Pusa hispida), ribbon (Histriophoca fasciata), bearded (Erignathus barbatus) and spotted (Phoca largha) seal. Variations in catch per unit effort were explored in relation to year, sea ice conditions, day of the year, and geographic location. In most years, the harvest was predominantly represented by ringed seals , followed by ribbon , spotted  and bearded seals . The struck-and-lost percentages were as high as 30–35% for ringed, bearded and spotted seals and 15–20% for ribbon seals. Catch per unit effort  for ringed, ribbon, and spotted seals had a similar seasonal pattern with a distinct spike in catches for spotted seals in the first week of May, for ribbon seals in the last week of May, and for ringed seals in the second week of June. Catches of bearded seals showed a less pronounced temporal structure with a gradual increase toward the end of the harvest season in the majority of years. Spatial distribution of harvest effort followed closely with seal distribution obtained from aerial surveys. These data could be used as a source of information on seal herd location throughout the breeding and molting seasons and for more complex demographic or life-table models. We did not find any evidence of the decline of catch per unit effort over the study period. Timely introduction of state regulations and efficient harvest management apparently prevented severe depletion of ice-associated seal populations in the Sea of Okhotsk during the periods of their intense exploitation.Sealing log books from 75 out of 79 commercial harvest cruises carried out between 1972 and 1994 in the Sea of Okhotsk, Russia, were analyzed to describe spatial and temporal allocation of ice-associated seal harvest effort, species composition of catches, total harvest rates, and related parameters for species including ringed ( Pusa hispida), ribbon (Histriophoca fasciata), bearded (Erignathus barbatus), and spotted (Phoca largha) seal. All four species have a long history of exploitation including periods of aboriginal and commercial harvests, shore- or vessel-based harvests, and unlimited or state-regulated harvests ) Data cleaning and mapping were performed in QGIS 2.12.0 –Lyon . Ice datThe vessel-based harvest in the Sea of Okhotsk during 1972–1994 was typically conducted from early April until mid-July with the majority of seals caught in May-June . In 1972The total numbers of seals harvested during the study period and reported in log books were: 231,879 ringed, 159,241 ribbon, 34,858 bearded, and 90,359 spotted seals . We compAccording to population estimates from aerial surveys in the second half of the 20th century  and the In most years , the harvest was predominantly represented by ringed seals , followed by ribbon , spotted , and then bearded seals .Out of 75 harvest cruises over the 22-year period, enforcement officers were present on 49 of them see . Their mStarting in 1975, harvested seals were divided into age classes. The proportion of pups, both white-coated and molted, taken in catches during 1975–1994 remained stable for all species throughout the study period  with verCPUE was estimated for all the data points where the number of skiffs working at sea was recorded in log books  as the number of seals of each species caught by one hunting brigade in one day. CPUE for ringed seals was the highest , followed by ribbon , spotted , and finally bearded seals . For all species combined, each skiff on average harvested a mean of 24.31 seals per day .p<0.05) effects in the GAM regressions on CPUE for all four species. CPUE for ringed, ribbon, and spotted seals appeared to have a similar pattern in relation to day of the year  of year class on harvest start date suggesting that, perhaps, on low ice-coverage years (ice coverage <51%) the harvest tended to start later than on medium and high ice-coverage years. The effect of the ice-coverage on harvest end date was not significant. There were no significant differences revealed in CPUE, number of vessel-days and total catches for each species per year in relation to ice-coverage class .In 1972–1994  there were 2 years with high ice coverage (1979 and 1980), 6 years with low ice coverage and 12 years with medium ice coverage  and reported by Okhotskrybvod, we possess data from approximately 94% of the sealing cruises. Nevertheless, we encountered certain discrepancies between catch rates for particular species recorded as the harvest progressed, and harvest rates reported later in the literature [Harvest and fishing vessels working at sea were obliged to accommodate an enforcement officer on board if one was provided. Based on log book records, officers were only present on some cruises and could work only on one of the skiffs, whereas most vessels had several skiffs. This officer, generally either a state official or biologist from a research institution, was empowered to enforce seal harvest regulations according to sealing areas and quotas as well as proper reporting. State officials could be involved in harvests directly as hunters . In these cases, we assumed that the reliability of the inspection results could have been compromised due to extra work duties performed and potential conflicts of interest.We did not discern any relationship between the presence of an enforcement officer on a vessel and the accuracy of the records, or the consistency in reporting of harvest losses. For instance, even though vessels had an officer on board, daily harvest rates for all species were apparently rounded  on some cruises during 1976. We also found several instances when the same person was listed as an enforcement officer on two different vessels simultaneously working at sea. Although we believe that the discrepancies described above do not diminish the overall value of the data preserved in the log books, they should be borne in mind when interpreting our results and drawing specific conclusions.We hypothesized that harvest start dates differed somewhat depending on ice coverage in April. Although we expected harvest to start later in years with heavier ice conditions, the model outcome showed the opposite trend. According to the recollections of the authors of the present study who participated in the harvest cruises, use of HFVs removed the limitations associated with ice conditions and the vessels began the harvest depending on organizational and technical aspects, mainly vessel maintenance, expedition supply, and crew recruiting. At the same time, the lack of a significant effect of ice conditions on catch rates implies that within observed ranges of ice coverage in 1972–1994 sealers always managed to take their quotas, apparently due to abundant seal populations.In the Sea of Okhotsk we did not find any pronounced trends in the frequency of high ice years or low ice years, neither during the study period nor in the following years. Based on our classification, the proportion of high ice years slightly decreased from 0.14 to 0.09, in low ice years from 0.41 to 0.32, and in medium ice years increased from 0.45 to 0.59 when comparing data from 1972–1994 with data from 1995–2013. Finer scale data are needed to demonstrate a possible effect of ice conditions on seal population abundances and harvest success.in utero [The beginning of sealing season in 1972–1994 typically coincided with peak pupping season for ringed seals, and early pupping season for ribbon, bearded and spotted seals in the Sea of Okhotsk –48. The in utero . Single Ringed seal CPUE increased gradually from the beginning of harvest to reach its maximum level only in mid-June, later than the CPUE of the other three seal species see . Ringed Ribbon seal CPUE see  followedLow bearded seal CPUE, in comparison to the other species, could be related to the ability of bearded seal females and pups to escape into the water during the nursing season, and a generally lower interest in this particular species from hunters. Bearded seal catches were slowly increasing, presumably due to the beginning of the molt, from late May to early June and continued to do so until the end of the harvest season see .Spotted seal harvest seemed to have been more pup-oriented compared with other species as it primarily took place on pupping grounds. Outside the breeding season, adult spotted seals on ice were characterized  as havinChanges in CPUE annual mean from year to year may be linked to organizational aspects of harvest operations, changes in harvest techniques, and staffing problems. Starting in 1975, new ice- reinforced vessels replaced the schooners and this change required additional staffing and training of new crews. The Korsakovskaya oceanic fishery government enterprise became responsible for vessel and crew preparation for the harvest season and the Okhotskrybvod later reported a highly unsatisfactory level of performance: there was not enough warm clothes, food supplies, and safety and shooting training provided to the newly employed vessel crews. Lack of sufficient hunting experience in a number of hunting brigades resulted in a two- to three-fold decline in CPUE , substantial increase in numbers of wounded seals and even led to an accidental injury of one of the hunters in 1977. The total time spent on-effort at sea also decreased due to testing and essential maintenance of new skiffs as well as crew at-sea training which took up to two weeks. The technical and staffing problems were gradually overcome after 1978 which resulted in increasing trends in catches see . A steadDeclining CPUE values in exploited populations might be a good indicator of negative changes in population size. Visual inspection of GAM outcomes did not provide clear evidence of the decline of this parameter over the study period. Temporary troughs in CPUEs are shown to be largely associated with harvest management.Spatial variation in catches was largely associated with vessel initial positions and harvest effort allocation. However, vessel harvest locations revealed significant differences in the distribution of catches of different seal species, which supported inferences about actual seal distributions on spring ice.The ringed seal stands apart from other ice-breeding species due to its ability to maintain a system of water access and breathing holes in thick fast ice throughout the ice-covered period (e.g. ). This aBased on aerial survey maps published in various years, ringed seals are fairly evenly distributed throughout their range in the Sea of Okhotsk –59. HoweHarvest data for 1972–1994 demonstrate that the main concentrations of ringed seals in April-May were closely associated with the near-shore fast-ice zone. A well-defined band of high seal density occurred along the coast from Shelikhov Gulf in the North to the northern Shantar Sea waters and the northern part of Sakhalin Island in the South. Fedoseev  describeThe spatial structure of the ribbon seal harvest corresponded well with this species’ distribution in the whelping and molting seasons. The ribbon seal is a typically pelagic species whose habitat is associated with zones of the continental slope. It is incapable of maintaining breathing holes in the ice and occurs primarily in the areas where vertical circulation of water masses leads to formation of large ice floes, which makes these areas rich in fish. According to Fedoseev , in the A dense concentration of ribbon seals was typically located north of 58°N and was considered as a separate breeding herd