More and more people pay attention to the printing speed and quality of 3D printing tools. In order to understand whether the 3D printing rehabilitation brace can play a role in the treatment and repair of joint trauma, we used 3D printing technology to print the rehabilitation brace and compared with the traditional rehabilitation brace. The printed parts were analyzed in detail. The experimental results prove that the rehabilitation braces made by the two methods can play a role in the repair of joint trauma. However, 3D printed rehabilitation braces can better meet the needs of patients with detailed patient data in application. The braces are more suitable, and their production speed is about 35% faster than traditional methods. Through the survey of patients and doctors, it is found that the satisfaction of patients and doctors with printed braces is above 89%, while the satisfaction with traditionally made braces is only about 60%. This shows that the rehabilitation brace based on the Internet of Things 3D printing technology has a more significant role in the treatment and repair of joint trauma, and the effect is better. With the continuous improvement of China's medical service system, rehabilitation medicine has been widely concerned . Sports Compared with the traditional processing and manufacturing technology in the past, 3D printing technology can not only realize “from scratch,” save a lot of time and labor, but also carry out “personalized manufacturing,” making the product unique . At presLiu Zheng thinks that the traditional rehabilitation brace manufacturing process is complex, and the accuracy is difficult to adapt to the patients and meet the needs of patients. Therefore, it is very important to use 3D printing technology to make rehabilitation support. 3D printing technology can print the rehabilitation support into a structure and shape suitable for patients according to the patient's situation. Qiang combined 3D printing technology with traditional rehabilitation technology, innovatively proposed the shape and appearance design of 3D technology in rehabilitation brace, introduced the difficulties and challenges faced in the application of 3D printing technology in practice, elaborated the important role of 3D printing in promoting the integration of medical and engineering technology, and highly praised the value of 3D printing technology in rehabilitation brace , 10. ZheIn this paper, through the establishment of 3D printing rehabilitation brace model, it is compared with the traditional rehabilitation brace in all aspects, to test whether it can play a role in joint trauma. Collect relevant experimental data to analyze the comparison results of the experimental data in detail. Finally, patient and expert reviews can conclude that the results supporting 3D printing repairs have high practical value.The Principle of Appropriateness. The principle of appropriateness refers to the fit between the supply chain management system based on the Internet of Things and the characteristics of the supply chain itself. As a new generation of information technology, the Internet of Things technology is regarded as the third wave of information technology and the key to grasp the competitive advantage of the information industry in the future. However, for the decision-makers in the supply chain, the realization of interests is the main goal, and the advanced technology should be treated calmly and objectively instead of blindly worshipping; only the supply chain management system which is consistent with the characteristics of the supply chain itself is the most reasonable and appropriate system. Any reasonable system must conform to the principle of cost and benefit. The construction of supply chain management system based on the Internet of Things technology must also conform to the principle of cost and benefit , from which the matching value The simulation parameters are calculated as follows:In this paper, based on the theoretical results of the Internet of Things 3D printing research, using the methods of literature, comparative research, mathematical statistics, logical analysis, and other methods, this paper analyzes the application of rehabilitation brace and the difference between 3D printing and conventional production and studies its application mode and characteristics.We investigated 100 doctors and 100 patients and asked them to score the rehabilitation brace. The score was divided into four stages, and the score was carried out according to the entropy method.Entropy method is a relatively objective evaluation index weight assignment method, which can effectively avoid the subjectivity of artificial scoring, and has high accuracy. But at the same time, this study also realized that the entropy method cannot directly reflect the knowledge, opinions, and experience judgment of experts and scholars, and the weight results may be contrary to the actual situation. Therefore, this paper uses AHP and entropy method to determine the weight coefficient of regional higher education evaluation index.Comprehensive Quantitative and Qualitative Analysis. Quantitative analysis is to analyze the data of the problem, using the intuitive and clear essence of mathematics to reflect the existence of the problem; qualitative analysis is to collect, read and sort out the relevant domestic and foreign research literature, and systematically summarize the relevant theoretical results. Some standards cannot be directly analyzed by quantitative method but can only be evaluated by qualitative analysis. The evaluation standard system is constructed by combining quantitative and qualitative analysis methods. At the same time, the calculation formula and evaluation standard of relevant standards are given.In this paper, Internet of Things, 3D printing, rehabilitation brace, etc., are used as keywords, and the starting and ending years are set as 2015–2019. The relevant literature is searched by CNKI, Baidu, and other network platforms, and the collected data are classified and sorted. 167 Chinese and foreign academic papers, 29 academic works, 34 newspapers and network materials, and 15 doctoral and master's theses related to this research are obtained. At the same time, through questionnaire survey and field interview, we collected the opinions of patients and doctors on 3D printing rehabilitation brace and traditional rehabilitation support.We have made statistics on the use effect of 3D printing rehabilitation brace (control group) and traditional rehabilitation support  by doctors and patients. The specific data statistics are shown in It can be seen from Figures We investigated the feelings of patients and doctors after using rehabilitation brace and counted their opinions on the use of 3D printing rehabilitation brace and traditional rehabilitation brace. The specific statistical data are shown in From Figures We have made statistics on the time required for 3D printing rehabilitation brace and traditional rehabilitation brace. Relatively speaking, in a small amount of cases, the time required for 3D printing and traditional rehabilitation brace is similar. However, in mass production, the production speed of 3D printing is extremely excellent. The specific data are shown in From Figures The production of rehabilitation brace requires a lot of materials and time, so it is very necessary to maintain a high success rate of brace fabrication. However, there is a certain risk of failure in both 3D printing and traditional production. Overall, the success rate of 3D printing is higher than that of traditional production. The specific data are shown in As can be seen from 3D printing based on virtual reality is based on the 3D CAD model created by 3D design software. The 3D digital model is divided into two-dimensional slicing models with several layers by the software. The contour information of these two-dimensional slice models is imported into the 3D printer to convert it into the moving track of the printing head. The special materials such as liquid, cell tissue, plastic, powder, etc., can be transformed into the moving track of the printing head. Finally, a solid body is formed by layer superposition and bonding by melt nozzle and laser beam.Through prototype design, production and debugging, and product modeling design, the design decision is implemented on the product. The process of joint rehabilitation brace product development was completed from investigation analysis, user research, demand acquisition, design decision, design, and development. Through the analysis of the special user group of rehabilitation support equipment, the reasonable user research method is selected, and the user research route in the design and development process of rehabilitation support equipment is summarized.In this paper, user interviews and user behavior observation methods are used to obtain user requirements. These methods are subjective. In the follow-up research process, we will use physiological signal measurement equipment, behavior analysis model analysis tools, product user test, and other quantitative attribute tools and research methods to conduct design research, information acquisition, and product verification, so as to improve the quality of user research.


The mediobasal hypothalamus (MBH) shapes the neural regulation of glucostasis by 5′-AMP-activated protein kinase (AMPK)-dependent mechanisms. Yet, the neurochemical identity and neuroanatomical distribution of MBH neurons that express glucoprivic-sensitive AMPK remain unclear. The neurotransmitters γ-aminobutyric acid (GABA) and nitric oxide (NO) act within the MBH to correspondingly inhibit or stimulate glucose counter-regulation. The current review highlights recent findings that GABA and NO, neurons located in the ventromedial hypothalamic nucleus (VMN), a distinct important element of the MBH, are direct targets of noradrenergic regulatory signaling, and thereby, likely operate under the control of hindbrain metabolic-sensory neurons. The ovarian hormone estradiol acts within the VMN to govern energy homeostasis. Discussed here is current evidence that estradiol regulates GABA and NO nerve cell receptivity to norepinephrine and moreover, controls the noradrenergic regulation of AMPK activity in each cell type. Future gains in insight on mechanisms underpinning estradiol’s impact on neurotransmitter communication between the hindbrain and hypothalamic AMPKergic neurons are expected to disclose viable new molecular targets for the therapeutic simulation of hormonal enhancement of neuro-metabolic stability during circumstances of diminished endogenous estrogen secretion or glucose dysregulation. Introduction: Insulin-induced hypoglycemia (IIH) is a relentless, worrisome complication of the meticulous therapeutic management of type I and long-term type II diabetes mellitus patients, and is a major impediment to optimal glycemic control. IIH poses a risk of neurological dysfunction and injury as decrements in glucose supply, the primary fuel source to the brain, impede vital