In the past two decades, microneedles (MNs), as a painless and simple drug delivery system, have received increasing attention for various biomedical applications such as transdermal drug delivery, interstitial fluid (ISF) extraction, and biosensing. Among the various types of MNs, porous MNs have been recently researched owing to their distinctive and unique characteristics, where porous structures inside MNs with continuous nano- or micro-sized pores can transport drugs or biofluids by capillary action. In addition, a wide range of materials, including non-polymers and polymers, were researched and used to form the porous structures of porous MNs. Adjustable porosity by different fabrication methods enables the achievement of sufficient mechanical strength by optimising fluid flows inside MNs. Moreover, biocompatible porous MNs integrated with biosensors can offer portable detection and rapid measurement of biomarkers in a minimally invasive manner. This review focuses on several aspects of current porous MN technology, including material selection, fabrication processes, biomedical applications, primarily covering transdermal drug delivery, ISF extraction, and biosensing, along with future prospects as well as challenges. Microneedles (MNs), having microscopic needle structures, were initially developed to facilitate transdermal drug delivery by piercing human skin and providing transport conduits across the stratum corneum with a thickness of 10–15 µm . MNs havThe first MNs, which aimed at enhancing the skin permeability for transdermal drug delivery, were of the solid type . Solid MConversely, the development of coated MNs overcame the issue of pore closure after the penetration and removal of solid MNs. By coating the drug material on the surface of the MNs, the drug can diffuse into the skin directly while maintaining the pathways formed by the penetration without leaving drug waste on the surface of the skin. Similar to the solid type, the coated MNs are fabricated using silicon, metals, or polymers –14. CoatHollow MNs, which are manufactured using silicon, metals, or non-dissolving polymers, enable the administration of a large dose of the drug –19. One Dissoluble MNs are generally made of biocompatible and biodegradable polymers or sugar-encapsulating active substances –23. ThusSwellable MNs are generally fabricated using physical crosslinking techniques such as exposure to ultraviolet (UV) light with photoinitiators . Water-sRecently, porous MNs, which have interconnected micro-sized pores throughout the entire structure of MN, have attracted significant attention –34. PoroFrom the application perspective, transdermal drug delivery utilising MNs has been widely researched. Drug materials such as insulin –38, antiFor further applications using MNs, the extraction of body fluids, including blood and ISF, has recently emerged as an alternative to conventional hypodermic needles for painless extraction, followed by monitoring as well as diagnoses. Among them, the skin ISF, which is primarily located in the epidermis and dermis layers of the human skin, was reported to have abundant biomarkers such as glucose, alcohol, lactate, cortisol, cholesterol, and other proteins . SeveralPorous MNs were developed only 5 years ago for collecting skin ISF. Porous MNs have interconnected micron-sized pores through which fluids can be transported because of the channel-like connected pores by capillary action, thereby transporting the collected fluid to the analysis system directly. Moreover, a wide range of biocompatible materials have been used for the fabrication of porous structures to extract skin ISF in a passive manner, enabling the collection of body fluids by detecting and analysing biomarkers using biosensors , 58. AltBoth non-polymeric and polymeric materials can be used for the fabrication of porous structures. In this chapter, the material properties, representative fabrication methods, biomedical applications, and characteristics of porous structures are described with respect to each material and listed in Table Initially, MNs were fabricated from silicon material by using conventional microelectromechanical system (MEMS) technologies. Solid, coated, and hollow MNs with various dimensions and aspect ratios were fabricated using a deep reactive ion etching (DRIE) process , 59, 60.Alumina is a first-generation bio-ceramic material and has been applied in biomedical and clinical applications such as implants owing to its biocompatibility and high mechanical strength . RecentlWhile considering the applications, alumina-based porous MNs are used to store and deliver antibodies , insulinThe benefits of using porous alumina include strong mechanical properties when compared to polymer MNs and improved controllability of porosity. In the case of the sintering process, porosity can be controlled by adjusting the sintering temperature. Similarly, the pore diameter and density can be modified by changing the applied voltage during the chemical etching process. Conversely, one of the limitations of using alumina is that it is brittle and can be easily broken inside the human body. Although such porous structures  are biocompatible, their brittleness is considered to be undesirable in biomedical and clinical devices.Meanwhile, other bio-ceramics such as calcium sulphate dihydrate (CaS) and calcium phosphate dihydrate (CaP) have also been applied to fabricate porous MNs as bone substitute materials because of their superior biocompatibility , 71. PorMetals such as titanium and stainless steel can be used to fabricate porous MNs. Among several types of metals, titanium has been extensively applied for biomedical devices such as dental and orthopaedic implants owing to its biocompatibility and excellent mechanical strength . By capiSimilar to the titanium material, medical grade stainless steel (316L) has been used for MNs for over several decades owing to its biocompatibility under increased clinical applications and acceptance of the patients , 78–80. Polymers have received considerable attention because they have unique mechanical characteristics such as the ability to withstand large bending forces without being fractured. More importantly, polymeric MNs have advantages over other materials as they can be easily fabricated without complicated micromachining processes as well as equipment and the requirement for a clean room environment. In the last decade, several types of polymers have been researched and developed as materials for porous MNs for various biomedical applications such as transdermal drug delivery and the extraction of biofluids.Synthesised polymeric materials such as poly (ethylene glycol-co-methacrylic acid) were developed to produce porous structures by using bulk polymerisation techniques with different porogens to achieve various fluidic characteristics and mechanical properties . Poly (eSimilar to poly (ethylene glycol-co-methacrylic acid), poly (glycidyl methacrylate) (PGMA) synthesised by photo-polymerisation in the presence of a porogenic solvent was employed to produce porous structures. Porous PGMA MNs are fabricated by using the monomer, glycidyl methacrylate (GMA), and crosslinkers, TRIM and triethylene glycol dimethacrylate (TEGDMA), mixed with poly (ethylene glycol) (PEG) solution as a porogen. After the MNs are produced by photo-polymerisation, micropores are formed by a leaching process to remove the PEG porogens , 86. A pPoly (lactic-co-glycolic acid) (PLGA) is a copolymer of poly (lactic acid) (PLA) and poly (glycolic acid) (PGA). For nearly three decades, it has been extensively employed in the field of MNs for biomedical and clinical applications (e.g. drug-encapsulated nano- and micro-particle delivery) because of its biocompatibility and biodegradability –89. SevePolydimethylsiloxane (PDMS) is a well-known polymeric material used in the field of microdevices, especially as biomedical devices, owing to its notable chemical inertness, processability, flexibility, and biocompatibility . As PDMSCellulose acetate is a synthetic polymer that is also regarded as a proper base material to fabricate MNs owing to its biocompatible characteristics . The porAs a widely used biocompatible and biodegradable polymer, PLA is extensively employed in the field of biomedical applications such as drug delivery using MNs , 93–95. In this chapter, current fabrication methods for porous structures as well as porous MNs are described. In addition, previous literature pertaining to each method are reviewed and discussed in detail.For the formation of porous silicon structures, electrochemical anodization process is commonly used owing to its accurate replicability and easy modification. Silicon MN arrays are prepared by employing microfabrication processes based on MEMS technologies , 61, 62.3PO4, where a platinum plate is used as the cathode. Here, with the positive potential applied to aluminium in the H3PO4 electrolyte, aluminium is oxidised to Al3+; then, it reacts with O2− to form aluminium oxide, which is a porous structure. Similarly, the pore size is controllable, ranging from 15 to 500 nm, by adjusting the concentration of the electrolyte, temperature, and anodization time.Similar to silicon-based porous MNs, a porous-structured anodised aluminium oxide (AAO) MN array can also be fabricated by an electrochemical anodization process . First, 3: HF: H2O = 5: 2: 7) is applied to etch each microcolumn into the tapered structures. In this process, continuous pores are first blocked by metal chips or dust during the cutting process. However, the pores are finally connected after the etching process using acids that eliminate chips and dust.Isotropic wet etching can be used to fabricate porous titanium MNs . In this3(PO4)2, 55 wt% Ca(H2PO4)2·H2O, and 0.5 M citric acid solution in 0.4 liquid/powder ratio. Subsequently, the mixed materials were poured, vacuumed, and cured in a PDMS female mould at 37 °C for 48 h . Thus, either drug loading or ISF extraction can be achieved by using porous MNs arrays. In addition, a wide range of materials, including non-polymers and polymers, can be chosen and used to fabricate porous structures for versatile applications.While considering drug delivery, solid and hollow MNs can only deliver liquid drug formulations, while drugs in the solid state are delivered by coated and dissoluble MNs owing to their inherent structure and used matrix materials . ConversFurthermore, it can be envisioned that porous MNs offering painless skin penetration and targeting the skin ISF by capillary action can replace existing painful blood sampling methods , 34, 58.However, owing to the inherent elastic property of human skin, the length of MNs that can be inserted into the skin is limited, which must be optimised . MoreoveRecently, the increasing population and rising incidence of chronic diseases such as diabetes and hypercholesterolemia have driven the rapid growth of the POC diagnostic devices market . With thAlthough certain technical breakthroughs have been achieved, the industrialisation and commercialisation of porous MN devices have still been impeded for the following reasons. The current porous MN patches are designed to be small in size for primary utilisation through in vivo experimental tests. Thus, large-sized MN patches should be considered for practical applications in the real world related to human beings. Moreover, the substrate of the porous MN patch should be thin and flexible to be attached to the human skin. In addition, several reported fabrication processes suitable for laboratory scale might not be transferable to the industrial scale for mass production. In particular, the relatively high cost of the matrix material and manufacturing could limit the scale-up in the industry. Furthermore, the complicated and time-consuming fabrications that require several steps might increase the cost while diminishing the efficiency of production. Further, when assembled with biosensors for early diagnosis, affordable price and adequate reliability should also be considered for a competitive advantage in the current POC devices market. In addition, several years are required to design MN-based sensing devices for commercialisation due to the regulations that require the evaluation of accuracy and reliability through long-term clinical tests. Moreover, the sterilisation, usage, and disposal should be standardised for consumers or patients to handle medical devices appropriately and safely at home. Nevertheless, there is no doubt that in the near future, MN-based healthcare POC devices would be commercialised and even integrated with wireless communication modules to achieve comprehensive personal healthcare, which will result in the rapid growth of the POC market aimed at home health devices.This review primarily focused on and described porous MNs. The different characteristics of several other types of MNs were summarised and compared in terms of their inherent structures and working principles. In particular, for interconnected porous MNs, the continuous nano- or micro-sized pores offer a driving force for fluid transportation by capillary action. A wide range of matrix materials from non-polymers to polymers, which can be selected to fabricate a porous matrix, were listed and described. Various fabrication methods for porous structures were extensively developed based on the materials used, of which the processes and their advantages and disadvantages were also summarised. It is significantly important to optimise porous MNs with sufficient mechanical strength to maintain the MN structures for successful penetration into the skin of the patient. Moreover, fabrication methods of porous MNs that are suitable for scaling up mass production still require investigation and development. Porous MNs show promising perspectives in drug/vaccine delivery  as well as ISF extraction/biosensing . In particular, it can be envisioned that portable and minimally invasive POC devices comprising porous MNs and miniaturised biosensors for early and rapid diagnosis of diseases would perform a major and crucial role in home healthcare for the increase of self-testing and preventive action.