isolated presumably by bathygenic inflow of the warm Pacific waters passing through the Kashevarov Bank towards St. Iona Island . Based oThe bearded seal is a typical benthic and epibenthic feeder that prefers shallow, continental shelf regions. In wintertime it breeds on grey, grey-white, and white ice with a thickness of typically less than 60 cm  that preCompared to ringed seals, bearded seals are less active in making and maintaining breathing holes in ice and require presence of water leads and polynyas. Thus, their breeding habitat in the Sea of Okhotsk is mostly located in the drifting ice zones in Shelikhov Gulf, Tauyskaya Bay and off eastern Sakhalin . Their bBearded seals in their molting season remain patchily distributed , but theSpotted seal breeding areas are very similar to the ones occupied by ribbon seals. Fedoseev  suspecteThere are two major molting concentrations of spotted seals , but maiCommercial seal harvests in the Sea of Okhotsk not only had a significant impact on the marine ecosystem as a source of mortality for ice-associated seals inhabiting the area, but also provided a large amount of data on species biology, demography, and seasonal distribution. The distributions and timing of the harvests corresponded well with the prevailing understanding of the different species’ natural histories, and therefore our harvest data should provide an enhanced basis for future assessment of impacts from the reduction of sea ice anticipated in the Sea of Okhotsk as the Arctic climate continues to warm.Original records contained in log books enhanced our understanding of the seal harvest and the populations that were exploited. Comparison of harvest logs with relevant information published elsewhere allowed cross-checking to identify and resolve discrepancies. The edited data set will support a planned quantitative analysis of relationships between biological patterns in ice-associated seal populations, harvest patterns, and environmental variability.Despite substantial struck-and-lost rates and high numbers of seals harvested throughout the study period, timely introduction of state regulations and efficient harvest management apparently prevented severe depletion of ice-associated seal populations in the Sea of Okhotsk.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Thermococcus onnurineus NA1 is an anaerobic archaeon usually found in a deep-sea hydrothermal vent area, which can use elemental sulfur (S0) as a terminal electron acceptor for energy. Sulfur, essential to many biomolecules such as sulfur-containing amino acids and cofactors including iron-sulfur cluster, is usually mobilized from cysteine by the pyridoxal 5′-phosphate- (PLP-) dependent enzyme of cysteine desulfurase (CDS). We determined the crystal structures of CDS from Thermococcus onnurineus NA1 (ToCDS), which include native internal aldimine (NAT), gem-diamine (GD) with alanine, internal aldimine structure with existing alanine (IAA), and internal aldimine with persulfide-bound Cys356 (PSF) structures. The catalytic intermediate structures showed the dihedral angle rotation of Schiff-base linkage relative to the PLP pyridine ring. The ToCDS structures were compared with bacterial CDS structures, which will help us to understand the role and catalytic mechanism of ToCDS in the archaeon Thermococcus onnurineus NA1. Thermococcus onnurineus NA1 is a sulfur-reducing hyperthermophilic organism living in a strictly anaerobic condition of a deep-sea hydrothermal vent area at high temperatures between 80 and 100°C  scaffolds or sulfur acceptor proteins like IscU, TusA, and CsdE  cluster synthesis under the adverse conditions of oxidative and heavy metal stress and iron starvation [Thermococcus onnurineus NA1's habitat is also physicochemically extreme with respect to temperature ranges of 2 to 100°C, oxygenation states, and fluid velocities [Recent studies on the archaeal sulfur metabolism revealed many novel enzymes and pathways . Howeverarvation . The envToCDS gene of Thermococcus onnurineus NA1 and determined the crystal structures of ToCDS alone (NAT) and in complex with catalytic intermediate ligands . ToCDS belongs to class II CDS based on the primary and tertiary structures and showed the highest similarity with EcSufS. ToCDS is a PLP-dependent enzyme and the crystallographic snapshots of catalytic intermediates of ToCDS showed the conserved dihedral angle rotation of Schiff-base linkage relative to the PLP pyridine ring as shown in EcNifS_CsdB and XometC, which implies the PLP-dependent catalytic mechanism of ToCDS is well conserved. This study intends to help on understanding the catalytic mechanism of ToCDS and archaeal sulfur-trafficking system for the synthesis of sulfur-containing biomolecules.We cloned the Figure S1. Omit maps of active site in ToCDS PSF, GD, and IAA structures.Figure S2. Active site in ToCDS GD and IAA structures.Figure S3. Proposed transaldimination mechanism from XometC structures.Figure S4. Tight recognition of alanine at the substrate-binding site of ToCDS.


The dorsal tangential (DT) view has been shown to improve the detection of dorsal screw perforation during volar distal radius fracture fixation. Here we performed a cadaveric imaging survey study to evaluate if the DT view was uniformly beneficial for all screws.p value < .05 was considered significant. Standardized placement of fixed-angle volar distal radius plates was performed on two cadavers. Fluoroscopic images depicting variable screw perforation of each of the four screw holes on the plate were generated. A 46-image survey was distributed at a large academic medical center. Respondents were asked to answer if the screw was perforating through the dorsal cortex in each image. Statistical analysis was performed using Fisher's exact test. A  The DT view offered a significantly more reliable determination of dorsal screw penetration than traditional lateral imaging for the radial-most screw at all degrees of perforation and the middle two screws at 2 mm of perforation. Residents and attendings had more accurate screw readings overall using the DT view. The DT view is superior to traditional lateral imaging in the detection of small amounts of dorsal perforation of the radial-most three screws of a fixed-angle volar plate.  Although volar plate fixation has become the gold standard in the treatment of unstable distal radius fractures, extensor tendon irritation and rupture are known complications of this procedure. The etiology of extensor rupture is believed to relate to dorsal screw prominence in the majority of cases , 2. IntrThe dorsal tangential view has recently received attention as an intraoperative fluoroscopic imaging technique that may better detect dorsal radius screw penetration , 5–7. ItHere we performed a cadaveric experiment and related survey study to compare the relative efficacies of the lateral and dorsal tangential (DT) views in the identification of dorsal screw prominence across training backgrounds. We hypothesized that the DT view is more effective than the lateral view in the detection of dorsal screw prominence at all experience levels.All protocol elements were approved by the Committee for Oversight of Research and Clinical Training Involving Descents (CORID #587) at our institution. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2008. A traditional volar approach to the wrist was performed on two fresh-frozen cadaveric forearms, permitting the placement of a fixed-angle distal radius plate  with 4 distal screw holes. Screws were inserted into each of the distal holes with their tips flush with the dorsal cortex, confirmed by visual inspection through a separate dorsal incision. Plate positioning and placement were performed by the senior investigator (JRF). A screw from a single hole was then removed and replaced with a longer screw, producing a collection of images with standardized single screw prominences of 0, 2, 4, or 6 mm. Lateral and DT fluoroscopic radiographs were obtained after each screw exchange Figures . A true p < .05.A total of 46 images were digitized into high quality digital image files and randomized using an online survey platform . Surveys were distributed to orthopedic surgery residents, fellows, and faculty. Instructions for each survey question asked the respondent to consider each image individually and determine if the screw had penetrated the dorsal cortex. All questions were in a binary yes/no format and asked the respondent to identify any screw protrusion in each successive image. Results were analyzed with GraphPad Prism 6.0  using Fisher's exact test for categorical data, as normality cannot by assumed given the limited sample size included in this study. In all cases, significance was defined as Completed responses were logged from a total of 18 respondents  out of a total of 52 faculty, fellows, and residents surveyed. A response was considered complete if it included answers to all questions. An additional five responses that failed to meet this criterion were removed from our sample size.p < .0001). Significant differences between DT and lateral view accuracy were also observed in the attending  and resident  subgroups  compared to the lateral view  and continued to be problematic at all screw prominence lengths at the radial styloid view (13 ± 1.8% at 4 mm and 49.4 ± 61.5% at 6 mm). However, more ulnar positions showed improved accuracy . In contExtensor tendon rupture after volar plate fixation of distal radius fractures may occur secondary to screw prominence. The protuberance of Lister's tubercle can mask dorsal screw penetration on lateral fluoroscopic views. The dorsal tangential (DT) view has been proposed as a method that can more sensitively detect dorsal screw prominence compared to standard lateral radiographs. Our work suggests that the primary advantage of the DT view compared to traditional lateral fluoroscopic views is in the evaluation of small degrees (2 mm) of screw protrusion on the radial three screws on a fixed-angle volar plate. Larger magnitudes of screw protrusion further limit the benefit of the DT view to the single most-radial screw on the plate. Overall, the DT view was found to be more reliable than the lateral view by all experience cohorts but active clinical fellows. However, the sensitivity and specificity of both techniques were still equivocal.Our findings appear to stand in contrast to existing work. Ozer et al.  comparedThis disagreement may be independent of the benefit of the DT view. Cadaveric work by Perry et al.  found thWhile Wall et al.  demonstrWhile the findings of this work appear to be largely supported by prior work, our study has several limitations. There is a small amount of unpreventable variability in positioning and obtaining the dorsal tangential and lateral fluoroscopic films. As demonstrated by Haug et al. , the accIn a survey study based on measurements made using a cadaveric model, we found that the DT view is superior to the lateral view of the wrist in the identification of any degree of dorsal cortex perforation of the radial-most screw in a volar locking plate. Further, the DT view may also be superior to the lateral view in the detection of 2 mm screw prominence in central plate holes. Further work that utilizes an increased sample size and image quantity and considers additional plate types and hole configurations is required to validate the findings of this pilot work.


