Edible crickets are among the praised insects that are gaining recognition as human food and livestock feed with a potential of contributing to food security and reduction of malnutrition. Globally, the sustainable use of crickets as food or feed is undermined by lack of information on the number of the edible crickets, the country where they are consumed, and the developmental stages consumed. Furthermore, lack of data on their nutritional content and the potential risks to potential consumers limits their consumption or inclusion into other food sources. We reviewed published literature on edible cricket species, countries where they are consumed, and the stage at which they are consumed. We further reviewed information on their nutritional content, the safety of cricket consumption, and the sensory qualities of the edible crickets. We also looked at other benefits derived from the crickets, which include ethnomedicine, livestock feed, pest management strategies, contribution to economic development, and livelihood improvement, particularly in terms of use as food preservatives and use within music, sports, and cultural entomology. Lastly, we reviewed information on the farming of edible crickets. In this review, we report over 60 cricket species that are consumed in 49 countries globally. Nutritionally, crickets are reported to be rich in proteins, ranging from 55 to 73%, and lipids, which range from 4.30 to 33.44% of dry matter. The reported amount of polyunsaturated fatty acids (PUFA) is 58% of the total fatty acids. Edible crickets contain an appreciable amount of macro- and micro-mineral elements such as calcium, potassium, magnesium, phosphorus, sodium, iron, zinc, manganese, and copper. Also, the crickets are rich in the required amount of vitamins such as B group vitamins and vitamins A, C, D, E, and K. Overall, the cricket species examined in this review are safe to be consumed, and they display high proximate content that can replace plant and livestock products. The crickets play valuable roles in contributing to the economies of many countries and livelihoods, and they have medicinal and social benefits. This review is expected to promote greater recognition of crickets as a source of food, feed, and other benefits in the world and encourage up-scaling by farming them for sustainable utilization. Brachytrupes membranaceus , 38Brachus icipe .Jongema  documentCrickets have been consumed as food in Asia, Latin America, and Africa as far back as prehistoric times. In Biblical scriptures, cricket consumption is recommended to the Israelites by God to be fit for their consumption: “these you may eat any kind of locust, katydid, cricket or grasshopper” (Leviticus 11: 22). In China, crickets have been consumed as food for over 2,000 years , 39. In A. domesticus to be two-fold as compared to that of broiler chickens and pigs, four-fold compared to that of sheep, and more than 12-fold compared to that of cattle. Moreover, crickets may be produced on locally available food substrates such as agro byproducts and weeds, and they thus aid in cleaning the environment  and polyunsaturated fatty acids (PUFA), mineral elements, carbohydrates, energy, and the ease of farming make crickets promising as a sustainable food source , 48. Theironment , 29, 51.ironment , 44, 45.ironment . House cWhile edible crickets are found to be rich sources of proteins and other nutrients , 53, theCrickets are non-wood wild products found in natural resources all around the globe apart from cooler regions at latitudes 55° and beyond; the greatest species abundance is found in the tropics where temperatures are warm and suitable for their faster development compared to cold regions . CricketEdible crickets are excellent sources of proteins, lipids, carbohydrates, mineral salts, and vitamins . HoweverBrachytrupes sp. and 83.9% for A. domesticus  per 100 g of dry weight . The enelt stage , 61  and arachidonic acid (50.43 g/100 g of dry matter) are the most abundant fatty acids in Brachytrupes sp. and B. portentosus, respectively , and palmitic acid (C16 fatty acid) as compared to other fatty acids , 73, 115ectively . The seccalities , 58, 114Tarbinskiellus portentosus, G. testaceus, G. assimilis, A. domesticus, A. confirmata, Brachytrupes sp., and B. portentosus have higher content of polyunsaturated fatty acids (PUFA) compared to pork and broiler chicken meat  compared to pork and broiler chicken meat. Most of the crickets in our review, except for G. bimaculatus and T. emma, have more essential fatty acids than the pork and broiler chicken. Crickets generally have more unsaturated fatty acids (UFA) compared to saturated fatty acids (SFA) , and some cancers affecting human beings, are due to the consuming of SFA  , 61. A nd to UFA . This dig of SFA . Consumpg of SFA .Gryllus bimaculatus cricket contains the highest amount while Brachytrupes sp. contains the least amount of calcium  is applied to patients suffering from athlete's foot for treatment  diet containing 100, 75, 50, 25, and 0% fish meal can be successfully changed to contain 0, 25, 50, 75, and 100% G. bimaculatus crickets, respectively . Gathering crickets from farms and consuming them as food can also help in reducing pesticide use in controlling these cricket pests. This, in return, can protect the environment from pollution, minimizing the killing of other useful insects and poisoning of consumers . This practice of allowing crickets to fight was later declared illegal during the Qing Dynasty (1644–1911 CE). Currently, however, cricket fighting is legal, and it has become a common sporting activity in many Chinese cities, such as Beijing, Guangzhou, Huwan, Hong Kong, Shanghai, and Tianjin, where cricket fighting clubs and societies have been formed . CricketCrickets have contributed a lot to the shaping of literature, art, and doctrine in societies around the world—referred to as cultural entomology . ContribThe crickets have also played different roles in folklore and superstition in different parts of the globe. In this perspective, some communities hold a lot of esteem for crickets since they believe that once you hear the song of the cricket it spells good fortune, although others say it is a bad omen when a cricket makes noise around you. In China, for instance, the crickets have been implicated to foretell the coming of rain, death, or the returning of a lover who has been away . MoreoveScapteriscus and Neocurtilla, Gryllotalpidae) that enters into the house of a Brazilian brings both good luck and rainfall  . The othA. domesticus and Brachytrupes. The results showed that various types of Enterobacteriaceae and sporulating bacteria can be identified and subsequently seperated from raw crickets entering them most likely during contact with the soil , the short-tail cricket B. portentosus, and T. portentosus  , 189. Extentosus , 189–191he globe .A. domesticus, G. bimaculatus. They, however, prefer rearing G. bimaculatus, which form a greater portion of the Thai production since G. bimaculatus has a short lifecycle and is stronger and hardy, though less popular than A, domestica , brown field cricket (G. assimilis), and two-spotted field cricket (G. bimaculatus) are the common crickets bred in Europe and for industrial processing . A grant from the Danish government facilitates the technology transfer of cricket farming through GREENiNSECT project that supports “the rearing and eating crickets as a delicious, affordable and healthy solution for malnutrition.” The project has accelerated, leading to the establishment of small-scale cricket farms in L. Victoria region, which was the initial point of introduction in Kenya and Uganda. From this point of introduction, cricket farming has spread to other regions, such as central Kenya and the coast of Kenya. Three cricket species, S. icipe, A. domesticus, and G. bimaculatus are reared (Scapsipedus icipe and A. domesticus are most popular among farmers because they are softer than G. bimaculatus. In Kenya, there are about 300 cricket farmers who produce 28,800 kg of crickets per year (In Africa, cricket farming is at its infancy stage in countries such as Kenya, Uganda, Mali, and Madagascar , 202. Cre reared , 204. Inper year . The criper year , the larper year ,. The yiFarming of crickets requires varying degrees of labor input during the rearing cycle . Each daicipe have already assessed hygienic and toxicological and microbial risks related to edible crickets, more research on their composition and nutrient profile should be carried out to fully implement edible crickets as food into the global legislation documents. Currently, there are only a few cricket species that are farmed. The farmers must be encouraged to start rearing other species of crickets that have not yet been confined. Also, animal breeders should try to find out whether it is possible to crossbreed the crickets with a long lifecycle with the ones with a short lifecycle.The current study has shown that consuming crickets as food by human beings is traditionally practiced in 49 countries around the world. Over 60 cricket species are known to be edible. Crickets are a highly nutritious food resource and may therefore be included in the list of the common diet of global consumers in the future. These crickets could also be used as nutritional supplements for special diets for schoolchildren, sick people, and athletes. Inclusion of potentially suitable species of crickets into the normal diet requires defined and standardized conditions of their rearing as well as the detailed monitoring of their composition, including