V) channels are one such group of ion channels that play a significant role in multiple pain pathways. This review will explore the literature around conotoxins that bind NaV channels and determine their analgesic potential.Chronic pain creates a large socio-economic burden around the world. It is physically and mentally debilitating, and many sufferers are unresponsive to current therapeutics. Many drugs that provide pain relief have adverse side effects and addiction liabilities. Therefore, a great need has risen for alternative treatment strategies. One rich source of potential analgesic compounds that has emerged over the past few decades are conotoxins. These toxins are extremely diverse and display selective activity at ion channels. Voltage gated sodium (Na V channels have potential as pain therapeutics, as reviewed previously [V channel inhibitors for pain management.Chronic pain is a major problem in the world today. The total cost of chronic pain in the United States was estimated to be between $560 and $635 billion per annum . This coeviously ,3. The pThe pain transmission pathway involves sensors known as nociceptors, which transmit the signal from the site of pain to primary sensory neurons in the spinal cord known as dorsal root ganglion (DRG) neurons. Central fibres of nociceptive DRG neurons project to the superficial laminae of the dorsal horn, particularly laminae I and II ,5.V channel inhibitors, where specific inhibition of neurotransmission in the central terminals of nociceptive primary afferent nerves may be achieved [DRG neurons process many sensory modalities, such as proprioception and touch, with the exception of small DRG neurons, which primarily conduct pain signals. Thus, DRG neurons can be characterised into four dorsal root fibre types. Aα fibres conduct information from muscle and skeletal mechanoreceptors. Aβ fibres relay signals from cutaneous and subcutaneous mechanoreceptors. Aδ and C fibres conduct nociceptive information . The cenachieved .V) and glutamate receptors located on presynaptic terminals of primary afferent fibres in the dorsal horn [V channel subtypes are expressed selectively in pain sensing neurons.Within DRG neurons, there is a multitude of ion channels, receptors and peptides ,9,10. Thsal horn . Among tV channels are large complex structures made up of a primary alpha (α) subunit along with one or two auxiliary beta (β) subunits. The primary α subunit is about 260 kDa in size with four homologous domains each with six transmembrane segments (S1–S6), [VAb [VAb has a central pore surrounded by four pore forming modules made of S5 and S6 segments and a pore loop. The outer edge of the pore is associated with S1–S4 voltage sensing modules. There is also a wide outer vestibule, a narrow ion selectivity filter, a large central cavity and an intracellular activation gate made by crossing of S6 segments [Na(S1–S6), ,12. Much6), [VAb . NaVAb hsegments .V channels are generally made of two β subunits [N-terminal extracellular immunoglobulin-like fold, a single transmembrane segment and a short intracellular segment. The primary component of the β subunit helps regulate channel kinetics such as voltage dependence of activation and inactivation. The extracellular immunoglobulin domains of β subunits function as cell adhesion molecules that interact with extracellular matrix proteins and other cell adhesion molecules [In contrast to the single α subunit, there can be one or two β subunits based on the location of the channel. The brain Nasubunits . The β solecules .V1.1 to 1.9. Despite different isoforms of NaV, they share at least 50 percent of their amino acid sequence [V1.1, 1.2, 1.3 and 1.7 show great similarity and can be found on human chromosome 2q23-24 [V1.5, 1.8 and 1.9 encoded on human chromosome 3p21-24 are also closely related with 64 percent similarity to NaV channels on chromosome 2. Distinguished from both these groups, NaV1.4 is primarily expressed in skeletal muscles and encoded by chromosome 17q23-25 while NaV1.6 is mainly found at nodes of ranvier and encoded by chromosome 12q13. However, both NaV1.4 and NaV1.6 show at least 84 percent similarity to NaV channels coded by chromosome 2 [There are nine different mammalian sodium channel α-subunits labelled, Nasequence . When ph 2q23-24 ,15. In cmosome 2 ,14.V channels play a role. Within the diverse DRG neuronal population, NaV channels are localised in various subgroups based on function. Therefore, the properties of each NaV channel needs to be explored. NaV1.1 is primarily found in large diameter A-fibre DRG neurons suggesting a possible role in proprioception [V1.1. In fact, mutations of the SCN1A gene that codes for NaV1.1 have been associated with familial hemiplegic migraine suggesting a likely role in nociception [V1.1 levels are downregulated. Hence it is unclear as to what role NaV1.1 may play in nociception [V1.2 is found in many regions of the central nervous system (CNS), its level remains low in adult DRG neurons. Therefore, NaV1.2 channels are unlikely to play a major role in nociception [In terms of neuropathic and inflammatory pain, it is unlikely that all Naoception . Howeveriception ,16. In riception ,17. Althiception ,17.V1.3 is extensively expressed in neonatal DRG neurons. However, only a little of it can be detected in adult naïve DRG neurons [V1.3 positive [V1.3 positive after SNL [V1.3 in neuropathic pain remains controversial, as neuropathic pain develops in conditional and conventional NaV1.3 knockout mice [V1.3 conventional knockout mice [V1.3 plays a major role in the induction of abnormal spontaneous activity in injured neurons. Alternatively, it is possible that the loss of NaV1.3 is compensated by other NaV channel subtypes. It therefore remains uncertain whether or not conotoxins that target NaV1.3 could be developed as pain therapeutics.Na neurons . Followipositive . Furtherfter SNL . In spitout mice ,21. MoreV1.4 is primarily found in skeletal muscles and NaV1.5 is mainly located in cardiac muscles [V1.4 and 1.5 unlikely candidates to play a role in nociception. In terms of analgesic potential, activity of conotoxins at NaV1.4 and 1.5 channels may lead to severe adverse effects.Na muscles . Both thV1.6 co-localised with the neurofilament-200 (NF200) marker, which binds to sensory neurons that gives rise to myelinated axons [V1.6 is an A-fibre specific channel, which is not surprising because it is associated with nodes of Ranvier. Following SNL, NaV1.6 levels are down-regulated [V1.6 levels proximal to the lesion site [V1.6 plays a role in the abnormal spontaneous activity of injured DRG neurons [In contrast, Naegulated . Howeverion site . Despite neurons .V1.7 shows a wide distribution across different DRG neurons but can be predominantly found in small DRG neurons [V1.7 was primarily co-localised with NF-200 negative neurons [V1.7 is unable to respond during high frequency stimulation [V1.7 [V1.7 is coupled to NaV1.8 to boost subthreshold stimuli that activates NaV1.8 which recovers from inactivation at a rapid rate and produces high frequency action potentials [V1.7 mRNA and protein levels are upregulated [V1.7 knockout mice do not develop hyperalgesia in pain models [V1.7 is linked to channelopathy-associated insensitivity to pain (CIP), while SCN9A gain of function mutations cause paroxysmal extreme pain disorder and primary erythromelalgia [V1.7 is important for the development of acute and inflammatory pain.Na neurons ,24. Sincmulation . Howeveron [V1.7 ,25. It mtentials . Followiegulated ,27. Furtn models . In humamelalgia ,30. ThisV1.7 in neuropathic pain seems more complex. In a rat diabetic neuropathy model it was found that both NaV1.7 protein and current was elevated [V1.7 protein levels [V1.7 protein levels following peripheral axotomy or traumatic central axotomy in the injured DRG neurons of humans [V1.7 [V1.7 protein [V1.7 are likely to have therapeutic benefits in at least some pain states. Additionally, as the only recognised deficit in CIP patients is a loss of smell (anosmia), NaV1.7 remains a promising analgesic target with a minimal side effect profile [The role of Naelevated ,31. Howen levels ,22,32. Ff humans . In micens [V1.7 . In cont protein ,35,36. N profile .V1.8 is primarily found in small diameter nociceptive DRG neurons [V1.8 mRNA and protein [V1.8 knockout mice displayed impaired thermal and mechanical pain hypersensitivity following carrageenan induced inflammation [V1.8 is widely accepted to play role in inflammatory pain. However, peripheral nerve injury decreased NaV1.8 mRNA and protein levels in small diameter injured DRG neurons [V1.8 in uninjured C-fibres of sciatic nerves [V1.8 levels as inhibition of synthesis and knockout of TNFα eliminated the up-regulation of NaV1.8 following nerve injury [V1.8 plays a role in the development in allodynia was reinforced by a study which revealed that MuO-conotoxin MrVIB selectively blocked Nav1.8 sensory neuron specific sodium channels and chronic pain behaviour without motor deficits [V1.8 selective blockade in rats with SNL induced neuropathic pain [V1.8 mutations isolated in patients with painful neuropathies enhanced the response of DRG neurons to depolarisation [V1.8 in neuropathic pain immerged in an investigation of saphenous nerve neuromas [V1.8 null mice. Furthermore, 10 days post-surgery, a significantly higher proportion of fibres were mechanosensitive in wild type mice in comparison to NaV1.8 null mice. Thus, it was concluded that NaV1.8 is critical for spontaneous activity and that it may induce ectopic mechanosensitivity in sensory axons that are damaged [V1.8 plays a role in painful neuropathies by promoting neuronal hyperexcitability. However, NaV1.8 knockout mice develop neuropathic pain suggesting a possible difference in the role of NaV1.8 in neuropathic pain between rats and mice [V1.8 polymorphisms have been linked to atrial fibrillation. Atrial fibrillation is an electrical disturbance in the heart leading to inefficiencies in cardiac activity [V1.8 block. Overall, NaV1.8 is a promising target for inflammatory analgesia. However, its usefulness for neuropathic pain relief is less clear due to differences between rats and mice as well as potential side effects linked to atrial fibrillation.Na neurons . Carrage protein ,39. Moreammation . Hence,  neurons . Additioc nerves . It is be injury . The posdeficits . Anotherhic pain . Additiorisation . More inneuromas . Results damaged . These rand mice . Moreoveactivity . TherefoV1.9 is selectively found in small-diameter DRG neurons [V1.9 was significantly elevated [V1.9 knockout mice show decreased pain behaviours to induced inflammation [V1.9 may also play an important role in inflammatory pain. However, immunohistochemical analysis after SNL in rats show decreased NaV1.9 levels in the injured DRG neurons [V1.9 knockout mice after spared nerve injury to the same extent as wild type mice [V1.9 mutations isolated from patients with painful neuropathies were expressed in NaV1.9 knockout mouse DRG neurons, the resting membrane potential was depolarised [V1.9 was found in two unrelated individuals with congenital inability to sense pain [V1.9 mutation was isolated in patients with autosomal dominant episodic pain [V1.9 mutation appears to lead to contrasting clinical outcomes. Overall these results indicate that NaV1.9 may play a role in neuropathic pain.Na neurons . These c neurons . In the elevated . Moreoveammation . Therefo