high-energy-demand nerve cell functions, including the maintenance of transmembrane electrolyte gradients. Hypoglycemia-associated neuro-glucopenia triggers counteractive, autonomic, neuroendocrine, and behavioral responses that heighten circulating glucose levels. The ventromedial hypothalamic nucleus (VMN), a distinct unique component of the mediobasal hypothalamus (MBH) and a key element of the brain’s gluco-regulatory network, processes nutrient, endocrine, and neurochemical cues on a metabolic status to control glucose counter-regulation -1H-pyrazole dihydrochloride (MPP) or ERβ antagonist 4-pyrimidin-3-yl]phenol (PHTPP) implicate these estrogen receptors (ERs) in forebrain mechanisms that govern VMN nNOS and GAD65/67 protein responses to hypoglycemia [gy fuels . In the gy fuels , as wellgy fuels . In thatgy fuels ,18,28. Ogy fuels . Documenglycemia .Estrogen regulation of noradrenergic input to VMN gluco-regulatory neurons. VMN NO and GABA neurons are presumptive objects of NE and estradiol action as each cell type expresses ERα, ERβ, and the transmembrane G protein-coupled estrogen receptor GPER/GPR30 proteins [1-AR protein expression in nitrergic (up-regulated) versus GABAergic (down-regulated) neurons, responses that are respectively mediated by ERβ alone versus ERα and ERβ signaling. Moreover, NO and GABA neuron β1-AR profiles are decreased during hypoglycemia by ER-dependent versus -independent mechanisms. We conducted studies to address the premise that estradiol may modulate NO and/or GAD neuron receptivity to NE. Using pharmacological and high-resolution microdissection tools in a characterized OVX, plus an estradiol replacement model in which plasma estradiol levels are reinstated within the physiological range [65/67-immunoreactive (-ir) – neurons for nerve cell type-specific measurements of α1-AR, α2-AR, and β1-AR protein expression. The study outcomes provide unique proof that both ERs up-regulate basal VMN nNOS, but not GAD protein profiles, and that NE suppression or enhancement of nNOS and GAD expression was, respectively, ER-independent or -dependent. In both neuron types, VMN ERβ activity inhibited baseline α1- and/or α2-AR protein expression, but ERα and -β signaling was paradoxically crucial for the noradrenergic up-regulation of α2-AR. This work importantly documents distinctive ERα and ERβ actions on the metabolic transmitter and AR protein expression in VMN nitrergic versus GABAergic neurons. The interesting observation that ER actions varied in the presence versus absence of NE bolster the novel concept that NO and GABA neurons are substrates for critical estradiol and noradrenergic regulatory interaction.proteins . ERs regal range ,33,34, wal range . The resEstrogen regulation of VMN gluco-regulatory nerve cell AMPK reactivity to NE. Using the same methodological approach for the selective procurement of VMN NO and GABA neurons and techniques for the administration of NE to the VMN [ the VMN , our lab the VMN .Noradrenergic regulation of VMN astrocyte glycogen metabolism. Brain astrocytes store glycogen as a vital metabolic fuel reserve; this depot exhibits a dynamic turnover during normal brain activity and metabolic homeostasis, and is a critical source of lactate equivalents during heightened neural activity or brain glucose deficiency [l-lactate for trafficking to neurons [1-AR, α2-AR, and β1-AR proteins, and that hypoglycemic patterns of AR variant expression are controlled by ERs [ficiency . Circula neurons  by astro neurons . Glycoge neurons , and inh neurons ,42. We f neurons . These dd by ERs , it is pNoradrenergic regulation of VMN astrocyte AMPK activity is controlled by estradiol. Recent studies indicate that the noradrenergic regulation of VMN energy sensor function is not exclusive to the nerve cell compartment. We carried out work involving the administration of the selective CA neurotoxin 6-hydroxydopamine (6-OHDA) into the caudal fourth ventricle of estradiol–versus non-hormone-replaced OVX female rats to address the premise that VMN astrocytes express AMPK, and that the reactivity of this sensor to hypoglycemia is estradiol- and/or hindbrain NE-dependent [1-AR protein expression, as well as in GS and GP profiles. Collectively, our research implicates AMPK in the noradrenergic control of VMN astrocyte glycogen synthesis and metabolism. Distinguishing effects of NE on distinctive VMN astrocyte adrenergic and estrogen receptor variants support the possibility that AMPK regulation of glycogen metabolism in the presence of NE may be mediated, in part, by one or more receptors characterized in our work by sensitivity to this catecholamine.ependent . Individependent . Data reependent  show tha1-AR signaling and that this receptor mediates acclimated energy sensor responses to RIIH [icv delivery of the α1-AR reverse-agonist prazocin (PRZ) or vehicle ahead of induction of a single versus final episode  of hypoglycemia. VMN neurons identified by immunocytochemical labeling as nNOS- or GAD65/67-ir were procured by laser-catapult microdissection for Western blot analysis of nNOS or GAD profiles as well as AMPK, pAMPK, α1-AR, α2-AR, and β1-AR protein expression. The study results show that the hypoglycemic activation of VMN NO and GABA AMPK involves the α1-AR signal up-regulation of pAMPK profiles in nitrergic neurons or the down-regulation of the total AMPK protein in GABAergic nerve cells. The outcomes also reveal that antecedent hypoglycemia represses sensor activation in both cell groups during re-exposure to hypoglycemia. In nitrergic neurons, an acclimated sensor function may involve the loss of α1-AR stimulation of AMPK activity as PRZ did not modify sensor activation from control range during RIIH. Prior bouts of hypoglycemia may elicit, in the absence of α1-AR signaling to these cells, α1-AR-independent mechanisms that protect cellular energy balance and thus stabilize AMPK activity, during ensuing hypoglycemia. On the other hand, α1-AR-reliant and -non-reliant mechanisms may converge on AMPK, such that the latter remains operational during the pharmacological blockade of α1-AR. Indeed, AMPK is controlled by various endocrine and neurochemical signals alongside nutrient cues. Meanwhile, GABAergic neurons exhibit a switch from α1-AR stimulation to inhibition of AMPK activity during single versus serial exposure to hypoglycemia; additional work is needed to elucidate mechanisms that underlie this change in direction of α1-AR influence on AMPK activity state. Evidence that NO and GABA neurons express α1-AR supports the notion that PRZ treatment effects involve the direct action of this drug on each nerve cell type. It is noted that as this study involved icv PRZ delivery, the blockade of upstream α1-AR that controls afferent input may also contribute to the VMN neuron responses to this drug. We theorize that demonstrable adjustment from positive to negative α1-AR influence on GABA AMPK activity may involve downstream receptor and/or signal transduction pathway mechanisms. Yet, it is plausible that antecedent hypoglycemia may alter the increased α1-AR-mediated inhibitory neurochemical cues to VMN GABA AMPK. Our results indicate that VMN NO and GABA neurons exhibit common and dissimilar AR protein responses to hypoglycemia, as well as discrepant AR acclimation to RIIH. Both neuron populations showed α1-AR-driven augmentation of α1-AR protein during acute hypoglycemia, but this up-regulated response persisted in nitrergic, but not GABA cells during RIIH. Expression of α2-AR protein was refractory to acute hypoglycemia in both cell groups, but NO neurons showed an α1-AR-dependent increase in this profile during chronic hypoglycemia. Interestingly, NO (up-regulated) and GABA (down-regulated) nerve cell β1-AR expression diverged during both acute and recurring hypoglycemia. Collectively, these outcomes imply that antecedent hypoglycemia may augment noradrenergic input to nitrergic neurons as a consequence of increased α2-AR expression. Alternatively, GABA nerve cell habituation to RIIH may involve diminished α1-AR signaling. Nevertheless, the likelihood that post-receptor signal transduction pathways may acclimate to recurring hypoglycemia, independent of changes in receptor expression, cannot be disregarded.Adrenergic receptor regulation of VMN gluco-regulatory nerve cell AMPK during acute versus recurring insulin-induced hypoglycemia (RIIH). RIIH can trigger hypoglycemia-associated autonomic failure in male type 1 diabetes patient, a pathophysiological mal-adaptation that manifests as diminished hypoglycemic awareness and defective glucose counter-regulation ,45. In v to RIIH . Our resEstrogen regulation of hindbrain metabolic-sensory noradrenergic neuron energy metabolic adaptation to RIIH. Evidence that estradiol-replaced OVX female rats exhibit no change in hypothalamic neuron transcriptional activation patterns and counter-regulatory hormone secretion between acute versus chronic exposure to hypoglycemia [glycemia ,27 suppoglycemia . Our resglycemia . InteresConclusions: Growing proof of the physiological relevance of hindbrain AMPK to the neural control of energy and glucose homeostasis has generated interest in the characterization of signal types, e.g., nutrients, hormones, and neurotransmitters that converge to regulate the net activity of this sensor and neurochemical mechanisms of communication of activation status. Our studies demonstrate that hindbrain AMPK provides regulatory input to VMN AMPK, a concept that challenges the current view that the metabolic sensors in various neural structures provide autonomous input to downstream integrative and pre-motor elements of the neural gluco-regulatory circuitry. Departure from the norm of exclusive focus on hypothalamic, hindbrain, or portal vein sensors is expected to yield a unitary approach that addresses the novel prospect of sensor interaction and cooperation. This ‘inclusionist’ perspective represents a paradigm shift from the customary view of the hypothalamus as a self-contained, singular source of sensory indicators of cellular metabolic imbalance. We predict research outcomes which highlight that metabolic sensors assimilate unique combinations of metabolic stimuli, e.g., they are not inter-changeable, and that neural communication between sensors is a mechanism for disseminating distinctive signals and ultimately, a collective assessment of the body’s metabolic status. Hindbrain and hypothalamic metabolic-sensory neurons may plausibly operate as unique substrates for the as-yet incompletely understood protective actions of estradiol with respect to the defense of cellular and systemic energy homeostasis. Further insights concerning the molecular mechanisms of such hormone action, which will require the implementation of complimentary molecular, genetic, and electrophysiological approaches, will undoubtedly identify potential therapeutic targets for the enhancement of a positive energy balance during states of estrogen deficiency in women, and for the alleviation of maladaptive neural acclimation to recurring exposure to bio-energetic stressors.