Periplaneta americana and Sitophilus oryzae. The knockdown activity of 2,3-dimethylmaleic anhydride was associated with in vivo inhibition of AChE. At KD99 dosage, the 2,3-dimethylmaleic anhydride showed more than 90% inhibition of AChE activity in test insects. A significant impairment in antioxidant system was observed, characterized by alteration in superoxide dismutase and catalase activities along with increase in reduced glutathione levels. Computational docking programs provided insights in to the possible interaction between 2,3-dimethylmaleic anhydride and AChE of P. americana. Our study reveals that 2,3-dimethylmaeic anhydride elicits toxicity in S. oryzae and P. americana primarily by AChE inhibition along with oxidative stress.Evolution of resistance among insects to action of pesticides has led to the discovery of several insecticides (neonicotinoids and organophosphates) with new targets in insect nervous system. Present study evaluates the mode of inhibition of acetylchlonesterase (AChE), biochemical efficacy, and molecular docking of 2,3-dimethylmaleic anhydride, against  Acetylcholine (ACh) is a one of major neurotransmitter involved in neurotransmission6. ACh is produced from acetylating reaction with choline and Acetyl-CoA by the enzyme choline acetyltransferase. In the synaptic cleft, ACh is degraded by an enzyme acetylcholinesterase forming acetate and choline, which are recaptured by the pre-synaptic neurons7. Organophosphates and carbamates insecticides are potent inhibitors of the enzyme acetylcholinesterase.Major insecticides developed in the last 60 years act on one of the following targets; acetylcholinesterase, an enzyme of critical importance in the transmission of nerve impulse (organophosphorus and carbamates), voltage-gated sodium channels across the nerve membrane (pyrethroids and DDT), and the acetylcholine receptor (neonicotinoids)10. Discovery of novel effective insecticidal compounds is needed to combat the increasing resistance rates. Botanicals contain active insecticidal phytochemicals, which are considered as alternatives to hazardous and non biodegradable chemical insecticides13. Essential oils and plant volatile organic compounds are good sources for developing insect pest control agents because they are known to have many bioactivities including insecticidal, repellents, antifeedants, ovicidal and insect growth regulators activity against insect pests17. The main advantage of plant volatile organic compounds is its highly volatile nature. High volatility reduces the residue problems and, easily degradable, eco-friendly and less toxic in mammals18 and used in the indigenous traditional medicines in most of tropical areas19.Insect pest management is facing the economic and ecological problems worldwide due to the human health, environmental hazards and pest species developing insecticides resistance caused by extensive use of chemical insecticidesColocasia esculenta var esculenta (L.) Schott, commonly known called as Taro, geographically occurs throughout India and it is cultivated worldwide. It is an annual herbaceous plant with a long history of usages in traditional treated medicine such as asthma, arthritis, diarrhoea, neurological and skin disorders and further, juice of corm is widely used for treatment of bodyache and baldness20. In North East India, people have practise to consume corm and root stock of C. esculenta as food from many centuries and there were no reports on adverse effect on health.The evaluations of insecticidal efficacy of plant derived products are based on use in the traditional practices in pest management, which forms a basis for an ideal approach in the development of newer insecticides from plants. One of the plants is et al.21 isolated 2,3-dimethylmaleic anhydride, a novel biofumigant molecule from root stock of C. esculenta (L.) Schott, which is highly toxic to various stored grain insects and house fly by fumigation21. Further, this molecule has no adverse effect on seed germination which makes it highly desirable for grain/seed protection against stored grain insect pests. The insect toxicity of 2,-3-dimethylmaleic anhydride in the fumigant bioassay was analyzed based on the symptoms and behaviours and the results indicated its neurotoxic nature similar to those produced by organophosphates. In this paper, we investigate the toxicity and the mode of inhibition of acetylcholinesterase by 2,3-dimethylmaleic anhydride. We also measured the effect of antioxidant defense system , involved in the toxicity aspect of its action in Sitophilus oryzae (L.) and Periplaneta americana, which leads us to postulate that these natural components represent the discovery of mode of action and oxidative imbalance. Subsequently, a molecular docking study was investigated to enlighten the possible molecular interactions between 2,3-dimethylmaleic anhydride and the enzyme acetylcholinesterase (AChE) of P. americana.Recently, Rajashekar 25, KD50 and KD99 doses of 2,-3-dimethylmaleic anhydride, was markedly suppressed in a dose-dependent in neural ganglion of American cockroach and homogenate of rice weevil  and P. americana (10 µg/L) treated with a single KD50 dose of 2,-3-dimethylmaleic anhydride at various exposure times (0–120 min), inhibition of acetylcholinesterase increased with time and correlated with the knock down effect  crude homogenate and the inhibition efficiency of AChE from isolated compound is comparable to that of standard AChE inhibitor pyridostigmine bromide . While for ure Fig. .S. oryzae, while in P. americana 360% and 475.3% respectively) compared with control was observed following 12 and 24 h exposure  at 3.4 Å resolution was chosen as the best available template to build the 3D structure for AChE with 99% query coverage and 73% similarity. Target-template alignment and its superimposed structure with the template 5HQ3 chain B are shown in Fig. AChE Catalytic Subunit of CASTp analysis of the predicted model of AChE identified three major pockets, named here as CP1 (CASTp Pocket 1), CP2, and CP3. Fig. . The det21,22. Among the many important insecticide classes, their origin is traceable to a natural product as in the case of pyrethoids, avermectins, decaleside; spinosads and the neonicotinoids25. Despite the diversity of natural chemistries, compounds with new modes of action have eluded discovery11,26. Although, insect control agents acting on targets other than nervous system such as insect growth regulators , have been developed, due to their lack of fumigant toxicity, they are not highly successful, but find a place in the integrated pest management scheme28.Natural products could be an excellent source of novel pest control agents. An important attribute of natural product- based pesticide compounds is that they are relatively eco-friendlyColocasia esculenta, were found to be toxic to a variety of insect species by the method of fumigation21. Our earlier studies showed that the insect toxicity of 2,3-dimethylmaleic anhydride was comparable to that of chemical insecticides (fumigants) and more potent than that of reported natural fumigants as evident by the LC50 values in fumigant bioassay21. Although, several natural compounds have been reported to exhibit fumigant toxicity, there is no comparative study of the toxicity of a natural compound with that of synthetic insecticides on insects in a fumigant bioassay. The nature of insect toxicity of 2,3-dimethylmaleic anhydride in the fumigant bioassay, based on the symptoms, indicated hyperactivity as seen in the increased movements followed by knockdown with legs showing over exciting movements indicating a neuro-muscular effect. The neurotoxic symptoms were similar to that of organophosphates.2,3-dimethylmaleic anhydride the natural insecticides isolated and characterized by us from the root stock of in vivo inhibition closely correlates with insect toxicity of compound on increasing the concentration and exposure period. In addition, the in vitro studies reveal that, the molecule 2,3-dimethylmaleic anhydride showed potent AChE inhibitor and inhibition was competitive. AChE is an enzyme which breakdown the neurotransmitter acetylcholine to acetate and choline at the neuromuscular junctions26,29. Therefore, the inhibition of AChE activity leads to persistence of acetylcholine at the synaptic cleft (the space between two nerve cells) and cholinergic stress30, which could be one of the possible reasons of fumigant toxicity of 2,3-dimethylmaleic anhydride. Another probable reason is behavioral systems similar to that of insect exposed to carbamates. These two similar supportive observations have been reported for the most commercial available conventional insecticides such as organophosphates and carbamates31.A complete understanding or elucidation of the mode of action is of practical importance because it may provide newer opportunities for development of eco-friendly insecticides. Therefore, in the present