biologically active compounds. Though the EFSA and Edible crickets have become popular in the past few years not only in the scientific literature but in other platforms as well. One of the major advantages of eating crickets is their impressive nutritional composition. Many sources report that crickets have better nutritional characteristics than traditional protein sources. In our research, we aimed to give a complete picture of edible crickets in the world, their nutritional profile and other benefits. The materials we used are published results of different authors from the past few years. The list of crickets provided by various authors' shows that there are 66 crickets that are consumed as food and feed in the world and crickets generally have a better nutritional profile than other meats. Based on our findings, crickets have a promising nutritional profile in terms of energy, protein, lipids and important fatty acids, mineral elements vitamins, carbohydrate and medicinal elements and may become part of many food products in future. As an enterprise, cricket farming, can mitigate food insecurity, act as a source of income when sold and a source of employment. The present review provides comprehensive information on the diversity of crickets, their nutritional values and their potential to contribute to the livelihood of mankind.HM, SN, MA, MM, SE, JE, EK, JO, SH, KF, MO, NR, and CT: conceptualization, writing—original draft, and writing—review and editing. HM, SN, EK, and SH: data curation. HM, SN, EK, SE, and MM: formal analysis. HM, SN, MA, MM, SH, NR, SE, JE, EK, and KF: methodology. HM, SN, and EK: software. SN, MM, JE, SE, SH, CT, and KF: validation. HM: investigation. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Compliance mismatch between an arteriovenous dialysis graft (AVG) and the connected vein is believed to result in disturbed haemodynamics around the graft–vein anastomosis and increased mechanical loading of the vein. Both phenomena are associated with neointimal hyperplasia development, which is the main reason for AVG patency loss. In this study, we use a patient-specific fluid structure interaction AVG model to assess whether AVG haemodynamics and mechanical loading can be optimised by using novel electrospun polyurethane (ePU) grafts, since their compliance can be better tuned to match that of the native veins, compared to gold standard, expanded polytetrafluoroethylene (ePTFE) grafts. It was observed that the magnitude of flow disturbances in the vein and the size of anastomotic areas exposed to highly oscillatory shear ( Haemodialysis (HD) is the main mode of renal replacement therapy for most patients with end-stage renal disease. During HD, a haemodialyser removes metabolic waste products and excess fluids from the patient’s blood stream. A functional vascular access (VA) is of vital importance for these patients, since this is the only location where the haemodialyser can be connected to the body during HD. VAs need to be created surgically, as no autologous vessel allows for easy and repeated cannulation, whilst also providing the high blood flow . Despite multiple interventions to maintain patency, these grafts have a typical lifespan of only 2 years. This short lifetime of grafts can mainly be attributed to venous neointimal hyperplasia (NIH) near the graft–vein anastomosis, resulting in stenosis, low flow and ultimately thrombosis and graft patency loss. Disturbed flow and resulting non-physiological or multi-directional wall shear stress (WSS) after AVG creation, as well as limited biocompatibility are believed to be the main triggers of NIH development  has emerged as a possible substitute to ePTFE for vascular grafts  models of the venous anastomosis of an ePTFE and an ePU graft were developed, that allowed for studying the impact of different graft materials on blood flow. In this respect, the modelling approach differs from more commonly applied computational fluid dynamics models that assume rigid vessels. Furthermore, haemodynamic and mechanical properties that would be hard to measure in experimental set-ups, could be straightforwardly computed from the FSI model.Maastricht, the Netherlands). A waiver for ethical approval for this study was obtained from the local medical ethical committee.The patient data used in this study were obtained from clinical follow-up data for monitoring graft function and was measured at the Maastricht University Medical Centre , vessels with a circular cross section were imposed onto the centrelines, representing the inner lumen of graft and the vein at zero transmural pressure. Zero-pressure graft diameter was set to 6 mm, whereas zero-pressure vein diameter was reconstructed from pre-operative ultrasound data and was set to A patient-specific axillary-artery to axillary-vein loop AVG geometry was reconstructed from 15-month post-operative diagnostic computed tomography angiography (CTA) data and 2-week pre-operative ultrasound diameter measurements of a single patient Fig. . Centrel17.2, Ansys, Canonsburg, PA, USA). A mesh independent solution was obtained for Geometries were meshed in ICEM CFD  FSI solver Unicorn  in the post-operative working range 30–60 mmHg  consisting of 24 CPU cores took approximately 15 days to complete.Simulations performed on a single node of the Cartesius supercomputing cluster  velocity perturbations in the vein was quantified using Reynolds decomposition. Here, flow velocity magnitude Mechanical stress in venous and graft walls was characterised by the von Mises stress:Flow in the graft was laminar during the complete cardiac cycle for both the ePTFE and the ePU graft simulations. After leaving the graft, flow impinged on the venous wall, giving rise to secondary flow and a jet along the venous floor Fig. a. In botmHg Fig. . At the The magnitude of velocity perturbations in the venous segment was generally higher in the ePTFE graft compared to that of the ePU graft Fig. b. In botentclass1pt{minimaBoth the anastomotic and the total venous area that were exposed to highly oscillatory shear , the magnitude of flow disturbances and the size of the anastomotic area exposed to very high or oscillatory wall shear stress can be considerably reduced by using more compliant electrospun polyurethane (ePU) grafts. Since AVG failure induced by neointimal hyperplasia is triggered by disturbed blood flow and non-physiological wall shear stress, the results from this study suggest that AVG longevity will be improved by the use of ePU grafts. The improved


We benchmarked Fusion-Bloom against the performance of five other state-of-the-art fusion detection tools using multiple datasets. Overall, we observed Fusion-Bloom to display a good balance between detection sensitivity and specificity. We expect the tool to find applications in translational research and clinical genomics pipelines.Presence or absence of gene fusions is one of the most important diagnostic markers in many cancer types. Consequently, fusion detection methods using various genomics data types, such as RNA sequencing (RNA-seq) are valuable tools for research and clinical applications. While information-rich RNA-seq data have proven to be instrumental in discovery of a number of hallmark fusion events, bioinformatics tools to detect fusions still have room for improvement. Here, we present Fusion-Bloom, a fusion detection method that leverages recent developments in https://github.com/bcgsc/pavfinder and released under a Creative Commons License , as described at http://creativecommons.org/licenses/by/4.0/.Fusion-Bloom is implemented as a UNIX Make utility, available at Bioinformatics online. Although developments in long read technologies may alter this assessment in the future, the current cost-benefit-value balance still favours short reads for many applications.Gene fusions have long been known as drivers for both development and progression in various tumour. Over the years, a number of software tools have been developed to detect gene fusions from RNA sequencing (RNA-seq) data (de novo transcriptome assembler, RNA-Bloom (Here, we describe a pipeline called Fusion-Bloom, which combines the use of a new Fusion-Bloom is implemented as a UNIX Make utility, which automates three analysis stages: assembly, alignment and analysis . In the We took a commonly-used benchmarking dataset consisting of 50 fusions to compare the performance of Fusion-Bloom against 5 other fusion detection tools: deFuse . Fusion-A publicly available dataset consisting of synthetic fusion transcripts spiked in at a wide range of molarity levels to total RNA provides another useful benchmarking test of sensitivity .To assess the tools’ specificity in experimental data, we analyzed three RNA-seq samples that are technical replicates of a whole-blood sample pooled from five healthy donors . Using ade novo assembly is a valuable approach in that it provides base-pair precision of fusion breakpoints, and can also be used for detecting other long-range transcriptome rearrangement such as tandem-duplications and splice variants (We benchmarked the computing performance of the tools using the 20 spike-in samples which contained 73 to 180 million read pairs . On averThis work was supported by Genome Canada and Genome BC [281ANV]; and the National Institutes of Health [R01HG007182]. Scholarship funding was provided by the Natural Sciences and Engineering Research Council of Canada. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding organizations.Conflict of Interest: none declared.btz902_Supplementary_DataClick here for additional data file.