neurons . Moreoveype mice . Howeverolarised . Thus, tnse pain . Howeverdic pain . TherefoV channels bind to the α subunit [V channels. These toxins have been identified in venoms from centipedes, spiders and sea dwelling cone snails. Due to the difficulties of isolating native toxins in large quantities, synthetic forms of the native toxins have been usually developed. The specific binding characteristics of these toxins have made them great tools to identify channel structures.Most pharmacological agents that interact with Na subunit . At leasV channels with a potential fifth family have been discovered recently. These are identified as µ, μO, δ, ι and µO§. µ and µO-Conotoxins inhibit NaV channels through targeting the channel pore and the voltage sensor, respectively. δ-Conotoxins cause over excitation by slowing inactivation. ι-Conotoxins induce excitotoxicity through shifting the voltage dependence of activation to more hyperpolarised voltages. µO§-Conotoxins cause NaV channel inhibition by mechanisms that are yet to be elucidated. As reviewed elsewhere, it is evident that conotoxins have very diverse mechanisms of action correlated with great structural variability [Conotoxins are potent ion channel inhibitors. While there are many different types of conotoxins, four families that target Naiability . TherefoV channel current as these P loops play a critical role in controlling permeation and selectivity [Site 1 binds tetrodotoxin (TTX), saxitoxin (STX) and μ-conotoxins. This site is largely comprised of the P loops between S5 and S6 in each domain that form part of the outer vestibule of the channel. In general, μ-conotoxins bind more superficially to the channel pore than TTX and STX due to their larger size . Howeverectivity . Whilst C. geographus. They are a family of 22-residue peptide amides that inhibit sodium channels [V channels and acetylcholine receptors, respectively. The first extensively studied μ-conotoxin was GIIIA. It had three hydroxyproline residues and three disulphide bridges. It was shown to compete with TTX and STX for binding at NaV1.4 channels but showed no affinity for NaV neuronal subtypes [V1.4 subtypes increasing from an IC50 value of 50 nM in rats to 1500 nM in humans [V current in rat DRG neurons [μ-conotoxins were first isolated from the venom of the fish-hunting snail channels . μ-contosubtypes . Neverthn humans . Moreove neurons .V channel site as TTX and STX hydroxyl groups [V1.4 as just the presence of R13 and K16 alone can cause channel block [V1.4 channels.In 1989, GIIIA was synthetically manufactured . The pepl groups . Howeverel block . Hence, V1.4 channel selective activity [GIIIB has a similar global fold to GIIIA along with very similar backbone confirmations and Naactivity . In factactivity . The chaactivity . Neverthactivity ,77.Conus geographus that selectively targets NaV1.4 channels [V channel activity.μ-conotoxin GIIIC is also another peptide from channels . It alsochannels . HoweverConus purpurascens, an Eastern Pacific fish hunting species of cone snails [V channels [V (rNaV) α subunits were expressed in Xenopus laevis (Xenopus) oocytes, it reversibly inhibited rNaV1.4 channels with an IC50 value of 41 nM. Unlike GIIIA, it also blocked rNaV1.2 channels at an IC50 value of 690 nM and rNaV1.7 channels at an IC50 value of 6.2 μM [V1.7 channel as seen from an IC50 value of 3.1 μM [V channels to distinguish different subtypes. This was illustrated in a nerve growth factor (NGF) treated PC12 cell line which had a transient upregulation of NaV1.7 in the first 24 h followed by an increase in NaV1.2 beyond 24 h. As a result, it was shown that within the first 24 h, only 13% of the observed NaV current was inhibited by PIIIA followed by 39% at 48 h and 63% at 72 h [V channel subtypes when used in conjunction with other selective toxins.μ-conotoxin PIIIA was isolated from e snails . Similarchannels ,80. Whenf 6.2 μM . Interesf 3.1 μM . Hence i at 72 h . Thus, PConus tulipa [V channels including Arg14 was conserved in TIIIA. TIIIA potently displaces STX from NaV1.2 channels in the rat brain and NaV1.4 channels in skeletal muscle. Consistent with low levels of NaV1.2 and the absence of NaV1.4 channels, 3 µM TIIIA showed no inhibition of NaV current in DRG neurons [V1.2 and 1.4 channels outside of DRG neurons.A µ-conotoxin with a similar single conformation in solution to PIIIA is TIIIA. It was isolated from the venom duct of s tulipa . At the  neurons ,66. TherV currents are BuIIIA, BuIIIB and BuIIIC from the cone snail species Conus bullatus [V1.4 channels expressed in Xenopus oocytes by about 87 percent while BuIIIB and BuIIIC inhibited about 96 percent of the current at a concentration of 1 μM. However, BuIIIA block of NaV1.4 was reversible with a koff value of approximately 0.021 min−1, while BuIIIB and BuIIIC showed little to no recovery in 50 min [C-terminal residues such as Trp16, Arg18 and His20 are crucial for NaV blockade [N-terminal extension forming part of a short α-helix that include Glu3 to Asn8. In fact, mutation of residues two and three in this N-terminal extension caused a 40 fold increase in BuIIIB potency from a KD of about 0.2 μM to 0.0048 μM at NaV1.3 channels [V1.3 channels [N-terminus region is critical to determine toxin potency. In terms of analgesic potential, these BuIIIA, B and C conotoxins do not show great promise due to their activity at NaV1.4 channels that may cause many side effects. As discussed above, the potent NaV1.3 block by mutated BuIIIB need to be further explored in pain research. But this may not produce analgesia since NaV1.3 knockout mice still developed neuropathic pain [Another set of μ-conotoxins that target TTX-sensitive Nabullatus . More spn 50 min . Similarblockade . Howeverchannels . Furtherchannels . TherefoV channel inhibitors, SmIIIA blocks TTX resistant NaV channel currents in amphibian DRG and sympathetic neurons [Conus stercusmuscarum. Despite having a different target, SmIIIA shares similar characteristics with other µ-conotoxins, such as the arrangement of cysteine residues in the primary sequence and the conserved Arg13. Nonetheless, structural differences in SmIIIA from other µ-conotoxins include the lack of a Hyp residue and the presence of a Trp15 side chain [V1.8 and NaV1.9 channels play a role in pain pathways, it was of interest to further explore this toxin. Unfortunately, later results revealed that 3 µM SmIIIA did not inhibit human NaV1.8 channels [V channels.A µ-conotoxin that is significantly different from the previously described toxins is SmIIIA. Unlike the other TTX sensitive Na neurons . µ-Conotde chain ,71. As Tchannels . This fiV channels include SIIIA and KIIIA from the fish-hunting cone snails Conus striatus and Conus Kinoshitai, respectively. Both toxins block TTX resistant NaV currents in neurons of frog sympathetic and dorsal root ganglia . However, these peptides show poor inhibition of action potentials in frog skeletal muscles. The primary reason for this weak inhibition is most likely due to these action potentials being mediated by TTX sensitive NaV channel currents [Other µ-conotoxins that inhibit TTX resistant Nacurrents .V current in mouse DRG neurons [V1.2, 1.3 and 1.4 channels expressed in Xenopus oocytes [V1.5, 1.7 or 1.8 channels. This irreversible activity and non-selective profile, limits the analgesic potential of µ-SIIIA. But activity at multiple TTX sensitive channels may be utilized to treat some forms of pain. For example, in an open label trial, intramuscular TTX provided analgesia in 17 out of 31 treatments in 24 patients with cancer related somatic, visceral or neuropathic pain. The achieved analgesia also lasted beyond two or more weeks [V channels may provide analgesia to some patients. However, the ability of µ-SIIIA to provide pain relief needs to be evaluated alongside the above mentioned, irreversible pharmacological profile at certain NaV channel subtypes.Similar to the conotoxin SmIIIA, μ-SIIIA does not have a Hyp residue and adopts a trans conformation . SIIIA s neurons . Further neurons . However oocytes . Additiore weeks . In a sure weeks . Moreovere weeks . TherefoV1.2 channels in Xenopus oocytes [V interactions including Lys7, Trp8, Arg10, Asp11, His12 and Arg 14 were all conserved on KIIIA [V isoforms expressed in Xenopus oocytes, it inhibited NaV1.2 channels irreversibly and NaV1.6 with partial reversal. Moreover, NaV1.5, 1.3 and 1.4 channels in oocytes were also inhibited by KIIIA with progressively increasing potency [V channels, KIIIA also showed analgesia in a mouse model of formalin-induced pain without motor impairment. Results revealed that systemic administration of the toxin, decreased the paw-licking frequency of these mice without impairment in motor performance on a rotarod test up to a concentration of 10 nmol [V1.5 may preclude further development due to a high likelihood of cardiac side effects.µ-KIIIA is a 16 residue peptide with three disulphide bridges. It inhibits Na oocytes . Previouon KIIIA . In mouson KIIIA . When KI potency . Despite 10 nmol . OverallV1.2 channels expressed in Xenopus oocytes there was a residual current that can be abolished by TTX application [V inhibition rate. Additionally, co-application of TTX alongside KIIIA in any order (KIIIA first or TTX first) accelerated the peptide dissociation rate following washout [V channels [An interesting aside to µ-KIIIA activity is its ability to bind site 1 and act alongside TTX. When high concentrations of KIIIA was applied to Nalication . However washout . Therefochannels .Site 2 binds lipid soluble toxins such as vertridine which amplifies channel activation. This site is primarily located around the S6 segments of domain 1 and 4 ,90. Moretityus zulianus binding, the C terminal loop of domain III has also been included as a β-scorpion toxin interaction site [Receptor site 4 binds β-scorpion toxins that also enhance channel activation similar to site 2 . This siion site . Outsideion site . Due to ion site .Conus marmoreus [V inhibition mediated by MrVIA in mammalian cells [V current in Xenopus oocytes was reversed back to baseline after repeated pulses to +120 mV during washout [The most extensively studied µO-contoxins are MrVIA and MrVIB, isolated from the snail hunting species armoreus . The µ carmoreus . It was armoreus . This rearmoreus ,100. Furan cells . In cont washout . DespiteV channels at high concentrations. In caudodorsal neurons of freshwater snail Lymnaea stagnalis both MrVIA and MrVIB inhibited NaV channels at an IC50 of 0.1–0.2 µM while they also blocked fast inactivating CaV currents above concentrations of 1 µM [V channel current inhibition required extensive washing to restore baseline levels while CaV inhibition was rapidly reversed [V current in the snail neurons was investigated. Results showed that MrVIA inhibited some of the sustained CaV current while MrVIB displayed no inhibition. In mice, MrVIA CNS injection led to extreme reactions such as ataxia and coma at a very low dose of 0.1 nmol. However, intraperitoneal injection showed no distinct change even at doses 100 times greater [µO-conotoxins might also inhibit mammalian Ca of 1 µM . Furtherreversed . A diffe greater .50 value of 82.8 nM in rat DRG neurons [V1.8 and 1.9 channels that play an important role in pain pathways, this finding was of great interest. However, it is important to note that µO-conotoxins also inhibited TTX sensitive currents in rat DRG neurons at an IC50 value