Direct compaction (DC) is considered to be the most effective method of tablet production. However, only a small number of the active pharmaceutical ingredients (APIs) can be successfully manufactured into tablets using DC since most APIs lack adequate functional properties to meet DC requirements. The use of suitable modifiers and appropriate co-processing technologies can provide a promising approach for the preparation of composite particles with high functional properties. The purpose of this review is to provide an overview and classification of different modifiers and their multiple combinations that may improve API tableting properties or prepare composite excipients with appropriate co-processed technology, as well as discuss the corresponding modification mechanism. Moreover, it provides solutions for selecting appropriate modifiers and co-processing technologies to prepare composite particles with improved properties. The tablet is still the most commonly used solid form due to its dose accuracy, convenience, stability, and ease of production in large quantities ,2,3,4. DIt was demonstrated that the materials for DC must possess good flowability, excellent compactibility, high bulk density, and low lubricant sensitivity, etc. ,15,16,17The physical properties of powders can be divided into fundamental properties and functional properties ,22,23,24A composite particle (CP) is defined as a specific combination of two or more established APIs and/or excipients at a sub-particle (even molecular) level designed to physically modify their properties in a way not achievable by simple physical mixing ,25,26,27Co-processing, a highly effective technique, is often employed to prepare CPs with improved surface modification of particle structure and excellent functional properties ,17,19,27According to the published literature, four aspects of CPs are mostly emphasized: (i) exploring and/or comparing the effect of different surface modifiers on CPs; (ii) characterizing the functional properties of CPs; (iii) developing multifunctional materials with enhanced performances and various co-processing methods; and (iv) analyzing the mechanisms of CPs property improvement from the powder level ,34,35,36Co-processed excipients are commonly considered as the combination of two or more pharmaceutical excipients co-processed by suitable co-processing methods . In co-processing, their properties are improved by physical modification rather than simple physical mixing ,45. The HPMC, a commonly used binder in tablets and a popular material in film coating, is made of alkali cellulose, propylene oxide, and alkane chloride, exhibiting a white or slightly yellow powder state with a tasteless, odorless, and non-toxic nature . HPMC is4HCO3), to form binary and ternary modifiers in order to improve multiple properties of the target drug powder or prepare functional composite excipients.Since the solution of HPMC has a good viscosity and surface tension, it is broadly used to prepare core-shell composite particles by fluid-bed coating and spray drying technology. The composite particles can effectively improve the tableting properties of drug powders. HPMC can be combined with other modifiers, such as porous mannitol, porous lactose, and ammonium bicarbonate , a representative of high dose drugs with poor compactibility, failed to be compacted into intact tablets during the compaction range of 74~444 MPa. Therefore, aqueous feed solutions of MH and HPMC E3 in ratios of 97.5:2.5 or 95:5 were spray dried. When compared to MH, both co-processed products showed improved compactibility: (i) the tablet tensile strength at a porosity of 0.15 increased from 0 MPa to 1.89~2.67 MPa; (ii) the work of compaction, which is related to the ability of a material to absorb work during compression, increased by 66.2~84.4%; (iii) the yield pressure and elastic recovery decreased by 4.5~6.2% and 7.6~21.2%, and the lower yield pressure and elastic recovery generally indicated plastic deformation and enhanced compactibility . This coSimilar studies were also reported for the preparation of functional excipients, such as starch, anhydrous dibasic calcium phosphate (DCPA), and mannitol as three types of commonly used tablet fillers . They arCo-freeze drying includes three main steps, freezing, primary drying, and secondary drying. It is widely used in the manufacturing and pharmaceutical fields . Furtherw/w, 3:1) excipients exhibited the best cushioning performance, excellent tabletability, and better dissolution. First, the disintegration time decreased by 41%, and second, the tablet tensile