As starch properties can affect end product quality in many ways, rice starch from Thai domesticated cultivars and landraces has been the focus of increasing research interest. Increasing knowledge in this area creates a high demand from the research community for better organized information. The Thai Rice Starch Database (ThRSDB) is an online database containing data extensively curated from original research articles on Thai rice starch composition, molecular structure and functionality. The key aim of the ThRSDB is to facilitate accessibility to dispersed rice starch information for, but not limited to, both research and industrial users. Currently, 373 samples from 191 different Thai rice cultivars have been collected from 39 published articles. The ThRSDB includes the search functions necessary for accessing data together with a user-friendly web interface and interactive visualization tools. We have also demonstrated how the collected data can be efficiently used to observe the relationships between starch parameters and rice cultivars through correlation analysis and Partial Least Squares Discriminant Analysis.Database URL: http://thairicestarch.kku.ac.th Thai rice is an important agricultural product: in 2019, the total amount of Thai rice exported was 7 580 505 metric tons, contributing $4.2 billion to the country’s GDP : the amyThe properties of rice starch from Thai domesticated cultivars and landraces have been increasingly investigated leading to the greater discovery of knowledge and data generation , 6, 9–15The present study has established the Thai Rice Starch Database (ThRSDB) aiming to serve the rice starch research community with a digitalized, well-organized and searchable version of important Thai rice starch data. The database is an open resource freely accessible through a web interface with several functions. Various analyses were performed to examine the relationships between starch variables among Thai rice cultivars addressing the potential application of the ThRSDB.The focus of ThRSDB is to collect the starch properties of Thai rice cultivars (indica subspecies) that have been reported in original research articles. The japonica rice and long-grain basmati rice grown in Thailand have not been included in the current database because they are not native Thai rice cultivars or landraces. We searched for publications indexed in the ISI, Scopus and Thai-Journal Citation Index Centre (TCI) databases using keywords such as Thai rice, Thai rice starch, Jasmine rice and cultivar names . There were 39 published articles in both English and Thai from the literature search. The data was manually curated to obtain the observed values of key starch properties including gel consistency, granule size, amylopectin chain length distribution, gelatinization, retrogradation, viscosity, hydrolysis, moisture content, percentage amylose, percent crystallinity, swelling power and percent solubility. The amylose:amylopectin ratio was calculated as the percentage amylose divided by (100—the percentage amylose) . From eahttps://d3js.org/) and DataTables (https://datatables.net/) were for rendering interactive graphs and tables, respectively.Thai Rice Starch Database (ThRSDB) is a relational database using MySQL to store and manage data. PHP 7.2 was used for data queries. The web interface was implemented using standard HTML, JavaScript and CSS running on the Apache web server. JavaScript libraries: D3.js (https://CRAN.R-project.org/package=psych). We demonstrated the usefulness of the curated data for rice starch research through two analyses: (i) we examined the associations between the starch parameters by calculating Pearson’s correlation coefficients using the R package ‘corrplot’  and ranges were summarized using the R package ‘psych’  and RD6 (the famous Thai sticky rice), respectively Figure . KDML 10s Figure . Of the s Figure . Many ofs Figure .The overall statistics of the different starch parameters are summarized in Table A user-friendly web interface was implemented containing several functions for data access: Browse, Search and Advanced Search Figure . The Brohttp://thairicestarch.kku.ac.th.The detailed information on each cultivar was presented on a separate page Figure E. This iThe composition and molecular structure of starch are key determinants of its functional properties , 5, 28. P-values of less than 0.05. It can be seen that the amylose content was significantly and positively correlated with the rheological properties, particularly the pasting temperature, final viscosity and starch setback. These relationships are consistent with those reported in previous studies on Thai rice cultivars, which showed that starch setback and pasting temperature increased with amylose concentration  were calculated for a filtered set of the rice cultivars, which indicated the associations between the starch parameters Figure . The corntration , 15, 30.ntration . This exRice starches from different cultivars possess distinct morphological, thermal and rheological characteristics , 33. TheTo demonstrate the utility of our curated data, we examined whether the Thai rice starch data collection in ThRSDB could correctly discriminate waxy from non-waxy rice. Therefore, PLS-DA was conducted to examine the variation among the Thai cultivars Figure . PLS-DA To the best of our knowledge, ThRSDB is the first platform where data on Thai rice starch composition, molecular structure and functionality from many sources of the scientific literature has been deposited and where this dispersed information has been made available in a well-organized and user-friendly version to the rice community. The database contains 28 essential parameters of 13 key starch properties, of which the parameters of percentage amylose, moisture content and starch viscosity were the most frequently studied. Of the two main types of cultivar, waxy and non-waxy rice, RD6 and KDML 105, respectively, were the most frequently reported.We have demonstrated the value of the curated data through two successful analysis cases. Correlation analysis demonstrated that, with the extensively curated data, significant correlations could be observed between grain compositional parameters, such as percentage amylose and starch functionality parameters, such as pasting temperature and setback viscosity. PLS-DA also showed that the waxy and non-waxy rice cultivars could be separated based on the values of key discriminant parameters such as percentage amylose, pasting temperature and viscosity.This digitalized, well-organized and searchable compendium of Thai rice starch data allows the rapid and efficient use of dispersed information on rice starch. It facilitates comparisons between rice cultivars and across research units. The database can be broadly applied in several aspects: for instance, it is possible to use the database in breeding programs through observing and comparing particular starch traits between two or more cultivars, through exploring the diversity of starch traits among different cultivars, or through preliminary screening for cultivars offering a specific starch trait. The broad continuum of industrial applications of starch also requires knowledge of its structural and functional properties, which can be easily obtained from ThRSDB. Besides, the source information shows the variety and availability of rice cultivars in different areas of Thailand. We believe that this information could be useful for research units, companies and other stakeholders and for economic planning. The raw data could be made available for download upon request, which allows extensive exploitation of the data with other information repositories.As a starting point, ThRSDB currently focuses on starch information from Thai rice cultivars, an indica subspecies, under normal growing conditions. In the future, we plan to include other Asian rice cultivars such as japonica rice and long-grain basmati rice and to take account of variations in growing conditions so that the database will become a more comprehensive resource for rice starch information. As more research into rice starch properties is undertaken, we will continuously collect this new data and update the database. We are currently investigating to embed a data analysis module , which would allow users to statistically analyze their own datasets. We believe that ThRSDB will be a valuable resource for the rice community worldwide and so will become a collaborative platform for sharing data in this research area.A key limitation of ThRSDB is the incompleteness of the curated data. Most of the original research articles have only reported a limited number of grain quality traits because of differences in their research focus and technological concerns. Nevertheless, the grain amylose content was the most frequently characterized parameter. This parameter alone is of critical value to users because (i) it has long been proved to be an important trait for determining rice functionalities and grain qualities and (ii) it is widely used to categorize rice cultivars into different groups such as waxy, low amylose and high amylose rice.Another limitation concerns the low amount of information on growth conditions. This is because the rice samples were mostly acquired from national rice research centers, private companies or markets. Experiments investigating a particular treatment such as elevated temperature, drought or soil salinity have been excluded from the current database.baaa068_SuppClick here for additional data file.


