The mediating role of innovation in the relationship between market orientation and university performance in Pakistan

Universities globally are going through a paradigm shift with a need to become more innovatively market-oriented to handle the issue of growing competition for funding, as well as attracting/retaining the international/local competent students and academicians. However, there appears to be a dearth of research on how such state of affairs could be addressed, particularly in the emerging economies like Pakistan. In the light of resource-based theory (RBT), as well as organizational-learning theory (OLT), literature suggests that market-orientation (MO) and innovation are to be the desirable unique resources, as well as the guiding philosophies, to enable universities for a more competitive performance. Hence, this study investigated how resources like marketorientation (MO), and innovation, can influence university performance (UP). The study also tested empirically the potential mediating effect of innovation on the MOUP relationship. In addition, how the dimensions of MO influenced the innovation and university performance (UP) were also tested empirically in the universities of Pakistan. Results of the PLS path modelling (with 369 respondents from the target public-sector universities) firstly confirmed significant effect of the “universal construct of MO” and two of its dimensions “the advising and mentoring, as well as the intelligencegeneration and response” on UP. However, one dimension of MO, which is the administration-leadership, was not significantly supported to directly influence the UP. Secondly, the study confirmed that there were significant direct effects of the “universal construct of MO”, as well as all of its dimensions, on innovation. Thirdly, the study also found that there was a significant effect of innovation on UP. Furthermore, the bootstrapping results found significant mediation of innovation between the MO-UP relationship. Hence, the results show that UP can be directly enhanced through MO and innovation. Even the use of innovation as a mediator can further strengthen the MO-UP relationship. Based on the findings, the study offers theoretical and practical implications, followed by its limitations and directions, for future research

Mapping the molecular surface of the analgesic NaV1.7-selective peptide Pn3a reveals residues essential for membrane and channel interactions

Compelling human genetic studies have identified the voltage-gated sodium channel NaV1.7 as a promising therapeutic target for the treatment of pain. The analgesic spider venom-derived peptide µtheraphotoxin-Pn3a is an exceptionally potent and selective inhibitor of NaV1.7, however, little is known about the structure-activity relationships or channel interactions that define this activity. We rationally designed seventeen Pn3a analogues and determined their activity at hNaV1.7 using patchclamp electrophysiology. The positively charged amino acids K22 and K24 were identified as crucial for Pn3a activity, with molecular modeling identifying interactions of these residues with the S3-S4 loop of domain II of hNaV1.7. Removal of hydrophobic residues Y4, Y27 and W30 led to a loss of potency (>250-fold), while replacement of negatively charged D1 and D8 residues with a positively charged lysine led to increased potencies (>13-fold), likely through alterations in membrane lipid interactions. Mutating D8 to an asparagine led to the greatest improvement in Pn3a potency at NaV1.7 (20-fold), whilst maintaining >100-fold selectivity over the major off-targets NaV1.4, NaV1.5 and NaV1.6. The Pn3a[D8N] mutant retained analgesic activity in vivo, significantly attenuating mechanical allodynia in a clinically relevant mouse model of post-surgical pain at doses 3-fold lower than wild-type Pn3a, without causing motor adverse effects. Results from this study will facilitate future rational design of potent and selective peptidic NaV1.7 inhibitors for the development of more efficacious and safer analgesics but also to further investigate the involvement of NaV1.7 in pain

Gating modifier toxin interactions with ion channels and lipid bilayers: Is the trimolecular complex real?

