The rheological properties showed that the dough development time increased significantly (p < 0.05) with increasing lupin flour in the formulation with higher water absorption. The consumer acceptability sensory results varied among the different treatments. However, the AF, CF, and EF doughnuts had the highest value for flavour, texture and crust colour, respectively. Different levels of lupin flour can be used in gluten-free doughnuts production to improve their quality and to enhance their nutritional value in the presence of inulin at a 6% level. These results may have significant implications for the development of novel, healthier food products for gluten-sensitive consumers.Celiac disease is an immune-mediated disease caused by ingestion of gluten-containing products. The main aim of this study was to develop novel gluten-free doughnuts with high nutritional value using inulin and lupin flour. Five different doughnuts were formulated. Lupin flour was used to replace the potato starch–corn flour composite at levels of 15, 30, 45, 60 and 75% in gluten-free doughnut formulations (AF), (BF), (CF), (DF) and (EF), respectively. Inulin was added to all blends at a level of 6%. Doughnuts made with 100% wheat flour (C1) and 100% corn flour–potato starch blend (C2) served as the controls. The results indicated that the moisture, ash, fat, protein and crude fibre contents of the doughnuts were increased significantly ( Celiac disease is an immune-mediated disease caused by ingestion of gluten-containing products in genetically susceptible individuals, which results in mucosal inflammation and malabsorption of micro and macronutrients . Gluten Replacement of wheat flour in baked products is a complex challenge because gluten is an essential structure-building protein with important physio-elastic properties. Moreover, gluten-free products are often not fortified and are generally prepared from refined flour and starch. Therefore, the market lacks gluten-free products of high nutritional value. In particular, gluten-free formulas and baked products are generally low in protein; thus, protein fortification of such products is important.Lupinus typically contains 36–52% protein, which is 2–3 times higher than the protein content of cereals. The seeds of white lupin have a protein content ranging from 33% to 47%, depending on genotype and location . Th. Thp ≤ 0p ≤ 0.05). The redness (a*) of the doughnuts’ crust colour ranged from 13.06 for BF to 31.76 for C2  received the highest L* value, while EF received the lowest L* value (6 for C2 . Yellown6 for C2  ranged fp ≤ 0.05), while blend C1 had the highest values for overall texture, crumb colour, hardness and aftertaste (p ≤ 0.05). Blend EF had the lowest scores for flavour and aftertaste (p ≤ 0.05). Blend CF had the lowest scores for overall acceptability, crust colour and crumb colour (p ≤ 0.05). Blend AF had the lowest score for texture (p ≤ 0.05), and blend BF had the lowest score of colour (p ≤ 0.05). It was evident that the overall acceptability, flavour, colour, crust colour and aftertaste scores declined as more of the potato starch–corn flour combination was substituted with lupin flour (p ≤ 0.05), and this was probably due to increasing levels of alkaloids and proteins in the blend. Alkaloids give a bitter taste, and high protein content leads to undesirable crust and crumb colour due to the Maillard reaction. The overall texture did not significantly (p > 0.05) decline as the level of lupin flour was increased. Hardness was significantly (p ≤ 0.05) improved as more of the potato starch-corn flour combination was substituted with lupin flour due to more protein in the flour blend. Previous studies on baked products, such as gluten-free cakes and muffins, documented that incorporation of lupin flour at up to 20–30% can be achieved without reducing the sensory quality and acceptability [The sensory evaluation results are shown in tability . In conclusion, it is possible to produce good quality, high-nutritional-value, gluten-free doughnuts from inulin (6%), potato starch, whole grain corn flour and whole lupin flour at various ratios. However, the gluten-free doughnut formulations yielded lower sensory attributes compared to controls. The doughnut blends with low levels of lupin flour (15–30%) have acceptable flavour and colour. The main challenge in preparing lupin flour-based doughnuts was the bitter taste and unacceptable crust colour. These results have significant implications for the development of healthier, novel gluten-free food products containing lupin flour.The results obtained from this study were promising, such that it is feasible to develop quality gluten-free doughnuts with an emphasis on nutritional value regarding increased levels of protein, fiber, and prebiotics and within a sensually acceptable degree by replacing wheat with different levels of lupin flour with the presence of inulin at 6% level. This may have significant implications for the development of a novel, healthier food product for gluten-sensitive consumers.


In this paper, a low-altitude wind speed estimation method based on the fuselage frustum conformal array system is proposed. Firstly, based on the signal model of the fuselage conformal array radar, the four-dimensional joint phase compensation of the echo data in the Doppler domain and three-dimensional space-frequency domain is performed by using the four-dimensional frequency domain compensation method. Secondly, the clutter covariance matrix is estimated by the compensated echo data, and a space-time Adaptive Processing (STAP) processor suitable for low-altitude windshear target is constructed to suppress clutter. Finally, the maximum Doppler value of each distance cell is extracted, and the wind velocity is estimated. Simulation results show that the proposed method can effectively suppress clutter and accurately estimate wind speed. Low-altitude windshear is an atmospheric phenomenon in which the airflow below 600 m in height hits the ground and spreads in all directions . Sudden Airborne weather radar is an important tool for pilots to detect weather phenomena ahead of the aircraft during flight missions, and pilots can avoid severe weather phenomena such as low-altitude windshear on time based on the display and alerts from the airborne weather radar to ensure the safety of the entire mission . With thWhen an airborne weather radar detects a low-altitude windshear target ahead, the widely distributed and strong ground clutter tends to drown out the low-altitude windshear signal, making it difficult to achieve wind speed estimation, so clutter suppression before wind speed estimation is especially important. In the forward-looking working state of airborne weather radar, the clutter distribution characteristics of the echo signal of each distance unit vary with the change of distance unit, especially in the near-ground distance unit performance is more dramatic, this phenomenon is called clutter range dependence. The inhomogeneity of clutter caused by range dependence makes the echo signal data samples of each distance unit no longer satisfy the independent identically distributed condition, which causes the deterioration in the performance of traditional clutter suppression methods and the degradation in target detection performance. At present, the compensation methods used for the clutter inhomogeneity problem are mainly Doppler compensation type methods, such as the DW method , the HODA method is proposed to estimate the wind speed of low-altitude windshear in the fuselage frustum conformal array system for the problem that the useful signal will be affected by strong ground clutter when the low-altitude windshear is detected by fuselage frustum conformal array weather radar. The method is based first on the essential reason for the difficulty of clutter suppression caused by the special array structure of the fuselage frustum conformal array, and then uses the four-dimensional frequency domain compensation method for joint phase compensation of the echo data of each distance unit in the Doppler domain and the three-dimensional space-frequency domain. Secondly, the compensated echo data samples are used to estimate the clutter covariance matrix and construct a Space-Time Adaptive Processing (STAP) processor for low-altitude windshear target to accumulate windshear signals while adaptively suppressing ground clutter. Finally, the maximum Doppler value of each distance unit is extracted to achieve the accurate estimation of wind field velocity. The simulation results show that the method can effectively achieve ground clutter suppression and accurate wind speed estimation.When the fuselage frustum conformal array detects the low-altitude windshear target, the echo mainly consists of three parts: ground clutter signal Assuming that the number of transmitted pulses in one coherent processing time of the radar is K, the space-time two-dimensional data received by the The ground clutter signal rd model  developeFirstly, the width of a single distance unit is determined based on the radar-related parameters of the airborne platform. Then, the horizontal azimuth is divided within the single distance unit, the ground clutter echo signal within the single distance unit is divided into multiple clutter patches, and the echo signal of each clutter patch is calculated. Finally, the echoes of all clutter patches on the single distance unit are summed to obtain the ground clutter echo signal Assuming that the ground clutter signal is undulation-free and unambiguous, the ground clutter signal Supposing that Suppose the radius of the bottom surface of the frustum is rived as :(4)p, it can be seen that the normalized space-frequency For the line array antenna, the geometric distribution of the array elements in space is one-dimensional. Assuming that the line array axis is in line with the positive direction of ion (13) :(15)Fd−lFor the planar array antenna, the geometric distribution of its array elements in space is two-dimensional. Assuming that the length of the planar array is in line with the positive direction of the ion (13) :(16)Fd−pAs for the fuselage frustum conformal array, the antenna array elements are geometrically distributed in space as three-dimensional space, so its clutter distribution characteristic needs to be described in four-dimensional space ion (13) :(17)Fd−fFrom Equation (17), it can be seen that the range of values of The analysis of the clutter characteristics of the fuselage frustum conformal array shows that the clutter characteristics are determined by the four-dimensional frequency The four-dimensional frequency domain compensation method requires joint compensation in the four spaces of Doppler domain In the Doppler domain Assuming that the azimuth of the main beam is The normalized Doppler frequency To make the Then the compensation matrix In the space domain At this point, the normalized space domain frequency The normalized space domain frequency To make the center of the clutter spectrum of the Then the space domain compensation matrix In which In the space domain At this point, the normalized space domain frequency The normalized space domain frequency To make the Then, the space domain compensation matrix In which In the space domain At this point, the normalized space domain frequency The normalized space domain frequency To make the Then, the space domain compensation matrix In which From Equations (21), (25), (29), and (33), the compensation matrix essed as ,27:(34)βIn which Suppose that the echo data of the After compensation, the inhomogeneity of the clutter in the echo data samples caused by the fuselage frustum conformal array structure is improved. The data of the The space-time adaptive processor technique is based on space-time coupling characteristics of the echo to perform clutter suppression and signal matching , and theIn which Taking the estimated By matching the filtering of target signals in different Doppler bands and solving the output signal power value of the optimal processor, the processor can effectively suppress ground clutter and accumulate windshear signals when the output power is maximum. Then, the Doppler frequency of the distance unit under detection is searched in one dimension, and the estimated Doppler frequency Finally, the echo data of all the distance units of the fuselage frustum conformal array are subjected to optimal space-time adaptive processing, and the wind field velocity is estimated, so that the variation of the low-altitude windshear wind speed can be derived for different distance units.Step 1: Compensate the echo data by four-dimensional frequency domain compensation method to remove the inhomogeneity of ground clutter caused by the structure of the fuselage frustum conformal array;Step 2: Estimate the clutter covariance matrix of the distance unit under detection from the echo data samples of the reference distance unit;Step 3: Construct the space-time adaptive processor, solve the optimal weight vector of the processor, complete clutter suppression and signal matching, estimate the center frequency of the low-altitude windshear signal within the present distance unit, then get the wind speed estimation result;Step 4: Update the distance unit under detection and deal with the echo data samples of all distance units within the radar operating range to get the final wind speed estimation result.The values of the main simulation parameters of the radar system are shown in As can be seen from After compensating the echo signal by the four-dimensional frequency domain compensation method proposed in this paper, the clutter range dependence of the near-ground distance unit has been suppressed in For the problem that the useful signal is affected by strong ground clutter when detecting low-altitude windshear, a method for estimating low-altitude windshear wind speed in the fuselage frustum conformal array system is proposed. The method introduces the fuselage frustum conformal array and analyzes the essential reasons for the difficulty of clutter suppression in the fuselage frustum conformal array. Then, it uses a four-dimensional frequency domain compensation method to combine the phase compensation of the echo data of each distance unit in the Doppler domain and the three-dimensional space-frequency domain. After that, it estimates the clutter covariance matrix from the compensated echo data samples and constructs a space-time adaptive processor applicable to the low-altitude windshear target. Finally, it extracts the maximum Doppler value of each distance unit after adaptively suppressing the ground clutter to achieve the accurate estimation of wind field velocity. The simulation results show that the method can effectively suppress the ground clutter and accurately estimate the wind speed in the fuselage frustum conformal array system.