Conch shell bio-filler (CSBF) was prepared by washing, ultrasonicating, and pulverizing of conch shells and then was applied in waterborne intumescent fire-retardant coatings. The influence of CSBF on fire resistance and anti-ageing properties of intumescent fire-retardant coatings were studied by using different analytical methods. The fire protection and smoke density tests showed that when the mass fraction of CSBF was 3%, the resulting FRC3 coating had the optimum synergistic flame-retardant and smoke-suppression effects concomitant with a flame-spread rating of 10.7, equilibrium backside temperature of 152.4 °C at 900 s, and smoke-density rating value of 10.4%, which were attributed to the establishment of a more dense and stable intumescent char layer against heat and mass transfer. Thermogravimetric analysis indicated that the presence of CSBF increased the thermal stability and char-forming performance of the coatings, and the char residue of FRC3 rose to 34.6% at 800 °C from 28.6% of FRC0 without CSBF. The accelerated ageing test suggested that the incorporation of CSBF reduced the migration and decomposition of the flame retardants and the yellowing, blistering, and powdering phenomenon, thus improving the structural stability of the coating, resulting in better durability of flame retardancy and smoke-suppression performance. Intumescent fire-retardant coating is mainly composed of an intumescent fire-retardant system, a synergist, a binder, and an auxiliary agent, and is considered one of the most effective materials to protect substrates from fire hazards . The intThe incorporation of synergists is an effective way to improve the properties of intumescent fire-retardant materials, and the addition of inert fillers may slow down the speed of combustion and diminish the flammability of the material. There may also be synergistic or antagonistic catalysis associated with the fillers, which can effectively improve the drawbacks of intumescent fire-retardant systems. At present, a large number of studies have been reported on the application of fillers as synergists in traditional intumescent fire-retardant systems. As an environmentally friendly material, bio-fillers can significantly enhance the material properties ,9,10,11.In this work, the intumescent fire-retardant coatings were prepared with APP-PER-MEL as an intumescent fire-retardant system, waterborne epoxy resin as binder, and CSBF derived from conch shell as a synergistic agent. The effect of CSBF content on the fire resistance and anti-ageing properties of intumescent fire-retardant coatings was studied by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TG), X-ray diffraction analysis (XRD), a cone calorimeter test, and scanning electron microscopy (SEM). In addition, a possible synergistic flame-retardant and smoke-suppression mechanism between the CSBF and the intumescent fire-retardant system was proposed.®-Resin ERE 2581) and waterborne epoxy hardener (Wanna-Resin ERC 2610) were obtained from Guangzhou Oupeng Chemical Co., Ltd., Guangzhou, China. The acrylate copolymer carboxylate used as a dispersant was supplied by Qingdao Xingguo Coatings Co., Ltd., Qingdao, China. The non-silicon defoamer was supplied by Qingdao Xingguo Coatings Co., Ltd., Qingdao, China. The MEL (purity ≥ 99.5%), APP , and PER (purity: 99.5%) were from Hangzhou JLS Flame Retardant Chemical Co., Ltd., Hangzhou, China.The conch shells were provided by the faculty canteen of Central South University, Changsha, China. The waterborne epoxy resin .An illustration of the preparation of the intumescent fire-retardant coatings is shown in Fourier transform infrared spectroscopy (FTIR) was used to characterize the coatings and their char residues with an iCAN9 FTIR spectrometer  using KBr pellets.Scanning electron microscopy (SEM) using a MIRA 3 LMU  was applied to observe the microscopic morphology of the CSBF and char layer after combustion at a voltage of 20.0 kV.X-ray diffraction (XRD) analysis was performed with an Advance D8 X-ray diffractometer  with scanning conditions of: Cu–Ka radiation, a range of 3 to 70°, and a rate of 5°/min.3 with a wet coating density of 500 g/m2.The smoke-generation performance was tested on a PX-07-008 smoke-density tester for building materials  according to the GB/T8627-2007 standard. The dimensions of each plywood board were 75 × 75 × 4 mm3 and a coating density of 500 g/m2, was exposed to a Bunsen burner with an approximately flame temperature of 900 °C. The cabinet method test used an XSF-1 apparatus ; each plywood board had dimensions of 300 × 150 × 4 mm3 and was coated at a density of 500 g/m2. The tunnel method test conducted with an SDF-2 instrument ; each plywood board had dimensions of 600 × 90 × 4 mm3 and was coated at a wet density of 250 g/m2.The big panel method test was carried out on a K-type thermocouple with a MT-X multiplex temperature recorder  according to GB12441-2005. The coated side of the samples, with dimensions of 150 × 150 × 4 mmThe adhesion classification test was performed on a QFH-HD600 adhesion tester  to determine the adhesion of the coating according to GB/T 9286-1998.The pencil hardness test of the coatings was carried out on a QHQ-A portable pencil scratch tester according to GB/T 6739-2006.3 was placed in the aluminum foil with a thermal flux of 50 kW/m2.The cone calorimeter test was carried out according to ISO 5660-2002 standard procedure, and the sample with the dimensions of 100 × 100 × 3 mmWtheo) of the sample was calculated using Formula (1):TG analysis was conducted using a TGA/SOTA 851 thermogravimetric analyzer . Samples of 2–3 mg were treated from 35 °C to 800 °C at a heating rate of 10 °C/min under a nitrogen atmosphere. The theoretical char residue . One ageing cycle lasted 12 h, during which the UV exposure was 8 h at 60 ± 3 °C, and the condensation time was 4 h at 50 ± 3 °C. The samples were subjected to accelerated ageing tests for 2, 6, and 11 cycles. In order to ensure the samples received the same irradiance in the same ageing period, the locations of the samples were exchanged every 12 h during the ageing process.The accelerated ageing test was carried out on a UV-accelerated ageing tester  according to ASTM G154-2006. The ultraviolet lamp was irradiated at 0.76 W/, it was clear that the presence of CSBF caused a decrease in PHRR2 and THR, and the time for PHRR2 was significantly less, so the existence of CSBF could better inhibit the decomposition of the wood substrate. Compared to FRC0, the intumescent fire-retardant coatings with CSBF presented lower HRR and THR, while FRC3 demonstrated the minimum HRR and THR, indicating that the CSBF behaved well in improving the fire resistance performance of the intumescent fire-retardant coatings during combustion. However, the synergistic effect of the CSBF in the coatings relied on the amount of CSBF, and was weakened when the amount of CSBF in the coating exceeded 3 wt %. These results may have been dictated by the reaction of the CSBF with the phosphoric acid produced by the decomposition of APP to form thermostable calcium phosphate and calcium metaphosphate in the char layer, which effectively isolated the heat and mass transfer, thereby improving the fire resistance performance [The cone calorimeter test is a feasible way to evaluate the fire performance of intumescent fire-retardant coatings. The heat release rate (HRR) and total heat release (THR) curves of the samples are depicted in formance . NeverthThe light absorption curves and smoke density rating values of FRC0–FRC4 are given in The TG and DTG curves of FRC0–FRC4 are depicted in T0 and Wexp values of FRC1–FRC4 increased with the addition of CSBF, and the PMLR value first decreased and then increased, among which FRC3 exhibited the best thermal stability. Moreover, the Wexp value of FRC3 was higher than the Wtheo value, which indicated that the waterborne epoxy resin and the intumescent flame retardant reacted more strongly under the synergistic effect of the CSBF. It is possible that the presence of CSBF induced the reaction of calcium carbonate and APP to produce thermostable calcium phosphate and calcium metaphosphate, which then contributed to the formation of an expanding protective carbon layer to block heat and material transfer, thus enhancing the amount of char residue. The maximum ∆W was 15.9% for FRC3, indicating that the addition of 3 wt % CSBF in the intumescent fire-retardant coating had the optimum char-forming efficiency [As revealed in ficiency ,14. The The light absorption curves of FRC0 and FRC3 after different ageing cycles are illustrated in The fire protection performance of FRC0 and FRC3 after the ageing process is depicted in −1), P=O (1249 cm−1), N–H (1552 cm−1), C=N (1654 cm−1), and –NH2  groups were significantly enhanced after two ageing cycles [2, and N–H groups decreased significantly, which suggests the decomposition of the coating components. In particular, the –NH2 and N–H groups disappeared in the spectra of FRC0 and FRC3 after 11 ageing cycles. Compared to FRC0, the main functional groups of the FRC3 coating had stronger absorption vibration peaks upon the identical ageing treatment, which indicated that FRC0 had a higher degradation degree. Based on the above results, it was revealed that the addition of CSBF was beneficial to strengthening the structural stability and anti-ageing properties of the coating, which contributed to reducing the migration and decomposition of the flame retardants, thus endowing the coatings with better durability of their flame retardancy and smoke-suppression performances [Intumescent fire-resistant coatings are subject to ageing factors such as oxygen, irradiation, humidity, and temperature in actual use, and the ageing process can be detected by FTIR spectra. The FTIR spectra of FRC0 and FRC3 after different ageing cycles are presented in 1), P=O 19 cm−1, Ng cycles ,28,29,30The intumescent process of the coatings in the cone calorimeter test was analyzed, and the results are illustrated in −1) and –NH2  groups disappeared due to the decomposition of APP [−1), C–O (1016 cm−1), and PO32− (1084 cm−1) groups disappeared [−1) group appeared [−1), P–O–C (994 cm−1), C–O–C (1139 cm−1), and P=O (1284 cm−1) groups, demonstrating that the FRC3 char residue with CSBF produced more aromatic structures and phosphorus-rich cross-linking structures [To further investigate the expansion pattern of the char layer, n of APP ,27,28. Tappeared ,31, and ructures ,36,37. I3 and H2O), calcium phosphate, calcium metaphosphate, and phosphate. The orthophosphate and phosphate compounds esterified with the PER to form a molten layer, while the inert gases diluted the fuel gases and reduced the intensity of combustion. With the rise in temperature, the ester mixture and MEL decomposed into carbon via cyclization, while releasing a supply of inert gases to promote the expansion of the char layer. The presence of CSBF prompted the coating to form more cross-linking and aromatic structures during combustion, and formed a more compact and intumescent char layer that effectively inhibited the further decomposition of the coating, thus achieving better fire resistance and smoke suppression.The flame-retardant and smoke-suppression mechanism of the CSBF in the intumescent fire-retardant coatings is demonstrated in In this paper, CSBF derived from conch shells was produced and then characterized thoroughly using FTIR, TG analysis, XRD analysis, and SEM. The intumescent fire-retardant coatings were prepared with APP-PER-MEL as the intumescent flame retardant, waterborne epoxy resin as a film-forming polymer, and CSBF as a synergist. The effect of CSBF on fire resistance and anti-ageing properties of the intumescent fire-retardant coatings was studied. The results suggested that the introduction of CSBF enhanced the flame retardancy and smoke-suppression properties of the coatings, exhibiting superior synergistic effects. The synergistic efficiency of CSBF in the coatings depended on the content of CSBF; an excessive content of CSBF diminished the synergistic effect. The FRC3 coating containing 3 wt % CSBF showed the best flame retardancy and smoke-suppression properties among the samples. By comparison with FRC0, the weight loss, char index, flame-spread rating, maximum light absorption rate, and smoke density rating value of the FRC3 coating decreased by 24.2%, 42.2%, 38.2%, 44.2%, and 46.7%, respectively, and the intumescent factor of the char layer increased by 88.3%. A char residue analysis showed that the presence of the CSBF contributed to the formation of more cross-linking and aromatic structures during the combustion process, which effectively enhanced the denseness of the char layer structure, and thus exhibited a higher thermal stability and residual weight, as supported by the TG analysis. In particular, the FRC3 coating exhibited the highest residual weight of 34.6% at 800 °C and onset decomposition temperature of 222.6 °C in the TG analysis. The accelerated ageing test showed that an appropriate amount of CSBF could weaken the yellowing, blistering, and powdering phenomena of intumescent fire-retardant the coatings, thus imparting better durability of the flame retardancy and smoke-suppression properties to the coatings. In summary, CSBF is an effective synergist in the preparation of intumescent fire-retardant coatings, with excellent fire resistance and anti-ageing properties.


