Inverted perovskite solar cells (PSCs) have been extensively studied by reason of their negligible hysteresis effect, easy fabrication, flexible PSCs and good stability. The certified photoelectric conversion efficiency (PCE) achieved 23.5% owing to the formed lead−sulfur (Pb−S) bonds through the surface sulfidation process of perovskite film, which gradually approaches the performance of traditional upright structure PSCs and indicates their industrial application potential. However, the fabricated devices are severely affected by moisture, high temperature and ultraviolet light due to the application of organic materials. Depending on nitrogen, cost of protection may increase, especially for the industrial production in the future. In addition, the inverted PSCs are found with a series of issues compared with the traditional upright PSCs, such as nonradiative recombination of carriers, inferior stability and costly charge transport materials. Thus, the development of inverted PSCs is systematically reviewed in this paper. The design and fabrication of charge transport materials and perovskite materials, enhancement strategies  and the development of all−inorganic inverted devices are discussed to present the indicator for development of efficient and stable inverted PSCs. At present, humanity is faced with serious energy and environmental issues ,2. Even Perovskite solar cells (PSCs), as a novel type of solar cells, have received a lot of attention and research because of their wide light absorption range, high optical absorption coefficient and high carrier mobility of metal halide−based perovskite materials. With a few short years, PSCs have made a significant breakthrough, and the certified PCE has already achieved 25.5% . To date3 )/ETL. The hysteresis effect of inverted PSCs can be significant reduced compared to that of traditional upright PSCs. Moreover, the inverted structure possesses good interface stability, fewer defect states between interfaces, and it is beneficial to prepare the flexible devices with low temperature process  (TAPC) as HTL in inverted PSCs [oc of 1.15 V. Li et al. used the micro−molecule organics (NPB) without dopants as HTL in MAPbI3 based inverted devices, leading to a prominent absorption layer with better crystallization properties, as well as a best PCE of 19.96% [2Br PSCs, which exhibited a greatest efficiency of 15.4% because of the enhanced photoelectric properties, superior interfacial energetics, matched energy band structures and reduced charge recombination [Compared to the conductive polymer materials with high cost and complex synthesis process, small−molecule hole−transport materials not only have the advantages of simple synthesis, low cost and easy purification, but also can optimize the chemical structure and photoelectric performance by molecular engineering, which has gradually attracted the interest of researchers . With thted PSCs . Smooth,ted PSCs . The insf 19.96% . In 2019f 19.96% . As a ref 19.96% . Not onlbination . Furtherx is a typical P−type inorganic material with a wide bandgap, usually representing a mixture of nickel (IV) and nickel (III) oxide (NiOx), which is the focus of current researches among P−type inorganic semiconductors. The researches show that NiOx has high transmissivity, efficient charge extraction capability, and the matched energy level with perovskite [x. Docampo et al. adopted NiOx as HTL in the inverted PSCs through calcination of nickel acetate tetrahydrate and monoethanolamine on a conductive substrate for the first time [x in inverted PSCs is difficult to support an enough thick perovskite absorption layer, leading to a relatively inferior covering surface of absorption layer on NiOx substrate. Thus, the enhancement of anchoring capability for NiOx is the key to improving the NiOx based devices. For this matter, Zhu et al. synthesized the NiOx layer with high transparency as HTL via a sol−gel process [x nanocrystals have a corrugated surface and well contacted with FTO substrates, which could support a sufficiently thick perovskite (300 nm) film. As a result, an excellent efficiency of 9.11% was acquired for the inverted devices on the basis of NiOx nanocrystals HTL. However, the photoelectric performances of inverted PSCs on the basis of the NiOx are still far behind that of inverted PSCs based on polymers materials and PSCs based on n−i−p structure. For this reason, in 2015, Park et al. improved the efficiency to as high as 17.3% with the FF value of 0.813 by preparing a well−ordered nanostructured NiOx with a pulsed laser deposition method, which was the highest efficiency achieved at that time for the inverted PSCs based on P−type inorganic materials [x film with cubic structure as HTL via a solution process with optimized concentration [x film, with high conduction band, exhibited a superior electron blocking property, leading to a cell with less hysteresis, better air storage stability and 14.42% PCE. Islam et al. employed the sputter−deposited method to prepare the NiOx film as HTL [x, the inverted devices based on the polycrystalline NiOx film showed a better stability and a high PCE of 15.2%. Tang et al. prepared the NiOx film with superior transmittance via sol−gel method, and its matched energy level and excellent optical transmittance led to a high PCE of 18.15% for the inverted devices [x (np−NiOx) film via a coprecipitation process, and an excellent efficiency of 19.1% was acquired for the inverted devices on the basis of np−NiOx [NiOrovskite ,140,141.rovskite ,144,145 rst time . Howeverntration . The obtm as HTL . With op(1)xDoping NiOx HTLs. Doping elements are usually alkali metals , alkaline earth metals , transition metals  and micro−molecule organics (F6TCNNQ), as is shown in x film are as follows: (1) Higher conductivity can effectively accelerate the extraction and transfer of photoexcited charges at NiOx/perovskite interface and reduce the series resistance of devices; (2) The work function of NiOx film is closer to top of the VB of perovskite film, resulting to the higher hole mobility; (3) The transmittance of NiOx film is improved, and absorption layer on NiOx layer has better crystallinity and higher coverage.Element doping is one of the most efficient ways to improve photoelectric performance of NiOx film as a dopant by Kim et al. [x, which accelerates extraction of photoexcited holes at NiOx/perovskite interface and reduces the series resistance of PSCs. It turned out that the efficiency of copper (Cu)−doped NiOx (Cu:NiOx) based device was increased to 15.4%, as well as the Jsc and FF. In 2015, Chen et al. added lithium (Li+) and magnesium (Mg2+) to conduct heavily doped the NiOx film to promote photoelectric performance of NiOx. Strategy of lithium−magnesium (Li+ − Mg2+) doping NiOx film (Li0.05Mg0.15Ni0.8O) enhanced the extraction of photoexcited charge, and avoid the morphology of pinholes, as well as the local structural defects of PSCs for the large area devices [2+ doping compensates the band shift caused by Li+ doping into the lattice, leading to a high PCE of 18.3% and a large−area (˃1 cm2) device with 16.2%. Cesium (Cs2+) doped NiOx film (Cs:NiOx) was conducted to prepare the HTL in inverted PSCs devices by them in 2017 [2+ ions improved the conductivity and work function of NiOx film, and finally realized a high PCE of 19.35%. In 2018, Wei et al. reported a novel inorganic HTL based on Ag+ doped NiOx (Ag:NiOx) [+ (AgNi) and behaving as the acceptor in NiOx lattice. Absorption layer on Ag:NiOx HTL show superior crystals, preferable covering surface and smoother topography, leading to a PCE of 16.4%, consequently. Yttrium (Y) was incorporated into the NiOx film as a dopant via sol−gel method by Hu et al. [x HTL exhibited high hole mobility and quality morphology of perovskite film, which promoted interface charge recombination and transfer, and achieved an efficiency of 16.31%. Chen et al. successfully introduced an organic small molecule (F6TCNNQ) into the preparation of NiOx film, which improved Fermi energy level of NiOx and reduced energy level offset between NiOx and absorption layer [Copper (Cu) was incorporated into the NiOm et al. . Doping  devices . In addi in 2017 . The intAg:NiOx) . Transmiu et al. . The obton layer . Consequ(2)Interface modificationx, which can avoid possible uneven doping and unmanageable disorders caused by element doping, as is shown in x film in the inverted devices [x film through spray pyrolysis method and introduced an inert mesoporous alumina (Al2O3) with high transmittance as the barrier layer, leading to a higher PCE of 13.5% [oc from 1.07 eV to 1.15 eV. Lian et al. used an insulating film of PTAA film to improve the contact at NiOx/absorption film interface to decrease defects, which led to a better energy level arrangement, a deeper HOMO of NiOx/PTAA, less interface energy loss and a superior Voc as high as 1.19 eV [x film, resulting in a highest PCE of 22.13% with an FF of 82.8% [Interface modification is another efficient way to promote photoelectric properties of NiO devices ,154. In of 13.5% . Howeverof 13.5% . The mod 1.19 eV . In 2020of 82.8% . Therefo3/PCBM/Au structure [Cu−based materials (CuX), as typical P−type materials, have high carrier mobility and carrier diffusion length. More importantly, the matched energy level of CuX HTLs can effectively prevent the leakage of photoexcited electrons and reduce the energy loss of perovskite solar cells, especially for copper thiocyanate (CuSCN) and copper iodide (CuI). Hence, several representative inverted devices on the basis of the Cu−based HTL structures were reported, as is shown in tructure . In 2017tructure . The traxO has a narrower bandgap (2.2 eV) and the work function of −5.4 eV in comparison to CuSCN and CuI. The CuxO crystal with small size is easy to get high quality film with uniform surface topography, which is conducive to the crystalline grow of perovskite film. Wu et el. prepared an ultrathin Cu2O thin films via thermal oxidation method and applied in the inverted devices with CuOx/MAPbI3/PCBM/Ag structure [2O film, and an efficiency of 11% was acquired with the thickness of 5 nm Cu2O films through precisely optimizing the thickness of Cu2O layer due to the high hole mobility, matched energy structure with perovskite film and long lifespan of photoexcited charges. In 2017, Yu et al. prepared CuOx film as HTLs in inverted PSCs via a solution process, and the high quality of perovskite film was achieved based the CuOx film [3PS4 [3PS4 nano materials as a novel HTL for inverted devices, which was conducive to growth of absorption film on the Cu3PS4 HTL and leads to a maximum PCE of 18.17% with less hysteresis [P−type Cutructure . The phouOx film . As a relm [3PS4 ,167. Raolm [3PS4 . Yin et oc and FF are relatively low, which can be ascribed to low charge collection at HTLs/absorption layer interface and large series resistance at the interface. Hence, interface modification, the hole transport bilayer and ion doping can be used to improve the photoelectric