NBPF19 cause two disorders that were formerly unlinked, namely neuronal intranuclear inclusion disease (NIID) [NBPF19, LRP12, and LOC642361, which cause NIID, oculopharyngodistal myopathy (OPDM), and a new overlapping disease oculopharyngeal myopathy with leukoencephalopathy (OPML), each designated as NIID1, OPDM1, and OPML1, respectively [Unstable tandem repeat expansions are important genetic motifs that underlie various neurological disorders. Two interesting aspects of noncoding CGG expansions have recently attracted attention: pleiotropy in which expanded CGG repeats in e (NIID) , 4 and ee (NIID) , and theLRP12  repeat associated non-AUG (RAN) translation that generates toxic, often ubiquitinated aggregation-prone proteins and 2) formation of RNA foci that sequester RNA-binding proteins. In the present autopsied patient with OPDM1, the inclusions in the skeletal muscles were not ubiquitinated and visible only by electron microscopy, whereas those in extra-skeletal muscle organs were ubiquitinated and detectable by routine histology. The mutually exclusive distribution and different natures of these two types of inclusion may reflect a difference in the pathomechanisms whereby they are recruited to skeletal muscles or extra-skeletal muscle organs. Interestingly, like OPDM1, no eosinophilic, ubiquitinated inclusions have been reported in skeletal muscles in OPML1 or NIID1 (Table LRP12 including the first reported genetically confirmed autopsy case showing histological evidence of systemic NIID-1 like lesions. Further clinicopathological and molecular studies will help to clarify how noncoding CGG expansions in different genes form the NIID-OPDM spectrum, and the pathomechanism underlying the similarities and differences among these disorders.In conclusion, we have presented an OPDM1 pedigree associated with Additional file 1.


Background: The purpose of this study was to evaluate the risk of peri-prosthetic fracture of constructs made with cephalomedullary (CM) long and short nails. The nails were made with titanium alloy (Ti-6Al-4V) and stainless steel (SS 316L).Methods: Biomechanical evaluation of CM nail constructs was carried out with regard to post-primary healing to determine the risk of peri-implant/peri-prosthetic fractures. Therefore, this research comprised of, non-fractured, twenty-eight pairs of cadaveric femora that were randomized and implanted with four types of fixation CM nails resulting in four groups. These constructs were cyclically tested in bi-axial mode for up to 30,000 cycles. All the samples were then loaded to failure to measure failure loads. Three frameworks were carried out through this investigation, Michaelis–Menten, phenomenological, and probabilistic Monte Carlo simulation to model and predict damage accumulation.Findings: Damage accumulation resulting from bi-axial cyclic loading in terms of construct stiffness was represented by Michaelis–Menten equation, and the statistical analysis demonstrated that one model can explain the damage accumulation during cyclic load for all four groups of constructs (P > 0.05). A two-stage stiffness drop was observed. The short stainless steel had a significantly higher average damage (0.94) than the short titanium nails . Long titanium nail group did not differ substantially from the short stainless steel nails (P > 0.05). Results showed gender had a significant effect on load to failure in both torsional and bending tests .Interpretation: Kaplan–Meier survival analysis supports the use of short titanium CM nail. We recommend that clinical decisions should take age and gender into consideration in the selection of implants. Nearly 30% of the population between the ages of 70–75 years experiences a femoral fracture . This raThe CM nail has been featured in literature describing the clinical outcomes for long  and shorin vivo. Since the stiffness has been related in the literature to have an effect on the life of the construct  with short or long nails have not reported in literature discussing the peri-implant and/or peri-prosthetic fractures and resulting construct efficacy. An attempt was made to mathematically predict the cyclic damage development using the stiffness parameter for these constructs. Such models do not exist in the literature that will be very beneficial in predicting and/or developing new experimental programs. This research motivated by that gap in knowledge yet to apply the results obtained in this experimental program in surgery. Damage development in the constructs was modeled in terms of stiffness, and prediction models developed. Since in pre-primary healing, fixation efficacy, and joint stability is demonstrated by how much the joint gap decreases over time or cycles, stiffness drop during cycling proposed to describe the process. The risk of peri-prosthetic fracture of long versus short nails using titanium and stainless-steel materials, in four groups, was determined from failure data for each gender probabilistically.The experimental work was performed at the biomechanics laboratory located at Miami Valley Hospital in Dayton, OH, United States to evaluate the risk of peri-prosthetic fracture of non-fractured femora constructs. Similar procedure was previously performed on synthetic femurs to investigate different types of fixation  using vaB testing type. This testing was performed by applying the load to the center of the femoral shaft on the medial side . One side of each pair was tested in simultaneous axial compression at 700N to simulate single-leg stance, where torque produced during walking was simulated by applying ±5 Nm  for 30,0ial side . StiffneThe failure criterion in this research is multi-fold:1.Under dynamic conditions, cyclic axial-torsional fatigue, the 30,000 cycles would best describe an additional lifetime of 14 weeks of recovery. At the end of this time the construct is assumed to have been healed, no longer susceptible to failure.2.Under static condition, the load to failure was carried out, upon the construct had been pretested in fatigue. Thus, pre-fatigue constructs, all underwent 30,000 cycles, were tested to failure and the load was determined. Such tests also enable the nature of the failure via oblique cracks running perpendicular to bending load application and femoral neck fractures. Therefore, these observations were directed to support the clinical usage in the operation room by recommending the efficacy of one device over the other based on the stiffness drop.3.Torque and flexural load to failure determined to simulate the rotation of the distal femur and bending of mid-shaft failure so that those critical parameters avoided during the rehabilitation.4.Stiffness drop 80% at the end of 1000 cycles was assumed to define the failure. Since the stiffness provides the construct stability and fixation outcome, where a healing had taken place, this parameter was used to control the 28 weeks of recovery.p-value < 0.05 was performed. Paired Student’s t-test was used to evaluate the significant differences between the four groups of CM nail fixation with respect to age, gender, length, and material properties. Kaplan–Meier analyses were performed to predict construct survival significant difference between the four groups.The statistical analysis was performed with JMP14  and MATLAB R2019a  programs. The stiffness, load to failure, number of cycles to failure, and damage accumulation were documented for each construct and checked for normality. One-way analysis of variance (ANOVA) with a statistical significance set at a Cumulative cyclic damage accumulation with respect to the number of cycles is very useful analysis to predict the construct efficiency. This data may be used to design rehabilitation schedules and restrict load bear. Such models do not exist in the literature as most efforts are directed by applications in surgery. We used various empirical approaches to predict composite damage for four constructs using the following:In order to achieve clinical relevance in the biomechanical testing of a bone and implant construct, current research protocols utilize high numbers of cycles to simulate months of use after a device has been implanted. For instance, average number of steps walked during a typical year is assumed to be one million. Therefore, assuming 20,000 steps per week will translate to 80,000 fatigue cycles per month for the femur construct. The majority of femur studies fall somewhere within this range of fatigue cycles. In order to help choosing an appropriate number of cycles to attain clinical relevance, it is important to understand the fatigue mechanism the bone/construct should resist.In order to analyze the damage accumulation, damage (D) was defined as:ξT is the instantaneous total stiffness at a given cycle and ξOT is the total stiffness under bi-axial, and flexural fatigue conditions.Where Equation 1 shows that the damage is a function of the stiffness ratio, prior to cycling it is 0, and as the cycles increase it increases and assumed to reach 1 at failure. Therefore, stiffness is very important to pre-healing and triggering union/non-union in the surgeries. If we control the stiffness, the fracture fixation and the stability of the fixation device will be ensured.During the initial load bear, the construct undergoes stiffness changes, where early cyclic action results in higher stiffness drop. When the stiffness behavior is modeled with respect to fatigue cycles; we observe a two stage behavior, where stage I represents the stiffness reduction stage and stage II represents stiffness steady state behavior. Therefore, the two stage stiffness drop, presents a potential parameter in which to represent damage, that accrues at different rates.A Regression model was developed for bone damage accumulation with respect to stiffness and bone mineral density using MATLAB. The regression model is,D denotes the damage accumulation of the bone, ξA is the axial stiffness, ρ is the bone mineral density, a0,a1,a2,a3,a4,a5,a6,a7, anda8 are empirical coefficients. A comparison between the prediction profiler of damage accumulation with respect to load to failure in both bending and tension tests was performed and found to be a function of stiffness parameters and density.Where A comparison between the prediction profiler of damage accumulation with respect to load to failure in both bending and torsion was performed and found to be a function of stiffness parameters and density. The prediction expression of damage accumulation with respect to load to failure in bending is as follows:Aξ is the instantaneous axial stiffness, ξOA is the original axial stiffness, ρ is the bone mineral density, ℒB is load to failure in bending, b0,b2,b3,b4,b5,b6,b7,b8,b9,b10,b11,b12, andb13 are empirical coefficients. Additionally, the prediction expression of damage accumulation with respect to load to failure in torsion is as follows:Where ξA is the instantaneous axial stiffness, AOξ  is the