Tachycineta bicolor, a model species of wild songbird, are well-studied during their breeding season; but our understanding of their biology at other times of the year, when they are not tied to the fixed location of a nest, is more limited. We developed a lightweight radio tag with no battery (solar nanotag) to study the movements of small animals, and we deployed it to explore the behavior of Tree Swallows after the end of their summer breeding season. We tagged 32 breeding adult swallows and 36 juveniles and monitored their presence and absence at the breeding site during the post-fledging period. Although our observations are based on very small sample sizes, the tags revealed previously unknown patterns in Tree Swallow behavior during the post-breeding season. Some Tree Swallow fledglings continued to visit the site repeatedly in the months following the nesting season, with the latest detection occurring on September 30th; by contrast, all adults had permanently departed by the end of July. These results inform future hypotheses about post-breeding movements in Tree Swallows. But, more generally, the detection of tagged swallows on their distant wintering grounds, seven months after tagging, indicates the potential of studying small passerine movements throughout their entire lifetimes, and suggests a rich array of applications for these “Life Tags” to study the movements of small animals world-wide.The availability of small, lightweight tracking devices enhances our ability to study birds during mobile phases of their lives. Tree Swallows  Zonotrichia leucophrys) who stayed at natal sites long after fledging were more likely to return to breed there after migration. A better understanding of movement patterns between fledging and territory establishment will lead to critical advances of understanding about dispersal, a highly consequential process for population dynamics and evolution. For example: Are dispersal decisions more strongly affected by the nestling period or the post-fledging period? What factors during the post-fledging period influence dispersal, and how do these vary by context? Although the importance of the post-fledging period is clear, it has historically been difficult to study [The time between fledging and the onset of migration  is a coto study ,10. Receto study  and the to study .We used solar-powered radio tags to investigate patterns of natal site visitation in adult and juvenile Tree Swallows during the post-fledging period. These solar nanotags were produced by the Technology for Animal Biology and Environmental Research (TABER) group at Cornell University, and they have been improved and are now being sold as “Life Tags" by Cellular Tracking Technologies, Cape May, NJ, under license to Cornell University. Because these tags are powered by solar cells and have no batteries, they have no set limit to their lifespan and, at 0.7 grams, they are light enough to be carried by many small passerines. A unique identification code is transmitted by each tag, allowing a large number of individuals to be monitored on a common frequency. The tags transmit their ID code approximately once every 2 seconds during daylight hours, and they are receivable from a range of approximately 2 km with base stations equipped with Yagi antennas.Tree Swallows are considered a model organism for wild passerines , but virWe studied Tree Swallows at the Cornell University Experimental Ponds Facility, Unit 1, in Ithaca, New York, between June and October of 2015. The site hosts a colony of Tree Swallows nesting in man-made nest boxes around a system of ponds. This colony has been monitored regularly each summer for over 30 years. Further details about the site, and the general methods used each summer, are described in [We tagged juvenile Tree Swallows while they were still in the nest. The nestlings used in this study were subjected to an experimental manipulation of the stress hormone corticosterone. This study is described in full in , but thaWe tagged nestlings between 10 and 18 June 2015. Tagged nestlings were between 14 and 17 days old, with 75% tagged on day 15. Nestlings are unable to fly at this age and were returned to their nest boxes after tagging.Before tagging, we weighed nestlings and attached tags to the heaviest nestling from each experimental group in each nest; thus, an equal number of corticosterone-manipulated and control individuals were tagged . We taggWe sexed nestlings using DNA from red blood cells and a standard protocol for sexing Tree Swallows . AdultsAdult male Tree Swallows average heavier than females  and, becWe tagged 32 adults  breeding at the site between 4 June and 14 July 2015. Adults were captured after their eggs hatched, usually when nestlings were 6 days old or older. We tagged adults opportunistically based on availability of tags during the periods when adults were being captured; adults did not come from the same nests where we tagged nestlings, except for one male at a nest with tagged chicks. We weighed adults before tagging to avoid tagging the few individuals in poor condition (those weighing < 17.5 g). Tags with a harness weighed 0.7 g; the 17.5 g cutoff insured that no birds had tags that weighed more than 4% of their body mass.We considered the “breeding site” to be a circle with a radius of 500 m centered over the nest box area. This circle, which also represents the area over which tags could be detected by our autonomous receiver, includes all of the nest boxes  and a small amount of surrounding area. We placed an autonomous base station receiver with a 1.67-m Diamond X50NA omnidirectional antenna  near the center of the experimental site on 7 June 2015 and removed it on 25 October 2015. During that time period, the station was not operational for ~18 days  because of battery and mechanical issues. For an additional 23 days , it was either not operational for part of the day or its operational status could not be confirmed  was heat-sealed between two sheets of mylar polymer with a protruding tongue. Pairs of pass-through holes were incorporated into the polymer tongue for leg loop attachment points of a harness [Details of these tags and how to make them are part of a proprietary manufacturing license with Cellular Tracking Technologies , but, in short, the tags consist of a small integrated circuit chip  that transmits a 32-bit digital signal. This signal is interpreted as an 8-digit code by the receiver, with each digit representing 4 bits of the 32-bit signal. The ID was coded and transmitted via amplitude-shift keying  whenever harness  on the bBecause they use solar panels rather than batteries, these tags were intended to have a working lifespan of more than one year. However, this first generation of tags had issues with antenna attachment because, to save mass, we used only solder to attach the antenna to the circuit board, believing that the mylar sheath would protect the attachment point; however, the adhesive between mylar layers near the attachment point was eroded by antenna movement over time, allowing strain on the antenna attachment that caused it to fail. This problem was remedied in subsequent versions of the tag by a reduction in antenna mass and stiffness, structural reinforcement of the antenna-board connection and a vibration damping connection between the mylar sandwich and antenna base.After fall migration had begun, we searched for tagged Tree Swallows in the field on migratory staging and wintering grounds. On 17 & 26 July, and 23 & 28 August 2015, TMP and ERGC brought receivers equipped with Yagi antennas  to Montezuma Wildlife Refuge, ~60 km north of the breeding site. Tree Swallows stage at this site as they prepare for migration, often forming large roosts . On 18–1Data from the base station included a received tag’s code and the date and time the code was received. Data were offloaded from the base station, processed in TextWrangler to remove unusual characters or corrupted reads, and analyzed in R 3.4.1 .Most of our analyses focused on whether a tag code was received on a given day. A tag was considered present at the breeding site on a day if its code was received at least once on that day, and unless otherwise noted, we made no analyses distinguishing the number of times a code was received. We used Wilcoxon rank-sum tests to compare mean values between the adults and fledglings because the data were not normally distributed. We compared the following variables:Fledgling latency to departure is the number of days between fledging and the first day on which no tag signal from that individual was received. For adults, we used the bird’s nest- fate day (either fledge day or death day) as the benchmark from which to measure latency to depart. However, we usually do not accurately measure fate dates at this breeding colony to avoid disturbing nestlings who are old enough to be at risk of premature fledging. For each nest, there is thus an interval of time between the last confirmed date the nest was active, and the date on which the fate of the nest was first discovered; we refer to this interval as the “fate interval” . We measNumber of returns for fledglings was the number of times (between June and October 2015) they returned to the site after being absent for one day or more. Number of returns for adults is similar; we only counted returns that occurred after the fate interval midpoint.For fledglings, this value represents the total number of days a fledgling was present at the site after fledging . For adults, this similarly represented the total number of days an adult was present after its fate interval midpoint.For fledglings, latest detection is measured as age in days (rather than days since fledging). For adults, latest detection is measured as the number of days since eggs hatched; thus, these two measures are directly equivalent between adults and fledglings, because both start counting at the hatch date at a given nest.PVEducation.org .We also estimated the lighting conditions under which the tags transmitted normally using light level information from In 7 of 15 cases where both siblings fledged, they fledged on the same day. Most other pairs of siblings fledged within three days of each other (mean difference = 1.2 days). In one nest, a single tagged nestling was found alive in a box six days after its siblings fledged ; at another nest, the fledge date of one sibling is unknown because the nest box was empty several days before that bird’s tag code appeared in the data collected by the receiver.Fledglings generally first departed the breeding site  within a few days of fledging, but there were some exceptions . For 11 Eighteen fledglings visited the breeding site after their initial departure. Most visits lasted only a day or a few days, although some visits lasted more than a week . AlthougSome adults left the breeding site for long periods within a day of being tagged, whereas others stayed until after their nestlings fledged . Though P = 0.0019). They were similar in number of returns (P = 0.076), but fledglings spent more time at the site after the breeding season and had later mean latest detection dates (P < 0.0001 for both tests).Adults (N = 32) and fledglings (N = 36) differed in latency to departure because many adults first departed before their nests' fate-dates (2). Assuming a clear day, illumination at solar noon at our field site is roughly 110,000 lux (0.869 kW/m2) and illumination drops below 7,000 lux within 0.5 hr of sunrise or sunset; this indicates that tag performance may be suboptimal in the 30 minutes after sunrise and before sunset. The tags performed as expected, with tag receptions spread broadly over the majority of daylight hours.The tags transmit steadily under lab conditions when illuminated at the equivalent of 7,000 lux , juveniles are sometimes known to defend nest cavities at their natal site during the post-fledging period [Only fledglings, not adults, returned to the breeding site after July. These fledglings may have returned to the site during the process of prospecting for their future breeding location: in the Purple Martin (g period . Across g period ), so it g period , while mg period . Swallowg period –32. In oRadio-tagging always presents some risk for birds , and theTagging adults may also make them less likely to produce fledglings; many nests at which we tagged adults failed to fledge young see . HoweverThe results presented here will serve as a starting point for future investigation. For example, actively searching for adult Tree Swallows between their departure from the breeding grounds and the onset of pre-migration roost formation may reveal where they go during this time period, lending insight into the habitats and resources they use as they begin to molt  and prepare for migration. Gathering information of this kind is increasingly important because Tree Swallows, like many other aerial insectivores, have experienced population declines in recent years . ConstrBecause the lifespan of these tags is not limited by the weight constraints of a battery, their theoretical lifespan is longer than that of the average passerine. The first-generation version of these tags lasted less than a year because of a weak attachment between the antenna and the tag, but we are confident that current improved versions of the tag will last far longer. This means that these tags can be used to find individuals which make natal or breeding dispersals away from the areas where they were captured. The ability to track individual songbirds for more than a year will facilitate attempts to experimentally manipulate dispersal distance, improving our understanding of this critical yet difficult-to-study event in avian life history.