Spider peptide toxins have attracted attention because of their ability to target voltage-gated ion channels, which are involved in several pathologies including chronic pain and some cardiovascular conditions. A class of these peptides acts by modulating the gating mechanism of voltage-gated ion channels and are thus called gating modifier toxins (GMTs). In addition to their interactions with voltage-gated ion channels, some GMTs have affinity for lipid bilayers. This review discusses the potential importance of the cell membrane on the mode of action of GMTs. We propose that peptide–membrane interactions can anchor GMTs at the cell surface, thereby increasing GMT concentration in the vicinity of the channel binding site. We also propose that modulating peptide–membrane interactions might be useful for increasing the therapeutic potential of spider toxins. Furthermore, we explore the advantages and limitations of the methodologies currently used to examine peptide–membrane interactions. Although GMT–lipid membrane binding does not appear to be a requirement for the activity of all GMTs, it is an important feature, and future studies with GMTs should consider the trimolecular peptide–lipid membrane–channel complex. This article is part of the Special Issue entitled ‘Venom-derived Peptides as Pharmacological Tools.

Lengths of the C-terminus and interconnecting loops impact stability of spider-derived gating modifier toxins

Spider gating modifier toxins (GMTs) are potent modulators of voltage-gated ion channels and have thus attracted attention as drug leads for several pathophysiological conditions. GMTs contain three disulfide bonds organized in an inhibitory cystine knot, which putatively confers them with high stability; however, thus far, there has not been a focused study to establish the stability of GMTs in physiological conditions. We examined the resistance of five GMTs including GpTx-1, HnTx-IV, HwTx-IV, PaurTx-3 and SgTx-1, to pH, thermal and proteolytic degradation. The peptides were stable under physiological conditions, except SgTx-1, which was susceptible to proteolysis, probably due to a longer C-terminus compared to the other peptides. In non-physiological conditions, the five peptides withstood chaotropic degradation, and all but SgTx-1 remained intact after prolonged exposure to high temperature; however, the peptides were degraded in strongly alkaline solutions. GpTx-1 and PaurTx-3 were more resistant to basic hydrolysis than HnTx-IV, HwTx-IV and SgTx-1, probably because a shorter interconnecting loop 3 on GpTx-1 and PaurTx-3 may stabilize interactions between the C-terminus and the hydrophobic patch. Here, we establish that most GMTs are exceptionally stable, and propose that, in the design of GMT-based therapeutics, stability can be enhanced by optimizing the C-terminus in terms of length, and increased interactions with the hydrophobic patch

Efficient enzymatic ligation of inhibitor cystine knot spider venom peptides: using sortase a to form double-knottins that probe voltage-gated sodium channel NaV1.7

Gating modifier toxins from spider venom are disulfide-rich peptides that typically comprise a stabilizing inhibitor cystine knot (ICK). These knottin peptides are being pursued as therapeutic leads for a range of conditions linked to transmembrane proteins. Recently, double-knottin peptides discovered in spider venom and produced by recombinant expression have provided insights into the pharmacology of transmembrane channels. Here, we use chemoenzymatic ligation to produce double-knottins to probe the effect of bivalent modulation on the voltage-gated sodium channel subtype 1.7 (Na1.7), which is implicated in pain signaling. Monovalent knottins were oxidatively folded and then biochemically conjugated using sortase A, to form double-knottins. The structural integrity of the peptides was confirmed using NMR, and fluorescence-based activity assays provided evidence suggesting that coincubated monovalent and bivalent knottins can cooperatively modulate Na1.7. We anticipate that double-knottins will provide novel tools for enhancing our understanding of, and design strategies for, therapeutically relevant voltage-gated ion channels

Gating modifier toxins isolated from spider venom: Modulation of voltage-gated sodium channels and the role of lipid membranes