To the authors’ surprise, self‐polycondensation of the asymmetric 1,2,4‐HPB‐NH2  also affords highly crystalline COF  similar to the symmetric 1,3,5‐HPB‐NH2 counterpart with identical topological structure. The triangular porous structures of both HPB‐based COFs are well resolved by powder X‐ray diffraction (PXRD), theoretical simulations, nitrogen sorption, and morphologies analysis. This work demonstrates the “two‐in‐one” type asymmetric building blocks can also produce highly crystalline frameworks and thus provides a new structural design strategy for reticular chemistry.Most of the reported covalent organic frameworks (COFs) so far are prepared from highly symmetric building blocks, which to some extent limits the expansion of COF diversity and complexity. Low‐symmetric building blocks can be designed through a desymmetrized vertex strategy, which might be used to construct new topological COFs. But reports of COFs constructed by asymmetric building blocks are thus far very rare. Here, a feasible strategy to design asymmetric building blocks for COF synthesis is introduced, by simply varying the positions of functional groups in the monomer. As a proof of concept, two isomeric hexaphenylbenzene‐based “two‐in‐one” type monomers . Self‐polycondensation of the asymmetric monomer 1,2,4‐HPB‐NH2 can also form highly crystalline triangular topological 1,2,4‐HPB‐COF, which is almost the same as that of 1,3,5‐HPB‐COF prepared by the C3‐symmetric 1,3,5‐HPB‐NH2 isomer. The structures and electronic properties of these two HPB‐COFs are systematically investigated.A branch‐exchange desymmetrization molecular design strategy is demonstrated by synthesizing two isomeric hexaphenylbenzene‐based “two‐in‐one” type monomers (1,2,4‐HPB‐NH Their promising application potentials benefit from the intriguing structural features, including highly ordered crystalline skeletons, large and permanent porosities, high stability, and tunable functionality and topologies. Since the first report by Yaghi et al., tremendous synthetic efforts have been devoted toward high‐quality COFs with fascinating structures and functions. Choosing suitable building blocks is crucial to precisely regulate and obtain the targeted COFs with desired topological structures. Up to now, the highly symmetric building blocks are most commonly used for the construction of COFs. For example, the hexagonal or quadrangular COFs can be readily prepared using 2‐Csymmetric linear linkers with 3C or 4‐Csymmetric vertices. However, reports on trigonal porous COFs through reacting 6C‐ and 2C‐symmetric building blocks are much sparse due to the limitation of suitable building units, while the triangular topology usually exhibits smaller aperture and higher π‐column density than those of 2D hexagonal and tetragonal structures as reported by Jiang et al. Low‐symmetric building blocks can also be utilized to construct diverse COFs. For example, Zhang et al. exploited a desymmetrized vertex design approach through lengthening or shortening the branches of high‐symmetry building blocks  or even asymmetric ones, which can form a variety of heteroporous COFs  in the same monomer  as a new class of crystalline and porous organic materials1 haves Scheme .[15]r Scheme .[16] The six external benzene rings and the almost fixed angle favor altering the position of the branch to regulate the symmetry. In addition, several synthetic routes to prepare HPB derivatives are available, such as Diels−Alder reaction, cyclotrimerization of diphenylacetylenes, and Suzuki–Miyaura coupling reaction. Among them, cyclotrimerization of asymmetric functionalized diphenylacetylenes can form two isomers, i.e., the 3‐Csymmetric 1,3,5‐substituted isomer and the asymmetric 1,2,4‐substituted counterpart. The asymmetric 1,2,4‐substituted isomer could be considered as position exchange between sites 3 and 5 and between sites 2 and 4 of the highly symmetric 1,3,5‐substituted isomer, which meets the aforementioned desymmetry strategy by altering the positions of the branches ‐based building blocks can be used as an ideal example to demonstrate the third desymmetrization approach by reversing the positions of the four branches. The six peripheral phenyl substitutes in HPB were arranged in the central benzene core with nearly identical angles between two peripheral branches (60°).17 Thes Scheme . Owing ts Scheme .2 and 1,2,4‐HPB‐NH2  results. Interestingly, the PXRD profiles of 1,2,4‐HPB‐COF are nearly the same as those of 1,3,5‐HPB‐COF. Because of the nonsymmetry of 1,2,4‐HPB‐NH2 and disorder of —C═N— in framework, it is too difficult to distinguish different possibilities and directly simulate the long‐range ordering structure of triangular 1,2,4‐HPB‐COF on Materials Studio software , the isomeric intermediates  were first prepared by cobalt‐catalyzed cyclotrimerization of the asymmetric tolan precursor aniline with 1,3,5‐HPB‐Br and 1,2,4‐HPB‐Br, respectively  in various simplex solvents . Better crystallinity can be achieved in five different alcohols as the reaction solvents using 300 µL of 6 m acetic acid as the condensation catalyst.To synthesize the two targeted isomeric monomers  13C NMR spectra of both HPB‐COFs  spectra  and transmission electron microscopy (TEM). Figure 20 Theg Figure . These rFigure 2 and disorder arrangement of —C═N— bonds in 1,2,4‐HPB‐COF , (200), (31¯0), (001), and (400) facets, respectively. Meanwhile, the six propeller‐like peripheral phenyl units are uniformly distributed around the central benzene core with dihedral angles of ≈66° . Therefore, self‐polycondensation of the asymmetric 1,2,4‐HPB‐NH2 also produces stable triangular 1,2,4‐HPB‐COF via with unit cell parameters similar as that of 1,3,5‐HPB‐COF. These results also suggest asymmetric “two‐in‐one” building blocks can readily be used to prepare COFs through rational molecular design.Furthermore, the skeleton of 1,3,5‐HPB‐COF was validated by means of Rietveld refinement on the program Topas v5. The refined PXRD profile was in good agreement with the experimental result of 1,3,5‐HPB‐COF and the unit cell parameters were determined as 2 adsorption‐desorption isotherms were measured at 77 K for these HPB‐COFs prepared from five different alcohols. All isotherms of 1,2,4‐HPB‐COFsolvents and 1,3,5‐HPB‐COFsolvents exhibited type I sorption characteristics with a sharp N2 uptake under low relative pressure range  surface areas of ≈1000 m2 g–1. According to the nonlocal density functional theory (NLDFT), the pore size distribution (PSD) of the HPB‐COFs was calculated. 1,2,4‐HPB‐COFsolvents showed micropores of 0.99–1.10 nm, which were in good agreement with the aperture sizes of 1,3,5‐HPB‐COFsolvents and the simulated AA stacking model  benzene  and asymmetric 1,2,4‐tris(4‐aminophenyl) benzene , which change the position of the branch compared with 1,3,5‐triphenyl benzene  with neopentyl glycol acetal protecting groups. To our great surprise, the asymmetric monomer 1,2,4‐HPB‐NH2 can still form highly crystalline 1,2,4‐HPB‐COFs with nearly identical triangular topological structures as that of 1,3,5‐HPB‐COF synthesized from the symmetric 1,3,5‐HPB‐NH2 counterpart. The successfully prepared the triangular 1,2,4‐HPB‐COF demonstrates the feasibility and new possibilities for preparing novel COFs via the branch‐exchange desymmetrization design strategy, which provides new insights into COF design from asymmetric monomers and enhances the diversity of the COF library.In conclusion, we designed and synthesized two isomeric HPB‐based “two‐in‐one” monomers  and organic solvent were sequentially added into the Pyrex reaction tube. The mixture was then sonicated and added with acetic acid as a catalyst. After further sonication and three freeze–pump–thaw cycles, the reaction tube was heated at 120 °C. Finally, the precipitated off‐white solid was collected via centrifugation after 3 days. Please refer to Supporting Information for detailed preparation steps of all intermediates, 1,2,4‐HPB‐NH2, and 1,3,5‐HPB‐NH2.The “two‐in‐one” type monomers (1,2,4‐HPB‐NHThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Single-molecule fluorescence spectroscopy and molecular dynamics simulations illuminate the structure and dynamics of PSD-95, a protein involved in neural plasticity. Related research article Hamilton GL, Saikia N, Basak S, Welcome FS, Wu F, Kubiak J, Zhang C, Hao Y, Seidel CAM, Ding F, Sanabria H, Bowen ME. 2022. Fuzzy supertertiary interactions within PSD-95 enable ligand binding. eLife11:e77242. doi: 10.7554/eLife.77242.To help you process this article, billions of neurons in your brain have been learning for years how to convert images and letters on the screen into words and ideas. The biochemical background for learning and memory is neuroplasticity: neurons emerge and die, form new synapses, and abandon old ones. The strength of each connection is fine-tuned by protein molecules communicating with each other inside a synapse.One of the most abundant proteins in the postsynaptic density – the area of the neuron where nerve signals are amplified or repressed – is a protein called postsynaptic density 95 (PSD-95). This protein modulates interactions between hundreds of other proteins , and itsNow, in eLife, a group led by Mark Bowen (from Stony Brook University), Feng Ding and Hugo Sanabria (both from Clemson University) – with George Hamilton, Nabanita Saikia and Sujit Basak as joint first authors – reports on a structural model of PSD-95 based on single-molecule fluorescence microscopy and computer simulations. The group also confirms the predictions of the proposed model with a complimentary biochemical technique called disulfide screening, and reveal how ligand binding by one protein domain within PSD-95 is assisted by the proximity of another domain .PSD-95 consists of five domains: three PDZ domains , an SH3 domain, and a GuK domain . Three oHamilton et al. used single-molecule fluorescence microscopy to track the conformational dynamics of PSG within full-length PSD-95, showing that the PDZ3 domain switches between two major conformations with respect to the SH3-GuK complex . In partThe conformations observed via smFRET are in good agreement with those that Hamilton et al. describe using discrete molecular dynamics simulations. In one conformation, the PDZ3 domain is close to the SH3 domain without contacting the GuK domain, while in the other, the PDZ3 domain is bound to the GuK domain. To validate the proposed atomic structures Hamilton et al. used disulfide screening. This technique relies on the fact that cysteine residues will form disulfide bonds with each other quicker when they are in close proximity. By substituting amino acid residues in PSD-95 for cysteines, Hamilton et al. were able to determine that residues that were presumed to be close together based on the proposed structures formed disulfide bonds faster than randomly selected pairs of residues.Often, in realtime structural investigations like the ones performed by Hamilton et al., if a protein switches between two conformations faster than the detector can capture, the observed conformation will be a ‘blurred’ average, equally distant from the two extreme protein states . PreviouThe observation by Hamilton et al. that PSD-95 fast-switches between its two conformations solves this almost decade-old contradiction: experiments that only capture slow dynamics result in an ‘averaged’ structure of the protein, while capturing the fast dynamics of PSD-95 reveals the two extreme conformations that lead to that average. Hamilton et al. captured both fast and slow protein dynamics using two types of single-molecule FRET microscopes, and their structural model containing two states for PSG now agrees nicely with data from other techniques.Finally, Hamilton et al. demonstrated that interactions between protein domains in the PSG supramodule are critical for the activity of PSD-95, and, in particular, for its interaction with neuroligin. Negative electric charges from the SH3 domain balance positive charges near the ligand binding pocket of the PDZ3 domain to allow PSD-95 to bind to the positively charged ligand. Additionally, Hamilton et al. showed that structural dynamics of PSG are fine-tuned by the two other domains (PDZ1 and PDZ2). This tuning results in complex local changes of structural dynamics on the timescales from microseconds to seconds.The techniques used by Hamilton et al. pave the way for future insights into natural interactions between domains in the same protein. This may facilitate the design of more selective drugs targeting inter-domain protein interfaces, as well as de novo design of multi-domain proteins for biotechnological applications. Furthermore, the work of Hamilton et al. builds a solid foundation for further intriguing investigations. For example, how are the conformations and structural dynamics of PSD-95 affected by ligands, protein partners, or post-translational modifications? It will also be important to determine whether there are protein partners and ligands that preferentially bind PSD-95 in the conformation in which the PDZ3 domain directly interacts with the Guk domain? This is, definitely, something for us and the neurons in our brains to learn in the future.


SHARPIN and RBCK1 (which encodes HOIL-1L), components of LUBAC, were recently identified as genetic risk factors of AD. A structural biological simulation suggested that most of the SHARPIN SNPs that cause an amino acid replacement affect the structure and function of SHARPIN. Thus, the aberrant LUBAC activity is related to AD. Protein ubiquitination and ubiquitin-binding proteins, such as ubiquilin 2 and NEMO, facilitate liquid-liquid phase separation (LLPS), and linear ubiquitination seems to promote efficient LLPS. Therefore, the development of therapeutic approaches that target ubiquitination, such as proteolysis-targeting chimeras (PROTACs) and inhibitors of ubiquitin ligases, including LUBAC, is expected to be an additional effective strategy to treat neurodegenerative diseases.In neurodegenerative diseases such as Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS), the progressive accumulation of ubiquitin-positive cytoplasmic inclusions leads to proteinopathy and neurodegeneration. Along with the seven types of Lys-linked ubiquitin chains, the linear ubiquitin chain assembly complex (LUBAC)-mediated Met1-linked linear ubiquitin chain, which activates the canonical NF-κB pathway, is also involved in cytoplasmic inclusions of tau in AD and TAR DNA-binding protein 43 in ALS. Post-translational modifications, including heterologous ubiquitination, affect proteasomal and autophagic degradation, inflammatory responses, and neurodegeneration. Single nucleotide polymorphisms (SNPs) in  Neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington’s disease (HD), and prion diseases, are fatal diseases caused by the progressive loss of structure and function of neurons in the central or peripheral nervous system, and accompanied by protein aggregation and ubiquitin-positive inclusion body formation . Importavia seven internal Lys (K) residues, K6, K11, K27, K29, K33, K48, and K63. The most abundant K48-linked ubiquitination predominantly induces proteasomal degradation, whereas the second most K63-linked chain functions signal transduction, DNA repair, and membrane trafficking. Importantly, linear ubiquitin chain assembly complex (LUBAC) is the only E3 that generates the N-terminal M1-linked linear ubiquitin chain through a peptide bond (Protein ubiquitination is one of the major PTMs. The human ubiquitination system comprises two ubiquitin-activating enzymes (E1s), ∼40 ubiquitin-conjugating enzymes (E2s), and >600 E3s, and regulates various cellular functions by producing multiple types of ubiquitin linkages, so-called “ubiquitin code” . The C-tide bond , and is ide bond . These dide bond . Most liide bond . These Uide bond . For exaide bond , whereaside bond . Currentide bond , and theide bond . TherefoUBB, UBC, UBA52, and UBA80), recycling ubiquitin prior to proteasomal degradation, editing the ubiquitin linkages, and maintaining the status of the free ubiquitin pool , and HOIP (RNF31)  Tokunag. The ubiHOIP has a PNGase/UBA or UBX (PUB) domain at the N-terminal portion, which plays an important role to recruit DUBs such as OTULIN  and the +1), are reportedly involved in AD  analyses showed that single nucleotide polymorphisms (SNPs) in SHARPIN and RBCK1 (which encodes HOIL-1L) are genetic risk factors for late-onset AD (Sharpin in mice (cpdm mice) causes early-onset severe dermatitis (RBCK1 are known to cause polyglucosan body myopathy type 1 (PGBM1), with or without immunodeficiency   is an intron variant  . Most of variant . Among t variant . Moreove variant . As an e variant . The siRSHARPIN, we will start from a structural biology point of view. The amino acid sequence alignment of SHARPINs from various species showed that some SNPs occur at evolutionarily conserved amino acids and/or in the functional domains  and the UBL domain (residues 219–302) of SHARPIN bind the UBL domain of HOIL-1L and the UBA1 domain of HOIP, respectively . These iM domain . The GlyM domain . The strM domain . G183 ofM domain . FurtherM domain . As the tivation , the G18The R274W, D291G, and P294S SNPs are located in the SHARPIN UBL domain  de Roja. The cryin vitro (2)  that binds to the proximal ubiquitin to enhance the binding affinity for M1-linked chains. Although the SHARPIN NZF does not contain an additional NZF tail, the Arg-Pro sequence is conserved between the NZF domains of HOIL-1L and SHARPIN (R365-P366 of SHARPIN) , except for the regions outside the NZF domain of SHARPIN (residues 377–387)   (Sato etitro (2) . The HOISHARPIN) . Since t HOIL-1L . The pre377–387) . T358A o377–387) . An alip377–387) . Furtherbiquitin . Since t 58-fold , the T35 58-fold . The Alp 58-fold . Therefoin vitro, it inhibits M1-linked chain assembly and NF-κB activation by LUBAC in the cell. However, the effects of the S17F, W379X, and P381R mutations on SHARPIN cannot be predicted, because residues 1–19 and 377–387 are poorly conserved and within flexible loop regions , SOD1, C9ORF72, OPTN, UBQLN2, and so on, have been identified (SOD1) was initially identified as a causative gene in fALS  shares significant sequence similarity with that of NEMO . We repopatients . Althougpatients . Therefopatients . Furtherpatients . These rNot only tau and TDP-43, but M1-ubiquitin is reportedly colocalized with protein aggregates formed by the overexpression of HD-derived polyglutamine proteins and Machado–Joseph disease-associated ataxin-3 . ImportaIn addition to proteasomal degradation, protein ubiquitination also plays an important role in aggrephagy, the adaptor-mediated autophagy of aggregated proteins. In this pathway, ubiquitinated protein aggregates are recognized by autophagy receptors such as p62 (SQSTM1), NBR1, TAX1BP1, NDP52 (CALCOCO2), and OPTN, through the ubiquitin-binding domains. These receptors further involve the LC3-interacting region (LIR) domains, and therefore bridge ubiquitinated cargo and lipidated LC3 for selective autophagy . In neurDuring inclusion body formation in neurodegenerative diseases, proteins with low-complexity domains are sorted by LLPS into protein/RNA-rich droplets and membrane-less organelles. Various PTMs, such as phosphorylation, ubiquitination, acetylation, arginine methylation, arginine citrullination, and PARylation, regulate LLPS . HeterolUBQLN2 cause dominant X-linked ALS and ALS/dementia  contains the N-terminal ubiquitin-like (UBL) and C-terminal UBA domains, and mutations in the Pro-rich (PXX) region of dementia   domains, which bind M1- and K63-ubiquitin chains, respectively, are crucial for the LLPS  antagonist, and monoclonal antibodies such as Aducanumab, Gantenerumab, and so on, which target and remove β-amyloid, are expected to be effective disease-modifying therapeutics  such as tacrine (tetrahydroaminoacridine), donepezil, rivastigmine, and galantamine are used for the symptomatic treatment of AD . Memantiapeutics .etc. are in clinical studies  and E3s such as CRL4-Cereblon (CRBN), VHL, IAPs, and MDM2, with an optimal linker, leading to the proteasomal degradation of target proteins . PROTACs studies . At pres studies . To trea studies . Similar studies . Importaal cells   and edaravone (an antioxidant) are used for ALS treatment, although their efficacies are limited . TherefoOPTN-deficient cells (Hoip or the treatment with the HOIPIN-8 reduced the aggregates of the ALS-associated A315T mutant of truncated TDP-43 in Neuro2a cells (in vivo drug efficacy analyses using ALS model mice are necessary.We screened 250,000 small molecular chemicals, and identified a thiol-reactive, α,β-unsaturated carbonyl-containing chemical compound, named HOIPIN-1 from HOIP inhibitor-1, as a LUBAC inhibitor . We devent cells . We foun2a cells , and the2a cells  (Zhang e2a cells . These rIn this review, we have summarized the findings detailing how various types of ubiquitination and deubiquitination, including M1-ubiquitin chains, are involved in aggregate formation and proteinopathy in AD and ALS. Ubiquitination causes changes in the physical properties and cell functions of aggregable proteins, and is deeply involved in the progression of neurodegenerative diseases. It is likely that the involvement of non-Lys ubiquitination and non-protein ubiquitination in these diseases will also be revealed. The development of E3 inhibitors functioning in ubiquitin-positive aggregate formation could suppress neurodegenerative diseases, and since M1-ubiquitin can only be produced by LUBAC, it is an important therapeutic target. PROTACs may also become effective tools in the future. If E3 proteins such as RNF182 and TRIM9, which are specifically expressed in the central nervous system, can be linked to target proteins, then it may be possible to develop therapeutic drugs for neurodegenerative diseases with minimal side effects . Further


