Solanum melongena L.) is known to cause food allergy in some Asian countries but detailed studies on eggplant allergy are lacking.Eggplant  by skin prick test, allergen-specific IgE, and immunoblots. Allergens were analyzed for glycoprotein nature by staining/lectinoblots, and in vitro stability in simulated gastric fluid.All the eggplant-sensitized subjects showed positive skin prick test with peel, pulp, raw, and cooked eggplant extracts; allergen-specific IgE to all these was positive. Raw eggplant contains 5 allergens in the range 36-71 kD. Most allergens are localized in the eggplant peel  than the pulp ; among these, the 26, 28, 36, and 71 kD allergens seem to be heat-stable. The 43, 45, 64, and 71 kD allergens are detected as glycoproteins; the 26, 64, and 71 kD allergens are stable displaying retention of IgE-binding ability in simulated gastric fluid digestion.Eggplant is a multiallergenic vegetable in the context of presence of allergens in all edible parts of eggplant having preponderance in the peel. Solanum melongena L.) has been cultivated in Asia, Europe, and other parts of the world  at 4°C overnight. The secondary antibody used was ALP-conjugated mouse antihuman IgE (Sigma-Aldrich) at 1:1000 dilution. The immunoblots were developed using the insoluble substrate BCIP-NBT (1: 5 dilutions in TBS-T without BSA).Allergenic profiling was carried out using sera from 6 eggplant-allergic subjects showing positive SPT to peel, pulp, raw fruit and cooked extracts of eggplant. Approximately 40 The eggplant peel extract was subjected to SDS-PAGE. After electrophoresis, one half was stained for glycoproteins by periodic acid-Schiff (PAS) staining  and the μg protein was dissolved in 60 μL of prewarmed distilled water. Twenty microliters of prewarmed 0.4 M HCl containing 8 g/L of NaCl and 1.28% of pepsin , were added. Digestion proceeded at 37°C with continuous shaking. Similarly, BSA, a protein that is considered to have a lesser allergenic potential,[μL) of the digest was periodically withdrawn, digestion was stopped with 6 μL of 0.2 M Na2CO3, and samples were mixed with 6.5 μL of 5 × sample buffer for SDS-PAGE analysis. The contents of the reaction were subsequently heated at 95°C for 5 minutes. Fifteen microliters of each sample were loaded per lane (~20 μg protein per well) for separation by SDS-PAGE; one set was visualized by silver staining and the other set (~40 μg protein per well) was transferred onto nitrocellulose membrane for IgE-immunoblot experiments with pooled eggplant-allergic sera.Proteins from eggplant peel extract were digested in simulated gastric fluid (SGF) according to a previously published protocol . BrieflyThe wheal/flare diameter (in millimeters) obtained in SPT for the 6 study subjects were in the range of 4.5-6.0/16-30, 4.0-4.5/0-16, and 4.5-5.0/16-20 for eggplant peel, pulp, and raw eggplant (raw) extracts, respectively  . Similarly, there was no significant difference between raw and cooked eggplant extracts in terms of specific IgE (A405) values  Table . Althouges Table .The protein content of extracts from different parts of eggplant fruit are as follows: 26 mg/100 g eggplant peel, 19 mg/100 g eggplant pulp, 21 mg/100 g eggplant fruit (raw), and 2.4 mg/100 g eggplant fruit (cooked), all expressed on fresh weight basis (data not shown). The protein patterns of the eggplant peel, pulp, raw, and cooked eggplant extracts are represented in Figure When immunoblots were performed using raw eggplant extract and individual eggplant-allergic sera, the IgE-binding patterns in all the 6 cases were similar to the pattern observed with raw eggplant extract using pooled eggplant-allergic sera shown in Figure From the foregoing analysis, it is clear that eggplant peel contains the majority of the allergens found in eggplant fruit. Therefore, eggplant peel extract was subjected to immunoblotting and probed with individual serum of eggplant-allergic subjects. The results are shown in Figure Because all the allergens present in eggplant pulp and eggplant fruit (raw) with the exception of 52 kD were also present in the eggplant peel, we analyzed the allergens present in eggplant peel extract for the presence of glycans in the allergenic proteins and, subsequently, their in vitro gastric stability using SGF.N-linked glycans) in these glycoproteins.PAS staining after SDS-PAGE of the eggplant peel extract detected the 43 and 45 kD as prominent glycoprotein bands whereas the 64 and 71 kD allergenic proteins appeared as faint, but sharp bands indicating the presence of glycans in all the 4 proteins , 38 kD, 64 kD (band b), and 71 kD (band a) allergens appeared as faint but intact protein bands at 120 minutes of digestion, whereas the 45 kD allergen band (band c) remained intact up to 30 minutes and disappeared later as visualized by silver staining  displayed uniform IgE-binding at all time intervals from 0 to 120 minutes of digestion Figure . ProteinEggplant is known to induce IgE-mediated hypersensitive reactions in sensitized individuals with a wide spectrum of symptoms varying from skin rashes, wheezing, itching of the eye, gastrointestinal abnormalities, and itching and hoarseness of the throat; prevalence is ~0.8% having a female predominance ,14,15. OThe most common clinical presentation of eggplant allergy in the study subjects is either localized or generalized skin rashes (urticaria) affecting different parts of the body within 1-2 hours of ingestion of eggplant in cooked form. In the present study, 4 of 6 eggplant-allergic subjects had other atopic conditions mostly related to respiratory allergies. Generally, food allergy tends to occur in subjects who also have other inhalant allergies, but not always so . EggplanThe SPTs with eggplant peel produced more pronounced wheal/flare response compared with pulp, indicating different parts of eggplant fruit can have different allergenicities. A similar trend was noticed in the case of SPTs with peel and pulp extracts of Rosaceae fruits, figs, and tomato -37. UnliAll the eggplant-sensitized subjects have shown specific IgE to both eggplant peel and pulp extracts, indicating the presence of IgE-binding components. The relatively higher specific-IgE values for eggplant peel than the pulp further demonstrate varying allergenicities of different parts of eggplant fruit. Allergen-specific IgE to both raw and cooked eggplant extracts showed similar IgE-binding patterns comparable to earlier studies,,13 whichImmunoblotting with pooled eggplant-allergic sera detected 9 IgE-binding proteins between 26-71 kD in eggplant peel, and 3 major IgE-binding proteins between 52-71 kD in eggplant pulp. However, in the raw eggplant extract, 5 IgE-binding proteins between 36-71 kD were detected. This clearly indicates that most of the allergens are concentrated in the eggplant peel. The prevalence of allergenic proteins in the external layer such as peel is a common feature in the case of figs  and other allergenic fruits of the Rosaceae family such as apple, peach, and pear ,35,39,40Gel electrophoresis by silver staining revealed the presence of 4-6 protein bands in cooked eggplant extract; it may be noted here that the 71 kD protein band was identified as a heat-stable protein in our earlier study . HoweverAmong the allergenic components identified in the eggplant peel, the 45, 64, and 71 kD allergens are glycoproteins as seen in lectinoblots using Con A, a lectin having mannose/glucose specificity. Although the 43 kD protein showed glycoprotein nature by PAS-staining, its nondetection in lectinoblots could possibly be because of the absence of mannose residues in the glycoprotein. Because many of the cross-reactive food allergens are glycated in nature,,33 it maThe relatively uniform IgE-binding of the 3 allergens  in eggplant peel after SGF digestion further strengthens the point that the allergenic proteins exhibit persistence in gastric environment that has been considered as an important criterion for allergenicity assessment ,46. In fSeveral plant allergens correspond to proteins having a defense role in plants . BecauseBheemanapalli N. Harish Babu, MSc (Agri), is currently at All India Coordinated Research Project (Safflower), Agricultural Research Station, Annigeri-582201, Dharwad District, Karnataka State, India.Source of support: Grant-in-aid project (No. BT/PR6281/AGR/16/574/2005) to Y.P.V. from the Department of Biotechnology, Government of India, New Delhi, India. Senior research fellowship to B.N.H. from the Council of Scientific & Industrial Research, New Delhi, India.