Moreover, 20-Hz MNS entrained neural oscillation over the dorsal STN, which correlated positively with motor disabilities. MNS allowed localization of the sensorimotor STN and identified neural characteristics under inhalational anesthesia. This paradigm may help identify an alternative method to facilitate STN identification and DBS surgery under inhalational anesthesia.Deep brain stimulation (DBS) improves Parkinson’s disease (PD) symptoms by suppressing neuropathological oscillations. These oscillations are also modulated by inhalational anesthetics used during DBS surgery in some patients, influencing electrode placement accuracy. We sought to evaluate a method that could avoid these effects. We recorded subthalamic nucleus (STN) neuronal firings in 11 PD patients undergoing DBS under inhalational anesthesia. Microelectrode recording (MER) during DBS was collected under median nerve stimulation (MNS) delivered at 5, 20, and 90 Hz frequencies and without MNS. We analyzed the spike firing rate and neuronal activity with power spectral density (PSD), and assessed correlations between the neuronal oscillation parameters and clinical motor outcomes. No patient experienced adverse effects during or after DBS surgery. PSD analysis revealed that peripheral 20 Hz MNS produced significant differences in the dorsal and ventral subthalamic nucleus (STN) between the beta band oscillation  and gamma band oscillation  ( Parkinson’s disease (PD) is a neurodegenerative disease characterized by loss of dopaminergic neurons, which causes abnormalities in the downstream basal ganglia such as abnormal firing patterns  and irregular oscillatory activity. Subthalamic nucleus (STN) deep brain stimulation (DBS) is an effective treatment for PD, possibly exerting its effects via various plausible mechanisms ,2. NeuroDBS surgery is typically performed under local anesthesia while the patient remains awake in order to ensure accurate electrophysiological mapping. This approach allows physicians to conduct intraoperative microstimulation tests to assess motor responses with minimal adverse effects. However, if a PD patient is experiencing marked off-medication symptoms such as anxiety, painful dystonia, and respiratory distress, the patient may not be amenable to this lengthy surgical procedure . In suchSomatotopic mapping within the STN during MER may facilitate optimal placement of DBS electrodes. Nevertheless, this procedure is time-consuming, particularly under inhalational anesthesia, and the STN topography may not be delineated by MER signals due to partial suppression by anesthetics . This maThus, in this study, we aimed to identify stimulation parameters and strategies by using peripheral MNS that would make it possible to avoid the unnecessary adverse effects of inhalational anesthesia in patients undergoing DBS surgery for PD; The characteristics of the PD patients are shown in Intraoperative recordings from 26 MER units in the dorsal parts and 29 units in the ventral parts were analyzed for spontaneous STN activity. All patients had similar motor disabilities before undergoing bilateral STN-DBS. Their symptoms were significantly improved by DBS treatment .p < 0.001; percentage of improvement from DBS off vs. DBS on, 57 ± 15.4; p = 0.002) and Part III scores .The effectiveness of STN-DBS did not differ among patients. Postoperative DBS substantially improved the clinical status of the patients. A follow-up examination at 14.0 ± 2.6 months after DBS surgery revealed that STN-DBS significantly improved the Unified Parkinson’s Disease Rating Scale (UPDRS) Part II scores , which was divided into theta-, alpha-, beta-, and gamma-band oscillations .When we compared the no-MNS condition with each of the MNS conditions for the whole STN, we found no significant differences. We then compared the dorsolateral and ventromedial STN activity under the different MNS conditions. In theta-band oscillations, a significant difference was found between no-MNS and MNS-5. In the alpha-band oscillations, a significant difference was found between the no-MNS, MNS-5, and MNS-20 conditions. The gamma-band oscillations differed significantly across all conditions. Moreover, we calculated the difference in percentage between the dorsal and ventral parts. The largest difference between beta- and gamma-band oscillations was observed for MNS-20 , while the no-MNS condition showed a larger difference in theta- and alpha-band oscillations  .p = 0.023), MNS-20 , and MNS-90  conditions. Moreover, these scores showed a negative correlation with gamma-band oscillations under the MNS-20 condition. In the ventral STN, only gamma-band oscillations under the MNS-5 condition showed a negative correlation with Part II scores . The Part III scores were strongly correlated with beta-band oscillations under the MNS-5 and MNS-20 conditions, and no correlation in the ventral STN. Firing rates were not correlated with any neurological symptoms.Spearman’s test was used to evaluate correlations between individual power bands in the dorsal or ventral parts of the STN under inhalational anesthesia with different MNS parameters and motor disabilities . We did In this study, we sought to identify new DBS stimulation parameters and strategies for PD patients by using noninvasive peripheral MNS to avoid unnecessary adverse effects of inhalational anesthesia in PD patients undergoing DBS surgery. We applied MNS at different frequencies  and observed neuronal activity during DBS under inhalational anesthesia. The firing rate showed a slight increase in the PDS in the dorsal STN for all MNS frequencies. In contrast, a nonsignificant decrease in firing rate occurred in the ventral STN under all MNS conditions.We analyzed the PSD to evaluate the effects of MNS at different frequencies on the oscillation frequency bands within the STN. The neuronal oscillations with no MNS under inhalational anesthesia and inhalational anesthetics were significantly different between the dorsal and ventral parts in terms of theta-, alpha-, and gamma-band oscillations, but not beta-band oscillations. MNS-20 produced beta-band oscillations with significantly higher power in the dorsal STN than in the ventral STN; this phenomenon did not occur under the MNS-5 and MNS-90 conditions. Beta-band oscillations (13–30 Hz) are the main feature distinguishing the dorsolateral STN and can be used to guide DBS electrode placement under local or inhalational anesthesia ,17,18,19An earlier study  demonstrOur study had several limitations. First, we did not include a control group receiving MNS without inhalational anesthesia  to compare differences in neural firings. However, our included patients underwent MNS under different conditions, but with the same anesthesia depth. This could help to elucidate how inhalational anesthetics have different effects on neural oscillation under different peripheral stimulation parameters. Second, we used desflurane or sevoflurane to maintain general anesthesia during neural recording of STN. We did not perform sub-group analysis between the different anesthetics due to limited case numbers. Finally, as this was a small case series study, the findings may be confounded by interpersonal variability. Future research with more patients might provide direct evidence without heterogeneity, along with additional insight into how peripheral neural stimulation modulates intracranial recordings and oscillations under various inhalational anesthetics.Eleven consecutive patients with PD who underwent bilateral STN-DBS from March 2018 were enrolled in this study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Tzu Chi General Hospital, . All patients gave their informed consent for inclusion before they participated in the study.All PD patients met the United Kingdom Parkinson’s Disease Brain Bank diagnostic criteria and demonstrated at least two main symptoms. Each patient underwent a levodopa test prior to surgery to confirm a positive levodopa response (>30% improvement in UPDRS Part II and Part III scores). Each patient underwent brain magnetic resonance imaging (MRI) preoperatively to rule out structural abnormalities. All patients underwent evaluation using the UPDRS in four different conditions: pre-operative on medication (Med on), pre-operative off-medication (Med off), post-operative off medication off DBS (Med off/DBS off) and post-operative on medication on DBS (Med on/DBS on). Details regarding the evaluation procedures (including UPDRS part II and part III) are described in our previous report ,32,33. TImages were obtained using a 1.5-T MRI scanner . The standard settings included T1-weighted axial images of 0.75-mm slice thickness and T2-weighted axial images of 2-mm slice thickness. Each sequence was conducted with contiguous slices. The images were transferred to the Digital Imaging and Communications in Medicine database and the Stealth Neuronavigation workstation . Image fusion software was used to fuse the two sets of MR images using 3D reconstruction. The tentative surgical target coordinates for placement of the tip of the permanent implantable electrode were set at the central and lowest border of the STN by direct visualization on MRI, as previously described . A LekseAll patients received inhalational anesthesia via endotracheal intubation. Anesthesia was initially induced by administering regular narcotic agents. In all patients, anesthesia was maintained using desflurane or sevoflurane inhalation during the surgical procedures. The depth of anesthesia was maintained at 0.5–1.0 minimal alveolar concentration, and each patient’s heart rate and blood pressure were monitored to ensure the absence of a cough reflex as well as changes in heart rate and blood pressure during the MER procedure.A constant current stimulator  was used to apply electrical stimulation to the contralateral median nerve during MER. A stimulation electrode was placed on the wrist . The stimulation parameters included a pulse width of 0.2 ms, an intensity of 30 mA, and frequencies of 5 Hz (MNS-5), 20 Hz (MNS-20), and 90 Hz (MNS-90).The microelectrode was 10–40 μm in diameter and 200 mm in length and had a <50-μm tungsten tip and recording impedance between 0.5 MΩ and 1 MΩ. The microelectrode signal was recorded using an intraoperative MER system  where the signal was amplified (×10) and filtered (300–3 kHz). Recording started at 10 mm above the planned target coordinates. The microelectrode was advanced in steps of 200–500 µm, with pauses at sites of robust neuronal firing. Firing at each depth was recorded for the no-MNS, MNS-5, MNS-20, and MNS-90 conditions. The latency of discharge from each depth was recorded for 10 s.Consecutive axial computed tomography slices of the brain  were obtained after surgery to exclude intracranial complications and localize the postoperative electrode coordinates through image fusion with the preoperative MRI . An acutMER traces were excluded based on the following conditions: (1) the duration of the recording trace was <10 s; and (2) the recording contained artifacts with amplitudes exceeding 300 µV or arm movements at the onset of the baseline “rest” condition. Each MER trace was classified as a dorsal or ventral trace based on the central point in STN-in and STN-out. To prove that the microelectrode recordings were under MNS, we extracted MER data about 4 to 8 s for analysis. In addition, we applied a notch filter to remove artifacts from the MNS which removed 5 Hz, 20 Hz, and 90 Hz signals under the MNS-5, MNS-20, and MNS-90 conditions, respectively.We detected a spike train by a threshold that was an amplitude >3.5 standard deviations every 1 s of recording and evaluated the firing rate over the entire session using 1-s bins. We analyzed the percentage change in firing rates at different MNS stimulation frequencies. Records were enveloped using the full-wave rectification method. We applied the PSD of the spike train with Thomson’s multi-taper method  to evaluU test was used to compare between no-MNS and the different frequencies of MNS, with p < 0.05 considered significant. Moreover, to evaluate the discrepancy between the dorsal and ventral parts in the four different frequency MNS conditions, Spearman correlations were used to estimate the association between STN neuronal activity parameters  and UPDRS scores (Part II and Part III) during off-medication. Statistical significance was set at p < 0.05.Statistical analyses were conducted using SPSS software 21  and MATLAB 2019 . The Mann–Whitney Anesthetics affect the neurophysiological features of PD under inhalational anesthesia, which may cause inaccurate placement of an implant electrode during DBS surgery. However, we found that MNS at 20 Hz may increase beta-band oscillations in the dorsal STN and increase gamma-band oscillations in the ventral STN to facilitate appropriate placement of the stimulating electrode during DBS surgery. Moreover, we showed that MNS may strengthen neuronal activity in the dorsal STN, which is related to motor symptoms.