of 1.02 µM [V1.4 channels were expressed in HEK 293 cells, MrVIA and MrVIB inhibited the NaV current at an IC50 value of around 265 nm and 222 nm, respectively. Thus both toxins block NaV1.4 channels to a similar extent. In comparison, TTX sensitive NaV1.2 channels expressed in HEK 293 cells only showed very weak levels of inhibition. Hence it is evident that µO-conotoxins distinguish between NaV1.2 and NaV1.4 channels.An intriguing aspect of MrVIA is its ability to inhibit TTX resistant currents at an IC neurons . As this 1.02 µM . When TTV β subunits are expressed alongside the α subunit. In fact, the dissociation constant of MrVIB decreased from 44 nm for the NaV1.8 α subunit alone to 1 nM when the β2 subunit was co-expressed in Xenopus oocytes. This dissociation constant was also different depending on the co-expressed β subunit type where β1, β3 and β4 had dissociation constants of 8.5 nm, 6.5 nm and 3.5 nm, respectively [V1.8 α subunits expressed in Xenopus oocytes was minimally altered by the co-expressed β subunit type. Therefore, two alternative explanations are that MrVIB interacts with some component of the β subunit or that the β subunit type allosterically modulates the MrVIB binding site on the α subunit to different extents [2 subunits is particularly interesting as the β2 subunit is upregulated in rat neuropathic pain models [Another distinction in µO-conotoxins activity arises when different Naectively . In cont extents . This inn models . A furthn models . Moreoven models . As discn models . Thus, MIt is important to note that it was difficult to chemically synthesise a sufficient yield of µO-conotoxins. In fact, the previously implemented two-step oxidative folding strategy only produced a very low yield of MrVIB. Moreover, MrVIB did not fold into the native peptide from its linear, reduced form. Therefore, selenocysteine, an uncommon amino acid was introduced as an isoteric replacement for cysteine. This chemical replacement based on the regioselective formation of a diselenide bond in place of a disulfide bond allowed efficient folding with a greater yield . More imConus magnificus, synthetically manufactured using a novel regioselective approach described elsewhere [V1.4, 1.5, 1.6 and 1.7 channels expressed in HEK 293 and CHO cell lines were recorded in the presences of MfVIA. These results revealed that this toxin inhibited, NaV1.4, 1.5, 1.6 and 1.7 at IC50 values of 81 nM, 431 nM, 1.2 μM and 2.3 μM, respectively. Additionally, NaV1.2 channels had an IC50 values around 5.1 μM MfVIA while NaV1.8 channels had an approximate IC50 value of 158 nM, when expressed in Xenopus oocytes [V1.4 and 1.8 channels. Since NaV1.8 is abundant in DRG neurons and show promise as an analgesic target, MfVIA mediated inhibition may provide pain relief. However, the presence of NaV1.4 channels in skeletal muscles make it an undesirable target limiting the analgesic potential of MfVIA.Outside of MrVIA and MrVIB, a µO-conotoxin that show some analgesic promise is MfVIA . It is alsewhere . Subsequ oocytes . TherefoConus radiatus venom, five different toxins labelled r11a (ι-RXIA), r11b, r11c, r11d and r11e were identified [Besides μO-conotoxins, site 4 is also suggested to bind ι-conotoxins due to functional similarities with β-scorpion toxins . This faentified . With thentified . The funin vivo experiments that demonstrated intracranial injections of ι-RXIA caused seizures in mice [These results found in the presence of r11b were replicated for ι-RXIA, r11c and r11e. However, ι-RXIA was the most potent and least reversible toxin from this group. ι-RXIA caused many more spontaneous action potentials and consistent action potential trains in comparison to the other toxins in this group . The inc in mice . V1.6 voltage dependence of activation to more hyperpolarised potentials with minimal effects on inactivation [V α subunits expressed along with β1 in Xenopus oocytes, NaV1.7, 1.2 and 1.6 channels were sensitive while NaV1.1, 1.3, 1.4 and 1.5 channels were insensitive [V1.8, the TTX resistant component of small mouse DRG NaV current was isolated. As this TTX resistant current is primarily composed of NaV1.8, any effect would be mediated through it. However, results indicated that NaV1.8 was fairly insensitive to ι-RXIA [Subsequent experiments set out to elucidate the mechanisms behind this excitotoxic profile. It was found that ι-RXIA shifted mouse Nativation ,109. Addensitive . To asseo ι-RXIA .V1.6, similar to β-scorpion toxin Cn2 [V1.2 channels, the G845N mutation of the S3–S4 loop in domain 2, decreased the affinity of β-scorpion toxin Css IV by almost 10 fold. This decrease in affinity was coupled with a loss of toxin modulation on channel activation despite activation remaining intact due to the mutation [V binding site 4 similar to β-scorpion toxins [In terms of the ι-conotoxin binding site, several lines of evidence need to be explored further. Firstly, ι-RXIA has a high affinity for Naoxin Cn2 . Moreovemutation . Thus, cn toxins . OverallSite 5 binds complex polyether toxins such as brevetoxin and ciguatoxin produced by dinoflagellates. In particular, brevetoxin was found to bind S6 of domain 1 and S5 of domain 4 . HoweverV channel inactivation rate. Although the exact location of binding site 6 is unknown, δ-SVIE conotoxin was found to interact with a conserved residue in the S3-S4 linker of domain 4 [Neurotoxin binding site 6 is linked to δ-conotoxins that slow the Nadomain 4 ,112. In domain 4 ,15. SeleV channel inactivation [Conus textile was the first δ-conotoxin to be isolated from a snail hunting species of cone snails [V channels [in vivo with the Conus striatus toxin [Similar to other conotoxins, δ-conotoxins are small peptides that are rich in disulphides. As mentioned earlier they increase the time course of Nativation . King Koe snails . The unichannels . This fuchannels ,114,115.Conus purpurascens and Conus striatus, respectively [V time course of inactivation. However, they differed in the reversal of inhibition where δ-SVIE mediated activity was mostly irreversible while δ-PVIA effects were reversed with washout. An additional difference between these two conotoxins was the level of toxin activity on inactivation as a result of an 80 mV conditioning depolarisation for 300 ms. These results showed that δ-PVIA activity on NaV inactivation was significantly decreased by the conditioning depolarisation in comparison to δ-SVIE activity which was largely unaffected [Xenopus oocyes, δ-PVIA inhibited rat NaV1.2, 1.4 and 1.7 channels at progressively higher concentrations [Two more intriguing δ-conotoxins are PVIA and SVIE isolated from the fish hunting, ectively ,122. Bottrations .V1.4 channels expressed in HEK 293 cells, it was found to be a potent gating modifier [D value of around 500 nM. The specific interaction of the toxin with NaV1.4 channels was believed to be dependent on 3 hydrophobic residues identified as Y1433, F1434 and V1435 on the S3/S4 linker of domain 4. However, the analgesic potential of δ-SVIE is very limited due to its activity at NaV1.4 channels.When δ-SVIE was tested on Namodifier . More spConus gloriamaris that induces convulsions in land snails [Aplysia abdominal ganglia cells, δ-GmVIA shifted the NaV voltage dependence of steady state inactivation to more depolarised potentials and the steady state activation curve to more hyperpolarised potentials. When the toxin was tested on NaV channels expressed in Xenopus oocytes it inhibited NaV1.2 and 1.4 channels. Upon structural analysis, a similar cysteine framework to the King Kong peptide and CaV inhibiting ω-conotoxins was identified. Overall this toxin was very unlikely to have any analgesic potential due to its activity on NaV1.4 channels [δ-GmVIA is a conotoxin from the mollusk-hunting snail d snails ,118. In channels .Conus ermineus is a 32 amino acid with a six cysteine and four-loop framework similar to omega conotoxins [V channel inactivation. Upon application, δ-EVIA was also found to act on rat NaV1.2, 1.3 and 1.6 channels while showing no activity at NaV1.4 or NaV1.5 channels expressed in Xenopus oocytes. Moreover, when injected intracerebroventricularly, low doses (40 pmol) of δ-EVIA produced hyperactivity and seizures within 1–3 min. High doses (1 nmol) of δ-EVIA were found to be lethal [V channel subtypes.δ-EVIA from notoxins . In amphe lethal . These rConus tessulatus [V current. Relative to TTX, δ-TsVIA demonstrated slow dissociation kinetics in calcium imaging experiments of mouse DRG neurons. Furthermore, when mouse NaV1.6 α subunits were co-expressed with rat NaVβ1 subunits in Xenopus oocytes, the rapid channel inactivation was inhibited by δ-TsVIA [V channel subtype specificity and reversibility of inhibition.Recently isolated δ-TsVIA conotoxin was sourced from the venom of ssulatus . It is a δ-TsVIA . Future Conus suturatus [V1.3, 1.4, 1.6 and 1.7 channels expressed in HEK 293 cells. Further electrophysiological analysis revealed that δ-SuVIA increased peak NaV1.7 channel current by about 75 percent and shifted the voltage dependence of activation to −15 mV from −25 mV. Half maximal activation voltage was also shifted in a hyperpolarising direction by about 9.5 mV [V1.7 current. Overall, δ- SuVIA does not show any analgesic potential due to the absence of inhibitory activity.δ-Conotoxin SuVIA was isolated from the worm hunting uturatus . It was t 9.5 mV . These cThis site is positioned in a hydrophobic cavity surrounded by the domain II S4–S5 linker and S5-helix as well as domain III, S6 helix. Site 7 is associated with binding insecticides such as pyrethroids and DDT . HoweverV pore loop to be disulphide tethered to Cys24 on the toxin. As µO§-GVIIJ conotoxin causes NaV channel inhibition, it should be noted that site 8 differs from the binding sites for µ-conotoxins and µO-conotoxin MrVIB which also cause NaV channel inhibition. To functionally verify different binding sites, it was shown that the residual current following µ-conotoxin KIIIA application on to NaV1.2 channels was blocked by 3 µM GVIIJSSG . In the simultaneous presence of GVIIJSSG and µO-MrVIB, depolarising pulses caused µO-MrVIB to dissociate from the NaV1.2 at an accelerated rate. The remaining component following depolarisation was identical to the residual current that was detected after the application of GVIIJSSG alone. Therefore, it was evident that GVIIJSSG was bound at a separate site to µO-MrVIB [Binding site 8 is located around the pore loop of domain II . This siµO-MrVIB .Conus geographus [SSG and GVIIJSH due to the limited availability of the native peptide. A primary difference between these two analogues is based around Cys24 which is in a disulphide linkage on GVIIJSSG in comparison to a free thiol form on GVIIJSH. Therefore, GVIIJSH served as a reference to discover the role of the disulphide linkage on Cys24. GVIIJSSG inhibited all TTX sensitive rat NaV channel subtypes expressed in Xenopus oocytes at KD values that ranged from 5 to 360 nm. More interestingly, the IC50 value at NaV1.5 channels was over 200 µM while no NaV1.8 block was detected even at concentrations of 100 µM [V1.2, GVIIJSSG block reversed slowly with washout while GVIIJSH had a two phase recovery which included a rapid phase followed by a slower one. In terms of recovery from inhibition, the native µO§-GVIIJ had a similar slow reversal of inhibition as seen in the presence of GVIIJSSG.