The crystal structure of a nucleotide exchange factor in white blood cells reveals an autoinhibitory mechanism that reinforces the switch-like behaviour of the signalling protein Ras. Related research article Iwig JS, Vercoulen Y, Das R, Barros T, Limnander A, Che Y, Pelton JG, Wemmer DE, Roose JP, Kuriyan J. 2013. Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1. eLife2:e00813. doi: 10.7554/eLife.00813Image A linker region between two domains of RasGRP1 (red) blocks the Ras binding site in the absence of calcium ions and diacylglycerol (not shown)Cells in many organisms can modify their behaviour after receiving signals from elsewhere in the organism. In principle, these signals can evoke two types of responses: a linear response or a switch-like response. The regulatory circuits that control various metabolic pathways within the body often involve linear responses: in other words, the strength of the response depends on the strength of the input signal. However, the signalling pathways that regulate the proliferation, death or survival of cells often involve switch-like responses: that is, there is no response when the input signal is below a certain threshold, and there is a full response when the input is above this threshold.One prototypical switch in the cell is a small protein called Ras that can exist in two different conformations (shapes), depending on whether it is bound to the regulatory small molecules GTP or GDP . When iteLife, a collaboration between the laboratories of John Kuriyan, of UC Berkeley, and Jeroen Roose, of UC San Francisco, reports new insights into one of these regulatory proteins, a factor important for the development of white blood cells , thus ensuring that signalling by Ras is tightly regulated under normal conditions. Now, in od cells .Two categories of proteins can regulate Ras . GAPs G. These GRasGRP1 has been found to consist of several domains. The catalytic Cdc25 domain is responsible for interacting with Ras and for enhancing the exchange of GDP for GTP. RasGRP1 also contains a C1 domain that can direct it to the membrane, where it can associate with Ras. Through another domain, the EF-hand domain, RasGRP1 can bind calcium ions, which results in significant changes in conformation. Finally, a C-terminal coiled-coil region can assist RasGRP1 in forming dimers.It has previously been shown that SOS can be prevented from binding to Ras by an autoinhibitory mechanism, in which the Ras binding site on the catalytic domain of SOS is blocked by another portion of the SOS protein. Interestingly, the activation of SOS requires activated Ras: GTP-bound Ras can associate with an allosteric binding site on SOS that is separate from the catalytic site of the protein. This induces a conformational change in SOS that un-blocks its catalytic Ras binding site. Additional Ras can then bind to the catalytic site and, in turn, be activated by SOS. However, this presents an enigma: if activated Ras is necessary to activate SOS, how is this Ras activated in the first place? The structural and functional investigations of RasGRP1 by the UC team offer an intriguing model for how this might occur. They reveal that RasGRP1 crystallizes in a dimeric, autoinhibited conformation. The autoinhibition occurs because the linker peptide that connects the Cdc25 catalytic domain with the EF domain obstructs the Ras binding site.But this is only one aspect of inhibition. As has been seen in the autoinhibited structures of many other proteins , autoinhThis autoinhibited conformation of RasGRP1 also suggests how the protein could become activated. Binding of diacylglycerol to the C1 domain and calcium to the EF domain would induce large conformational changes that are incompatible with the closed, autoinhibited conformation. Functional studies carried out by the UC team have indeed shown that both calcium and diacylglycerol can activate RasGRP1. This leads to a compelling model for how the activity of Ras is regulated in developing white blood cells. Activation of RasGRP1 activates a small amount of membrane-associated Ras, which can then interact with the allosteric binding site on SOS. Activated SOS, in turn, strongly amplifies the activating signal for Ras, thus establishing the switch-like behaviour required to regulate this protein. This interplay of a weak initiator and a strong enhancer protein leads to a safe, and at the same time very effective, biological switch that ensures that white blood cell development is adequately controlled. It will be exciting to see if this model can be further verified by functional analysis of the interactions among these three proteins in cells.


From 2001-2003 monodon slow growth syndrome (MSGS) caused severe economic losses for Thai shrimp farmers who cultivated the native, giant tiger shrimp, and this led them to adopt exotic stocks of the domesticated whiteleg shrimp as the species of cultivation choice, despite the higher value of giant tiger shrimp. In 2008, newly discovered Laem-Singh virus (LSNV) was proposed as a necessary but insufficient cause of MSGS, and this stimulated the search for the additional component cause(s) of MSGS in the hope that discovery would lead to preventative measures that could revive cultivation of the higher value native shrimp species.FJ498866). In situ hybridization probes and RT-PCR tests revealed that ICE and Laem-Singh virus (LSNV) occurred together in lymphoid organs (LO) of shrimp from MSGS ponds but not in shrimp from normal ponds. Tissue homogenates of shrimp from MSGS ponds yielded a fraction that gave positive RT-PCR reactions for both ICE and LSNV and showed viral-like particles by transmission electron microscopy (TEM). Bioassays of this fraction with juvenile giant tiger shrimp resulted in retarded growth with gross signs of MSGS, and in situ hybridization assays revealed ICE and LSNV together in LO, eyes and gills. Viral-like particles similar to those seen in tissue extracts from natural infections were also seen by TEM.Using a universal shotgun cloning protocol, a novel RNA, integrase-containing element (ICE) was found in giant tiger shrimp from MSGS ponds (GenBank accession number ICE and LSNV were found together only in shrimp from MSGS ponds and only in shrimp showing gross signs of MSGS after injection with a preparation containing ICE and LSNV. ICE was never found in the absence of LSNV although LSNV was sometimes found in normal shrimp in the absence of ICE. The results suggest that ICE and LSNV may act together as component causes of MSGS, but this cannot be proven conclusively without single and combined bioassays using purified preparations of both ICE and LSNV. Despite this ambiguity, it is recommended in the interim that ICE be added to the agents such as LSNV already listed for exclusion from domesticated stocks of the black tiger shrimp. P. monodon since 2002. It has been suggested that an infectious agent may be the cause  and thein situ hybridization reactions for LSNV in retinal lesions that were not present in large shrimp from MSGS ponds or in shrimp from normal growth ponds that gave positive RT-PCR reactions for the presence of LSNV [Using this strategy, one new RNA virus (LSNV) was identified  but its  of LSNV . This le-3)[Based on previous work suggesting that the causative agent(s) of MSGS were viral in nature , the gil-3) . This was subjected to a Blastn search that revealed no significant identity to known nucleic acid sequences except for 100% identity in 46 bases near the 3' end of the sequence with thrombospondin of Penaeus (Marsupenaeus) japonicas (GenBank AB121211). A frame +1 translation of the 2233 bp sequence yielded a single uninterrupted sequence of 744 deduced amino acids that had no known homology to any protein or translated protein sequence at GenBank except for an integrase protein of bacteriophage SH046 of Acinetobacter johnsonii  near the 3' end, but at low identity (29%). The deduced amino acids at positions 1894-2025 (44 amino acids) in the C-terminal portion of the sequence had homology to a conserved domain for phage integrases and DNA breaking enzymes in the DNA_BRE_C family, as outlined in grey in Figure From a total of 180 clones obtained from shotgun cloning of the nucleic acid extract from the 21% CsCl density gradient band, 31 did not hybridize with labeled DNA from normal shrimp in dot-blot hybridization assays, and these were selected for nucleic acid sequencing. Partial sequencing from both ends revealed that 10 clones contained shrimp sequences and 4 bacterial sequences. These clones were not studied further. Of the 17 remaining clones, CLUSTAL W analysis of insert end-sequences revealed that all contained common sequence regions. Thus, 2 of the largest inserts of approximately 2.2 kb called B004 2250 bp) and C010 (2223 bp) were subjected to single-pass sequencing. This revealed that the sequence of C010 fell completely within the sequence of B004. Primers were designed based on the consensus sequence for C010/B004 to produce amplicons of 397 bp from the 5' end (Probe 1), 642 bp from the middle (Probe 2) and 399 bp from the 3' end (Probe 3) Table . An addi0 bp and Total nucleic acid extracts from the 21% CsCl gradient band also gave positive RT-PCR reactions for LSNV. Attempts to remove LSNV reactivity by further density gradient centrifugation failed. Therefore, it is important to understand that the inocula used for all challenge tests consisted of a mixture of at least LSNV and ICE. In addition to LSNV, the extract was tested and found negative for IHHNV by PCR.When RNA extracted from the 21% CsCl gradient band was subjected to northern blot analysis using ICE probe 3, a positive hybridization signal at approximately 3.0 kb was obtained for ICE, while no signal was detected in the RNA extracted from SPF shrimp .When one-step RT-PCR protocols for ICE Probe 3 and for LSNV were used with template RNA extracted from gills of shrimp from ponds that fit the MSGS case definition, it was revealed that most of the shrimp tested gave positive reactions for both LSNV and ICE Table . By contin situ hybridization tests with lymphoid organ (LO) tissue of shrimp from MSGS ponds resulted in positive results for both LSNV and ICE  prepared from ICE and a fluorescence probe (green fluorescence) prepared from a fragment of LSNV for E Figure .When parallel tissue samples from shrimp showing positive hybridization with ICE probes were subjected to examination by transmission electron microscopy (TEM), non-enveloped, icosahedral viral-like particles approximately 25 nm and 15 nm diameter were seen, similar to those previously shown for samples from MSGS ponds  for a second challenge test. In the second challenge test, the secondary inoculum was injected into a second set of test shrimp, also maintained for 60 days before analysis. The size of the