Ascompared with the lowest quintile, the highest quintile of cystatin C was associatedwith a significantly increased risk of death from cardiovascular causes (hazard ratio[HR] 2.27 [1.73 to 2.97]), myocardial infarction (HR 1.48[1.08 to 2.02]), and stroke (HR 1.47 [1.09 to 1.96]) aftermultivariate adjustment. However, cystatin C is typically known as a marker of renalfunction, being roughly correlated with glomerular filtration rate in early stages ofkidney diseases.8 Reasonably, since glomerular function is a strongsurrogate marker of cardiovascular disease, it suggests an obvious association betweencystatin C and cardiovascular outcomes. A mechanism to avoid the impact of thisinexorable bias was to study only individuals with normal kidney function. Yet,additional studies have shown inconsistent magnitudes of effect between cystatin C andcardiovascular outcomes.In the 9 in this issue of the Brazilian Archives ofCardiology investigated the role of plasma cystatin C levels on the risk ofall-cause mortality and other softer endpoints by pooling studies of individuals withnormal renal function. Unfortunately, only two studies compared quartiles of cystatin Cwith multivariate regression analysis, hence providing a sample size that is not too farfrom the original Ludwigshafen Risk and Cardiovascular Health (LURIC)study.10 The meta-analysissuggested a robust association between high levels of cystatin C and the risk ofall-cause mortality in individuals with normal renal function . Heterogeneity among studies was substantial (I2> 50%) and no sensitivity analysis was provided. Besides the critical limitations inmeta-analysis data, authors also provided substantial elements in a systematic review ofstudies on the same topic.In that context, Einwoegerer and Domingueti12Although a first step for a candidate biomarker is to show strong association with aclinical outcome, this is not sufficient to prove its complementary clinicallyusefulness beyond traditional cardiovascular risk factors, such as age, gender, smoking,hypertension, diabetes, hyperlipidemia, obesity and aortic stenosis. A next fundamentalstep is to show whether cystatin C could improve risk prediction of cardiovascularoutcomes in Receiver operating characteristic (ROC) curves models, net reclassificationindex (NRI) and integrated discrimination index (IDI) compared-to or added-to theFraminghan Heart Risk, ASCVD risk score, or any validated cardiovascular riskscores/engines.Cystatin CMendelian Randomization Consortium no association could be found.13 This finding in no way suggests thatwe should abandon the use of cystatin C for risk stratification purposes in kidneydiseases, but there are two key messages in it: (i) it alerts against the chase oftherapeutic strategies that target at lowering plasma cystatin C levels; (ii) it alsoindicates a low likelihood of association between cystatin C as a surrogatecardiovascular marker on top of classical risk factors. However, the last word in favoror against the use of cystatin C in clinical practice for cardiovascular riskstratification of individuals with normal renal function should be based on studiesevaluating detrimental effects of this marker on established risk scores/engines.Besides the potential mechanistic link between cystatin C and atherosclerotic disease,this association is unlikely to be causal. By using a Mendelian randomization approach,which takes into account both the genetic association with cystatin C and CVD totriangulate the causal effect, and combining a set of cohorts of over 250,000individuals with 63,000 cases of cardiovascular events from the


P = 0.003), shorter surgery time (P = 0.001), and less blood loss (P = 0.037). Compared to the conventional free-hand technique, 3D printed model–assisted is helpful to screw placement in atlantoaxial internal fixation, which can be used as a common tool to provides important guidance for upper cervical surgery.The aim of this study was to evaluate the efficacy and feasibility of a life-size 3-dimensional printing assisted posterior internal fixation. We performed a retrospective review of 138 patients who received posterior atlantoaxial internal fixation from October 2009 to March 2015 with a minimum follow-up period of 12 months. Group A included 76 patients who received the conventional free-hand technique. Group B included 62 patients who were treated with internal fixation assisted by 3D printing. The placement accuracy of the screw was evaluated in the computed tomography images according to the methods of Hojo and clinical outcomes were evaluated using the visual analogue scale, the Japanese Orthopedic Association Score, and the Neck Disability Index score. There were no significant differences in the clinical results at any of the follow-up time points regarding the JOA, VAS, or NDI scores between two group. However, compared to Group A, Group B had better results for screw installation ( Unlike in the transarticular screw method, a free-hand transpedicle screw may migrate into the vertebral artery (VA) foramen and lead to vertebral artery injury, which may cause cerebellar infarction or brain stem infarction. In conventional surgery, 3D-reconstructed preoperative computed tomography (CT) images can only be visualized on a computer screen or X-ray view box, and the images must be memorized for use during surgery. This approach meets the demand for surgery with higher precision and efficiency.Posterior atlantoaxial screw fixation is the standard operative treatment for atlantoaxial internal fixation7.CT navigation can lead to the more precise placement of screws. However, the navigation system is too expensive for most hospitals; therefore, the application of this technology is limited due to factors such as a lack of equipment, insufficient training, and high costs10. However, posterior atlantoaxial internal fixation has rarely been studied in the context of surgery assisted by 3D printing. This study aimed to evaluate the accuracy of screws position during posterior atlantoaxial surgery, which was assisted by 3D-printed patient-specific models.Recently, 3D printing technology has been more widely used to develop precise and personalized surgical treatmentsFrom October 2009 to March 2015, 138 patients with atlantoaxial lesions received posterior C1–C2 fixation treatment in our hospital. Indications for surgery were reducible atlantoaxial fracture and dislocation (99 cases), congenital odontoid nonunion (12 cases), atlantoaxial instability due rheumatoid arthritis (10 cases) and other causes of atlantoaxial instability (17 cases). Exclusion criteria: (1) Other internal fixation techniques. Such as lamina screw, atlas lamina hook. (2) Atlantoaxial malignant tumor. (3) Atlantoaxial pedicle is thin, and the screws cannot be fixed accurately. All patients went through static and dynamic X-ray inspection, CT scanning evaluation, and magnetic resonance imaging (MRI).In the early stage, free-hand technique was used more frequently. 3D models were used according to doctors’ habits and patients’ wishes. In addition, some patients cannot use 3D models because they need emergency surgery. The patients were divided into 2 groups based on inclusion and exclusion criteria. Group A (n = 76) included 36 men and 40 women who treated with the traditional free-hand technique. There were 51 cases of atlantoaxial fracture accompanied dislocation, 7 cases of congenital odontoid nonunion, 4 cases of atlantoaxial instability due rheumatoid arthritis and 9 cases of atlantoaxial instability due rheumatoid arthritis. Group B (n = 62) included 26 men and 36 women treated with the assistance of 3D-printed models. There were 48 cases of atlantoaxial fracture accompanied dislocation, 5 cases of congenital odontoid nonunion, 6 cases of atlantoaxial instability due rheumatoid arthritis and 8 cases of atlantoaxial instability due rheumatoid arthritis. Group A had an average age of 51.3 years and Group B had an average age of 49.8 years. No statistically significant difference between the two groups in diagnosis and general data  examination before operations to conduct 3D printing of a VA simultaneously. DICOM data were saved as STL files through MIMICS software , and then 1:1 models were printed using 3D printers . It took approximately 4–6 hours from the initial CT scan to obtain the models, with general costs of roughly 1000 RMB. The cost of the 3D model is included in the hospitalization.3D models with a scale of 1:1 were used as templates to help surgeon diagnose fracture types and make preoperative plans, select the best screw entrance point and direction. The 3D models were sterilized at low temperatures before the operation and were used during the operations to assist surgeons with identifying anatomy,which were provided more intuitive information Fig. .Figure 211. The C1 screws  were inserted via the posterior arch of C1 into the lateral mass. The entry point of the C1 screw was at least 2 mm below the superior rim of the C1 posterior arch and aligned with the center of the C2 lateral mass. The trajectory was approximately 10° in the medial direction and 5° in the cephalad direction. The screw trajectory for the C2 pedicle screw placement was conducted using the method described by Harms and Melcher12. The entry point for the C2 screw  was the cranial and medial quadrant of the isthmic surface of C2 in line with the trajectory of the pedicle. The screw trajectory was placed approximately 20° to 30° medially and in the cephalad direction along the C2 pedicle.The screw trajectory for the C1 pedicle screw fixation was inserted using the method described by ResnickIn the atlantoaxial fracture and dislocation group,3D model can well reflect the specific location and severity of the fracture, show the direction of dislocation. In congenital odontoid nonunion group, 3D model allow surgeons to visually understand the true shape of the odontoid and extent of atlantoaxial dislocation. In atlantoaxial instability due rheumatoid arthritis group, 3D model show the extent of atlantoaxial dislocation and the area of bone destruction. In conclusion,The 3D printed model was utilized to assist the surgeon understand the degree of atlantoaxial injuries of patients, and determine the location and direction of screw placement during the operation, particularly to reduce the rate of VA injury. In surgery, the model can be reused from multiple perspectives and provide a more 3D concept and a more realistic view for the surgeon than can 3D computed tomography pictures.13. Grade 0 (G0): correct position. Grade 1 (G1): position of less than half of the screw diameter was incorrect. Grade 2 (G2): position of more than half of the screw diameter was incorrect , Japanese Orthopedic Association (JOA) and the Neck Disability Index (NDI)t tests. Quantitative data were analyzed with t tests for independent samples, whereas qualitative data were analyzed with chi-square tests or rank-sum tests. Pvalues less than 0.05 were considered statistically significant.Statistical analyses were conducted using SPSS 17.0 software. Differences in clinical and imaging data before and after the operations were studied using paired P = 0.037) and longer operation times (P = 0.001) than Group B.A total of 552 screws were placed in 138 consecutive patients during the study period. The patient distribution is summarized in Table P > 0.05) between the temporal points at 1 week, 3 months, 6 months, and 12 months after their operations.Detailed data are listed in Table Two independent observers measured CT imaging data using ACDSee Canvas 11 software one week after surgery, and they were unaware to which group the patients belonged. If the grade of screw is controversial, more significant degree of breach will be used in the final analysis.Detailed measurement data of the screw positions are listed in Tables All patients were prepared with conventional neck supports within 8 weeks to restrict their neck movements. There were six patients in group A  