Epoxy is considered to be the most popular polymer and is widely used in various engineering applications. However, environmental considerations require natural materials-based epoxy. This necessity results in further utilization of natural materials as a natural reinforcement for different types of composites. Corn cob is an example of a natural material that can be considered as an agricultural waste. The objective of the present work is to improve the economic feasibility of corn cob by converting the original corn cob material into powder to be utilized in reinforcing epoxy-based composites. In the experiment, the corn cob was crushed and ground using a grain miller before it was characterized by scanning electron microscopy (SEM). The corn cob powder was added to the epoxy with different weight fractions . In order to prevent corn cob powder agglomeration and ensure homogeneous distribution of the reinforcement inside the epoxy, the ultrasonic technique and a mechanical stirrer were used. Then, the composite’s chemical compositions were evaluated using X-ray diffraction (XRD). The mechanical experiments showed an improvement in the Young’s modulus and compressive yield strength of the epoxy composites, increasing corn cob up to 8 wt% by 21.26% and 22.22%, respectively. Furthermore, tribological tests revealed that reinforcing epoxy with 8 wt% corn cob can decrease the coefficient of friction by 35% and increase wear resistance by 4.8%. A finite element model for the frictional process was constructed to identify different contact stresses and evaluate the load-carrying capacity of the epoxy composites. The finite element model showed agreement with the experimental results. An epoxy containing 8 wt% corn cob demonstrated the optimal mechanical and tribological properties. The rubbed surfaces were investigated by SEM to identify the wear mechanism of different composites. Recent global challenges and improvements associated with the utilization of different materials in tribological applications and polymer composites’ performance have attracted the attention of many researchers. Polymer composites are lightweight materials used in various applications such as automotive, satellites, marine, aerospace, etc. . FurtherOne of the most popular polymers used in different engineering applications is the epoxy thermoset, which has many advantages that encourage industrialists to use it. Epoxy-based thermosetting resin can be used for its chemical and heat resistance , high co2. They found that 4 vol% TiO2 could considerably decrease the specific wear rate. Utilizing the same additives with varying weight fractions, TiO2, Srivastava et al. [2O3 with different weight fractions into epoxy matrix. They could prove that 1 wt% Al2O3 can improve the mechanical and tribological characteristics of the composite. Still, with the increase in Al2O3 weight fraction up to 3%, all the composite properties have deteriorated due to the additives’ agglomeration inside the matrix. Fouly et al. [2O3. They found that a 0.4 wt% Al2O3 could improve the epoxy composite’s mechanical and tribological properties.To enhance the properties of epoxy, researchers have tried to add some additives as a reinforcement and investigate the different characteristics of epoxy composites, including mechanical, tribological, electrical, and thermal qualities . The meca et al.  evaluatea et al.  investigy et al.  studied The reinforcements used with different composites can be classified as natural and synthetic additives . SynthetCorn cob is considered one of the most common agricultural wastes, and some researchers have tried to turn it into a useful material. Chen et al.  extracteAccording to the above literature survey, using natural materials as a reinforcement could affect polymer composites’ mechanical and tribological properties. Although many researchers investigated the effect of corn cob as a reinforcement additive with polymers, there is a lack of research on the impact of a corn cob on the tribological properties of polymer composites. Consequently, the objective of the present investigation is to evaluate the performance of epoxy composite after incorporating corn cob powder with different weight fractions, 2, 4, 6, 8, and 10 wt%. The density, hardness, Young’s modulus, and compressive yield strength were determined based on the corn cob content. The tribological characteristics are introduced by evaluating the friction coefficient and specific wear rate of different composites. A finite element model was constructed based on the mechanical and tribological results to assess the load-carrying capacity. The wear mechanisms of each composite were identified after evaluating the worn surfaces using scanning electron microscopy (SEM).The matrix material utilized in the current study was the commercial-grade Kemapoxy 150  epoxy resin kit consisting of crystal-clear resin, a density of 1.11 ± 0.02 kg/L crystal-clear epoxy hardener. As mentioned before, corn cob is considered one of the most common agricultural wastes. Corn cob is the backbone part of the corn that the kernels grow around. After removing the kernels, corn cobs were dried under the sun for three months to decrease the moisture. Then, the corn cobs were initially crushed into smaller particles utilizing hammering before transferring the crushed powder to the mono chamber of a grain miller at a speed of 250 rpm for 3 h, with 15 min grinding and 15 min rest. Finally, the achieved powder was dried again in an oven at a temperature of 70 °C for 5 h to ensure the removal of all moisture.The fabrication of tested samples passed through different stages. First, the corn cob powder was added to ethanol and mixed ultrasonically for 15 min to prevent powder agglomeration. Second, the liquid epoxy was added. To ensure homogenous dispersion of the corn cob powder inside the liquid epoxy, mixing using a mechanical stirrer was applied to blend the whole mixture for 30 min at 150 rpm. Thirdly, the epoxy/ethanol/corn cob mixture was inserted into a vacuum chamber to remove any voids and to fumigate the ethanol for 24 h. Fourthly, the hardener was added to the mix in a ratio of 1:2 to the epoxy weight and mixed again for 5 min. Eventually, the mixture was discharged into a mold taking the shape of needed specimens and placed again in a vacuum at 35 ◦C. A schematic diagram illustrating the fabrication process is shown in The mechanical characteristics of the fabricated epoxy/corn cob composites were evaluated to investigate the effect of adding corncob powder with different weight fractions. According to ASTM D2240 , the D-iSWR) was calculated using Equation (1), dependent on sliding distances (L), the difference in weight before and after the test (Δm), the density of each composite sample measured by Archimedes’ principle (ρ), and the applied normal load (nF).The tribological performance of epoxy/corn cob composites was evaluated in dry conditions according to ASTM G99-95 . A pin-oAfter the grain milling process, the corn cob particles’ morphology was characterized utilizing a scanning electron microscope (SEM). As shown in To investigate the effect of incorporating corn cob powder into epoxy resin on the chemical composition of the composites, X-ray diffraction (XRD) was utilized. The density of the produced composite is one of the most important properties that can affect the weight of the new generated composite. The density of composite materials usually depends on the density of both the matrix and reinforcement materials. Consequently, the current study used Archimedes’ principle to calculate the density of the fabricated epoxy/corn cob composites. In the same figure, The experimental results of the compression test of epoxy corn cob composites are shown in On the other hand, incorporating 10 wt% corn cob resulted in the deterioration of Young’s modulus and the compressive yield strength. This result could be attributed to the ease of debonding corn cob particles from the epoxy matrix because of the high corn cob weight fraction. Consequently, the load transmission from the epoxy to the corn cob particles failed; hence the mechanical characteristics diminished.The variation in the friction coefficient of neat epoxy and 2–10 wt% corn cob particles-reinforced composite under different normal loads (3–12 N) is illustrated in The finite element results show the generated equivalent and shear stresses variation due to the friction between different epoxy composite samples and the stainless steel disk, as shown in To validate the finite element results and evaluate the influence of different corn cob weight fractions on the tribological performance of epoxy composites, the specific wear rate of the epoxy composites under different normal loads was calculated. The microscopic analysis of epoxy composites rubbed surfaces were utilized to identify the wear mechanism during the friction process, as shown in The results revealed that corn cob could be used as a natural reinforcement for polymer-based composites.Without using an agent to enhance the bonding between the epoxy and the corn cob, the weight fraction of the corn cob is limited to 8 wt%.Increasing the corn cob weight fraction to 10 wt% decreased both density and hardness of the composite by 22.87% and 17.24%, respectively.For 8 wt% corn cob, Young’s modulus and compressive yield strength of the epoxy composite reached 21.26% and 22.22%, respectively.For 8 wt% epoxy/corn cob, the composite recorded a frictional coefficient of 35% less than pure epoxy, as well as enhanced wear resistance.The finite element results showed an enhanced load-carrying capacity due to corn cob incorporation.In conclusion, we conducted an experimental investigation on reinforcing epoxy-based polymer using corn cob, a natural material. The physical, mechanical, and tribological characteristics of the fabricated epoxy/corn cob composites were evaluated at different corn cob weight fractions, and the following conclusions are drawn:Finally, corn cob particles could be modified chemically to improve the interfacial bond between the epoxy and corn cob particles and to reinvestigate the new composite characteristics.