µO§-GVIIJ is a conotoxin extracted from ographus . Activitf 100 µM . Upon anV channel subtypes were expressed in HEK 293 and CHO cell lines, both GVIIJSSG and GVIIJSH caused inhibition of NaV 1.2, 1.4, 1.6 and 1.7 channels at nm ranges. At a concentration of 10 µM, GVIIJSSG inhibited NaV1.5 by 19% while GVIIJSH only showed 0.3% inhibition. In contrast, NaV1.1 and 1.3 channels were only inhibited by GVIIJSH. Overall, GVIIJSH was more potent at all the channels tested in comparison to GVIIJSSG [When human NaGVIIJSSG .SSG activity [V1.7 α subunit was expressed with rat β1 or β3 subunit, it was inhibited by GVIIJSSG while the presence of β2 or β4 subunit, led to toxin resistance. Additionally, chimeras of β1 and β2 subunits were made by joining the extracellular and transmembrane domains of one β subtype with the intracellular domain of the other β subtype. When co-expressed with NaV1.7, these chimeras provided the means to identify the β2 region that induced resistance. Thus, results showed that the extracellular domain of β2 was critical to induce toxin resistance [2 and β4 subunits that induce resistance compared to β1 and β3, is the presence of a disulphide bond with NaV1 in the resistant subunits. Therefore, the Cys910 identified earlier may be disulphide linked to its β subunits. Overall, µO§-GVIIJ conotoxin provides another pharmacological tool to study NaV activity.Subsequent experiments set out to identify the effect of β subunits on GVIIJactivity . These rsistance . The autV binding site is designated for local anesthetics. It is located in the α helix of domain IV segment 6 [V subtype selectivity and yet produce analgesia without a severe side effect profile [V1.1 to 1.7 channels [The final Naegment 6 . Local a profile . In autochannels . Despitechannels . Side efchannels .V1.4 and 1.5 channels create significant risks.Multiple factors are attributed to the absence of major side effects following lignocaine treatment. For example, channel block is enhanced at more depolarized voltages. This suggests that the local anesthetic would only be primarily active during pain transmission. Moreover, channel block accrues following a train of brief depolarizing pulses. Therefore, repeated firing neurons would be inhibited to a greater extent in comparison to regular firing neurons. Additionally, channel block is dependent on the frequency of stimulation . OverallV channels, conotoxins bind voltage gated calcium (CaV) channels, voltage gated potassium (KV) channels and many other targets [V channels. In fact, the only conotoxin approved for clinical use is ω-conotoxin MVIIA  which mediates its activity through CaV2.2 channels [V channels are involved in nociceptive neurotransmission throughout sensory nerves, conotoxins appropriately targeting subtypes of these channels should have the advantage of activity via systemic administration or targeted administration to peripheral tissues.Outside of Na targets . Therefochannels . Unfortuchannels . Since Pchannels . As a reV channels are intimately involved in all different forms of pain. From the NaV targeting conotoxins, it is evident that μ, μO and µO§-conotoxins are more likely to produce analgesia through inhibition of NaV channels involved in nociceptive transduction and transmission. However, within each conotoxin family there is a great number of NaV targets that will need to be refined through structural modification before therapeutically useful agents can be developed. In general, combinations of NaV1.1, 1.3, 1.7, 1.8 and 1.9 channels should be targeted for pain treatments. It may eventuate that the optimal analgesic NaV inhibitors target multiple NaV subtypes among this group. However, different NaV channels play a more substantial role based on the location and type of pain as highlighted in section four. So pain type specific agents may be needed. Perhaps one pitfall of conotoxins that are significantly more selective for one NaV channel subtype is the possibility of redundancy within the system that allows other NaV channel subtypes to compensate for the loss of activity. The studies on pain relief mediated by TTX and local anesthetics such as lignocaine which affect a broad range of NaV channels suggest that inhibiting multiple NaV channels may be an effective approach. In fact, non-selective NaV channel inhibitors such as mexiletine have been shown to be effective in the treatment of neuropathic pain due to peripheral nerve damage and diabetic neuropathies [V channels have a significant risk associated with them, as they affect skeletal muscle related NaV1.4, cardiac contraction associated NaV1.5 and nerve conduction linked NaV1.6 channels. Therefore, a potential analgesic conotoxin should avoid NaV1.4, 1.5 and possibly NaV1.6 channels to not to minimize the possibility of adverse side effects. In general, future studies should explore conotoxins that target combinations of NaV channels to attain effective pain relief with a minimal side effect profile.As discussed here, there is evidence to suggest that Naopathies ,132,133.


Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignancies in gastrointestinal tract cancers and even patients with early ESCC have a high metastatic potential. Difficulties are associated with clinically predicting tumor progression and prognosis based on conventional tumor markers determined from preoperative blood examinations. The aim of the present study was to measure plasma fibrinogen levels and the neutrophil–lymphocyte ratio (NLR) in blood and compare the clinical impacts of their combined values (fibrinogen and neutrophil–lymphocyte ratio score—F-NLR score) and the modified Glasgow Prognostic Score (mGPS) in patients with ESCC.We classified 238 patients with ESCC based on cut-off values for hyperfibrinogenemia (>400 mg/dL) and high NLR (>3.0) as F-NLR scores of 2 , 1 , or 0 . We also categorized patients based on cut-off values for high C-reactive protein (CRP) (>0.5 mg/dL) and hypoalbuminemia (<3.8 g/dL) as mGPS of 2 , 1 , or 0 .P < 0.05). Prognoses among the groups based on the F-NLR score and mGPS significantly differed . A multivariate analysis identified the depth of tumor invasion, lymph node metastasis, and F-NLR score as independent prognostic factors .The F-NLR score correlated with the depth of tumor invasion, lymph node metastasis, lymphovascular invasion, tumor size, and stage (all The results of the present study showed that the F-NLR score is a promising blood predictor for tumor progression and outcomes in patients with ESCC. Furthermore, difficulties are associated with clinically diagnosing the depth of tumor invasion and lymph node metastasis, which are established prognostic factors, based on preoperative examinations. Carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), and p53 are now clinically used in the management of patients with ESCC. However, these tumor markers are inadequate for identifying subclinical patients with early tumors and predicting disease recurrence.4Esophageal cancer is the tenth most common malignant disease and seventh leading cause of cancer death in Japan, with an estimated 17,497 new patients and 11,746 deaths in 2008.5 Previous studies reported the clinical impact of GPS as a predictor of tumor progression and prognosis in patients with various malignancies, including ESCC.10 The modified GPS (mGPS), based on adjusted cut-off values of CRP and albumin, has recently been introduced as a renewed predictive marker of disease outcomes.12 On the other hand, hyperfibrinogenemia and a high neutrophil–lymphocyte ratio (NLR) have been associated with an advanced tumor stage and poor prognosis in patients with various types of malignancies.18 Therefore, we proposed a novel prognostic marker based on a combined analysis of plasma fibrinogen values and the NLR in patients with malignant diseases. However, the clinical significance of the combined score determined by plasma fibrinogen levels and the NLR (the F-NLR score) in blood specimens from patients with ESCC has not yet been determined.The Glasgow Prognostic Score (GPS), constructed from C-reactive protein (CRP) and albumin, is an inflammation-based blood marker.The aim of the present study was to assess the relationship between the F-NLR score and clinicopathological factors, including prognosis, in patients with ESCC. We also compared the F-NLR score with mGPS in order to determine its clinical utility as a prognostic marker.19Table Between January 1998 and December 2012, 309 consecutive patients with ESCC underwent esophagectomy with lymphadenectomy at Kagoshima University Hospital . The inclusion criteria of this study were as follows: ESCC confirmed by histopathology; patients without endoscopic treatments; patients without palliative resection; patients without preoperative chemotherapy and/or radiotherapy; patients without synchronous or metachronous cancer in other organs; and blood specimens obtained within 2 weeks before surgery. Two hundred thirty-eight patients  who fit the criteria were enrolled in the present study. Patients were classified and staged based on the Union for International Cancer Control tumor-node-metastasis (TNM) classification for esophageal carcinoma.The Ethics Committee at Kagoshima University approved this study and all patients provided written informed consent to the use of their information.Blood specimens were obtained within 2 weeks before surgery. Plasma fibrinogen concentrations were determined using an STA-R coagulation analyzer . Neutrophils and lymphocytes were counted using an XE-2100 automated hematology analyzer  and the NLR was then calculated as the neutrophil count divided by the lymphocyte count. CRP and albumin were measured using a JCA-BM automatic analyzer .P-value of <0.05 was considered significant.Differences in the relationships between fibrinogen concentrations or the NLR and categorical clinicopathological features were assessed using the Wilcoxon rank sum test. Relationships between the F-NLR score or mGPS and categorical clinicopathological features were assessed using the Chi-squared test. Differences in survival generated using the Kaplan–Meier method were examined using the log-rank test. Prognostic factors were determined by univariate and multivariate analyses . All data were statistically analyzed using SAS statistical software . A P < 0.001). The mean concentrations (±SD) of plasma fibrinogen in 113 and 125 patients with N0 and ≥N1 nodal statuses were 351.6 ± 90.2 and 385.1 ± 85.0 mg/dL, respectively. Patients with lymph node metastasis had significantly higher fibrinogen levels than those without lymph node metastasis (P = 0.003). Moreover, plasma fibrinogen concentrations correlated with lymphatic and venous invasion, as well as tumor size . The mean concentrations (±SD) of plasma fibrinogen in 86 and 152 patients with stages I and ≥II cancer were 325.0 ± 72.1 and 394.1 ± 87.9 mg/dL, respectively. Categorical stage-related differences in plasma fibrinogen levels significantly differed (P < 0.001).Plasma fibrinogen concentrations ranged between 50 and 640 mg/dL in 238 patients with ESCC. The mean concentration (±SD) of plasma fibrinogen was 369.2 ± 88.9 mg/dL. The mean concentrations (±SD) of plasma fibrinogen in 145 and 93 patients with pT1–T2 and pT3–T4 tumors were 336.4 ± 73.4 and 420.2 ± 87.1 mg/dL, respectively. Hyperfibrinogenemia correlated with deeper tumor invasion  and low  plasma fibrinogen values. This binary classification of plasma fibrinogen levels was applied in subsequent prognostic analyses.We