domesticated shrimp used for the first challenge was 5.132 ± 0.767 g while that for the second was 5.633 ± 0.798 g. Some of the stock shrimp were positive for LSNV by RT-PCR .In an attempt to fulfil Koch's postulates as modified by Rivers (1937), two challenge tests were carried out. In the first challenge, primary inoculum was prepared from the original 21% CsCl gradient band and injected into juvenile At the end of 60 days in the first challenge experiment, there was no significant difference in survival between the test (61.4%) and control 64.3%) groups. One-step or nested RT-PCR  and control (78.6%) groups. As with the first test, there was a tendency for abnormally dark coloration and brittle antennae in the test but not the control group , while those negative by RT-PCR gave negative hybridization results for both ICE and LSNV  sequences and the lack of an RT sequence from ICE made such a comparison impossible. ICE also contained no group-specific antigen-like (gag-like) sequence for structural proteins, another common feature of viral-like members of the families Pseudoviridae and Metaviridae. If the problem of unsuccessful 3' and 5' RACE reactions could be resolved with ICE, RT and gag sequences might be revealed and allow standard phylogenetic comparisons. On the other hand, the northern blot result indicated that the full size of the RNA fragment containing the ICE target sequence was not a great deal larger than the ICE clone raising the question as to whether it would be sufficiently large to contain RT and gag sequences. Although ICE clearly contained an integrase domain with a conserved catalytic triad H-R-H and a conserved tyrosine (Y) in a binding region that may be involved in host genome insertion [Pseudoviridae and Metaviridae. This is also problematic. One might ask whether the ICE sequence arose from bacteria in the shrimp homogenates. We believe this to be unlikely since inspection by light microscopy and TEM revealed no bacteria in the parallel tissue sections that were positive for ICE by in situ hybridization. Thus, the nature of ICE and its phylogenetic relationship to other viral-like entities known to occur in invertebrates is an enigma.Here we have shown the presence of an integrase containing element (ICE) that is located together with Laem-Singh virus (LSNV) in the cytoplasm of cells of the lymphoid organ (LO), gills and the sub-retinal, fasciculated zone of the eyes of black tiger shrimp exhibiting gross signs of disease referred to as monodon slow growth syndrome (MSGS). Since ICE was isolated from a density gradient band derived from gill tissue homogenates of shrimp from an MSGS pond and since the gradient band contained two sizes of viral-like particles (approximately 25 and 15 nm diameter) it is possible that ICE originated from these particles. Since the extracts containing ICE also contained genomic material from LSNV, and since the diameter of unenveloped LSNV particles has been estimated at approximately 27 nm from thin tissue sections [enbergii  as a posenbergii ,13, the enbergii . Similaraviridae  that areoviridae . In the nsertion ,19, the in situ hybridization analysis revealed that ICE and LSNV occur together in LO and eyestalk tissues previously shown to give positive reactions for LSNV in stunted shrimp from MSGS ponds . In no case were shrimp found that were positive for ICE in the absence of LSNV. Since LSNV was proposed to be a necessary but insufficient cause of MSGS based on its association with retinal lesions only in small shrimp from MSGS ponds, the co-association of ICE with LSNV in such lesions suggests that it may also be causally linked to MSGS. Although the inability to clearly separate ICE from LSNV for laboratory challenge tests makes it difficult to prove this causal relationship, our results using inoculum positive for ICE and LSNV to challenge shrimp already positive for LSNV did support the argument that they may be co-causes of MSGS. This is because the control shrimp already infected with LSNV did not show retarded growth, darkening or in situ positive retinal tissues that were seen in the challenged shrimp, even though the LO of the control shrimp were positive for LSNV. It is true that the ICE and LSNV positive reactions in the test shrimp were not associated with retinal lesions in our challenge tests, but this may have been due to the age of the shrimp at the time of challenge or other experimental parameters that differed from those in shrimp ponds. In addition, it cannot be ruled out that the inoculum contained yet another viral-like agent(s) that was not identified but moved together with LSNV and ICE. Thus, final proof that LSNV and ICE are causally associated with MSGS will probably have to await the advent of infectious clones of ICE and LSNV so that perfectly controlled single and dual challenge tests can be carried out. Unfortunately, complete sequences of neither ICE nor LSNV are yet available.Despite the uncertainty regarding the nature and phylogenetic relationships of ICE, we have shown that there is a strong tendency for it to be associated with LSNV in shrimp samples from MSGS ponds, and in shrimp challenged with inoculum from a density gradient band derived from tissue homogenates of shrimp from MSGS ponds. Furthermore, in situ hybridization probe derived from that sequence gave positive hybridization results in the cytoplasm of cells of the lymphoid organ and eyes of shrimp where no bacterial cells were seen by either light microscopy or by electron microscopy, but where viral-like particles were seen by electron microscopy. Finally, the inoculum for the challenge trials was prepared with ultrafiltration steps that would have removed bacterial cells. Altogether, we may conclude that the ICE element is not from a bacterium, but that its nature remains elusive. Despite this ambiguity, we found that ICE occurred together with LSNV only in shrimp from MSGS ponds and only in shrimp showing gross signs of MSGS after injection with a preparation containing ICE and LSNV. ICE was never found in the absence of LSNV although LSNV was sometimes found in normal shrimp in the absence of ICE. This association between ICE and LSNV suggests that they may act together as component causes of MSGS. However, conclusive proof that they cause MSGS is not possible without single and combined bioassays using purified preparations of both ICE and LSNV.An ICE clone was derived from a process of differential centrifugation that would have eliminated bacteria from the final CsCl gradient, and the gradient band contained no bacterial cells. The nucleic acid clone derived from that band had no sequence identity to any known bacterium, and PCR and RT-PCR tests with that nucleic acid revealed that the clone arose from RNA, not DNA. Further, an Penaeus monodon in Thailand and it has been recommended that shrimp farmers avoid stocking post larvae RT-PCR positive for LSNV to avoid MSGS. In the same way, we recommend that ICE be added to the list of excludable agents in development of SPF stocks, since it is not highly prevalent in wild stocks of P. monodon that might be sourced as founders for such stocks. In addition, the availability of RT-PCR methods makes it possible to carry out continual monitoring in research programs to determine whether there is a positive correlation between MSGS and simultaneous presence of LSNV and ICE in cultivated P. monodon.Since the discovery of LSNV in 2006, it has been added to the list of viruses to be excluded from domesticated, specific pathogen free (SPF) stocks of th August 2004. The mean shrimp weight was approximately 13 g  and the CV was 46% CV at 120 days cultivation (average daily weight gain of 0.1 g/day). A total of 5 kg were collected. Most of the gills were removed from the living shrimp for immediate homogenization and gradient centrifugation, but some were fixed, together with the remaining body for normal histological examination by light microscopy [Live shrimp from a shrimp pond that fit the MSGS case definition were obtained from Laem-Singh district Chanthaburi province, on the eastern coast of Thailand on 202PO4, 100 mM Na2HPO4, 170 mM NaCl, 15 mM sucrose, pH 7.4). The pieces were prefixed with 4% glutaraldehyde in 0.15 M Millonig's buffer at 4°C overnight, and then post-fixed in 1% OsO4 in the same buffer at 4°C for 1 h prior to being processed routinely for conventional embedding in Spurr's resin. Ultrathin sections were stained with uranyl acetate and lead citrate before viewing with a Hitachi H-500 transmission electron microscope at 75 kV.Natural or experimental shrimp were stunned in ice water, dried with a towel and surface sterilized using 70% ethanol before aseptic dissection and removal of tissues for cutting into small pieces and washing with 0.15 M Millonig's phosphate buffer , sucrose gradient for centrifugation at 200,000 g for 6 h.Pooled gills were collected from natural or experimental living shrimp and stored in separate cryotubes under liquid nitrogen. Following rapid thawing, gill samples were homogenized in TN buffer  using a glass homogenizer. The resulting suspension was clarified 3 times by first-round centrifugation at 600 g for 2 h before the pellet was re-suspended again after overnight incubation in TN buffer. The final purification was performed by layering the pellet fraction onto a 10 to 40% (w/w) CsCl gradient and centrifuging at 200,000 g for 18 h. The bands obtained (presumptive viruses) were pelleted by ultracentrifugation at 300,000 g for 2 h and pellets were dissolved in TN buffer for further tests.Each fraction containing presumptive viral particles was diluted in TN buffer and re-centrifuged at 300,000 ® reagent . RNA from the suspension was then purified using a Perfect RNA kit . The first and second strand cDNA synthesis was carried out using the total nucleic acid extract as template and following the recommended protocol of a Marathon cDNA Amplification Kit .Total nucleic acid (DNA and RNA) was extracted from each band collected from the initial sucrose gradient separation using a High Pure viral nucleic acid kit . For RNA extraction, samples were homogenized in TrizolTaqStart Antibody for hot start PCR combined with Marathon kit primers that included an adaptor sequence for later cloning, as described in the system Advantage®2 PCR enzyme user manual (Clontech).Target templates  were amplified by touch-down PCR with an Advantage 2 Polymerase mix that included Direct cloning was achieved using either a TOPO T/A cloning kit  or a pGEM-T Easy  cloning kit as directed by the manufacturer. Selected clones were screened by colony blots and dot-blot hybridization as previously described  using DIin situ hybridization assays using candidate fluorescein-labelled probes selected for negative hybridization with shrimp DNA and lack of significant