Escherichia coli (E. coli).Leukemia inhibitory factor (LIF) is a multifunctional cytokine which plays multiple roles in different biological processes such as implantation, bone remodeling, and hematopoiesis. The buESCs are difficult to culture due to lack of proper understanding of the culture conditions. LIF is one of the important factors which maintain the pluripotency in embryonic stem cells and commercial LIF from murine and human origin is used in the establishment of buffalo embryonic stem cells (buESCs). The LIF from a foreign origin is not able to maintain pluripotency and proliferation in buESCs for a long term which is contributed by difference in the binding sites on LIF; therefore, culture medium supplemented with buffalo-specific LIF may enhance the efficiency of buESCs by improving the environment of culture conditions. The high cost of LIF is another major drawback which restricts buESCs research, thus limits the scope of buffalo stem cell use. Various methods have been developed to produce human and murine LIF in prokaryotic system. However, Buffalo leukemia inhibitory factor (BuLIF) has not been yet produced in prokaryotic system. Here, we describe a simple strategy for the expression and purification of biologically active BuLIF in Bubalus bubalis) was cloned into pET22b(+) and expressed in E. coli Lemo-21(DE3). The expression of BuLIF was directed into periplasmic space of E. coli which resulted in the formation of soluble recombinant protein. One step immobilized metal affinity chromatography (IMAC chromatography) was performed for purification of BuLIF with ≥ 95% of homogeneity. The recombinant protein was confirmed by western blot and identified by mass spectroscopy. The biological activity of recombinant BuLIF was determined on murine myeloid leukemic cells (M1 cells) by MTT proliferation assay. The addition of BuLIF increased the reduction of MTT by stimulated M1 cells in a dose-dependent manner. The BuLIF induced the formation of macrophage like structures from M1 cells where they engulfed fluorescent latex beads. The recombinant BuLIF successfully maintained pluripotency in buffalo embryonic stem cells (buESCs) and were positive for stem cells markers such as Oct-4, Sox-2, Nanog, and alkaline phosphatase activity.The BuLIF cDNA from buffalo (E. coli through single step purification. BuLIF effectively maintained buffalo embryonic stem cells pluripotency. Thus, this purified BuLIF can be used in stem cell study, biomedical, and agricultural research.The present study demonstrated a simple method for the production of bioactive BuLIF in The online version contains supplementary material available at 10.1186/s43141-022-00328-1. Leukemia inhibitory factor (LIF) is a secretory multifunctional glycosylated protein which belongs to interleukin-6 (IL-6) cytokine family , 2. LIF Embryonic stem cells are promising therapeutic agents for use in regenerative medicines and the use of buESCs in livestock species have potential applications in the field of agriculture, pharmaceutical, and biomedical research –16. hLIFE coli expression system [. The recombinant protein is expressed either in cytoplasm or periplasm space of E coli. The over-expression of recombinant protein in cytoplasm leads to the formation of insoluble aggregates [Various researchers have successfully expressed and produced biologically active hLIF and mLIF in n system , 20, 21.gregates . Therefogregates –21, 23.E. coli expression system. In the proposed strategy, BuLIF was expressed as soluble protein in periplasmic space with the help of pelB sequence. One-step purification was done using IMAC . Finally, biological activity of purified BuLIF was confirmed by dehydrogenase activity of M1 cells followed by phagocytic assay. Finally the efficacy of BuLIF was tested on buffalo embryonic stem cells for its ability to maintain pluripotency.LIF is expressed by various cell types, thus serves as a promising therapeutic target for various diseases and disorders including infertility, cancers. LIF role in pregnancy is well studied in human and murine , 9; howe18 primer . The primers were designed from GenBank Acession No.: NM_001290925 nucleotides 67-606 to excluding the signal peptide coding sequence. The coding region of BuLIF was amplified using a forward primer containing NcoΙ restriction site (underlined) 5′-TAGTTCCCATGGCAAGCCCCCTTCCCATCACCCCG-3′ and reverse primer containing XhoІ restriction site (underlined) 5′-CAGTCTCGAGGAAGGCCTGGGCCAGCAC-3′. The BuLIF was amplified using Q5 High-Fidelity DNA Polymerase , in a GenePro Thermal Cycler . The thermocycling conditions were pre-incubation at 98 °C for 30 s and 30 cycles at 98 °C for 7 s, 62 °C for 30 s, 72 °C for 30 s, and final extension at 72 °C for 5 min. BuLIF was subcloned into pET22b(+) expression vector using NcoΙ and XhoІ restriction endonuclease sites and the resulting vector named pET22b(+)-pelB/BuLIF. This vector encodes coding sequence of N-terminal pelB leader sequence for periplasmic expression fused with BuLIF containing His6 for affinity chromatography. The vector pET22b(+)-pelB/BuLIF was confirmed by sequencing for correct and inframe cloning of coding sequence BuLIF with pelB sequence. The recombinant plasmid was confirmed by colony PCR, double restriction enzymes digestion using NcoΙ and XhoІ enzymes followed by nucleotide sequencing.Total RNA isolated from buffalo mammary epithelial cells (BuMEC)  by RNA pE. coli cells. The positive recombinant transformants were identified by colony PCR; briefly, overnight grown colonies were picked after transformation and mixed with PCR master mix . The gene specific primers were used to amplify the target BuLIF insert in the pET22b(+) construct. After confirmation of recombinant plasmids, a single colony was grown overnight at 37 °C in 5 ml LB medium containing 50 μg/ml ampicillin. The overnight grown culture was then sub-cultured into 5 ml LB broth containing 50 μg/ml ampicillin. The expression of target protein was induced with 1 mM IPTG when the optical density at 600 nm (OD 600) reached 0.6–0.8. The cells were harvested at different time intervals: 4 h, 6 h, 8 h, 12 h, and o/n. The expression of the target protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The maximum expression was observed at 12 h after IPTG induction. For purification, 5 ml of culture was inoculated into the 500 ml LB broth containing 50 μg/ml ampicillin. The culture induced with 1 mM IPTG and harvested by centrifugation at 3500×g for 30 min at 4 °C. The target protein was extracted from periplasmic space of E. coli via osmotic shock as previously described with slight modification [g. The supernatant was collected and cell pellets were re-suspended in hypotonic solution  and incubated at 4 °C for 30 min. The supernatant was collected in a clean 50 ml falcon tube after centrifugation and dialyzed against binding buffer  16 h at 4 °C. The dialyzed protein filtered through 0.45 μm syringe filter  and purified by immobilized metal ion affinity chromatography (IMAC). The 1 ml of Ni-NTA resin (BioRad) was loaded onto the gravity flow column and equilibrated with binding buffer . The filtered lysate was loaded onto the equilibrated resin and allowed to bind for one hour. The column was washed with washing buffer  to remove non-specific protein. The protein was eluted with elution buffer . The eluted protein was dialysed against phosphate buffer saline for overnight at 4 °C to remove imidazole and salts. The purity of recombinant protein was analysed by SDS-PAGE followed by coomassie staining. The concentration of protein was measured using Bradford method. The purified protein was filtered through the 0.22 μm syringe filter and stored at − 80 °C.The vector pET22b(+) pelB/BuLIF was transformed into Lemo21 (DE3) competent Silver staining is very sensitive method of detection of protein; it can detect 0.5–5 ng protein, so we performed silver staining to check the purity of BuLIF (23 kDa). SDS-PAGE was run and the gel was incubated in fixer , 50% water) for 1 h. Gel was washed repeatedly by water for 30 min and sensitized in 0.02% sodium thiosulfate for 1 min followed by washing with water. The gel was incubated in cold 0.1% silver nitrate solution for 20 min at 4 °C followed by washing with water. Gel was developed by 3% sodium carbonate and 0.05% formaldehyde and washed with water to avoid the excess of stain. Staining was terminated by 5% acetic acid by incubating for 5 min and gel was stored in 1% acetic acid at 4 °C.The recombinant protein was confirmed by the western blot using antiLIF antibody. Briefly, the purified protein was separated by electrophoresis and transferred on to the PDVF membrane  in transfer buffer  carried out on semi-dry blotting unit . The membrane was blocked using blocking buffer  for 2 h at room temperature. The membrane was further incubated at 4 °C for overnight with primary antibody  in dilution 1:1000 with blocking buffer. The washing was done with TBST three times at the interval of 15 min. The membrane incubated with secondary HRP conjugated antibody  in dilution1:2000 for 2 h at room temperature followed by three times washing with TBST. The membrane was developed using 3, 3-Diaminobenzidine tetrahydrochloride .4HCO3) and 40% ACN  in the 1:1 ratio. The gel pieces were dehydrated by adding 200 μl of 100% acetonitrile and incubated for 15 min at room temperature. The rehydration solution (5 mM DTT in 40 mM ABC) was added to the sample, incubated at 60 °C for 45 min to cleave the disulfide bonds. The sample was treated with 200 μl alkylation solution (20 mM Iodoacetamide) and incubated for 10 min in dark followed by addition of 200 μl of 100% acetonitrile. The gel pieces were treated by digestion buffer (12.5 ng/μl of trypsin in 50 mM ammonium bicarbonate buffer) followed by incubation at 37 °C for overnight. The reaction was stopped by adding 5% formic acid, and the peptides were extracted from gel by adding 100 μl of extraction buffer (5% formic acid in 40% ACN). The final extraction was carried out by adding 100 μl of 100% ACN, and the extracted peptides were dried in vaccum concentrator. Peptides were desalted using Zip tip  and eluted with 20 μl of 0.1% formic acid + 60% ACN). The peptides were analyzed by Q-TOF Bruker Mass Spectrometer (Maxis HD). The generated data was interpreted by ProteinScape with Mascot.The identification of purified recombinant BuLIF was verified by matrix assisted laser desorption/ionization-time of flight Mass spectroscopy (MALDI-TOF MS) as described previously . The pur2/ 95% air mixture for 24 h. Ten microliters of freshly prepared MTT solution was added to each well. The MTT solution was prepared in a concentration of 5 mg/ml in phosphate-buffered saline (PBS) and filtered through a 0.2 μm filter. The plates were incubated at 37 °C in a humidified 5% CO2/ 95% air mixture for 4 h. After formation of Formazan crystals, 75 μl DMSO was added to solubilize the MTT formazan and kept for 15 min