The persistent left superior vena cava (PLSVC) is a congenital heart anomaly reported in 0.3–0.5% of the general population and can be associated with congenital heart diseases in up to 8% of cases. Prenatal identification of PLSVC is important to prompt an extended cardiac and extracardiac fetal examination. We retrospectively reevaluated anomaly scans performed in our unit in a 2-year interval according to the national guidelines to evaluate the incidence of PLSVC and its association with prenatal morbidity. In our population, the incidence of PLSVC was 0.31%, and we found a low association with cardiac and extracardiac anomalies. The standard sections  are insufficient to exclude cardiac anomalies whenever PLSVC is found. In our case series, only one newborn required postnatal surgery for total pulmonary vein anomaly, and at 2 years of life all babies had a normal evolution. Prenatal diagnosis of PLSVC can raise counseling issues; therefore, awareness of its good outcome when isolated and need for an extended examination to rule out other anomalies is very important. Persistent left superior vena cava (PLSVC) is a frequent congenital heart anomaly reported in 0.3–0.5% of the general population. Generally, is a benign finding, without hemodynamic consequences, but its association with congenital heart diseases (CHD) in 4–8% is significant and requires a detailed prenatal cardiac evaluation and a systematic follow-up of the apparently isolated PLSVC to exclude unfavorable outcomes ,2.Cardio-vascular system development starts early in embryonic life, and around the 23rd day the primitive heart starts beating. The embryonic venous system is formed from three pairs of primitive veins, i.e., the vitelline, umbilical and cardinal veins. There are four cardinal veins, i.e., two pairs of anterior and posterior (left and right) veins that are responsible for the systemic venous return. In week 7, the innominate vein  is formed by the anastomosis of the left and right anterior cardinal veins. From the anterior part, the internal jugular veins are derived, and from the inferior part, the superior vena cava is formed. The pair of posterior cardinal veins are responsible for inferior vena cava and iliac veins appearance . In someThe isolated PLSVC has no hemodynamic consequence, even when the superior right vena cava is missing, since the venous blood from the upper part is drained in the right atrium. Considering the increased amount of blood accommodated through the coronary sinus (CS), the PLSVC is always, in variable degrees, associated with CS dilatation .There are is mention of social or environmental factors that could be responsible for PLSVC in the literature, which otherwise is scarce regarding that condition, reporting studies mainly regarding its prenatal diagnosis. Postnatal studies focus on the importance of being aware of that anomaly at the time of cardiac surgery  or when it is associated with other cardiac anomalies such as heterotaxy, aortic coarctation absence of Right Superior Vena cava and dilated coronary sinus. Awareness of PLSVC existence is important whenever an interventional procedure requires venous cardiac catheterization , or when associated cardiac or extracardiac anomalies are present ,5,6.The aim of this study was to evaluate the incidence and significance of the prenatal diagnosis of PLSVC in our clinic and to review the literature data regarding PLSVC diagnosis and association with cardiac and extracardiac anomalies.We conducted a retrospective study on the ultrasound scan anomalies performed in our unit in the last 2 years. The study started in February 2017 and ended in March 2019 due to the COVID 19 pandemic, when our section was closed for general obstetrics. As a tertiary maternity center, we receive referred pregnancies from the southern part of Romania . The ultrasound scans were performed according to the National Protocols approved by the Ministry of Health during the second (20–23 weeks) and the third trimester (and 30–32 weeks) by two fetal medicine certified specialists, using equipment with a probe of 3.5 MHz. The patients were informed about the scans prior to examination and provided consent for examination and medical data use for research and teaching purpose. Standard sections of the heart obtained from all patients included: thoracal transverse sections in 4-chamber view, three-vessel and trachea view (TTVV) and images of the left and right outflow tract. As a teaching and referral center, we also usually perform a thorough cardiac assessment including a sagittal view of the arches and a bicaval section. The PLSVC is diagnosed in three main sections: 3-vessel view, where a small additional vessel is found at the main pulmonary artery (MPA) left, 4-chamber view (4 CV), where the PLSVC is detected as a round shape adjacent to the wall of the left atrium and a dilated coronary sinus is present, and in a sagittal view that shows the “pipe”-like aspect of the PLSVC. Whenever a suspect image is found, an extracardiac detailed scan is also performed. Images and clips are stored for further examination and as a proof if required. The cardiac images were reviewed, and for the cases with PLSVC, we retrieved from the patients file the following data: mothers’ age, pregnancy age, parity, history of previous congenital anomalies, cardiac or extracardiac associated anomalies, pregnancy and fetal outcome, age and type of delivery, neonate outcome and baby evolution at 12 months of life.2) patient, while three patients were overweight (BMI over 25 kg/m2). No correlation between smoking status and incidence of PLSVC could be made. The main characteristics of the cases are indicated in In 24 months, 1897 pregnancies were scanned according to our records, and 6 cases of PLSVC were identified (incidence of 0.31%) . Two casThis case, a 23 years primigravida scanned at 22 + 1 weeks, presented the most commonly encountered and typical appearance of PLSVC. The diagnosis was made on four main images, three-vessel and trachea view , where fThis case was an uncommon situation defined by the association of PLSVC with the absence of RSVC. A 33-year-ols secundigravida was scanned at 21 weeks of pregnancy. In the three-vessel trachea view, PLSVC was identified, but no RSVC was observed ; a dilatCase 3 was found in a dichorionic diamniotic pregnancy of a 29-year-old primipara. One fetus had multiple venous anomalies: PLSVC, absent infradiaphragmatic IVC and hemiazygos vein , absent This case underlines the importance of careful examination and follow-up, whenever an isolated PSLVC is found. In this case, a 34-year-old woman was scanned at 22 + 1 weeks in another center and referred at 26 weeks to our unit for isolated PSLVC . We idenNo particular image was available for cases 5 and 6; no associated cardiac and extracardiac anomaly was observed. The baby of case 5 had at 3 days of life a supraventricular recurrent tachycardia that required cardiac surveillance but, under arrhythmic drugs, she recovered and presented no other events until 12 months of life.With an incidence of 0.3–0.5%, PLSVC seems to be a frequent anomaly in the general population; 4–8% of patients with CHD also have PLSVC, as reported in the last two decades. The anomaly is considered benign and without consequences on the cardiac function .However, more recent studies found that 60% of CHD patients have PLSVC, which is also associated with extracardiac anomalies in 37% of cases . The samIn our study, PLSVC incidence was 0.31% , similar to that reported in the literature. Considering the low association in our group with cardiac and extracardiac anomalies and the absence of aortic coarctation, we suggest that isolated PLSVC is more frequent than thought. The above-mentioned studies reported a higher incidence of association probably due to the postnatal diagnosis of already symptomatic patients, while in asymptomatic patients, isolated PSLVC was overlooked.Concerning the association of chromosomal anomalies in fetuses with PLSVC, some studies found a rate of 12.5% . ObviousAs a consequence, prenatal invasive diagnosis (at least CMA) is indicated whenever PLCVS is not isolated or whenever the first trimester screening calculated risk was above the cut-off. Chromosomal microarray analysis (CMA) is recommended because it can identify small deletions or duplications that can be missed by a conventional karyotype. A study from 2015 that evaluated the importance of CMA CHD-affected babies found an incremental yield of 7.0% in the overall population of CHD and of 3.4% in isolated cases .In our group, we performed only three genetic analyses in cases that had associated anomalies and were at a higher risk of aneuploidy. No chromosomal anomaly was found, and the follow-up of the babies until up to 2 years did not yield concerns about such associations.Since 2019, the standardized international and national guideline for second- and third-trimester ultrasound has been of paramount importance in soaring the prenatal detection of CHD. The protocols include two crucial sections also for PLSVC diagnosis: “two-duct” (TD) and “three-vessel and trachea view” (TVT) . These sPrenatal diagnosis of the PLSVC is not difficult, especially if we use the Doppler effect and analyze the flow direction with a peculiar interest in the innominate vein (IV) . As showSuch a challenging situation was found in case 4, where the fourth vessel was identified on the left side of OPS, but we saw also the IV that theoretically excludes a PLSVC diagnosis. Extending the examination in the sagittal section, we identified a vertical vessel behind the left atria, with a flow directed opposite to the flow in SVC. The vessel collected the pulmonary veins and was directed to the IV. In making the diagnosis, the Doppler effect was crucial in the context of CS normal size and image. The case illustrated how fallacious can be a superficial standard examination of the main sections and the importance of an extended scan whenever an anomaly is found, leading to a correct prenatal diagnosis. This can be life-saving and spare time and invasive late procedures.In our opinion, for PLSVC prenatal diagnosis, a standard TVT section must be completed with the IV examination in a higher more cranial transverse section. The absence of IV confirms the diagnosis, but its presence does not exclude it in all cases. If the IV has an inverted flow toward the PLSVC, then the RSVC is absent. Other helpful images are to assess the CS where PLSVC drains and also the presence of the RSVC.Other imaging tools such as computerized tomography, MRI or angio MRI are not used currently in prenatal diagnosis of PLSVC but can be useful in the postnatal period mainly for symptomatic babies and before surgery .Forensic necropsy performed in the same period for intrauterine or peripartum demises did not identify overlooked cases of PLSVC.Despite the reported association of chromosomal anomalies with PLSVC , in the Considering the latest studies finding a PLSVC in prenatal anomaly screenings, we must keep searching for intra and extracardiac anomalies, as Minsart et al. showed recently . In a stThe retrospective analysis and the reduced number of cases that did not allow a systematic statistical analysis are the limitations of our study. In order to assess properly the true incidence of PSLVC in normal pregnancies and its association with chromosomal, cardiac and extracardiac anomalies, more studies, on a larger unselected population, are required.Our study was based on a systematic, extended cardiac examination of fetuses and underlines the importance of not relying only on basic standard sections when an anomaly is found.Prenatal diagnosis of PLSVC is feasible and relatively easy if standard protocols are respected at the time of a second-trimester anomaly scan. Once a suspicion of PLSVC is raised, an extended examination of the fetal heart is mandatory. Coronary sinus and innominate vein are clue markers in order to rule out some rare conditions such as the absence of RSVC or TPVRA.Isolated PLSVC has no significant consequences in utero and in infancy, but its presence can require further cardiac surgery.