The Ganz’ periacetabular osteotomy (PAO) consists of four technically challenging osteotomies (OT), namely, supraacetabular (saOT), pubic (pOT), ischial (iOT), and retroacetabular OT (raOT).We performed a proof of concept study to test (1) the feasibility of augmented reality (AR) guidance for PAO, (2) precision of the OTs guided by AR compared to the freehand technique performed by an experienced PAO surgeon, and (3) the effect of AR on performance depending on experience.A 3D preoperative plan of a PAO was created from segmented computed tomography (CT) data of an anatomic plastic pelvis model (PPM). The plan was then embedded in a software application for an AR head-mounted device. Soft tissue coverage was imitated using foam rubber. The 3D plan was then registered onto the PPM using an anatomical landmark registration. Two surgeons (one experienced and one novice PAO surgeon) each performed 15 freehand (FH) and 15 AR-guided PAOs. The starting point distances and angulation between the planned and executed OT planes for the FH and the AR-guided PAOs were compared in post-intervention CTs.p = 0.0027). AR guidance increased the accuracy of the performance of the novice surgeon for iOT (p = 0.03). An intraarticular osteotomy performed by the novice surgeon with the FH technique could be observed only once.AR guidance did not affect the performance of the expert surgeon in terms of the mean differences between the planned and executed starting points, but the raOT angle was more accurate as compared to FH PAO (AR guidance of osteotomies for PAOs is feasible and seems to increase accuracy. The effect is more accentuated for less-experienced surgeons.This is the first proof of concept study documenting the feasibility of AR guidance for PAO. Based on these findings, further studies are essential for elaborating on the potential merits of AR guidance to increase the accuracy of complex surgical procedures. Periacetabular osteotomy (PAO) is a well-established procedure for the treatment of hip dysplasia , 2 and fTraditional PAO techniques are based on orientation on the bony anatomy alone with or without the addition of intraoperative fluoroscopy as help in orientation to execute and verify the accuracy of the osteotomies. However, fluoroscopy may add to the overall surgical time, as well as increase the patient’s and operating room personnel’s exposure to radiation. Furthermore, it is not sufficient for the control of certain osteotomies due to its two-dimensional nature. Therefore, more sophisticated techniques for the navigation of PAO have been suggested and investigated, including optical tracking systems –10 and pTherefore, there is a need for a simple and radiation-free system to safely perform all the four types of 3D planned osteotomies with greater accuracy. Augmented reality (AR) is an evolving technology with the potential to overcome this challenge. AR can be described as the real-time integration of computer-generated information in the user’s environment. In medicine specifically, AR technology is capable of superimposing a preoperative plan for the user to view. This concept has already aroused interest in the field of medicine since the 1990s , but theWe performed a proof of concept study to find the potential of AR for the guidance of the surgeon while performing PAOs with oscillating saw and OT-chisels using an AR-HMD.Is it feasible to execute a 3D preoperative planning of a PAO with AR guidance?Is there a difference in accuracy between AR-guided PAO as against the freehand (FH) technique?Is there a difference between the performance of an experienced PAO surgeon and a novice both assisted by AR guidance?The study questions were the following:Sawbone models, consisting of the femur and pelvis, manufactured by Sawbones® , were manually embedded in synthetic foam  to simulate skin and soft tissue Fig. a, b.FigPrior to the AR-guided procedure, a Computed Tomography (CT) scan of the sawbones was acquired with a 64-detector row scanner  with a tube voltage of 120 kV and a slice thickness of 1 mm. Then 3D surface models of pelvis and femur were extracted from the CT data using commercially available segmentation software . Thereafter, the 3D models were imported into the preoperative planning software CASPA  to preoperatively simulate the PAO osteotomies on the 3D bone models  and another hip consultant with no PAO experience  performed 15 AR-guided and 15 FH PAOs each on the sawbone models according to Ganz  for the FH vs. 1.5 mm [± 1.0] in the AR-guided PAO. In the pOT, the difference was 2.1 mm [± 1.1] in the FH vs. 3.5 mm [± 2.3] in the AR PAO. In the iOT, the difference was 2.4 mm [± 3.1] in the FH vs. 2.7 mm [± 1.7] in the AR-guided PAO. Lastly, in the raOT, the difference was 2.0 mm [± 1.7] in the FH vs. 1.8 mm [± 1.6] in the AR-guided PAO. The differences did not reach statistical significance.p = 0.0027). No intraarticular osteotomies or interruption of the posterior column occurred in any of OTs performed by the experienced surgeonWith AR navigation, the raOT angle had a significantly better correlation with the pre-intervention plan than in the FH group . The retroacetabular angle was without significant difference between FH and AR-guided PAOs: 8.8° [± 3.7] vs. 7.2° [± 3.2], p = 0.554), respectively. One intraarticular osteotomy was encountered in the FH group.The novice surgeon achieved the following mean distances between planned and the executed starting points: In the saOT, the difference was 2.4 mm [± 1.3] in the FH vs. 1.5 mm [± 1.6] in the AR PAO. In the pOT, the difference was 6.1 mm [± 2.7] in the FH vs. 4.5 mm [± 3.6] in the AR-guided PAO. In the iOT, the difference was 3.2 mm [± 3.0] in the FH vs. 1.3 mm [± 1.6] in the AR-guided PAO. In the raOT, the difference was 2.6 mm [± 1.5] in the FH vs. 1.8 mm [± 1.9] in the AR-guided PAO. The iOT was the only osteotomy showing a statistical difference (Detailed information is provided in Table While the PAO technique has been well-described since its introduction , it remaThis proof of concept study is the first to prove the feasibility and high accuracy of AR-guided PAOs and investigate the stability of the results in the dependence of different levels of surgical experience. Therefore, the answer to the first question posed in this study is answered in the affirmative. Even though no significant differences were apparent for the OT starting points for the experienced PAO surgeon, the most difficult ischial OT, however, was significantly more accurate with AR guidance for the novice surgeon. Interestingly, the angle of the AR-guided raOT was only more precise for the PAO surgeon and not different for the novice surgeon when compared to FH. This observation can be interpreted to have statistical but not clinical relevance because a deviation of 11° from a correct starting point and along a short path does not necessarily result either in an interruption of the posterior column or an intraarticular OT. While the experienced PAO surgeon did not make a malpositioned OT reaching the posterior column or the joint, the novice surgeon, while working FH, i.e., without AR guidance, performed one intraarticular osteotomy. Additionally, the ischial OT, considered technically as the most challenging, had a significantly higher correlation with the pre-intervention plan with AR guidance compared to FH for the novice surgeon. Therefore, the second and the third study questions are not answered unequivocally in the affirmative or negative. However, the results were discussed in the section above.Even though the proof of concept of AR-guided OTs for PAOs has now been established, further alternatives need to be discussed to decide whether further research and development of such technologies for bringing them into clinical practice is justified by their potential.To improve the accuracy and reliability of osteotomy, several supplementary tools have been employed, including navigation systems with fluoroscopy, with infrared tracking, and with patient-specific guides –12. The In our opinion, the guiding system with the AR technique that we tested has very great potential because of its three advantages. First, OT poses a 3D problem and AR technology facilitates making a 3D plan to solve it and execute it in 3D, taking the joint loading forces into account. Second, the guidance is directly in the surgeon’s field of vision and does not need the surgeon to take his sight away from the incision. Third, due to the overlay of the osteotomies and also the guidance of the chisel, it is possible to guide the most important osteotomy in PAO, namely, the first ischial partial osteotomy.Therefore, we believe that further research and development of AR-guided OTs are needed to capitalize on its potential merits as a cost-effective and more intuitive navigation method.This proof of concept study does not claim for the direct application of the technology in clinical care because the limitation of the presented setup needs consideration: Certainly, the surgical setup with the sawbone pelvis/femur models covered with foam does not resemble the in vivo situation. Therefore, the next step is the execution of cadaver studies. Additionally, in this study, only the osteotomies were AR-guided, and the feasibility of navigating the reorientation and fixation of the acetabulum in a new, corrected position was not investigated. However, this study could be a new platform to establish other merits of AR guidance. As the surgeons performed the AR-guided and FH PAOs alternately and in one setting, there probably was a memory effect, especially for the FH group. But despite the memory effect that may have helped in the FH group, the osteotomies in the AR group were more reliable.This is the first proof of concept study documenting the feasibility and reporting on the accuracy of AR-guided osteotomies in PAO. Our study showed that the holographic surgical navigation of the PAO surgery is feasible and precise in a realistic pelvis-hip model using the Hololens AR headset. The study shows that AR-guided OTs are safe to perform, showed a high correlation with the preoperative plan, and were as precise as osteotomies performed in an FH fashion by an experienced PAO surgeon.