on an orbital shaker. The absorbance was measured at 570 nm in microplate spectrophotometer. For the control, the cells were cultured in the absence of BuLIF. The data represent mean ± SD of three independent experiments.3--2, 5-diphenyltetrazolium bromide (MTT) assay was carried out to measure the activity of BuLIF. M1 cells (ATCC-TIB-192) were purchased from ATCC and routinely maintained in RPMI 1640 medium (Gibco) containing 10% FBS. Serial two-fold dilutions of different BuLIF preparation (10 to 50 ng/ml) were made in flat bottom 96-well microtitre plates  using multichannel pipette. M1 cells were harvested by centrifugation and seeded per well at the density of 10,000 cells . The plates were then incubated at 37 °C in a humidified 5% CO5 cells/ml in 24-well dishes. The fluorescent latex beads were added in dilution 1:300 into the cells and incubated for 24 h at 37 °C. The cells were analyzed using Inverted Fluorescence microscopy .The phagocytic assay was carried out as per manufacture’s instruction . In brief, the unstimulated M1 cells were treated with 40 ng/ml BuLIF for 4 days and seeded at 1 × 102 incubator (5% CO2 in air) in medium containing Tissue Culture Medium-199 (TCM-199) supplemented with 10% fetal bovine serum (FBS), porcine follicle-stimulating hormone , estradiol-17b (1 μg/mL), 0.81 mM sodium pyruvate, and 50 μg/mL gentamicin sulfate. The frozen semen straws were collected from ABRC, NDRI. The semen straws were thawed for 30 s in a 38.5 °C water bath. Sperms were washed twice by centrifugation (1200 rpm) for 7 min in 10 ml of Brackett and Oliphant medium (BO medium) containing 3 mg/ml of BSA supplemented with 10 mM caffeine. The washed pellet of sperms was resuspended in 0.5–1 ml BO medium. The matured cumulus-oocyte complexes were washed twice and transferred into a 50-μl drop of BO medium (20–25 oocytes/drop) containing 6 mg/ml of BSA and 10 mg/ml of heparin, and 50 μl of sperm suspension were added to each drop. Oocytes were incubated with sperm for 16–18 h at 38.5 °C in 5% CO2 in humidified air. After fertilization oocytes were cultured in RVCL media for 7 to 8 days.Buffalo embryonic stem cells were (buESCs) were cultured as described previously . The embFor culture of embryonic stem cells, the inner cell masses (ICMs) from hatched blastocyst were isolated mechanically by using microblades under zoom stereomicroscope . The ICMs were separated from trophoectoderm and seeded on to the mitomycin-C inactivated bovine fetal fibroblast cells. The ICMs were cultured in embryonic stem cells medium , 1% non-essential amino acids, 0.1 mM β-mercaptoethanol and 50 μg/ml gentamycin sulphate). Instead of mLIF or hLIF, homemade BuLIF was used in this assay. The putative ES cells with a uniform, undifferentiated morphology were selected individually using a micropipette, mechanically dissociated into two to six clumps and replated. The ES cells were passaged every 11–14 days after replating.After 5–10 passages, in addition to morphology, buESCs were characterized by the expression of embryonic stem cell markers. The immunocytochemistry was done with specific antibodies for Oct-4 , Sox-2 , Nanog  and a commercially available kit for detecting alkaline phosphatase .P values < 0.05 were considered statistically significant.Experiments were performed in triplicate. One-way analysis of variance (ANOVA) was performed using GraphPad Prism version 8.00  ). ) in frame with N-terminal pelB signal sequence that directed the expression of BuLIF into the periplasmic space of E. coli. The BuLIF was fused to a C-terminal hexahistidine (His6-tag) and the whole expression of pET22b(+) pelB/BuLIF vector was regulated by strong T7 promoter. The constructed pET22b(+)-pelB/BuLIF vector encodes a N-terminal pelB sequence in frame with the BuLIF sequence followed by a poly-histidine tag  . The recovery of bioactive protein is low from IBs, consequently to enhance the solubility of protein; various parameters are optimized such as temperature, IPTG concentration [The production of recombinant protein was achieved by transforming the pET22b(+)-pelB/BuLIF construct into Lemo-21 (DE3) host cell. The expression of BuLIF was observed at 4 h and 12 h post-IPTG induction, a band of approximately ~ 23 kDa was present in SDS-PAGE as shown in Fig. ntration . Severalntration , 29.Fig.2+ affinity chromatography. Supernatant was loaded on to the affinity column, after washing; the bound BuLIF was eluted into different fractions with elution buffer. SDS–PAGE analysis of purified recombinant BuLIF was corresponding to ∼ 23 kDa . The buESCs were cultured in media containing 40 ng/ml of purified BuLIF. After 7 passages 40–50 days) of culture, the buESC colonies showed normal morphology  [In the present study, we have successfully expressed BuLIF in the periplasmic space of .8 mg/L) –21. LIF We have tested the biological activities of BuLIF through different biological assays. The MTT proliferation assay on M1 cells showed that BuLIF inhibited the growth of M1 cells in dose-dependent manner, as the concentration of BuLIF increased, the growth of M1 cells was restrained. MTT is a dye which is reduced by mitochondrial dehydrogenases to a purple formazan. The viability of cells determines the amount of MTT reduction in to formazan crystals and has been correlated to lymphocyte cell growth stimulation or inhibition . M1 cellFurther, we evaluated the efficacy of BuLIF on buffalo embryonic stem cells (buESCs). The maintenance of buESCs in pluripotent state is difficult because of poor understanding about the culture requirements. Buffalo embryonic stem cells (buESCs) cannot be passaged in vitro continuously and it gradually loses its pluripotency during culture , 38. HerE. coli peripalsm via pET-based expression system. The recombinant BuLIF was purified by a single step chromatography and exhibited dehydrogenase activity on M1 cells. The purified BuLIF effectively maintained the pluripotency in buffalo embryonic stem cells. The quantity of purified protein recovered is enough for in vitro and in vivo studies.In conclusions, we report a simple protocol for the production of soluble and proper folded recombinant buffalo leukemia inhibitory factor in Additional file 1. Nucleotide sequencing of BuLIF. The in frame cloning of BuLIF into pET22b(+) vector was analyzed by sequencing. The sequence was in frame with pET22b(+) vector, the nucleotide sequence contained pelB sequence followed by NCoΙ restriction site which have ATG sequence to start the translation. The BuLIF gene was present containing 540 bp nucleotides followed by XhoІ restriction site, His6X tag and stop codon.Additional file 2. Mass spectrometry analysis of recombinant BuLIF protein. The purified BuLIF was digested and peptide were searched in database using PSM, protein, site decoy fraction FDR values of 0.01 minimum peptide length 7 amino acids. Confidences of the identification of peptides were shown by lowest PEP, score, Intensity, and high MS/MS counts.


The results show that the EPM can simultaneously achieve 99% H2O2 clearance and doubled glucose consumption rate. This strategy can be extended to the preparation of other dual- or multi-enzyme therapeutic nanoreactors, showing great promise in the treatment of metabolic disorders.Enzyme therapy has important implications for the treatment of metabolic disorders and biological detoxification. It remains challenging to prepare enzymatic nanoreactors with high therapeutic efficiency and low emission of cytotoxic reaction intermediates. Here, we propose a novel strategy for the preparation of enzymes-loaded polypeptide microcapsules (EPM) with concentrically encapsulated enzymes to achieve higher cascade reaction rates and minimal emission of cytotoxic intermediates. Mesoporous silica spheres (MSS) are used as a highly porous matrix to efficiently load a therapeutic enzyme , and a layer-by-layer (LbL) assembly strategy is employed to assemble the scavenging enzyme  and polyelectrolyte multilayers on the MSS surface. After removal of the MSS, a concentrically encapsulated EPM is obtained with the therapeutic enzyme encapsulated inside the capsule, and the scavenging enzyme immobilized in the polypeptide multilayer shell. Performance of the concentrically encapsulated GOx-catalase capsules is investigated for synergistic glucose metabolism disturbance correction and cytotoxic intermediate H Ilication . Inspirelication . To impr3 cores and CaCO3 separation layers, in which the cascade reaction starts from β-glucosidase on the outer compartment to GOx in the first peripheral, and to horse radish peroxidase (HRP) in the central [To date, various multienzyme nanoreactors with different spatial arrangements have been developed. These nanoreactors are mainly designed based on liposome or polymer-encapsulated enzymes including polymersomes and layer-by-layer (LbL) microcapsules ,20,21,22 central . However2O2 clearance since these enzymes have been frequently employed in therapeutic nanoreactor for blood glucose redressing [2O2 scavenging (99% clearance rate) in the EPM system is much more efficient than previously reported multienzyme nanoreactors, such as the co-localized multienzyme nanocomplex (about 85%) [Mesoporous silica spheres (MSS) have been widely used in biomedical applications due to their many distinct characteristics, such as highly tunable particle and pore size, high loading capacity, and easy surface chemical modification . In thisdressing ,18,37. Oout 85%)  and the −1), glucose oxidase from Aspergillus niger , acetic acid (HAc), 4-morpholineethan-esulfonic acid (MES hydrate), poly(acrylic acid) , tetraethyl orthosilicate (TEOS), poly-L-lysine hydrobromide , poly-L-glutamic acid sodium salt , 4-morpholineethan-esulfonic acid (MES hydrate), glutaraldehyde , 3-aminopropyl triethoxysilane (APTS), hexadecyltrimethylammonium bromide (CTAB), 3,3’,5,5’-tetramethylbenzidine (TMB), 7-(diethylamino)coumarin-3-carboxylic acid N-succinimidyl ester (DCCA), sulforhodamine B acid chloride (SRB), and fluorescein isothiocyanate isomer I (FITC) were purchased from Sigma-Aldrich . Horseradish peroxidase  and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Aladdin Reagent Company . All chemicals were of at least analytical grade and used without further purification. Fluorochrome-labeled enzymes and PLL  were prepared by adding 50 μL of dye (10 mg mL−1) to 1.0 mL of enzyme or PLL solution (10 mg mL−1) in 0.1 M sodium bicarbonate buffer at pH 9.0. After stirring at room temperature for 1 h, the mixture was sufficiently dialyzed against deionized water to remove free dyes.Catalase from bovine liver  included three steps. (1) GOx loading: 5.0 mg of amine-modified MSS  was washed with HAc buffer  twice to remove ethanol. Then 2.0 mL of GOx solution (1.0 mg mL−1) was added to the MSS suspension. After incubation at room temperature for 12 h, GOx-loaded MSS were collected by centrifugation . To crosslink the amino groups in GOx with the