Previous studies demonstrated that Kalirin lacking its Sec14 domain fails to maintain cell morphology or dendritic spine length, yet whether and how KalbSec14 interacts with lipids remain unknown. Here, we report the structural and biochemical characterization of KalbSec14. KalbSec14 adopts a closed conformation, sealing off the canonical ligand entry site, and instead employs a surface groove to bind a limited set of lysophospholipids. The low-affinity interactions of KalbSec14 with lysolipids are expected to serve as a general model for the regulation of Rho signaling by other Sec14-containing Rho activators.Gated entry of lipophilic ligands into the enclosed hydrophobic pocket in stand-alone Sec14 domain proteins often links lipid metabolism to membrane trafficking. Similar domains occur in multidomain mammalian proteins that activate small GTPases and regulate actin dynamics. The neuronal RhoGEF Kalirin, a central regulator of cytoskeletal dynamics, contains a Sec14 domain (Kal A subset of Sec14 domain proteins use their gated hydrophobic pocket to bind and transport lipids. Here the authors provide structural and biochemical data revealing how the Sec14-fold of a Rho-GEF forms a surface groove that interacts with lysolipids. The functions of family members that contain only a Sec14 domain range from coordinating lipid metabolism and vesicular trafficking in yeast to providing 11-cis-retinal to retinal pigment epithelial cells and Mueller glia for delivery to photoreceptor cells7. A third of the ~30 human Sec14 superfamily members also contain enzymatic domains that control activities of the Rho family small GTP-binding proteins9.The Sec14 superfamily is defined by the presence of a CRAL_TRIO domain , best known for its ability to bind small lipophilic molecules1. Structural, biochemical and functional studies of yeast Sec14p, the prototypical CRAL_TRIO sub-family member, have guided subsequent studies12. Despite relatively limited sequence identity, members of the CRAL_TRIO sub-family for which structural information is available adopt a characteristic two-lobed globular structure. The CRAL_TRIO motif harbors a large hydrophobic pocket in the center of its α/β fold with a mobile helical gate controlling access of lipophilic ligands to the internal pocket11. A small N-terminal lobe, referred to as a CRAL_TRIO_N domain , closes off one end of the lipid-binding pocket with an all-helical tripod-like fold that can interact with bound lipid head groups. The CRAL_TRIO_N domain also participates in subcellular targeting of Sec14 domain proteins14. The only BCH sub-family member for which structural data are available is Schizosaccharomyces pombe p50RhoGAP, in which intertwined asymmetric monomers contribute to a uniquely different lipid/RhoA-binding pocket15.Based on their sequences and structural features, Sec14 superfamily members fall into two sub-families, CRAL_TRIO and BCH8. SESTD1, a synaptic regulator of Rho GTPases, falls into this sub-group, but lacks a RhoGEF domain16. Each of these six proteins lacks a CRAL_TRIO_N domain but contains a putative CRAL_TRIO domain followed by multiple spectrin-like repeats (SRs), a common structural element consisting of a three-helix bundle.The Sec14-like domains of five mammalian Rho GDP/GTP exchange factors (RhoGEFs) share limited sequence identity with both BCH and CRAL_TRIO sub-family members17 , SH3 and protein kinase domains17 Fig. . The abi20. Attachment of the Ex1C front peptide to the CRAL_TRIO domain of Kalirin enables its direct interaction with liposomes in a phosphoinositide-dependent manner; attaching it to EGFP localizes EGFP to the Golgi region20. When preceded by the Ex1B front peptide, the ability of the CRAL_TRIO domain to interact with phosphoinositide-containing liposomes is eliminated20. The ability of these front peptides to alter Sec14 domain-mediated membrane interactions is essential to their function.The tissue-specific and developmentally regulated use of alternate initiation exons (Ex1) places different peptides immediately upstream of the Kalirin CRAL_TRIO domain. Ex1B encodes a hydrophilic negatively charged front peptide, producing bKal7, while Ex1C generates a positively charged front peptide that forms an amphipathic helix, producing cKal7bSec14), the only Sec14 structure for a RhoGEF. KalbSec14 alone adopts a Sec14p homologous CRAL_TRIO domain fold, but with a closed conformation impeding access to the canonical ligand entry site. Our biochemical, biophysical and mutagenesis studies demonstrate that the amino acid residues lining a surface groove on KalbSec14 are directly involved in the binding of specific lysophosphatidylcholine (LPC) lipids and that the Ex1B peptide enhances lipid binding affinity. Both secondary-structure predictions and structure-based alignments identify KalbSec14 as the prototype of a Sec14 fold sub-class, with a unique ligand-binding site and ligand specificity . Coupled with interactions mediated by their other domains, linear peptide motifs and numerous phosphorylation sites, our findings suggest that the low-affinity interactions of SecSR domains with membrane lipids play an important role in the ability of Kalirin and other SecSR-containing RhoGEFs to participate in rapid regulation of their subcellular localization and signaling functions.In this report, we present the crystal structure of the CRAL_TRIO domain of Kalirin preceded by the Ex1B front peptide , the putative CRAL-TRIO domain (residues 26–182) and a part of the predicted A-helix (residues 183–192) of the first SR  on one side and by two α helices, H8 and H9, on the other. An N-terminal 10-amino-acid extension (residues 18–27) upstream of the H1 helix projects ~18 Å away from the αβα core, confirming the notion that the Sec14 domain of Kalirin lacks the CRAL_TRIO_N domain found in many stand-alone Sec14 proteins. Interestingly, Ile186 and Leu190 of helix H9 engage in van der Waals interactions with Ala34, Val37, Leu38, Ile40 and Leu41 of helices H1 and H2. In addition, the bulky side chains of Tyr180, His182 and Trp185 on the H9 helix stack with the benzene ring of Phe47 from strand S1. As such, the interaction of the C-terminal H9 helix, which is part of the first SR, with the central αβα fold buries 441 Å2 of solvent-accessible surface area. In the absence of the H9 helix, diffraction-quality crystals of the Kalirin Sec14 domain were not obtained, suggesting that this interaction stabilizes the core fold of the Kalirin CRAL_TRIO domain.Kalres Fig. . The cenbSec14 and several CRAL_TRIO sub-family proteins can be superimposed with a Cα rmsd of <5.0 Å  in the center of its αβα fold, none of which is large enough to accommodate even a single phospholipid molecule  and phosphatidylinositol 4,5-bisphosphate (PIP2), α-Toc is bound deep in its hydrophobic core, while PIP2 is found in close proximity to the gating helix25 , each CRAL_TRIO_N domain is distant from the location of the presumed surface groove but adjacent to the bottom of the gating helix, allowing it to interact with the head group of the bound ligand  mimetics  spectroscopy, which allows detection and characterization of weak, transient interactions27.Expanding on the finding of the KalbSec14 in the absence and presence of PC mimetics to determine ligand-interacting residues and ligand binding affinities. As expected for a well-folded structural unit, well-dispersed signals with adequate intensity were detected in the acquired two-dimensional (2D) 1H-15N heteronuclear single quantum coherence (HSQC) spectrum of KalbSec14 in native-like conditions  made it difficult to unambiguously identify individual cross-peaks. To simplify NMR data collection and subsequent data analysis, a shorter construct referred to as KalSec14 was generated. KalSec14 (residues 25–192) consists of the CRAL-TRIO domain and the A-helix of the first SR but lacks the Ex1B front peptide. The 2D 1H-15N-HSQC spectrum of KalSec14 features an extremely well-dispersed set of resonances  measurements of Kal1H and 15N resonances of the 160 non-proline residues, and 92% of the 13Cα, 91% of the CO and 88% of the expected 13Cβ resonances were unambiguously assigned for KalSec14 based on a standard set of triple resonance spectra  or dodecylphosphocholine (FC12), indicative of specific interactions between KalbSec14 and FC14/FC12 and fast exchange between the free and bound states on the NMR timescale  of 17.1 ± 4.9 μM and 245 ± 36 μM for FC14 and FC12, respectively, revealing a 14-fold higher binding affinity for FC14  suggests a role for the front peptide in ligand binding  are those forming the afore-described surface groove 20. Unilamellar liposomes formed by mixing synthetic dioleoyl phospholipids and cholesterol in proportions mimicking the membranes of the trans-Golgi network (Golgi-mixture) were incubated with KalbSec14. An Accudenz gradient was used to separate liposome-bound proteins, which were recovered from the top of the gradient (fraction 1), from unbound proteins, which remained at the bottom (fraction 4). Protein levels in each fraction were analyzed by Western blotting with an antibody to the Sec14 domain of Kalirin. As observed previously, <2% of the total KalbSec14 bound to the control liposome  lipids, 1-lauroyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC12:0) and 1-myristoyl-2-hydroxy-sn-glycero-3- phosphocholine (LPC14:0)  possesses a positively charged head group , a phospholipid with two acyl chains and a PI head group20. Strikingly, KalbSec14 does not interact with liposomes that contain PI4P , 1H and 15N chemical shifts in KalbSec14 change continuously upon titration with either LPC14:0 or LPC12:0, but not with LPC16:0  that carry alanine substitutions lining each side of the groove. These four amino acid residues were chosen not only because of their large CSPs during NMR titrations, but also for their locations in loops, thus minimizing potential disturbance of protein folding  are located in four regions surrounding the rear portion of the surface groove  than it does with other Sec14 family proteins  and SESTD1. With the KalStructural studies of multiple stand-alone Sec14-domain proteins have been integral in understanding how ligand binding induces