeks  examined2 area coastal lagoon located southeast of Cape Hatteras in the outer banks of North Carolina . The bight waters in the semi-enclosed basin are influenced by both the back barrier island lagoon system as well as water from offshore [Cape Lookout Bight  is a 2 koffshore . The geooffshore . The suroffshore . Sedimenoffshore . No specSediment cores were collected in April, June and August of 2013, using a piston core, close to the study location of Martens et al. . Sedimen2 using a LiCor 7000 CO2 detector [Sediment pore water was collected by centrifugal extraction and filtered with a 0.2 μm filter. Sulfide concentrations in porewater was preserved in 20% Zn acetate solution and analyzed with the spectro-photometric method . Sulfatedetector . The meaThe Wormcam system similar to Sturdivant et al.  was usedPhotoshop (Adobe Systems Inc.) was used to rotate and scale images, and ImageJ (NIH) was used for digital measurements of sediment properties and biogenic structures. Quality control on image data was accomplished by reanalysis of a random selection of 25% of the images by a second analyst. A 5 hr interval, totaling 547 images, was used to comparatively assess macrofauna activity and sediment properties. Oxidation state of the sediment and depth of the apparent-color redox-potential discontinuity (aRPD) was determined by color: tan-brown sediment was considered oxidized and grayish-black sediment was considered reduced . During Polydora tetrabranchia); 2) straight–burrows that are relatively straight in form ; and 3) curved–burrows had at least one meander . When assessing burrow lengths from SPI it was accepted that there may have been unknown confounding variance due to the two-dimensional assessment (the profile image) of a three-dimensional structure (the sediment column). If animals were burrowing away from the faceplate the length of burrows could have been underestimated. To limit the potential for this confounding factor in burrow length measurements, only those burrows with a distinct connection to the sediment surface were included in calculations.To determine burrow lengths and rates, 88 individual burrows were followed through time at 1 hr intervals. These individual burrows were assigned a unique ID and the burrow shape, burrow depth, burrow length, burrow duration (time burrow was actively inhabited), and cause of vanishing were all recorded. Burrow rates were recorded and defined as the plus or minus change in burrow length per hour, and accounted for the burrowing activity of worms. The shape and form of a burrow can be important in species identification. Burrow shape was classified in three categories: 1) U-shaped–burrows were connected with two openings at the sediment water interface  and infauna bioturbation (burrow depth and burrow length) were tested. Because of the experimental design, the residuals were not assumed to be random,  distribution. The residuals for each regression were plotted against time to determine if a pattern existed or they were randomly distributed. If a pattern existed, the autocorrelation structure would have been accounted for in the estimation of the regression coefficients. However, no pattern of autocorrelation existed for the tested variables, and general linear models were employed. To assess the variability in sediment geochemistry (aRPD depth), and bioturbation (burrow depth), Kruskal-Wallis H test, a non-parametric analysis of variance (ANOVA), was conducted. Dunn’s test, a pairwise comparison following ranked-based ANOVA, was performed to assess significant differences. Non-linear regression was used to assess the relationship between bioturbation (represented by burrowing rates) and rates of deposition, where an exponential curve was fit to the data. For all analyses normality was checked with Shapiro-Wilk’s test, and homogeneity of variance was assessed with Bartlett’s test . All of oC (SD ±5), mean salinity was 35 psu (SD ±3), and mean DO concentration was 5.8 mg l-1 (SD ±1.1). The sediment grain-size major mode was >4 phi, which represents fine silt-clay mud sediments typical of organic-rich high deposition areas.Our study period was a 5-month interval from April to September 2013. Mean bottom water temperature was 25 Polydora tetrabranchia, identified by its unique U-shaped burrows  as sediment DIC increased inversely with sulfate reduction [To characterize the spring-summer changes in the geochemical profiles of sediment porewater, sulfate, sulfide, and dissolved inorganic carbon (DIC) concentrations were measured. In the top 10 cm of the sediment we found decreases in sulfate concentrations and increases in sulfide and DIC concentrations from April to August . These reduction . In JuneDuring our observation period burrowing activity was detected within the first hour of deployment of the Wormcam system. Small spionid-like worms in U-shaped burrows were observed above the aRPD near the sediment-water interface (SWI). On average the initial burrow lengths were ≥ 70% of the maximum length , indicating that the majority of burrow formation was completed within an hour–the time interval between images. The initial length of less than a quarter of the burrows were ≥ 90% of the maximum length. Burrow morphology was significantly correlated with aRPD depth, which is a boundary proxy for changes in sediment biogeochemistry that can be influenced by burrowing activity . Burrow Burrows generally had two distinct sections, the portion of the burrow above the aRPD and the portion below the aRPD. When assessing burrow and sediment dynamics, the aRPD is used as a delineating demarcation. In our study 65% of the burrows analyzed extended from the surface to below the aRPD. On average burrows extended 1.0 cm (SD = 0.6 cm) below the aRPD. Worms did not appear to favor either side of the aRPD and were observed moving throughout the vertical extent of their burrows during all conditions. Of these burrows 100% were oxic above the aRPD, and 98% were oxic below the aRPD. Burrows that had oxic portions below the aRPD produced an “oxic halo” along the length of the burrow below the aRPD, which extended from the burrow walls into the reduced sediment on average 0.2 cm (SD = 0.06 cm).We found visual analysis of the sediment biogeochemistry matched the biogeochemistry trends in our core samples. From the SPI we determined 3 distinct temporal periods, pre-reduced, reduced, and post-reduced periods of sediment biogeochemistry. The major sedimentary biogeochemical change occurred during the mid-summer (07/15/2013–08/24/2013) when the sulfate reduction zone was just below the sediment surface . This pe-1) compared to the pre-reduced (1.0 cm h-1) and post-reduced (0.9 cm h-1) periods (-1). Infauna burrowing rates significantly correlated with both the timing and magnitude of erosion and accretion events  during the reduced period  as sediment DIC increased inversely with sulfate reduction [The general temporal trend observed in the sediment biogeochemistry of Cape Lookout Bight during our study was unchanged from what was described previously: in the upper sediment column (< 20 cm) sulfate concentrations decreased and sulfide concentrations increased from spring to summer . This reeduction ; increaseduction ,28. In oeduction , or may eduction .The source of sulfide is sulfate reduction , and incDuring the summer, the sediment of Cape Lookout Bight is a unique system. High organic matter input results in sulfate depletion near the sediment surface and results in a severely reduced sedimentary environment where high concentrations of sulfide reside near the surface . Despite3, resulting in the inhibition of aerobic respiration [Hydrogen sulfide is generally a highly toxic substance . Its bespiration ,75. Howepiration , though piration ,77, but piration .In the natural environment chemical and biological processes can reduce/remove sulfide concentrations produced in the sediments and macrofauna organisms benefit from association with these areas . SulfideBottom water oxygen saturations were normoxic throughout our study, and burrows with connections to the sediment surface were fully oxidized. This oxygenation allows for oscillations between oxic and anoxic conditions inside of burrows. Cyclical redox oscillation is common within individual burrow structures and is accompanied by rapid switching in dominant metabolic processes . As a reDespite the ability of worms to survive the harsh sediment conditions of the bight, bioturbation was significantly limited during periods of reduced sediment biogeochemistry, as indicated by the relationships between aRPD depth and burrow depths and lengths. The aRPD depth, the boundary layer between aerobic and anaerobic sediments , is an eUsing what we know from our sediment cores and image analysis, we are able to hypothesize a mechanism by which macrofauna function was perturbed and limited in Cape Lookout Bight. As the seasons progressed and temperatures increased, microbial activity was enhanced contributing to expanded reduced conditions in the sediment . This caA reduction in burrow depths and lengths diminishes the area of influence of bioturbators , limitinDecreases in burrow depths and lengths during the summer when the sulfate reduction zone was shallower was to be expected, but the decrease in the rate at which sediment was reworked suggests a macrofauna response to increased sediment toxicity at either an individual or community level. For example, the fitness of organisms could have decreased accounting for the reduction in burrowing activity, or the community could have shifted to smaller organisms due to the toxic disturbance; a common occurrence in disturbed systems ,91. The During our observation period burrow depths at Cape Lookout Bight were relatively shallow, averaging less than 3 cm in depth during the non-reduced periods, and less than 1 cm in depth during the reduced period. The depth and type of burrows provides context about the state of the benthic community as represented by successional stage . ChangesThough it is clear sediment biogeochemistry limited macrofauna function in CLB, biogeochemistry and bioturbation are coupled interactions in the sediment . The preS1 FileThis excel file contains two tabs that compose all of the raw data for the study.(XLSX)Click here for additional data file.