strength increased by 2.50~5.20-fold. These could be attributed to the following aspects: (i) porous mannitol has disintegration-enhancing effects [Co-processed excipients also had widespread applications in multiple-unit pellet systems (MUPS) ,67. Howe effects , which mZingiberis rhizoma extracted powder (ZR) was chosen as the model drug that had poor flowability and tabletability, and HPMC E3 was selected as the coating material [(0.5)) of composite particles was 3.95-fold larger than parent ZR. The uniformity of composite particles was decreased from 2.3 to 1.7; (ii) HPMC coated on the surface of raw ZR particles formed spherical agglomerates, increasing the distance and decreasing the contact area of the cohesive ZR particles; (iii) HPMC as the shell layer had the better bonding capability and plastic deformation; (iv) the core-shell composite particles exhibited a 250.80-fold higher hardness and 22.27-fold larger cohesiveness, which were beneficial for tabletability [Fluid-bed coating is widely used in pharmaceutical industry to physically modify powders to obtain targeted particles, e.g., masking unpleasant tastes and improving functional properties of APIs ,68,69,70erial [0.) of comptability ,72,73;  is a novel particle-engineering technique that adopts appropriate modifiers and suitable solvents for co-processing with the drug, producing spherical crystal products with improved micromeritic and mechanical properties, solubility, and dissolution ,92,93. CIn the paper by Rosenbaum T et al., an API  was selected as the model drug, dissolved in water, and HPMC was add as a modifier to the API solution. They were co-processed by CCA, and the ratio of API to HPMC was 3:1 . CompareIn the report from Wang ST et al., lactose (pharmatose 90 M) was chosen as the core material, and HPMC E3 and cross-linked polyvinylpolypyrrolidone (PVPP) were selected as the shell materials. They were co-spray dried to prepare a novel ternary composite excipient. The ratio of lactose to HPMC and PVPP was 89.5:7:3.5 . ComparePolyvinylpyrrolidone (PVP) is a water-soluble polymer formed by the polymerization of N-vinyl-pyrrolidone . In the In the study by Sadeghi F et al., acetaminophen, a representative drug with high crystallinity, failed to be compacted into intact tablets by direct compression. Therefore, aqueous feed solutions of acetaminophen and PVP K30 in ratios of 95:5 were co-spray dried . When coIn another similar study reported by Al-Zoubi N and coworkers, metformin hydrochloride (MH), a representative of high dose drugs with poor compactibility, failed to be compacted into intact tablets during the compaction range of 74~444 MPa. Therefore, aqueous feed solutions of MH and PVP K30 in ratios of 95:5 were co-spray dried. When compared to MH, the co-processed products showed improved compactibility: (i) the tablet tensile strength at a porosity of 0.15 increased from 0 to 2.00 MPa; (ii) the work of compaction, which relates to the ability of a material to absorb work during compression, increased by 72.87%; (iii) the yield pressure and elastic recovery decreased by 19.23%; the lower yield pressure and elastic recovery are generally associated with enhanced plastic deformation and compactibility . This coIn addition, PVP can be used as the templating agent to prepare porous particles. In a study reported by Zhu WF et al., the PVP K30 was utilized as the templating agent to prepare porous lactose, which was successfully applied to improve the dissolution behavior of curcumin. The ratio of lactose to PVP was 99:1, 98:2, and 97:3, respectively. First, they were separately prepared by co-spray drying. Then, the PVP was removed to prepare porous lactose . When coIn a recent work reported by Volkova TV et al., leflunomide was co-freeze dried with PVP to improve dissolution . The resZingiberis rhizoma extracted powder (ZR), which was unable to be compacted into tablets directly due to the poor compactibility and flowability. The ratio of ZR to PVP K30 was 93:7, which were then co-processed by a laboratory fluid-bed coating machine [Poria, Puerariae Lobatae Radix, Zingiberis rhizoma), three water-extracted powders , and three directly pulverized powders  were chosen as the model drugs with poor flowability and tabletability, and PVP was employed as the modifier. They were co-processed by fluid-bed coating.In a report conducted by Li Z et al., PVP K30 was selected as the unitary modifier to improve the flowability and tabletability of  machine . Compare machine , where tThe mechanism of the modification mentioned above could be summarized into two parts. First, it improved the