set the cut-off value of plasma fibrinogen concentrations at 400 mg/dL based on previous findings.P < 0.001). The mean NLR (±SD) in 113 and 125 patients with N0 and ≥N1 nodal statuses were 2.0 ± 0.9 and 2.5 ± 1.4, respectively. Patients with lymph node metastasis had significantly higher NLR than those without lymph node metastasis (P = 0.001). Furthermore, the NLR correlated with lymphatic and venous invasion as well as tumor size . The mean NLR (±SD) in 86 and 152 patients with stages I and ≥II cancer were 1.8 ± 0.8 and 2.5 ± 1.3, respectively. Categorical stage-related NLR significantly differed (P < 0.001).The NLR ranged between 0.6 and 10.6 in 238 patients with ESCC. The mean NLR (±SD) was 2.2 ± 1.2. The mean NLR (±SD) in 145 and 93 patients with pT1–T2 and pT3–T4 tumors were 1.9 ± 0.8 and 2.8 ± 1.4, respectively. The NLR correlated with the depth of tumor invasion  and low  NLR. This binary classification of the NLR was applied in subsequent prognostic analyses.The cut-off value of the NLR was set at 3.0 based on previous findingsP < 0.001; Figure P < 0.001; Figure The 5-year survival rate was significantly lower in patients with high fibrinogen values than in those with low fibrinogen values .We classified 238 patients with ESCC as follows: F-NLR score of 2, with hyperfibrinogenemia (>400 mg/dL) and high NLR (>3.0); F-NLR score of 1, with one of these abnormalities; F-NLR score of 0, with neither hyperfibrinogenemia nor high NLR. According to the criteria of the F-NLR score, 147 (61.8%), 64 (26.9%), and 27 (11.3%) patients had F-NLR scores of 0, 1, and 2, respectively. Table 11 According to the criteria of mGPS, 168 (70.6%), 54 (22.7%), and 16 (6.7%) patients had mGPS of 0, 1, and 2, respectively. Table P = 0.007, P < 0.001, P < 0.001, P = 0.041, and P = 0.021, respectively).We also categorized patients according to established mGPS criteria as follows: mGPS of 2, with elevated CRP (>0.5 mg/dL) and hypoalbuminemia (<3.8 g/dL); mGPS of 1, with one of these abnormalities; mGPS of 0, with neither elevated CRP nor hypoalbuminemia.P < 0.001). Figure P < 0.001).Figure P < 0.001, P < 0.001, P < 0.001, P = 0.004, P < 0.001, and P = 0.013, respectively). The multivariate analysis identified the depth of tumor invasion, lymph node metastasis, and F-NLR score as independent prognostic factors  . The incidence of lymph node metastasis in patients with an F-NLR score of 2 was higher than that in patients with mGPS of 2 (77.8% vs. 56.3%). Consequently, a relationship may exist between lymph node metastasis and the F-NLR score rather than mGPS. These results suggest that the F-NLR score is superior to mGPS for predicting the presence or absence of lymph node metastasis in patients with ESCC.GPS and mGPS are both established prognostic markers in various malignant neoplasms.We compared the prognostic impact of the F-NLR score and mGPS in the present study. The log-rank test revealed significant survival differences among patients with ESCC classified by F-NLR scores as well as mGPS. The 5-year survival rate of patients with an F-NLR score of 2 was 18.4%. Furthermore, none of the patients with an F-NLR score of 2 had stage I tumors. These results indicated that patients with an F-NLR score of 2 had more malignant tumors than those with F-NLR scores of 0 and 1. Furthermore, the 5-year survival rates of patients with high NLR and mGPS of 2 were 24.4% and 22.4%, respectively. These results demonstrated that patients with an F-NLR score of 2 have a poorer prognosis than those with high NLR or mGPS of 2. If the F-NLR score is preoperatively determined in patients with ESCC, we may be able to discriminate patients with the worst outcomes from all patients. Therefore, a classification system based on the F-NLR score may assist in the selection of patients with ESCC for neoadjuvant systemic chemotherapy or chemoradiotherapy in preoperative management. Moreover, the multivariate analysis selected the F-NLR score, but not mGPS as an independent prognostic factor. The hazard ratio for the F-NLR score was higher than that for mGPS (1.94 vs. 1.08). Therefore, the F-NLR score was found to be superior to mGPS for predicting survival in patients with ESCC.The present study had several limitations. The median follow-up period was only 26 months. Furthermore, this study was based on a retrospective analysis designed by a single institution. These limitations may have resulted in biases. Accordingly, larger validation studies are needed in order to strengthen the results obtained herein in patients with ESCC.In conclusion, we suggest that the F-NLR score determined by plasma fibrinogen values and the NLR serves as a prognostic blood marker in patients with ESCC. Our study demonstrated that a close relationship existed between cancer progression and the F-NLR score assessed from preoperative blood specimens. The F-NLR score may be more useful than mGPS as an economical biomarker to plan therapeutic strategies in preoperative patients with ESCC.


HLA genotyping by next generation sequencing (NGS) requires three basic steps, PCR, NGS, and allele assignment. Compared to the conventional methods, such as PCR-sequence specific oligonucleotide primers (SSOP) and -sequence based typing (SBT), PCR-NGS is extremely labor intensive and time consuming. In order to simplify and accelerate the NGS-based HLA genotyping method for multiple DNA samples, we developed and evaluated four multiplex PCR methods for genotyping up to nine classical HLA loci including HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DQB1, and HLA-DPB1.We developed multiplex PCR methods using newly and previously designed middle ranged PCR primer sets for genotyping different combinations of HLA loci, (1) HLA-DRB1/3/4/5, (2) HLA-DQB1 (3.8 kb to 5.3 kb), (3) HLA-A, HLA-B, HLA-C, and (4) HLA-DPB1 (4.6 kb to 7.2 kb). The primer sets were designed to genotype polymorphic exons to the field 3 level or 6-digit typing. When we evaluated the PCR method for genotyping all nine HLA loci (9LOCI) using 46 Japanese reference subjects who represented a distribution of more than 99.5% of the HLA alleles at each of the nine HLA loci, all of the 276 alleles genotyped, except for HLA-DRB3/4/5 alleles, were consistent with known alleles assigned by the conventional methods together with relevant locus balance and no excessive allelic imbalance. One multiplex PCR method (9LOCI) was able to provide precise genotyping data even when only 1 ng of genomic DNA was used for the PCR as a sample template.In this study, we have demonstrated that the multiplex PCR approach for NGS-based HLA genotyping could serve as an alternative routine HLA genotyping method, possibly replacing the conventional methods by providing an accelerated yet robust amplification step. The method also could provide significant merits for clinical applications with its ability to amplify lower quantity of samples and the cost-saving factors.The online version of this article (doi:10.1186/s12864-015-1514-4) contains supplementary material, which is available to authorized users. B. B25]. BIn this paper, we describe the development and evaluation of four types of multiplex PCR methods that genotype multiple HLA loci to the field 3 level (6-digit typing) using combinations of locus specific PCR primers for up to nine classical HLA loci . We evaluated the uniformity and accuracy of NGS-based HLA genotyping among the nine HLA loci and between HLA alleles obtained by one of the multiplex PCR methods (the nine loci [9LOCI] multiplex method) in a single NGS run with the Ion PGM sequencer using 46 genomic DNA reference samples from Japanese subjects who represented a distribution of more than 99.5% HLA alleles in each of the HLA locus in the Japanese population. In addition, we investigated template DNA amounts as low as 1 ng to evaluate the smallest amounts of genomic DNA samples that we could use successfully in our multiplex PCR methods for NGS-based HLA genotyping.Four types of multiplex PCR methods were developed after optimization of primer composition and PCR conditions such as annealing and extension temperatures using the HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DQB1, and HLA-DPB1 specific primer pair sets Figure . These fWhen we applied the nine loci multiplex PCR (9LOCI) method using 46 genomic DNA samples JPN01 to JPN46), the PCR products with similar band patterns were observed in all samples, although weak bands were also observed in some samples such as JPN23, JPN24, JPN25, and JPN29  on average) that were high quality sequence reads with more than 10 quality values (QV) and an average QV of 28.0 ± 0.2 in the high quality sequence reads. The draft read bases in total were 1,447 Mb with a range between 21.7 Mb in JPN021 and 43.4 Mb in JPN041 (31.4 ± 6.0 Mb on average), with an overall average read length of 273.3 ± 9.9 bases and an overall mode read length of 358.6 ± 16.7 bases . Through this process one synonymous substitution was newly detected in exon 4 of HLA-C*07:04 of JPN16.To evaluate the 9LOCI method, we compared the depth of redundancy derived from the sequence read numbers between HLA alleles and among HLA loci. An observed average depth and range for six HLA loci was as follows: 78.5 ± 42.0 from 31.6 to 225.1 for HLA-A, 116.5 ± 51.1 from 33.7 to 341.8 for HLA-B,130.0 ± 59.3 from 62 to 331.3 for HLA-C, 209.1 ± 115.9 from 44.1 to 712.4 for HLA-DRB1, 194.7 ± 104.9 from 74.9 to 614.4 for HLA-DQB1, and 59.2 ± 34.6 from 25.3 to 161.4 for HLA-DPB1  that is based on single molecule real-time (SMRT) technology (unpublished data).The high density in the average depth of sequences by NGS suggests that an increase in the DNA sample numbers for sequencing beyond 46 per run as described here is likely to contribute to even lower costs. For example, 85,879 sequence reads for sample JPN33 that was imbalanced at DRB1*04:06:01 was assigned with a sequence depth of 58.9. When we assume that an average of 85,879 sequence reads was obtained from a total of 5,284,570 reads that have a similar quality to those described in Additional file Although, a few samples like JPN15 (DPB1*05:01:01/DPB1*135:01 and DPB1*25:01) were not fully resolved by the multiplex PCR-NGS method, this problem could be solved in future by determining the full gene nucleotide sequence for the DPB1 gene with the *25:01 allele. Hence, it is necessary to comprehensively collect the HLA allele sequences for all of the PCR regions of all the HLA genes to avoid misidentifying the true locus because of potential problems of allele sharing between different loci or PCR amplification of sequences in the wrong gene regions. In this study, only five ambiguous HLA alleles, DRB1*04:07:01/*04:92, DRB1*04:10:01/*04:10:03, DRB1*09:01:02/*09:21, DRB1*12:01:01/*12:10, and DPB1*13:01/*107:01, were observed at 17 loci. In these cases, the ambiguities were not solved because the informative SNPs for these genes are outside of the PCR regions such as in some of the introns or the 5′ and 3′ non-coding regions or because the informative SNPs that differentiate between ambiguous alleles are located outside of the PCR regions such as within exon 1 or exon 4 of HLA-DRB1 or HLA-DPB1  was for HLA-DPB1 among the six loci with more than a read depth of 25 , but the 9LOCI method is likely to be the more valuable method for future routine genotyping for the following reasons and technical advantages. 