study effect of 2,3-dimethylmaleic anhydride on AChE and antioxidant enzyme system was studied. Results clearly showed that AChE activity is drastically inhibited in insects exposed to 2,3-dimethylmaleic anhydride in fumigation bioassay. The 32. Therefore, in the present investigation the effect of 2,3-dimethylmaleic anhydride on antioxidant enzyme system of insects were studied with dose-response and time-course dependent. The present result reveals that 2,3-dimethylmaleic anhydride causes significant impairment/imbalance in the antioxidant enzyme system (SOD and CAT) of both insects. In an organism, the SOD-CAT antioxidant system provides the first defense against oxygen toxicity. A delicate balance exists between the rate of formation of H2O2 via dismutation of superoxide by SOD activity and the rate of removal of H2O2 by catalase. Any impairment in this pathway, will affect the activities of other enzymes in the cascade34. Induction of antioxidant enzyme activities enhances metabolic detoxification and antioxidant defenses of the tissue, while their reduction results in oxidative stress35. In our study, 2,3-dimethylmaleic anhydride treated to insect led to reduced antioxidant capacity of the neural ganglion as evident from the decreased activity of all the antioxidant enzymes. However, John et al.36 and Aslanturk et al.37 reported that, variations on antioxidant enzyme system of treated insect due to the difference in biochemical and physiological response of treated insects. Further a significant increase in GSH level was recorded for the S. oryzae exposed to 2,3-Dimethylmaleic anhydride. GSH also serves as a substrate for glutathione peroxidises (GPx) and GST, primarily involved in the detoxification of toxic electrophilic metabolites via formation of GSH-conjugate38. During the enzymatic reaction catalyzed by GPx, GSH is oxidized to GSSG which is reduced back to GSH by GR. GR, therefore, plays a central role in maintaining the cellular GSH level. GSH plays an important role in protecting cells against reactive oxygen species (ROS) mediated injury by detoxification of lipid hydro peroxides formed due to oxidative damage39. Our results show drastic reduction in the activity of GSU in both test insects by 2,3-dimethylmaleic anhydride which could compromise the biochemical antioxidant defenses of the brain. Marked depletion of GSH and its dependent enzymes in the test insect by 2,3-dimethylmaleic anhydride indicates a major deleterious effect on the neural ganglian of insect. The results lead us to conclude that the oxidative imbalance may be involved in the toxic effects of 2,3-dimethylmaleic anhydride. Our results are the first report of a natural insecticide (biofumigant) might be associated inhibition of AChE activity and oxidative imbalance. The molecular mechanisms involved in the associated inhibition of AChE activity and oxidative imbalance that leads to the knockdown effect, the toxic outcome finally leading to mortality, are not clear at present. Further, our results imply that 2,3-dimethylmaleic anhydride may be associated inhibition of AChE activity and oxidative imbalance. However, further downstream events leading to the AChE inhibition need to be understood.Further, it has been reported that oxidative stress is one of the possible mechanisms of the non target of commercial available pyrethriods, organochlorine and organophophates to the mammalian systemP. americana. Since AutoDock program40 has been successfully applied for publishing docking studies with AChE41, it is utilized for our binding study. Absence of crystal structure of AChE protein of P. americana compelled us to perform the homology modelling to predict its 3D structure using SWISS-MODEL automated server. Best model generated from the server was further validated as described in results section. Although the ultimate availability of crystal structures of AChE protein of P. americana would improve the results of docking, the homology model predicted here acted as the preliminary step for testing 2,3-dimethylmaleic anhydride binding. CASTp finding of surface accessible pockets plus internal remote openings in the predicted model suggest that there are three major binding pockets covering very large volume (2982.2 Å3), with the largest pocket CP1 (1140.5 Å3) having most of the substrate binding sites and catalytic triad residues. By means of whole protein docking simulation we conclude that out of four major clusters formed, AC1 cluster of ligand conformations coincides with the pocket CP1 sites having most of the substrate binding site residues and catalytic triad. In this study, the intriguing finding is the different binding sites for 2,3-dimethylmaleic anhydride at AChE of P. americana. Focused docking on the CP1 binding site obtained a refined IE for the ligand. Evaluation of binding site residues involved in pocket configuration and those involved in hydrogen bond formation with 2,3-dimethylmaleic anhydride indicated that the ligand binds near the substrate binding site. By analysing the docking solutions, binding free energy and IE, we suggest that the 2,3-dimethylmaleic anhydride can attach to the substrate binding site of AChE through both electrostatic and van der Waals interaction as well as hydrogen bond, where the GLU199 played as main facilitator. As our experimental and computational findings corroborate each other, we can articulate that 2,3-dimethylmaleic anhydride is a potent inhibitor of AChE in P. americana for its activity. Nevertheless, this study reports the first binding analysis of 2,3-dimethylmaleic anhydride with AChE of P. americana.In addition, molecular docking may be reducing the understanding of the factors determining biological activity. Hence, we have investigated the possible binding modes of the 2,3-dimethylmaleic anhydride with AChE of C. esuclenta. The knock down activity 2,3-dimethylmaleic anhydride is concomitant in vivo inhibition of AChE in both test insects are dose-response and time dependent. In addition, this study is first to explore the constructed atomistic AChE model of P. americana, and its possible mode of inhibition by 2,3-dimethylmaleic anhydride through in-silico binding analyses. Further, the molecule showed significant impairment in the antioxidant enzymes. Therefore, our study reveals that 2,3-dimethylmaeic anhydride elicits toxicity in S. oryzae and P. americana primarily by AChE inhibition along with oxidative imbalance.The findings of present investigation reveal that, the, 2,3-dimethylmaleic anhydride is potent AChE inhibitor extracted from Sitophilus oryzae L.) were collected at ware house of Food Corporation India, Imphal, Manipur, India and reared on whole wheat (Triticium aestivum). Whereas American cockroaches (Periplaneta americana L.) were collected from Insect Culture centre, at Department of Food Protectant and Infestation Control, CSIR-Central Food Technological Research Institute, Mysore, Karnataka, India and reared in plastic tubes with harborages, containing broken wheat and biscuits, and water was provided ad libitum. The cockroaches were maintained at 23.6 ± 2.5 °C, 70% relative humidity and a photoperiod of 12:12 (Light: Dark). Adults of S. oryzae (3–5d) were used for the experiments. Experiments were carried out in the laboratory at 27 ± 2 °C and 70 ± 5% r.h.42.Mixed-age cultures of rice weevil , 5,5-dithio-bis-2-nitrobenzoic acid (DTNB), reduced glutathione (GSH), pyridostigmine bromide, 1-chloro-2,4-dinitrobenzene (CDNB) and Folin’s reagent were procured from Sigma chemical Co. . Trichloroacetic acid (TCA), hydrogen peroxide, sodium di-hydrogen phosphate, sodium hydroxide and sodium carbonate were procured from Sisco Research Laboratory, Mumbai, India. 