original axial stiffness, ρ is the bone mineral density, ℒT is load to failure in torsion, and c0, c1, c2, c3, c4, c5, c6, c7, c8, c9, c10,c11,andc12 are empirical coefficients.Where The Monte Carlo simulations were performed in JMP14 to produce a set of 500 random variables that are lognormally distributed about a mean and standard deviations. In this study, we assumed that each damage mode had a lognormal distribution with damage. For a given group, the percentile of the damage determines the probability of failure. The simulation shows that group #2 is more susceptible to failure while group #4 the least. A MATLAB code was written to compute the probability of failure.The results were summarized in  showing p < 0.05). Additionally, there was no significant difference in average load-to-failure between long titanium group and stainless-steel groups (p > 0.05) regardless of the length, as shown in The mean torsional load to failure of long titanium and stainless steel nail groups were 76.29 ± 20.23 and 56.11 ± 24.11 Nm, respectively. In the short nails, the mean load to failure of titanium group and stainless steel group were 40.74 ± 15.05 and 56.25 ± 23.53 Nm, respectively. In general, the long titanium group had a significantly higher average load-to-failure than the short titanium (p < 0.05). Long stainless-steel nails did not differ substantially from the short titanium nails when loaded to failure in bending (p > 0.05) in 4-point bending tests per ASTM protocols. Length of the nail and gender had significant effects on the average force to failure in bending for all the four groups , as shown in Similar results were found when the matched pair was tested in four-point bending. The mean flexural force to failure of long titanium and stainless-steel groups were 10,095.44 ± 1,825.06 and 8,823.47 ± 2,389.71 N, respectively. In the short nails, the mean flexural force to failure of titanium group and stainless-steel group were 6,245.91 ± 2,273.03 and 5,071.17 ± 3,240.29 N, respectively. In general, the flexural force to failure using long titanium and stainless-steel nails were approximately 160% higher than the short nails. Furthermore, the bending strength of long titanium nails were significantly higher than the short titanium nails . Furthermore, long titanium nails did not differ significantly from the long and short stainless-steel nails (p > 0.05), as shown in The mean instantaneous stiffness of long titanium and stainless-steel groups were 8,217.51 ± 4,520.05 and 9,731.39 ± 2867.89 N/mm, respectively. In the short nails, the mean initial instantaneous stiffness of titanium and stainless steel groups were 12,535.97 ± 6,124.35 and 6,794.27 ± 2671.34 N/mm, respectively. On the other hand, the average total stiffness of long titanium and stainless steel groups were 655.33 ± 343.37 and 675.75 ± 225.22 N/mm, respectively. In the short nails, the mean total stiffness of titanium group and stainless steel group were 699.77 ± 291.25 and 629.84 ± 262.64 N/mm, respectively. In general, the short titanium CM constructs had a significantly higher average instantaneous stiffness than the short stainless steel nails (p < 0.05). Furthermore, long titanium nail group did not differ substantially from the short stainless steel nails (p > 0.05), as shown in The damage  at the end of 30,000 cycles, of long titanium and stainless steel groups were 0.92 ± 0.02 and 0.93 ± 0.01, respectively. In the short nails, the mean damage of stainless steel group and titanium group were 0.94 ± 0.02 and 0.90 ± 0.04, respectively. In general, the short stainless steel had a significantly higher average damage (0.94) than the short titanium nails . Two values are found from this model, including Dp = 0.64), results showed gender had a significant effect on load to failure in both bi-axial and 4-point bending tests , as shown in Short stainless steel nail group showed the highest mean damage accumulation compared to the other three groups. On the other hand, short titanium group showed the least mean damage accumulation. Despite the non-significant difference in damage accumulation with respect to genders in the four groups  of pre-fatigued constructs under bi-axial and bending conditions. These conditions simulated cyclic axial and torsion loading, and femoral bending as a result of load bear. The reduction of fracture-gap was proposed to be a function of construct cyclic, instantaneous stiffness. This study was designed to investigate the effect of dynamic stiffness under cyclic loading and that stiffness reduces abruptly with the application of cycles to about 1000 cycles, then stabilizes and achieves a plateau. An empirical equation (1) proposed to describe damage development, which was assumed to be 0 at the beginning of the cycling and reaching a critical value at the end of 30,000 cycles. This behavior was modeled using Michaelis–Menten modeling, phenomenological modeling that uses critical test parameters, and probabilistic Monte Carlo simulation.Literature shows that clinical advantages of the constructs made with short nails with their counterparts, namely long nails; present shorter operation time that is beneficial for patients with multiple trauma, special anesthesia requirements, or patients with severe medical conditions. Literature indicated that long and short CM nails had similar benefits for older patients (>65) despite the biomechanical differences . We founIt can be seen from We found that group #3 had significantly higher load-to-failure than the group #4 when loaded to failure in torsion. Additionally, there was no significant difference in average load-to-failure between groups #1 and 3 regardless of the length. Long stainless-steel nails (#1) did not differ substantially from the short titanium nails (#4) when loaded to failure in bending. Furthermore, length and gender had significant effects on the average load to failure in bending for all the four groups. Analysis of the stiffness shows that stage I of cyclic fatigue, where the majority of stiffness reduction occurs, is completed within the first 1,000 cycles and the forces applied did not produce a significant change in torsional stiffness. Kaplan–Meier analysis indicated that there is no significant difference between the four CM fixation groups and only 20% of the fixation with the four groups is estimated to survive after 30,065 cycles. In general, bone with no device had x-2.64 lower load to failure in both bending and torsion than the bone with long nail constructs and x-1.79 lower than the bone with short nail constructs.Group #2 indicated the highest mean damage accumulation compared to the other three groups. On the other hand, Group #4 showed the least mean damage accumulation using the three frameworks in which damage accumulation was modeled. Despite the non-significant differences in damage accumulation with respect to gender in the four groups, results showed gender had a significant effect on load to failure in both torsional and bending tests. Even though no significant difference was observed in damage accumulation results when age was the factor, Group #3 showed higher load to failure in both bending and torsion and damage accumulation with ages younger than 60 years were more susceptible to failure compared to other fixation groups. Michaelis–Menten model was found to be applicable to all four groups of testing, where the composite model developed here can predict the damage development in femoral constructs and delay load bear if needed. While this study is the first study that observes damage accumulation of different fixation devices using cadaver bone samples and considering age and gender factors, some limitations were encountered. The male and female bone samples have not been distributed evenly between the four groups because the samples were randomized to receive fixation. Because of that, long titanium had only male sample and was excluded from the evaluation of gender factor on damage accumulation. Another limitation of our study is due to performing the experiments in non-fractured state. As our investigation focused on evaluating CM nail constructs with regard to post-primary healing to determine the risk of peri-prosthetic fractures, no fracture was introduced to the cadaveric femora. Because of that, the effect of fracture type and the severity of bone failure on damage accumulation were not included in our investigation, as our study focuses on the device performance rather than fracture treatment.The Monte Carlo simulations produced a set of 500 random variables that are lognormally distributed about a mean and standard deviations. The simulation shows that short titanium CM nail is the least susceptible to failure and short stainless steel is the most susceptible to failure. The probability of failure was compared with age for both genders. The comparison illustrates that the device probability of failure increased for the age group 70–90 years old for both genders with higher probability to failure.Our study illustrates the efficacy of short titanium CM nails, group #4, compared to short stainless steel, long titanium, long stainless steel nails for femoral fixation and the effect of age and gender on damage accumulation. We recommend that clinical decision should take age and gender into consideration before the implant selection is made.1.In vitro biomechanical models of the CM nail constructs that were investigated with regard to post-primary healing to determine the risk of peri-prosthetic fractures offer insights in understanding the underlying mechanisms of injury and dysfunction, leading to improved prevention, diagnosis, and treatment of bone problems.2.Analysis of the stiffness shows that stage I of cyclic fatigue, where the majority of stiffness reduction occurs, is completed within the first 1,000 cycles, and forces applied did not produce a significant change in torsional stiffness.3.Our investigation supports the use of short titanium CM nail, as Kaplan–Meier survival analysis illustrated that this nail type is the least susceptible to failure for the fixation of femoral fractures when the use of long or short nails is possible.4.Our analysis demonstrated that the clinical decision should take age and gender into consideration before the implant selection is made.5.Mathematical models and regression equations would be beneficial in developing a novel procedure to predict the failure of the bone/device constructs to make clinical decisions in the operating room.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Clinical Ethical Committee of the Miami Valley Hospital. The cadaver material was procured from Anatomical Gift Program of Wright State University, Dayton, OH, United States to the Orthopaedic Surgery, Sports Medicine and Rehabilitation Department for the investigation. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.in vivo behavior and gait. All authors contributed to the article and approved the submitted version.MP designed the clinical aspects of the research, obtained the devices from Smith and Nephew, and supervised resident research (MS) who prepared the device constructs. TG led the biomechanical evaluation of the project and necessary fatigue interpretations along with advising resident, as well as Ph.D. student AW who performed the tests. FH, Ph.D. student of TG, reduced the data, performed all the modeling and simulations, and prepared the manuscript with TG. JR provided necessary data on the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This Reflection starts from the ongoing COVID-19 pandemic as unprecedented occasio to reflect on the approach to international law, which—it is contended—is anthropocentric, and its inadequacy to respond to current challenges. In the first part, the Reflection argues that there is, more than ever, an undeferrable need for a change of approach to international law toward ecocentrism, which puts the environment at the center and conceives the environment as us, including humans, non-human beings, and natural objects. To encourage the incorporation of ecocentrism in the entire discipline, the Reflection will rely on some insight of ecofeminism, whose potential has not been fully investigated in international legal scholarship. In the second part, the Reflection illustrates what an eco-centric international law would mean, imagining three possible applications: first, what the author has called environmental global health, which is connected to the current pandemic and puts into question the proposals dealing with global health that completely miss the theorization of the environment as a whole; second, how actors of international law would change according to an eco-centric perspective; and, third, how the rules prohibiting the use of force might be reconceptualized. The analysis contained in these pages cannot itself exhaust all the possible nuances of the legal reasoning, but it is aimed at being a provocative starting point for a change in the mindset and approach of international legal scholarship. The Last Man, the least known of her works. It was a novel, but one with a high power of evocation. In The Coming Plague, in which she scientifically talked about deforestation and disruption of natural balances, viruses whose existence becomes notorious with the outbreak of epidemics, and social disparities in the access to health services. What can international lawyers learn from these Cassandran books?Back in occasioThe pandemic that spread around the world in 2020 has affected human rights and raised human security issues. It represents, above all, a major environmental issue. International law, as all fields of law, is facing enormous challenges. How relevant is international environmental law in the debate? Are states, along with international organizations, capable of providing appropriate answers? This Reflection does not aim to suggest possible ways of surviving  this global pandemic or to determine the responsibility for the spread of the coronavirus. It rather starts from the pandemic as unprecedented In a second part, the Reflection illustrates what an eco-centric international law would mean, imagining three possible applications: first, what I have called “environmental global health,”Pachamama as part of the 2008 constitutional revision.Ecocentrism is not new to international law. Over the past decades, there have been some eco-centric moves in international environmental law, which disrupted its initial anthropocentric biases based on the idea of protecting the environment “not for its own sake, but because of its value to humans – its importance for human health, economics, recreation, and so forth.”What if we consider international law in its entirety as anthropocentric and blind to the dynamics of powers and domination that are present in the international community, within human communities and between the humans and the environment? Patterns of discrimination and oppression characterized by both intra-species and inter-species hierarchies are entrenched in international law and can be appreciated by developing some ecofeminist concerns and arguments.The Boundaries of International Law, the exclusion of women is “an integral part of the structure of the international legal order, a critical element of its stability,” and the silences of the discipline are “as important as its positive rules and rhetorical structures.”13International feminist legal scholarship has already pointed out the patriarchal structure of international law as we know it today, characterized by the marginalization of women in the international legal system. As Chinkin and Charlesworth wrote in Patterns of oppression and domination are however not only intra-species but also inter-species, in the relation between humans and the nature. The human/nature dichotomy has been used to determine patterns of oppression and discrimination beyond humans, including non-human animals and “natural objects.”20Ecofeminism has played a pivotal role in denouncing the patterns of oppression within humans and of a part of humanity on nature, despite presenting different streams of thought. As acknowledged by the philosopher Plumwood, “ecological feminists differ on how and even whether women are connected to nature, on whether such connection is in principle sharable by men, on how to treat the exclusion of women from culture, and on how the revaluing of the connection with nature.”26Ecofeminism does not simply mean to combine environmental and feminist issues. As Puleo argued, it is “an attempt to outline a new utopian horizon, addressing the environmental issue from the categories of patriarchy, androcentrism, care, sexism and gender.”The eco-centric approach to the international law that is encouraged in these pages could be informed by some of the main findings of ecofeminism, most importantly the need for a disruption of patterns of oppression and of the dichotomy human/nature or culture/nature, and the emphasis placed on the intersections of grounds of oppression.A change of approach in international law cannot be thoroughly explained in few pages. This paragraph will sketch some possible trajectories which will constitute a solid starting point for future research. It starts from what I have called “environmental global health,” before moving to two selected, and admittedly limited, issues of international law, namely, actors of international law and the prohibition of the use of force.The current framework of global health law, mainly based on World Health Organization (WHO) law, seems inadequate to respond to current challenges, because it does not appreciate the importance of taking into consideration the environment as “us.”An environmental global health governance should focus on prevention, ensuring campaigns to raise awareness, for example, of how wet markets represent ticking bombs for the environment, but also of how increasing emissions, plastics, disruption of ecosystems, land grabbing, water grabbing, weak public health systems characterized by inequalities and lack of participation, fuel the spread of pandemics and/or the effects of them. As the reality has ruthlessly shown us, the prevention of pandemics cannot be merely based on the WHO law. What we need is the sharing of responsibilities and liabilities for a plurality of actors44Animal Liberation, argued that discrimination of living beings “solely on account of their species is a form of prejudice, immoral and indefensible in the same way that discrimination on the basis of race is immoral and indefensible.”59States represent anthropocentrism in their reproduction of rooted schemes of subordination. Scholarship has emphasized the male nature of the state as traditionally conceived and identified the elements of oppression, including the principle of territorial integrity.Decentering states and the humans does not simply mean to re-center the legal debate on nature, most importantly because states and (a part of) human beings have been the cause of the ongoing environmental disasters and ecological imbalances. An eco-centric approach would rather entail that human policies and perspectives are reoriented toward a common end, with the guarantee of the integrity of the environment, to which humans constitute an integral, though no longer dominant, part. It would also stress the importance of non-governmental organizations and environmental (and not only human) rights defenders in the disruption of the predominance of states in the international realm.An eco-centric reading of the prohibition of the use of force can follow two  directions.62It is also the main argument of this paragraph that when authorizing the use of force, the UN Security Council should include the respect for the environment, especially due to air and soil contamination caused by military strikes that affects the ecosystems and the human beings as part of them. It would be even possible to say, evoking the call for disarmament and peace of the International Congress of Women of 1915, that if we consider the environment as a whole, the quest for peace manifests itself as expression of ecocentrism.The second direction concerns the use of weapons. In this sector, the eco-centric approach to international law can be appreciated at its best, suggesting an eco-centric, inspired by ecofeminism, rewriting of the highly criticized Advisory Opinion on the legality of the threat or use of nuclear weapons of 1996.64We did not need the pandemic to highlight the weaknesses of the global health governance and international law more generally as we conceive them today. New ways of framing international law have been advocated for a long time. The pandemic represents however the game changer, which unveils existing problems and pushes for a change. The interconnection between global health and the environment must be appreciated in all ideas of reform of the WHO system as a starting point, but this does not seem enough. There is an urgent need of new creative approaches to international law to grasp the complexity of the environment, which includes human beings as part of a holistic whole. This argument does not only lead to the adoption of new rules of international law but also to apply and interpret existing ones in an eco-centric way. This Reflection has provided a few examples of existing practice in the described direction. Its invitation to endorse an eco-centric approach to international law, which is critical of existing patterns of discrimination and oppression, is not devoid of risks, including the resistance states could show to new rules informed by eco-centric concerns. Nonetheless, as international lawyers, we should respond to the crisis and demonstrate that international law, starting from its basic concepts, can evolve and embrace the environment, which is, in the end, nothing less than a pivotal issue to work on in all the fields of the discipline.