particle structure and fundamental properties, e.g., spheroidal shape and smoother surface, increased particle size, more uniform size distribution, lower bulk, and tapped densities, which were conducive to the functional properties. Second, the shell material could increase the distance and decrease the contact of the cohesive raw particles, which leads to the reduction in van der Waals forces between composite products. Moreover, the enhanced compactibility could also be the result of excellent plastic deformation of shell materials, and the phenomenon appeared in the process of forming core-shell particles, e.g., the particle–particle collisions, solid and liquid bridge formation, and interlocking between particles.In the research reported by Vanhoorne V et al., paracetamol was used as a model drug that had poor flowability and a high tendency for capping during compaction. PVP and lactose were selected as the surface modifiers to improve the properties of paracetamol. The ratio of paracetamol, PVP, and lactose was 75:5:20, respectively. The composite products of the three materials were prepared by co-spray drying . CompareZingiberis rhizoma to PVP and mannitol was 93:1.4:5.6, respectively, and the composite particles were produced by fluid-bed coating [Zingiberis rhizoma, the AR, Hausner ratio, and Carr’s index of composite particles decreased from 46.60°, 1.4, and 29.2 to 30.11°, 1.3, and 22.4, respectively; and the area under the tensile vs. compaction force curve of composite particles increased from 0 MPa·kN to 5.498 MPa·kN. The mechanism of modification was similar to that concluded in In a report studied by Li Z et al., PVP K30 not only acted as the unitary modifier, but could also serve as a binary modifier with mannitol. The ratio of In the research reported by Qu L et al., a binary modifier containing PVP and magnesium stearate was used to improve the tensile strength of the ibuprofen tablets. The ratio of ibuprofen to PVP and magnesium stearate was 89:10:1, respectively. The composite powder was prepared by dry coating . Compare2), a form of silicate that contains silicon and oxygen, is abundant on earth and can be found in a variety of forms. It needs to be processed or manufactured using synthetic chemical processes before being used in pharmaceutical and other fields [Silica  [Due to its small size, silica has a large specific surface area (about 100–400 mn grade) ,133. Thun grade) ,136,137.Dry coating is widely regarded as a promising method to substantially improve the flowability, dispersibility, and hygroscopicity of cohesive powders in the pharmaceutical field ,135. DurIn the paper reported by Kunnath K et al., three cohesive APIs, such as micronized acetaminophen, coarse acetaminophen, and micronized ibuprofen, were chosen as the model drugs. They were dry coated with 1% nano-silica (hydrophobic or hydrophilic) to improve their flowability and tabletability . CompareIn another similar piece of research published by Huang ZH and colleagues, micronized acetaminophen, α-lactose monohydrate Pharmatose 450, and microcrystalline cellulose Avicel PH-105 were selected as the host materials. They were co-processed with nano-silica R972P to improve their direct compaction properties by dry coating . The weiIn other similar research, microcrystalline cellulose PH102 was dry coated with nano-silica to prepare a composite excipient in order to improve the direct compaction properties of acetaminophen . The weiLiquid dispersion is a method of mixing liquids with each other, which could cause the modifier to evenly distribute within and/or on API particles for modifying their surface and texture. It was considered to be a suitable surface modification method, especially for the liquid extracts of traditional Chinese medicine and chemical drug solutions .Zingiberis rhizoma extracted powder (ZR) by liquid dispersion [3 vs. 0.79 g/cm3, 0.80 g/cm3, 0.79 g/cm3, 0.75 g/cm3), hardness , cohesiveness , and tensile strength . All of these demonstrated that the composite particles showed improved flow properties and tablet-forming properties. The possible modification mechanisms could be concluded as follows: (i) the structure of composite particles showed a nearly spherical shape, which improved the flowability; (ii) the coating material effectively and uniformly distributed on the surface of ZR particles, which, as a result, could reduce contact area and increase inter-particle distance between cohesive primary ZR particles, hence, decreasing the van der Waals force; (iii) the distribution of the silica on the composite particle surface could form a