2,3-dimethylmaleic anhydride is isolated and characterised  from root stock of Colocasia esculenta var esculenta (L.) Schott as reported earlier21.50 (50% knockdown in 24 h exposure) were determined from the dose-response data using probit regression analysis43.Knockdown of insects defined as the state of intoxication and partial paralysis with lack of movement which usually precedes death by exposure to 2,3-dimethylmaleic anhydride was done by fumigation using adults of the cockroach and rice weevil. Five adult cockroaches or 30 adults of rice weevil of known age were released into 0.85-l desiccators that served as the fumigation chambers. In each desiccator, a Whatman No. 1 filter circle (9 cm size) was placed to serve as an evaporating surface for injecting the isolated compound. For each species, there were four replicates for each dose of the isolated compound, with equal number of untreated control replicates. The dosages ranged from 0.2 to 20 µg/L, and the effective dosages were chosen based on trial experiments. Four replicates were used for each dosage. KDIn vivo inhibition of AChE in relation to the toxicity of 2,3-dimethylmaleic anhydride was investigated in cockroaches.25, KD50, and KD99  doses of 2,3-dimethylmaleic anhydride in the fumigant bioassay21. The doses were selected based on the results of toxicity of 2,3-dimethylmaleic anhydride to American cockroach. Respective batches of solvent treated controls were also employed. After 60 min exposure insects were removed. In case of cockroach the neural ganglion were dissected from head out and frozen for enzyme assay, where as in case of stored grain insects whole insect was homogenate and stored at −20 °C for enzyme assay.Insects were exposed to KD50 (10 µg/L for P. americana and 3.0 µg/L for S. oryzae) dose of 2,3-dimethylmaleic anhydride in the fumigation bioassay and removed at various exposures time intervals . Solvent (ethanol) treated groups served as control. After various time intervals of exposure, insects were removed and the insect tissues were dissected as described above for enzyme assay. Thereafter, homogenate was centrifuged at 10000 rpm for 10–12 min at 4 °C in a table-top refrigerated centrifuge, and the supernatant was separated and used for the further experiments.Insects were exposed to single KD44 which is based on the method of Ellman et al.45. The reaction was carried out in a 96 vial micro plate and reaction mixture containing the suitable amount supernatant and DTNB in Tris-HCl buffer  was prepared. Thereafter, acetylcholine iodide (ATCI) (10 µl of 0.1 M solution) was added to reaction mixture and change in absorbance was monitored over 3 min in a microplate reader at 405 nm and at 25 °C. The primary AChE inhibition assay was replicated at least four times. The percentage AChE inhibition of the bioactive compound was calculated according to the following formula:Inhibition of Acetylcholinesterase (AChE) was estimated by ELISA microplate reader following the modified method of Galgani and BocqueneIn vitro inhibition of AChE by 2,3-dimethylmaleic anhydride in the neural ganglion of cockroaches, whole insect homogenate of stored grain insects (crude preparation) was studied. The enzyme was pre incubated with 2,3-dimethylmaleic anhydride (0.5 µM–10 µM) along with or without inhibitor (pyridostigmine bromide) at 37 °C for 30 min and the inhibition of AChE was determined.46.The type of enzyme inhibition was studied kinetically, in neural ganglion tissue homogenate. The reaction mixture as described above was preincubated with various concentration of with or without inhibitor . The residual activity at each of the inhibitor concentration was assayed at various substrate concentrations. The nature of inhibition was determined from the data by double-reciprocal or Linweaver-Burk plot47. The reaction mixture contained pyrogallol in 0.1 M Tris buffer (pH 8.2) and the insect homogenate. The reaction was started by adding the substrate and the absorbance was read at 420 nm for 3 min at an interval of 1 min and results were expressed as unit/mg protein. The amount of the enzyme that inhibits auto-oxidation by 50% is referred as one unit of enzyme activity.Superoxide dismutase (SOD) activity was measured using pyrogallol (2 mM) auto-oxidation as described by Marklund and Marklund48, 10% insect tissue homogenates were prepared in 5% w/v TCA, centrifuged at 2000 rpm for 10 min and supernatant (GSH) was mixed with 10 mM DTNB in 0.1 M phosphate buffer (pH 8.0). The mixture was incubated for 10 min at room temperature and the color was read at 412 nm. Glutathione content was calculated by a standard curve and expressed as µg/mg protein.Glutathione (GSH) content was estimated by the method of Ellman49. The reaction mixture contained 3% H2O2 in 0.05 M phosphate buffer (pH 7.0). The reaction was started by the addition of 100 μl of enzyme and the change in absorbance was read at 240 nm for 3 min and activity was expressed as n mole H2O2/min/mg protein.Catalase (CAT) activity was assayed by the method of Aebiet al.50 using BSA as the standard.Protein content was measured by the method of Lowry P. americana was retrieved from GenBank in FASTA format, scanned for conserved domains and other putative substrate binding sites using NCBI CDD51. SWISS-MODEL (http://swissmodel.expasy.org/), an automated comparative protein modelling server, was used to generate the 3D model by querying the protein sequence as target. The server generated homology models by performing a target-template sequence alignment via searching template structures in Protein Data Bank (PDB) and SWISS-MODEL template library (SMTL) repositories using blastp and HHBlits algorithms52. The best structural model was selected based on Global Model Quality Estimation (GMQE) and QMEAN scoring functions, which has been trained specifically for SWISS-MODEL53. Stereochemical quality and accuracy of the predicted models were analysed using PROCHECK at PDBsum server (http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html), ProQ54 and ProSA-web56.Acetylcholinesterase (AChE) protein sequence (Accession Number: ALJ10969.1) of https://pubchem.ncbi.nlm.nih.gov/) in SDF format and converted to PDB format using Open Babel57. The predicted structural model of AChE was analysed for possible ligand binding pockets using CASTp server58. Docking simulations were performed using AutoDock 4.2.659 incorporated in MGL Tools version 1.5.6 (The Scripps Research Institute). Rigid docking was used in this study, where protein is treated as a rigid molecule and ligand as flexible. Polar hydrogen atoms were added to the protein and non-polar hydrogen were merged. Gasteiger charge was added to the macromolecule. The grid size was set to 100 Å × 100 Å × 100 Å with a spacing of 0.636 Å to cover entire binding surfaces and pockets of AChE. During the docking, 100 conformations were considered with Lamarckian genetic algorithm search, other assorted parameters were set to the default values of the AutoDock 4.2.6 program. Cluster analyses of these conformations were performed on the basis of their respective root mean square deviation (RMSD) in BIOVIA Discovery Studio visualizer60. Top populated conformation clusters from the analysis were selected for comparison with major CASTp pockets and previously designated substrate binding site with catalytic triad of the predicted AChE model. Impending binding cavity was selected based on the top cluster of AutoDock and largest pocket by CASTp lie on top with the substrate binding site. Another set of docking simulations on this resulted binding site was performed by setting up a smaller grid box, 50 Å × 48 Å × 48 Å with a spacing of 0.375 Å and by keeping the rest of the procedures same as earlier. The best docked conformation of 2,3-dimethylmaleic anhydride was selected based on lowest binding energy. Furthermore, CHARMm force-field with smart minimizer (2000 steps) was used to minimize the docked complex, considering Generalized Born with Molecular Volume (GBMV) implicit solvent model. Total and individual interaction energy (IE) possessed by the amino acid residues was calculated. UCSF Chimera61 package was used to perform molecular graphics and analyses on the resulted docked complex. Schematic diagrams of the interactions were depicted using LIGPLOT program62.The structural coordinates of 2,3-dimethylmaleic anhydride (PubChem CID: 13010), was downloaded from NCBI PubChem compound database  observed by Newman-Keuls test using Statplus 2007 software and computer program SAS .KDSupplementary Information


The smoothened (SMO) receptor is an essential component of the Sonic hedgehog (SHH) signalling, which is associated with the development of skin basal cell carcinoma (BCC). SMO inhibitors are indicated for BCC patients when surgical treatment or radiation therapy are not possible. Unfortunately, SMO inhibitors are not always well tolerated due to severe side effects, and their therapeutical success is limited by resistance mutations.We investigated how common are resistance-causing mutations in two genomic databases which are not linked to BCC or other cancers, namely 1000 Genomes and ExAC. To examine the potential for combination therapy or other treatments, we further performed knowledge-based simulations of SHH signalling, in the presence or absence of SMO and PI3K/Akt inhibitors.The database analysis revealed that of 18 known mutations associated with Vismodegib-resistance, three were identified in the databases. Treatment of individuals carrying such mutations is thus liable to fail a priori. Analysis of the simulations suggested that a combined inhibition of SMO and the PI3K/Akt signalling pathway may provide an effective reduction in tumour proliferation. However, the inhibition dosage of SMO and PI3K/Akt depended on the activity of phosphodiesterases (PDEs). Under high PDEs activities, SMO became the most important control node of the network. By applying PDEs inhibition, the control potential of SMO decreased and PI3K appeared as a significant factor in controlling tumour proliferation.Our systems biology approach employs knowledge-based computer simulations to help interpret the large amount of data available