properties of HTL. In 2018, Wang et al. incorporated the CuSCN into the CuI HTL by a solution process method, resulting in high quality HTLs with higher conductivity and an impressive PCE of 18.76% [Compared with NiOx, Cu−based materials have a relatively narrow band gap (2~3 eV), which is beneficial to inhibit the recombination of photoexcited charge due to the reverse transport of photoexcited holes. However, the Cu−based materials have higher requirements on the quality, thickness and transparency of HTLs. Compared with other hole transport materials, Vf 18.76% . Javaid f 18.76% .x and Cu−based materials, other inorganic semiconductor materials were used as the HTL for inverted devices, for instance V2O5, MoOx and CoOx, as is shown in 3/PCBM/BCP/Ag exhibited an enhanced PCE of 10.8% with good reproducibility and desirable stability. Chen et al. employed oxo−functionalized (oxo−G1) as HTL in inverted devices by a solution process to solve the stability issues of PSCs [1−based inverted devices illustrated an obviously enhanced stability and an efficiency of 15.2% with the outstanding Voc of 1.1 V. Wang et al. treated GO with ammonia via a general strategy to prepare the HTL for the inverted devices, considering the acidic nature of GO [2 and the perovskite layer and used infrared annealing (IRA). The final PCE was 18.30%, and the hysteresis of the devices was significant reduced [In addition to NiO of PSCs . As it tre of GO . Consequ reduced .2O5 has a narrow band gap (2.0 eV) and a low work function (−5.2 eV), which could gain the effective hole extraction junction with absorption film and has been investigated to replace the frequently used HTL in inverted PSCs [2O5 as the hole transport materials for inverted devices with the construction of V2O5/MAPbI3−xClx/PCBM/TiOx/Al, and the PCE was less than 1% [2O5 thin films through a solution process method and obtained device with PCE of 5.1%. After that, they fabricated thin V2O5/PEDOT nanoribbons via a cocoon−to−silk−fiber reeling process as HTL for inverted devices, resulting in a high PCE of 8.4% [2O5 and P3CT−K as HTL in the inverted PSCs, which gave rise to the boosting efficiency of 19.7% [2 for more than one month. Anizelli et al. reported that luminescent−down−shifting quantum dots enable filtering of UV radiation with increased solar cell stability [Vted PSCs ,175,176. than 1% . Guo et tability . The PCEtability . They fotability .x has been developed as HTL by Tseng et al. [x film as the effective HTL in inverted PSCs by using the solution processing [x layer has been used to effectively improve the perovskite layer with uniform and well−packed film and decrease charge recombination synchronously. More importantly, CoOx−based devices exhibited an enhanced efficiency of 14.5% and outstanding long−time stability, which retained a PCE about 12% for over 1000 h.To avoid the issue of stability, the thermal evaporated MoOg et al. . Furtherocessing . IntroduIt is well−known that the HTL are essential interfacial contact layers between perovskite and electrode for inverted devices. Selective transport of holes by HTLs can not only accelerate the extraction of photoexcited holes at HTL/absorption layer interface and reduce the energy loss, but also efficiently inhibit electrons leak for photoexcited carriers. At present, an increasing number of effective and low−cost conductive polymer materials, such as polymethyl methacrylate (PMMA) and self−assembled monolayers (SAM), were developed as alternative HTL for inverted devices, which are wettable with perovskite materials or their precursors and beneficial to the growth of perovskite films with high quality. In addition, their softness makes them more compatible with flexible devices. Compared with conductive polymer materials, most of inorganic P−type semiconductors have low work functions, superior carrier mobility, excellent optical transparency and stability. The extraordinary advantages are conducive to the fabrication of high efficiency inverted PSCs with exceptional stability.3, as 3NH3+ (MA+) and HC(CH2)2+ (FA+), Cs+ and Rb+. B ions are usually divalent metallic ions, including Pb2+ and Ge2+, etc. X ions are halogen ions, including Cl−, Br− and I−. MAPbI3 is the most widely used perovskite material in the early stage. However, MAPbI3 decomposes easily in hot and humid environment, which affects the long−term stability of the device [3 has good resistance to high temperature and humidity, and a narrow band gap can expand the absorption region [3 phase is unstable in the cubic crystal structure and easily transforms into the hexagonal crystal structure, which greatly weakens its capacity of light absorption. Adding some ions with small radius, such as MA+, Cs+ or Rb+, to obtain other combinations, such as RbCsFA, RbMAFA, CsFAMA and RbCsMAFA, is an effective method to stabilize FAPbI3 phase and inhibit the phase transformation [+ ions, such as CsPbI3, CsPbI2Br, CsPbIBr2 and CsPbBrThe best B−site metal ion is Pb2+, but its toxicity is not suitable for commercial production in the future. Replacing the Pb2+ ion with other non−toxic metal ions is an effective method to solve this problem. However, Sn2+ is unstable and can easily be oxidized into Sn4+ in the air to form self−doping, which leads to the decomposition of absorption layer and the low efficiency of the PSCs [2+ and Bi2+ are used to replace Pb2+, it will lead to the low efficiency due to the serious recombination of carriers. The radius of halide ions can increase the lattice constant of perovskite materials. The diffusion length of carriers can be increased from 100 nm to 1 μm after Cl− doping in MAPbI3, and the formation of defects can be reduced [sc and increase the Voc [Inverted PSCs and the traditional upright PSCs have the same structure and requirements for the perovskite absorption materials, so the inverted PSCs can adapt to the perovskite absorption materials that the traditional structure can adapt to. The perovskite absorption material of PSCs should satisfy the structure of ABXe device . Insteadn region . Howeverormation ,185. Altthe PSCs . If Ge2+ reduced . The int the Voc , and a s2+ ions, Pb−I anti−site, MA vacancies, halide ions vacancies and shallow level defects. Moreover, surface or grain boundary passivation is a common method to solve above defects of perovskite materials. In addition, various processes have been used to inhibit non−radiative recombination of carriers, such as the increase of grain size, ion compensation, secondary growth, heterojunction engineering and 2D/3D mixing, which are the most common methods [Apart from composition of perovskite film, preparation and processing of perovskite films also play a decisive part in the properties of perovskite film. According to reports, defects on the surface or crystal boundary of perovskite crystals are caused by the solution process, which leads to non−radiative recombination of carriers and degradation of device efficiency . The pri methods ,191,192.60 and its derivatives as well as other non−fullerene electron transport materials.In inverted devices, separation and transfer of photoexcited electrons at perovskite/ETL interface play a decisive part in photoelectric properties of devices. In order to meet the development trend of large−area inverted and flexible devices, the ETL ought to conform following conditions: (1) Meet energy level matching of the perovskite/ETL and the ETL/metal electrode to promote the separation and transfer of photoexcited electrons. (2) High electron mobility to promote the fast transfer of electrons. (3) The deposition of ETL does not affect the properties of perovskite layer. 60) and its derivatives are the most frequently applied ETL materials for inverted PSCs because of their applicable energy levels, excellent electron mobility and simple film forming process, as is shown in 60 and C60−derivatives as ETL in the inverted devices firstly, demonstrating the first inverted PSCs based on PCBM with a PCE of 3.9% [61BM and improved the efficiency of inverted devices from 3.9% to 11.5% [61BM with PC71BM, making the PCE up to 16.3% [Fullerene Ox between PCBM and Al, achieving a PCE of inverted devices up to 18.3% and retaining over ninety percent of initial efficiency under 1000 h of illumination [Functions of interface modification are follows: (1) Enhancing the ohmic contact at ETL/metal film interface, promoting charge transport of photoexcited electrons. (2) Preventing the leakage of photoexcited holes. (3) Eliminating the local structural defects of PCBM layer to achieve the fast migration of carriers. In 2014, Wang et al. introduced an ultra−thin film of bathocuproine (BCP) at PCBM/metal electrode interface, which could not just improve direct contact between PCBM and metal film, but effectively prevent recombination of holes and electrons at the interface . HoweverM/C60/Al . On the  PCBM/Ag . Lee et  PCBM/Ag . Furthermination . Ren et mination .oc and a highest PCE of 18.1% [61BM:SnO2 as ETL in inverted devices [2 were inhibited, which led to an outstanding efficiency of 19.7%. Yang et al. doped PCBM with n−type polymer material (F8TBT) to achieve a good coverage of PCBM on the perovskite film, achieving an efficiency up to 20.6% [70BM with ultra−thin Bi2Te3 flakes [70BM with Bi2Te3 flakes was 2% v/v, which resulted in a PCE of 18.0%. After that, they also used Bi2Te3 flakes as the interlayer. They formed two spin coatings of the Bi2Te3 flakes dispersion onto the PC70BM, leading to a PCE of 18.6%. Finally, they combined two engineering approaches, obtaining a PCE of 19.46%. This was the highest record for inverted PSCs at that time. In another study, Rueda−Delgado et al. fabricated C60 interlayers and deposited either at the interface between SnO2 and MAPbI3−xClx absorber layer in a n−i−p architecture. C60 interlayers reduced the charge carrier accumulation at the interface. The final optimal PCE was 17.3% [In addition to the interface modification, doping modification, such as n−type dopant and surfactant dopant, is another method to promote the electrical conductivity and electron mobility of electron transport materials, strengthen surface coverage of PCBM on the absorption layer and reduce the charge recombination. Ji et al. used the graphene−doped PCBM as ETL for inverted devices improve the transmission performance of photoexcited electrons for the first time, pursuing the PCE of 14.8% . Afterwaof 18.1% . Chen etof 18.1% . Kakavelof 18.1% . Wang et devices . As a reto 20.6% . In anot3 flakes . The optas 17.3% . Compare60 and its derivatives have been comprehensively applied as electron transport materials for typical configuration of inverted PSCs, as is shown in 60 ETL interface achieved the high PCE nearly 19%. Organic small−molecule material can also tune the energy gap and increase the electron affinity. The small−molecule material is promising in the PSCs [Novel organic small−molecule materials have been developed as ETL even though Cthe PSCs . However2S3, as the ETL via a thermal evaporation method for