and four patients in group B  had migraines, which were significantly reduced after 3 months of symptomatic treatment. A total of sixteen patients with cerebrospinal fluid leakage and no infection after the operations, including ten patients in group A , six patients in group B . One patient in Group A  experienced cerebral infarction Fig. . The myo6, the placement of cervical pedicle screws has a considerable risk of injury, either to nerves or to the VA. Abumi et al.17 reported that 45 out of 669 inserted screws (6.7%) were misplaced in their early series. Since then, numerous reports have been published regarding the malposition rates of cervical PSs. Neo et al.18 reported that their malposition rate was 29% in patients with degenerative conditions. Onishi et al.19 reported on a patient who suffered cerebral infarction due to brain embolism after the placement of cervical PSs.Atlantoaxial surgery presents a considerable challenge because it has substantial risks, such as limb paralysis, VA injury, and even life-threatening emergencies. The traditional surgical approaches include the posterior approach, the anterolateral retropharyngeal approach, and the transoral approach. C1-C2 posterior screw fixation is currently the most popular technique. Despite the biomechanical superiority of cervical pedicle screws20. For example, during the operation the small and deep operational sight makes surgeon difficult to accurately measure the patient’s anatomy with trunk bone measurement methods or image data, and individual differences among patients, some of whom suffer from posterior arch deformities. When the variation occurs in the VA, the relationship between the top of the VA loop and the pedicle may change internally and externally, which could result in serious squeezing of the pedicle by the VA and difficulty in setting the screws. Although CT can be used for 3D reconstruction before operations, it provides only a general understanding of partial anatomical structures, and the limited pictures cannot fully and truly reflect the details of many partial anatomic structures. As a result, traditional 3D images can be viewed on a 2D computer screen or exposed to actual anatomical structures during surgery, which may affect the doctor’s placement of screws in the operation.Usually the surgeon based on preoperative imaging data and intraoperative anatomic measurements to determine screw trajectory. However, The anatomic structure of the atlantoaxial pedicle has not changed as much as that of the thoracolumbar anatomical structure, and there are more factors of anatomic variation for anatomic structures of the atlantoaxial pedicle. When applying the pedicle screw technique to atlantoaxial vertebrae, individual differences should be considered22. Intraoperative navigation has been used to help screw placement in the upper cervical spine. CT navigation can lead to more precise placement of screws. Compared with the preoperative three-dimensional CT navigation system, the navigation in the operation can reduce the difference between the data in the prone position during surgery and the preoperative CT data24. Yu et al.7 used intraoperative navigation in 23 cases  for screw placement. The authors point out that, despite the benefits of using navigation technology, there are potential pitfalls. Wide application of navigation system is limited by a higher level of radiation (the highest dose = 40 mGy o arm two scan), cost of establishing a navigation system and daily running cost, the additional time needed for preoperative image acquisition, data transmission, and the interrupt of surgery by intraoperative navigation performance24. Another problem is potential navigational errors. Uehara et al.25 used the CT navigation system in 359 consecutive patients, with PS insertion of C2–L5. The perforations have a rate of 5.0% in C2 screw fixation. The author indicated that because the anatomy is highly variable, although CT-based navigation systems can result in a more precise procedure, there are still some problems at the upper cervical spine levels27.To avoid serious complications such as VA injury, researchers attempted to improve atlantoaxial screw placement. For example, navigation systems have been used28. Furthermore, the rates of navigation system use in developing countries are lower than those in developed countries29. Therefore, it is necessary to develop a better method with improved accuracy and an easier operation.In addition, the navigation system is too expensive for most hospitals and the application of this technology is limited due to factors such as a lack of equipment, insufficient training, and high costs. Training, technical difficulties and learning curve related issues are commonly believed to be the main barriers to acceptance of navigation surgery32. In orthopedics, 3D printing materials can be made into implants, prostheses, prosthesis production, and the creation of life-size anatomical models34. Individual 3D printing technology can contribute to surgical planning by depicting precise anatomy and thus a potential improvement in surgical outcome.At present, 3D printing technologies are commonly used in product design industries, and their use is growing in all fields, including medicine, such as orthopedic surgery, orthopedics, urology, transplant surgery, cardiothoracic surgery and pediatric surgery35. In the discussion and consent of complex surgical cases, 3D printed model made surgeon predicted the difficulty of the operation, assisted the preoperative plan, and selected the best surgical option36. In addition, the models can be sterilized by low temperature plasma and taken into the surgical region. Local anatomical structures could be intuitively revealed during the operation from multiple perspectives. During the operation, the presence of 3D model did not change that technique of screw insertion. However with individualized 3D printed model-assisted, surgeons obtain visual and tactile help, the same proportion of the anatomical mode assist surgeons select the screw insertion point, direction of screw,length of screw, and improve the accuracy of screws. To some extent, it can improve the safety of surgical procedure, reduce muscle tissue exposure and shorten operation time. The operation time and blood loss of group B were statistically significant compared with group A.The 3D printed model provides a clear sense of anatomical structure, and 3D perspective photograph that provides much more information than 2D perspective photograph, help surgeon understand the characteristics of patients’ specific bone anatomiesAdditionally, in atlantoaxial internal fixation surgery, many patients have serious complications, such as VA infarction. However, these complications may not the result of direct damage, are caused by VA spasm due to local stimulation. A 3D printed model could facilitate the combination of screw trajectory and proper diameter and minimize the impact on the VA. The pathway of the VA and its relationship with surrounding bone structures are revealed intuitively, and thus the interference with the VA in the exposure approach at the beginning of surgery could be minimized. The use of 3D printed model may be responsible for improvement of the clinical symptoms.31. The cost of 3D printed model was 350 RMB per segment of the vertebra in our hospital. For atlantoaxial diseases, we usually choose 2–3 segments to print. So each patient will cost about 1000 RMB. Compared with the expensive cost of navigation technology, the usage fee of the navigation system is 5000 RMB per time , and the cost of 3D printed model is more acceptable to patients. The use of 3D model did not increase the economic burden of patients through statistical analysis of two groups of hospitalization expenses, further explained that when navigation is not available, 3D printed model could be used as a common tool for upper cervical surgery.With the development of 3D printing technology, production cost of the model is not expensive, it is relatively simple to makeThe disadvantage of this technology is that approximately 4–10 hours must be spent on obtaining CT data and producing the 3D-printed life-size model. Therefore, this approach cannot be used for patients who urgently require an operation.The study had some limitations. First, this was a retrospective study, where patient selection existed subjective factors. In the future, prospective, randomized controlled, multicenter, large sample clinical studies will be designed. Secondly, no comparison with intraoperative navigation technology, which is research hotspot of atlantoaxial screw placement at present. More research will be done in this area in the future. In addition, no further imaging studies were conducted, such as lack of postoperative CTA examination, which can reflect the malpositioned screws and determine the exact cause of postoperative complications. Postoperative CTA should be increased in future studies.Our data showed that the application of 3D printing technology can provide accurate data and rich information for surgeons, improve the precision of the fixation, reduce bleeding during operations, shorten surgical time, and reduce the rate of malposition complications. It can be used as a routine preoperative planning, assisted surgical simulation, act as an orientation aid during surgery. In our experience, 3D model has potential value for medical institution where cannot afford to use expensive navigation systems.Both 3D-model assisted group and free-hand technique group had significant improvements in the aspects of VAS, JOA, and NDI score before the operation. Compared to the conventional free-hand technique, application of 3D-printed model improved correctness of screw placement according to Hojo grading, reduced the operation time and blood loss. Unlike the navigation system, which is too expensive for most hospitals, a 3D-printed model could be used as a common tool to provides important guidance for upper cervical surgery.


In the presence of IgE, the hairpin structure and G-quadruplex would be destroyed, resulting in the inactivation of DNAzyme and subsequent reduction of its absorbance. This cost-effective approach detected IgE in the linear range from 5.0 pg/mL to 500 ng/mL, with the limit of detection (LOD) of 2.0 pg/mL, under optimal conditions. Moreover, the developed method was successfully applied to the rapid detection of IgE in human urine, indicating a great potentiality of this approach in clinical diagnosis and other biomedical applications.In this work, we have developed a simple and rapid colorimetric assay for the detection of immunoglobulin E (IgE) using functional nucleic acids (FNAs) and a solid-phase competition enzyme-linked immunosorbent assay (ELISA). The FNAs including aptamer of recombinant IgE, G-quadruplex and its complementary fragments were immobilized on 96-well microplates to achieve recognition and detection of IgE in biological samples. The G-quadruplex DNAzyme catalyzed 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS)-hemin-H Proteins, the natural biomolecules, often serve as biosignaling molecules, molecular indicators, and biomarkers. Proteins have attracted enormous research interest because of their crucial roles in clinical diagnosis, proteomics, and metabolomics. Detection and quantification of proteins during disease prevention and treatment can assist the clinicians in selecting proper treatments and monitoring patient’s responses, adjusting the therapeutic regimens timely and properly. In general, several traditional immunoassays, such as enzyme immunoassay , fluoroiRecently, FNAs, containing nucleic acid aptamer (single-stranded oligonucleotides from DNA/RNA libraries that can bind specific molecular targets) and DNA enzymes (DNAzymes) have become an important tool in protein detection assay . Due to In general, serum and urine are prevalently used to determine the protein concentration in the human body . While b2O2 system was