sequence identity with shrimp sequences or known pathogen sequences at GenBank. Random primed DNA labeling for LSNV was achieved following the protocol of a PCR fluorescein isothiocyanate (FITC) Fluorescein-High prime labelling kit  using a plasmid containing a 600 bp sequence of LSNV . Briefly, the 2 oligo fluorescein labeled probes were mixed and applied to the specimens and incubated overnight at 42°C. The negative control comprised fluorescein without a labeled probe. After incubation, the specimens were washed and viewed with a confocal microscope.Tissues of specimens originating from MSGS ponds or experimental challenge tests and embedded in paraffin blocks were sectioned (5 μm) and subjected to NV Table . Candidahttp://www.ncbi.nlm.nih.gov/BLAST/) and protein domain searches using InterPro 17.0 [http://www.ebi.ac.uk).The clones were sequenced on both strands by Macrogen Inc. (South Korea). Fragments larger than 500 bp were sequenced from both ends, after which new primers were sequenced from the preliminary leads to fill gaps for both strands. Multiple alignments were performed using the programs DNA Baser  and CLUSTAL W  to identPro 17.0  and EMBLRNase A (Fermentas) and RNase-free DNase I (Fermentas) were used according to the manufacturer's instructions to determine whether the template nucleic acid for clones of interest was DNA or RNA. The nuclease digestions were performed in 10 μl volumes using 250 ng total nucleic acid from shrimp incubated for 30 min at 37°C. The nucleic acid from each treatment was extracted and purified using High Pure viral nucleic acid kits  according to the manufacturer's direction. Nucleic acid templates were eluted from the columns using DEPC-treated water and used as for RT-PCR and PCR assays with the primers shown in Table RNA was extracted from the 21% CsCl gradient band and subjected to 0.8% agarose-formaldehyde gel electrophoresis before blotting onto a Biodyne nylon membrane . RNA extracted from SPF shrimp gills was also run in a parallel lane. The membrane was hybridized with DIG-labelled DNA probe prepared from ICE Probe 3. After incubation with anti-DIG antibody conjugated with alkaline phosphatase, hybridized signals were detected with NBT/BCIP substrate  according to the supplier's protocol.Penaeus monodon  obtained from the quarantine facility of the Shrimp Genetic Improvement Center at Chaiya in Surathani province were used for bioassays with inoculum derived from gradient centrifugation fractions of the tissue homogenates described above. These stocks had a history of freedom from HPV, MBV, yellow head virus (YHV) and WSSV for more than 6 generations. Using pleopod samples, they were tested again and found negative for the same viruses before challenge tests using standard PCR detection kits . Although tests for HPV and MBV are often carried out using DNA from hepatopancreatic tissues, our experience has shown that they can also be detected in DNA extracts of pleopod samples from infected shrimp. In addition, subsequent tissue sections prepared in parallel with those for our in situ hybridization reactions were stained with hematoxylin and eosin and showed no histopathology characteristic MBV or HPV in the hepatopancreas. The collected shrimp were acclimatized in the laboratory for 5 days in 65 L aquaria containing continuously aerated artificial seawater at 15 ppt and 30 to 35°C. There were 7 shrimp in each aquarium and 4 aquaria in each of the challenge and control groups . The control shrimp were each injected with 100 μl TN buffer only. The test shrimp were each injected with 100 μl of a 10-6 dilution of a centrifuge fraction (21% CsCl w/w) that had been confirmed by one step RT-PCR to be positive for ICE using the primer set described in Table WP did most of the experimental and investigative work and assisted in preparation of the manuscript. SaS assisted WP with molecular methods and helped to design experiments and interpret the results. SiS did the electron microscopy and interpreted its results. BW helped with conceptualization of the work, experimental design and provision of shrimp for bioassays. TWF conceived the work, assisted in the experimental design and assumed the major role in interpretation of the results and preparation of the manuscript. All authors read and approved the final manuscript.Nortrhern blot analysis of RNA extract from the 21% CsCl gradient band. Northern blot analysis of RNA extracted from the 21% CsCl gradient band (ICE) and from shrimp gills of specific pathogen free P. monodon (SPF) hybridized with DIG-labeled ICE Probe 3. M, Perfect RNA marker 0.2-10 kb (Novagen). The marker bands (length in nucleotides) are indicated to the left.Click here for fileShrimp survival and growth from the 1st bioassay. Details of survival, weight and length of test and control shrimp from the 1st bioassay using inoculum from the 21% CsCl gradient band containing ICE.Click here for fileIn situ hybridization negative control from bioassay #1. Example of confocal photomicrographs of LO tissue from a buffer-injected control shrimp specimen from challenge test 1, negative for both LSNV and ICE by RT-PCR.Click here for filein situ hybridization of LO from bioassay #1Low magnification . Low magnification example of an in situ hybridization reaction using a rhodamine-labeled ICE probe (red) together with an FITC-labeled LSNV probe (green) with lymphoid organ (LO) tissue of a P. monodon specimen from challenge test 1 that was positive by RT-PCR for both ICE and LSNV.Click here for fileIn situ hybridization negative control of eye from bioassay #2. Example of confocal photomictographs of the fasciculated zone of the eye of a buffer-injected control shrimp specimen from challenge test 2, negative for both LSNV and ICE by RT-PCR.Click here for file


Paris polyphylla Sm. var. yunnanensis (TSSPs) have been widely used in China for the treatment of abnormal uterine bleeding. We previously studied the main active constituents of TSSPs and their structure-activity relationships with respect to rat myometrial contractions. Tg (pennogenin tetraglycoside) was identified as one of the active ingredients in TSSPs able to induce rat myometrial contractions. However, the mechanisms underlying the pharmacological actions on uterine activity have not been described clearly.Total steroidal saponins extracted from the rhizome of 2+]i) was monitored under a confocal microscope using Fluo-4 AM-loaded myometrial cells.Here Tg was screened for effects on contractile activity in isolated uterine strips from estrogen-primed rats and on MLC20 phosphorylation and related signaling pathways in cultured rat myometrial cells as determined by Western blot. Intracellular calcium . Use of Ca2+ channel blockers and kinase inhibitors demonstrated that Tg-induced myometrial contractions are mediated by activation of the phospholipase C (PLC)-inositol triphosphate (IP3) signaling pathway, resulting in increased MLC20 phosphorylation. Furthermore, Y27632, a specific inhibitor of Rho kinase (ROK), notably suppressed Tg-stimulated myometrial contractions and decreased MLC20 phosphorylation.Tg dose-dependently stimulated rat myometrial contractions as well as MLC20 phosphorylation These data provide evidence that rat myometrial contractility induced by Tg results from enhanced MLC20 phosphorylation, while both PLC-IP3 and RhoA/ROK signaling pathways mediate the process. These mechanisms may be responsible for the therapeutic effects of TSSPs on abnormal uterine bleeding. Paris polyphylla Sm. var. yunnanensis (TSSPs). Steroidal saponins from the rhizome of Paris polyphylla var. yunnanensis have been isolated and studied by several groups Gongxuening capsule (GXN) based on the yuannanbaiyao formulation, is a single preparation of total steroidal saponins prepared from Paris polyphylla Smith var. yunnanensis Paris polyphylla Smith var. yunnanensis and steroidal saponins with similar structure using a varity of chemical methods. The chemical foundation of the steroidal saponins was then investigated by activity screening and analysis of structure-activity relationships In our previous study, the strengthening of uterine contraction and promotion of hemostasis were found to be responsible for the therapeutic effects of GXN on AUB Although we have defined the general treatment effects of TSSPs on AUB and investigated to some extent the structure-activity relationship and possible function via activation of platelets, the exact mechanisms of the pharmacological actions, especially the signaling transduction pathways, on uterine contractions are still unclear.19 of MLC20 has been the primary interest in studies of regulation of SM contractile activity. This phosphorylation reaction can be mediated by MLCK, which is predominantly regulated by the concentration of free calcium ions (Ca2+) and the presence of calmodulin (CaM) MLC20, also known as “regulatory light chain”, has a pivotal role in regulating muscle contraction in vascular and uterine smooth muscles (SM) Chemicals used in the study, 2-aminoethoxydiphenyl borate (2-APB), ML-7, W-7, U73122, thapsigargin and Y27632, were purchased from Sigma (St. Louis. MO). Stock solutions of these inhibitors were prepared in dimethylsulfoxide (DMSO). Myosin light chain-2 antibody, phospho-myosin light chain-2 (ser19) antibody and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG were obtained from Cell Signaling Technology . Tg was isolated from the TSSPs, and dissolved in DMSO Animal welfare and experimental procedures were carried out in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. This study was approved by Beijing Experimental Animal Ethics Committee (2006) No. 5118 set by the Beijing People's Government. Animals in this study were sacrificed by cervical dislocation. All efforts were made to minimize discomfort, distress, pain and injury.All experiments were performed with female Sprague Dawley (SD) rats, weighing 120–140 g, from the Laboratory Animal Center, Chinese Academy of Medical Sciences. The animals were kept in cages at 22±2°C with free access to pellet food and water on a 12-h light/dark cycle.The rotary shaker (TS-2000), capillary electrophoresis system (DYY-5) and RJ-TDL-5A centrifuge were purchased from Ruijiang Instruments Co., Ltd . The tension transducer connected to a polygraph system was purchased from Beijing Microsignalstar Techology Development Co., Ltd .3 strips along the longitudinal axis. The uterine strips were suspended vertically in 5-ml organ baths of Krebs’ solution , aerated continuously with 95% O2/5% CO2 and maintained at 37±0.2°C. Muscle tension was recorded isometrically with a tension transducer connected to a polygraph system. The preload condition was 1.0 g. The solution for each strip was first changed to 40 mM K+ for 10 min to ensure contractile viability and to determine