sandwich with the two adjacent composite particles, which results in the formation of solid and liquid bridges; and (iv) larger hardness and cohesion, which are often used to characterize the particle strength and bonding strength of material [In a recent published study, colloidal silica N20 was used as the unitary surface modifier to improve direct compaction properties of spersion . The ratCitri Reticulate Percarpium powder (CB) was co-processed with hydrophilic or hydrophobic nano-silica by liquid dispersion in order to improve the direct compaction properties [In another similar research reported by Gao YT et al., the operties . The ratoperties .w/w) silicon dioxide (Aerosil 200) was chosen as the unitary surface modifier. They were co-processed by co-milling technology to improve the flowability and bulk density of ibuprofen particles [3 vs. 0.50–0.56 g/cm3). The modification mechanisms could be summarized by the following two points: First, the silicon dioxide particles with well-dispersing properties distributed on the surface of ibuprofen particles, which could effectively increase the distance and decrease the contact area of separately coated cohesive host powders, and thus, reduces the van der Waals forces between composite particles and ultimately exhibit better flowability. Second, the silicon dioxide particles inhibit the cohesive attractions among host particles, which allows them to rearrange more easily, ultimately increasing the bulk density of composite particles.In the research reported by Mullarney MP et al., ibuprofen, a cohesive with poor flowability, was selected as the model drug. One percent (articles . The co-Zingiberis rhizoma extracted powder (ZR), when compared with the unitary modifier of HPMC [Zingiberis rhizoma extracted powders, a representative of traditional Chinese medicine materials, which cannot be compacted into tablets directly due to quite poor compactibility and flowability. HPMC and the combination of HPMC and silica were used as the coating excipients. The mass ratio of drug to modifier were 93:7 and 92:7:1, respectively. They were co-processed by fluid-bed coating. Comparing to the raw powder, the co-products formed a core-shell structure and exhibited improved DC properties via fluid-bed coating. First, the composite particles exhibited significantly  improved flowability  compared to parent ZR . Second, the composite particles also showed improved compactibility. The tensile strength under the compaction force of 10 kN of the parent ZR, composite particles with unitary modifier and composite particles with binary modifiers were 0 MPa, 2.1 MPa, and 3.2 MPa, respectively. Furthermore, the area under the tensile strength vs. compaction force (2~10 kN) curves were 0 MPa·kN, 8.79 MPa·kN, and 14.40 MPa·kN, respectively. Third, the equilibrium hygroscopic moisture content was 18.21%, 15.12% and 14.42%, respectively, which indicated that the composite particles exhibited a significantly lower hygroscopicity. All of the above demonstrated that silica can further improve the functional properties of ZR. Silica acting as an isolation layer could effectively improve the defective surface properties of ZR, such as hygroscopicity and cohesiveness, thus, reducing agglomerations. Additionally, silica adsorbed onto the surface of a composite particle could further improve the flowability by reducing inter-particulate friction. Furthermore, the binary coating liquid exhibited higher viscosity (195.70 mPa·s vs. 178.90 mPa·s) and lower surface tension (39.70 mN/m vs. 40.30 mN/m) than unitary liquid. The higher viscosity and lower surface tension of the modifier liquid were conducive to enhance the bonding force of materials and contributed to modifier spread ability and wettability on the surface of core materials, thus, forming a more perfect core-shell structure. The applications of SiO2 were summarized in Li Z et al. revealed that the binary modifier of the combination of HPMC and silica has more advantages in improving the DC properties of  of HPMC . In theiMicrocrystalline cellulose (MCC) is a commonly used dry binder that is obtained from cellulose and was first discovered by Battista and Smith in 1955 ,153. MCC2 to 9.8 kg/cm2, and the AR was decreased from not being able to be measured to 31.4°. These improvements could be attributed to the following two factors. First, the composite particles display a spherical shape and smooth surface, which are crucial for better flowability. Second, MCC exhibits excellent plastic deformation characteristics and distributes well on the surface of mannitol particles, which can