considered as an effective strategy for the colorimetric assay for proteins [DNAzymes are DNA enzymes which can catalyze chemical reactions. G-quadruplex DNAzyme exhibit peroxidase-like activity. Efforts to develop catalytic DNAzyme sensors have been made, which allowed the biocatalytic detection in the hybridization process. In addition, it was reported that DNAzyme was coupled with nanoparticles as a colorimetric sensor for thrombin detection . Moreoveproteins ,23.Inspired by these facts and considerations, we hypothesized a simple, cost-effective, rapid, ultrasensitive assay for the detection of critical proteins based on FNAs. In this hypothesis, FNAs including aptamer and DNAzyme sequence can be modified on the surface of microwells and then be used to detect protein via absorbance measurements on a microplate reader. Meanwhile, applying the DNAzyme as a catalytic unit and aptamer can improve the sensitivity and selectivity, respectively.2O2. As a result, the colorimetric assay was easily achieved. Moreover, the developed method was validated in terms of linearity, sensitivity, repeatability, and accuracy. For clinical applications, the IgE would be successfully detected in a urine sample, with great capability and merit. Thus, we believe that the proposed simple and rapid strategy useful for IgE assay will be promising in disease diagnosis.As a proof-of-concept, IgE has been selected as a model analyte because it is an important indicator related to allergy-mediated disorders, atopic dermatitis, and other immunodeficiency-related diseases. IgE was analyzed by FNA-based fragment on competition law using a microplate reader. Initially, the surface of a micro-well was modified with aptamer exploited to specifically recognize IgE. The FNA fragment was designed with a hairpin structure including IgE aptamer, G-quadruplex sequence and its complementary sequence. The hairpin and G-quadruplex structure would be destroyed in the presence of IgE, leading to the reduction of the absorbance signal, based on G-quadruplex catalyzed ABTS–hemin–H165), epidermal growth factor (EGF), immunoglobulin G (IgG), immunoglobulin E (IgE), tween-20, ABTS and hemin were purchased from Sigma Co. Ltd. . Bovine serum albumin (BSA) was provided by Xiamen Tagene Biotechnology Co. Ltd. . Recombinant IgE Kit was purchased from Immuno Clone Co. Ltd . Tris (Hydroxymethyl) Aminomethane, NaCl, Na2HPO4, KH2PO4, Na2CO3, KCl and NaHCO3 were purchased from Sinopharm Chemical Reagent Co. Ltd. . The coating solution was prepared by 0.05 mol/L sodium carbonate buffer, with a pH of 9.6. The washing solution was prepared by adding 0.05% tween-20 (v/v) to PBS. The FNA was dissolved in TE buffer . The blocking solution was prepared by adding 1% BSA (w/v) to PBS. The assay solution was prepared by adding 0.15 mol/L NaCl to 0.02 mol/L TE buffer, with a pH of 8.0. Biotin was dispersed in Na-phosphate buffer . Streptavidin was dissolved in 0.1 M phosphate buffer saline (PBS) buffer (pH = 7.4).Platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor 165 (VEGFG GGT AGG GCG GGT TGG AAG CTT TAA CTC AGG GGC ACG TTT ATC CGT CCC TCC TAG TGG CGT GCC CCG CGC-biotin-3′. The underlined fragments refer to the first complementary sequences to the fragment (GCG) in G-quandruplex. The italicized fragment was the aptamer of IgE and the dissociation constants (Kd) of 10 nM aptamer ranged from 0.01 nM to 0.3 nM [The FNAs were synthesized from Sangon Biotech. Co. Ltd.  as following. 5′-GTo 0.3 nM ,24. The A protein stock solution was prepared by dispersing IgE at a concentration of 50 μg/mL in the assay solution, stored at −20 °C. The working solution of IgE was prepared in assay buffer. Other proteins were also prepared by dispersing them respectively in the assay buffer. Urine samples were donated by group members and were centrifuged at 1000 g for 10 min to remove the deposition immediately. After centrifugation, the urine samples were stored at −20 °C. Before the experiment, the urine temperature was restored to room temperature.The captured aptamer was immobilized in the microwell through the streptavidin-biotin technique by following steps. Briefly, the biotinylated microwell was washed three times using ultrapure water, and then the microwell was incubated by adding various concentrations of streptavidin in PBS buffer for 30 min in the dark, washed with PBS containing 0.1% SDS and water in sequence and then allowed to air dry. Various concentrations of biotinylated FNAs from 5 to 2500 fmol/µL were suspended in TE buffer and injected into microwells at different concentrations. Furthermore, the coating was performed by depositing 200 µL of biotinylated FNA solutions in each of the microwells. After evaporation, biotin solution  was injected into microwell for 15 min to fill the unoccupied biotin binding sites. Non-specifically bound FNAs were removed by three 15-min washes in Na-phosphate buffer at 37 °C. A standard curve was determined by coating similar solutions in additional microwells but without washing.2O2 mixture. After 10 min of incubation at room temperature, the corresponding absorbance values were detected using the microplate recorder. It should be noted that the prepared microplates and all the immunoreagents must be equilibrated to room temperature before the assay for each analysis.Initially, 100 μL of various concentrations of IgE solutions were added into corresponding microwels and incubated at 37 °C for 5 min. After the reaction, the wells were washed three times using the washing solution (200 μL). Subsequently, 100 μL of hemin (100 μM) was injected into the microwell, followed by adding 100 μL of ABTS and H0, B0 was the absorbance of the assay solution, B was the absorbance of samples, and X was the IgE concentration.The assay solution and samples were run in wells, and the absorbance was measured. Calibration curves were obtained by plotting Y against the logarithm of IgE concentration and fitted to the linear equation of Y-logX, where Y = B − B2O2 were strong. On the other hand, in the presence of IgE at 50 pg/mL, the absorbance values of FNAs were significantly low, demonstrating G-quadruplex structures were destroyed. These results indicated that the proposal is feasible, and the designed FNAs include G-quadruplex fragments that can be effectively used for the determination of IgE.In this work, we hypothesized that the formation of a stable hairpin structure based on the aptamer of IgE, G-quadruplex and its partial complementary sequence could be efficiently used for detection of protein A. The haIn this strategy, the hairpin structures, FNAs were fixed on the surface of microwell by streptavidin-biotin to capture the target IgE. First, the binding properties of the coat streptavidin into biotin coating microplate were investigated. It was evident that streptavidin can interact with the biotin modified microwell, leaving two additional free sites to bind biotinylated FNAs ,26. IndeWe further optimized the immobilized conditions of FNAs in microwells. It should be noted that the appropriately fixed FNAs conditions are important for the sensitivity and IgE homogeneity assay, as well as the time of incubation. We investigated the binding characteristics of FNA inside streptavidin-coated microwells in 2O2) and ABTS. Therefore, the high concentration of hemin will lead to a high background value and may eventually affect the accuracy of the analysis. However, the low concentration of hemin could not reflect the change of the concentration in time and extend the analysis time. Indeed, the catalytic conditions of G-quadruplex affect not only the detection time but also the detection accuracy. In addition to G-quadruplex DNAzyme, the high concentration of hemin can accelerate the reaction rate of hydrogen peroxide  was chosen as the optimum concentration ratio for the assay.Further, we explored the color signal produced by our system. It was evident that the color signal was also produced dependent upon the concentration ratio of chromogenic reagents including hydrogen peroxide and ABTS. Hydrogen peroxide-mediated oxidation of ABTS generated ABTSn by IgE B. Thus, 2O2-hemin and the results were plotted in 2O2-ABTS and hemin-DNAzyme catalysis. Therefore, the reaction time of 10 min was considered as the shorter analysis time.We further studied the effect of produced color signal with time as the time of chromogenic reaction could affect the absorbance and the detection time. We recorded the reaction time of DNAzyme-ABTS-H165, EGF, IgG, and BSA were spiked into PBS buffer with a concentration of 500 pg/mL. The blank was used as a control, which is a PBS buffer without any protein. The concentration of other proteins (500.0 pg/mL) was 10-folds higher than of that of IgE (50.0 pg/mL). The absorbance by the non-specific binding was much higher than that of the absorbance by IgE, demonstrating that the developed system resulted in high selectivity towards IgE in More often, the selectivity, as well as specificity, is considered as the most important factor for effective protein analysis. To investigate the selectivity of the developed system, five other proteins including PDGF-BB, VEGF3, 5.0 × 104, 5.0 × 105 pg/mL). As shown in 2 = 0.988) in the range from 5.0 to 5.0 × 105 pg/mL with low relative standard deviations (RSDs) between 1.84% and 2.67% (n = 3). We deduced the main reasons for different RSDs would be the roughness of the microporous surface and the deviation of the coating process. By the calculated signal to noise ratio of the, the limit of detection value was around 2.0 pg/mL . The sensitivity of the proposed method is comparable to that of the ELISA assay and other methods, such as luminescent, fluorescent, and electrochemical techniques in The absorbance of IgE was further considered to explore the quantitative analysis at different concentrations  The usage of FNAs instead of antibodies to immobilize the surface of microwells in this work, which can be achieved target capture and detection.These could not only reduce the analysis procedure but also reduce the cost-effective, making IgE detection simple and fast, (ii) the utilization of microplate reader without changing or updating the instrument made the detection user-friendly and straightforward, (iii) the utilization of competition law and DNAzyme catalyzed ABTS-hemin-H