Nelumbo nucifera) caused by the nematode Hirschmanniella diversa is an emerging agronomic problem. In this study, the authors documented the early infection processes of H. diversa in the apices of young rhizomes of Indian lotus by electron microscopy analysis using an artificial inoculation method. Nematodes were attracted to young rhizome apices, invading them via narrow indentations by 4.5 hr after inoculation. Host cells adjacent to the cavity around the invading nematodes were absent and appeared to have disintegrated during infestation. Following contact with the nematodes, host cell walls and cellular contents became electron-dense and less defined, likely due to digestive enzymes secreted by the invading nematodes. Nematodes invaded to a depth of about 1 mm by 24 hr after inoculation, but did not penetrate the plant any further vertically, similar to the observation of browning in mature rhizomes in the field. The authors propose that the invasion sites of young rhizome apices become blackish-brown blotches as rhizomes mature in the field due to oxidation.Browning rhizome  in Indian lotus ( Nelumbo nucifera Gaertn), cultivated in Japan since ancient times, includes both the flowering lotus, with high ornamental value, and the edible lotus, the rhizomes of which are consumed as a vegetable  Luc and Goodey, 1964, H. imamuri, and H. diversa (H. oryzae and H. imamuri parasitize rice (Oryza sativa L.) roots and cause root rot  were excised and placed in the center of 3.0-cm-diameter petri dishes containing 4.0 ml of 0.4% water agar, with the apex in contact with the bottom of the dish . ApproxiInoculated (or uninoculated control) specimens were immersed in 2.5% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.2, for 12 to 72 hr at 4°C. Specimens were sectioned into 90-μm-thick slices with a microslicer  and observed utilizing a light microscope.Specimens inoculated with nematodes were immersed in 2.5% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.2, for 12 to 24 hr at 4°C. Postfixation with 1% osmium tetroxide was performed in the same buffer for 12 hr at 4°C. Specimens were dehydrated in a graded series of ethanol: 50, 70, 80, 90, and 100%. Thereafter, ethanol was exchanged with 100% t-butyl alcohol (2-methyl 2-propanol), and specimens were freeze-dried . The specimens were coated with approximately 8 nm of platinum by ion spatter  and observed by field emission SEM  at 25 kV. To observe the inner tissues of rhizomes that had been invaded by nematodes, specimens were vertically cut into two pieces with a razor blade. The pieces were coated with platinum and observed by SEM as described above.Specimens inoculated with nematodes were immersed in 2.5% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.2, for 24 hr at 4°C and then postfixed in 1% osmium tetroxide in the same buffer at 4°C for 12 hr. The specimens were rinsed with distilled water for 10 min and dehydrated in an ethanol series: 50, 70, 80, 90, and 100%. Absolute 100% ethanol was then replaced with QY-1 , and specimens were embedded in Quetol 651 resin mixture . For light microscopy, 0.5-μm-thick sections were cut using glass knives and stained with 1% toluidine blue in 1% sodium borate solution. For transmission electron microscopy, ultrathin sections were cut from resin blocks with an ultramicrotome  using a diamond knife. Ultrathin sections were collected on 200 × 75mesh Formvar-coated grids, stained with saturated uranyl acetate in distilled water for 10 min, and then stained with lead citrate for 10 min. A Hitachi H-7650 transmission electron microscope was used to observe the interfaces between invading nematodes and host cells.Nematodes began to move towards the apices of young rhizomes when nematode suspensions were poured into the surrounding water agar in petri dishes. After 4.5 hr of inoculation, many nematodes gathered around the surface of the apices and invaded them . After tWe observed many invading nematodes in the epidermis and in a cavity where a few layers of cortical cells had disappeared at 24 hr after inoculation . NematodNematodes penetrating the epidermis were observed 4.5 hr after inoculation . We obseSEM images of cross sections of nematodes reached a depth of at most 1 mm by 24 hr after inoculation . We obseArtifacts generated by freezing in liquid nitrogen during sampling included the collapse of the cytoplasm and fragmentation of plasma membranes in uninoculated apices . HoweverCell walls facing the cavities were degraded and sometimes completely absent . RemainiH. diversa in the apices of young rhizomes of Indian lotus by use of an artificial inoculation method. Most of the nematodes were attracted to the apices of young rhizomes within a short period and invaded the rhizomes. Radopholus oryzae (H. oryzae) nematodes invaded roots of rice plants through previous nematode entry points. Such an invasion mode was also observed in the root-knot nematode with light microscopy and SEM  “salivation zone” was formed around the stylet tip, and granules from the dorsal esophageal gland flowed down the gland duct to the ampulla, which opens just behind the stylet knob during salivation. In our study, we observed with TEM a layer of fine granular materials, about 2 μm in thickness, close to the nematode. We propose that this region is the salivation zone reported for P. penetrans at the cell penetration site  invades the roots of rice plants and lives in the cavities between the parenchyma lamellae of the cortex. Hirschmanniella diversa lives in root cortex cells for almost all their lives; in contrast, when invading the apices of young rhizomes, the nematode does not penetrate beyond a depth of about 1 mm. We presume that this difference is due to different conditions; the cell walls of roots were markedly thinner and softer than those of young rhizomes, and the volumes of cortical cells were more than eight times larger , corresponding to the depth at which invading nematodes stopped progressing in the apices of young rhizomes in this study. In addition, nematodes can burrow between the epidermis and cortex in mature rhizomes, but cannot invade cortical cells . Therefore, the blackish-brown blotches observed in mature rhizomes in the field appear to arise from the invasion sites at the young rhizome apices and change color due to oxidation . We sugg


After publication of this supplement , it was The name has now been corrected in the supplement and, furthermore, can be found in the author list of this correction.For reference, please find the body of the abstract in question below:Introduction: Catecholamine hypo-responsiveness is a hallmark of sepsis, but its pathogenesis remains elusive. The adrenergic pathway, from catecholamine binding to the adrenergic receptor (AR) to the final physiological effect is a multistep process. Knowledge about protein expression and localisation of different constituents of the adrenergic signalling in sepsis, including adrenergic receptors themselves, is sparse.Objectives: Using our well-characterised 72 h fluid-resuscitated rat model of faecal peritonitis (1), where accurate prognostication can be made as early as 6 h, we analysed expression and membrane localisation of the b2-AR protein in heart tissue at early (6 h) and late (24 h) timepoints.Methods: Sepsis was induced in male Wistar rats by i.p. injection of faecal slurry under general anaesthesia. The animals were then recovered and i.v. fluid resuscitation was started 2 h after sepsis induction. Sham-operated rats (SH) were treated identically but did not receive the slurry. At 6 h, an echo-measured heart rate cut-off of 460 bpm was used to classify animals into predicted survivors (SR) and non-survivors (NSR). Rats were culled at either 6 or 24 h with heart tissue collected and snap frozen in liquid nitrogen. To study b2-AR expression, either protein lysates of whole heart tissue or membrane fractions were prepared and analysed by Western Blot with antibodies against b2-AR and PFK or Caveolin 3. Specific signals were nalysed in Image Studio Lite 5.2. Results are presented as mean ± SE and considered significant at p < 0.05 (Student’s t-test).Results: The level of expression of b2-AR myocardial protein expression did not change in survivors but declined in non-survivors being significantly lower than survivors at 24 h (Table 1). Representative results of membrane localisation of b2-AR within different membrane fractions are presented in Fig. 1. Lipid rafts were observed in only two of the 12 gradient fractions, namely fractions 4 and 5. This was confirmed by cholesterol distribution and caveolin 3 staining. In sham operated animals and in predicted survivors at 24 h, b2-AR appeared strongly in fraction 5, associated with the lipid rafts. In predicted non-survivors, b2-AR was not detected in fractions 4 and 5 either at 6 or 24 h post-sepsis.Conclusion: More severe sepsis, with corresponding poor prognosis, is accompanied by increasing catecholamine hyporesponsiveness. One cause could be a decrease in adrenergic receptor protein expression. Localisation of the b2-AR within lipid rafts is also a prerequisite for their normal functioning. In predicted non-survivors myocardial b2-AR was dissociated from the lipid rafts. Further studies are necessary to identify strategies that restore adrenergic signalling in sepsis.


The optimized batch was further loaded with RSV and evaluated for in vitro and ex vivo permeation and for in vivo hepatotoxicity. The developed ITT-SNEDDS exhibited globule size below 300 nm, up to 272.27 ± 7.12 mcg/cm2.h and 61.27 ± 2.83% of steady-state flux (JSS) and permeability % respectively. Optimum formulation consisted of 0.15 g oil mixture, 0.6 g of surfactant (Labrasol), and 0.250 g co-surfactant (Transcutol). Permeability studies confirmed the enhanced permeation percentage of ITT (40.77 ± 1.18%), and RSV (29.94 ± 2.02%) from optimized formulation, with enhanced steady-state flux (JSS). In vivo studies demonstrated the superior hepatoprotective activity of optimized formulation compared to a different drug formulations and marketed product. Therefore, RVS loaded ITT-SNEDDS might be a successful strategy for acne management with improved action, and minimum side effects.Acne Vulgaris is one of the most common chronic inflammatory skin disorders that affect majority of teen-agers worldwide. Isotretinoin (ITT) is the drug of choice in the management of acne, but, it suffers from serious side-effects including hepatotoxicity, and some psychological disturbances following its oral intake. The objective of this study was to develop and optimize ITT loaded nanoemulsions (ITT-SNEDDS) and to incorporate resveratrol (RSV)in optimum formulation to decrease ITT side effects The ITT solubility was first tested in various essential oils, surfactants, and co-surfactants to select the essential nanoemulsion ingredients. Mixture design was applied to study the effect of independent variables and their interactions on the selected dependent responses. The developed ITT-SNEDDS were characterized for their globule size and  Food and Drug Administration) in 1982 to treat severe acne , chemically 3,4,5-trihydroxystilbene, available in two different forms,  Baxter, . RSV has Baxter, , anti-in Baxter, , cardio- Baxter, , anti-ox Baxter, , and ant Baxter,  effects. Baxter, . RSV has Baxter, . Drug-meThe potential application of RSV as a therapeutic agent has been limited due to photosensitivity and partial intestinal absorption, thus decreasing bioavailability and causing extensive elimination and catabolism through ABC transporters , on dilution, produce nano-sized transparent systems called nanoemulsions . Essential oils were obtained from Hayward, CA, USA. RSV, was procured from Water Import and Export Co., Ltd (China). Triglycerides, propylene glycol monolaurate, and glycerol monooleate, Labrasol, and transcutol were generously gifted by Gattefosse (France). All other reagents and chemicals used were of analytical grades.2.2.The solubility of ITT in various oils, surfactants, and co-surfactants was determined. An excess amount of ITT was dissolved in about 2 ml of each vehicle individually. The mixture was then shaken on a water bath  for 24 h, and the temperature was maintained at 37 ± 0.5° C. Once the equilibrium was attained, the resulting mixture was centrifuged  at 8° C for 1 h at 15,000 rpm. The formed supernatant was then diluted with a suitable quantity of methanol and analyzed quantitatively for ITT concentration using a UV-Visible spectrophotometer at 362 nm. The experiment was repeated three times, and the values were noted as mean ± standard deviation.2.3.Nine nanoemulsion formulations were designed with different proportions of the selected oils, surfactant, and co-surfactant using different concentrations of ITT (20–60 mg) in the first formulation only, then ITT concentrations from 20 to 40 were used in formulations 2 and 3. After that, the drug concentration that yielded the lowest globule size in formulations from 1 to 3 was used in the rest of the formulations. The composition of the 9 formulations was presented in The SNEDDs were prepared as follows; ITT was added to the oil phase mixture; subsequently, a mixture of surfactant and co-surfactant was added. The formed mixture was further sonicated using Ultrasonic Processors . Finally, the formed dispersion was added to distilled water drop by drop, and nanoemulsion was formed spontaneously on mixing at room temperatures  , surfactant [labrasol] (X2), and cosurfactant [transcutol] , steady-state inflow (JSS) (Y2), and penetrability percentage (Y3) was studied  has proven a proficient tool in optimizing the various parameters in a multifarious process to obtain the desired responses; thus, it has been applied to formulate ITT-SNEDDS. Among several models, the mixture