significantly enhance the tableting properties of integrated particles.In a study, mannitol, a water-soluble excipient, was selected as the model material that had poor tableting and flow properties, leading to poor direct compaction . MCC wasPuerariae Lobatae Radix was selected as the model drug, and MCC PH101 was chosen as the modifier. The liquid dispersion method was used to prepare composite particles with 1:0.06, 1:0.25, and 1:0.5 (w/w) of the model drug with a modifier of high, medium, and low drug loading, respectively [In a recent work by Zhang Y et al., ectively . CompareMCC is also a commonly used surface modifier. It improves powder properties by employing physical modification techniques, which not only provide a possible solution for drugs that cannot be compressed into integrity tablets but also increase the loading of drugs into tablets while enhancing their disintegration. Consequently, these functionalities are extremely useful for both applications and reducing the use of excipients in tablets. Traditional tablets (that are not modified by surface modifiers) often need a higher percentage of binder, filler, lubricant, and disintegrant to improve the direct compaction properties, resulting in low drug loading. On the other hand, composite powders exhibit improved hygroscopicity, glass transition temperature, and tableting behavior, which are beneficial to form qualified tablets and ensure their stability during storage and transportation.Lactose, a fragile material, was modified by a binary modifier, i.e., MCC and cornstarch . The proMannitol, a versatile excipient with low hygroscopicity and inertness to APIs, is widely employed in tablets and other dosage forms. It has a sweet and cool taste that can cover the unpleasant taste of drugs ,162. Thew/w) with NH4HCO3  was co-processed by co-spray drying [In the research reported by Al-Khattawi A et al., The aqueous combined mannitol  for mannitol and 2% (w/w) for sucrose. The composite products were washed with ethanol to remove sucrose and produce a porous mannitol carrier. As a result of the application of the carrier, nifedipine and indomethacin showed improved drug loading and dissolution. The results were as follows: (i) the drug loadings improved from 3.2% (w/w) to 9.1% (w/w) and 4.1% (w/w) to 12.6% (w/w); and (ii) the cumulative drug release improved from 58% to 99% and 50% to 90%. The mechanism of improved dissolution and drug loading could be summarized as follows: (i) the disordered reorganization and dispersion of drug crystals leads to a reduction in particle size of the drug loaded inside the porous mannitol to the nanometer scale; (ii) corresponding improvement in wettability and dispersibility of co-treated particles; (iii) the larger pore volume of porous mannitol is conducive to the entry and penetration of the dissolution medium and improves the contact area and contact rate of the medium, thus, improving the dissolution of the drug. The applications of MCC were summarized in Adsorption is a common method for improving the dissolution properties of insoluble drugs by using a carrier with sufficient surface area and pore spaces for particle dispersion and deposition ,169,170.Polyvinylpolypyrrolidone (PVPP) is a resin with a highly cross-linked form of the water-soluble polyvinylpyrrolidone polymer. PVPP is insoluble in water and is used in many fields, e.g., DNA extraction, beverage clarification, super-disintegrant, and so on ,174. In w/w, 95~97.5/5~2.5) by co-spray drying [In order to improve the compactibility of MH, Al-Zoubi et al. also employed PVPP VA 64, sodium alginate, and sodium carboxymethylcellulose as surface modifiers and prepared core-shell CPs with varying ratios  is a commonly used porogen that can be gasified and converted into H2O, NH3, and CO2 when the temperature is above 50 °C [4HCO3. The application of NH4HCO3 in producing porous particles could simultaneously solve the tableting and dissolution problems of APIs [Ammonium bicarbonate  the AR decreased from 49.3° to 44.7°, 44.2°, 43.2°, respectively; (ii) the tensile strength was increased from 0.75 MPa to 2.9 MPa, 3.5 MPa, 4.5 MPa, respectively; and (iii) the corresponding tablets dissolution rates improved to twice that of the raw tablets. The improvement reasons could be summarized as follows: first, the morphology of co-processed products exhibited loose, hollow, porous, and spheroidal structures; second, the spheroidal and porous/hollow structures were easily broken under pressure, which could increase the interparticle and intraparticle contact area and result in the formation of a tight combination