the inverted devices [2SIn 2017, Tan et al. introduced the CdSe quantum dots (QDs) as ETL and LiF as buffer to achieve prominent photoelectric properties of inverted PSCs [x layer as ETL for the inverted PSCs via a solution process [x could prevent moisture from corroding the perovskite film and the metal electrode, leading to an enhanced stability with more than 90% of initial efficiency under 200 h of illumination. In 2019, Hossain et al. developed a scalable low−temperature TiO2 ETL based on presynthesized crystalline nanoparticle (np−TiO2). Nb5+ doping increased the conductance through the np−TiO2 amd reduced the series resistance. The optimal PCE they obtained was 19.5% [2 QDs ETLs. They found that the bilayer ETL composed of lithium−doped compact SnO2 (c(Li)−SnO2) at the bottom and potassium−capped SnO2 nanoparticle layers (NP−SnO2) at the top enhanced charge transport properties of PSCs, and significantly reduced the degree of ion migration. This resulted optimal PCE reached 20.4%, and strongly reduced J−V hysteresis for PSCs [The removal of organic components and the application of inorganic ETL are effective methods to improve thermally stable of inverted PSCs. The n−type semiconductor materials were surveyed and employed to replace organic ETL for inverted devices, such as ZnO and CdS, as is shown in  devices . Moreoveted PSCs . The int process . The denas 19.5% . In 2022for PSCs .2O5 layer as ETL and the polyacetylene derivatives doped perovskite as a absorber layer to fabricate the inverted PSCs, acquiring an outstanding efficiency of 20.41% and the prominent stability of devices [2O3 and Sn:In2O3 nanoparticles as ETL in inverted PSCs through a low temperature preparation method, which led to an impressive PCE up to 20.65% due to the enhanced built−in field, efficient electron extraction and decreased charge recombination [2Br film to improve long periods of stability, maintaining over 90% of initial PCE at 85 °C without illumination and continuous under 45 °C with 1000 h of illumination [In 2021, Jiang et al. adopted a Nb devices . Yang etbination . All−inomination .+) are ideal light absorption materials for the construction of stable PSCs because of their excellent photothermal stability and comparable photoelectric properties to hybrid perovskite materials, including CsPbI3, CsPbI2Br, CsPbIBr2 and CsPbBr. Combined with inorganic charge transport materials, the design and preparation of all−inorganic PSCs are expected to break through the bottleneck of PSCs, as is shown in Though the PCE of inverted PSCs has exceeded 23%, it is expected to replace traditional crystalline silicon solar cells. However, the stability of hybrid PSCs has seriously hindered their commercialization. The key element influencing stability is existence of organic cations that decompose easily under high temperature and ultraviolet light. Therefore, the preparation of devices by inorganic materials is the key to solving the problem. Inorganic perovskite materials formed by substituting organic groups with inorganic cations  to decrease non−radiative recombination, which gave rise to a highest efficiency of 16.1% with enhanced photostability, as is shown in Among the above inorganic perovskite materials, CsPbI devices . Howeverectively . PSCs wi devices . Consequ61BM/ZnO . ConsequThe inverted PSCs emerged in with the improvement of traditional upright devices. Properties of inverted devices have also made significant progress in recent years, with more than 24% efficiency being achieved. The architecture and the preparation process of inverted PSCs have been systematically investigated, including electron transport layer, perovskite absorption layer, interface modification layer, doping, etc. With the research of underlying mechanism, the stability of devices has been significantly enhanced in recent years. Furthermore, the photoelectric performance of the device is getting better and better with development of all−inorganic inverted PSCs.sc. (3) It is related to the design of the series−parallel structure and the component technology, resulting in an increase in the series resistance of the components and a decrease in the parallel resistance. Now, there are many processing technologies, such as solvent treatment, moisture assisted growth, hot spin−coating and additives, which have been confirmed as an effective way. Exploring new methods or combining these existing technologies to further improve the film quality should be our next direction. Moreover, encapsulation technology is another significant component necessary to enhance the long stability of inverted PSCs. Fullerenes can forbid ion movement and enhance the charge transfer to reduce the hysteresis. A fullerene/metal oxide such as PCBM/ZnO bilayer system shows promise in terms of reduced hysteresis and improved highly stability. Moreover, the small−molecule materials we introduced in front of the review are continuously developed by researchers. They play a significant role in enhancing the performance of the inverted PSCs. So, we think we can pay attention to the small−molecule materials.Up to now, the highest PCE of inverted devices has achieved 24.3%, which will catch up with the highest PCE of 25.5% for the traditional upright PSCs, and the hysteresis effect can be reduced with the application of fullerene derivatives ETL, leading to valid and reliable tested results. In addition, most of preparing technology for the inverted PSCs involves a low−temperature preparation process, which is beneficial to reduction of production costs and the preparation of flexible PSCs. Reports of superior PCE, long periods of stability and inexpensive for preparation make the inverted PSCs more competitive during the process of solar cells commercialization. Moreover, the inverted PSCs are more suitable for 2T perovskite−silicon tandem solar cells than the traditional upright PSCs . BecauseIn addition, more stable and environmentally friendly PSCs without lead or with less lead can be developed by replacing or reducing the content of Pb in perovskite film with non−toxic metal elements, such as Sn and Bi. Therefore, if the development of new methods, new materials or new ideas can solve the issues of efficiency, stability, large area, inexpensive and environmental protection during the process of commercialization, it will effectively promote the industrial application of inverted PSCs.


Miscanthus sinensis. Poly (ethylene glycol) 2000 (PEG 2000) was found to be more efficient in SAP than in other tested surfactants. Compositional and structural analysis revealed that the SAP process with 1% of PEG 2000 produced more efficient lignin removal and microstructure disruption of the pretreated sample, thus indicating much higher reducing sugar yields of 544.4–601.2 mg/g compared to the samples that were untreated or pretreated by alkali alone. Moreover, SEH with 1% Tween 80, which could block the lignin-enzyme interactions, produced a substantial reduction of 33.3% in the enzyme loading to achieve a higher sugar recovery from the SAP sample.Surfactants play a vital role in the delignification and saccharification of lignocellulosic biomass. A strategy for coupling surfactant-assisted alkaline pretreatment (SAP) with surfactant-assisted enzymatic hydrolysis (SEH) has been proposed for improving sugar recovery from a potential energy crop,  Its attractive merits include effective nutrient cycling, high genetic variation, easy propagation, and high yield (27–38 t/ha)  . TherefoBioconversion of lignocellulosic biomass remains limited due to its recalcitrant structure. A key step in bioconversion is pretreatment . An ideaEucalyptus samples, and pine foliage, was obtained by alkaline pretreatment . The composition and microstructure in response to the pretreatments were then investigated to better illustrate the exact roles of the pretreatment in biomass recalcitrance changing and subsequent improvement of the enzymatic hydrolysis. Moreover, considering the high enzyme cost of bioethanol production  and surfactant-assisted alkaline pretreatment (SAP) were performed in glass bottles in an autoclave. In brief, the dried samples were added to glass bottles containing 0–2.0% (w/v) surfactants ), and 0.6–1.0% (w/v) NaOH solutions, respectively, based on a solid loading rate of 10% . DiffereThe enzymatic hydrolysis of the raw and pretreated samples was conducted in a 250-ml conical flask using 50 mM of sodium acetate buffer (pH 5) containing 40 μl of tetracycline hydrochloride (25 mg/ml) . The rawM. sinensis was observed by SEM . The specimens were then examined with a JEOL JSM-5600 LV scanning electron microscope  under a high vacuum. An accelerating voltage of 5.0 kV was used for the observations.The microstructure of the raw and pretreated stalk samples of d by SEM . The driThe sample mixtures were centrifuged after AP and SAP. Solid yields were calculated based on the residual total solid of the stalk samples after all pretreatments. The contents of cellulose, hemicellulose, and lignin were measured gravimetrically following the standard method of Goering and Van-Soest . In brieThe determination of cellulose accessibility of untreated and pretreated stalks was done according to the direct red dye (DR28) adsorption method . In brieM. sinensis, which contains the original recalcitrant structure of the plant cell wall, had the lowest yield of reducing sugars after 72 h of the enzymatic hydrolysis. The digestibility of the pretreated stalks was significantly enhanced by AP using 0.6% NaOH alone.Different surfactants including PEG 400, PEG 2000, PEG 4000, PEG 6000, PEG 20000, Tween 60, and CTAB were tested in SAP. The untreated and treated stalk samples were subjected to subsequent enzymatic hydrolysis and the results are shown in The addition of the above surfactants enhanced the efficiency of the AP process. In cases of SAP assisted by 0.5% of Tween 60 and CTAB, obvious improvements in the enzymatic hydrolysis were recorded. As the surfactant concentrations in SAP became elevated (1 vs 0.5%), the corresponding sugar yields also increased. As shown in Previous studies have reported that CTAB and PEG 6000 were most effective for enhancing enzymatic hydrolysis of pine fallen foliage  in acid Results of the compositional analysis of the untreated and pretreated samples are shown in Different pretreatments decrease the recalcitrance of lignocellulosic wastes by reducing the lignin and hemicellulose components and exposing the cellulose to cellulases, thereby improving their bioconversion . The hemThe SEM at a magnification ratio of ×500 was used to observe morphological changes of the untreated and pretreated samples. As shown in A strong disruption of the plant cell wall was also observed in the different biomass samples pretreated with acid/alkaline . The hydInterestingly, PEG 2000 assisted alkaline pretreatment, which help to produce better structure modification and/or extraction of dissolved lignin, enabled much more efficient saccharification compared to other tested