maximum contraction, the recorded value of which was taken as the control The animals were pretreated intraperitoneally with estradiol benzoate (0.1 mg/kg) for 2 days before the experiments. Rats were sacrificed by cervical dislocation. Uteri were excised, cleaned of adhering fat and mesentery tissues, and then cut into 10×2×2 mm3 pieces and placed in culture flasks containing DMEM medium supplemented with 15% fetal bovine serum (FBS) and maintained at 37°C in 5% CO2 atmosphere. Cells were subcultured every 3–4 days prior to reaching confluence. The semi-dispersed tissues were washed with PBS twice, and trypsin (0.25% w/v) was added. When most of the cells had contracted and become rounded, FBS was added to neutralize the effect, and then the suspension was centrifuged. The cells were resuspended with DMEM medium containing 10% FBS and plated on glass coverslips for 24 h at 37°C in 5% CO2 atmosphere before the experiments.Myometrial SM cells were isolated from estrostilben primed rats. After the uteri were removed and dissected free of fat and endometrium, the tissues were cut into 1×1×1 mm2+]i, the myometrial smooth muscle cells were loaded with the fluorescent calcium indicator by incubation of the cells in Fluo 4-AM  plus Pluronic F-127 for 60 min and Hanks’ balanced salt solution (PH = 7.4) alone for an additional 30 min to allow de-esterification of the dye. Changes in cytosolic free calcium concentration were monitored by detecting changes in fluorescence of single myometrial cells under a confocal scanning laser microscope . Fluo 4- AM was excited with the 494 nm line of an argon ion laser, and the emitted fluorescence was measured at 510 nm. A single myometrial cell was exposed to 2.5 µM Tg or 0.1% DMSO (vehicle control) for 120 s and the fluorescence was continuously recorded using time course software .To assess the i). Considering the involvement of MLCK in myometrial contractions and Tg-induced MLC20 phosphorylation, a specific MLCK inhibitor, ML-7, was used to determine the mechanism of Tg-induced myometrial contractions in the following experiments. As shown in P<0.01). Overall, these results support an essential role for MLCK in Tg-stimulated MLC20 phosphorylation during uterine contractions.The regulation of MLC20 phosphorylation is known to be mediated by the enzyme MLC20 kinase (MLCK), which is predominantly regulated by the intracellular concentration of free calcium ion ([Ca2+) and the presence of calmodulin (CaM). The alterations in [Ca2+]i in response to Tg were then detected in the Flu-4-AM loaded myometrial cells. Flu-4-AM, a cell-permeable acetoxymethyl ester, produces a large increase of fluorescence intensity upon binding Ca2+. As shown in 2+]i in rat myometrial cells. Furthermore, the increase of fluorescence was not reversed for at least 5 min, and retreatment with Tg did not produce an additional peak in the fluorescence (data not shown).Phosphorylation of MLC20 in rat myometrial cells can be mediated by MLCK, which is predominantly regulated by the concentration of free calcium ions  is a calcium-binding messenger protein expressed in myometrial cells and CaM-dependent myosin light chain kinase (MLCK) is considered the primary regulator of MLC20 phosphorylation. To investigate the role of CaM in Tg-induced myometrial contractions and MLC20 phosphorylation, W-7 was used to prevent CaM from binding target proteins. In the contraction experiments, W-7 was applied at concentrations of 0, 20, 100 and 500 µM, while a single concentration of 500 nM was chosen for the Western blot analysis. W-7 could strongly inhibit the Tg-induced myometrial contractions P<0.01,  in a dos2+ were investigated in subsequent experiments. As shown in 2+. Addition of Tg or PGF-2α (as a positive control) did not restore contractility in Ca2+-free buffer, but resulted in some contraction waves at high concentrations. However, when CaCl2 was subsequently added at the physiological concentration, phasic contractions resumed in Tg or PGF-2α pretreated myometrial strips. The role of extracellular Ca2+ in Tg-induced MLC20 phosphorylation was also examined . The effect of nitrendipine, a voltage-operated L-type calcium channel blocker, which inhibits the influx of extra cellular calcium The roles of extracellular as well as intracellular Caexamined . The res2+]i in Tg-induced MLC20 phosphorylation. Thapsigargin, a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) class of enzymes 2+ concentrations by blocking the ability of the cell to pump the ion into the sarcoplasmic and endoplasmic reticula, causing these stores to become depleted. As shown in P<0.01). Furthermore, ruthenium red, a ryanodine receptor inhibitor which inhibits Ca2+-induced Ca2+ release from the ryanodine-sensitive intracellular stores P<0.01)  .2+ through voltage-operated Ca2+ channels may play an important role in Tg-induced uterine contractions. During Tg-induced myometrial contractions, the release of intracellular Ca2+ also plays an essential role. The increase in [Ca2+]i is known to result in formation of the Ca2+-CaM complex in myometrial cells, which activates muscle MLCK. The subsequent phosphorylation of regulatory myosin light chains allows them to rapidly bind to and detach from actin filaments, thereby generating tension Hence, it was proposed that the influx of Ca2+ from internal stores P<0.01) (2+) and U73122  could significantly suppress Tg-induced MLC20 phosphorylation (P<0.01) (Previous reports have demonstrated that phosphoinositide-specific PLC (PI-PLC) is an important component of the myometrial intracellular oscillator. PLC hydrolyzes PIP2 to yield IP3, which binds specific receptors on the endoplasmic reticulum (ER) to mobilize Ca P<0.01) . In addi P<0.01) . By West(P<0.01) . These rRhoA is a small monomeric G-protein and a member of the Rho subfamily of the Ras superfamily of monomeric GTPases Paris polyphylla Smith var. yunnanensis in uterine contractions 2+ channel blockers and several kinase inhibitors demonstrated that Tg-induced myometrial contractions involve activation of the PLC-IP3 signaling pathway, resulting in MLC20 phosphorylation. Finally and importantly, the Tg-induced myometrial contractions and MLC20 phosphorylation could be inhibited by Y27632, a specific ROK inhibitor, suggesting that the RhoA/ROK signaling pathway is also involved in the process. These mechanisms may be responsible for the therapeutic effect of TSSPs on AUB.Steroidal saponins are widely distributed in the botanical kingdom, possessing a broad range of biological and pharmacological properties, such as hypocholesterolemic, anti-tumor, anti-diabetic, anti-inflammatory, anti-fungal and platelet agonistic and inhibitory activities 2+]i. We also observed that ML-7 significantly reduced the amplitude but not the frequency of myometrial contractions induce by Tg, as MLC20 activity but not [Ca2+]i signals was inhibited.The term ‘myosin’ refers to a large superfamily of genes that share the ability to bind to actin and possess ATPase activity 2+]i signals within the myometrium have important functional consequences, as they determine contractility 2+]i can be increased by influx from the extracellular space, release from intracellular storage sites, or both 2+-dependent cytosolic protein which binds four Ca2+ ions 2+-CaM complex activates the key enzyme MLCK, causing an immediate and marked increase in phosphorylation of MLC20, which activates the contractile machinery 2+ ions and thapsigargin, which would affect the [Ca2+]i of rat myometrial cells, significant inhibiton or enhancement of Tg-induced MLC20 phosphorylation was observed.In common with other SM tissues, changes in [Ca2+]i, is extremely important in Tg-induced MLC20 phosphorylation and the resulting phasic myometrial contractions.Previous reports have demonstrated that agonist-induced myometrial contractions are coupled to phosphoinositidespecific phospholipase C (PI-PLC) activation and inositol 1,4,5-trisphosphate (IP3) production. IP3 induces a rise in free cytosolic calcium via the release of intracellular calcium from IP3-sensitive stores and the calcium-induced calcium release 2+/calmodulin-dependent myosin light chain kinase. The RhoA/ROK pathway has been implicated in the tonic phase of force maintenance in response to various agonists, with no evident role in the phasic response 2+]iA variety of contractile agonists trigger activation of the small GTPase RhoA. An important target of activated RhoA in smooth muscle is Rho-associated kinase (ROK), one of the downstream targets that is the myosin binding subunit (MYPT1) of myosin light chain phosphatase (MLCP). ROK phosphorylates the myosin targeting subunit (MYPT1) of MLCP at two potential sites , thus results in a decrease in phosphatase activity of MLCP and an increase in myosin light chain phosphorylation catalyzed by Caq protein. Recent data demonstrate that Gα12/13 can induce Rho-dependent responses. Furthermore, Gα12/13 can bind and activate Rho-specific guanine nucleotide exchange factors, providing a mechanism by which GPCRs that couple to Gα12/13 could activate Rho and its downstream responses The intracellular PLC-IP3 signaling pathway is known to be activated by G-protein coupled receptor (GPCR) signaling, which is mediated by the Gin vivoin vivo, and pennogenin glycosides with a spirostanol structure may be responsible for the therapeutic effects on AUB. These mechanistic insights will be helpful for identifying molecular targets of steroidal saponins for the purpose of drug development in the treatment of AUB.Steroidal saponins, present in plants and some marine animals, have many pharmacological and biological activities 2+]i regulated by PI-PLC pathway, while the other via Ca2+ sensitization of the contractile proteins signaled by the RhoA/Rho kinase pathway. We speculate that specific GPCRs may be targeted in the response to Tg, which may be responsible for the therapeutic effect of TSSPs on AUB. These findings may help facilitate the drug discovery process for AUB therapies.In summary, this study demonstrates that rat myometrial contractility induced by Tg results from MLC20 phosphorylation in myometrial cells, while the PI-PLC and RhoA/ROK signaling transduction pathways mediate the process. Furthermore, the two signaling pathways act under different mechanisms, one occurs via the changes of [Ca


Spike trains with temporal correlations have been related to cognitive and sensory processing , and extIt is known that excitatory and inhibitory activities are typically balanced , and maiIn this study, we use a Leaky-Integrate-And-Fire neuron with inputs that model a canonical disynaptic feedforward-inhibitory circuit Figure . We showOur findings provide insight in the role of inhibitory STDP, in maintaining detailed balance and enhancing transmission efficacy in disynaptic neural circuits.