Records of the ice pressures on the joggle skirt of the coal quay in Spitsbergen were performed over winter seasons in 2013 and 2015. The ice thickness below the quay was above 2 m. Ice temperature over the ice thickness and water pressure at the sea bed were measured synchronously. It was discovered that sea water migrates through the ice confined inside the joggle skirt under the influence of tidal changes of the water pressure below the ice. It leads to the formation of floods on the ice surface during high tide. The ice surface becomes dry during low tide. Ice temperature and ice pressure on the joggle skirt are changed according to the semidiurnal cycle. Spectral analysis and correlation analysis are performed to analyse the loads caused by thermal expansion of the ice. A thermo-mechanical model of ice based on elastic-plastic rheology with thermal effect is used in numerical simulations of the observed phenomena. Numerical simulations are performed with finite element software.This article is part of the theme issue ‘Modelling of sea-ice phenomena’. Effects of thermal loads are mostly considered for the design of hydraulic structures in rivers, lakes and reservoirs . Thermalhickness –6. Therm−5 K−1 [n GPa reaches 55n kPa when the ice temperature increases on 1°K. Processes of heat transfer and stress relaxation influence the significant reduction of this pressure if the temperature changes in the ice are caused by weather changes. In this case, the time for the increase of ice temperature at 1°K over entire ice thickness is big enough for significant relaxation of the stresses [The linear coefficient of thermal expansion (CTE) of freshwater ice 5.5 × 10−5 K−1  shows thstresses . Therefostresses .Saline ice is a composite material including solid ice matrix containing liquid and gas inclusions. Liquid brine may exist inside sea ice in closed brine pockets and permeable channels ,11. Phaset al. [Migration of brine through the ice is caused by the pressure gradient. The permeability appears as a coefficient in Darcy's law describing the dependence of brine flow from the pressure gradient (e.g. ). The peet al.  show thaet al. .https://en.wikipedia.org/wiki/Cape_Amsterdam) are investigated and ice loads on the joggle skirts (JSs) of the quay are analysed. The paper is organized as follows. Physical processes in the confined ice and deformations of JSs are described in the second section of the paper. The third section is devoted to the description of the research site, installed scientific equipment and results of the field observations. Results of numerical simulations are performed in the fourth section. Results of modelling and field observations are compared and discussed in the last section.In natural conditions, the formation of water overpressure below the ice can be caused by the action of tidal variations of the water pressure on confined ice with limited capacity to move in a vertical direction. Typical examples are related to ice foot and hinge zone near the shore line –24, part2.The coal quay at Kapp Amsterdam owned by Store Norske Spitsbergen Kullkompani (SNSK) was designed and reconstructed in 2000 by AF Anlegg Harbour for loading operations on vessels with dead weights up to 70 000 t. Coal boats provided the transport only during the ice-free season in the Van Mijen Fjord, and during the ice season the quay needed to survive without damage. The total length of the quay is 195 m; it is keyed in the seabed with vertical piles of 80 cm diameter and four steel JSs connecting some of the piles . The pila). During low tide, the middle part of the ice inside JSs can be displaced downwards up to 0.6 m in comparison to its position during high tide (figure 2b). Displacements and deformations of JS sections perpendicular to the shoreline in the alongshore direction have been registered in different locations of the quay since 2008 [a). Although the physical reasons for loads causing the JS deformations can be associated with both ice and soil actions the main focus of this study was on ice loads because of visual observations of ice deformations and floods inside JSs.Sea ice in the fjord moves up and down together with sea water due to the semidiurnal tide. The ice inside JSs becomes frozen to the walls during the winter season and does not move like water. Synchronous measurements of the water pressure inside and outside JSs show similar semidiurnal changes of the pressure at the seabed . Tide-infigure 2nce 2008 . The proc). Migration of sea water in the presence of vertical temperature gradient over the ice thickness gives a reason to assume that ice loads on JSs are caused by thermal expansion of the ice. At the same time, the ice inside JSs has visible vertical deformations which can lead to mechanical loads of the ice on JSs.The ice thickness measured in different locations inside JSs was 2.2 m in April 2009 , 1.5 m ifigure 3The hinge zone separating floating sea ice from the ice frozen to JS sections parallel to the shoreline is formed from the marine side and extended on approximately 5 m distance from JSs. Depending on the tidal amplitude, floods are observed also in the hinge zone during high tide. The surface of the hinge zone becomes dry during low tide. Usually, two cracks separating the hinge zone from sea ice and the ice frozen to JSs are clearly visible in the hinge zone. Ice blocks between the cracks are bent and rotated over the tidal cycle. In the ice-free season it was discovered that JS sections on the marine side of the quay are not mounted fast and move cyclically with the amplitude of a few centimetres perpendicular to their plane direction due to wave actions. Vertical piles supporting the quay are mounted fast and not moving.3.http://www.geokon.com/4850) LC1 and LC2 were screwed to two Π-shape beams, and each of the beams was mounted by four bolts on the JSR section parallel to the shore line from its marine and inner sides (a). Similar pressure cells LC3 and LC4 were screwed to the Π-shape beam and the beam was mounted on the section of JSR perpendicular to the shore line from its inner side (a). This section had the highest deformations, shown in figure 3a,b. LC1, LC2 and LC3 were mounted at the same distance from the water level, and LC4 was mounted 1 m deeper than LC1–LC3. It is assumed that LC1–LC3 measure pressure in the surface ice layer with a thickness of 0.5 m, and LC4 measures pressure in the middle layer of the ice (b). The works were performed during spring tide in November 2012. For the mounting of the Π-shape beams, JSR was drilled manually from a plastic rowing boat placed on the water below the quay. Cables from pressure cells LC1–LC4 were grouped together on the surface of the horizontal beams mounted to vertical piles and connected to the data logger fastened on the horizontal beam near the vertical pile N (a). Programming of the data logger and upload of the data were performed using LabView software with the laptop connected to the data logger.The research site was organized in the JS on the west part of the quay in the autumn of 2012. Further, this JS is named JSR. Geokon Pressure Cells (er sides a. Similaner side a. This s the ice b. The wol pile N a. Prograhttp://www.geoprecision.com/en/produkte-en/temperaturmesskette-en) with 21 thermistors was placed inside a vertical steel pipe and the pipe was strapped to the vertical pile S in November 2012 (a). The first thermistor located inside the logger was extended 1 m from the next thermistor; 18 thermistors were distributed with 20 cm distance between neighbouring thermistors along the temperature string. The last two thermistors at the end of the temperature string extended from each other by 1 m. Vertical distances from thermistor 1 (TS1) and thermistor 2 (TS2) to the surface of ice near the pile S were around 1.5 and 0.5 m, respectively. Thermistors 1–4 (TS1–TS4) were above the ice surface. Thermistors 5–10 (TS5–TS14) were within the ice thickness when the ice inside JSR was thick enough (March–May). Thermistors 15–20 (TS11–TS20) were below the ice in the water and on the sea bottom. The temperature string supports wireless data transmission to the laptop via a USB dongle. Programming of the data collection was performed using GP5Shell software.Temperature string Geoprecision (ber 2012 a. The fihttps://www.seabird.com/moored/sbe-39plus-temperature-depth-recorder/family?productCategoryId=54627473774) placed on the sea bottom near the quay and below the quay inside JSR. Monitoring of floods on the ice surface inside JSR was performed with a Reconyx HC600 camera (www.reconyx.com) mounted on the vertical pile inside JSR. Four Geokon Pressure Cells (http://www.geokon.com/4800) were used in March 2015 for the monitoring of ice stresses in the central part of JSR synchronously with LC1–LC4.Registration of the tidal variations of sea level was performed with pressure and temperature autonomous recorders SBE 39 (http://www.kartverket.no/en/), and a 40 min phase shift was added to get the tide data for Svea Bay [Figures Svea Bay . Samplina). These ice pressure increases were of short duration, lasting about 2 h. They can relate to local effects of ice interaction with JSR in the hinge zone. Except for these two events, the ice pressures registered by LC1 and LC2 were much smaller than the ice pressures registered by LC3 and LC4. This can be explained by the damping of ice loads by the hinge zone and high compliance of JSR section on the marine side of the quay. The air temperatures in the beginning and middle of April were lower in 2013 than those in 2015 by almost 10°C. Therefore, sea ice was colder and stronger in 2013. Large ice pressures registered by LC3 and LC4 are attributed to the low temperature in 2013. At the same time, high ice pressures were registered in 2013 and 2015 when the air temperature was above −10°C. LC3 recorded ice pressures above 0.4 MPa at the end of April to the beginning of May 2013, and LC3 and LC4 recorded ice pressures above 0.15 MPa, respectively, at the end and at the beginning of April 2015.The highest ice pressures above 0.9 and 1.3 MPa were registered by LC1 on the marine side of JSR in two consequent tidal cycles at the beginning of May 2013 inset in a. These a). It corresponds well to the analysis of tidal constituents performed by Kowalik (private notes) based on the tide measurements in the Van Mijen Fjord from October 2007 to August 2009.Tidal changes of the water pressure below the ice and air temperature are the main factors influencing a change in the ice temperature and ice loads on JSR. The tidal signal has a sinusoidal shape corresponding to dominating semidiurnal tide M2 with a period of 12.42 h. Spectral analysis shows the existence of tidal constituents K1, M4 and 2M6 with periods of 24, 6.2 and 4.1 h a. It cori = 1–18) calculated with the formulai, and i-temperature averaged over 24 h. Spectral analysis shows that fluctuations of the air temperature (b). The highest spectral maximum at diurnal frequency corresponds to the air temperature variations caused by daily variations of sun radiation. The maximum at the semidiurnal frequency is explained by the influence of the floods on the ice surface on the air temperature below the quay. Spectral analysis of the temperature fluctuations recorded by TS4–TS6 shows the existence of spectral maxima at a diurnal frequency . Figure 10b shows that in 2015 LC3 records have a positive correlation with the tide pressure. Records of LC4 showing relatively high pressure have a negative correlation with the tide pressure. In 2015 the records of LC1 and LC2 also show mostly positive correlation with the tide pressure.Since the ice pressures recorded by LC1 and LC2 were very small in 2013 (except two high pressure events measured by LC1) the information in figure 10May 2013 b. Figurea and located between high and low tide in figure 11b. Moreover, figure 11b shows that a significant part of ice pressures recorded by LC3 is spread around low tide. These events correspond to two columns showing a negative correlation of LC3 records with the tide records (a). Local maxima of ice pressures recorded by LC4 correspond to low tide in figure 11a and spread between low and high tides in figure 11b. Ice temperature records show local maxima of the temperatures during high tide and local minima during low tide in figure 11a. They are visible only on the records of TS5 and TS6 in figure 10b because of the higher air and ice temperatures (c). The ice temperatures at depths greater 0.5 m were almost at freezing point.figure 11 records a. Local eratures c. The ica shows the flood times reconstructed by photographs taken with the Reconyx camera mounted inside JSR. Time lapse photographs were made with 15 min interval. The flood intervals are marked in red. The floods were observed during high tides. There is a tendency of flood duration increase with the increase of the tide amplitude. The depth of the flood water inside JSR was measured from 5 to 25 cm [b). A decrease of ice salinity during low tide in comparison with high tide reaches several parts per thousand.Figure 12to 25 cm . Ice cor4.E is the elastic modulus of ice and Thermo-mechanical ice loads caused by temperature changes in the ice are estimated using Bergdahl's rheological model . The eququal to  . The absqual to  . In the −6 s−1, reference creep stress 1 MPa and stress exponent 3. The elastic modulus and Poisson's ratio are, respectively, 2 GPa and 0.33. The computational domain is a rectangular block with sizes 20 m along the x-direction, 10 m along the y-direction and 2 m along the z-direction. The origin is located in the low corner of the computational domain, and