surfactants; therefore, it was chosen for further studies. Effect of alkali concentrations of SAP assisted by PEG 2000 on enzymatic hydrolysis of the pretreated sample was investigated. Distinct differences in reducing sugar recovery were observed between the PEG 2000 assisted SAP and AP using alkali alone . When 0.The surfactant addition in SAP significantly improved the alkali disruption of the cell walls and resulted in more cracks, porous structure, and lignin removal , which wIt is well known that the industrial application of bioethanol production is impeded by the high cost, in which the enzyme cost accounts for 20–30% . To furtAs shown in 3 and ethanol  in the sugar yield using the same enzyme loading (15 FPU/g); or using a much lower enzyme loading (10–12 FPU/g) for a small margin of increase (4.3–5.5%). Recent studies have reported that the addition of Tween 80 greatly shortens the hydrolysis time with a 50% reduction of enzyme loading to obtain the same level of glucose yield for pretreated sugarcane bagasse using FeCl ethanol . The mas ethanol . Surpris ethanol .M. sinensis sample pretreated by SAP was comparable to those observed in previous studies  of the SAP sample, which could modify lignin surface properties and block lignin-cellulose interactions, achieved a higher sugar recovery, even with a substantially reduced cellulase loading. These results indicate that the strategy of coupling SAP with SEH indeed has the potential to achieve the effective bioconversion of M. sinensis, which makes this material a promising candidate for bioenergy production.The impact of surfactants on alkaline pretreatment and enzymatic hydrolysis of


SHANK3 being one of the high-risk genes for ASD. In recent years, knowledge has been growing regarding the neuroplasticity effect induced by hyperbaric oxygen therapy (HBOT) and its potential use for ASD. Here, we characterized the effect of HBOT on a mouse model for ASD with the human genetic condition of InsG3680 mutation in the Shank3 gene. As compared to placebo, HBOT improved social behavior and reduced neuroinflammation in the cortex of the InsG3680(+/+) mice. Specifically, HBOT induced upregulation of Insulin-like growth factor 1 (Igf1) expression levels and reduced the number of Iba1-positive cells in the mouse model for ASD compared to placebo control. Together, our research suggests that HBOT has the potential to improve the clinical outcome of ASD by ameliorating some of the core pathophysiological processes responsible for the development of the disorder.Autism spectrum disorder (ASD) is a multifactorial neurodevelopmental disorder (NDD) characterized by impaired social communication and repetitive behavior, among other symptoms. ASD is highly heritable, with  SHANK3 gene, which is one of the high-risk genes for monogenic ASD [SHANK3 are known to cause Phelan McDermid syndrome (PMS), characterized by autistic-like phenotype, developmental delays, hypotonia, and other symptoms [Autism spectrum disorders (ASDs) are neurodevelopmental disorders (NDDs) characterized by impaired social behavior and communication, restricted interests, and repetitive patterns of behavior, among other symptoms . Accordienic ASD ,4. Microsymptoms .SHANK3 is an insertion of guanine in position 3680 of the SHANK3 gene [SHANK3 is a protein that takes part in the postsynaptic density (PSD) of glutamatergic synapses . It is aNK3 gene . Based oNK3 gene ,10, demoNK3 gene .Although the mutation’s effect on synaptic dysfunction is well-established in the InsG3680 mouse model , its effSince there is a wide clinical spectrum of symptoms and pathophysiological heterogeneity among patients with ASD ,19,20, nOne of the well-defined deficits in brain physiology related to ASD is hypoperfusion ,22 and tSHANK3 gene (unpublished data). Single photon emission computed tomography (SPECT) results before HBOT showed impaired brain perfusion in the orbital-frontal region, dorsa-lateral cortex, medial area of temporal lobes, and in the cerebellum. SPECT analysis after HBOT showed improved brain perfusion and function in the right orbital-frontal cortex, right motor cortex, left frontal temporal lobe, Broca’s area, left dorsal-lateral and dorsal-medial frontal cortex, left occipital lobe, right thalamus nucleus, and the cerebellum compared to the pre-HBOT state. Additionally, the patient’s parents self-reported an improvement in the patient’s social behavior and cognitive performance (these aspects were not measured quantitatively and are not presented).The beneficial effects of HBOT were studied mostly in regenerating previously healthy brain tissue exposed to acute insult, while the data on the beneficial effects in brain function abnormalities related to specific born-related DNA mutations are still lacking. Regarding the use of HBOT for ASD, the data are still conflicting, using different protocols and variable cohorts that are not focused on a specific pathophysiological-related subtype of ASD ,35,36,37Motivated by the beneficial outcomes HBOT had on ASD pathology, this study aimed to evaluate the molecular and behavioral effects of HBOT on the InsG3680 mouse model, to better understand the mode of operation HBOT has on the autistic brain.Shank3 as in the human patient. Our motivation was to test whether the improved behavior in the autistic human patient could also be found when proper statistical power is tested, and to study neurobiological properties that can explain the improved behavior reported.The physiological and behavioral improvements demonstrated in the human HBOT study prompted us to study the neurobiological properties of HBOT on a mouse model for ASD that harbors the same insertion mutation in (+/+) mice as compared to InsG3680(+/+) mice in the placebo control group exposed to air   on InsG3680 levels) . All mic levels) .(+/+) HBOT group and the InsG3680(+/+) placebo group spent significantly more time with the stranger mouse than with the object, as opposed to previous findings from this mouse model [(+/+) HBOT group showed significantly increased exploration time close to the novel mouse than the familiar mouse, whereas the InsG3680(+/+) placebo group did not show any preference between them, similar to previously reported social novelty deficits [Because social behavior deficits are hallmark features of ASD, we studied whether HBOT has the potential to alleviate social behavior dysfunction. In the three-chambers sociability test, both the InsG3680se model   placebo group spent a significantly longer time in the margins compared to the InsG3680(+/+) HBOT group ((+/+) HBOT group compared to the InsG3680(+/+) placebo group.Because elevated anxiety levels are known in ASD compared to typically-developed (TD) individuals, we next sought to characterize the effect of HBOT in ASD on anxiety-like behavior. In the open field test, no significant difference in overall exploration properties was found, except in the one-time bin (25 min), in which the InsG3680OT group C. In theOT group D and theOT group E, no sigTo seek neurobiological properties that could support the improved brain perfusion in the human patient and the improved behavior in our mouse model, we examined whether HBOT affects hypoxia and neuroinflammation properties. The indications of a hypoxic state and neuroinflammation in ASD ,17,22 su(+/+) HBOT group compared to InsG3680(+/+) placebo group  HBOT compared to InsG3680(+/+) placebo. We found a reduced number of Iba1-positive cells in the motor cortex of the InsG3680(+/+) HBOT group compared to InsG3680(+/+) placebo group  HBOT group compared to the InsG3680(+/+) placebo (Igf1 expression levels in both InsG3680(+/+) HBOT and InsG3680(+/+) placebo (CD11b (CD68 ((+/+) HBOT group compared to InsG3680(+/+) placebo group.Since previous studies showed that IGF1 administration is effective in ameliorating ASD deficits ,39 and c placebo D. Intere placebo E. No sigo  mice compared to InsG3680(+/+) placebo control group mice, indicating that HBOT is effective in adults and not necessarily in the early postnatal developmental stage only. Specifically, HBOT improved social novelty preference, a subtype of social behavior tested in the three chambers social interaction test. The improved preference of a novel mouse compared to a familiar mouse in the social novelty test could also indicate improved cognition and memory. It may demonstrate that InsG3680(+/+) treated mice remember the familiar mouse better than the InsG3680(+/+) placebo group [(+/+) mice compared to controls [We found that HBOT improves the social behavior of adult InsG3680bo group . Neverthcontrols . The lac(+/+) placebo group. To determine whether HBOT ameliorated the brain’s neuroinflammation, we studied the properties of microglia, resident macrophages of the brain. We found a reduced number of microglia (Iba1-positive cells) in the InsG3680(+/+) HBOT group compared to the InsG3680(+/+) placebo group. Previous studies indicated elevated immune response in the brain of autistic patients [Igf1 mRNA expression level following HBOT. IGF1 is a neurotrophic factor essential for central nervous system development [Igf1 transcript in the whole cortex of the P120 InsG3680(+/+) HBOT group compared to the InsG3680(+/+) placebo group.In search of molecular and cellular evidence that will explain how HBOT affected neurobiological properties, we characterized parameters related to the hyperoxic-hypoxic paradox . Along tpatients ,43; therpatients  and reduelopment ,45,46,47SHANK3 micro-deletion, IGF1 rescued synaptic transmission deficits in excitatory neurons [Shank3 deficient mice rescued abnormal motor skills and reversed major deficits related to excitatory synapse signaling [Igf1 expression level we measured following HBOT may lead to a cascading effect [Previous studies found significantly reduced levels of IGF1 in the cerebrospinal fluid (CSF) of autistic children and infants compared to TD controls ,49. Of c neurons . Similarignaling . Based oignaling ,52,53,54g effect ,56,57 thIgf1, we found a significant negative correlation between the two parameters. This correlation suggests that higher levels of Igf1 are correlated with a potential reduction in neuroinflammation following HBOT and that Igf1 may induce tissue repair, as was also found in previous studies [Interestingly, when testing for a correlation between the number of Iba1-positive cells and the expression level of  studies ,59,60. NIgf1 expression, and improves social behavior in adult mice.Overall, our data suggest that HBOT is an effective treatment in the ASD genetic condition tested, reduces neuroinflammation, increases The molecular and cellular effects we characterized following HBOT were examined 1 month after the HBOT session, suggesting a long-term effect of HBOT. Moreover, while HBOT was mainly studied in acute neurological conditions or chronic conditions in adulthood, our study shows the efficacy of HBOT in treating a genetic condition associated with chronic NDD starting from embryogenesis.This research gives hope for the potential new biological intervention for a subtype of ASD, and future research should better dissect its long-term clinical effect. Looking forward, the use of treatments for ASD that are specifically proven to be effective on a specific genetic condition rather than on the phenotype of behavior hopefully marks the future of effective treatments for ASD. Since other subtypes on the ASD spectrum have overlapping pathophysiology, such as hypoperfusion, this study may be relevant to these other conditions as well.Terminology:(+/−)—mice that are heterozygous to the mutation.InsG3680(+/+)—mice that are homozygous to the mutation.InsG3680WT—mice without the mutation on both chromosomes.