in public databases, and provides application-oriented solutions for improved cancer resistance treatments.The online version of this article (10.1186/s12885-018-4451-1) contains supplementary material, which is available to authorized users. Smoothened (SMO), a 7-pass transmembrane protein, is a component of the Hedgehog (HH) and Patched (PTCH) signalling network responsible for the regulation of cell growth and embryonic development. Mutations that affect the members of this signalling network are associated with the development of skin basal cell carcinomas (BCC)  through ®;, FDA-approved in 2012) and sonidegib [®;, FDA-approved in 2015) that block SMO are available [Amino acid mutations in SMO that lead to an unrestrained receptor activity are called activating mutations . Althougonidegib  , D473N (p=4.13·10−5) and D473H . This indicated that normal genetic variation was enough to confer resistance to SMO inhibitors. Of note, in the vast majority of cases, resistance mutations lead to a decrease in the drug’s affinity towards its molecular target, which cannot be offset by a higher dose of the drug due to toxicity and intolerance. Combination therapy therefore represents a potential solution to treatment-resistant cancers. Simulation and sensitivity analysis of the SMO signalling network were used to identify optimal signalling targets for a combination therapy against drug-resistant BCC.We further searched for the presence of resistance mutations in two genomic databases that are not linked to BCC or any other cancer, namely 1000 Genomes and ExAC. This analysis (carried out on February 2017), revealed that three resistance mutations were found in the population: W281C , signalling to the tumour proliferation end-point is mediated principally by the pathway involving the PI3K and Akt kinases  is a PDE4 inhibitor also used to treat B-cell malignancies [Several, selective and non-selective PDEs inhibitors are currently available and have the effect to increase intracellular cAMP , 21. PDEgnancies .β(cAMP) from 0.001 to 0.1), the SMO network became less branched: the nodes GSK3b and SUFU became less involved in conveying signalling from SMO to the tumour proliferation end-point (TumPro). In addition, the steady-state activity of the tumour proliferation end-point was reduced  assumed in the network simulation, revealed a bottleneck where the only relevant control node for the signal flow in this extreme situation is cAMP , tumour proliferation had a high steady-state activity (not shown), and was rather insensitive to variation in PI3K and SMO =0.001 and β(cAMP)=0.1, respectively), the system’s highest sensitivity values were obtained by variations in SMO activity, i.e., the network end-point’s controllability primarily depended on variations in SMO activity ; and secondarily on PI3K activity variations . In contrast, when the cAMP level was high (corresponding to a very strong PDEs inhibition), the end-point’s controllability showed a stronger dependence on PI3K than on SMO activity variations (0.02538 versus 0.01380). Therefore, under no and moderate PDEs inhibition (β(cAMP)≤0.1), SMO was ∼20 more influential than PI3K for controlling tumour proliferation, while at high PDEs inhibition (β(cAMP)=10) PI3K was ∼20% more determinant than SMO for the end-point control.Network control analysis suggested that the effect of dual inhibition of SMO and PI3K differed depending on the PDEs activity  sensitivity values in Table Principal component analysis (PCA) was used to detect co-activity and co-regulatory patterns  between the signalling components at the different cAMP levels applied in the network control analysis dataset presented in section “Signalling pathway analysis identified PI3K/Akt and PDEs as potential targets for treatment of BCC”. Sensitivity PCA showed that by increasing cAMP from a low level, a cluster formed that included cAMP and PKA . Our results indicate a complex regulation of SMO and PI3K as a function of PDEs activity. SMO was the most important control node of the network at no and moderate inhibition of PDEs. However, under strong PDEs inhibition, the systems became more sensitive to both SMO and PI3K, but PI3K became more relevant than SMO for controlling tumour proliferation. PI3K was previously suggested to be involved in acquired resistance to treatments against refractory tumours , 17. Ourαi proteins decrease the level of cAMP upon Guanosine-5’-triphosphate (GTP) hydrolysis (indicated as GaiGTP); and consequently prevent the inhibitory phosphorylation of Gli by protein kinase A (PKA). In parallel, G βγ (Gbg) subunits inhibit PKA through the PI3K/Akt (phosphoinositide 3 kinase / protein kinase B) pathway [β (GSK3b)-mediated inhibition of Gli proteins [A signalling network model of SMO and its principal interaction partners described in the literature was constructed based on the current state of knowledge , 28–36 . SMO is  pathway , 33. Thiproteins , 35. Furproteins , 36.The interaction network model of SMO signalling was simulated with the computational method developed by us previously , 14. Theβ) is summarised in Additional file The simulation procedure yielded steady-state activity levels of the different network components according to a given set of parameters. The range of independent activities of the different network components (high (β=0.1) or low (β=0.001) initial activity state  denotes the steady-state activity of a node N and β(N) its independent activity state. The arrow (→) indicates a change in condition.where sn conditions that represent all possible states of the network .Without considering the combined activity change of multiple control nodes simultaneously, but only the changes occurring subsequently one after another (as it would be expected by point mutations affecting the activity of a protein), Eq. vice versa). This resulted in a set of calculated sensitivities derived from the coarse-grained simulations that comprises Sensitivity is subsequently computed for each pair of simulated conditions that differ by a single parameter  indicates that upon the increase of the activity of A, B’s activity will also increase. Similarly, a negative sensitivity indicates that upon an increase of A’s activity, B’s activity will decrease. Sensitivity values close to 0 indicated independence between nodes. Signal flow graphs see Fig.  were buiβ) in small steps  activity, while few nodes, identified by signalling pathway analysis as relevant for controlling the network behaviour, were varied over a range of activities  and represented in a tree-like structure (dendrogram) whose branches grouped network components according to their similarity over the different simulations . ItAdditional file 1Supplementary Material. PCA at different levels of cAMP and model parameters. (PDF 1380 kb)


This study shows that at pressures >1 GPa the crystallisation behaviour of VHDA and uHDA is basically the same for all heating rates. That is, parallel crystallisation is almost entirely suppressed with mainly ice XII forming. This contrasts former results reporting parallel crystallisation to approximately levelled phase mixtures of ice IV and ice XII even at higher pressures for uHDA. We speculate this to be due to formation of microcracks upon decompression in earlier works, but not in the present one. Crystallisation temperatures Tx are up to 16 K higher than previously reported, raising the low-temperature border to no man’s land and opening a considerably larger window for future studies on non-crystalline water. The results indicate uHDA to contain heterogeneities on the nanoscale, but VHDA to be rather homogeneous with nano-crystallites being largely absent. Upon transforming uHDA to VHDA, the nano-scale heterogeneities disappear for >1 GPa whereas microcracks do not.The crystallisation behaviour of very high-density amorphous ice (VHDA) and unannealed high-density amorphous ice (uHDA) has been studied  One of its puzzling qualities is the great variety of solid phases it can form. Besides the numerous solid crystalline types (polymorphism)5 also different solid amorphous phases have been identified (polyamorphism)7. Figure h and at lower temperatures of ice XI, i.e., ~0–0.2 GPa. Intermediate pressures8  range from ~0.2–2 GPa. This is the richest pressure range, in which water exhibits a broad variety of stable and metastable crystalline phases . The higher pressure range is accordingly located at ~p > 2 GPa where the “symmetric” ices VII, VIII and X can be prepared8. The amorphous ices have been labelled according to their densities and may occur below T ≈ 180 K and p < 3.5 GPa. Amorphous ice of low density can be obtained by water vapour deposition on a cold substrate10, hyperquenching of micrometer-sized droplets onto a cryoplate11 or by decompression of high-density amorphous ice (HDA) at elevated temperatures12. The low-density amorphous ices (LDAs) prepared by these different routes are very similar in