specific biological outcomes within this large and diverse protein family. Sec14 domains also play essential roles in a number of highly-conserved multidomain mammalian RhoGEFs known to participate in the protein complexes that control signaling, membrane trafficking and cytoskeletal organization. The results presented here provide structural and biochemical evidence that Kalirin possesses a CRAL_TRIO domain with a unique ligand binding site. Preceded by alternate front peptides and followed by multiple SRs, the Sec14 domain of Kalirin contains structural and functional features evolving from both CRAL_TRIO and BCH sub-group proteins and serves as a prototype for other SecSR sub-class family members.bSec14 exhibits clear structural homology to the αβα fold of canonical CRAL_TRIO domains; a similar fold is observed in the BCH domain of p50RhoGAP even though one of its five strands is contributed by the other monomer present in its intertwined dimer15. However, KalbSec14 adopts a closed conformation in which H7, the presumed gate helix, blocks access to its fragmented hydrophobic interior, leading to the discovery of its alternate ligand binding site. By assigning NMR resonances of KalSec14, we developed a powerful tool to study the interactions of KalbSec14/KalSec14 with ligands in solution. Our NMR CSP data and mutagenesis studies demonstrate that the residues lining a distinct surface groove of the Kalirin Sec14 domain are responsible for its interaction with selected detergents and LPC lipids. Pocket distribution analyses using the structures of six CRAL_TRIO sub-family proteins confirm the notion that each contains an internal hydrophobic pocket and lacks an exposed groove similar to that observed in KalbSec14. Two exposed pockets were identified in the BCH domain of p50RhoGAP, one in a location similar to that of KalbSec14 and the other close to the canonical gating helix  are located in both the CRAL_TRIO_N and CRAL_TRIO domains and are conserved in Sec14p homologs12 and in α-tocopherol transfer protein25, indicating that these two domains function as a unit. As both the CRAL_TRIO_N domain and the phospholipid binding barcodes are absent from Kalirin, our structural and biochemical studies are consistent with the inability of KalbSec14 to recognize phosphatidylinositides20. KalbSec14 binds LPC12:0 and LPC14:0 about twice as tightly as KalSec14. Comparisons of the NMR spectra of KalbSec14 and KalSec14 indicate that the Ex1B front peptide might mimic at least some of the functions attributed to CRAL_TRIO_N domains by populating the KalbSec14 core fold in a conformation optimal for ligand binding.Unlike many stand-alone Sec14 domain proteins, the Kalirin CRAL_TRIO domain lacks a helical CRAL_TRIO_N lobe. In both phosphatidylinositol- and phosphatidylcholine-bound structures of yeast Sfh1, the lipid head groups are oriented towards the N-terminal lobe while the acyl chains remain sequestered in the hydrophobic pocketbSec14 and KalSec14 led to the identification of lysophospholipids with a single, relatively short acyl chain and a positively charged head group, with KDs in the micromolar ranges. The ~20 μM KD of KalbSec14 for LPC14:0 is distinctly different from the nanomolar KD values determined for the interactions of stand-alone Sec14 proteins such as α-TTP/Sec14p/SPF/CRALBP and their ligands31. Identification of an exposed groove suggests a major role for protein-lipid interactions on the KalbSec14 surface, rather than lipid transfer activity via the canonical buried hydrophobic pocket observed in stand-alone Sec14 proteins11.Our search for lipids that bind to Kal36. Functional studies using knockout mice and knockdown technologies indicate that Kalirin can play a role in downstream signaling from receptor tyrosine kinases, GPCRs and ion channels20. Consistent with these observations, tissue-specific, regulated phosphorylation of multiple Ser/Thr and Tyr residues in Kalirin has been documented20. In addition, multiple protein-protein interactions involving the Sec14, SR, PH and SH3 domains of Kalirin have been identified, along with interactions of its short, linear peptide motifs with PDZ-, SH3- and SH2-domains. Coupled with these additional interactions, the low-affinity interaction of KalbSec14 with lysolipids may contribute to the essential role the Kalirin Sec14 domain plays in controlling spine length and endocytic trafficking20. While the SRs and PDZ-binding motif of Kal7 may ensure its stable enrichment in the post-synaptic density, changes in the lipids accessible to the Kalirin Sec14 domain at the post-synaptic membrane or in tension on its SRs may contribute to the inhibitory effect these regions have on its GEF activity (see below).It is widely recognized that rapid responses require low affinity (1–100 μM), readily reversible interactions amongst multiple proteins; modularity of recognition domains and the simultaneous use of multiple low-affinity interactions ensures more readily modified, specific interaction3. Lysophospholipids, integral components of the plasma membrane, are usually present only in small amounts37. They can be generated rapidly by phospholipases (A1 and A2), serving as intermediates in phospholipid metabolism and as second messengers in GPCR-mediated signaling pathways39. With a relatively large hydrophilic head group and a cone shape, incorporation of lysolipids into membrane bilayers introduces packing stresses and drives lipid assemblies into structures with more positive curvature41. Interestingly, it should be noted that KalbSec14 and KalSec14 bind selectively to a very narrow range of the LPC species (with hydrocarbon tails from 12:0 to 14:0). While our data suggest a role for phospholipase activation in generating a lysophospholipid that might bind to KalbSec14, acyl chains with 14 carbons are generally not prevalent. Whether membrane curvature or membrane disruption associated with vesicular trafficking might expose a limited region of longer acyl chains has not yet been assessed. New methods are needed to allow the assessment of lipid/protein interactions in a cellular environment with subcellular specificity42.For the vast majority of Sec14 superfamily members, the identities of their physiologically relevant ligands remain unknown43. Unlike conventional CRAL_TRIO domain containing proteins, SecSR family members share high sequence homology and are implicated in control of actin cytoskeletal organization and dynamics by modulating the activity of small GTPases. None of the other Sec14 sub-groups contains a RhoGEF, and RhoGAPs are found exclusively in BCH domain sub-families1. Besides a role in subcellular localization, the function of the SecSR domain in five RhoGEF proteins seems to be inhibition of GEF activity. The catalytic activity of the isolated GEF domain of Kal7 is much higher than that of intact Kal7 or Kal7 lacking its Sec14 domain and first four SRs17. In agreement with this observation, Sec14 domain and SR removal reveals the full transforming activity of MCF2 and MCF2L, identifying an autoinhibitory intramolecular interaction of the Sec14 domain with the GEF PH domain45. A subset of BCH-domain containing RhoGAPs, much like their GEF counterparts, use an autoinhibitory interaction of their BCH and GAP domains to control activity47. As the postulated non-protein ligands for the SecSR-containing proteins are currently unknown, an exciting possibility is that the SecSR domain may directly interact with the small GTPase or its post-translationally modified protein terminus to regulate signaling cascades. Further structural and functional studies are needed to understand the potential implications for small GTPase signaling mediated by the previously uncharacterized SecSR domain-containing proteins.Our structure-based sequence alignment analyses demonstrate that the Kalirin CRAL_TRIO domain can serve as the prototype for a distinct functional subclass of the Sec14 superfamily. The six members of the SecSR sub-group are multidomain proteins with an N-terminal CRAL_TRIO domain followed by a variable number of SRs. Kalirin, TRIO, MCF2, MCF2L and MCF2L2 are Dbl family RhoGEFs, while SESTD1 partners with multiple GAPs and GEFs to modulate RhoA GTPase activitiesKALRN sequence [NCBI Accession #: U88156.1 with two single nucleotide sequence corrections (C197G and A244T) that lead to two amino-acid changes (T66S and N82Y)]; constructs were verified by DNA sequence analysis. Amino acid numbers are based on the initiator Met encoded by Ex1B as residue 1. Inserts encoding KalbSec14  and KalSec14  were cloned into a modified pET15b vector containing a removable tobacco etch virus (TEV) protease recognition site. Primer sequences are: 5'-ATATGCGGCCGCAACCCCCCGGAGGGGGCA , 5'-ATATGGTACCTCACAGGGAGAGGCGCAACTCAA , and 5'-ATATGCGGCCGCGGATCTTTTCGGAATGATGGTTT . The surface groove mutations of KalbSec14 were introduced using an overlap PCR method and confirmed by sequencing. Each of the constructs was expressed in E. coli BL21(DE3) and purified by Ni2+-nitriloacetic acid  affinity chromatography. After removal of the N-terminal His6 tag by overnight cleavage with TEV protease, the proteins were further purified by anion-exchange  and gel-filtration  chromatography. For crystallization, KalbSec14 was concentrated to 1.1 mM by ultrafiltration in a buffer containing 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 5 mM dithiothreitol (DTT). SeMet-substituted KalbSec14 was produced following established procedures48 and purified as described above. For NMR chemical shift perturbation experiments, each protein was expressed in M9 medium supplemented with 15NH4Cl  as the sole nitrogen source. For NMR backbone resonance assignment experiments, KalSec14 was expressed in M9 medium prepared with 15NH4Cl and [13C]-D-glucose  as the sole nitrogen and carbon sources, respectively. The 15N- and 15N/13C-labeled proteins were purified as described above and concentrated to 0.4 mM in NMR buffer containing 20 mM Na/K phosphate (pH 7.2), 120 mM NaCl, 2 mM β-mercaptoethanol, 0.2 mM EDTA, 8% D2O and 0.02% NaN3, immediately prior to data acquisition.The Kalirin constructs were designed based on the rat bSec14 was crystallized in a solution consisting of 100 mM sodium