(+/−) mice were crossed with InsG3680(+/−) (Het-Het mating). InsG3680 and WT mice are with a C57 B6/S129 Sv mixed background. The resulting mice are either homozygous to the mutation (referred here as InsG3680(+/+)), heterozygous to the mutation (referred here as InsG3680(+/−)), or do not have the mutation (referred here as WT). The mutation occurs in all the cells of the body.Breeding: To test the efficacy of HBOT on the InsG3680 mutation, InsG3680Housing: Mice of the same gender were contained in cages with 2–4 littermates with random genotypes. The environmental conditions were stable with 20–24 °C under a 12 h light/dark cycle (lights at 07:00 until 19:00), with food and water available at all times. All experimental procedures conformed to the guidelines of the Institutional Animal Care and Use Committee of Tel Aviv University, Tel Aviv, Israel. All efforts were made to minimize animal pain and suffering and the number of animals used.(+/+) HBOT, InsG3680(+/+) placebo, WT HBOT, and WT placebo. The HBOT group received 100% oxygen gas in 2 ATA pressure levels, while the placebo group received air (containing 21% oxygen) with 1 ATA pressure levels. The HBOT chamber was filled with 100% oxygen prior to compression for 5 min to enrich the oxygen in the chamber. Compression and decompression were carried out gradually to reduce the risks of changing pressures.Treatment was performed on 1-month-old mice for 40 sessions for 2 months, 5 days a week, 1 h a day. A total of 30 mice were divided into 4 groups: InsG3680Behavioral tests were performed and analyzed with the experimenter blinded to the type of treatment and genotype. Mice in the experiment went through habituation in the test room for 1 h before all tests. Each group of test mice was used for 4 behavioral tests and had at least 3 days between tests. The mice were tested at the age of 3 months (2 months after the start of the treatment).To perform the test, C57-black mice were ordered from the Jackson Laboratory with similar ages and body weights. Habituation for the stranger mice in the social preference test was performed by placing the mice inside an inverted wire cup for 30 min, 2 sessions per day for 3 consecutive days prior to the test. The test apparatus (65 cm long × 44 cm wide × 30 cm high) was divided into three sections , which were connected via a lever-operated door that was opened 5 cm from the floor. The object, stranger, and familiar mice were placed inside an inverted wire cup , and on top of it, a weighted cup was placed to block the test mice from climbing over it. Following each trial, the wire cup was cleaned with ethanol and water. The test was comprised of three phases (15 min each): habituation, sociability, and social novelty. In the habituation phase, the test mouse was placed in the center and explored the environment of all three sections with the inverted wire cups empty. Next, in the sociability test, the tested mouse was placed in the center section with the doors closed while placing a stranger mouse and an object inside the wire cups of the left and right sections; Afterwards, the doors were lifted to allow the mouse to explore. Finally, in the social novelty test, the mouse was placed again in the center section with the doors closed while placing a novel mouse instead of the object, and afterward, the doors were lifted to allow the mouse to explore. The place of the object and stranger mouse were switched between trials to exclude place preference. Each of the stimulation mice were used only twice a day. Following the test, the experimenter analyzed the time spent with the object compared to a stranger mouse and the time spent with a familiar mouse compared to a novel mouse, blinded to treatment type, using the EthoVision XT 14.0.1326 software .Mice were placed in the center of a Plexiglas box (40 cm long × 40 cm wide × 30 cm high) for 1 h (one mouse in each box). Motor activity and exploration were videotaped and were measured by the time spent in the margins of the box while the experimenter was blind to the treatment type.The maze contains 2 sections—open and closed arms (Height 60 cm). Mice were placed in the closed arms of the zero maze and explored for 5 min. Movement between the sections was videotaped and was measured by the time spent in the open arms, while the experimenter was blind to the type of treatment.Mice were placed on the apparatus of an accelerating rotarod, and motor coordination was measured by latency to fall. Each subject was tested three times with a time interval of 30 min between each test.After treatment and behavioral experiments, male mice were deeply anesthetized with isoflurane. Consciousness was tested via a response to a pinch on the foot, and tissue samples from one ear were taken for genetic verification. Then, mice were perfused with 15 mL of ice-cold PBS solution, followed by brain dissection. The brain was separated into two hemispheres. One was kept in 4% paraformaldehyde (PFA) diluted in PBS for 4 consecutive days, and one was taken to a petri dish with PBS for dissection. The Cortex was separated from other brain tissue using cleaned surgical tools and a stereomicroscope . Cortices were placed in 200 μL of RNA-later solution  and preserved at 4 °C for 24 h. RNA-later was taken out of the tubes, and samples were frozen at −80 °C. All tools and equipment were sterilized with ethanol and sprayed with RNAse inhibitor .Cortices were placed in safe-lock tubes with stainless steel beads and 200 μL of cold TRIzol reagent  and were homogenized in TissueLyser 2 (QIAGEN) for 40 s in 24,000 Hertz. After the tissue was fully homogenized, an additional 800 μL of TRIzol was added, and the samples were incubated at room temperature (RT) for 5 min. 200 μL of chloroform  was added to each of the tubes, and they were shaken by hand for 15 min and incubated once more for 3 min at RT.g  . The homogenate was separated into 3 layers-protein, DNA, and RNA, which was the top layer. The clear RNA layer was transferred to a new tube and was diluted with a proportion of 1:1 with isopropanol . The tubes were manually shaken, incubated for 5 min at RT and centrifuged for 15 min at 4 °C, 800× g . After the centrifuge, the RNA was precipitated in the bottom of the tubs, isopropanol was taken out, and the remaining pellet was washed with 1 mL of 80% ethanol  diluted in DEPC-treated water , followed by centrifugation for 15 min at 4 °C at 13,800 rpm.Then, samples were centrifuged for 20 min at 4 °C, 800× This was repeated twice, while the ethanol was replaced the second time. All the ethanol was removed, and the tubes were placed upside down and opened on Kim wipes to let the remaining ethanol aspartate for approximately 30 min. 35 μL of DEPC-treated water was added to each sample, and then the sample was heated for 5 min at 60 °C. Finally, samples were pipetted to create homogenous concentration, measured using the Thermo Scientific NanoDrop One device , and kept frozen at −80 °C.RNA was diluted to a concentration of 20 ng/µL according to the sample’s original concentrations after RNA extraction. Reverse transcription was performed with random primers and the High-Capacity cDNA Reverse Transcription Kit . The protocol used with the C1000 Touch thermal cycler  was the following: 10 min at 25 °C, 120 min at 37 °C, 5 min at 85 °C, and a final step of 4 °C. cDNA was frozen at −20 °C.(+/+) placebo). Primers were programmed in the lab and ordered from Hy Laboratories Ltd. . The final dilution of the primers was done with 10 mM in DEPC-treated water  and the Bio-Rad CFX Connect Real-Time PCR Detection System . The protocol proceeded as follows: 20 s at 95 °C, 40 amplification cycles , and a melt curve: 60 °C for 5 s, and an increase of 0.5 °C every 5 s (including a plate read) until reaching 95 °C. mRNA levels were calculated based on the comparative cycle threshold (Ct) method . To normBrains were extracted as described above and then sectioned at a 100 μm thickness using a vibratome . Sections were stained using the free-floating method as follows: from each mouse, a section from the motor cortex was chosen  and was washed 3 times in 1 mL PBS for 5 min each. Then, sections were permeabilized with 1.2% Triton X-100 in PBS for 15 min. Following permeabilization, sections were washed in 1 mL PBS for 5 minutes each and blocked with 5% normal goat serum (NGS), 2% bovine serum albumin (BSA), and 0.2% Triton X-100 in PBS for 1 h. Afterward, sections were placed in a 96-well plate with 250 μL of primary antibodies diluted in blocking buffer (described above) overnight at 4 °C. The next morning, sections were washed in 1 mL PBS, once for 5 min and twice for 15 min each. Slices were then incubated with secondary antibodies conjugated with Alexa Fluor 488, 555, and 647  diluted in blocking buffer for 1 h. sections were washed in 1 mL PBS, once for 5 min and twice for 15 min each. Finally, for the mounting process on glass slides, VECTASHIELD Hardset Antifade Mounting Medium with DAPI  was used. Images were captured using a light microscope  with the experimenter blind to the treatment type. For quantification of cellular properties in the motor cortex, images were taken with ×10 or ×20 magnification according to the type of staining and analysis. Cell number and intensity were quantified manually using the ImageJ program.Commercial antibodies used: anti Iba1 , anti Hif1a p-values were calculated using Student’s t-test, 3-way repeated measures ANOVA, Kolmogorov-Smirnov and Pearson’s correlation coefficient, with p < 0.05 considered significant . The normality of distributions and equality of variances were checked and addressed accordingly using the appropriate statistical analysis. Outliers were determined via the extreme studentized deviate (ESD) method (p < 0.05).Data are presented as the mean ± standard error of the mean (s.e.m.), as calculated by GraphPad Prism 9.4.1 for Windows