A large portion of the global wheat crop is milled to produce flour for use in the production of foods such as bread. Pressure to increase food supplies sustainably can be address directly by reducing post-harvest losses during processes such as flour milling. The recovery of flour in the milling of wheat is genetically determined but difficult to assess in wheat breeding due to the requirement for a large sample. Here we report the discovery that human selection for altered expression of putative cell adhesion proteins is associated with wheats that give high yields of flour on milling. Genes encoding fasciclin-like arabinogalactan proteins are expressed at low levels in high milling wheat genotypes at mid grain development. Thirty worldwide wheat genotypes were grouped into good and poor millers based flour yield obtained from laboratory scale milling of mature seeds. Differentially expressed genes were identified by comparing transcript profiles at 14 and 30 days post anthesis obtained from RNA-seq data of all the genotypes. Direct selection for genotypes with appropriate expression of these genes will greatly accelerate wheat breeding and ensure high recoveries of flour from wheat by resulting in grains that break up more easily on milling. It provides 20% of the total calories and proteins consumed worldwide2. Wheat is ground to flour by the process of dry milling to make various food products such as bread which are suitable for human consumption. From a milling perspective wheat grain has three main components; endosperm, bran and germ, each with different mechanical properties3. In milling, the endosperm of the grain is scraped from the bran by passing the grain between metal rollers and sieving out the flour derived from the small particles of endosperm released. Clean separation of endosperm from bran and germ is desired in this process4. The efficiency of the process as measured by the yield of flour is the subject of selection by wheat breeders developing new wheat varieties for these products. The recovery of flour is largely determined by genotype and milling process with recoveries usually in the range 70 to 80%5. However, the maximum theoretical yield expected is around 85% which is the percentage of endosperm in the wheat grain4. In wheat breeding, small scale laboratory test mills are widely used for the selection of wheat genotypes that deliver a higher flour yield. However, this process is difficult because the test has poor repeatability and still requires more than 1 kg of grain. These quantities of grain are not available from single plants to allow early generation selection for this key trait.Wheat is the leading crop in the temperate world8. Three main factors which influence milling performance of the wheat are grain hardness9, endosperm to bran ratio and ease of separation of endosperm from bran3. Soft wheats usually produce ~2% less flour than the hard wheats due to differences in mechanical properties of the grains4. Several QTLs associated with milling yield have been reported, on chromosome 6A10, 3B11, 1DL, 2 AS, 2BS, 2DS, 3DL, 4AC, 4DS, 5AL, 6 AC, 6DS12, 1B, 2A, 2B, 3B, 6A, 7 A, 7B13. Co-relation between starch granule size distribution (SGSD) and milling yield has been demonstrated by Edwards et al.14 in hard wheats. It was suggested that an even size distribution  of starch granules is genetically controlled and associated with higher flour yield. Edwards et al.9 showed that 68% of the variation in hard wheats (Pina-D1a/Pinb-D1b) could be explained by combined effect of grain hardness and SGSD. Endosperm strength and stiffness measured by SKCS has also been shown to correlate with milling yield15. Much of this may explain environmental rather than genetic variation in milling performance. Despite of all this analysis the genetic and environmental components of variation and their molecular basis remain unclear.Flour yield is a complex trait controlled by interaction between genotype, environment and processinget al.16 (2017) reported that genotype was a large contributor to variation in yield of wheat in Germany (1983–2014). Knowledge of the molecular basis17 of the intense human selection for high flour yield in breeding modern wheat genotypes would greatly accelerate the breeding of wheat combining high productivity and the grain quality required by consumers. This capability will be of great value in rapidly adapting wheat to changing and variable climates. The main objective of this study was to identify candidate genes which may control flour yield of wheat. RNA Seq of 30 diverse worldwide wheat genotypes was conducted at 14 and 30 days post anthesis (DPA) and gene expression was analysed relative to flour yield obtained on laboratory milling of the