the vertical coordinate z is directed upwards.Numerical simulations were performed with finite element software (Comsol Multiphysics 5.2a). The program was prepared in a Structural Mechanics module using a model of linear elastic material with creep and thermal expansion implementing the model of Cox  for 3D sFixed constrains are used as the boundary conditions at the lateral faces of the computational domain. The lateral faces correspond to the surfaces of ice in contact with JSs. The free surface boundary condition is used on the ice surface. The ice bottom is subjected by the action of hydrostatic pressure imitating tidal pressureb. Numerical simulations were performed using the 60 h temperature records corresponding to the temperature records from 1150 to 1210 h in figure 11b. Temperature profiles measured by the temperature string in this time are shown in figure 13a. In numerical simulations, the temperature measured by TS5 specifies the surface temperature of the ice sheet. Temperatures measured by TS8–TS18 are almost constant. Therefore, thermal expansion is most significant in the surface ice layer of 0.5 m thickness. In numerical simulations, the ice temperature is assumed to equal to −1.7°C by b). The insets in Thermal loading is specified according to the example shown in figure 11a the stress range extends from −0.1 to 0.1 MPa in the bottom layer of ice and from −0.25 to 0.25 MPa in the surface layer of ice. In figure 14b the stress range extends from −0.2 to 0.2 MPa in the bottom layer of ice and from −0.6 to 1.35 MPa in the surface layer of ice. The difference of the stress ranges near the ice surface and ice bottom is explained by the influence of the thermal expansion. Computed tensile stresses are much higher than the tensile strength of sea ice (less than 0.2 MPa) measured in the full-scale tests near the coal quay [Figures oal quay . Therefox,y,z) are shown in the legends. According to the definition the ice pressure equals x = 10 m and y = 5 m. In figure 15a the ice pressure phases follow the phase of the vertical displacement. Therefore, positive thermal expansion . Thus, negative thermal expansion in the surface ice layer explains the formation of ice pressure on JSs shown in figure 11b.Amplitudes of computed vertical displacements of the ice are below 0.5 mm. This is much smaller than the observed values up to 0.6 m. The large displacements are related to the formation of ice failure zones in the ice. The model should be modified to take into account ice failure effects. Another correction of the model is related to the more accurate formulation of the creep rheology of sea ice taking into account its granular structure .5.Ice pressures on JSR in the coal quay in Spitsbergen and ice temperatures were recorded during two winter seasons in 2013 and 2015. The ice loads are created by confined ice sheet trapped inside JSR. The ice thickness was about 2 m. Three pressure cells recorded the ice pressure on JSR in the surface ice layer of 0.5 m thickness, and one pressure cell recorded the ice pressure on JSR 1 m deeper than three other cells. Two ice pressure increases above 0.9 and 1.3 MPa were registered in the surface ice layer on the marine side of JSR at the beginning of May 2013. Except for these two events with less than 2 h duration the recorded ice pressures oscillate with semidiurnal frequency. The amplitudes of the pressure oscillations were below 0.2 MPa in most of the records except the record of approximately 200 h duration at the end of April to the beginning of May 2013 when the pressure amplitude exceeded 0.4 MPa in the surface ice layer. Spectra of the pressure records correspond to tidal constituents M2, M4 and 2M6 with periods of 12.42, 6.2 and 4.1 h. Correlation analysis shows that highest pressures in the surface ice layer are realized during high tide except for the 200 h record in 2013 when the highest pressures in the surface ice layer were recorded during low tide.Spectra of temperature fluctuations have maxima corresponding to the same tidal constituents visible up to a depth of 1.7 m below the ice surface. The temperature fluctuations are explained by sea water migration through the ice under the action of tidal pressure on the ice bottom. Spectral maxima of the ice temperature at diurnal frequency are visible in the surface ice layer up to a thickness of 0.5 m. They are related to diurnal variations of the air temperature and sun radiation. Correlation analysis shows that the ice temperature increases when the water level in the sea increases and decreases when the water level decreases. Migration of sea water through the ice influences floods on the ice surface during high tide when the tide amplitude is big enough. The flood durations increase with the increase of the tide amplitude. The ice surface becomes dry during low tide. The ice salinity during low tide is smaller than the ice salinity during high tide by several parts per thousand.Observations show that the ice inside JSR bends downwards during low tide and the ice displacement in the middle of JSR reached 0.6 m. This influences the pressure of the bottom ice layer on JSR during low tide, and the surface ice layer during high tide. Similar loads are caused by thermal expansion of the ice in the case of the positive CTE. Numerical simulations with the elastic-plastic model of ice taking into account thermal expansion are performed when the ice temperature changes according to the 60 hour temperature records during April 2013. Amplitudes of ice pressure on JSs are estimated below 0.25 MPa when ECTE of sea ice equals to CTE of fresh ice. The increase of ice pressure amplitudes up to 0.6 MPa during low tide was reproduced by numerical simulations with the elastic-plastic model of ice where ECTE of sea ice was negative in the surface ice layer, and its absolute value was 10 times greater than the CTE of fresh ice.−4 K−1. Recent in situ investigations of the influence of ice compression on ice permeability performed on sea ice near the coal quay demonstrated strong reduction of the permeability in compressed ice [b). The compression influences a reduction of ice permeability and a shift of ECTE to negative values. Negative thermal expansion of the surface ice layer influences high ice pressure on JSR during low tide.Negative thermal expansion of saline ice with salinities of 6 and 9.4 ppt was reproduced in laboratory experiments when the ice temperature was higher than −5°C ,20. The ssed ice . Based o


Ki’s) for natural inhibitors AMP and fructose-2,6-bisphosphate  compared with the Wild-type enzyme (WT). A separate mutation (M248D) was performed in the active site of the enzyme to investigate whether the enzyme could be activated. This mutant displayed an approximate seven-fold increase in Ki for F2,6-BP. Interfacial mutants L56A and L73A exhibited an increase in Ki for F2,6-BP by approximately five-fold. Mutations in the AMP-binding site (K112A and Y113A) demonstrated an eight to nine-fold decrease in AMP inhibition. Additionally, mutant M248D displayed a four-fold decrease in its apparent Michelis constant (Km), and a six-fold increase in catalytic efficiency (CE). The importance—and medical relevance—of specific residues for FBPase structural/functional relationships in both the catalytic site and AMP-binding site is discussed.Specific residues of the highly regulated fructose-1,6-bisphosphatase (FBPase) enzyme serve as important contributors to the catalytic activity of the enzyme. Previous clinical studies exploring the genetic basis of hypoglycemia revealed two significant mutations in the coding region of the FBPase gene in patients with hypoglycemia, linking the AMP-binding site to the active site of the enzyme. In the present study, a full kinetic analysis of similar mutants was performed. Kinetic results of mutants Y164A and M177A revealed an approximate two to three-fold decrease in inhibitory constants ( Fructose-1,6-bisphosphatase (FBPase) is a complex tetramer and a highly regulated enzyme. FBPase activity is regulated synergistically by the allosteric inhibitors AMP and fructose-2,6-bisphosphate . As an enzyme, FBPase functions in the degradation of fructose-1,6-bisphosphate (FBP), which hydrolyzes to fructose-6-phosphate (F6P) and inorganic phosphate -thiazol-2-yl}-methanol) binds; To determine if specific mutations cause disruption in enzymatic activity and structural changes, site-directed mutagenesis was done on selected residues. Site-directed mutagenesis was performed using seven different pig kidney FBPase primer sequences via PCR, amplifying single strands or plasmids of interest ,15. The In order to overexpress, isolate, and purify recombinant mutant enzymes, transformations and purification of mutant plasmids was performed. Following site-directed mutagenesis, each of the mutant plasmids underwent transformation with XL Blue super-competent cells ,19. CellEscherichia coli cells were removed after being frozen at −80°C and thawed on ice. A 50-µl volume of competent cells was added to an empty Eppendorf tube with 2.0 µl of plasmid (1–20 ng plasmid DNA) . ,33. 1,3350 for K112A and Y113A mutants  is shown in close proximity to an activating manganese metal in the crystal structure. This motivated us to change the methionine to aspartic acid to see if, in the presence of magnesium activating metal, the mutant would be more active than the WT.50 in 50 value for the active site inhibitor F2,6-BP is 7× greater, corresponding to a lower affinity for inhibition. With reference to Km decreases by nearly four-fold relative to the WT. This implies an occurrence of tighter binding action, and is further supported by an observed six-fold increase in CE, suggesting that the mutant enzyme is more efficient than the WT. These results are consistent with a 6.25 decrease in Ka for magnesium binding, as seen in From the data presented, M248D can be classified as an ‘activated’ mutant. Although the AMP IC50 of F2,6-BP and 5× higher Ki  in vivo, the complexity of the signaling system regarding the transport of glucose out of the liver may limit the ability of researchers to directly correlate mutant FBPase activity with blood glucose levels. More work needs to be done in vivo in correlation to FBPase mutations, enzymatic activity, and the effects these mutations have on blood glucose levels. If active site mutations such as M248D exist in the FBPase gene product , then new regimes for drug treatments for this disorder may be explored as based on previous drug studies [However,  studies ,37.Interpretation of kinetic data tells us that a presence of specific mutations in the coding region of this gene may possibly deregulate the enzyme such that the rate of production of F6P increases dramatically, leading to enhanced gluconeogenesis (glucose production) in direct correspondence to activation of the enzyme where AMP inhibition is lost.