In the event of an acute ischemic stroke, saving the penumbra is the most important aspect of early treatment. The rapid and accurate identification of ischemic penumbra plays a key role in its comprehensive treatment. At present, the identification method and evaluation standard of ischemic penumbra have not been unified. Numerous pieces of software identifying ischemic penumbra have been developed, such as rapid processing of perfusion and diffusion (RAPID), Sphere, Vitrea, and computed tomography perfusion+ (CTP+). The RAPID software, analyzing and integrating multi-mode image data (mainly based on perfusion weighted imaging (PWI) or computed tomography perfusion (CTP) images, shows good performance in identifying ischemic penumbra and has been utilized for the assessment of ischemic penumbra in many ischemic stroke clinical studies, achieving good outcomes and promoting the transition from “time window” to “tissue window” in the treatment of early stage AIS. To obtain a comprehensive understanding of the RAPID software and its accuracy in evaluating ischemic penumbra, this paper reviews the background and development of the RAPID software, summarizes the published acute cerebral infarction trials using the RAPID software, generalizes the threshold parameters in different time windows, and further discusses its application and limitations. Moreover, in recent years, the RAPID software has been proved and applied in many international clinical studies to provide the software with a good chance for optimizing an appropriate parameter to identify penumbra. The current paper reviews the background, development, application, and limitation of the RAPID software and summarizes the history of clinical trials using RAPID to identify penumbra.Acute strokes pose the greatest threat to human health. They are characterized by high morbidity, disability, death, and recurrence rates. Acute ischemic stroke (AIS) is the most common subtype of acute stroke characterized by the reversible or irreversible loss of cerebral tissue and neurological function caused by an inadequate supply of cerebral blood flow . In 2019In 2006, the diffusion and perfusion imaging evaluation for understanding stroke evolution (DEFUSE) study , based oTmax>6s lesion volume/DWI lesion volume > 1.2, DWI or PWITmax>8s lesion ≤ 100 mL, and |PWITmax>6s lesion volume − DWI lesion volume| ≥ 10 mL) achieved a better clinical prognosis following intravenous tissue plasminogen activator (tPA) reperfusion therapy, which could slow down the increase in infarction volume.In 2011, Lansberg MG et al.  continue−6 mm2/s and critically hypoperfused tissue as Tmax in PWI > 6 s. The criteria for identifying target mismatches were as follows: critically hypoperfused tissue/ischemic core ≥ 1.8; a |critically hypoperfused tissue − ischemic core| ≥ 15 mL; ischemic core volume < 70 mL; and lesion volume Tmax>10s ≤ 100 mL. Among a total of 138 enrolled patients, 104 patients had reliable MR image quality (MRI profile cohort) and the consistent rate between automatic analysis by the RAPID software and the manual identification of lesion range reached 97% (101/104), which proved that the RAPID software accurately identifies hypoperfused and ischemic lesions and their locations [In 2011, the DEFUSE 2 study defined the ischemic core as ADC < 600 × 10In 2013, the RAPID software obtained the FDA 510 K clinical use license and is, at present, a medical image processing software package approved by the FDA. In 2015, the extending the time for thrombolysis in emergency neurological deficits (EXTEND) study confirmed that the automatic analysis of CTP-based hypoperfusion/infarction mismatches by the RAPID software was also rapid and reliable, and the processing time of RAPID was quicker with CTP data than MR data [In 2017, several large trials of acute cerebral infarction successively used the RAPID software to select patients with target mismatches, and further explored the threshold parameter adjustment of the software in different time windows. The timeline of the main RAPID-related clinical trials is summarized in RAPID has been used as an image processing platform for hypoperfusion/infarction mismatches in large clinical studies, such as DEFUSE1, 2, 3; DAWN; SWIFT PRIME; EXTEND; and EXTEND-IA using MR or CT perfusion images.When the Tmax threshold is 6 s, it is the most accurate value used to determine the hypoperfused area based on the voxel ,9. Recen−2 mm2/s, which was most consistent with the literature data [−2 mm2/s has a low segmentation sensitivity and an ADC higher than 640 × 10−2 mm2/s has a low segmentation specificity [Based on CTP, when the rCBF threshold was 0.30–0.34, the predicted infarct range presented the greatest consistency with the infarct range 27 h later . The MR-ure data ,19. In oThe DEFUSE3 study  enrolled3, and NIHSS ≥ 10; age < 80 years, infarction area 0–31 cm3, and NIHSS ≥ 10; and age < 80 years, infarction area 31–51 cm3, and NIHSS ≥ 20. The results show that patients with anterior circulation AIS with a small infarct size and mismatched clinical function scores can still take advantage of endovascular therapy within 6–24 h of the last normal time.DAWN study  defined −6 mm/s [In SWIFT PRIME study ,20, pati−6 mm/s , which p−6 mm/s . Within −6 mm2/s for patients undergoing an MR scan [The EXTEND study  was desi MR scan . The resThe EXTEND-IA study  determinhttps://doi.org/10.3760/cma.j.issn.1005-1201.2019.11.002, accessed on 18 October 2022) and Chinese Stroke Association guidelines for clinical management of cerebrovascular disorders: executive summary and 2019 update of clinical management of ischaemic cerebrovascular diseases [The accurate estimation of the extent of the ischemic zone and infarct core area is the core problem and key purpose in the evaluation of ischemic penumbra. Compared with traditional image techniques, RAPID has apparent advantages. It only spends several minutes to evaluate an AIS patient, which could reduce the clinical decision time and improve the prognosis of AIS patients (demonstrated by the above-mentioned RCTs). RAPID also has good performance compared with other recently developed evaluation software. Rava et al. divided patients with AIS into endovascular and conservative treatment groups and compared the assessment of the ischemic zone and infarct core zone using RAPID, Sphere, Vitrea, and CTP+ software, respectively. The results show that, in the endovascular treatment group, RAPID has the least chance to overestimate the volume of the infarct core area, which presents patients with the greatest possibility for receiving endovascular treatment ,26. Nowadiseases  suggest diseases . Severaldiseases . (3) SevThis paper summarized the background, development, application, and limitations of the RAPID software in clinical research. Several studies have shown that RAPID software can rapidly and accurately judge the state of ischemic penumbra at the early stage of AIS, providing a histological basis for thrombolysis or thrombectomy. However, in the future, more clinical randomized controlled studies are needed to verify the evaluation power of RAPID software on ischemic penumbra and further optimize the “mismatch” parameter and its threshold, which may allow the software to be formally recommended by relevant guidelines and lay a foundation for extensive introduction into medical institutions and cost reduction. Moreover, it remains to be further explored whether RAPID can also accurately identify ischemic penumbra in most patients who have exceeded the time window for vascular recanalization. At present, our research team is exploring the use of RAPID software to evaluate the effects of butylphthalide  ,30,31 on


ACAN, COL10A1, and COL2A1 expression compared to pellet culture alone and allowed GAG deposition. The soluble portion of the scaffold did not affect chondrogenesis. Furthermore, they promoted the increase in COL1A2, showing a slight tendency to differentiate towards fibrous cartilage. The results also showed that Colombian silk could be used as a source of biomedical devices, paving the way for sericulture to become a more diverse economic activity in emerging countries.The development and evaluation of scaffolds play a crucial role in the engineering of hyaline cartilage tissue. This work aims to evaluate the performance of silk fibroin hydrogels fabricated from the cocoons of the Colombian hybrid in the in vitro regeneration of hyaline cartilage. The scaffolds were physicochemically characterized, and their performance was evaluated in a cellular model. The results showed that the scaffolds were rich in random coils and β-sheets in their structure and susceptible to various serine proteases with different degradation profiles. Furthermore, they showed a significant increase in  Hyaline cartilage is a tissue located at the end of bones and serves as an adequate soft surface for joint movement . CartilaThe main disease associated with hyaline cartilage damage is osteoarthritis. Knee and hip osteoarthritis is one of the most important causes of chronic disability and pain worldwide and is responsible for substantial financial costs in health care . This isAmong the strategies used to recover lost or damaged tissue, those based on tissue engineering, which uses mesenchymal stem cells and scaffolds, stand out for their results. However, even with the latest advances, there are currently no effective treatments available to most people. Therefore, searching for cells and scaffolds for cartilage damage treatment is still ongoing .There are different types of scaffolds or biomaterials, such as films, porous structures, hydrogels, sponges, and membranes, among others . These pThe structural and physicochemical characteristics of the biomaterial have an impact on the quality of the graft. However, it is still challenging to develop functionally equivalent scaffolds with relevant clinical results . One of Bombyx mori, composed of a heavy and a light chain bound by disulfide bridges [Many scaffolds are fabricated from biological compounds such as proteins, polysaccharides, or polyesters. An example is silk fibroin-based scaffolds, a fibrillar protein extracted from some arthropods, such as  bridges . Silk fi bridges . Their b bridges .Silk fibroin-based hydrogels are manufactured by subjecting fibroin solutions to vortexing, sonication, electric current, pH changes, or the addition of cross-linkers . The proIn the last decade, research on the use of silk fibroin for cartilage treatment has increased significantly ,15,16. HIn Colombia, commercial silk is obtained only from the Pílamo 2 hybrid silkworm cocoons. This hybrid was developed by classical genetic improvement through crosses of breeds that could adapt to the environmental and climate conditions of Colombia. Recently, our research group explored the mechanical characteristics of Colombian hybrid silk fibroin hydrogels, finding that the Young modulus and the rheological behavior satisfied the necessities of tissues with high mechanical requirements .All this considered, this work aims to evaluate the performance of silk fibroin hydrogels fabricated from the cocoons of the Pílamo 2 Colombian hybrid in the regeneration of hyaline cartilage. To achieve this, the scaffolds were physicochemically characterized, and their performance was evaluated in an in vitro model.Bombyx mori L. var. Pílamo 2) were obtained from the experimental farm “El Pílamo”, administered by the Universidad Tecnológica de