mature grain.Genetics controls a large part of the variation in flour yield. For example, Laidig Milling yield of the thirty wheat genotypes measured as % of total flour yield is shown in Table Pina-D1a/Pinb-D1a alleles of puroindoline genes within which 7 genotypes showed soft grain texture (SKCS HI < 50) and 7 showed hard grain texture (SKCS HI > 50), 8 genotypes were hard with Pina mutation (Pina-D1b/Pinb-D1a), and 7 were hard wheats with Pinb mutation (Pina-D1a/Pinb-D1b) . GH726097 and TC379245 were expressed at much lower levels in wheat genotypes with high flour yield; 28.2 and 6.1 fold, respectively  p-value of <0.01 Tables  and 3. SThe expression of GH726097 and TC379245 was significantly up-regulated in all of the genotypes of the PM-group but significantly down-regulated in all genotypes from HM-group except for the genotype Jing Hong No.1. Jing Hong No.1 showed very high up-regulation of β-amylase genes  compared with all other good millers. CA713114 and CA717472 were 707 and 80 fold up-regulated in Jing Hong No.1.http://www.wheatgenome.org/). NCBI BLASTn identified DQ872381 as a highly similar gene to GH726097, TC379245 and TC429180. DQ872381 has been annotated as a fasciclin-like arabinogalactan-8 19.The alignment of three differentially expressed putative FLA genes GH726097 (536 bp), TC379245 (1267 bp) and TC429180 (1252 bp) is shown in  showed that TC379245 shares 99% sequence identity with Traes_2BS_D44E4B43A and DQ872381 in the coding region .Analysis of published wheat transcriptome data was used to examine the tissue specificity of expression of these genes. In the grain, both transcripts have been reported in pericarp (inner and outer) but not in endosperm tissue  and also at a later stage, close to maturity (30 DPA) in genotypes that gave high flour yield  encoding cell adhesion proteins, 22. AGPs that contain putative cell adhesion domain known as fasciclin domain are known as fasciclin like arabinogalactan proteins. FLA has been implicated in cell adhesion and may link the cell membrane and cell wall. AGPs carry glycosylphosphotidylinositol (GPI) anchor at the C-terminus which signals the peptide to the plasma membrane. However, wheat FLA-8 has a shorter GPI-anchor which raises a question about its binding to plasma membrane19. But it might have a role in cell to cell and cell to extracellular environment interaction as suggested by Faik et al.19. Modification of a FLA in poplar23 has been shown to reduce the mechanical strength of plant tissues25. Human selection for higher flour yield has apparently resulted in the selection of wheat genotypes with greatly reduced levels of expression of this gene. Nanospherical arabinoxylan proteins have been recently identified as the adhesive component secreted by the climbing plant, English ivy26.The arabinogalactan proteins (AGPs) are highly glycosylated proteins that are rich in hydroxyproline and found in plant cell wall and plasma membraneFLA-8 was expressed at higher levels in high milling wheats at 30 DPA. The tissue specificity of expression is not known in this system but varies significantly in other plants25. Most gene expression at 30 DPA may be in the embryo as the endosperm is terminally differentiated by this stage.A third transcript  with homology with et al.27 (2017) reported that the product of the granule softness protein gene was an AGP. This would complicate any attempts to relate FLA levels to milling yield. Methods for distinguishing the FLAs from other AGPs would be required. Hard wheats are generally considered to be better milling wheats14 and a reduced expression of AGP encoded by the Gsp-1 gene at the hardness locus might contribute to improved flour yield. However this study showed that flour yield was not associated with the pin genotype at the hardness locus . A major QTL for flour yield has been reported from chromosome 2B12. The QTL on chromosome 2BS had the highest overall LOD score (13.1) in analysis of the progeny of a Sunco X Tasman cross, explaining 14.4–31.3% of the variation in flour yield at different sites7. Sunco was shown to have low expression of the FLA gene in the current study and was the source of the high flour yield in the Sunco X Tasman cross7. A QTL on chromosome 4B was the second most significant flour yield QTL in this cross. However in another cross, Katepwa X CD87, the 4B QTL was the most important and explained 13.6–23.8% of variation in flour yield. The coincidence of the structural genes encoding FLA and the chromosomal locations of the major know QTLs for milling yield in these crosses on chromosomes 4B and 2 A provides strong confirmation of the significance of these associations.28 that fill the endosperm cells and account for most of the grain content. The expression of storage protein genes was significantly altered in wheat with high flour yield. Several genes may contribute to changes in protein synthesis in the better milling wheat genotypes. The expression of 40 S ribosomal protein was significantly higher in the better milling wheat