The double-bundle technique with two points of patellar fixation in the upper half of the patella replicating the broad attachment site of the native medial patellofemoral ligament (MPFL) is the most commonly performed procedure for MPFL reconstruction. Complete transverse patella tunnels pose a threat to the integrity of the patella. We present an implant-free, double-bundle technique for MPFL reconstruction with gracilis autograft, overcoming the problem of complete patella bone tunnels and over-drilling.After standard gracilis graft harvesting, the anteromedial side of the patella is exposed. With the guidance of an anterior-cruciate-ligament (ACL) tibia-aiming device, two 2-mm parallel guide pins are inserted from medial to lateral at the upper half of the patella. The two guide pins are over-drilled with a cannulated 4.5-mm drill bit 2-cm deep, to create two transverse blind semi-patellar tunnels. For the femoral fixation, a 2.4-mm guide pin with an eyelet is drilled at the Schöttle point and over-reamed with a 6-mm cannulated reamer to a depth of 30 mm. The two free ends of the graft (with two running Krakow sutures placed) are pulled into the two patella tunnels and the graft sutures are tied together with tension for stable graft fixation at the lateral patella rim. With the help of a femoral suture loop , the graft-loop is advanced into the femoral bone tunnel and the graft is finally fixed with a 7-mm interference screw at 30° of knee flexion.The utilization of blind transverse tunnels (not trans-patellar tunnels) offers the advantage of avoiding stress risers at the patella. Thanks to the ACL tibia aiming device, multiple drilling, and breaching of the anterior patellar cortex or articular surface of the patella is avoided.This implant-free, and consequently affordable technique, isolated or combined with bony procedures, minimizes possibilities for perioperative bony complications at the patella fixation site. Μedial patellofemoral ligament (MPFL) reconstruction is currently the first-choice soft-tissue procedure for patients requiring surgery after more than 1 or 2 episodes of patellar dislocation . An isolWe present a double-bundle technique for MPFL reconstruction, without patella implants, overcoming the problem of complete patella bone tunnels and overdrilling.The patient is placed in a supine position, and a tourniquet is applied to the upper thigh. Arthroscopy would be warranted prior to MPFL reconstruction, in case of suspected intra-articular pathology . Gracilis tendon autograft is harvested through a vertical or horizontal incision which is placed 2 cm medially to the pes anserinus. The overlying sartorial fascia and the pes anserinus bursa are exposed and incised Fig. . AdhesioA solution of 100 ml of sterile saline is prepared in a tray and mixed with 500 mg of vancomycin powder. The prepared graft is immersed in the tray and is then wrapped in gauze that has been saturated with the vancomycin solution beforehand to eliminate graft contamination. The graft is kept there until the implantation.With the knee flexed at 90°, a longitudinal incision 2–3 cm) is performed on the anteromedial side of the patella and the medial aspect of the patella is exposed all the way to the bone surface by electrocautery, without penetrating the capsule  are encouraged from the first week. Full range of motion is allowed after 6 weeks. Light running is initiated after 8 weeks. Patients can return to contact sports at 9 months after surgery.The use of screws and anchors in the patella fixation is less time-consuming and easier to use than hardware free-fixation techniques, but it has been accused of causing pain and irritation at the insertion side . On the A useful tip one should bear in mind when choosing this technique is to ensure graft length of at least 20 cm, since the anatomical length of the native MPFL has an average length of 5.3 cm  and moreWe have been using this technique in our department for several years, isolated or combined with bony procedures, without perioperative or postoperative complications and with good clinical results. This is a useful modification of an established technique , 30, whi


Human norovirus (HuNoV) is the leading cause of nonbacterial gastroenteritis worldwide with the GII.4 genotype accounting for over 80% of infections. The major capsid protein of GII.4 variants is evolving rapidly, resulting in new epidemic variants with altered antigenic potentials that must be considered for the development of an effective vaccine. In this study, we identify and characterize linear blockade B-cell epitopes in HuNoV GII.4. Five unique linear B-cell epitopes, namely P2A, P2B, P2C, P2D, and P2E, were predicted on the surface-exposed regions of the capsid protein. Evolving of the surface-exposed epitopes over time was found to correlate with the emergence of new GII.4 outbreak variants. Molecular dynamic simulation (MD) analysis and molecular docking revealed that amino acid substitutions in the putative epitopes P2B, P2C, and P2D could be associated with immune escape and the appearance of new GII.4 variants by affecting solvent accessibility and flexibility of the antigenic sites and histo-blood group antigens (HBAG) binding. Testing the synthetic peptides in wild-type mice, epitopes P2B (336–355), P2C (367–384), and P2D (390–400) were recognized as GII.4-specific linear blockade epitopes with the blocking rate of 68, 55 and 28%, respectively. Blocking rate was found to increase to 80% using the pooled serum of epitopes P2B and P2C. These data provide a strategy for expanding the broad blockade potential of vaccines for prevention of NoV infection. Noroviruses cause most of acute gastroenteritis cases in children and adults worldwide . AccordiPrevious studies described three conformational epitopes A, D, and E within the P2 domain of the GII.4 capsid protein ,15. In pPeptide-based vaccines currently represent a novel strategy for addressing challenges associated with the prevention of viral infections. In this way, in silico analysis may apply to predict new B- and T-cell epitopes for in vivo applications. This approach has the ability to incorporate several epitope peptides directed against multiple virus strains into one single delivery system to induce broad immune responses in a single administration. In this study, we described linear antigenic epitopes which can elicit blocking antibodies against NoV GII.4 genetotype in vaccinated mice. Molecular dynamics (MD) simulation has been widely used in structural biology investigations and can serve as a powerful tool to determine the atomic-level behavior of biological events, such as protein conformational changes under different physiological conditions . We applA total of 600 HuNoV GII.4 capsid protein sequences available in GenBank were collected. For each year, a phylogenetic tree was first constructed with maximum likelihood algorithm in the MEGA7.0.14 package  . Based oLinear B-cell epitopes were predicted using the Immune Epitope Database Analysis Resource (IEDB), Epitopia27  and BepiPred epitope prediction tools  ,20. Potehttp://mordred.bioc.cam.ac.uk/~rapper/rampage.php). Putative B-cell epitopes and variable sites that were potentially exposed on the surface of the capsid were mapped on the structures and analyzed for conformation changes over time from.Capsid protein secondary structures were predicted using highly accurate prediction tools Jpred 4  and PSIPRED  ,29. Homohttps://software.broadinstitute.org/morpheus/).Molecular dynamics (MD) simulation was conducted using GROMACS 4.5 package  . The prea trisaccharide and Lewis b tetrasaccharide, as the two main types of Lewis antigens, was performed using the COACH-D web server. The 3-D structures of the Lewis a and Lewis b were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov).Norovirus GII.4 variants are capable of binding Lewis HBGA types . To undeg. The VLP-containing fractions were pooled, dialyzed, and stored at 4 °C until further use. Purified VLPs were characterized on Sodium dodecyl sulfate (SDS)-polyacryamide gel electrophoresis (PAGE), followed by Western blotting to confirm the presence of the capsid protein with an apparent molecular weight of 58 KDa. To ensure that the particles were intact, each preparation was examined by negative-stain electron microscopy (EM), using 1% ammonium molybdate. Protein concentration was determined by the Pierce BCA Protein Assay Kit .Recombinant baculovirus expressing NoV GII.4 Sydney 2012 capsid protein was