cacodylate (pH 6.5), 0.2 M ammonium sulfate, 20%-25% PEG 3350, 0.2 M NaCl at 4 °C using the hanging-drop vapor diffusion method. Crystals were cryoprotected in reservoir solution supplemented with glycerol to a final concentration of 10% and flash-cooled in liquid nitrogen. Diffraction data were collected using the Life Science Data Collection (LSDC) software at the Stanford Synchrotron Radiation Lightsource (SSRL) beamlines 14-1 and 12-2 and 17-ID-1 and 17-ID-2 of National Synchrotron Light Source II (NSLS-II). The diffraction data were processed with autoPROC49 and Fast DP51. The crystals contain eight molecules in the asymmetric unit. The structure of KalbSec14 was determined by single-wavelength anomalous dispersion using the data collected at the selenium peak wavelength. About 16 selenium sites were located by HySS as implemented in AutoSol/PHENIX52. Initial phases calculated from these sites were improved by density modification using Phaser/PHENIX52. The resulting electron density map was readily interpretable and used to build >90% of the molecules with the program COOT53. Iterative cycles of refinement using REFMAC5/CCP454 and BUSTER  followed by manual rebuilding in COOT were carried out until no further improvement of the Rfree factor was observed. X-ray data collection and phasing and refinement statistics are summarized in Supplementary Table 55. Molecular graphics were rendered using PyMOL (Schrödinger LLC).Kalhttps://openvnmrj.org/) at 25 °C on an Agilent VNMRS 800 MHz (1H) spectrometer equipped with a cryogenic probe. A set of standard double and triple resonance experiments were performed using 0.4 mM uniformly 13C/15N-labeled KalSec14 in NMR buffer. The spectra used for sequence specific assignment  included 2D 1H-15N HSQC, and 3D HNCO, HNCACB, HN(CO)CACB, HN(CA)CO and 15N-filtered NOESY-HSQC58. All spectra were processed with NMRPipe59 and analyzed using CARA (http://cara.nmr.ch) and CcpNmr Analysis60, which were made available through NMRbox61. The near-complete resonance assignment was obtained with 92.7% of all expected chemical shifts assigned . The assignments for KalSec14 peaks in the 1H-15N HSQC spectrum were readily transferred to the cross peaks for KalbSec14. Secondary structure predictions for KalSec14 were performed using chemical shift assignments of five atoms  for a given residue in the sequence with TALOS-N62. The predicted ρ(α) and ρ(β) values for each residue are provided in the Source Data file.NMR spectra for backbone assignment were recorded using VnmrJ  spectrometer equipped with a cryogenic probe. 1H-15N HSQC spectra of the uniformly 15N-labeled KalbSec14 mutants were recorded using the TopSpin software (https://www.bruker.com/en/products-and-solutions/mr/nmr-software/topspin.html) at 25 °C on a Bruker Avance NEO 600 MHz spectrometer equipped with a cryogenic probe. All detergents and lipids used in this study were purchased from Cube Biotech Inc.  and Avanti Polar Lipids, Inc. , respectively; details are listed in Supplementary Table 59, and peak heights and locations were analyzed in CcpNmr Analysis60.H and ΔδN are the 1H and 15N chemical shift differences between the free and bound states, respectively, and α denotes the chemical shift scaling factor for 15N63. The scaling factor α, which was set to 0.15464 for this study, was determined from the ratio of the average variances of the 1H and 15N chemical shifts observed for the 20 common amino acid residues in proteins as deposited with the Biological Magnetic Resonance Bank (BMRB)65.For each titration series, observed CSPs for a single amino acid residue were calculated using the combined shift change (Δδ) of the backbone amide nitrogen and proton based on the equation KD) was extracted by nonlinear least-square fitting of the following equation: 66. The bound fraction plotted as the y-axis in the Figures was defined as KD calculations are provided in the Source Data file.In each titration series, the peaks for all residues were ranked based on the CSP observed upon titration at the highest ligand concentration. For those peaks that showed a significant CSP, defined as those larger than two standard deviations (2σ) of the average shift for all residues, nearest neighbor chemical shift difference analysis using custom python scripts was performed to confirm shifted peak locations. The well-resolved and non-overlapped CSPs, usually including most of the 2σ CSPs and some of the 1σ CSPs, were then plotted as a function of the ligand concentration and the dissociation constant . Docking of the lipids into the KalbSec14 structure was performed using the iterated Local Search Global Optimization algorithm provided by AutoDock Vina68. The PDBQT format files (required as input) of both the lipids and KalbSec14 were generated using the AutoDock Tools package provided by AutoDock 4. The flexible ligand docking protocol was adopted in this work to allow rotation of any rotatable bonds in the lipid when it is docked to the binding site in the protein. Once the rotatable bonds are identified, the ligand is allowed to be flexible by moving different rigid parts through the iterated local search global optimizer. During the global energy optimization, the ligand binding score is evaluated by an empirical scoring function, which is a weighted sum of terms representing van der Waals, hydrogen bonds, electrostatic and desolvation contributions. The entire surface of the KalbSec14 structure was searched for possible binding sites without bias. All other parameters were set at the default values defined by AutoDock Vina. In particular, a cutoff distance of 5 Å for van der Waals interactions and 3.7 Å for hydrogen bonds between ligand atoms and protein acceptors were used. The resulting poses were scored based on the free energy of binding in which the least energetic poses would be ranked as the top hits. Total ten hits were outputted and top four hits were shown in Supplementary Fig. Three-dimensional coordinates of the lipids were generated by Open Babel20. In brief, stocks of synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), -phosphoethanolamine (DOPE), and -phospho-L-serine (DOPS) were mixed with cholesterol in molar proportions mimicking those observed in secretory pathway membranes (control lipid mix). Individual test lipids were dried together with the control lipid mix under a nitrogen stream and lyophilized overnight. Each dried lipid film was reconstituted to a final concentration of 1 mm in HN buffer containing 20 mm HEPES (pH 7.4) and 150 mm NaCl. Each final lipid mixtures consisted of 52% DOPC, 20% DOPE, 5% DOPS, 15% cholesterol and 8% tested lipid; inclusion of a trace amount of fluorescent lipid (Liss Rhod PE) allowed for localization of liposomes following gradient fractionation (see below). Each lipid mixture underwent five freeze-thaw cycles separated by vigorous vortexing each time it was thawed. Using a mini-extruder, unilamellar liposomes were prepared by passing each lipid mixture 21 times through a polycarbonate membrane of 100-nm pore size. Hydrated lipid mixtures were stored at −20 °C for up to 1 month; liposomes were freshly prepared for each experiment.The synthetic and natural lipids used in flotation assays were purchased from Avanti Polar Lipids, Inc.  solutions (80 and 20% weight/vol) were prepared in HN buffer. To remove any particulate material, the protein to be tested was first centrifuged at 436,000 g at 4 °C for 15 min in a TL-100 ultracentrifuge using a Beckman TLA100.2 fixed angle rotor. Freshly prepared unilamellar liposomes (50 μl) were combined with 0.2 μg of the centrifuged protein and incubated at room temperature for 30 min. The entire liposome/protein mixture was then combined with 50 μl of 80% Accudenz at the bottom of an ultracentrifuge tube and carefully mixed to completion by pipetting. The resulting mixture was overlayered with 250 μl of 20% Accudenz and then with 50 μl of HN buffer. Samples were centrifuged at 259,000 g at 4 °C for 30 min in the same ultracentrifuge using a Beckman TLS55 swinging bucket rotor. Gradients were then divided into four 100-μl fractions, which were carefully collected from the bottom of the tube using gel loading micropipette tips. Each fraction was analyzed for liposome and protein content using spectrophotometry (546-nm excitation/571-nm emission) and Western blot, respectively. Only those assays with at least 90% of the Liss Rhod PE marker recovered in the top fraction (fraction 1) were used for Western bloting analysis.Flotation assays were conducted following the protocol previously described69, 1:3000) and an HRP-tagged goat anti-rabbit IgG (H + L) secondary antibody . SuperSignal West chemiluminescent substrate (Thermo Scientific) and a Syngene PXi digital imaging system (Synoptics Group) were used to visualize blots, with exposures adjusted to be in the linear range. After background subtraction, bands were quantified using the GeneSys software provided with the imaging system, and data were represented as a proportion of total protein recovered. Uncropped blots, quantification of each data point and exact P-values are provided in the Source Data file.For Western bloting analysis, equal aliquots of each gradient fraction were denatured in 1× Laemmli sample buffer, fractionated on 15% acrylamide gels, and transferred to PVDF membranes. The protein content was detected using a custom-made rabbit antibody to the Sec14 domain of Kalirin (CT302Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Antisense oligonucleotides (ASOs) are an important tool for the treatment of many genetic disorders. However, similar to other gene drugs, vectors are often required to protect them from degradation and clearance, and to accomplish their transport in vivo. Compared with viral vectors, artificial nonviral nanoparticles have a variety of design, synthesis, and formulation possibilities that can be selected to accomplish protection and delivery for specific applications, and they have served critical therapeutic purposes in animal model research and clinical applications, allowing safe and efficient gene delivery processes into the target cells. We believe that as new ASO drugs develop, the exploration for corresponding