terms of structure and density, and thus they represent a thermodynamically well defined metastable phase13. HDA was discovered in 1984 by Mishima et al.12 by pressurisation of hexagonal ice Ih at liquid nitrogen temperatures. This process of pressure induced amorphisation (PIA) was then observed for the first time on the example of water. The latest amorphous phase described is very high-density amorphous ice (VHDA14). It can be obtained by annealing of HDA at intermediate pressures (p > 0.8 GPa). Just like all stable and metastable crystalline ices, the amorphous ices can be quenched and recovered at ambient pressure and stored in liquid nitrogen. At atmospheric pressure and 77 K the amorphous ices’ densities are 0.94 ± 0.02 g cm−3 (LDA15), 1.17 ± 0.02 g cm−3 (HDA15) and 1.25 ± 0.01 g cm−3 (VHDA14). LDA is located in the lower pressure regime, while HDA and VHDA are situated in the intermediate pressure regime and cease to exist at pressures exceeding ~3 GPa16. Just like between crystalline ices it is possible to switch back and forth between amorphous ices by varying pressure. One has to note, though, that the pressure-induced interconversion of LDA into HDA and HDA into LDA is accompanied by a hysteresis, i.e., there is a range of metastability in the low-pressure regime for HDA and likewise in the intermediate-pressure regime for LDA18.Water is a fascinating substance and in many ways its behaviour eludes scientific expectations. These aberrations from the ‘norm’ have been labelled the anomalies of water and have been described many times in literature19. It aims to explain the abnormal evolution of various thermodynamic quantities in a region of the phase diagram which has been rather elusive to experiments. This region has been labelled ‘no man’s land’ of water20, a term originally “used to define a contested territory”21 and especially connected to the unoccupied fighting ground between the opposing forces in World War I. Regarding water, no man’s land is bordered by the homogeneous nucleation at higher temperatures TH where liquid water freezes inevitably and at lower temperatures by the crystallisation temperature of the amorphous ice phases TX 17 and on HDA by in situ volumetry32. In the study performed by Seidl et al. mainly in the low-pressure regime32 it was shown that the pressure annealed variant of high-density amorphous ice – expanded HDA (eHDA) – is up to 11 K more stable against crystallisation than the HDA obtained from pressure-induced amorphisation of ice Ih . It was reasoned that the lower thermal stability of uHDA is due to nano-crystalline domains that remain in the amorphous matrix when it is produced from ice Ih31. When uHDA is annealed at elevated pressures >1 GPa and decompressed at elevated temperatures to form eHDA the crystalline domains might disappear. In this scenario crystalline seeds are already prevalent in uHDA and only have to grow upon heating. In eHDA however, they first have to nucleate and then to grow, resulting in a higher thermal stability against crystallisation.Different routes have been taken to breach or overcome the boundaries to no man’s land, both in experiments as well as in simulationset al. was extended to higher pressures by investigating crystallisation of uHDA and VHDA in the intermediate pressure range. The motivation is to study whether or not the pressure-annealing - that likely results in the disappearance of nano-crystalline domains responsible for the shrinking of the no-man’s land at low pressures - also helps to shrink the no-man’s land at intermediate pressures. Alternatively, the structural inhomogeneities related to nano-crystalline domains in uHDA may survive the process of annealing and still be prevalent in VHDA. As eHDA is prepared from VHDA when decompressing at elevated temperatures the nanocrystallites may very well disappear during the transformation of VHDA to eHDA. Similar to the experiments on uHDA and eHDA32 the crystallisation behaviour of VHDA was investigated in situ by volumetry and ex situ by powder x-ray diffraction. As we will demonstrate below, in the range p = 0.7–1.8 GPa VHDA behaves rather differently from uHDA as indicated in results previously reportedin literature30. Notably, the process of parallel crystallisation appears to be much more suppressed in VHDA and crystallisation temperatures Tx seem to be considerably higher. By conducting an own set of experiments on uHDA under comparable conditions it can be shown however, that uHDA and VHDA exhibit almost the same crystallisation behaviour at p > 1 GPa. That is, uHDA reaches a state very similar to that of VHDA prior to transformation. At pressures p ≤ 0.8 GPa though, the results provide evidence for the suggestion of there being nano-crystalline ‘seeds’ in the amorphous matrix of uHDA. Effectively, due to these novel results water’s low-temperature boundary to the no-man’s land is raised by up to 16 K as compared to values formerly presented in literature30.In the present study the work of Seidl in situ (software TESTXPERT 7.1). Displacement curves were converted into curves of volume change (ΔV) by taking into account the bore geometry. Temperature was controlled by heating elements inserted into separate bores in the cylinder close to the sample. Additionally, a stream of liquid/gaseous nitrogen pumped through copper loops surrounding the steel cell was regulated accordingly. The temperature was recorded via a temperature sensor (Pt-100) inserted into the steel cylinder in another separate bore close to the sample33.For the sample preparation a custom made piston cylinder setup (8 mm bore diameter) was employed. Uniaxial pressure was applied using a computerised commercial material testing machine (ZWICK model BZ100/TL3S) which also allows the tracking of the piston displacement, and hence sample volume, via uHDA, which itself was obtained by pressure-induced amorphisation of hexagonal ice Ih. In detail, ice Ih was compressed to 1.6 GPa at 77 K to yield uHDA, then decompressed to 1.1 GPa and subsequently heated isobarically at 1.1 GPa to 160 K. Then the so-produced VHDA was quenched to 77 K and finally brought to selected pressures between 0.7 and 1.8 GPa. At these low temperatures VHDA does not reconvert to HDA even when going to pressures p ≤ 0.8 GPa34. VHDA  was observed. Due to the difference in density between VHDA and the obtained crystalline phases (ρ (VHDA) < ρ (cryst. ice)), a given transformation could be identified by a rather steep ‘step’ in the displacement curve indicating sudden densification. Onset temperatures of crystallisation Tx were determined by the intersection of two straight lines .Crystallisation temperatures of very high-density and unannealed high-density amorphous ice were determined by conducting isobaric heating experiments. The starting material VHDA was prepared HDA Fig.  was heatθ-θ geometry . For quantitative analysis of the powder x-ray diffractograms the most intensive Bragg peaks  and eHDA (0.1–0.5 GPa32) VHDA experiences parallel crystallisation at intermediate pressures 0.7–1.8 GPa. That is, more than one polymorph of crystalline ice is formed upon crystallising VHDA. Figure p < 1.6 GPa VHDA mainly transforms to ices IV and XII, with some ice V forming at 0.7 GPa (0.5 K min−1). At pressures p ≥ 1.3 GPa ice VI is also found upon crystallisation  crystallisation occurs at a temperature 18.5 K lower (164.5 K) as compared to heating with a high rate .Similar to results formerly obtained on uHDA  and fast heating (30 K min−1). Furthermore, analogous to the former results on eHDA32 and uHDA32, different mixtures of crystalline polymorphs are obtained. Especially at lower pressures (0.7 and 0.8 GPa) there is a considerable difference in the relative yields of crystalline phases when changing from slow to intermediate and fast heating. At 0.7 GPa ice IV (50%) and ice XII (43%) are formed in roughly equal amounts after heating with 0.5 K min−1. However, at 5 K min−1 mostly ice XII is obtained (92%) and far less ice IV (8%). At 30 K min−1 formation of ice IV is entirely suppressed, and ice XII can be obtained purely. While the observation of parallel crystallisation kinetics at elevated pressures is in qualitative agreement with previous results32, the suppression of all competing crystallisation channels even at low heating rates in the amorphous matrix and especially at pressures p ≥ 1.1 GPa is a novel result. Parallel crystallisation from the amorphous matrix is generally governed by processes with different kinetics: slower processes starting at lower temperatures and faster ones commencing at higher temperatures (labelled type 1 and 230). Accordingly, at 0.7 GPa formation of ice IV (and V at a rate of 0.5 K min−1) on the one hand is governed by a process with slower kinetics, thus starting at lower temperatures and predominantly when heating with lower rates. On the other hand, formation of ice XII is determined by a process with faster kinetics. It