The control over ice crystal growth, melting, and shaping is important in a variety of fields, including cell and food preservation and ice templating for the production of composite materials. Control over ice growth remains a challenge in industry, and the demand for new cryoprotectants is high. Naturally occurring cryoprotectants, such as antifreeze proteins (AFPs), present one solution for modulating ice crystal growth; however, the production of AFPs is expensive and inefficient. These obstacles can be overcome by identifying synthetic substitutes with similar AFP properties. Zirconium acetate (ZRA) was recently found to induce the formation of hexagonal cavities in materials prepared by ice templating. Here, we continue this line of study and examine the effects of ZRA and a related compound, zirconium acetate hydroxide (ZRAH), on ice growth, shaping, and recrystallization. We found that the growth rate of ice crystals was significantly reduced in the presence of ZRA and ZRAH, and that solutions containing these compounds display a small degree of thermal hysteresis, depending on the solution pH. The compounds were found to inhibit recrystallization in a manner similar to that observed in the presence of AFPs. The favorable properties of ZRA and ZRAH suggest tremendous potential utility in industrial applications. Ice growth and nucleation play a significant role in life on earth, for example, in the freezing of environmental water resources, as well as the freezing of water in plant and animal tissue, both of which can result in fatal damage. Organisms living in cold environments must respond appropriately to survive when temperatures drop below the freezing point. Such organisms have evolved a series of mechanisms for controlling ice growth and protecting themselves from freezing damage, either by avoiding freezing or by tolerating the freezing process. Control over ice growth is crucial in industrial settings as well. Processes such as ice templating are used in the engineering of porous materials Ice growth may be controlled by inhibiting ice recrystallization, in which the growth of large ice crystals is favored at the expense of small ones  are a set of natural proteins that may be used to control ice growth and recrystallization. This expanding group of proteins interacts with ice for various biological purposes such as freeze avoidance, ice recrystallization inhibition (IRI), and ice nucleation promotion, thereby protecting organisms from ice damage Non-protein cryoprotective agents (CPAs) that act in a colligative manner such as glycerol, dimethyl sulfoxide (DMSO), and certain sugars are widely used in cryopreservation. These materials can protect cells from damage caused by the formation of intracellular ice during freezing and thawing. CPAs require high concentrations to be effective and some display high cytotoxic effects Here, we have expanded the study of ZRA and ZRAH by measuring their potential to act as cryoprotectants through direct ice shaping, modulating the growth rate of ice, and inhibiting ice recrystallization. ZRA and ZRAH induced hexagonal ice shaping, and both materials significantly reduced the rate of ice crystal growth compared to buffer, depending on the solution pH. ZRA and ZRAH were also shown to inhibit ice recrystallization in a manner similar to AFPs.3CO2)xZr(OH)y, x+y∼4. The pH of the ZRAH solution was adjusted using 100 mM acetic acid for pH values of 3.3 and 4.2, and 100 mM sodium acetate for pH 4.7. The ZRA is provided by the manufacturer as 16% solution in dilute acetic acid with the formula Zrx+(CH3COOH)x. Using the given density of 1.279 g/ml, this solution contains 200 g/L or 2.2 M Zr. The solution was diluted either with pure water (for pH of 3.3) or with 100 mM sodium acetate  to obtain a final Zr concentration of 13.3 g/L (150 mM). The pH was adjusted using HCl. A sample of a recombinant type III AFP from Ocean pout was kindly provided by Peter L. Davies (Queen’s University) ZRAH powder and a ZRA solution were purchased from Sigma-Aldrich Ltd. ZRAH was dissolved in 100 mM acetate buffer to a final concentration of 30 g/L. This concentration provides 100–150 mM of Zr in the solution, as calculated using the empiric formula (CHm) was determined as the lowest temperature at which melting was observed. The temperature was then reduced to the desired supercooling temperature and held at this temperature while growth was recorded. The experiment was repeated at least three times on three different samples for each temperature.The ice growth rate was measured using a custom-designed nanoliter osmometer controlled by a LabView interface developed in our lab. The system and procedure are described in detail elsewhere c and/or the a axis was measured and the difference between the measured distances divided by a factor of 2 was taken as the elongation of the crystal along the measured axis. The growth rate was determined as the division of the elongation by the time that passed during the growth course, and a mean value was calculated from the different snapshots for each experiment. The directionality of the ice crystals was determined according to the crystal symmetry (twofold symmetry in the c direction and six-fold symmetry in the a-direction). ZRA and ZRAH were tested for their effects on ice crystal growth over the pH range 3.3–4.7. pH 4.7 was the highest pH at which measurements could be collected. At higher pH values, the solutions polymerized and turned into gels. Even at pH 4.7, the solution became viscous and gelated after 4–6 days.Growth rates were determined from recorded images using an ImageJ analysis routine IRI experiments were performed using a Nikon optical microscope equipped with a LC-PolScope (Abrio IM™ Imaging System) and a Linkam MDBCS 196 cold stage . A sample of 5 µL was inserted into a 15 mm inner diameter quartz sample holder and covered with a circular glass coverslip. The quartz dish was then placed on a temperature-controlled silver block inside the cold stage. A flow of dry nitrogen was subjected on top to prevent humidity in the ambient air from condensing on the upper window. The experimental procedure was as follows: Samples were cooled from room temperature to –20°C at a cooling rate of 50°C/min. During this cooling phase, ice crystals spontaneously nucleated from the supercooled solution. Samples were kept at –20°C for 2 min, and during this time, images of the frozen samples were collected. The temperature was then warmed to –3°C at a constant rate of 5°C/min, and thereafter the temperature was maintained at –3°C for 10–60 min to permit recrystallization. During this time period the development of the ice crystals was recorded. With the LC-PolScope image system, multiple images with different polarization settings are recorded by a digital camera. These images were used to determine the optical axis in the sample fractions. A color gradation is used to indicate the orientations of the optical axis in each ice crystal. Three replicate experiments were performed, at a minimum, for each determination.a-direction and twofold symmetry in the c direction  revealed slow but steady growth at a rate of 0.01 µm/s along the a-axis. Notably, this growth rate was about 600 times slower than the growth rate observed in a buffer solution for the same supercooling level.Crystals in ZRAH and ZRA solutions at pH 3.3 did not significantly affect the ice growth rate along the ecreased –3. Growt.02 µm/s . This slc-axis in the presence of ZRA and ZRAH was measured. The relatively high growth velocities along the a-axis in the solutions at pH 3.3 resulted in flat shapes, which forced the crystals to float such that the c-axis was parallel to the view direction. This orientation limited our ability to measure the growth rate along the c-axis , and recrystallization appeared immediately in the acetate buffer sample , but nota-axis compared to the growth in buffer over the given temperature range. Nevertheless, whereas AFPs can completely halt ice growth over a range of temperatures, ZRA and ZRAH could not. The observation of continuous growth along the direction of the c-axis indicated that ZRA and ZRAH did not produce true TH activity, as is observed in the presence of antifreeze proteins. We note that although the hyperactive AFPs inhibit ice growth along the basal plane a-axis was reduced to less than 0.02 µm/s, which is 10 times below the rate that was previously considered to be “no growth” and, therefore, considered to be TH activity a direction was observed also within this temperature range, indicating that the interactions between ZRA or ZRAH and ice are not as strong as the interactions of AFPs. The mechanism by which ZRA inhibits growth must, therefore, differ from the mechanism by which AFPs interact with ice, as noted by Deville et al. The findings of this study showed that the inorganic synthetic materials ZRA and ZRAH exhibited certain traits that are characteristic of AFPs. A comparison of the growth of ice in a buffer solution or in the presence of ZRA or ZRAH indicated a hundred-fold reduction in the growth rates along the m, which is ten times slower than the melting rate of ice in an acetate buffer at the same temperature (2.1 µm/s). Such melting effects are characteristic of AFPs, as have been demonstrated by Celik et al. In addition to growth rate reduction, melting inhibition was observed. ZRAH at pH 4.7 produced a melting rate of 0.2 µm/s for ice at a temperature of 0.01°C above the TThe presence of ZRAH or ZRA dramatically slowed or completely stopped ice recrystallization compared to the buffer. Similar IRI results were obtained for the AFP type III positive control. Although recrystallization was observed for only a relatively short period of time, the effects of the compounds on IRI were clear.a-axis direction, suggested that these compounds were active on surfaces parallel to the ice prism planes or some other planes that were slightly tilted with respect to the prism planes. Moderate AFPs have been shown to interact with the prism planes of ice and surfaces slightly tilted with respect to these planes c-axis. As growth along the c-axis progresses, the basal planes shrink until they are reduced and the crystal eventually acquires a bipyramidal shape and is protected from growth in any direction. Despite