design was selected to study the interaction of independent variables. Three factors were selected, namely, percentages of oil mixture [tea tree oil/baobab oil in ratio 1:1]  with multiple response optimization and RSM. Optimized formulation was selected based on the constraints of selected responses.2.8.After the optimization of experimental runs, an optimized formulation was prepared according to the concentrations proposed by the software, and its various parameters were evaluated. The rationale for the selected experimental design was validated by comparing the experimental values with predicted values and calculating relative error (%). The following equation was used to calculate relative error  was utilized. First, the dialysis membrane was soaked in phosphate buffer (pH 7.4) for 15 min at 25 ± 0.5° C. The receptor compartment was filled with phosphate buffer, and glass tubes covered with the dialysis membranes and filled with certain volume of the optimized formulation equivalent to 5 mg of each drug were placed in the medium with continuous rotations (75 rpm). The temperature was maintained at 37 ± 0.5° C throughout the experiment. Samples were withdrawn at various time intervals , and ITT and RSV concentrations were analyzed at 362 and 299 nm, respectively.In vitro, drug release studies were performed to calculate the amount of ITT and RSV released for the optimized formulation according to a previously described method . Low hea2.10.The permeation study for optimum formulation was carried out as previously described in Section 2.6.2, however the permeation parameters for both ITT and RSV were calculated.2.11.Thirty-six male adult mice weighing 20.5 ± 1.5g were selected and adapted for seven days before the study. The animals were divided into six groups consisting of six animals in each group. Animal groups received the following treatments; group 1 received normal saline orally and served as negative control, while group 2 received oral aqueous dispersion of ITT. Moreover, group 3 received topical aqueous dispersion of ITT, group 4 received topical optimized ITT-SNEDDS formulation, group 5 received optimized topical ITT-RSV-SNEDDS and finally group 6 received the marketed topical cream. The dose of ITT in tested formulations was 5 mg/kg, and the treatment period was 14 days. The tested rats were maintained at temperature of 25 ± 0.5 °C and 30 − 70% of relative humidity and subjected to 12 h darkness and 12 h light cycle. At the end of the study period, animals were sacrificed with cervical dislocation, blood samples were instantly withdrawn and finally dysfunction of the liver was estimated by measuring AST, ALT, superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px).2.12.in vivo results obtained were expressed as mean ± S.D and analyzed using Statistical Package for the Social Science, version 26 . One-way ANOVA was followed by a post-hoc Tukey test to determine significant differences between quantitative variables. p Value <.05 was noted as statistically significant.The 3.3.1.The solubility of ITT in assorted vehicles is depicted in Also, ITT exhibited highest solubility with transcutol co-emulsifier (24.34 ± 14.28 µg/ml). Transcutol is known for its great miscibility in polar and non-polar solvents, as well as its competence to increase the absorption and degree of permeability , maximum JSS (Y2), and high permeability percent (Y3)  and the sequential sum of squares (Type-I), different models were selected , the model reduction could have been required. The Lack of Fit F-value (0.97) was non-significant, with only a 1.25% chance that the Lack of Fit F-value this large could occur due to noise.The selected model was statistically significant, F = 41.64, Furthermore, Adeq Precision measures the signal to noise ratio. In general, a ratio greater than 4 is desirable. The obtained ratio (17.910) indicated an adequate signal to navigate the design space  was affected significantly by the synergistic effect of liner mixture (p < .0001)  = 481.52 ×1 + 110.81×2 + 260.11×3Globule size  and Lack of Fit F-value (0.7014) for Response (Y2) confirmed that the selected model is significant and the lack of fit is not significant, relative to the pure error. Adeq Precision (17.910) indicated an adequate signal to navigate the design space. Based on ANOVA results  factors affected Response II antagonistically at a p value <.0001, and all remaining factors showed a synergistic effect. The generated polynomial equation shows that all these selected variables significantly affected JSS. Mixture design contour plots and 3D plots were similar to those for Y1. As discussed in the previous sections, higher concentrations of surfactants and oil mixture account for elevated and decreased JSS, respectively.The model F value  = − 12298.76 ×1 + 3087.44 ×2 − 494.73 ×3 + 17529.79 X1X + 25207.50 X1X3 − 4205.41 X2X3 − 21726.95 X1X2X3 +17752.15 X1X2 (X1−X2) + 13269.82 X1X3 (X1−X3) − 5101.24 X2X3 (X2−X3)J3)% permeated . The selected three components are depicted in the corners of the triangle, and 3 D contour plots were drawn to study interaction effects. The fitted linear special regression equation was generated, as shown below.Adequate Precision for the linear model of YFinal Equations with Coded Factors1 + 69.94 ×2 + +45.46 ×3% Permeated = 5.02 ×The above results showed a higher significant effect of surfactant concentration compared to other variables. As the higher surfactant proportions can reduce the globule size, this can enhance the permeation percentage  and 62.5% for globule size, Jss and permeation percent, respectively.The process was optimized by setting the goals for every response and simultaneously applying the Global desirability function (D). Based on these criteria, the desirability plot was generated with a D value of 0.918 . In conc3.4.SS, and percentage permeation. Relative error was calculated by comparing the predicted and practical values, as shown in In the end, the experimental design was further validated to confirm accuracy. Optimized formulation was prepared with the given conditions and evaluated for globule size, J3.5.SS values further supported this finding. ITT had more JSS values, resulting in higher permeation. In general, RSV has a slow permeation, but it can exhibit and retain antioxidant and anti-inflammatory effects on the skin.The permeability percentage through cellulose membrane for RSV and ITT was found to be 42.52 ± 2.02% and 58.66 ± 0.77% after 12 h . JSS val3.6.Ex vivo studies were conducted using simple, low head dissolution apparatus to establish the effect of ITT-RSV formulation. The permeation was studied using rat full-thickness skin, and the results are summarized in ex vivo permeation diagram of ITT and RSV from optimized RVS-ITT-SNEDDS formulation.3.7.Despite several notable advancements, hepatic safety and hepatic cell regeneration products are inadequate for treating several liver disorders. Numerous herbal extract formulations were also used in this approach  emerged between RSV-ITT SNEDDS formulation and the control formula for all tests. To conclude, the advanced formulation was developed relative to the marketed product in terms of hepatoprotective efficacy.The pretreatment with a formulation containing ITT + RSV for 14 days significantly increased the antioxidant property and reduced the lipid peroxidation, which reduced overall oxidative stress. All changes obtained with RSV formulation (Group 5) were more beneficial in ameliorating hepatotoxicity compared to other groups. Additionally, these two groups differed significantly from a marketed product (Group 6). No significant difference  were selected to formulate the nanoemulsion. A low concentration of ITT favored the formation of globules with smaller particle sizes. Mixture design was successfully applied to optimize the concentrations of various parameters according to the desirability approach. Formulation with 0.15 g of oil mixture, 0.6 g of surfactant (labrasol), and 0.250 g of co-surfactant (Transcutol) achieved the optimum formulation. The range of relative error was acceptable (<5%), confirming the preciseness of the design. Click here for additional data file.


Gastric cancer (GC) is one of the most common malignancies worldwide, and the tumor metastasis leads to poor outcomes of GC patients. Long noncoding RNAs (lncRNAs) have emerged as new regulatory molecules that play a crucial role in tumor metastasis. However, the biological function and underlying mechanism of numerous lncRNAs in GC metastasis remain largely unclear. Here, we report a novel lncRNA, lnc-TLN2-4:1, whose expression is decreased in GC tissue versus matched normal tissue, and its low expression is involved in the lymph node and distant metastases of GC, as well as poor overall survival rates of GC patients. We further found that lnc-TLN2-4:1 inhibits the ability of GC cells to migrate and invade but does not influence GC cell proliferation and confirmed that lnc-TLN2-4:1 is mainly located in the cytoplasm of GC cells. We then found that lnc-TLN2-4:1 increases the mRNA and protein expression of TLN2 in GC cells and there is a positive correlation between the expression of lnc-TLN2-4:1 and TLN2 mRNA in GC tissue. Collectively, we identified a novel lncRNA, lnc-TLN2-4:1, in GC, where lnc-TLN2-4:1 represses cell migration and invasion. The low expression of lnc-TLN2-4:1 is associated with poor overall survival rates of GC patients. These suggest that lnc-TLN2-4:1 may be a tumor suppressor during GC metastasis. Gastric cancer (GC) is the fifth most common cancer and the third leading cause of cancer mortality worldwide . PatientLong noncoding RNAs (lncRNAs) are a class of single RNAs with more than 200 nucleotides in length and fail to encode protein . In the Talin (TLN) plays a crucial role in cell migration, invasion, and cancer metastasis . TLN genIn the present study, we found a novel lncRNA, lnc-TLN2-4:1, located in the cytoplasm of GC cells, whose expression is decreased in GC tissue compared with matched normal tissue and is involved in poor overall survival rates of GC patients. We further found that lnc-TLN2-4:1 overexpression inhibits GC cell migration and invasion, but does not affect GC cell proliferation. These suggest that lnc-TLN2-4:1 may be a tumor suppressor during GC metastasis.Forty-nine pairs of fresh human GC samples in this study were collected from the consenting individuals based on the instructions approved by the Ethics Review Board at Xinqiao Hospital, Army Medical University , from 2013 to 2017. The GC tissues were processed in the operating room and stored in liquid nitrogen within 10 min. The matched normal tissues were collected at a distance of >5 cm from the tumor tissues, and all tissues were identified histologically. None of the patients underwent chemotherapy or radiotherapy before operation. A four-year follow-up of the 49 GC patients was performed.2.Six human GC cell lines  were purchased from BeNa culture Collection (BNCC). AGS cells were cultured in the F12 medium  supplemented with 10% FBS (Gibco BRL), and the other cells were cultured in the DMEM/HIGH GLUCOSE medium  supplemented with 10% FBS (Gibco BRL) at 37°C in an atmosphere of 5% COβ-actin, using the 2–ΔΔCt method. The primer sequences are as the follows: TLN2 sense: 5′ACGGCGGAACCAGAGGAGAT3′, TLN2 antisense: 5′GGTGTCCAGGTCGGCAATGAT3′; lnc-TLN2-4:1 sense: 5′GCTGGCTGCTTCTGAGACTTAC3′, lnc-TLN2-4:1 antisense: 5′TGGAGCAACAGACTGAGGACAT3′. The parameter of PCR running is 95°C for 1 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec. For immunoblotting, the anti-TLN2 antibody (ab108967) was purchased from Abcam, China , and HRP-conjugated secondary antibody was purchased from Zhongshan Biotechnology , and all antibodies were used according to the manufacturer's instructions.The procedures and reagents of RNA extraction, qRT-PCR, and immunoblotting are described in our previous study . For qRTThe LV5-V6256-1 vector containing lnc-TLN2-4:1 cDNA sequence was synthesized from a company, GenePharma . The lentivirus construction is described in our previous study .The procedures and reagents of cell migration, invasion, and proliferation experiments are described in our previous study . BGC823 t test or Mann–Whitney U test. The one-way ANOVA was used to analyze the difference among three or more groups. Receiver operating characteristic curve (ROC) was used to assess the power of distinguishing two groups. The patient survival was analyzed using the Kaplan–Meier method and log-rank test. P < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 19.0  and GraphPad Prism 8.0 .All data are presented as the means ± standard deviation or standard error. The difference between two groups was analyzed using Student's P < 0.01, and the lengths of lncRNAs are between 1000 nt and 2000 nt), we found an unidentified lncRNA, AF070527, whose expression was decreased in three GC tissues compared with the matched normal tissues (https://bigd.big.ac.cn/lncbook/index). Lnc-TLN2-4:1 is an intergenic lncRNA and shown to have no encoding capacity (LncBook).To find a novel lncRNA which may be of regulatory function in the development of GC, we analyzed the data from an lncRNA microarray (GSE58828) that was performed in our previous study . Based o tissues . The namTo further determine the exact expression of lnc-TLN2-4:1 in GC, we collected 49 pairs of GC tissues and matched normal tissues from the enrolled GC patients. QRT-PCR experiments revealed that the expression of lnc-TLN2-4:1 is significantly decreased in GC tissue versus matched normal tissue . We nextWe next determined whether the expression of lnc-TLN2-4:1 in GC tissue has a diagnostic power for GC. Receiver operating characteristic curve (ROC) analyses revealed an AUC of 0.7071, with the sensitivity of 65.31% and the specificity of 81.25% and the cutoff value of 1.246, in discriminating GC tissues from matched normal tissues , and an in vitro (Figures As abovementioned, aberrant expression of lnc-TLN2-4:1 was associated with GC metastasis; therefore, we directly investigated whether lnc-TLN2-4:1 could influence the migration and invasion of GC cells. To perform the gain-of-function, we measured the expression of lnc-TLN2-4:1 in six GC cell lines, including AGS, MKN45, MGC803, BGC823, SGC7901, and MKN74 and found that BGC823 and SGC7901 almost have the lowest expression of lnc-TLN2-4:1 ; thus, w Figures . BecauseTo well understand the underlying mechanism of lnc-TLN2-4:1 in GC metastasis, we determined the location of lnc-TLN2-4:1 in GC cells because the regulatory mechanism of the lncRNA is constrained by its location. We found that lnc-TLN2-4:1 is mainly located in the cytoplasm of BGC823 cells, and the expression of lnc-TLN2-4:1 is significantly increased in the cytoplasm of BGC823 cells with the ectopic expression of lnc-TLN2-4:1 compared to those with wild-type expression of lnc-TLN2-4:1 , suggestLnc-TLN2-4:1 is predicted to have no encoding capacity and has 1558 nt in length. In this study, we found that lnc-TLN2-4:1 expression is significantly decreased in GC tissue versus matched normal tissue and is associated with the GC cell lymph node and distant metastases. ROC analyses revealed that lnc-TLN2-4:1 expression has a potentially predictive power in distinguishing GC tissue from matched normal tissue, and the decreased expression of lnc-TLN2-4:1 is closely involved in poor overall survival rates of GC patients. So far, there are hundreds of lncRNAs that have been identified to have aberrant expression in GC development and also be considered as potential biomarkers for GC detection. For example, Zhuo et al. report an lncRNA, GMAN, which is overexpressed in GC tissue versus nontumor tissue and its upregulation is also associated with poor overall survival rates of GC patients ; Zhang eTo determine the biological function of lnc-TLN2-4:1 in GC, we selected two GC cell lines which have low expression of lnc-TLN2-4:1 and constructed BGC823 and SGC7901 cells with stably ectopic expression of lnc-TLN2-4:1 using a lentivirus containing lnc-TLN2-4:1-overexpressing vectors. This effect of ectopic expression of lnc-TLN2-4:1 was identified by qRT-PCR and immunofluorescence. Stably modified expression of lncRNAs using lentivirus is widely used in studying their biological functions, such as in our previous study . The bioTo address the underlying mechanism by which lnc-TLN2-4:1 represses GC metastasis, we searched for the candidate target genes of lnc-TLN2-4:1. Because lnc-TLN2-4:1 is located in the cell cytoplasm, we considered that lnc-TLN2-4:1 has a possibility of regulating TLN2 mRNA stability. A large number of studies have reported that lncRNAs can protect mRNAs from degradation , 22. TheIn conclusion, we identified a novel lncRNA, lnc-TLN2-4:1, which is downregulated in GC tissue versus matched normal tissue and whose low expression is associated with GC metastasis and poor overall survival rates of GC patients. We further found that lnc-TLN2-4:1 represses the ability of GC cells to migrate and invade, and lnc-TLN2-4:1 promotes the expression of TLN2 in GC cells and there is a positive correlation between the expression of lnc-TLN2-4:1 and TLN2 in GC tissues. These data suggest that lnc-TLN2-4:1 may be a therapeutic target for GC.


IIbβ3 receptor, converting it to a low affinity state for adhesion and aggregation processes. In the second mechanism, Dab2 is released extracellularly and interacts with the pro-aggregatory mediators, the integrin αIIbβ3 receptor and sulfatides, blocking their association to fibrinogen and P-selectin, respectively. Our previous research indicated that a 35-amino acid region within Dab2, which we refer to as the sulfatide-binding peptide (SBP), contains two potential sulfatide-binding motifs represented by two consecutive polybasic regions. Using molecular docking, nuclear magnetic resonance, lipid-binding assays, and surface plasmon resonance, this work identifies the critical Dab2 residues within SBP that are responsible for sulfatide binding. Molecular docking suggested that a hydrophilic region, primarily mediated by R42, is responsible for interaction with the sulfatide headgroup, whereas the C-terminal polybasic region contributes to interactions with acyl chains. Furthermore, we demonstrated that, in Dab2 SBP, R42 significantly contributes to the inhibition of platelet P-selectin surface expression. The Dab2 SBP residues that interact with sulfatides resemble those described for sphingolipid-binding in other proteins, suggesting that sulfatide-binding proteins share common binding mechanisms.Disabled-2 (Dab2) is an adaptor protein that regulates the extent of platelet aggregation by two mechanisms. In the first mechanism, Dab2 intracellularly downregulates the integrin α Two Dab orthologs, Dab1 and Dab2, are present in mammals. Dab1 is primarily expressed in the brain2, whereas Dab2 is ubiquitously expressed in different tissues4. Dab2 expression levels have been reported to exhibit a significant effect on cancer initiation and progression. Indeed, Dab2 expression is lost in breast, ovarian, prostate5, bladder6, and colorectal cancer cells7, suggesting that Dab2 can act as a tumor suppressor8. Two alternative spliced forms of Dab2 are expressed in humans, p96 and p67, with the latter lacking a central exon3. Dab2 contains a phosphotyrosine-binding (PTB) domain located at the N-terminus, clathrin-binding sites, NPF and DPF motifs, and a proline-rich SH3 domain located at the C-terminus, indicating that Dab2 primarily functions as an adaptor protein.The adaptor protein Disabled-2 (Dab2) is a multimodular signaling molecule involved in a variety of cellular processes including protein trafficking, cell growth and differentiation, cell adhesion, and modulation of platelet aggregation1. Modulation of inside-out signaling by Dab2 is mediated by its cytosolic S24-phosphorylated form, which binds to the β3 subunit of the integrin αIIbβ3 receptor, downregulating fibrinogen-mediated adhesion9. In outside-in signaling, ligand-receptor complexes on activated platelets promote platelet spreading, granule secretion, stabilization of the adhesion and aggregation of platelets, and clot retraction10. Some of the components of the intracellular granules are required to limit the extent of platelet activation, adhesion, and aggregation. Platelet function must be tightly modulated, as uncontrolled platelet activation can trigger unwanted clinical complications such as thrombocytopenia and thrombosis11. Dab2 is released from α-granules and relocates to the platelet surface where it modulates outside-in signaling12. To do this, Dab2 presents two extracellular targets. At the platelet membrane surface, Dab2 associates with the extracellular domain of the αIIb subunit of the integrin receptor via its RGD motif, preventing integrin-fibrinogen interactions. Dab2 also binds to sulfatides13, a class of negatively charged sphingolipids found on most eukaryotic cell surfaces14. Interestingly, cleavage of Dab2 by the platelet agonist thrombin may be prevented when Dab2 is associated with the sphingolipid13. Binding of Dab2 to sulfatides limits P-selectin association to sulfatides15, which is required to prolong platelet aggregation16.Recently, Dab2 was characterized as a negative regulator of platelet aggregation by modulating both pro-aggregatory inside-out and outside-in signaling pathways13. Combined mutations in residues K25, K49, K51, and K53 markedly reduce sulfatide binding and platelet aggregation13. Further studies showed that an α-helical-rich region of 35 amino acids within the Dab2 N-PTB, which was predicted to contain two potential sulfatide-binding motifs , mimic the inhibitory platelet-platelet interaction effects of Dab2 N-PTB17. In this report, by using a combination of structural, molecular docking, and functional approaches, we refined the mode by which SBP, the minimal sulfatide-binding unit derived from Dab2, associates with sulfatides. Molecular docking showed that the Dab2 SBP residues around α-helix 1 are required for interactions with the sulfatide head group, whereas α-helix 2 mediates sulfatide acyl chain interactions. Furthermore, we predict that R42, located in the first α-helix of Dab2 SBP, forms hydrogen bonds with the OS atoms of the sulfate group in the sulfatide. We demonstrate that both the charge and stereochemistry of R42 are critical for Dab2 SBP’s sulfatide recognition and inhibition of platelet P-selectin surface expression. Results from this report provide details of how Dab2 interacts with sulfatides, which can be used for the design of a Dab2-derived peptide that can block sulfatide interactions, and, consequently, prevent undesired platelet aggregation events.Initial studies reported that Dab2 binds sulfatides at its N-terminus including the PTB domain 17. Molecular docking was performed to refine potential residue interactions between a single sulfatide molecule and the model of the DPC-bound structural conformation of Dab2 SBP (PDB ID: 2LSW) containing vector residues (19-GPLGS-23), which were added to the N-terminus to match the experimental peptide used in this work. After energy minimization, the structure of Dab2 SBP was validated and showed favorable energetics and side chain positioning using Ramachandran plots, ProSA and QMEAN analyses  of DPC-associated Dab2 SBP, showed very flexible N- and C-termini and a rigid structure spanning residues E37-I56; such flexibility was not altered by sulfatides  and employed to screen for sulfatide binding using the lipid-protein overlay assay. Although this assay does not mirror the physiological organization of sulfatides in biological membranes, it is still useful for obtaining a first screening for the binding of the Dab2 SBP mutants to the sphingolipid. As expected, Dab2 SBP bound sulfatides and closely reached saturation at 800 pmol of lipid Fig. a, b. Con17, SPR data showed that Dab2 SBP bound sulfatide liposomes with a KD of 49.8 ± 5.4 µM and the association displayed saturation of binding . In agreement with previous