generated according to the instructions for the Bac-to-Bac Baculovirus Expression System . The virus-like particles (VLPs) were expressed in baculovirus-transformed Spodoptera frugiperda Sf9 insect cells, and then were purified on a step gradient of sucrose (15 to 60%) for 3 h at 112,700× There is evidence that the proteolytic degradation of short peptides can be markedly inhibited by N- and C-terminal residues modifications ,37. ThusLinear B-cell epitopes were predicted on the basis of hydrophobic residues on NoV GII.4 capsid protein. As presented in Amino acid residues in P2 domain epitope sites were found to be differently surface accessible over years A. In conStructural analysis of NoV GII.4 capsid proteins was conducted to find key amino acids affecting the conformation and accessibility of the putative epitopes. A secondary structure analysis revealed mutations in variable sites caused notable changes in the P2 domain ß-strands, as presented in We also analysis the 3-D homology models of the GII.4 norovirus proteins. For this aim, 3-D homology models were generated. Structural alignment between the models and real capsid protein crystal structure revealed that all modelled protein structures have RMSD < 2.0 A indicating that all the models were in the range of native conformation. The modelled protein structures were energy minimized using Modrefiner and steric clashes were removed using Chiron tools . After refinement of each modelled protein structures were validated using ProSA-web server. A Z-score of −6 to −6.44 was measured for the modelled proteins confirming that they are in the range of native conformations . ProteinTo delineate protein motions at the atomic level, we conducted molecular dynamics (MD) simulation. After 20 ns of simulation, analysis of the RMSD, RMSF, Rg and SASA parameters revealed changes in the P2 domain antigenic epitopes at molecular level over the years. RMSD is an indicator of structural stability, which is widely used for evaluating the structural differences between proteins. The average RMSD of the structures ranged from 0.3 to 0.5 nm during trajectory. A comparison of RMSD values revealed distinct patterns of instability in the epitope sites in different years A. We perMolecular docking of the reconstructed models to the HBGA oligosaccharides was conducted to understand the effect of amino acid substitutions in surface-exposed epitopes on HBGA binding. The data revealed changes in binding pattern and binding affinity to HBGA carbohydrate  as a resNoV GII.4 VLP were generated in ExpSf9 insect cells using the BAC to BAC baculovirus expression system and then were purified on sucrose gradient A; the prNorovirus GII.4 genotype exhibits epochal evolution, whereby major circulating strains give rise to emergent strains, likely due to escape from herd immunity ,43. It aPrior to this work, a number of discreet amino acids, in particular residues 296–298, 393–395 and also surface residues at 333, 340, 356, 368, 372, 407, and 412–413 have been described as potential evolving GII.4 antibody epitopes which change between GII.4 variants ,11,14,16In present study, we predicted five linear antigenic epitopes (P2A-E) in the P2 domain with surface-exposed areas which can potentially be used for improvement of NoV GII.4 vaccines. We propose a model of antigenic evolution for GII-4 NoV strains based on sequence diversity, structural modelling and dynamic evolution of these surface-exposed antigenic regions over time. They accumulated sequential amino acid mutations that correlated with increased prevalence of emerging GII.4 variants which sequentially change suggesting that these sites may play role in the escape from herd immunity. These sites undergo selective amino acid changes that coincide with the appearance of new epidemiologically significant GII.4 variants in the population. Evidence supporting this includes greater positive selection pressure on the amino acids in the P2 domain than in the remaining capsid protein. Although it is hard to determine how changes in amino acids will contribute to the specific antigenicity without further laboratory investigation, such changes will undoubtedly affect recognition between antibody and epitopes. We explored the value of modelling the predicted surface area derived from the different amino acid substitutions in variable sites as predictors of significant structural changes correlated with the emergence and/or switch of epidemic strains. This analysis revealed marked conformational changes in the P2 subdomain with major surface area motifs.Analysis of surface accessibility revealed that the predicted P2 domain epitopes expose to the surface at varying threshold over time. For antibody recognition and binding, the antigenic sites must be surface accessible and flexibility in these sites allows the formation of an antigen-antibody interface since flexible regions can adjust their conformation upon interaction with an antibody. Therefore, flexibility of a region is an indicator of the existence of a potential antigenic site . Positioa and Lewis b. The results revealed two binding pockets for interaction of the capsid protein with HBGA. In 1987, 1995, and 2002 variants, the HBGA binding pocket shares a part of epitope P2B  and a residue 374 in epitope P2C. It seems that mutations at positions 340 and 367 caused a clear increase in binding affinity in 1995 variant compared to the 1987. Later, in 2004/Hunter, 2006/Minerva and 2009/New Orleans, due to mutations in epitope P2B  and epitope P2C (aa 368 and 376) and subsequent conformational changes in the binding region, binding sites have shift to an alternative binding pocket which shares residues 391–393 in epitope P2D and residues 442, 443 and 444 in the surface-exposed P1C epitope within the P1 domain. Mutations in neighboring sites of the binding pocket within epitope P2C and P2D may likely change the number of H-bonds through residue-residue approximation constraints with adjacent amino acids. In Sydney 2012, similar binding pocket as early variants 1987–2002 was predicted. It can be due to a notable decrease in the accessibility of HBGA binding sites 391–393 in epitope P2D, and significant conformational changes in epitope P2C as a result of three new substitutions A368E, N373R, and T377A. Interestingly, in Sydney 2012 and 2015 variants, the flexibility of the protein was considerably reduced in P2A and P2D. It seems that T294 substitution in epitope P2A, and new E268H, R373N and A377T mutations in epitope P2D resulted in increased protein rigidity which can make the epitopes unsuitable for interacting with antibodies. Robust data exhibit altered antigenicity and HBGA binding over time in VLPs of major outbreak and pandemic GII.4 strains, and identify strain-specific potentially neutralizing antibodies in GII.4 NoVs [The binding specificity of the norovirus capsid protein to different HBGAs differs among the genotypes and genogroups , leadingI.4 NoVs . InteresHBGA blocking ability is suggested to be an important factor in achieving protective immunity against NoV 19,52) as neutralizing antibodies raised against NoV have been shown to block the binding of NoV VLPs to the synthetic HBGA receptor ,50,51. F as neutrThese data demonstrate the potentials of the identified linear epitopes P2B, P2C and P2D as blockade epitopes in generation of chimeric GII.4 VLPs, polyvalent synthetic and/or DNA-based peptide vaccines against NoV infection. Here, based on our findings, we suggest generating chimeric GII.4 VLPs harboring blocked epitopes from GII.4 variants with different HBGA binding pattern predicted with molecular docking. The use of such vaccines will allow broad coverage of the target NoV GII.4 variants as the most prevalent genotype in outbreaks. Previous studies indicated three loops  on the surface part of the NoV P particles , which cIn this work, we investigated surface-exposed linear antigenic epitope within NoV GII.4 capsid protein at sequence and structural level. Along with bioinformatics analysis, including molecular dynamic stimulation and molecular docking, the characteristic HBGA blocking rates in mice demonstrates the potential of the three identified linear epitopes P2B, P2C and P2D as blockade epitopes in generation broad reactive vaccines against NoV GII.4. The use of such vaccines will likely reduce the challenges associated with vaccine development against norovirus and allow broad coverage of new emergent variants.


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