genotypes at both 14 and 30 DPA  may allow tissue specific alteration of function. Recent analysis of expression of defence genes and response to pathogen infection has been shown to result in highly sub-genome specific expression31.The tissue specificity32 and new pathways for carbon assimilation33. Although the gene controlling bread quality has at least five copies in the wheat genome with high sequence homology, the cause of the differential expression of this gene was identified to be the promoter region of a specific allele of this gene. A simple PCR-test was develop which allowed the identification of wheat genotypes for the presence or absence of the specific allele of gene controlling bread quality32 which has been used by CIMMYT34. A similar strategy can be used to identify the presence or absence of the specific FLA allele which corresponds to high FLA-8 expression and thereby select for wheat genotypes for good milling.Analysis of the wheat grain transcriptome has identified a gene controlling bread quality35 on the rate of genetic gain in wheat breeding and assist the selection of new genotypes to support the adaptation of wheat production to climate change37.Selection for these genes and the flour yield genes identified in the present study provides a new set of tools for wheat breeders that should reduce the constraints of selection for wheat qualityhttp://www.seedpartnership.org.au/associates/agg). Plants of all thirty wheat genotypes were grown under field conditions at Narrabri  and harvested at maturity. Another trial was grown at Biloela . The conditions at this site were harsher resulting in some varieties not performing well. Milling of these samples gave flour yield data that showed a highly significant (P = 0.01) correlation with that from the Narrabri site but the data was not included in the association analysis because it was considered a far less reliable measure of the flour milling qualities of these genotypes when grown in unfavourable environments.Wheat grains of a worldwide set of wheat genotypes were sourced from Australian Winter Cereal Collection (AWCC), Tamworth, NSW, Australia; now known as Australian Grains Genebank (AGG), Horsham, Victoria, Australia  wheats and high milling (HM) wheats if their milling yield was equal to/above or below 77%, respectively.Milling yield of harvested grain, identified by as % of total flour yield, was determined using a Buhler mill as explained by Edwards et al.18. Puroindoline alleles in all the genotypes were also characterized as described in Nirmal et al.18.Grain hardness for all the genotypes was measured using Single Kernel Characterization System (SKCS) as described by Nirmal 38. cDNA was prepared and used to produce indexed Illumina NGS libraries which were then multiplexed to allow the sequencing of eight indexed libraries in one lane on a GA IIx Illumina sequencing platform to obtain 100 bp paired-end reads. RNA sequencing of the thirty wheat genotypes generated a total of ~2.5 million to ~7.2 million reads varying with the genotype.Total RNA was isolated from the whole caryopsis at 14 days post anthesis (DPA) and at 30 dpaftp://occams.dfci.harvard.edu/pub/bio/tgi/data/Triticum_aestivum/) which contains 221,925 tentative consensus sequences obtained from EST, cDNA and mRNA libraries was used as a reference database for RNA-seq analysis. Read alignment parameters length fraction and similarity fraction were set to 0.8 and 0.9, respectively. Paired reads were counted as two. Gene expression was normalised by calculating reads per kilo base per million mapped reads (RPKM).All NGS data was analysed using CLC Genomic Workbench Version 9.0 . All the reads were trimmed for quality to exclude calls with Fred quality scores less than 20. Following trimming all sequence data was subjected to quality analysis. After quality check reads were subjected to RNA-seq analysis. Triticum aestivum gene index (TAGI) database  by RNA-Seq analyses using the TAGI reference in CLC genomics workbench. The data included three biological replicates and since there is was no significant difference between the replicates the mean values are shown in Fig. Tissue specificity of expression of 39 within CLC workbench to identify differentially expressed genes (DEGs) in the HM-group when compared to the PM-group. Differentially expressed genes with FDR  p-value of < 0.01 were considered as statistically significant candidate genes associated. A further sorting of the DEGs was undertaken based on Baggerley’s weighted proportion difference, to identify those genes with consistent expression difference in genotypes within the PM and HM wheat genotypes.RNA-seq data of the PM-group and the HM-group was subjected to proportion-based Baggerley’s test40.Differentially expressed genes were functionally annotated using Blast2GO PROAll NGS sequence data as raw data was submitted to NCBI at the Sequence Read Archive (SRA) and is available as SRA Submission # SUB2954840 (under BioProject# PRJNA392390 and BioSample #SUB2843912).