during compaction; and third, the porous particle compacted to tablets could have higher tablet porosity, leading to the improvement in dissolution by facilitating water infiltration into tablets.In the report written by Zhou MM et al., the ethanol extract of ectively . CompareSodium lauryl sulphate (SLS) is a commonly used surfactant that has the advantage of controlling inter-particle cohesion force and is widely used to increase the wettability and solubility of drugs . In the Magnesium stearate (MgSt) is the most widely used pharmaceutical lubricant and is known for its friction and sticking reducing effects and cost efficiency ,180. MgSThe dry coating method is extensively reported as a promising approach for improving the flow, dispersion, and fluidization of the selected cohesive drug powders ,181,182.w/w), respectively. They were co-processed by the dry coating method. Compared to the raw ibuprofen powder, the modified products showed improved flow properties: (i) the cohesion decreased from 1.24 kPa to 0.94 kPa, 0.48 kPa, and 0.41 kPa, respectively; and (ii) the flow function (ffc) value increased from 4.0 to 5.0, 8.8, and 10.6, respectively. The ffc is an indicator of powder flowability . (iii) The Carr’s index decreased from 0.37 to 0.27, 0.16, and 0.21, respectively. Additionally, both of the corresponding tablets of raw powder and co-processed powders were dissolved in 5 min. The above phenomena can be analyzed from the following aspects: (i) an extensive MgSt coating layer was formed on the host particle surface, which facilitated enhanced packing of the powder, resulting in the reduction in interparticle cohesion; and (ii) the co-processed particles exhibited relatively smooth surfaces, which were beneficial to enhance the flow properties of modified products.In the research reported by Qu L et al., a fine ibuprofen powder with remarkable cohesive properties and a low melting point, which can cause sticking and picking during tableting, was chosen as the model drug . MgSt waw/w) MgSt was selected as the modifier [In another published research, a cohesive lactose monohydrate powder was chosen as the model material, and 1% (modifier . They weHydroxypropyl cellulose (HPC), is the product obtained after cellulose hydroxypropylation and is widely used in food and pharmaceutical fields. It is popular in the medicine field for its multifunctionality, e.g., stabilizer, thickener, disintegrant, binder, sustained release material, and so on ,186,187.w/w HPC HXF was utilized as the unitary modifier, and they were co-processed by co-spray drying [In a study reported by Shi LM et al., micronized acetaminophen  was chosen as the model active pharmaceutical ingredient with poor direct compaction properties, 1–10% y drying . The co-In another reported by Lin X et al., 5–15% HPC EF was dissolved in lactose (pharmatose 80 M) suspension to produce composite core-shell particles by co-spray drying . When cow/w) HPC was dissolved in an acetone–water solution to serve as the crystallization system for improving the flowability and compactibility of naproxen [Spherical crystallization is a particle design technique by which crystallization is conducted in one step. It is widely utilized for improving the flowability and compactibility of crystalline drugs . For exanaproxen . Comparenaproxen ,193. Thenaproxen ,202,203.Particle properties can be divided into two types: fundamental and functional properties. The fundamental properties include such as particle morphology, size, surface area, porosity, shape, density, and moisture content. The functional properties of the particles include flowability, lubricant sensitivity, tabletability, disintegration, and hygroscopicity, and they are interdependent and determined by fundamental properties ,22. In a(i) Core-shell structure, which is one of the most common and important structures for direct compaction can be seen as a structure with internal (core) and external (shell) layers or host (core) and guest (shell) ,9,18,124(ii) Porous structures have gained a lot of attention for their tremendous potential in improving the dissolution behavior and compactibility of tablet formulations. In terms of their high porosity and high specific surface area, particles with porous structure are widely used in the pharmaceutical field ,165,169.