A heterohexameric complex composed of minichromosome maintenance protein 2–7 (MCM2–7), which acts as a key replicative enzyme in eukaryotes, is crucial for initiating DNA synthesis only once per cell cycle. The MCM complex remains inactive through the G1 phase, until the S phase, when it is activated to initiate replication. During the transition from the G1 to S phase, the MCM undergoes multisite phosphorylation, an important change that promotes subsequent assembly of other replisome members. Phosphorylation is crucial for the regulation of MCM activity and function. MCMs can be phosphorylated by multiple kinases and these phosphorylation events are involved not only in DNA replication but also cell cycle progression and checkpoint response. Dysfunctional phosphorylation of MCMs appears to correlate with the occurrence and development of cancers. In this review, we summarize the currently available data regarding the regulatory mechanisms and functional consequences of MCM phosphorylation and seek the probability that protein kinase inhibitor can be used therapeutically to target MCM phosphorylation in cancer. DNA is replicated via a multi-protein machinery comprising DNA polymerase, helicase, primase, circular sliding clamps, a pentameric clamp loader, single-strand binding protein (SSB) and other components –5. This Multiple phosphorylation sites were distributed on the MCM2–7 subunits. The biological and functional consequence of MCM phosphorylation appears to be correlated with specific kinases and their phosphosites. Some MCM subunits undergo dynamic phosphorylation in a cell cycle-specific manner, which may be consistent with their cell-cycle-specific functions –25. AberCell division cycle 7 (Cdc7) is an evolutionary conserved serine-threonine kinase that promotes the initiation of DNA replication by targeting the functional substrate MCM2–7 protein –35. SimiCdc7 preferentially phosphorylates MCM2 as well as other MCM subunits Table . AlthougCell cycle progression through each phase is tightly regulated by Cdks and their regulatory proteins, cyclins. Alterations in these proteins, lead to uncontrolled cell division, which is a characteristic of many cancers. Several lines of evidences have shown that MCMs are substrates of Cdks and some Cdk-dependent phosphosites on MCMs have been identified and characterized Table . MoreoveReplication stress, commonly known as the slowing down or stalling of replication forks, is a major source of mutations that contribute to genomic instability and tumorigenesis , 60. ThiLyn-, Akt-, and integrin-linked kinase (ILK)-mediated MCM7 phosphorylation primarily correlates with the development of human cancer. MCM7 phosphorylation at Tyr-600 mediated by epidermal growth factor receptor (EGFR)-p56Lyn-axis promotes MCM complex assembly and chromatin loading, consequently enhancing DNA synthesis and cancer cell proliferation [Other than the three classes of kinases Cdc7, Cdk, and ATM/ATR, several additional kinases were also shown to be involved in MCM phosphorylation Table . Among tferation . Furtherferation . Similarferation . In contferation . Howeverferation , MCM3 waferation , and MCMferation . Among tferation . The autferation . In addiferation . Howeverferation . In contferation .MCM proteins are regulated by multiple kinases and the consequences of these phosphorylation events reflect the critical integration of DNA replication with cell cycle and the checkpoint response Fig. . However


Celtis africana, namely trans-N-coumaroyltyramine (1), trans-N-feruloyltyramine (2), trans-N-caffeoyltyramine (3), lauric acid (4), oleic acid (5), palmitic acid (6), lupeol (7), β-sitosterol (8) and oleanolic acid (9), respectively. Their structures have been elucidated by different spectroscopic techniques. The isolated compounds were screened for their antioxidant, anti-inflammatory and acetylcholinestrease enzyme inhibitory activities. Compounds 1–3 showed significant antioxidant and anti-inflammatory activities and weak to moderate acetylcholinestrease enzyme inhibition activity.Nine compounds have been isolated for the first time from  Celtis africana Burm. f. is common and widespread in South Africa where the leaves are used as a traditional human and veterinary medicine for the treatment of indigestion and edema  (DTNB) and galanthamine were purchased from Sigma . Buffers and other chemicals were of analytical grade. Acetylcholinesterase inhibiting activity was measured according to a slightly modified spectrophotometric method. Acetylthiocholine iodide was used as substrates in the assay. 5,5'-Dithiobis[2-nitrobenzoic-acid] (DTNB) was used for the measurement of cholinesterase activity. 0.1 mM sodium phosphate buffer , 3 mM DTNB (10 μL), test compound solution  and 15 mM acetylcholinesterase solution (20 μL) was mixed and incubated for 15 minutes (25 °C). The reaction was then initiated by the addition of acetylthiocholine (10 μL). The hydrolysis of acetylthiocholine was monitored at 412 nm after 20 min. All the reactions were performed in triplicate in 96-well microliter plates in SpectraMax 340 (Molecular Devices)  (16), 176, 164, 147 (100); 1H-NMR (CD3OD): δ 7.06 , 7.41 , 6.73 , 6.80 , 7.44 ; 6.38 , 3.46 , 2.75 ; 13C-NMR (CD3OD): δ 169.2 (C-9), 160.5 (C-4), 156.9 (C-4'), 141.8 (C-7), 130.7 , 131.3 (C-1'), 130.5 , 127.7 (C-1), 118.4 (C-8), 116.7 , 116.2 , 42.5 (C-8'), 35.8 (C-7'). trans-N-Feruloyltyramine (2). White solid (25 mg); m.p. 145–147 °C; EIMS m/z (rel. int.): 313 [M]+(15), 177 (100), 145 (22), 120 (18), 107 (19); 1H-NMR (CD3OD): δ 7.44 , 7.13 , 7.07 , 7.048 , 6.81 , 6.73 , 6.41 , 3.85 , 3.47 , 2.76 , 13C-NMR (CD3OD): δ 169.2 (C-9), 156.9 (C-4'), 149.8 (C-4), 149.3 (C-3), 142.0 (C-7), 131.3 (C-1'), 130.7 , 128.2 (C-1), 123.2 (C-6), 118.7 (C-8), 116.4 (C-5), 116.2 , 111.5 (C-2), 56.4 (OCH3), 42.5 (C-8'), 35.8 (C-7'). trans-N-Caffeoyltyramine (3). White solid (26 mg); m.p. 215–217 °C; EIMS m/z (rel. int.): 299 [M]+ (25), 180 (48), 163 (100). 120 (17); 1H-NMR (CD3OD): δ 7.41 , 7.06 , 7.02 , 6.98 , 6.79 , 6.75 , 6.40 , 3.47 , 2.76 , 13C-NMR (CD3OD): δ 169.3 (C-9), 156.7 (C-4'), 148.5 (C-4), 147.7 (C-3), 141.6 (C-7), 131.0 (C-1′), 130.7 , 127.1 (C-1), 122.1 (C-6), 118.5 (C-8), 116.3 (C-5), 116.4 , 114.5 (C-2), 42.4 (C-8'), 35.7 (C-7').C. africana. These compounds showed potent anti-inflammatory and antioxidant potential, and moderate acetylcholinestrease enzyme inhibition activity.In summary, we have evaluated the anti-inflammatory, antioxidant and acetylcholinestrease enzyme inhibitory activities of three phenolic amides isolated for the first time from


Caenorhabditis elegans. The expression of fish (NfeIBPs)- and fungus-derived IBPs (AnpIBPs and TisIBP8) in C. elegans improved its survival rate during exposure to 0 and −2 °C (cold shock) and −5 °C (freezing). The observed cold tolerance of C. elegans after cold shock is attributable to the stabilization of cell-membrane lipids with IBPs, and the freezing tolerance at −5 °C can be attributed to the inhibition of ice-crystal growth by the IBPs. Significantly, the survival rate of C. elegans at −5 °C was improved by expression of wild-type AnpIBP and maximized by that of TisIBP8, whereas it was lowered when a defective AnpIBP mutant was expressed. These results suggest that the ice-binding ability of IBP has a good correlation with the survival rate of C. elegans during freezing.Ice-binding proteins (IBPs) are capable of binding ice crystals and inhibiting their growth at freezing temperatures. IBPs are also thought to stabilize the cell membrane at non-freezing temperatures near 0 °C. These two effects have been assumed to reduce cold- and freezing-induced damage to cells and tissues. However, knowledge regarding the effects of IBP on the living animals is limited. Here, we characterized the relationship between the IBP effects and the physiological role by using the nematode  These IBPs ultimately modify the crystal into a needle-like shape. In contrast, “hyperactive” IBPs identified in insects and fungi bind the prism, pyramidal, and basal planes of ice crystals, modifying the crystals into a rounded-hexagonal morphology3. Ice-growth inhibition is thought to reduce damage to tissues and cells of living organisms under freezing conditions. Ice-growth inhibition is closely related to the freezing point depression activity of IBP solution, which is easily evaluated as the difference between melting and freezing temperatures or “thermal hysteresis (TH)”5. In addition to these IBP-ice interactions, IBPs are also thought to stabilize cell-membrane lipids at hypothermic temperatures6. This membrane stabilization effect is useful for protecting cells and tissues from cold-induced damage. Actually, several fish IBPs dissolved in Euro-Collins solution significantly improved the survival rate of rat insulinoma RIN-5F cells following a 5-day incubation at 4 °C7. Hirano et al. reported that fish NfeIBPs protect cultivated mammalian cells (HepG2) during a 4 °C cold shock, suggesting that the membrane stabilization effect is correlated with TH activity8. Ice-growth inhibition and membrane stabilization are thought to facilitate the survival of living organisms in cold and freezing environments, and these effects of several IBPs are correlated with TH activity8. However, knowledge regarding the relationship between TH activity and its physiological roles in living organisms during cold shock and freezing is limited.Ice-binding proteins (IBPs) inhibit the growth of ice crystals with their surface-bound waters that are spatially organized to bind an ice crystal. Fish type I IBPs bind to a specific pyramidal planeCaenorhabditis elegans is a valuable model used to characterize the functions of proteins at the behavioural and cellular level. A combination of molecular genetic manipulations and fluorescence imaging techniques has been used to identify the molecular mechanisms regulating behavioural responses and cellular functions in worms10. No ice-growth inhibiting substance, such as IBP, has been identified in C. elegans, but they employ several intrinsic mechanisms for survival upon lethal cold exposure. Light and pheromone-sensing neurons in C. elegans detect the temperature decrease in the cultivated environment and release insulin, which is received by the intestine and other neurons. The expression of DAF-16/FOXO regulating genes, which are involved in lipid production, is regulated by insulin signalling, resulting in the formation of cold tolerance in C. elegans12. Actually, the survival rate of worms at 2 °C cold shock was 10% or less when cultivated at 22 to 27 °C. However, most worms survived the 2 °C cold shock when cultivated at 15 °C for only 3 hours. Among nematode species, the Antarctic nematode Panagrolaimus davidi and Plectus murrayi intrinsically have strong tolerances to sub-zero and freezing temperatures15. P. davidi has an ice-active protein, and showed that its extracts produce an hexagonal ice crystals16. An expressed sequence tag (EST) analysis identified that P. murrayi has a protein with homology to a fish type II IBP17 and showed that its extracts inhibit ice-crystal growth15. The strategies used for survival in cold environments probably vary among nematode species. The Antarctic nematode employs a protein, such as IBP, for ice-growth inhibition, whereas C. elegans regulates the expression of genes involved in saturated phospholipids to protect cell-membrane lipids. Although IBPs have the potential to protect tissues and cells from cold- and freezing-induced damage, whether their effects improve the survival ability of C. elegans during cold shock and freezing is unclear.The nematode 18. Type III IBPs exhibit overall hydrophobicity at ice binding surfaces similar to type I IBPs and are approximately 6 kD in size19. Type IV IBPs are alpha helical proteins rich in glutamate and glutamine, consist of a 4-helix bundle and are approximately 12 kD in size20. NfeIBPs isolated from Zoarces elongatus Kner (Notched-fin eelpout) is a type III IBP that has at least 13 isoforms. The ice-binding effect differs among its isoforms21. The TH activity of NfeIBP6 is approximately 0 °C (at 0.4 mM), whereas that of NfeIBP8 is approximately 0.5 °C (at 0.4 mM)22. Although NfeIBPs are involved in cellular protection, as previously mentioned, how these differences in TH activity affect freezing tolerance in living animals is unclear. The fungal proteins AnpIBP and TisIBP were recently isolated from Antarctomyces psychrotrophicus23 and Typhula ishikariensis24, respectively. Whether these fungal IBPs improve cold or freezing tolerance in living cells, tissues or animals remains unclear.Many types of IBPs have been isolated from several polar fishes, microbials, plants and insects and analysed to characterize their biochemical properties. Fish IBPs are categorized into four types (I–IV) or antifreeze glycoprotein (AFGPs). Fish type I IBPs are alanine-rich proteins consisting of a single amphipathic alpha helix and are approximately 3.3–4.5 kD in size. Type II IBPs are cysteine-rich globular proteins containing five disulphide bondsin vivo through behavioural and