observations18. P-selectin promotes platelet aggregation in a sulfatide-dependent manner19. To establish whether Dab2 SBP can modulate P-selectin pro-aggregatory activity, we evaluated the cell surface expression of P-selectin. Washed platelets were previously activated with ADP, which represents a critical step for sulfatide-mediated platelet activation19. Consistent with previous observations15, incubation of activated platelets with sulfatide-containing liposomes led to an increase in the median fluorescence of the anti-P-selectin antibody bound to the platelet surface  were relevant for lipid interactions. Previous results suggested that the last 20 residues of Dab2 SBP are involved in sulfatide interactions21. Interestingly, a basic residue (H38) plays a role in accommodating one of the alkyl chains in the binding pocket of CD1a.Very few protein-sulfatide complexes have been structurally characterized. The interaction of the cluster of differentiation 1a (CD1a) with sulfatide involves hydrogen bonds to its 3′ sulfate group with R76 and E154 and the galactose moiety with R76 and S77, whereas the sulfatide fatty acid chains display van der Waals interactions mostly with hydrophobic residues24, predominantly exhibit a consensus helix-turn-helix fold with an aromatic residue, being solvent-exposed, and either a G or P residue contributing to the turn between the α-helices and several positively charged residues25. Intriguingly, a consensus sphingolipid-binding motif has been identified in α-synuclein26, which shares several features with Dab2 SBP  located upstream of Y39, also displays a major role for α-synuclein interactions with GM326. However, the equivalent residue in Dab2 SBP, K34, did not exhibit an important role in sulfatide binding as concluded from molecular docking and NMR assays. In contrast to GM3, sulfatides are negatively charged. Given that  α-synuclein has a preference for GM3 over sulfatides, it is possible that α-synuclein K43 may play a similar role to that observed for Dab2 R42 in sulfatide interactions. Considering that R42K mutation in Dab2 SBP reduced the activity of the peptide but did not abolish it, the presence of K43, instead of R43, might also reduce the preference of α-synuclein for sulfatides.Sphingolipid-binding domains, such as those described in viral, bacterial, and mammalian proteins19. Accordingly, Dab2 modulates the extent of platelet aggregation by targeting P-selectin function. The association of Dab2 to sulfatides impairs P-selectin-sulfatide interactions, thereby blocking homotypical and heterotypical cell–cell interactions15. Upon platelet activation, Dab2 is transiently secreted from α-granules to the cell surface13. Upon sulfatide binding, Dab2 likely undergoes a conformational change27, a state in which the protein is more tolerant to thrombin proteolysis13. It has been suggested that, once secreted, Dab2 modulates the extent of platelet aggregation by two independent mechanisms1. First, Dab2 downregulates the integrin receptor activity by interfering with its interaction with fibrinogen. Second, extracellular Dab2 blocks platelet-platelet P-selectin-mediated sulfatide-dependent interactions. Other sulfatide-binding proteins can be exported out of the cell for extracellular interactions with sulfatides. For example, the mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum protein that is secreted under stress-related stimuli28. Binding of secreted MANF to sulfatides, through its α-helical saposin-like domain, provides MANF cellular uptake and alleviates stress responses driven by the endoplasmic reticulum29.Sulfatides are found in discrete patches on activated spread platelet membranes, reminiscent of lipid rafts. Platelet surface expression of the sulfatide-binding pro-aggregatory protein P-selectin increases the stability of platelet aggregationIn conclusion, our results indicate how sulfatides interact with Dab2 SBP and identify the critical binding residues. Our data is in line with what was earlier defined as the minimal α-helical-turn-α-helical unit for sphingolipid binding. Furthermore, in the case of Dab2, we suggest that R42 mediates the recognition of the negatively charged sulfatide headgroup, and the α-helical lysine residues at the C-terminus provide an adequate hydrophobic environment through their flexible hydrocarbon spacers.The list of chemicals and their suppliers are: brain sulfatides, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) (Avanti-Lipids), N-dodecylphosphocholine (DPC) (Anatrace), cholesterol (Sigma), and isopropyl β-D-thiogalactopyranoside . All other chemicals were of analytical grade.30. Site-directed mutagenesis of Dab2 SBP was carried out using the QuikChange (Stratagene) protocol.A cDNA, representing residues 24–58 in human Dab2, was cloned into a pGEX6P1 vector . A GST fusion and untagged Dab2 SBP were expressed and purified as described31, using the NMR-derived, Dab2 SBP DPC-micelle embedded structure [PDB ID: 2LSW17] as a template. To align with the experimental peptide sequence in this work, residues 19–23 (GPLGS) were built onto the N-terminus of PDB ID: 2LSW using Schrödinger-Maestro’s 3D-Builder. Following the addition of residues 19–23 to the template structure, energy minimization was performed using the OPLS3e forcefield32 and default settings in Schrödinger-Maestro v2018-3. After energy minimization, the Dab2 SBP structure was exported into PDB format and model quality was assessed using several validation methods. Ramachandran plots generated using the Rampage webserver33 assessed model quality with respect to favorable and allowed φ and ψ angles of residues, and SWISS-MODEL35 calculated a QMEAN plot to quantify model quality with respect to solvation and torsion angle. ProSA37 compared the model to experimentally derived structures similar in composition. Five mutant peptides were individually created using the energy minimized Dab2 SBP structure as the starting template. Each mutation  was built using Schrödinger-Maestro31 by altering the residue(s) of interest using the built-in mutagenesis tool. Following residue alteration, energy minimization was performed as stated above. Structures were exported as PDB files and model quality was assessed using the same procedure as Dab2 SBP, with varying validation quality as influenced by the point mutations.Energy minimized Dab2 SBP and its mutant structures were created with Schrödinger-Maestro v2018-338. Sulfatide and Dab2 SBP and point mutation structures were prepared in AutoDockTools v1.5.639 and a sulfatide was docked to each Dab2 SBP energy minimized structure using AutoDock Vina v1.1.240. The same grid box and center was used for each docking experiment and covered the entire Dab2 SBP structure. The center coordinates for the grid box  were . The grid box dimensions  were , using a 1 Å grid space. Nine poses were created from each docking run, with the lowest energy pose used for further analysis on residue interaction and fingerprint analysis. Molecular visualization of docked poses was performed in PyMOL41. Sulfatide pose volume occupancy was visualized using UCSF Chimera38. Schrödinger-Maestro’s Interaction Fingerprints analysis tool was used with default parameters on each lowest energy docked pose to determine potential peptide-sulfatide interactions. The output matrix was converted manually to a table format and organized by interaction type.The structure of sulfatide was obtained from the ZINC database (ZINC96006133) and converted from the mol2 to the PDB format using UCSF Chimera15N-labeled Dab2 SBP (1 mM) was prepared in 90% H2O, 10% 2H2O, 10 mM d4-citrate (pH 5.0), 40 mM KCl, 1 mM NaN3, and 200 mM d38-DPC in the absence or presence of 8 mM sulfatide. A Bruker Avance III 800 spectrometer with cryoprobe and standard Bruker two dimensional 1H–15N HSQC-type pulse sequences were used to obtain 15N longitudinal (R1) and transverse (R2) relaxation rates, and 1H–15N NOEs of Dab2 SBP in the absence and presence of sulfatide at 25 °C. The pulse sequences were hsqcnoef3gpsi (Het-NOE), hsqct1etf3gpsi3d (R1), and hsqct2etf3gpsi3d (R2) [all from Bruker]. Relaxation delays for the R1 experiments were 5, 50, 150, 250, 400, 550, 750, 1200, and 2000 ms. Relaxation delays for the R2 experiments were 17, 34, 51, 68, 119, 153, 203, and 254 ms. The recycle delay for R1 and R2 experiments was 2 s. 1H–15N NOEs were measured by comparing intensities of 1H–15N correlation spectra with either 5 s of 1H saturation or a 5 s delay preceding 1H–15N correlation.Sulfatide strips were prepared by spotting 1 μl of the indicated amount of sulfatides, dissolved in chloroform/methanol/water (1:2:0.8), onto a Hybond-C extra membrane . Membrane strips were blocked with 3% fatty acid-free BSA (Sigma) in 20 mM Tris–HCl (pH 8.0), 150 mM NaCl, and 0.1% Tween-20 for 1 h at room temperature. Then, membrane strips were incubated with the indicated GST fusion peptide in the same buffer overnight at 4 °C. Following four washes with the same buffer, bound fusion peptides were detected with rabbit anti-GST antibody (Proteintech) and donkey anti-rabbit-horseradish peroxidase antibody . Binding of fusion peptides to sulfatides was probed using the Supersignal West Pico chemiluminescent reagent (Pierce).13. Briefly, lipids including DPPC:DPPE:cholesterol , or DPPC:DPPE:cholesterol:sulfatide (1:1:1:4), were solubilized in chloroform/methanol/water mixtures. The resulting mixture was dried under N2 followed by vacuum to remove solvent traces. The lipid mixture was then resuspended at 0.8 mg/ml in 20 mM Tris–HCl (pH 6.8) and 100 mM NaCl to reach a final concentration of 0.5 mM sulfatide, sonicated, and extruded for 100-nm liposome size at 68 °C. L1 sensorchips were first equilibrated with 20 mM Tris–HCl (pH 7.4) and 100 mM NaCl as a running buffer. Then, L1 sensorchips were pretreated with 40 mM octyl β-D-glucopyranoside. At a 30 µl/min flow rate, control liposomes were immobilized onto one of the L1 sensorchip channels whereas a second channel was loaded with sulfatide-containing liposomes. Typical liposome loading was ~ 4,000 response units/sensorchip channel. The apparent KD values were estimated using BIAevaluation software, version 2.0 .Binding of Dab2 SBP peptides to sulfatide liposomes were monitored by surface plasmon resonance (SPR) at room temperature using a BIAcore X-100 instrument . One-hundred nanometer size-calibrated liposomes were generated as previously describedg to separate a platelet-rich plasma (PRP) fraction from erythrocytes. Then, the PRP fraction was further centrifuged for 5 min at 1,100×g. The resultant platelet-containing pellet was resuspended in 10 mM HEPES (pH 7.4), 134 mM NaCl, 12 mM NaHCO3, 2.9 mM KCl, 0.34 mM Na2HPO4, and 1 mM MgCl2 (Tyrode’s buffer) containing 0.5 µM prostaglandin (PGI2). Platelets were then washed in PGI2-free Tyrode’s buffer, containing 5 mM dextrose and 0.3% BSA, and counted in a haemocytometer.Blood collection, obtained from healthy volunteers by venipuncture, and further processing was carried out following the Virginia Tech Institutional Review Board guidelines. All methods involving manipulation and processing of the blood samples were approved by the Virginia Tech Institutional Review Board. All subjects provided informed consent. Blood samples were collected into vacutainer ACD solution A blood tubes  and centrifuged for 20 min at 200×5 platelets/µl) were maintained in Tyrode’s buffer (unactivated) or treated with 30 µM ADP (activated). Both unactivated and activated platelets were incubated for 6 min at room temperature with either control liposomes  or sulfatide-containing liposomes  in the absence and presence of 50 µM of one of the following peptides: Dab2 SBP, Dab2 SBP R42A, or Dab2 SBP R42K. Platelets were fixed with 1% formalin in phosphate buffered saline and incubated with phycoerythrin-labelled anti-CD62P  antibody following manufacturer’s instructions. Antibody-bound platelets were quantified using a FacsAria flow cytometer.Washed platelets (1.5 × 10Supplementary Information.