crystallises to a greater extent at higher temperatures and faster heating rates. Similar behaviour is observed at 0.8 GPa where considerable amounts of ice IV crystallise only when heating with a low rate ., while ice V does not form at all. At higher rates (5 and 30 K min−1) ice XII forms almost exclusively with a relative yield of >90%. The suppression of parallel crystallisation processes in VHDA is also evident at pressures above 0.8 GPa. Ice XII forms predominantly at 1.1, 1.3 and 1.6 GPa, no matter the heating rate. Its relative yield is >95% in nearly every case. Furthermore, at p ≥ 1.3 GPa also ice VI is obtained at a high heating rate. At 1.8 GPa .At one given heating rate 30. That is, the process of parallel crystallisation can be characterised by two (or more) different kinetics associated with polymorphic formation. At lower heating rates crystallisation occurs at lower temperatures and phases that form with slower kinetics will be obtained to a greater extent. These slower kinetic processes govern the formation of ices IV and V at lower pressures . Two density steps are observable in the ΔV curve, one attributable to the formation of ice IX and the other to the formation of ices IV and V. In VHDA ices IV, V and XII form simultaneously. To elucidate these differences between the amorphous ices, uHDA was investigated in a likewise manner in a pressure range from 0.7 to 1.3 GPa employing heating rates of 0.5, 5 and 30 K min−1. The results demonstrate that notable discrepancies between uHDA and VHDA as starting materials in fact only occur at pressures p < 1 GPa. In Fig. −1), with a maximum difference of about 3.5 K at 0.7 GPa and heating with 5 K min−1. However, when high heating rates of 30 K min−1 are employed Tx is considerably lower for uHDA compared to VHDA, with a difference ΔTx of 12.5 K at 0.8 GPa and even 14 K at 0.7 GPa . This indicates that the structural states of the amorphous phases at one given pressure and when heating with one given rate is very similar, if not the same just before the transformation. A further evidence for this is that the densification steps upon transformation in the volume curves are of almost the same size, meaning that the density of the amorphous phases just prior to crystallisation is in fact very similar. This seems sensible, as one would expect uHDA to transform to VHDA at pressures p > 0.8 GPa already at temperatures T < Tx. The uHDA to VHDA transition has been shown to take place at 130 K and 1.1 GPa by Amann-Winkel  a different protocol of preparation was used to prepare uHDA, which was first decompressed to ambient pressure and then recompressed before isobaric heating rather than directly decompressed as done in this study.Inspecting the literature data on uHDA crystallisation more carefully, two differences to the present study surface: (i) different definitions for Tx for instance at intermediate heating rates  become almost negligible. However, at low heating rates of 0.5 K min−1 the variation in crystallisation temperature is still considerable even when taking into account different evaluation methods. One has to bear in mind that uHDA in literature30 and in this study were heated isobarically under nearly identical conditions , evaluating the old data with the tangent method also used in the present study the differences in 30 does not follow a stringent pattern. Even at pressures p > 1 GPa the relative yield of ice XII may be approximately 50% or less cracks’35 are introduced. These microcracks increase the surface area and may act as nucleation spots in the amorphous matrix and could very well be a cause for inhomogeneous crystallisation behaviour. In other words, the ice IV that was found to crystallize almost purely in literature in the slow heating experiments starts to crystallize from the additional surfaces within the microcracks. By contrast, ice IV cannot crystallize in the present work in similarly slow heating experiments because the microcracks and hence the nucleation sites for ice IV are absent.That is, the different evaluation method in item (i) cannot explain these variances. Hence, the difference has to be sought for in the experimental protocol, i.e., in item (ii) mentioned above. While in this study uHDA was prepared from ice ITx can be identified with ease for experiments at 1.1 GPa: the volumetric curves shown in Fig. Tx. The two straight lines indicate linear thermal expansion of VHDA and sudden densification caused by crystallisation. This is evidenced in Fig. ex situ X-ray diffraction experiments marked by arrows. Clearly, Bragg peaks suddenly appear at the kink, but not before the kink. At 0.7 and 0.8 GPa Tx can also be identified readily. The linear thermal expansion at low temperatures is followed by a stronger expansion at higher temperatures, where the amorphous matrix relaxes structurally and volumetrically in the experimental time scale. The sudden change from expansion to densification signals Tx. After the complete transformation to a crystalline phase/phase mixture the sample thermally expands upon further increase of temperature. Determination of Tx is less straightforward at 1.3 and 1.6 GPa. The reason being that relaxation of the amorphous matrix is associated with densification (rather than the expansion seen at 0.7 GPa and 0.8 GPa). That is, densification may signal either relaxation of VHDA itself or crystallisation of VHDA. In most cases, the time scale for relaxation is much longer than the time scale for crystallisation. As a result one can identify two kinks in the volumetric experiment  show that transformation takes place just before the slope of the ΔV curve becomes positive again. That is, the expected step in the volume curve indicating crystallisation is masked by the densification of VHDA due to thermal relaxation. It is evident from the broad halo peak in the diffractograms that even at 170 K the sample is mainly amorphous. Only at 171 K sharp Bragg reflexes indicate the start of crystalline ice XII growth from the amorphous matrix. The change of slope in the ΔV curve from negative to positive reflects the completion of thermal relaxation and subsequent crystallisation of VHDA. The temperature, at which crystallisation commences can be extracted in this curve by the method of tangent intersection  next to ice IV.We have demonstrated in this study of isobaric heating experiments that VHDA exhibits a considerably different crystallisation behaviour from that previously reported for uHDA in literaturep > 1 GPa uHDA and VHDA exhibit almost the same crystallisation behaviour. That is, the discrepancy in crystallisation temperature is ΔTx ≤ 2 K and apparently becomes smaller the higher the pressure. Also, both uHDA and VHDA transform to ice XII almost exclusively no matter the heating rate. This can be reasoned by the circumstance that at pressures p > 0.8 GPa HDA will transform to VHDA at elevated temperatures and before crystallising. Thus, at 1.1 and 1.3 GPa the different amorphous phases should be very similar prior to crystallisation. This is also indicated by the fact that the densification step in the volume curves during transformation is of approximately the same size - keeping in mind that both uHDA and VHDA transform almost entirely to ice XII, which will have the same density at a given pressure regardless of the prephase. This observation is supported by relaxation studies with dielectric spectroscopy36, indicating that very high-density amorphous ice transforms to an ultraviscous liquid state prior to crystallisation at 1 GPa and T > 140 K. Similarly, recent isobaric in situ volumetry studies on the relaxation of eHDA at higher pressures have yielded glass transition temperatures Tg of approximately 125 ± 5 K at p ≥ 1 GPa37.Here it could be shown that at pressures of p ≤ 0.8 GPa notable differences in the transformation behaviour can be found. While at low and intermediate heating rates values for Tx are very similar for uHDA and VHDA, they are up to 14 K lower for uHDA when heating with 30 K min−1. A possible explanation may be the existence of nano-crystalline domains acting as nucleation seeds within the uHDA matrix which had been proposed to be responsible for the differences in crystallisation behaviour between uHDA and eHDA at lower pressures32. It was reasoned that in case of uHDA the crystalline domains only need to grow, whereas in eHDA they first have to form and hence the higher thermal stability against transformation in eHDA of up to 11 K. For VHDA, while at low and intermediate heating rates the time scales may be low enough for crystalline seeds to disappear at 0.7 and 0.8 GPa, this may not be the case when heating with high rates. In this case the nano-crystalline domains may trigger transformation already at much lower temperatures than would be expected.At lower pressures et al. and the present work is shown in Fig. The size of the no-man’s land and how it was shrinked by the previous work of Seidl