the fact that these AFPs do not directly interact with the basal planes c-axis. The differences between the growth patterns of ice in the presence of AFPs, ZRA, or ZRAH can be explained according to differences in the adsorption planes or sites on the ice surfaces. Whereas AFPs may bind to more than one plane a-direction observed after hours of incubation.The ice shaping properties of ZRA and ZRAH, which led to hexagonal columnar growth patterns, along with the growth inhibition activities to the Hexagonal column-shaped crystals and continuous slow growth were previously observed in the presence of mutated AFPs While little is known about the actual structure of ZRA in an aqueous solution 4(OH)8(H2O)16]8+The pH dependence of ice growth inhibition by ZRA and ZRAH solutions may be due to differences in the complex structures, stoichiometry, or oligomerization in solution, as indicated above. Solutions containing a Zr:acetate ratio of 1∶2 (at concentrations of 0.01 or 0.02 M) were found to exhibit a dramatic change in the main complex species between pH 3 and 4 Deville et al. observed hexagonal shapes in the ice crystals at pH 4 but not at pH 4.5 or 3.3. The author suggested that the decrease in ice shaping activity at these pH values arose from surface charge repulsion between the ice and ZRA The functional groups that may participate in ice recognition by the hydroxyl-bridged ZRA oligomer are the acetate and hydroxyl moieties. The acetate group is linked to the Zr(IV) ion through an oxygen atom, leaving a methyl group and another oxygen or hydroxyl group exposed to the solvent. This arrangement is similar to the side chain of the threonine amino acid, which plays a significant role in the ice binding faces of many AFPs On this basis, we propose two structural arrangements by which ZRA can bind to an ice surface . In the Note that although the models presented here consist of chains and assume that preliminary polymerization steps have occurred, the interactions are valid for other ZRA structures that may be present in solution, such as a tetramer in which four zirconium atoms are bound by hydroxo The effects of the ZRA compound on the ice crystal growth or melting may be advantageous for cryopreservation. The efficient inhibition of ice recrystallization and the induction of smooth growth patterns without spontaneous “burst” growth, as are observed in the presence of AFPs, could prevent fatal damage to cells, tissues, and organs. For example, the addition of AFP type I This work examined the extent to which ice growth could be controlled in the presence of ZRA and ZRAH. These compounds were shown to share many of the properties of natural AFPs, including IRI activities, ice-shaping activities, and a reduction in the ice growth and melting rates. The structures of ZRA complexes and polymers suggested that ZRA interacts directly at the ice:water interface, although further studies are needed to elucidate the mechanism of action underlying the ZRA effects. While ZRA do not have high TH activity, it presents several practical advantages over AFPs, including a low production cost, good stability, and abundance in nature. ZRA is an excellent candidate agent that warrants further study for possible use in applications in which ice growth control remains a challenge.Figure S1Ice crystal growth as truncated pyramidal shapes in a solution containing ZRAH at pH 4.7. The images were taken at a temperature 0.040°C below the melting point.(TIF)Click here for additional data file.Figure S2Hopper shapes induced by ZRAH. Ice crystal grown at a high supercooling rate in the presence of ZRAH at pH 4.7 developed hopper shape with increased basal planes that appeared to be hollow. The sequence of images was taken during the growth course. Frame A was collected before the temperature had been reduced, at the melting point. The other three images were taken during temperature decline. The actual temperature below the melting point and the time lapse is presented. The arrows in A denote the crystal orientation in the sample.(TIF)Click here for additional data file.


Elevation of cardiac troponins and creatinine kinase is frequently observed in setting of systemic inflammatory response syndrome (SIRS), sepsis, or septic shock. Underlying pathophysiologic mechanism for such troponin leak, its clinical significance, and what different could be done in such settings remain elusive. In this paper we have briefly overviewed the proposed pathogenic mechanisms for SIRS, sepsis, or septic shock-related troponin elevation (SRTE) and have provided brief overview on its clinical significance. Upon review of the relevant literature we found that majority of patients with the SRTE with no prior history of coronary artery disease (CAD) upon testing are found not to have any CADs. We have also briefly discussed the possible pharmacologic agents and potential targets which are important from pathophysiologic and pharmacologic point of view that may alter the outcomes of SRTE-related myocardial depression in near future. Elevation of cardiac troponins and creatinine kinase (CK) is observed in 31%–80% of patients in setting of systemic inflammatory response syndrome (SIRS), sepsis, or septic shock ) [Majority of SRTE patients without any prior history of coronary artery disease (CAD) on testing are found not to have any significant CADs –5 Table. TroponiThe purpose of this paper is to briefly review three basic questions: what causes SRTE, what is its clinical significance and what different can be done in such cases?Troponin is a complex of three regulatory proteins   and TnT Most popular theory for explanation of SRTE has always been the demand and supply mismatch theory . In the There are two storage forms of cardiac troponins: cytosolic and myofibril. Quantity of troponins in cytosol is 35 times lower than in myofibril . It has β, IL-6, and TNFα), nitric oxide, endotoxins [α increases the permeability of endothelial cells to macromolecules and lower molecular weight solutes, researchers have proposed that a similar increase in permeability of myocardial cell membrane may be responsible for SRTE [β, IL-6, and TNFα are believed to play a central role in sepsis-mediated myocardial depression [α-mediated activation of neutral sphingomyelinase, suppression of nitric oxide and calcium transient pathways, modulation of intracellular proteases [α causes myocardial depression and SRTE [Bacterial myocarditis  leading dotoxins , and actdotoxins  in settidotoxins  is anothfor SRTE , 21. Frefor SRTE , 6. Tropfor SRTE . IL1β, Ipression . TNFα-meroteases , effect Altmann et al.  in a stuActivation of NADPH oxidase complexes and mitochondria (18) in setting of sepsis leads to a formation of free radicals. These free radicals along with leucocyte-derived superoxide radicals (19) are thought to cause myocardial cell damage and apoptosis leading to SRTE (19). Increased cardiac filling pressures and increased wall stress in setting of sepsis have been proposed to activate intracellular signaling cascade leading to cardiac myocytes apoptosis , myocyteGram negative as well as gram positive bacterial  and fungOn review of the literature we found that majority of patients with SIRS, sepsis, and septic shock-related deaths had positive cardiac troponins at time of death . Thus itIn setting of sepsis, heart undergoes different physiologic and metabolic changes which normalize within 7–10 days  in survi3, and presence of regional and global hypokinesia and a left ventricular fractional area contraction (LVFAC) of <0.4 under inotropic support as diagnostic criteria for SRTE-related myocardial dysfunction. Fernandes et al. [SRTE has been proposed as a biomarker of underlying myocardial dysfunction (a major contributor to the worse outcomes in the setting of SRTE)  in settis et al.  compareds et al.  transesoContribution of right ventricle in sepsis-related myocardial dysfunction is not known, though similar sepsis-related physiologic changes are believed to affect both ventricles .The concept of myocardial depression/dysfunction was for the first time described by Parker et al. , who perBetter imaging techniques which could assess true systolic and diastolic dysfunction in setting of sepsis, could assess myocardial wall strain and perfusion may serve as a better biomarker for detection of myocardial dysfunction in setting of sepsis.Different authors have used different cutoff values for TnT and TnI to define an elevation; in this review we have taken troponins positive or negative according to each author's criteria . FurtherStudies have shown that presence of cardiovascular dysfunction in setting of sepsis is associated with a significant increase in mortality rate to 70%–90% as compared to 20% with no cardiovascular compromise . On the Very few studies have considered the impact of preexisting risk factors on incidence of SRTE. Smith et al. in their study showed that diabetes was more prevalent in SRTE study population  and thatIn the current practice, patients found to have elevated troponins in settings of SIRS, sepsis, or septic shock are either observed or undergo some form of noninvasive or invasive cardiac testing to rule out CAD. History of chest pain is difficult to obtain in most of SRTE patients as majority is intubated or sedated or too sick to communicate . By reviAcute coronary syndrome medications like aspirin, beta blockers, and ACE inhibitors in setting of SRTE have not been shown to improve prognosis . α inhibitors [α [α was withdrawn from market due to lack of survival benefit in setting of septic shock. Role of TNFhibitors –35 and Ihibitors , 37. Simhibitors , 39 and hibitors . Nitric hibitors ; inhibithibitors . Blockadhibitors , 44. Bethibitors  and stathibitors  may alsohibitors  and low-hibitors . Previouitors [α  was showIn conclusion, future prospective studies with a large patient population are needed to define the role of cardiac testing in the setting of SRTE and to define the exact pathogenesis of SRTE. Randomized controlled trials are needed to determine the optimal treatment strategy in septic patients with SRTE. Addition of cardiac troponins in sepsis protocol may help risk-stratify patients so that appropriate measures like echocardiograms and cardiac consults could be taken early in the course of the disease.