Interestingly, UCH-L1 (known to be enriched in neurons and interacting with the Parkinson’s disease-associated protein α-synuclein) appeared to be a prognostic indicator of unfavorable outcome in endometrial and urothelial cancer, while increased expression of UCH-L3 and UCH-L5 was associated with poor survival in liver and thyroid cancer, respectively. In normal tissues, UCH-L1 was found to be strongly expressed in the cerebral cortex and hypothalamus, while UCH-L3 expression was somewhat higher in the testis. The occurrence of mutation rates in UCHs also suggests that BAP1 and UCH-L5 may play a more dominant role in cancers than UCH-L1 and UCH-L3. We also characterized the functional context and configuration of the repeat elements in the promoter of DUBs genes and found that UCHs are highly discriminatory for catabolic function and are mainly enriched with LINE/CR1 repeats. Regarding the thesis of an inverse association between cancer and NDD, we observed that among all DUBs, UCHs are the one most involved in both entities. Considering a putative therapeutic potential based on presumed common mechanisms, it will be useful to determine whether other DUBs can compensate for the loss of UCH activity under physiological conditions. However, experimental evidence is required to substantiate this argument. Emerging evidence suggests an inverse association between cancer and neurodegenerative diseases (NDD). Although phenotypically different, both diseases display a significant imbalance in the ubiquitination/deubiquitination processes. Therefore, we particularly investigated the expression of ubiquitin C-terminal hydrolases (UCHs Ubiquitination, the covalent attachment of ubiquitin (ub) to protein substrates, is achieved by the sequential action of three enzymes: E1 ubiquitin-activating enzyme; E2 ubiquitin-conjugating enzyme; and E3 ubiquitin ligase. This attachment can occur at one or more lysine sites, resulting in monoubiquitination, which can remain as it is or be further extended by additional ubiquitin molecules to form elongated chains (polyubiquitination). This ultimately leads to the degradation of unwanted proteins, altered subcellular localization/function and protein interactions. Deubiquitinating enzymes (DUBs) can efficiently remove ubiquitin from the protein and thus help to maintain a dynamic balance between ubiquitination and deubiquitination within the cellular environment which is essential for the efficient maintenance of cellular functions.BAP1 (BRCA1-associated protein 1) possesses unique ability as a tumor suppressor, while others  are characterized as tumor promoters [DUBs play several other functional roles besides deubiquitination, especially in cancer . For insromoters ,3,4,5,6.romoters . Severalromoters ,9,10. Inromoters . Though romoters . NeverthSince most studies published thus far are from mouse and cell lines, herein, we put emphasis on the cancer-specific characteristics of UCHs by using publically available databases of healthy humans (GTEx) , cancer patients (TCGA) and NDDs. With regard to cancer and neurodegeneration, this is the first study to show that UCHs may contribute significantly to cancer. The highly discriminatory catabolic function of UCHs and the enrichment of LINE/CR1 (CR1 subfamily of LINE elements) repeats have not been reported before.UCH-L1  was previously found to be enriched in neurons, and was shown to promote alpha-synuclein neurotoxicity in Parkinson’s disease (PD) patients [UCH-L1 and PD was mainly supported by the I93M mutation in a German family [GLU7ALAUCH-L1) was found in a Turkish family, mainly related to the occurrence of a neurodegenerative syndrome in childhood, not exclusively PD [UCH-L1 gene (S18Y) may play a protective role in Parkinson’s disease, but recent studies have shown contradictory results [UCH-L1 expression in the neuroretina and in lens epithelial cells of atopic cataracts [UCH-L1 has also been associated with cancer and therefore was proposed as an oncogene [UCH-L1 expression in 35 tissues of healthy individuals using the publicly available GTEx database. Our results show that a high expression of UCH-L1 is related to CNS-related tissues, which is consistent with previous studies  and urothelial cancer (p = 0.00019). Hence, it may be concluded that the altered expression of UCH-L1 is a disease-related consequence, particularly in cancer. It is noteworthy to mention that we cannot determine if the mutations/loss of UCH-L1, which is associated with cancer, has an additional effect on neurodegenerative diseases  or vice versa.patients . Primarin family ,15. The  results . Therefo results . FurtherL1 gene SY may plaataracts . It is noncogene ,21. ConsUCH-L1, the expression of the other two members, UCH-L3 and UCH-L5 , was found not to be restricted to CNS, but was also observed in other anatomical regions  and thyroid cancer (p = 0.000071)  A. Howeveo others .p = 1.6 × 10−18 and 9.3 × 10−6, respectively). In particular, UCH-L5 was involved in the splicing pathway by binding to the proteins PLRG1 and CDC5L, which are the components of the cell division cycle 5-like complex and are required for pre-mRNA splicing. Meanwhile, UCH-L1 and UCH-L3 were found to be mainly involved in the proteasome pathway through highly associated ubiquitins, such as UBA52, UBB, UBC, RPS27A (UBA80), and UBE2S  in addition to uveal (eye) melanoma [BAP1 (2032 mutations) and UCH-L5 (1276 mutations) harbor many more mutations than UCH-L3 (556 mutations) and UCH-L1 (456 mutations), respectively. In summary, DUBs, especially UCHs, seem to be nonspecifically altered in different types of cancer, while they show more specific alterations in NDDs . To further verify this, we accessed the differential expression of DUBs in NDDs (USP36 and USP21) were found to be altered in PD, while only a few also showed variable expression in AD samples compared to healthy controls. However, the changes in NDDs were not as noticeable as in cancer.With regard to melanoma . Herein, in NDDs . As expeUCH-L5 gene lacks repeats; similarly, in the MJD, only the JOSD2 gene showed the same pattern. The limited pattern of repeats was also visible in one unique gene, ZUFDP  and UCH-L1 (in upstream promoter region) contain a significantly high number of LINE/CR1  in the structure of these LINEs, with 28 being the most significant. Among the regulatory elements Cdx-1, C/EBPalpha, Ncx, and C/EBPdelta were found to highly distinctive to all LINE/CR1, especially the UCH-related LINE/CR1 was found to be associated with embryonic development and morphogenesis.The functional analysis of the DUBs family using gene ontology reveals two main associations: protein or histone deubiquitination as a ubiquitous function for all DUBs (with specific targets for each) and metabolic processes for specific subfamilies such as the UCHs A. Althoue, ZUFDP B. We have, ZUFDP C. FurtheLINE/CR1 . In contIt is well known that the families and subfamilies of LINE elements, which are abundant in the genome and often found in the vicinity of prime genes associated with human diseases, are also associated with cancer ,23. The Among all DUBs, UCHs are the ones most involved in cancer and NDD. Importantly, the contribution of highly discriminatory catabolic function of UCHs and the enrichment of LINE/CR1 repeats needs further investigation. In a broader prospective, it will be useful to determine whether other DUBs can compensate the loss of UCH activity under physiological conditions. The findings we have presented here require experimental evidence to pave the way for a deeper understanding of the mechanisms connecting cancer and neurodegeneration.UCH-L1, UCH-L3, UCH-L5 in 34 healthy tissues was extracted from the GTEx database. Briefly, the RNA-seq tissue data generated by the Genotype-Tissue Expression (GTEx) project are reported as mean pTPM (protein-coding transcripts per million), hence corresponding to the mean values of the different individual samples from each tissue. String analysis was performed using online server (https://string-db.org/). First, UCHs (UCH-L1/3/5) were specified and the functional partners of UCH-L1, UCH-L3, and UCH-L5 were identified using the online server. Only the experimentally-validated interactions were selected to establish the network. In the first level, only the ten most important interaction partners were retained, while five partners were kept for the second level. Then, the network was clustered with the k-means method and enrichment was further carried out for these selective proteins and their functional partners using STRING function.The expression analysis of http://www.ncbi.nlm.nih.gov/geo/). The p-value was called with two-sample t-test between normal samples and disease samples and the plot of p-value (-log10) against fold change (log2) was drawn.Gene expression datasets to evaluate DUBs in NDDs  were retrieved from GEO (Gene Expression Omnibus) (http://cancer.sanger.ac.uk) was used to check the mutational rate among UCHs.For cancers, The Cancer Genome Atlas (TCGA) data was accessed. Briefly, the RNA-seq data in 17 cancer types reported as median FPKM (number Fragments per Kilobase of exon per Million reads), generated by TCGA. In addition, the comprehensive cancer mutation database COSMIC (https://www.genenames.org/). The functional analysis was performed using gene ontology categories, all GO levels were included and normalized by using hypergeometric distribution for subfamilies. For repeats in the promoter of the DUB genes, the position of genes and repeats annotated in the Human Genome Reference (GRCh38.p7) were used. Hierarchical clustering was applied by using simple linkage and Pearson correlation methods. The standard maximum parsimony method was used to grade conservation and homology among these UCH-related LINEs for the phylogenetic tree. The possible regulatory sites in the structure of these LINEs were identified by using the PROMO dataset, using the link http://alggen.lsi.upc.es/.In order to perform a functional and structural analysis of the DUB genes, the list of genes by subfamily was retrieved from HUGO (