Gating modifier toxins (GMTs) are venom-derived peptides isolated from spiders and other venomous creatures and modulate activity of disease-relevant voltage-gated ion channels and are therefore being pursued as therapeutic leads. The amphipathic surface profile of GMTs has prompted the proposal that some GMTs simultaneously bind to the cell membrane and voltage-gated ion channels in a trimolecular complex. Here, we examined whether there is a relationship among spider GMT amphipathicity, membrane binding, and potency or selectivity for voltage-gated sodium (NaV) channels. We used NMR spectroscopy and in silico,/i> calculations to examine the structures and physicochemical properties of a panel of nine GMTs and deployed surface plasmon resonance to measure GMT affinity for lipids putatively found in proximity to NaV channels. Electrophysiology was used to quantify GMT activity on NaV1.7, an ion channel linked to chronic pain. Selectivity of the peptides was further examined against a panel of NaV channel subtypes. We show that GMTs adsorb to the outer leaflet of anionic lipid bilayers through electrostatic interactions. We did not observe a direct correlation between GMT amphipathicity and affinity for lipid bilayers. Furthermore, GMT-lipid bilayer interactions did not correlate with potency or selectivity for NaVs. We therefore propose that increased membrane binding is unlikely to improve subtype selectivity and that the conserved amphipathic GMT surface profile is an adaptation that facilitates simultaneous modulation of multiple NaVs

Enzymatic ligation of a pore blocker toxin and gating modifier toxin; creating double-knotted peptides with improved sodium channel NaV1.7 inhibition

Disulfide-rich animal venom peptides targeting either the voltage-sensing domain or the pore domain of voltage-gated sodium channel 1.7 (NaV1.7) have been widely studied as drug leads and pharmacological probes for the treatment of chronic pain. However, despite intensive research efforts, the full potential of NaV1.7 as a therapeutic target is yet to be realized. In this study, using evolved sortase A, we enzymatically ligated two known NaV1.7 inhibitors PaurTx3, a spider-derived peptide toxin that modifies the gating mechanism of the channel through interaction with the voltage-sensing domain, and KIIIA, a small cone snail-derived peptide inhibitor of the pore domain with the aim of creating a bivalent inhibitor which could interact simultaneously with two non-competing binding sites. Using electrophysiology, we determined the activity at NaV1.7 and to maximize potency, we systematically evaluated the optimal linker length, which was nine amino acids. Our optimized synthetic bivalent peptide showed improved channel affinity and potency at NaV1.7 compared to either PaurTx3 or KIIIA individually. This work shows that novel and improved NaV1.7 inhibitors can be designed by combining a pore blocker toxin and a gating modifier toxin to confer desired pharmacological properties from both the voltage sensing domain and the pore domain

Spider peptide toxin HwTx-IV engineered to bind to lipid membranes has an increased inhibitory potency at human voltage-gated sodium channel hNa(V)1.7 (vol 1859, pg 835, 2017)

The authors would like to correct Fig. 4. The electrostatic surface potential maps of HwTx-IV and gHWTx-IV are not correctly represented in the published Fig. 4B. Please find below the correct Fig. 4. [Formula presented] Although the electrostatic maps were not correct, they did not affect the discussion of the results and the conclusions. The paragraph referring to the Figure in the manuscript is correct: “Furthermore, a global profile of the electrostatic surface potential on the surface of gHwTx-IV containing the four mutations (E1G, E4G, F6W and Y33W) is more positive than the same surface on HwTx-IV (Fig. 4B), and gHwTx-IV has a higher net charge and residual dipole moment than HwTx-IV (Table 3)”. The authors would like to apologize for any inconvenience caused

Weaponisation ‘on the fly’: convergent recruitment of knottin and defensin peptide scaffolds into the venom of predatory assassin flies

Many arthropod venom peptides have potential as bioinsecticides, drug leads, and pharmacological tools due to their specific neuromodulatory functions. Assassin flies (Asilidae) are a family of predaceous dipterans that produce a unique and complex peptide-rich venom for killing insect prey and deterring predators. However, very little is known about the structure and function of their venom peptides. We therefore used an E. coli periplasmic expression system to express four disulfide-rich peptides that we previously reported to exist in venom of the giant assassin fly Dolopus genitalis. After purification, each recombinant peptide eluted from a C18 column at a position closely matching its natural counterpart, strongly suggesting adoption of the native tertiary fold. Injection of purified recombinant peptides into blowflies (Lucilia cuprina) and crickets (Acheta domestica) revealed that two of the four recombinant peptides, named rDg3b and rDg12, inhibited escape behaviour in a manner that was rapid in onset