1) The 9LOCI genotyping method is capable not only for typing the specific HLA loci such as HLA-A, HLA-B, and HLA-DRB1, but also other classical HLA loci such as HLA-C, HLA-DRB3/4/5, HLA-DQB1, and HLA-DPB1 at the same time   between 2006 and 2010 were retrospectively genotyped for HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, and HLA-DPB1 alleles to the field 2 level (4-digit typing) as described elsewhere . Of theshttp://www.1000genomes.org/) , 1.6 μL of 2.5 mM of each dNTP and 3.2-5.1 μL (10 pmol/μL) of each primer mixture. The cycling parameters were as follows: primary denaturation 94°C/2 min., followed by 30 cycles for 98°C/10 sec. and 70°C/4 min. The PCR reactions were performed using the thermal cycler GeneAmp PCR system 9700 . The DNA size was measured by using a 1 kb DNA ladder marker . The PCR products were purified by the Agencourt AMPure XP  and quantified by the PicoGreen assay  with a Fluoroskan Ascent micro-plate fluorometer .For PCR amplification of the multiplex PCR methods, the 20 μL PCR amplification-reaction-volume contained 1–25 ng of genomic DNA, 1 unit of PrimeSTAR GXL DNA polymerase , 4.0 μL of 5 × PrimeSTAR GXL Buffer . One hundred nanograms of the multiplex PCR products were used for the preparation of each DNA library. DNA samples were fragmented with a M220 Focused-ultrasonicator . Each DNA library was amplified by eight cycles of PCR. The DNA size and quantitation for each library was measured by an Agilent 2100 Expert Bioanalyzer using the Agilent High Sensitivity DNA Kit . Each barcoded-library was mixed at equimolar concentrations then diluted according to the manufacturer’s recommendation. Emulsion PCR (emPCR) was performed using the resulting pooled library with the Ion PGM Template OT2 400 Kit on an Ion OneTouch 2 automated system (Life Technologies) with the following cycling parameters: primary denaturation 95°C/10 min., followed by 20 cycles for 95°C/30 sec., 66°C/4 min., 20 cycles for 95°C/30 sec., 66°C/6 min. and 10 cycles for 95°C/30 sec., and 66°C/20 min. After the emulsion was automatically broken with the OneTouch 2 instrument, the beads carrying the single-stranded DNA templates were enriched according to the manufacturer’s recommendation. Sequencing was performed using the Ion PGM Sequencing 400 Kit and Ion 316 and 318 Chip Kit v2 with a flow number of 850 for 400 base-read [http://genome.ucsc.edu/), and thereafter, mapping of the sequence reads and the selected reference sequences were performed automatically with the GS Reference Mapper Ver. 3.0 software (Life Technologies). The mapping parameter was set to a perfectly matched condition between the read sequences and the reference sequences to avoid mismapping among the HLA loci and contamination of in vitro generated PCR crossover products [de novo assembly using the Sequencher Ver. 5.0.1 DNA sequence assembly software , and used it as a reference sequence.The raw data processing and base-calling, trimming and output of quality-filter sequence reads that were binned on the basis of the Ion Xpress Barcodes into 46 separate sequence fastq files, were all performed by the Torrent Suite 4.2.1 software (Life Technologies) with full processing for shotgun analysis. These files were further quality trimmed to remove poor sequence at the end of the reads with QVs of less than 10. The trimmed and barcode-binned sequence reads were used for HLA allele assignment to the field 3 and 4 levels by Sequence Alignment Based Assigning Software (SeaBass) . HLA allele candidates and/or reference sequences used for mapping of the sequence reads were selected by nucleotide similarity searches with HLA allele sequences in the IMGT-HLA database using the BLAT program (products . If a reAfter assignment of the HLA alleles we calculated uniformity among the HLA loci and between alleles using the sequence reads that separated to each allele. The read depth is the number of individual sequence reads that align to a particular nucleotide position . An aver


Furthermore, enhanced TKTL1 expression was put into context with resistance to chemotherapy and ionizing radiation. Here, a panel of human malign and benign cells, which cover a broad range of chemotherapy and radiation resistance as well as reliance on glucose metabolism, was analyzed 17 malign and three benign cell lines were characterized according to their expression of TKTL1 on the protein level with three commercially available anti-TKTL1 antibodies utilizing immunohistochemistry and Western blot, as well as on mRNA level with three published primer pairs for RT-qPCR. Furthermore, sensitivities to paclitaxel, cisplatin and ionizing radiation were assessed in cell survival assays. Glucose consumption and lactate production were quantified as surrogates for the “Warburg effect”.Considerable amounts of tktl1 mRNA and TKTL1 protein were detected only upon stable transfection of the human embryonic kidney cell line HEK293 with an expression plasmid for human TKTL1. Beyond that, weak expression of endogenous tktl1 mRNA was measured in the cell lines JAR and U251. Western blot analysis of JAR and U251 cells did not detect TKTL1 at the expected size of 65 kDa with all three antibodies specific for TKTL1 protein and immunohistochemical staining was observed with antibody JFC12T10 only. All other cell lines tested here revealed expression of tktl1 mRNA below detection limits and were negative for TKTL1 protein. However, in all cell lines including TKTL1-negative HEK293-control cells, antibody JFC12T10 detected multiple proteins with different molecular weights. Importantly, JAR and U251 did neither demonstrate an outstanding production of lactic acid nor increased resistance against chemotherapeutics or to ionizing radiation, respectively.in vitro. The presence of TKTL1 was unrelated to either the rate of glucose consumption/lactic acid production or resistance against chemo- and radiotherapy.Using RT-qPCR and three different antibodies we observed only exceptional occurrence of TKTL1 in a panel of malignant human cell lines The online version of this article (doi:10.1186/1471-2407-15-2) contains supplementary material, which is available to authorized users. TKT, together with the transaldolase enzyme, reversibly links the PPP to glycolysis [Transketolase TKT; EC: 2.2.1.1) is a thiamine-dependent key enzyme involved in the non-oxidative branch of the pentose phosphate pathway (PPP), which catalyzes the transfer of a 2-carbon fragment  as monolayers and harvested at 80-90% confluence using a cell-scraper (Biochrom) for further experiments. HEK293 cell transfectants stably producing full-length TKTL1 protein (293pCAG TKTL1) were used as positive control cells. HEK293 cells transfected with empty expression vector (293pCAG Δ) do not produce TKTL1 protein and were used as negative control cells. Both transfectants have been previously described in detail [The cell lines investigated herein were directly obtained from the companies given in Table 6 cells per cell line were harvested upon trypsinization, washed twice with PBS and fixed for 1 h in 4% PBS-buffered formalin at room temperature. All centrifugation steps were performed at 280x g at 4°C for 10 min. The pellet was then re-suspended in 150 μl PBS and mixed with the same quantity of pre-cooled 2% high-melting agarose . The mixture was cooled down on ice. The resulting clot was first transferred to a sample-embedding capsule and then to a 4% PBS-buffered formalin solution. The dehydration with graded alcohols and xylene, and the embedding into paraffin  were done automatically in parallel to tissue biopsy samples at the Institute of Pathology, University of Würzburg, in a Leica ASP200 S embedding unit. The resulting paraffinized cell clot samples were then set into paraffin blocks. Paraffin blocks with cell samples were cut into 2 μm thick sections and mounted up on aminopropylethoxysilane (APES)-coated slides. Slides for immunohistochemistry were rehydrated in descending concentrations of ethanol before being heated for antigen unmasking in 10 mmol/l sodium citrate buffer (pH 6.0) in a microwave oven at 600 W for 5 min. After rinsing in distilled water, inhibition of endoperoxidase was performed by incubating sections for 10 min in 3% H2O2 in methanol. Slides were washed in PBS and incubated with 1% human immunoglobulin  in PBS for 15 min to block FC-receptors [Approximately 1x10eceptors . Subsequeceptors , 40–43. 6 cells each were lysed in pre-cooled RIPA buffer  containing phosphatase inhibitors , proteinase inhibitors  and 2,5 mmol/l Dithiothreitol (DTT) reducing agent (Sigma-Aldrich). The mixture was incubated for 30 min on ice, combined with a thorough shaking every 10 min. Cell lysates were clarified of cell debris by centrifugation at 14,000x g for 5 min through a QIAshredder spin column assembly  at 4°C. Protein concentration was determined using the Bradford method [2O2[For protein extraction, 1x10d method  and coomthod [2O2 with sub6 cells each were disintegrated in Trizol reagent . Total RNA was extracted from Trizol as recommended by the manufacturer. RNA integrity was verified using the Experion automated electrophoresis station  and the RNA concentration was measured at 260 nm. For first strand cDNA synthesis, 1 μg total RNA was employed using the iScriptcDNA synthesis kit from BioRad. The cDNA synthesis of 1:2 diluted cDNA was performed by heating at 25°C for 5 min, at 42°C for 30 min, and at 85°C for 5 min.1x10TKTL1 primer pairs were used: primer pair 1 (150 bp), forward: TAACACCATGACGCCTACTGC, reverse: CATCCTAACAAGCTTTCGCTG [primer pair 2 (80 bp), forward: AAGCCTTTGGGTGGAAC ACTTA, reverse: CTGAGAAGCCTGCCAGAATACC [primer pair 3 (151 bp), forward: GGTATCTGTTGGTGACGATGGT, reverse: GCACATCCCCTTGGCATTGG [peptidyl propyl isomerase A , forward: TGTCCATGGCAAATGCTGGACCC, reverse: GCGCTCCAT GGCCTCCAC AA (140 bp); β-actin (access. No. NM_001101), forward: CCTTGCCATC CTAAAAGCC, reverse: CACGAAAGCAATGCTATCAC (96 bp). qPCR reactions were performed on a CFX96 real-time PCR system (Bio-Rad) operated by CFX Manager Software (version 3.0). The cycler protocol was 5 min at 95°C, 40 cycles of 15 sec at 95°C, 60 sec at 60°C, and 5 min at 72°C. Gene of interest expression was normalized to the reference genes PPIA and β-actin [Quantitative PCR (qPCR) was performed with MESA Green qPCRMasterMix Kit for SYBR Green containing Meteor Taq hotstart polymerase . Three sets of TTTCGCTG primer paAGAATACC ; primer GGCATTGG . TKTL1 p β-actin . Fold ex β-actin . Post-am5 cells/ml in 48-well flat bottom tissue plates  with culture medium and were grown for 24 h. Afterwards, medium was completely replaced, and glucose consumption and lactate production at 21% oxygen were analyzed 24 h later. Cell-free supernatant was snap frozen for analysis and cell numbers were calculated with the crystal violet assay [Cells were seeded at 1x1050 values.Adherently growing cell lines were seeded at 5,000 cells per 100 μl of cell culture medium in a 96 well flat bottom tissue culture plate  and grown for 24 h prior to treatment with chemotherapeutic drugs. Then, 100 μl cell culture medium containing paclitaxel or cisplatin (both from Sigma-Aldrich) were added, resulting in final drug concentrations ranging from 1% to 200% of the reported peak plasma concentrations achievable in patients . The max2. Medium was completely replaced after 60 min to start with fresh growth conditions. In addition, cell medium was replaced on day 4 and day 8. Ten days after irradiation, the CCK-8 assay was performed as described above.Cell lines were seeded at 100 cells in 200 μl cell culture medium in a 96 well flat bottom tissue culture plate  24 h prior to radiation. Graded X-irradiation  was then performed at room temperature using a 6 MV linear accelerator  at a dose rate of 3 Gy/min. Control cells were treated in a similar way, but without irradiation. Each radiation dose was applied in triplicates. 