amine-modified MSS, 1.0 mL of fresh GA solution  was added to the GOx-loaded MSS suspension and incubated at room temperature for 1 h. After that, the particles were centrifuged and washed with HAc buffer twice. (2) PLL infiltrating: 1 mL of s-PLL  was used to infiltrate into the porous particles for 12 h. After the infiltration of s-PLL and removal of excess s-PLL, 2.0 mL of freshly prepared EDC solution  was added to crosslink the residual carboxyl groups on GOx and amino groups on s-PLL. After 2 h of incubation, the obtained materials were washed with MES buffer twice. (3) LbL shell assembling: Three PLL/PGA bilayers were deposited on the MSS surface by mixing 2.0 mL of alternative PGA/PLL (1 mg mL−1 in MES buffer) and MSS for 10 min. Twice washing with MES buffer was applied to remove excess polypeptide after each step of polypeptide deposition. After that, EDC was added to crosslink the assembled enzymes and polypeptides. The EPMGOx was obtained after removal of the MSS template with 0.3 mL of HF/NH4F solution (2 M HF: 8 M NH4F) for 1 min. The loading and leaching of Gox during EPMGox preparation were monitored by UV-vis spectrophotometry at the band of 276 nm (Gox-Cat) was prepared by the same procedure for EPMGox preparation except that the second enzyme  was sequentially assembled on the Gox-loaded MSSs before the PLL/PGA multilayer coating.Briefly, the preparation of GOx-loaded EPM (EPMf 276 nm . The con−1). The color reaction was initiated by adding 20 μL of 10 mM glucose solution at 37 °C. After 20 min, 50 μL of H2SO4 solution (2.0 M) was added to end the reaction and the UV-vis absorbance at 450 nm was recorded. Enzymatic activity of catalase was monitored by adding 10 μL of sample to 2.0 mL of 20 mM H2O2 solution in 50 mM PB buffer (pH 7.0) and the decomposition of H2O2 with time was measured spectrophotometrically at 240 nm. Catalase active unit was defined as the amount of catalase which decomposed 1 μmol of H2O2 per min at pH 7.0 at 25 °C. The changes of glucose and H2O2 concentration catalyzed by EPM in sealed glucose solution were monitored by a combining GOx-HRP color reaction in a 96-well microplate. Firstly, 20 μL of EPM (~8.6 μg of GOx) was added into 1.0 mL of glucose solution (10 mM) sealed with 1 mL of n-hexane in a water bath at 37 °C. Secondly, 10 μL of sample was extracted from glucose solution at a certain time intervals and added into a 10 μL of 2.0 M H2SO4 solution to deactivate the EPM. Finally, distinguishing measuring samples were added into 96-well microplates with 200 μL of HAc buffer (pH 6.0), 10 μL of GOx , HRP (0.1 mg mL−1), and TMB (10 mg mL−1) solution. After incubating at 37 °C for 30 min, 10 μL of H2SO4 solution (2.0 M) was added to each well and the absorbance at 450 nm was recorded. The concentration of H2O2 was calculated based on the color reaction in absence of GOx compared to standard H2O2, while that of glucose was calculated by the difference value in the presence and absence of GOx.Enzymatic activity of Gox was monitored by combining HRP and TMB-based color reactions. Briefly, 10 μL of sample was added to 2.0 mL of HAc buffer (pH 4.0) containing 15 ng of HRP and 10 μL of TMB  containing 200 µL of DMEM media (high level of glucose) at 37 °C in 5% CO2. After 12 h of incubation, 1.0 µL of EPM, EPMGOx, or EPMGOx-Cat suspension was added, and the sample was further incubated for 3 h before the 3--2,5-diphenyltetrazolium bromide (MTT) assay to test the cell viability.HeLa cells were seeded into a 96-well plate (~10Transmission electron microscope (TEM) images of EPM were collected from the Hitachi H-600 microscope . The high-resolution TEM images of MSS were obtained from Tecnai G2 F20 S-Twin . Scanning electron microscope (SEM) images were collected from a field emission SEM . Fourier-transform infrared (FTIR) spectra were measured on a Nicolet 6700 Spectrometer . The fluorescence images were obtained from a Leica DM4000 B microscope . The UV-vis spectra were recorded on a Shimadzu, UV-2600 spectrophotometer . The blood glucose concentration was monitored by a one-touch blood glucose meter .The MSS with an average particle size of 800 nm was synthesized according to a method reported earlier . High reAs GOx has an isoelectric point (IEP) of 4.2, the amino-modification of the MSS is essential to obtain a high enzyme loading through the electrostatic interaction of the negatively-charged GOx and the positively-charged MSS surface. The loading of GOx in the MSS particles could be adjusted by changing the mass ratio of MSS to GOx in the incubation solution. When the MSS/GOx mass ratio was set at 2.5, 250 mg of GOx could be loaded in one gram of MSS particles , which corresponded to an enzyme immobilization rate of 62.5%. The GOx loading could be increased to 35 wt.% when the MSS/GOx mass ratio was set at 1, however, the enzyme immobilization rate was decreased to ~36%. Thereafter, the mass ratio of MSS to GOx was fixed at 2.5 in following experiments.As the GOx molecules were adsorbed into the mesopores through electrostatic interaction, changing of the solution environments  could cause leakage of the physically adsorbed enzyme molecules. It was found that up to 90% of the loaded GOx could be released during the procedure to assemble a PLL/PGA multilayer shell on the MSS to encapsulate the loaded enzyme. To minimize the leakage of loaded GOx, 0.2 wt.% GA solution was used to crosslink the enzyme molecules to form a covalently connected enzyme network inside the mesopores. There is only ca. 20% of GOx released during the GA crosslinking and the subsequent PLL infiltrating steps .GOx in PBS at 37 °C for 24 h  and 58 μg mL−1 of catalase (2651 U mL−1). In a similar leaching test in PBS buffer, about 98% of catalase was retained in EPMGOx-Cat. The enzyme retention rate in the capsules was directly related to the structural stability of EPM. The high retaining rate of GOx and catalase in the EPM clearly suggested the structural stability of the crosslinked EPM.GOx was immobilized steadily in the MSS after GA crosslinking, however, 80% of the immobilized enzymes would be released provided the MSS template was removed. Inspired by previous work that enzymes could be stably immobilized inside nanoporous polymer particles by bridging proteins with infiltrated polymer ,40, we cfor 24 h . The fin32@GOx-loaded MSSs (EMPM), and EPMGOx-Cat were detected (−1), Si−O−Si symmetric stretching (801 cm−1), surface Si−OH groups (957 cm−1), and Si−O−Si asymmetric stretching (1000–1200 cm−1) [−1 displayed in the MSSs and EMPM was mainly attributed to the overlap of the amino group (1630 cm−1) and water molecules (1652 cm−1) [− symmetric stretching vibration (1401 cm−1), CH3/CH2 in-plane bending vibration (1450 cm−1), COO− antisymmetric stretching vibration and NH3+ symmetric stretching vibration (1548 cm−1) [To monitor component changes of the samples, FTIR spectra of relative intermediates including MSSs, (PGA/PLL)detected . The FTI00 cm−1) . These p52 cm−1) ,42. Afte48 cm−1) . These pGOx-Cat in solution. The merged fluorescent image shown in GOx-Cat.Homogeneity of the enzyme-loaded EPM was studied via fluorescence microscopy images. For distinguishing different species in the EPM, three fluorescent dyes with distinguishing excitation and emission wavelengths, including FITC (green), DCCA (blue), and SRB (red), were used to label GOx, catalase, and PLL, respectively. The brightly colored particles seen in the images a–c refle2O2 and cycling the cascade reaction were studied. To this purpose, EPMGOx-Cat was used to oxidize glucose in PBS solution in an enclosed environment. The concentration of glucose decreased by EPMGOx and EPMGOx-Cat showed approximately linear reduction (GOx (10.8 μM min−1) and EPMGOx-Cat (20.0 μM min −1) were significantly different, exhibiting a synergistic effect of EPMGOx-Cat. Catalase catalyzed the decomposition of cytotoxic intermediates H2O2 and generates O2, which could be further reused in the GOx catalyzed glucose oxidation reaction. The cascade reaction was performed circularly based on Equations (1) and (2), in which the cycle ratio could be significantly enhanced by the rapid H2O2 scavenging and the concentrically arranged bioreactor compartments in a microenvironment.Roles of the concentric compartment on scavenging the toxic reaction intermediate Heduction a, with a2O2 generated from glucose decomposition by EPMGOx exhibited a linear increase reaching up to 4 mM in 6 h  [2O2 generated during the glucose oxidation , in which no H2O2 was generated.HM in 6 h b. On conout 85%)  or supraout 85%) . The culxidation c. After 2O2 scavenging enzyme for therapeutic purposes, the synergistic effect of GOx and catalase on nanoreactor design has been rarely reported [2O2 and regenerative O2 will keep free diffusion and only part of these is involved in the cascade enzymatic reaction, resulting in compromised H2O2 scavenging. Herein, we designed the concentric compartment of EPMGOx-Cat to limit the release of toxic H2O2 and further utilized it as a useful intermediate for O2 generation. On the whole, during the enzymatic reaction, all substrates and products form a certain concentration gradient  required a treatment time of around 15, 30, and 60 min, respectively, to redress the high blood glucose (10.5 mM) to normal blood glucose (~5 mM).The therapeutic application of nanoreactors on metabolic disorder redressing was demonstrated through fast blood glucose oxidation by EPMsolution a. This eGOx-Cat and blood could be adjusted to avoid the over-consumption of blood glucose, as the dose of EPMGOx-Cat was directly proportional to the amount of glucose consumption  in 60 min, while those of 0.5% and 0.25% EPM declined around 7 times and 4 times, respectively. The reduction of reaction rate was mainly caused by the decreased concentration of substrates. This typical enzymatic reaction feature was further amplified by concentration-dependent diffusion of substances in EPM.It must be pointed out that over-consumption of blood glucose will cause hypoglycemia, which is dangerous for health. Even taking account of fast glucose supplement from hepatic glycogen, therapeutic nanoreactor should also consider the problem of over-consumption of glucose or other biomolecules. The ratio of EPMsumption . Interesv/v) EPMGOx-Cat can potently redress hyperglycemia from 10.5 to 5 mM in 15 min with an initial glucose consumption rate of 400 μM min−1 in mouse whole blood. Meanwhile, 99% of the cytotoxic reaction intermediates (H2O2) scavenging ratio and double glucose consumption rate were realized. The highly efficient and concentrically compartmented features of EPM holds enormous promise in developing more multienzyme therapeutic nanoreactors for addressing metabolic disorders and scavenging toxic intermediates.In this work, we have designed and fabricated potent therapeutic capsules by loading GOx inside MSS, assembly of catalase on the shell, and subsequent encapsulating the loaded enzymes with a PLL/PGA multilayer shell. After crosslinking, GOx and catalase were stably trapped inside the concentric multicompartments of EPM with uniform distribution and high enzyme loading. Owing to the concentrically encapsulated GOx and catalase and their deliberately arranged locations, 1% (