cellular observations of transgenic C. elegans expressing IBPs. We show that IBPs recently discovered in fish NfeIBPs and fungal AnpIBP improve the survival rate of worms and protect their cells from lethal cold and freezing exposure. The fungal AnpIBP T156Y mutant exhibiting low TH decreased freezing tolerance in worms at −5 °C. The fungal TisIBP8 exhibiting high TH activity was observed to dramatically improve the survival rate of worms and strongly protect muscle cells. The relationship between the ice-binding abilities of IBPs and their in vivo roles in tolerances and cellular protection in living animals are discussed.In this study, we investigated the functions of IBPs C. elegans expressing IBPs in cells after cold shock and freezing , improve cold and freezing tolerance in living animals, we examined the survival rate of transgenic ing Fig. . The suring Fig. . Howevering Fig. . The suring Fig. . The neming Fig. . The woring Fig. . Each IBals Fig. . During als Fig. . IBPs mials Fig. . These rC. elegans after each cold shock and freezing. Our results suggest that these tolerances are caused by two abilities of IBPs. One ability is ice-growth inhibition, and other ability is cellular protection. Several studies have reported that these abilities of IBPs are correlated with their TH activity22. First, to elucidate whether differences in TH activity affect the survival rate of worms after cold shock and freezing, we focused on the fungal protein AnpIBP1a and examined the relationship between its structural properties and TH activity. The overall structure of AnpIBP1a is a right-handed β-helical fold with a parallel α-helix, which is similar to that of other microbial IBPs, such as LeIBP25  and AnpIBP1a N55D/T156Y (AnpIBP T156Y). After −5 °C exposure, the survival rate of the worms expressing AnpIBP T156Y was significantly lower than that of those expressing AnpIBP  on the AnpIBP1a B-face Fig. . To elucIBP Fig. . The NGMIBP Fig. . AnpIBP IBP Fig. . The NGMIBP Fig. , suggestin vivo function. To elucidate whether the expression level of the transgenes depends on the survival rate of C. elegans, we generated different transgenic lines using the same constructs of NfeIBP6, NfeIBP8, AnpIBP, and AnpIBP T156Y. The copy number and expression level of the transgenes vary among the transgenic lines. We generated multiple lines (#1-#3 or #1-#4) for the 4 IBP constructs and found that the expression level could differ among the different transgenic lines; however, the correlation between TH activity and the survival rate was largely similar regardless of whether the worms were derived from the same strain or different strains expressing the same construct , which is similar to the hyperactive IBP observed in insects2, although its crystal structure is similar to that of AnpIBP 34 that likely result from the inability of these cells to maintain their shape. Here, we examined whether TisIBP8 protects C. elegans cells from the intracellular side during cold shock and freezing. Using transgenic worms expressing the fluorescent protein wrmScarlet35 in the muscles in a nucleus-bound fashion, we evaluated the ability of IBPs to protect cells by comparing the number of cells in the wild-type worms and worms expressing AnpIBP and TisIBP8. The wrmScarlet is localized at the cell nucleus. The individual muscle cells are easily identified as small fluorescent puncta  caused by the phase transition of the cell membrane. This membrane stabilization effect of IBPs likely improved the survival rate of C. elegans during the −2 to 5 °C cold shock. To achieve this membrane protection activity, IBPs should be able to bind the cell membrane. The results of several studies investigating IBP-ice interactions suggest that a polypentagonal ice-like water network at ice-binding sites binds a quasi-liquid layer of ice crystal, resulting in the formation of an IBP-ice crystal complex. This ordered water molecular network, which rapidly and completely regulates the ice-binding process, can fit the quasi-liquid layer and may exist on ice-binding residues with favourable arrangements in IBPs. One possible explanation for the cellular protection conferred by IBPs is that water molecules arranged on the C. elegans cell membrane form an ordered hexagonal structure as a quasi-liquid layer allowing IBPs exhibiting high TH activity to strongly bind worm cell membranes through an ordered hexagonal water arrangement. The increased stability of cell membranes and TH activity, including the specific mode of ice binding, may be key factors responsible for cold- and freezing-tolerance under cold conditions. In this study, we did not examine the details of IBP-membrane interaction. Our future studies should provide a better understanding of the relationship between the cell membrane protection function and IBP characteristics, including IBP expression levels and its interaction with cell membranes, using several transgenic worm strains, such as those expressing intra- or extra-cellular IBP. We believe that these in vivo analyses could shed light on the mechanisms through which ice crystals interact with cell membranes, resulting in leaky contents and the ability to maintain a supercooled state dependent on TH activity in living animals.The cell membrane (lipid bilayer) changes from liquid to gel at cold temperatures, resulting in the leakage of the intracellular contents, subsequently leading to cell destructionC. elegans worms may be a step towards establishing a simple preservation method involving the freezing of fresh living cells.The proposed genetic expression approach could be of interest for high-performance preservation as IBP cold preservation could be applied to many materials ranging from various industrial materials, such as raw foods, to medical materials, such as cells and tissues. Our investigation using molecular genetic manipulation of living http://salilab.org/modeller/) with the following structures in the PDB as templates: TisIBP8 (PDB ID = 5B5H), LeIBP (3UYU), FfIBP (4NU2), ColIBP (3WP9), and IBPv_a (5UYT). The AnpIBP1a structure was visualized using PyMOL38.The 3D structure of AnpIBP1a was developed using the software MODELLER . One resulting transformant was precultured in 80 mL of BMGY medium , 1.34% YNB, 4 × 10−5% biotin and 1% glycerol) for 3 days at 22 °C. Once the OD600 of the culture reached 10, the cells were harvested by centrifugation at 7,500 rpm for 10 min at 20 °C and then resuspended in 450 mL of 2 × BMMY medium , 1.34% YNB, 4 × 10−5% biotin and 0.5% methanol). The cells were cultivated in a 1-L BMJ-01P fermenter  for 5 days at 20 °C. Methanol was continuously added to the fermenter through a peristaltic pump at a flow rate of 0.5–3.5 mL/h. After 5 days of cultivation, the cells were removed by centrifugation at 7,500 rpm for 10 min at 4 °C, and the culture supernatant was dialyzed against buffer A (20 mM Tris-HCl buffer (pH 8.0) and 0.5 M NaCl). The dialysate was loaded onto a 30 mL Ni-NTA Superflow column  equilibrated with buffer A, and then, the bound protein was eluted with buffer A containing 200 mM imidazole. The eluted fraction was dialyzed against 20 mM Tris-HCl buffer (pH 8.0). Subsequently, the dialyzed sample was applied to a High Q column , and the bound protein was eluted with a 0–300 mM NaCl gradient. The antifreeze-active fractions were recovered and loaded onto a Superdex-200 gel-filtration column  equilibrated with buffer A. The antifreeze-active fractions were recovered, dialyzed against water, and frozen at −20 °C until use.cDNA encoding AnpIBP1a (accession number LC378707 in DDBJ/EMBL/GenBank) was inserted into the plasmid pPICZα from an EasySelectTM Pichia Expression Kit  using the restriction enzymes 39. IBP was dissolved in 20 mM Tris-HCl buffer (pH 8.0). The sample (1 µL) was rapidly cooled in a glass capillary until frozen and then slowly melted until a single ice crystal formed. The melting point (Tm) is reported as the temperature at which the ice crystal began to melt. The single ice crystal was maintained at Tm − 0.1 °C for 5 min and then cooled again at a rate of 0.1 °C/min. The non-equilibrium freezing point (Tf) of the solution is reported as the temperature at which the ice crystal grew rapidly. The TH values at each concentration of the IBPs are reported as the difference between Tm and Tf .The TH activities of AnpIBP1a and its mutants were measured using a photomicroscope system following an established protocolmyo-3, rimb-1 (H20), and elt-2 promoter regions for the specific expression of IBPs in body wall muscles, neurons, and intestinal cells, respectively. The region in each promoter was PCR amplified and inserted at the XbaI and ApaI sites of the plasmid pPD95.79 (a kind gift from Andrew Fire). The coding sequences of the IBPs were PCR amplified using the following primer sets:We used the NfeIBP6: 5′aaagGAATTCATGGGCGAGTCCGTGGTGGC3′ and 5′atgcGAATTCCTACTTTGCCGGGACGTACG3′; NfeIBP8: 5′atgcGAATTCATGAACCAGGCGTCCGTGGT3′ and 5′aagcGAATTCCTAAGCCGGGGTGTACCCTT3′; AnpIBP1a N55D: 5′ggggGAATTCATGACTGGTTTGGATTTGGGTGC3′ and 5′gggcGAATTCCTAAACCTTGAAGAACTTAG3′; and TisIBP8: 5′cggcGAATTCGAAGGAGATATACATATGGCTGGT3′ and 5′ataaGAATTCCTCGAGTGCGGCCGCCTATTTTTGC3′. An AnpIBP1a N55D/T156Y fragment was created by overlap PCR fusion using the AnpIBP N55D primers listed above and the following primers: 5′atgcAGTTCCCAAATAAGCGGAGG3′ and 5′atgcCCTCCGCTACTTTGGGAACT3′. Each IBP fragment was inserted between the EcoRI sites of the myo-3p::Venus construct. For the cell counting analyses, the NLS::wrmScarlet construct was PCR amplified using the following primers: 5′agttGGTACCatgccaaagaagaagcgtaaggtcA3′ and 3′atgaGAATTCgctcgccaaggaggtcgagaactag5′.NLS::wrmScarlet PCR product was replaced with Venus using the KpnI and EcoRI sites of the myo-3p::Venus construct.The lin-15 (n765ts) mutant animals at a concentration of 5–100 ng/uL with a pbLH98 lin-15 (+) injection marker using a standard microinjection method40. The marker DNA rescues the multivulva phenotype in the lin-15 mutant animals. The plasmid DNA rearranges to form extrachromosomal arrays that are stably inherited although not at the same efficiency as actual chromosomes41. Extrachromosomal arrays have been estimated to contain hundreds of copies of injected plasmids42. The details of the construction of the transgenic worms are shown in Table To generate the transgenic animals, the resulting plasmids were injected into Escherichia coli OP50. Twenty to fifty adult worms were placed on the test plates. These adult worms were cultivated for 1 day under each cold shock  or freezing conditions (−5 °C), and the plates were subsequently transferred to room temperature (RT). After 2 hours at RT, the numbers of dead and living animals in the plate were counted, based on movement in response to touch stimulus43. A Bonferroni t-test was performed for multi-comparison. Student’s t-test was performed to compare two samples. If one of the two samples had unequal variances, Weltch’s t-test was performed. All statistical analyses were performed using R version 3.3.1.Well-fed animals were used for the cold- and freezing-tolerance assay. The worms were cultivated from eggs to the adult stage at 24 °C on nematode growth medium (NGM) plates with sufficient food C. elegans has a transparent body. To demonstrate the effects of IBPs at the cellular resolution level in living worms, we used transgenic worms expressing the fluorescent protein wrmScarlet in muscle cells in a nucleus-bound fashion. The wrmScarlet localizes to the cell nucleus via nuclear localization signal (NLS) as small puncta  with a 10× objective lens. A heating/cooling stage  mounted on the microscope was used to hold the agarose pad at −2 or −5 °C. Time-lapse images were obtained using an ORCA-Flash 4.0 CCD camera (Hamamatsu Photonics) controlled by HCImage software (Hamamatsu Photonics). The images were captured at a rate of 5 frames/sec.One- or two-day-old adult worms were used for this observation following an established protocolSupplementary Information