20 min after irradiation cells were cultivated at 37°C in a humidified incubator at 5% COSo far, TKTL1 protein expression was described primarily in paraffin-embedded tumor tissue. Hence, in order to produce comparable datasets, paraffin-embedded cell pellets of all selected cell lines (n = 20) served to analyze the expression of TKTL1 protein by immunohistochemistry. The cell lines assessed in the present study are summarized in Table Immunohistochemical staining with JFC12T10 and Sigma Prestige antibodies showed a strong cytoplasmatic staining in HEK293-TKTL1 transfectants, with an additional nuclear staining by the Sigma Prestige polyclonal antibody. JFC12T10, but not the Sigma Prestige antibody also stained the cytoplasm of TKTL1-negative HEK293-control cells. The staining intensity of JFC12T10 in HEK293-control and HEK293-TKTL1 transfectants was comparable indicating an unspecific staining pattern of JFC12T10 in HEK293-control transfectants  and published previously [Quantitative RT-PCR (RT-qPCR) was performed with three different primer pairs specific for the three eviously , 28, 35.High expression of TKTL1 was put into context of enhanced glucose consumption within the pentose-phosphate pathway, resulting in increased lactic acid production . In orde50 values were calculated , whereas U251 and MCF-7 were resistant to paclitaxel and Mel2A, and 23132/87 and HepG2 were resistant to cisplatin (IC50 > 35 nmol/ml). JAR cells displayed intermediate and high sensitivity towards placlitaxel and cisplatin, respectively  were subjected to treatment with paclitaxel and cisplatin in concentrations ranging from 0.2-31.8 nmol/ml and 0.1-25.4 nmol/ml, respectively. Representative results of dose–response curves after treatment with paclitaxel and cisplatin are shown  characterized by weak endogenous tktl1 mRNA expression (JAR and U251). Immunohistochemical proof of TKTL1 protein in cytoplasm, the expected location of TKTL1 as a protein which is supposed to be instrumental in the pentose-phosphate pathway, was successful in these two cell lines with JFC12T10. A very faint staining was seen for JAR cells while U251 cells were negative using Sigma Prestige polyclonal antibody, thus confirming negative Western blot results. All other cell lines demonstrated much lower tktl1 mRNA expression levels and no detection of TKTL1 protein in cell lysates by anti-TKTL1 antibodies 1C10 and Sigma Prestige, but multiple detections of proteins with apparent molecular weights above or below the calculated size of TKTL1 by anti-TKTL1 antibody JFC12T10. A relation of glucose consumption, lactic acid production and chemo- and radioresistance with expression levels of TKTL1 was not observed in any cell line tested in the present study. JAR and U251 cells did not show outstanding “aggressiveness” as measured by their resistance to both chemotherapeutic drugs and radiation or their ability to produce lactate  has gained increased attention due to several studies showing a TKTL1-positive staining of malignant tumor tissue of different origins , 43, 51 In this study, we tested three commercially available anti-TKTL1 antibodies for Western blot and immunohistochemistry staining as well as three primer pairs used in previously published work for the detection of tktl1 mRNA by RT-qPCR , 28, 35.in vitro.HeLa cells , colorecb anti-TKTL1 antibody clone JFC12T10, first described in 2005 [E. coli with putative smaller cleavage products. Furthermore, JFC12T10 showed a unique expression pattern of five different tumor cell lines with a predominant protein band at 75 kDa by Western blot analysis [Interestingly, the vast majority of studies based on antibody-based techniques published so far used the commercially available mouse monoclonal IgG2 in 2005 . JFC12T1analysis . This inanalysis .The second anti-TKTL1 antibody used for Western blot analysis was the monoclonal antibody clone 1C10 that detected a distinct single TKTL1 protein with approximately 65 kDa in HEK293-TKTL1 transfectants, without the additional protein bands seen with JFC12T10. Also, polyclonal Sigma Prestige antibody did not recognize additional proteins with different molecular weights in any of the cell lines. The marginal signals in a few cell lines seen with monoclonal 1C10 antibody most probably represents unspecific background, which was eventually similar in quality but inferior in quantity as compared to JFC12T10. Thus, monoclonal 1C10 and polyclonal Sigma Prestige antibody detect exogenously expressed TKTL1 in a highly specific manner and as a protein of expectedly 65.4 kDa.However, using a rabbit polyclonal antibody (Gene Tex), Li et al. detected endogenous TKTL1 expression in MCF-7 and HeLa cells by Western blot . This reSimilarly, immunohistochemistry analyses presented here support a rather rare expression of TKTL1 protein in cancer and normal cell lines. In 2 out of 3 benign and 8 out of 17 malign cell lines tested in the present study, TKTL1 protein was detectable in the cytoplasm with the two antibodies JFC12T10 and SigmaPrestige polyclonal antibody by immunohistochemistry. For only one of these positively stained cell lines (JAR) a weak expression of tktl1 mRNA was detectable. Therefore JAR appears to be positive for endogenous TKTL1 expression by RT-qPCR and immunohistochemistry, however failed to be detected by Western blot. The other cell line expressing tktl1 mRNA, U251, was positive in immunohistochemistry with the JFC12T10 antibody only and also showed no specific signal in Western blot.in vitro (Warburg effect) nor greater resistance against taxane and cisplatin or ionizing radiation than cells without clear TKTL1 detection. These findings are inconsistent with the hypothesis of TKTL1 being a predictor of an increased “Warburg effect” and special robustness of tumor cells against chemotherapy and radiation in vitro.In summary, two cell lines  were found positive for both endogenous mRNA expression as well as TKTL1 protein by immunohistochemistry with one or two different anti-TKTL1 antibodies. Intriguingly, these cell lines did neither exhibit an extraordinary production of lactic acid at 21% oxygen in vitro. TKTL1 expression emerged as a rather rare event in cultured cell lines. The two out of 17 human cancer cell lines JAR and U251, positive for tktl1 mRNA and TKTL1 protein by immunohistochemistry and Western blot for at least one antibody showed neither an outstanding production of lactic acid nor increased resistance against the chemotherapeutic drugs paclitaxel and cisplatin or to ionizing radiation, respectively, compared to the other cell lines.In this study we have demonstrated the necessity to carefully prove TKTL1 expression on mRNA and protein levels with multiple analytic tools  to identify cells with robust endogenous TKTL1 expression Additional file 1: Figure S1: Long time exposure of Western Blot. To make the WB results comparable, we exposed the x-Ray film to an extent, that the positive control cells (HEK293-TKTL1) resulted in a comparable intensity of band signal at 65.4 kDa. The negative HEK293 control cells as well as the tktl1 mRNA positive cell lines JAR and U251 were also shown. (PPT 10 MB)5 cells each. Summary of three independent experiments. (DOC 40 KB)Additional file 2: Table S1: Concentrations of glucose and lactate. Mean and standard deviation is shown for glucose consumption and lactate production of cell lines analyzed after 24 h of culture calculated per 10


Mesp1-expressing anterior mesoderm during heart development. Conditional ablation of Chd7 in this lineage results in major structural cardiovascular defects akin to those seen in CHARGE patients, as well as a striking loss of cardiac innervation and embryonic lethality. Genome-wide transcriptional analysis identified aberrant expression of key components of the Class 3 Semaphorin and Slit–Robo signalling pathways in Chd7fl/fl;Mesp1-Cre mutant hearts. CHD7 localises at the Sema3c promoter in vivo, with alteration of the local chromatin structure seen following Chd7 ablation, suggestive of direct transcriptional regulation. Furthermore, we uncover a novel role for CHD7 activity upstream of critical calcium handling genes, and demonstrate an associated functional defect in the ability of cardiomyocytes to undergo excitation–contraction coupling. This work therefore reveals the importance of CHD7 in the cardiogenic mesoderm for multiple processes during cardiovascular development.CHARGE syndrome is caused by spontaneous loss-of-function mutations to the ATP-dependant chromatin remodeller chromodomain-helicase-DNA-binding protein 7 (CHD7). It is characterised by a distinct pattern of congenital anomalies, including cardiovascular malformations. Disruption to the neural crest lineage has previously been emphasised in the aetiology of this developmental disorder. We present evidence for an additional requirement for CHD7 activity in the  •Chd7 knockdown in the cardiogenic mesoderm leads to major structural heart defects.•Cardiomyocyte excitation–contraction coupling is disrupted after Chd7 ablation.•Components of Semaphorin, Slit–Robo and calcium handling pathways are dysregulated.•Sema3C promoter.CHD7 directly binds and alters the local chromatin structure at the  The impCHD7 causes human CHARGE syndrome in over 90% of clinical cases 10.5 due to p53-dependant growth failure  and interrupted aortic arch type B (IAA-B)  , althougChd7 in the early cardiogenic mesoderm, driven by Mesp1-Cre, results in major structural defects and gene dysregulation, leading to cardiac failure and embryonic lethality around E15.5. Endocardial-specific ablation of Chd7 also results in septation and great vessel defects, indicating disruption to endocardium development is contributing to these malformations. CHD7 action lies upstream of key extracellular signalling molecules, including components of the Semaphorin and Slit–Robo pathways, as well as cardiac calcium handling genes, with consequences for excitation–contraction coupling in cardiomyocytes. Together, these results indicate that disruption to CHD7 activity in the cardiogenic mesoderm significantly contributes to the cardiovascular malformations seen in CHARGE patients.There are three known developmental origins for the cells that make up the mature heart: the cardiogenic mesoderm, from which the myocardium and endocardium are derived ; cardiac22.1Chd7 allele (Chd7fl/fl) were crossed with Chd7fl/+;Mesp1-Cre mice, to homozygously ablate Chd7 expression in the anterior mesoderm  confirmed loss of Chd7 mRNA throughout the heart in E11.5 Chd7fl/fl;Mesp1-Cre embryos  showed severe oedema and/or haemorrhaging, indicative of cardiac failure  of Chd7fl/fl;Mesp1-Cre embryos had interrupted aortic arch type B  malformations seen at E10.5, and Chd7 expression in the pharyngeal surface ectoderm – but not the mesoderm – was shown to be required for this early PAA morphogenesis  had double outlet arising from the right ventricle  staining of sections showed major structural defects in le DORV, B, and co.5 n=10, C. WhilstChd7fl/fl;Mesp1-Cre hearts  to form at the venous pole of the heart. It is this spine that