Xenopus anteroposterior axis specification, Rspo2 functions as a Wnt antagonist, both morphologically and at the level of gene targets and pathway mediators. Unexpectedly, the binding to RNF43/ZNRF3 and LGR4/5 was not required for the Wnt inhibitory activity. Moreover, Rspo2 did not influence Dishevelled phosphorylation in response to Wnt ligands, suggesting that Frizzled activity is not affected. Further analysis indicated that the Wnt antagonism is due to the inhibitory effect of Rspo2 on TCF3/TCF7L1 phosphorylation that normally leads to target gene activation. Consistent with this mechanism, Rspo2 anteriorizing activity has been rescued in TCF3-depleted embryos. These observations suggest that Rspo2 is a context-specific regulator of TCF3 phosphorylation and Wnt signaling.The Wnt pathway activates target genes by controlling the β-catenin-T-cell factor (TCF) transcriptional complex during embryonic development and cancer. This pathway can be potentiated by R-spondins, a family of proteins that bind RNF43/ZNRF3 E3 ubiquitin ligases and LGR4/5 receptors to prevent Frizzled degradation. Here we demonstrate that, during  Secreted Wnt proteins associate with Frizzled (Fzd) receptors and LRP5/6 coreceptors to stabilize β-catenin and promote β-catenin/T-cell factor (TCF)-dependent transcription. TCF3, also known as TCF7L1, is a predominant embryonic TCF that functions as a transcriptional repressor during early development5. In the presence of Wnt ligands, TCF3 is phosphorylated, followed by its dissociation from target promoters and transcriptional activation that can involve other TCF/LEF transcription factors including TCF1/TCF77. Whereas many studies of the Wnt pathway mainly focused on the control of β-catenin stability, the regulation of TCF protein activity has been less understood.The Wnt pathway is a key conserved developmental pathway that is utilized multiple times during animal development and frequently misregulated in disease8. The R-spondin (Rspo) family consists of four secreted proteins that share high similarity of amino acid sequence and structural organization and play critical roles in development, stem cell biology and cancer10. Mice lacking the rspo2 gene die at birth due to lung, limb and craniofacial defects, illustrating its essential functions in embryogenesis14. Additionally, Rspo2 has been implicated in fish skeletogenesis15 and frog muscle development16. The closely related Rspo3 functions in early angiogenesis in mouse and Xenopus embryos18. These observations highlight the important functions of R-spondins during embryonic development.R-spondins are prominent extracellular modulators of Wnt signaling in vertebrates16. R-spondins bind LGR4/5 receptors and the E3 ubiquitin ligases ZNRF3/RNF43, thereby stabilizing Frizzled and promoting Wnt signaling22. Recent analysis revealed that the mechanisms used by R-spondins to modulate Wnt signaling are more complex24. R-spondins can affect the Wnt pathway independently of LGR4/526, indicating the existence of multiple receptors and alternative signaling pathways. Supporting this view, the interaction with heparan sulfate chains is sufficient for R-spondins to modulate Wnt signaling in cells lacking LGR4/5/6 receptors27.R-spondins are thought to exert their effects by potentiating Wnt/β-catenin signaling30. Wnt signals promote posterior structures in the embryo, whereas secreted Wnt antagonists in the anterior region are responsible for head development32. One of the reported gain-of-function phenotypes for Rspo2 in Xenopus is the formation of ectopic cement gland16, an anterior mucus-secreting organ34. Notably, this phenotype is a common property of Wnt antagonists including GSK331, Axin-related protein35 and is exhibited in embryos with depleted β-catenin36. Since this observation is contrary to what is expected of a Wnt coactivator, we decided to reevaluate a role of Rspo2 in the Wnt pathway during Xenopus anteroposterior patterning. We show that Rspo2 inhibits Wnt signaling in a manner that is independent of the LGR4/5 and ZNRF3/RNF43 interactions. Mechanistically, we find that Rspo2 downregulates TCF3 phosphorylation that is necessary for target gene activation. Our findings indicate that the same R-spondin can function in a context-dependent manner to either stimulate or inhibit the Wnt pathway.The Wnt pathway plays crucial and specific roles during anteroposterior axis specification and patterning37, the injected embryos developed enlarged cement gland and other head structures  and splicing-blocking (RMOSB) morpholino oligonucleotides (MOs)37. Both MOs strongly reduced otx2 and ag1 levels , were expressed at similar levels and induced enlarged or ectopic cement glands in the majority of the injected embryos Vlemx, that contains a multimerized Wnt response element driving the expression of destabilized GFP57. Coinjection of Rspo2 RNA into the transgenic embryos with mRFP RNA as a lineage tracer suppressed GFP fluorescence at the injected side  and the C-terminus enriched in basic amino acid residuesons Fig. A to inteFirst, we asked which constructs retain the ability of full length Rspo2 to anteriorize the embryo. Rspo2 lacking the TSP domain (Rspo∆T) had a strong cement gland-inducing activity  during gastrulationect Fig. D. These Furthermore, TCF3 phosphorylation became prominent in the dorsal marginal zone explants isolated from Rspo2 morphants Fig. E. This eIf Rspo2 modulates Wnt target genes by inhibiting TCF3 phosphorylation, the depletion of TCF3 should prevent Rspo2 gain-of-function phenotype. For gain-of-function we overexpressed Rspo∆T RNA that consistently produces an ectopic cement gland , and posterior (cdx4 and msgn1) markers were partially rescued in the morphants by ∆βTCF3 CF3 Fig. A.Figure 61. Notably, Rspo2 did not downregulate axin2 induction by tcf1 RNA in ectoderm cells, whereas it significantly reduced Wnt8 activity in the same experiment  was constructed by inserting PCR-amplified coding region of Rspo2 into the EcoRI and BamHI sites of pCS2-Flag. pCS2-Rspo∆F-Flag lacks amino acids 37–134. pCS2-Rspo∆T-Flag lacks amino acids 147–204. Alanine substitutions have been made in pCS2-Rspo2-Flag or pCS2-Rspo∆T-Flag in the furin-like domain 1 (R65A or Q70A), and furin-like domain 2 (F105A or F109A) to generate Rspo2 that does not bind ZNRF3/RNF43 or LGR4/5 as described41. Various Rspo2 constructs containing deletions or alanine substitutions have been generated from pCS2-Rspo2-Flag using single primer-based mutagenesis as described31. Primer sequences are listed in Supplementary Table The DNA clone 6,988,843 encoding 81, pSP64T-Wnt882, pCS2-Wnt8, and pSP64T-Wnt5a83, ∆βTCF33, pCS2-TCF17, pCS2-mRFP (membrane-targeted), pCS2-Rspo-Flag, pCS2-Rspo∆F, pCS2-Rspo∆T, pCS2-RspoR65A-Flag, pCS2-RspoQ70A-Flag, pCS2-RspoF105A-Flag, and pCS2-RspoF109A-Flag. The following MOs have been purchased from Gene Tools : RMOATG, 5′- AAAGAGTTGAAACTGCATTTGG -3′, RMOSB, 5′- GCAGCCTGGATACACAGAAACAAGA-3′, control MO (CoMO), 5′-GCTTCAGCTAGTGACACATGCAT-3′. TCF3MO has been described previously3.Capped mRNAs were synthesized using mMessage mMachine kit . The following linearized plasmids have been used as templates: pSP64T-Wnt3aXenopus laevis embryos were carried out as described84. All manipulations have been carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experimental protocols have been approved by the IACUC of the Icahn School of Medicine at Mount Sinai. Staging was according to Nieuwkoop and Faber85. The Wnt reporter pbin7LefdGFP transgenic line of Xenopus laevis embryos Xla.Tg(WntREs:dEGFP)Vlemx57 have been obtained from the National Xenopus Resource . We routinely pooled 5 embryos or 10 explants for each experimental condition to minimize variability of transgene expression. For microinjections, four-cell embryos were transferred into 3% Ficoll in 0.5 × Marc’s Modified Ringer’s (MMR) buffer 86 and 10 nl of mRNA or MO solution was injected into one or more blastomeres. Amounts of injected mRNA and MOs per embryo, indicated in figure legends, have been optimized in preliminary dose–response experiments. Control MO was injected as at a dose that matched the highest amount of any other MO used in the same experiment.In vitro fertilization and culture of p values, e. g. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.Embryos were imaged at the indicated stages using Leica Wild M10 stereomicroscope using the OpenLab software. Unless otherwise specified, each experiment has been carried out at least three times. Statistical analyses were performed using GraphPad Prism 6 software. Data are shown as means ± s.d. and statistical significance was assessed using an unpaired two-tailed Student’s t-test or Fisher's exact test. Significant differences are indicated by Ectoderm explants were prepared at late blastula stages and cultured until the indicated time to observe morphological changes or lysed for RNA extraction or immunoblotting. Marginal zone explants were dissected at early gastrula stage and cultured until stage 12.5 when they were lysed for immunoblot analysis.To inhibit FGF receptor activity, ectoderm explants or marginal zone explants have been cultured with SU5402  from the time of isolation until they were lysed for immunoblot analysis.ef1a1 served as an internal control. Means + /− s. d. are shown. Statistical significance was assessed using the Student’s t-test.For quantitative PCR (RT-qPCR) and RNA sequencing, RNA was extracted from a group of 4–5 embryos, ten animal caps or ten marginal zone explants, at stages 10 or 12.5, using RNeasy kit (Qiagen). RNA sequencing was carried out using the HiSeq PE150 platform  and analyzed by Novogene . cDNA was made from 1 µg of total RNA using iScript (Bio-Rad). qPCR reactions were amplified using a CFX96 light cycler (Bio-Rad) with Universal SYBR Green Supermix (Bio-Rad). Primer sequences used for RT-qPCR are listed in Supplementary Table 87. Briefly, 10 animal caps or 7 marginal zone explants at stage 12.5 were homogenized in 50 µl of the lysis buffer . After centrifugation for 3 min at 16,000 g, the supernatant was subjected to SDS-PAGE and western blot analysis following standard protocols87. The following primary antibodies were used: mouse anti-FLAG , mouse anti-non-phosphorylated β-catenin , rabbit anti-XTCF3N88, rabbit anti-Dvl287. Staining with rabbit anti-Erk1  was used as loading control. Chemiluminescence was captured by the ChemiDoc MP imager (BioRad).Immunoblot analysis was carried out essentially as describedkrt12.489, foxg1/BF190, and cdx437, was carried out as described91.Whole-mount in situ hybridization with the digoxigenin-labeled antisense RNA probes for Supplementary Information.