Pereira, Colombia. The silk fibroin solution was prepared following the protocol described by Kaplan et al. [2CO3 for 30 min. After three washes with deionized water, silk fibroin was dried at 45 °C for 8 hours in an oven. The silk fibroin was then solubilized at a concentration of 20% in 9.3 M LiBr, at 60 °C for 8 hours. The residual solution was dialyzed against deionized water for 54 h using a 6000–8000 MWCO cellulose membrane, changing the water every 6 hours. Dialysis timing was determined by measuring the conductivity in the permeate. The resulting silk fibroin solution was vapor sterilized at 121 °C for 15 min and centrifuged twice at 9000× g for 20 min. TGA analysis was used to determine the effect of sterilization on silk fibroin mass. Finally, the concentration of silk fibroin was measured by comparing the mass of the solution with the mass of silk fibroin after drying at 70 °C for 12 h.Colombian hybrid silkworm cocoons , parallel β-sheets (1637–1613 cm−1), and antiparallel β-sheets (1637–1613 cm−1).Hydrogels were analyzed on a Cary 630 spectrometer coupled to an ATR modulus (Agilent Technologies). The secondary structure was quantified following the methods proposed by Goormaghtigh  and Belt−1 monitoring the absorbance at 214 nm.Proteolysis was performed as described by Brown et al.  with cer−1 using azocasein digestion.For the assay, we used solutions of the following enzymes: proteinase K (Sigma), protease XIV (Sigma), and serralysin . The asFor each incubation time, the chromatographic profiles were corrected by subtracting the chromatographic protease profile from the supernatant chromatographic profile, which resulted from the proteolysis of silk fibroin hydrogels. Additionally, the areas under the curve were analyzed for 24 h enzymatic digestions.2. The medium was changed every 2 days. The cell surface profile was tested using the MSC Phenotyping Kit Human  on a Guava EasyCyte flow cytometer .Mesenchymal stem cells (hMSCs) were cultured in MSCGM medium (Lonza) and incubated at 37 °C, 5% COEvaluation of cell metabolic activity was performed using the Alamar Blue assay  using hM5 hMSCs were cultured in hMSC Human Mesenchymal Stem Cell Chondrogenic Differentiation Medium (Lonza) supplemented with TGF-β3 (Lonza) in 15 mL conical tubes. Three types of samples were evaluated in triplicate: cell pellet, cell pellet on silk fibroin hydrogels (2D), and cells encapsulated inside silk fibroin hydrogels (3D). For cell pellet culture, cells were centrifuged at 400× g for 10 min [g for 10 min. In the case of 3D culture, cells were seeded and mixed by pipetting after 10 min of sonicating the silk fibroin solution (50 μL). The cells were cultured in a medium that was changed every two days for 28 days under standard conditions. The resulting cultures were stained with Alcian Blue. Furthermore, cells were frozen in liquid nitrogen, and their total RNA was extracted using TRIzol Reagent  to evaluate the expression levels of ACAN, COL2A1, COL10A1, SOX9, COL1A2, and the housekeeping control gene GAPDH. Total RNA was diluted in water, quantified using a NanoDrop2000, and kept at −80 °C. RT-qPCR was performed with the One-Step RT-PCR Kit and TaqMan Gene Expression Assays  using the 2−ΔΔCt method [5 × 10r 10 min . In the t method , taking p < 0.05 was considered significant.The results are shown as the mean value ± standard deviation (SD), and the experiments were carried out with at least three replicates. Statistical tests were conducted using GraphPad Prism, version 6.0 (GraphPad Software). The study was approved by Comité de Bioética at the Universidad Tecnológica de Pereira . Human Mesenchymal Stem Cells (hMSCs) were obtained from Lonza Bioscience, Basel, Switzerland, Catalog: PT-2501).The silk fibroin solution was obtained from the Colombian hybrid silkworm “Pílamo 2”. Although there are different silk fibroin fiber dissolution systems, such as organic acid complex compounds, ionic liquids, cuoxam metal complex systems, inorganic acids, alkalis combined with enzymes, and organic compounds, we used lithium bromide for silk fibroin solubilization. Lithium bromide is an expensive reagent, but it is neutral and does not hydrolyze the silk fibroin peptide bonds , key forw/v). The thermal analysis demonstrated for the solution that the silk fibroin of the Colombian hybrid is resistant to sterilization temperature (121 °C). The analysis shows that the maximum local degradation rate after sterilization did not change, and it took place at around 311 °C (g), a typical endothermic annealing peak representing the transition of amorphous silk fibroin from hard to soft. These values are close to those reported by Kasoju et al. [Sterile silk fibroin solutions were obtained at a concentration of approximately 7% , amide II (1520–1540 cm−1) and amide III (1200–1350 cm−1), which are associated with the lengthening of the C=O, C-H, and C-N bonds, the bending of the N−H bonds, and to side chain vibrations, respectively , anstrated .Bombyx mori L. in hydrogels is dominated by random coils (46–54%), which is consistent with the previously reported low crystallinity of silk fibroin in hydrogels.A deconvolution of the amide I band was performed to determine the secondary structure of silk fibroin in solution and in hydrogels. Results analysis C showed 2O2 [β-sheets were the second most abundant secondary structure in hydrogels, induced by sonication and the interactions between domains rich in the small amino acid residues Ala and Gly . It woul2O2 .The formation of hydrogels in the present work was achieved through a sol–gel transition by sonication. As previously reported by Kaplan et al. ,44, this214) in the chromatographic profile of the supernatant leached by the hydrogel without proteases showed that the silk fibroin hydrogels were stable for 24 h. This demonstrates that the mechanical cross-linking process was adequate, confirming the results of the physicochemical characterization. In the presence of proteases n motifs [The three enzymes did not appear to have distinct targets and resulted in similar degradation patterns in terms of molecular weight, possibly related to the highly repetitive sequence of silk fibroin, rich in (Gly-Ala-Gly-Ala-Gly-Ser)n motifs .To the best of our knowledge, this is the first time that size exclusion chromatography has been used to analyze the enzymatic degradation of silk fibroin hydrogels. This technique has been used regularly to study the thermal degradation of silk fibroin  and to dThe evaluation of the biodegradability of a biomaterial is a keystone process directly related to its biocompatibility, mechanical stability, and capacity for de novo tissue formation and tissue regeneration. Silk fibroin biomaterials have been observed to degrade slowly, both in vivo and in vitro , even inMesenchymal cells showed an adequate phenotype according to the minimal criteria established by the International Society for Cellular Therapy. They presented fibroblast-type morphology, were adherent, showed molecular markers CD73, CD90, and CD105 on their surface, and did not possess other transmembranal proteins such as CD45, CD34, CD14 (CD11b), CD19 (o CD79a) or HLA–DR .Other researchers have reported successful encapsulation of mesenchymal stem cells in 4% silk fibroin hydrogels 10 min after sonication . They reAlthough desired, the proliferation of hMSCs does not guarantee the success of the construct. Cell proliferation and differentiation are inversely related , implyinACAN and COL2A1 compared to the pellet culture alone. These two genes are directly related to the microarchitecture of the ECM of hyaline cartilage, which is fundamental for the tissue’s mechanical and biological properties of the tissue. COL2A1 expression is related to the synthesis of type II collagen fibrils that characterize ECM and limit chondrocyte hypertrophy [ACAN expression is implicated in the production of aggrecan, a fundamental proteoglycan in the water catchment, essential for the distribution of mechanical charge on the surface of the joint [Chondrogenic genes were clearly expressed after 28 days of culture, demonstrating successful cell differentiation . Our resertrophy . ACAN exhe joint .COL10A1 was highly overexpressed in hMSCs differentiated inside silk fibroin hydrogels, which has been previously reported for other types of scaffolds [COL10A1 is generally associated with hypertrophic cartilage, it is also a critical component of endochondral bone formation during skeletal development and the regeneration of osteochondral lesions [caffolds . Althoug lesions .SOX9 was overexpressed in all samples compared to hMSCs cultured in a nonchondrogenic medium. This gene is a master regulator of chondrogenesis, regulating the differentiation of hMSCs into chondrocytes and promoting cell proliferation. Its expression is related to the regeneration of hyaline cartilage [artilage .The soluble portion of the silk fibroin hydrogels did not affect chondrogenesis. This suggests that the biomaterial degrades safely without producing chondrogenesis inhibitors. Some authors have described that degradation could even increase the content of type II collagen and improve nutrient diffusion .COL1A2). This finding could negatively affect hyaline cartilage regeneration because this would result in mechanically inferior fibrocartilage [An unexpected result was that silk fibroin hydrogels promoted differentiation to fibrous cartilage (increased artilage . This suartilage . This tiartilage .A limitation of our study is the absence of information concerning the abundance of chondrogenic proteins using immunofluorescence or Western blotting techniques. However, several studies support the fact that differentially expressed mRNAs directly correlate with their protein product, although there might be a slight delay ,67. In tAlcian Blue staining can be observed in Pellet cultures A,B show Glycosaminoglycan production was highly evident in the pericellular space, with low diffusion of molecules through the material, which could be related to hydrogel porosity . These rChondrogenic differentiation assays agree with the results of other authors that constructed 4% hydrogels from other sources of silk fibroin ,74,75. TSilk fibroin hydrogels from the Colombian hybrid can be fabricated under sterile and stable conditions, which is fundamental for standardization approaches for biomedical device production. Furthermore, they were shown to be rich in random coils and β-sheets in their structure and susceptible to various serine proteases with different degradation profiles.The results demonstrated the high potential of Colombian hybrid Pílamo 2 silk fibroin hydrogels for the engineering of hyaline cartilage tissue. However, it is necessary to evaluate the in vivo formation of fibrocartilage and biomaterial performance when there is articular damage. The results also showed that Colombian silk could be used as a source of biomedical devices, which paves the way for sericulture to become a more diverse economic activity in emerging countries.