drives the separation of the AV canal into its left and right components and carries forward the inferior ends of the venous valves . To invee hearts A. This ie hearts B. These nd E10.5 . Despiteor E11.5 , indicatChd7fl/fl;Mesp1-Cre hearts correlate well with the reported clinical CHARGE malformations  in the heart at E11.5 or E13.5 following mesodermal deletion of Chd7 are available in Supplemental 2 FC>0.5, adj. p<0.05-see Chd7fl/fl;Mesp1-Cre hearts  for CHD7 followed by qRT-PCR performed on chromatin from dissected E11.5 hearts showed enrichment for CHD7 specifically at the promoter region of Sema3c  and Chd7fl/fl;Mesp1-Cre cardiomyocytes: 95% (n=22) of control cells responded to electrical pacing with regular Ca2+ transients recorded at the expected 1 s intervals, but only 39% (n=23) of Chd7fl/fl;Mesp1-Cre cells could be paced in this way  as cardiogenesis progresses, from transcription factors such as NKX2-5, to extracellular signalling and Ca2+ handling genes. Furthermore, Sema3c is identified as a novel direct target of CHD7 in the developing heart in vivo. Therefore, disruption to transcriptional networks in both the cardiac neural crest and cardiogenic mesoderm likely contribute to the cardiovascular defects seen in CHARGE patients due to haploinsufficient loss of CHD7 activity.We have demonstrated a crucial role for CHD7 activity in the mesodermal lineage for the alignment, septation and maturation of the heart, as well as cardiac innervation, vascularisation, and remodelling of the great vessels. Ablation of 4Further details of Materials and methods are included in the 4.1Chd7fl Wtsi), Mesp1-Cre Ysa), and Tie2-Cre 1Ywa). All lines were maintained on a C57Bl/6 J background. CD1 mice were used for CHD7 immunofluorescence and ChIP experiments. The date of observation of a vaginal plug was considered embryonic day E0.5.Animal maintenance, husbandry and procedures were carried out in accordance with British Home Office regulations. The mouse lines used were: conditional 4.2Embryos were fixed overnight in 4% PFA/PBS and mounted in low-melting agarose (Life Technologies). Samples were then trimmed and washed in 100% methanol followed by clearing in 2BA:1BB , before analysis using a Bioptonics OPT Scanner 3001M . NRecon software (Skyscan NV) was used for image reconstruction from projections using a back-projection algorithm, and ImageJ was used for image analysis.4.3Chd7fl/fl;Mesp1-Cre hearts was compared to cDNA from four Chd7+/+;Mesp1-Cre hearts. The data was normalised with the RMA normalisation algorithm using the Affy R package (E11.5 or 13.5 hearts were dissected into RNase-free PBS and stored in Buffer RLT (Qiagen) at −80 °C until genotyped. Total RNA was isolated using RNeasy Mini kits (Qiagen), including the optional on-column DNase digestion step. Sense-strand cDNA was generated using the Ambion® WT Expression Kit and processed for hybridisation onto the Affymetrix Mouse Gene 1.0 ST Array according to manufacturer's protocols. Scanning was performed on the Affymetrix GeneChip Scanner. For each time point four biological replicates were run in each group: cDNA from four individual  package  provided package  and anal package . Data ha package .4.4Sema3a and Sema3c from Christiana Ruhrberg; Robo2 from Gail Martin; and Slit2 from Bill Andrews . The Chd7 probe was generated using TOPO® TA Cloning® Kits (Life Technologies) with previously reported primers  glutarate (Sigma) for 45 min and 1% formaldehyde for 15 min. Hearts were quenched in 0.125 M glycine (Sigma) and washed in cold PBS. Tissue was lysed and passed through 21G and 23G needles five times each. Cells were pelleted by centrifugation and chromatin was released in 1% SDS buffer and sheared using a BioRuptor Sonicator (Diagenode), to an average size of 500 bp. All steps included 1× protease inhibitor cocktail (Roche).ChIP was performed as described previously  with ant4.62, 0.1% NP40). Nuclei were treated with increasing concentrations of DNase I (Promega) at 37 °C for 10 min. 25 mM EDTA was added to stop reactions and samples were RNaseA and Proteinase K treated, before purification using a Qiaquick PCR purification kit. Eluted DNA was quantified using a Qubit fluorometer (Life Technologies) and diluted to the same concentration, before RT-PCR analysis using primers designed around gene promoters  containing 10 µg/ml DNase I (Sigma) at 37 °C for 45 min. Trypsin was inhibited with 10% FBS (Life Technologies) and samples passed through a 23G needle three times. Cells were pelleted by centrifugation and nuclei released in lysis buffer  containing 10 µg/ml DNase I (Sigma) at 37 °C for 45 min. Trypsin was inhibited with 10% FBS (Life Technologies) and samples passed through a 23G needle three times. Cells were pelleted by centrifugation and resuspended in 1 ml culture medium  before plating for 1.5 h to allow adherence of fibroblasts. The supernatant containing cardiomyocytes was then plated onto laminin-coated (Sigma) coverslip dishes, and cells cultured at 37 °C, 5% CO2 for 36 h. Plating efficiency and survival of Chd7fl/fl;Mesp1-Cre cells did not appear compromised.Individual 4·7H20, 1.25 mM K2HPO4, 2 mM CaCl2, 10 mM HEPES, 10 mM d-Glucose, pH7.4) before incubation at 37 °C for 10 min in imaging buffer/Opti-MEM®  containing the Ca2+-sensitive fluorogenic dye 5 µM Cal-520™ (Stratech). A Leica SP5 confocal inverted microscope performed rapid line scan analysis (400 Hz) of Ca2+ transients in individual cells . Cells producing Ca2+ transients were chosen randomly and were electrically paced by field stimulation at 1 Hz using platinum electrodes. Data was collected and analysed by averaging values across the line, over time, using Leica LAS AF software. “Paced” cells were classified as those in which Ca2+ transients were observed every 1 Hz over a 40 s interval, whereas in “non-paced” cells, Ca2+ transients were absent or unsynchronised. p-values were calculated using two-tailed Fisher's exact test.For imaging, cells were washed once in imaging buffer (156 mM NaCl, 3 mM KCl, 2 mM MgSO2+ assays and RHA provided assistance with anatomical analysis of mutant hearts.SP, KM and PJS designed experiments, SP performed experiments and prepared the manuscript. MJB designed and performed ChIP and DNase assays, and analysed the microarray data. NP performed experiments and provided technical support. SD helped establish CaThe authors declare that they have no conflict of interest.


Different carbohydrate diets have been administrated to diabetic patients to evaluate the glycemic response, while Poor-controlled diabetes is increasing world wide. To investigate the role of an alternative carbohydrate diet on glycemic control, we explored the effect of a low glycemic load (Low GL)-high fat diet on glycemic response and also glycated hemoglobin (HbA1c) of poor-controlled diabetes patients. Hundred poorly-controlled diabetes patients, HbA1c > 8, age 52.8 ± 4.5 y, were administrated a low GL diet, GL = 67  for 10 weeks. Patients did their routine life style program during intervention. Fasting blood glucose and HbA1c before and after intervention with significant reduction were: 169 ± 17, 141 ± 12; 8.85% (73 mmol/mol) ± 0.22%, and 7.81% (62 mmol/mol) ± 0.27%; respectively (P < 0.001). Mean fasting blood glucose reduced by 28.1 ± 12.5 and HbA1c by 1.1% (11 mmol/mol) ± 0.3% (P=0.001). There was positive moderate correlation between HbA1c concentration before intervention and FBS reduction after intervention , and strong positive correlation between FBS before intervention and FBS reduction . This study demonstrated that our alternative low glycemic load diet can be effective in glycemic control. Poorly-controlled diabetes that is characterized with increased glycated hemoglobin (HbA1c) > 8% (64 mmol/mol)  is increTherefore the aim of the present study was to investigate the role of low glycemic load diet having lower amount of carbohydrate and higher fat content than traditionally introduced diets as an alternative approach to reduce glycemic response to carbohydrate and also reducing HbA1c concentration of poor-controlled diabetes. We hypothesized that carbohydrate-based low glycemic load diet (GL ≈ 67), with 36% fat, and 42% carbohydrate suppress glycemic response and reduces HbA1c concentration in poor-controlled diabetes.One hundred and twenty Poor-controlled (HbA1c > 8%)  diabetes2 who had recruitment criteria took part in this study. Fifteen persons had BMI ≤ 25, while 85 persons had BMI > 25. The mean values for data are shown in P <0.001). Mean fasting blood glucose reduced by 28.1 ± 12.5 mg/dl (16.6%), HbA1c by 1.1% (11 mmol/mol) ± 0.3%, weight by 3.3 ± 1 kg and BMI by 1.2 ± 0.4 kg/m2 after diet intervention (P <0.001). There were positive weak correlation between BMI kg/m2 before intervention and HbA1c level reductions , between BMI kg/m2 reduction and HbA1c reduction , and between HbA1c concentration before intervention and HbA1c reduction . Also there was positive moderate correlation between HbA1c concentration before intervention and FBS reduction , and strong positive correlation between FBS before intervention and FBS reduction , , aged 52.8 ± 4.5 y, weight 74.0 ± 5 kg, BMI = 27.2 ± 1.9 kg/m= 0.70), . ObserveThis study showed a significant effect of low glycemic load diet on FBS and HbA1c. In our study as we hypothesized, the administrated low glycemic load diet suppressed the HbA1c of poor-controlled diabetes patients to 7.8% (62 mmol/mol) ± 0.3% level which does not considered as poorly-controlled level  and was This study revealed that the more sever the dysglycemia, the greater effect of low GI diet on glycemic control was observed. This finding was parallel with point view of conducted workshop by Howlett and colleagues . Similarin weight management of type 2 diabetes, replacing carbohydrate with monounsaturated fats reduces post prandial glycemia and triglyceridemia” and recommends that carbohydrates and monounsaturated fat together should provide 60-70% of the energy intake in which their ratio should be individualized. However, alternatively, there is statement from ADA which limits carbohydrate intake to 45-65% of the calories intake  and consequently increases insulin sensitivity. Weight loss of 5-10% of initial body weight may significantly improve glycemic and other metabolic abnormalities, and prevents the development of diabetes in high risk populations . We observed 3.3%, 4.6% and 4.4% weight loss in persons having BMI ≤ 25, BMI > 25, and in total subjects respectively. Due to observed poor correlation between BMI kg/m2 and HbA1c and lack of any correlation between BMI reduction and FBS reduction, the weight loss in our study, less likely affected on glycemic profile improvement. We believe that the effect of administrated low glycemic load diet was dominant for weight reduction, appetite and also suppress postprandial blood glucose through slow absorption and resulting in reducing HbA1c. We did not have control group which was the limitation of our study.Different factors in current study may affected on glycemic control including: moderate energy intake (24 kca/per kg bod weight), low glycemic load diet, and consumption of monounsaturated fatty acids. Moderate energy intake lowers body weight (Freedman Our provided meal plan for glycemic control of poor-controlled diabetes subjects is appropriate and further investigation for long term effect of low GI diet for glycemic control of poor-controlled diabetes patients is suggested.