MK, compared to the concentration of the target substrate. We also find for a closely related model that bistable systems tend to have a specific kinetic conformation. Using deficiency theory and other methods, we provide a number of necessary conditions for bistability, such as the presence of multiple phosphorylation sites and the dependence of the scaffold binding/unbinding rates on the number of phosphorylated sites.The phosphorylation of a substrate at multiple sites is a common protein modification that can give rise to important structural and electrostatic changes. Scaffold proteins can enhance protein phosphorylation by facilitating an interaction between a protein kinase enzyme and its target substrate. In this work we consider a simple mathematical model of a scaffold protein and show that under specific conditions, the presence of the scaffold can substantially raise the likelihood that the resulting system will exhibit bistable behavior. This phenomenon is especially pronounced when the enzymatic reactions have sufficiently large  The modification of a protein at multiple sites can result in a number of interesting behaviors at the cellular level, such as all-or-none responses to an external input, or two different stable cellular states in otherwise identical environments. Such behaviors can aid in many different forms of cellular decision-making, e.g., cell differentiation or cell division. In this paper, we show that bistable behavior can be greatly enhanced by the presence of a scaffold protein, which binds to the substrate protein and either relocates it or otherwise affects the action of the modifying enzymes. The scaffold protein substantially widens the range of parameters for which bistability is observed when Protein phosphorylation is a ubiquitous form of post-translational modification Multisite phosphorylation is also a very common occurrence. For example, Epidermal Growth Factor (EGF) receptor activation involves phosphorylation at multiple tyrosine residues by another EGF receptor ultrasensitivity, consisting of a steep, switch-like response of output to increasing input concentrations. Bistability refers to the ability of a deterministic system to have two stable steady states. This property is useful in all-or-none cell fate decisions, such as the decision to differentiate, or to progress through the cell cycle It has long been known that the presence of multiple ligand binding sites in a protein can give rise to cooperative binding through allosteric interactions between binding sites Biochemical models of multisite phosphorylation have been studied in the literature with an eye towards ultrasensitivity and bistability, see for instance Gunawardena In this paper, we focus on the bistability of multisite phosphorylation systems with scaffold proteins. Four mathematical models with different topology and assumptions are developed. An analytical study using deficiency theory i phosphorylated sites, to form the complex The multisite (de)phosphorylation system is modeled using a standard sequential mechanism . To intrcomplex  . We alloWith regard to dephosphorylation, it has been proposed that some scaffold proteins may protect bound proteins from the action of phosphatases To quantify the dynamics of multisite phosphorylation, we have explored two types of commonly used mechanisms: full mass action kinetics (MA) trations . In the echanism . The fulechanism , the simechanism , the fulechanism , and theechanism .It is worth pointing out that a distributive mechanism is assumed for (de)phosphorylation on scaffold, that is, that the enzymes tend to unbind from the substrate after each modification. There is evidence that some scaffold proteins may behave in this way. For example, the Ste5 scaffold protein binds weakly to its associated kinases Before investigating the parameter patterns of bistable multisite (de)phosphorylation systems with scaffold, we first explore network topologies that exclude bistability regardless of kinetic parameter values. To this end, we employ the deficiency theory developed by Feinberg and others complexes. For instance, the reaction network deficiency of the network is defined as The deficiency theory of chemical reaction networks is able to predict under certain circumstances that a given system is incapable of having multiple steady states, regardless of the parameter values used . In order to do this, it only makes use of qualitative graphic-theoretic properties of the network, such as the number If the scaffold association and dissociation rates y states . In otheProof sketch: define the variables If phosphorylation and dephosphorylation only take place for scaffold-bound substrates, then the system can only have one steady state  . The samProof sketch: the two statements follow directly from the Deficiency Zero Theorem – however notice that We point out that even though the model in Even in the presence of a scaffold, a substrate with a single phosphorylation site is incapable of producing bistable behavior for several possible network configurations . This reWe take a closer look into the parameter values of the linear rate scaffold system LR-S, in search for patterns that might make bistability more likely. We simulate this system using a large set of phosphorylation, dephosphorylation, and scaffold binding and unbinding parameters. In particular, we randomly sample each of those parameters over a range of several orders of magnitude, consistent with experimental measurements For two-site (de)phosphorylation systems with scaffold, our numerical simulations suggest : (1) forIn summary, the above features in parameters indicate a fast flow nd state . Similaronly common conformation giving rise to bistability under the chosen parameter regime. Due to the mass conservation of total substrate, a conformation in which all variables are present in either high or low concentrations is precluded. The fact that we do not allow for phosphorylation off scaffold also breaks some of the possible symmetries. It is also significant that bistability is rarely observed when It is remarkable that this is the It is worth pointing out that although the cell membrane was considered a suitable scaffold for ultrasensitive behavior in We now consider the full mass action (MA) models and to what extent the addition of a scaffold facilitates bistable behavior. First we show that, at least for some sets of parameters, a scaffold allows a monostable multisite system to become bistable. By examining the dose-response curve as a function of the total kinase concentration em MA-NS . However MA-NS  is several times that of the model without a scaffold (2.2%). For n = 5, which is significant considering that the on and off rates as well as the total protein concentrations are randomly varied over several orders of magnitude. If the phosphatase acts only off the scaffold, the probability for bistability further increases to 29.8%. Similar results as in However, for It should be noted that the value of  see the  section,Notice also that these results hold regardless of the dimensionality of parameter space or of the geometry of the set of bistable parameters, since we are merely measuring the proportion parameter sets that yield bistable systems. As a matter of reference, if the fraction of bistable parameter sets under given conditions is around 10% and 500 samples are taken, one can expect about 1.3% of standard deviation between the sampled result and the actual fraction.In cellular signal transduction, multiple, consecutively-acting components of a signaling pathway are often physically organized into complexes by scaffold proteins. Here, by exploring various models of multisite (de)phosphorylation with scaffold, we conclude that under the following specific conditions the presence of a scaffold can enhance bistability of multisite phosphorylation systems.Non-processive multisite substrate phosphorylation. The signaling proteins that bind to scaffolds are often phosphorylated at multiple sites and believed to act in a non-processive manner, for instance in the case of the MAPK cascade . Many such proteins are organized by scaffold proteins A substrate concentration in the same order of magnitude or higher than the scaffold concentration. This assumption is also reasonable; for instance, in the case of the yeast mating MAPK cascade, several measurements indicate that the cellular concentrations of the Ste5 scaffold and its associated kinases Ste11 and Ste7 are all around 30–50 nM , and the ultimate substrate of Ste5 scaffolding, Fus3, has a cellular concentration that is at least 5-fold greater Kinases with a relatively high  value. For such enzymatic reactions the kinase-substrate complex is relatively transient. This assumption can be explained on the grounds that high A scaffold-substrate dissociation constant that varies with the substrate phosphorylation state. A possible implementation of this assumption is through a bulk electrostatic mechanism for substrate binding. If the scaffold is naturally negatively charged near its binding domain to the substrate, then the presence of negatively charged phosphorylations at the scaffold might prevent its binding and accelerate its unbinding. In principle, this can take place for a multisite scaffold protein in the absence of allosteric behavior. For instance, in the yeast pheromone-response pathway, protein Ste5 (itself a scaffold protein but here viewed as a substrate) binds to the membrane in part due to bulk electrostatic interactions that are modified by multisite phosphorylation Notice that certain relations among the various parameters are also consistently preserved. For instance, the larger phosphorylation rate for the second site suggests an allosteric behavior between the substrate and kinase.Several different models of bistability in protein networks are described in Another important aspect to consider in these chemical reaction systems is the effect of noise and stochastic behavior. If chemical reactions are allowed to take place in a non-deterministic way, the variables in a bistable system might switch spontaneously from one steady state to another. Here the fold-change measure introduced in the Notice that the simulations in We have concluded that adding a scaffold has a large likelihood of turning a monostable multisite system into a bistable one, for large Scaffolds typically do not possess any enzymatic activity themselves, but facilitate signaling between their bound components. One way in which they are thought to do this is by tethering their ligands in close spatial proximity to each other The mathematical model of scaffolding employed herein features these two key elements of sequestration and rate partition. Sequestration is achieved in our model by accounting for the second order mass action binding of scaffold and substrate. Rate partition is achieved by allowing different rates of substrate modification depending on whether the substrate is bound to the scaffold or not. Our simple model does not incorporate other potentially interesting features of scaffold-mediated signaling, such as combinatorial inhibition, processive on-scaffold phosphorylation, and multi-tier scaffolding (our model just has two tiers: a single kinase and its substrate). For other theoretical treatments of scaffold action, the reader is referred to the following references: There has been considerable interest in understanding how common biochemical modules and motifs can be flexibly tuned to achieve a variety of desired outcomes de facto output, we verified in hundreds of independent trials that the system is only bistable if Throughout the computational modeling, we used mass action kinetics to construct the systems of differential equations associated with each individual model. Given a parameter set, in order to test for bistability we first reduced the problem to a 3-variable system of equations involving A key aspect of the analysis is the choice of random parameter sets over several orders of magnitude. Rates of substrate binding to an enzyme or scaffold are normally in the range of All parameters were chosen under a logarithmic distribution—that is, using a uniform distribution for their natural logarithm. For the tables, in order to ensure that all Table S1Parameter set for . Under these parameter values the system MA-S is bistable but MA-NS is not.(PDF)Click here for additional data file.Table S2Parameter sets for . Derived using the analysis in (PDF)Click here for additional data file.Table S3Probability of bistable behavior for arbitrary fold ratio. In (PDF)Click here for additional data file.Text S1In this supplementary text we provide more information on the mathematical analysis of the various models involved. In Section 1, we describe the concept of deficiency including a precise statement of the Deficiency Zero Theorem, then proceed to prove several of the theorems stated in the (PDF)Click here for additional data file.