nonviral vectors is inevitable. Intensive development of nonviral vectors with improved delivery strategies based on specific targets can continue to expand the value of ASO therapeutic approaches. Here, we provide an overview of current nonviral delivery strategies, including ASOs modifications, action mechanisms, and multi-carrier methods, which aim to address the irreplaceable role of nonviral vectors in the progressive development of ASOs delivery. In human disease treatment, antibody-based and conformation-corrected therapies that focus on the clearance of certain proteins associated with genetic diseases are being developed , 2, partAntisense oligonucleotides (ASOs) are synthetic small single-stranded nucleic acid polymers (approximately 18 ~ 30 nucleotides) with diverse chemical properties that can be employed to regulate gene expression via various mechanisms. Unlike small-molecule drugs, antisense drugs work through Watson–Crick base pairing with the target RNA sequence . This diASOs are synthetic oligonucleotides or oligonucleotide analogs that can be designed to bind to protein-coding RNAs as well as noncoding RNAs. They regulate RNA function through a variety of different mechanisms, depending on the types of chemical modifications, modification sites, and binding sites by which they target RNAs. Moreover, ASOs can be designed to regulate the processing of RNA molecules, including the regulation of RNA splicing and the selection of polyadenylation sites , 12, to O-methoxyethyl-(2′-O-MOE) and 2′-O-[2-(methylamino)-2-oxoethyl] improve the binding affinity of ASOs and provide resistance to enzymatic degradation [Due to hindered cell uptake , 16, offradation .ASOs are theoretically designed to regulate the transfer of genetic information to proteins specifically, but the mechanisms by which ASOs induce biological effects are subtle and complex Fig. . Based oASOs bind to target RNA to form a conjugate that recruits RNase H to degrade RNA for silencing , 28. DegASOs with steric blockage function are designed to bind to target transcripts with high affinity. Still, they do not induce degradation of the target transcripts due to their lack of RNase H recruitment capacity , 31. ThiTo deliver ASOs to the target site by different routes of administration, nanocarriers of cationic polymers are usually preferred because of their ability to form polyelectrolyte complexes by facilitating ionic interactions between the positively charged functional groups and the negatively charged phosphate fraction . The forLNPs have a suitable particle size (diameter range of 10–500 nm) combined with their own biocompatible and biodegradable lipids, which enables LNPs-ASOs to escape uptake by the mononuclear phagocyte system (MPS), subsequently prolonging the circulation time of LNPs-ASOs and allowing the particles to passively and efficiently target cells through an enhanced permeability and retention effect to release ASOs –47. TheyHiroki et al. initially hypothesized that the optimal composition of ssPalmO-Phe/Chol for siRNA delivery could be applied to ASOs, but it was revealed that LNPssPalmO-Phe containing ASOs are highly unstable and susceptible to aggregation [Several prior studies have reported LNPs to be potentially exploitable. Although some studies have not used LNPs as traditional formulations of nucleic acid drugs, the addition of lipid components alone reduces positive charge toxicity and can significantly improve biocompatibility . Howeverregation . With anLiposomes are used in the pharmaceutical and cosmetic industries to transport a wide range of molecules. They are spherical vesicles composed of phospholipids and sterols, usually in the size range of less than 500 nm . LiposomGuan et al. functionalized liposomes with a tumor-homing and -penetrating peptide, iRGD, as a carrier of an ASO against androgen receptor (AR) for prostate cancer treatment, and these iRGD-liposomes markedly improved the ASO efficacy in suppressing the growth of tumor [Liposomes are potentially more enriched in the liver and spleen than other carriers, so it is essential either to develop different types of liposomes to counteract this property or to take advantage of this property to deliver ASOs that are expected to work in these organs , 71. Theof tumor . The modof tumor .Polymers have been one of the most widely used drug delivery systems since being discovered. In addition to proteins and small molecules, polymer-drug systems are also essential for the delivery of nucleic acid drugs , 75. TheLinear cationic compounds have long been shown to be effective in delivering nucleic acids. In the 1960s, these polycationic derivatives of dextran were shown to enhance the transfection of viral RNA and DNA , 89. TheMicelles, self-assembled from block copolymers, have a unique core–shell structure with a size distribution in the range of 10–100 nm –92. AlthSynthetic polycations such as PEI and PAMAM dendrimers, and some other polycations, such as poly(amine-co-ester) (PACE) are included in this category –106 FigA-C. Due Staphylococcus aureus (MRSA): the system was prepared by the sequential modification of gold nanoparticles with PEI and maintained antibacterial ability with reduced low cytotoxicity [Marcel developed a nanoparticle-based delivery system for ASOs targeting the antibiotic resistance of methicillin-resistant toxicity . Yoshidatoxicity .PAMAM is another cationic dendrimer used to deliver ASOs. A co-delivery system is based on a cationic dendrimer core that encapsulates fluorouracil and oligonucleotides within a hydrophobic lumen, modified with hyaluronic acid and cell-penetrating peptides. The codelivery complex showed efficient cellular uptake and consequently improved intracellular distribution and enhanced cytotoxicity on cells , 109.Cui loaded solid PACE nanoparticles (PACE-NPs) with oligonucleotides designed to knockdown Nogo-B, a protein that has been implicated in the progression of alcoholic liver disease and liver fibrosis, and demonstrates that PACE-NPs can effectively deliver oligonucleotides therapeutics to the liver to mediate protein knockdown in vivo [Polymeric nanoparticles, due to their tunable architecture (10–1000 nm), nontoxicity, biocompatibility, and controlled drug release are promising options for targeted drug delivery platforms , 110. Wi in vivo .The synthesis reactions of polymers are quite mature, so chemical precision and flexibility in designing synthetic strategies are considerable; accordingly, a highly functionalized nucleic acid polymer (HFNAP) library is usually designed as needed, and the target compounds are screened by parallel experiments , 123. InNaturally derived structural proteins and polysaccharides, such as cationic collagen derivatives, cyclodextrins (CDs), and chitosan, have been developed as gene carriers –129. ColKolonko et al. developed a nonviral delivery system based on the natural aminopolysaccharide chitosan (CS) for the transport of ASOs against ENaC to specifically address Na + hyperabsorption and confirmed the successful uptake of the nanocomplex by human airway epithelial cells, demonstrating the possibility of targeted transport of ASOs with chitosan [Due to the poor specificity, low stability, and low permeability of ASOs through cell membranes, an effective nucleic acid carrier system for most studies usually requires cationic materials. Chitosan is a strong candidate due to its cationic properties, biodegradability, and excellent biocompatibility . Chitosachitosan .The naming of extracellular vesicles is extremely chaotic. Extracellular vesicles (EVs) are vesicles that are released from cells into the extracellular space, and can be subdivided into microvesicles (100 nm to 500 nm in diameter) or exosomes (30 nm to 100 nm in diameter) by their specific diameter , 137, EVThe ASO is usually loaded into the exosome by electroporation , 139. ThWhile delivery systems for artificial materials are not immune to compatibility and clearance problems, biological vesicles represented by exosomes are not immune to another challenge: low nucleic acid loading rates and the potential safety threat of carrying their own nucleic acids. The loading of nucleic acids onto vectors is low in efficiency and their functional activity may be compromised . It was Anna Graczyk et al. invented a conjugate of gold nanoparticles and structural RNA that was successfully used as a tool for gene expression regulation successfully [Gong et al. constructed MALAT1-specific ASO and nucleus-targeting TAT peptide cofunctionalized Au nanoparticles, namely, ASO-Au-TAT NPs, which stabilized fragile ASOs, enhanced nuclear internalization, and exhibited good biocompatibility [Metal nanoparticles are widely used and recognized in the fields of biotechnology and bioengineering . Currentessfully . Gong ettibility  (Fig. 8Atibility  (Fig. 8BSensitive materials have a rich history of use in the treatment of infections and tumors with superior results , 156. SiThermosensitive materials can also help retain ASO in local tissues without a serious off-target effect . As confASOs are readily degraded by nucleases in body fluids and are enriched in metabolic organs such as the liver and kidney, where they are rapidly cleared, and the half-life of unmodified and unencapsulated ASOs is usually less than 10 min –170. In Although ASO drugs continue to come to market, safety has been a stubborn factor preventing them from expanding their impact . An inveThe human system is way more complex than the in vitro culture systems or even model animals , 183; deAt this time, no ASO drugs using drug delivery systems have been approved by the FDA for marketing, and there will be no substitute for ASO therapeutic technologies for rare diseases for a significant period. It is foreseeable that the emergence of ASO drugs delivered by carriers is inevitable, but the timing depends on innovations in delivery systems. This will require breakthroughs in the development of materials, evaluation systems, synthesis methods, ethical safety, and many other aspects. Given the rapid progress in this field, nonviral delivery systems will certainly play an irreplaceable role in the progressive development of gene therapy.