Respiratory distress syndrome (RDS) is a common disease that seriously endangers the life and safety of newborns, especially premature infants. Exogenous pulmonary surfactant (PS) is the specific agent for the treatment of neonatal RDS. Lung ultrasound (LUS) has been successfully used in the diagnosis of RDS, but its value in guiding the application of PS is still unclear. This paper explored whether the application of PS under LUS monitoring has some advantages, including (1) decreasing the misdiagnosis rate of RDS and decreasing probability of using PS, and (2) reducing the dose of PS without reducing the therapeutic effect.This study included two parts. Part 1: To decide whether the LUS is good to differentiate RDS from other lung diseases in the premature infants. All patients who were diagnosed with RDS and required PS treatment based on conventional criteria were routinely examined by LUS. Then, according to LUS findings, we decided whether they needed to receive PS treatment. Part 2: To see the dose reduction of surfactant is applicable. In RDS patients diagnosed based on LUS presentation and treated with Curosurf , the dose of Curosurf was compared with that recommended by the European RDS management guidelines.(1) Since March 2017, 385 newborn infants admitted to our neonatal intensive care unit met the traditional diagnostic criteria of RDS. Of these, only 269 cases were diagnosed with RDS and needed PS treatment according to LUS manifestations. The other 116 infants who did not meet the criteria for ultrasound diagnosis of RDS did not receive PS supplementation but obtained good outcomes, that is LUS findings decreased a misdiagnosis rate of RDS by 30.1% and subsequently resulted in a 30.1% reduction in PS use. (2) Among the 269 RDS patients diagnosed based on LUS findings, 148 were treated with Curosurf (another 121 RDS infants who received domestic PS treatment were not included in the study group), and the average dose was 105.4 ± 24.3 mg/kg per time, which is significantly lower than the dose of 200 mg/kg per time recommended by the European RDS guidelines. (3) The mortality rate of RDS patients was 0%, and no patients had ventilator-associated pneumonia or bronchopulmonary dysplasia in this study.LUS can decrease the misdiagnosis rate of RDS, thereby decreasing the probability of using PS and decreasing the dose of PS, and can help RDS infants to achieve better outcomes. Respiratory distress syndrome (RDS) is a common and severe pulmonary disease in newborn infants caused by primary or secondary deficiencies in pulmonary surfactant (PS), which result in the formation of an eosinophilic hyaline membrane and atelectasis of the lungs from the alveolar wall to the terminal bronchiole wall. As a result, progressive dyspnea, expiratory moaning, cyanosis, and respiratory failure occur shortly after birth. The high incidence, rapid progression, and high mortality of RDS not only causes serious harm to premature infants but also damages the health of full-term infants –3. RDS nThis study was approved by the Ethics Committee of Beijing Chaoyang District Maternal and Child Healthcare Hospital (No. 2011-LC-Ped-01). All methods were performed in accordance with the relevant guidelines and regulations. Written informed consent was obtained from the participants’ parents.Since March 2017, patients admitted to the Department of Neonatology and NICU at Beijing Chaoyang District Maternal and Child Healthcare Hospital diagnosed with RDS and requiring PS treatment based on conventional criteria were included in this study.This study included two parts:Part 1: To decide whether the LUS is good to differentiate RDS from other lung diseases in the premature infants: All patients who met the above criteria underwent LUS examination to determine how many of them still needed PS treatment based on their LUS presentation , the Curosurf dose used in this study was compared with that recommended by the European RDS management guidelines  Nonuse of surfactants: Patients who did not meet the ultrasound diagnostic criteria for RDS. (2) The PS agent used was domestic Calsurf  but not Curosurf [because of (1) absence of internationally recognized therapeutic dose as a control and (2) lack of a strict case–control study].LUS examinations: The GE Voluson S10  ultrasound system with a linear array probe (frequency >10 MHz) was used for LUS examinations in this study. All examinations were strictly performed in accordance with published guidelines and specifications  24.0 software was used to statistically analyze the data. Count data are expressed as percentages (%), and measurement data are expressed as the mean ± standard deviation (mean ± SD). A value of p < 0.05 indicated statistically significant differences if needed.During the study period, 385 newborn infants met the traditional diagnostic criteria of RDS. Of these patients, only 269 infants met the ultrasound diagnostic criteria for RDS and required PS treatment, while the other 116 patients did not receive PS treatment but had good outcomes. This result means that LUS decreased the misdiagnosis rate of RDS by 30.1% and correspondingly decreased the application of exogenous PS by the same proportion . The addCase 1: This baby was G1P1 with a gestational age (GA) of 30 weeks, cesarean delivery, and birth weight (BW) of 1,800 g. The patient was admitted to our NICU because of progressive dyspnea and expiratory moans for 2 h. Significant retraction was noted on physical examination (2: 57 mmHg, PaO2: 44 mmHg, and SaO2: 77%. Reduced transmissibility of both lungs and a bronchial air sign were noted on CXR in a local hospital. After admission, LUS examination showed SFS-like consolidation in the bilateral lungs, which was consistent with the ultrasound imaging characteristics of RDS (mination . Arterias of RDS , 17, 23 s of RDS . TherefoCase 2: This baby was G2P2 with a gestational age of 31 weeks, IVF, twin 2, and birth weight 1,620 g. The patient was admitted to our NICU because of progressive dyspnea and expiratory moans for 45 min. Significant retraction was noted at physical examination (2: 55 mmHg, PaO2: 47 mmHg, and SaO2: 80%. Reduced transmissibility of both lungs and a bronchial air sign were noted on CXR. Therefore, the baby was also clinically diagnosed with RDS. However, LUS examination showed confluent B-lines in both lungs at admission, which was consistent with the ultrasound imaging characteristics of TTN but not RDS (mination . Arteria not RDS –19, 24 ( not RDS . TherefoThe demographic data are shown in Of the 148 RDS patients who received Curosurf treatment, 24 infants received repeat doses of PS supplementation. The repeated application dose was 71.6 ± 31.5 mg/kg per time. Therefore, the repeat dose was 64.2% lower than that recommended by the International Guidelines .No patient died, and no VAP or BPD complications occurred among these patients, which resulted in a mortality rate of 0% and a VAP and BPD incidence rate of 0%.As mentioned above, the misdiagnosis rate of RDS based on the so-called traditional “Gold Standard” diagnosis can be as high as 62%–77% , 11. AccPS is an effective agent for the treatment of RDS, but the dose of PS has been controversial. The recommended dose of PS is 200 mg/kg per time according to the European guidelines . The resIt has been reported that the LUS score should be used to determine whether a newborn infant with dyspnea should receive PS treatment , 27. TheThere are some limitations in this study. The GA and BW of the infants are relatively large. The proportion of infants with GA <27 weeks and BW <1,000 g accounted for only a small proportion, which may reduce the chance of poor prognosis; further research needs to focus more on babies at lower gestational ages and birth weights. If one wants to perform LUS well, he or she needs to receive systematic training. In addition to mastering the basic theory of LUS, he or she needs to master operating skills and instrument adjustment skills . In addiIn summary, the result of the present study showed that LUS can help us to decrease the misdiagnosis rate of RDS, thereby decreasing usage and dose of PS, and can help RDS infants achieve better outcomes. Thus, it deserves widespread application in clinical practice.