(iii) Porous core-shell structures are novel structures containing core-shell and porous structures. Particles with porous core-shell structures can be prepared by co-processing with suitable coating modifiers and porogens. The major co-processing methods are co-spray drying and fluid-bed coating. It has the advantages of both core-shell and porous structures. These benefits and modification mechanisms were discussed in detail above in (i) and (ii).Crystallization is one of the strategies for particle formation and provides a method by which pharmaceutical materials can be controlled and optimized for specific processing applications, e.g., direct compaction ,214. It (i) Crystal forms: Crystalline forms often display irregular shapes , which are not good for the flow properties of particles . Crystal(ii) Crystal habits: Crystal growth and the relative orientation of crystallites are important crystal habits, which are the key factors in deciding the size and shape of crystals and increasing the number of interparticle contact points. The increase in inter-particle contact points could improve the degree of particle deformation, densification, and magnitude and the extent of bond formation during compaction. A small number of effective excipients/modifiers in the crystallization medium can dramatically change the crystal size and shape by blocking the surface of a growing crystal, thus, resulting in preventing its growth and production. Therefore, crystal growth modifiers are often used to modify the structure of particles at particle and crystal levels.(iii) Crystal states: The crystal states can be divided into two types, such as crystalline and amorphous states. Compared to the crystal state , the amorphous state  generally shows improved tabletability due to the tendency for plastic deformation . The addThe amorphous phase usually displays disordered structure and exhibits higher free energy (thermodynamic driving force), thus, resulting in higher apparent water solubility, dissolution rate, and oral absorption. In contrast, the crystalline phase shows poor dissolution behaviors due to its lattice energy barrier. Some modifiers, for example, PVP, a water-soluble polymer, could effectively increase the wettability of active pharmaceutical ingredient particles in addition to inhibiting crystal formation and growth, thus, improving the dissolution of the target drugs.Surface modifiers  were widely utilized as crystal growth inhibitors and effective additives for improving the compression properties of target drugs. The improvement in tabletability of composite particles obtained by co-processing techniques  is mainly due to the agglomerate structure  and the presence of crystal habit, state, and form .A surface modifier co-processed with the drug or excipient can effectively improve the physical properties of the drug or prepare high functionality excipients with excellent properties and better application . Co-procAccording to the excipient classification system, surface modifiers can be divided into sugars , salts , silica , polymers  and others. Sugars, for example, lactose and mannitol, are frequently used as template agents to produce porous carriers, which can significantly improve the drug loading and dissolution of target drugs. Silica is a fine and easily dispersed powder that can notably enhance the flowability of the substances by forming a homogenous coating. Polymers, such as HPMC, PVP, HPC, and their solutions have remarkable viscosity; thus, they are widely utilized in the preparation of core-shell composite structure particles by co-spray drying and fluid-bed coating.The molecular weight of the surface modifier can affect the applications of modifiers. For example, HPMC E3 may be used as a coating material for preparing composite particles with improved DC properties. Additionally, HPMC F4M can serve as a cushioning agent in multi-unit pellet system tableting. HPMC K100M is often selected as a controlled release agent to prolong the release of the drug. Generally, polymers with lower molecular weights exhibit better plasticity, smaller viscosity, and lower glass transition temperatures.When polymers are used as crystalline inhibitors, the effect tends to become more pronounced as the dosage increases. Furthermore, hydrophilic PVP shows a greater impact on drug crystallinity than HPMC and copovidone . PVP canThe particle size of the surface modifier may affect the flow properties, compaction, and disintegration of target substance. For example, nano-silica, a nanoscale powder, can effectively disperse on the surface of another material forming an agglomerate by dry coating that can remarkably improve the flow properties. Different particle sizes of MCC also exhibit different modification effects, such as, the micronized MCC displays excellent flowability and tabletability.The thermodynamic properties of the surface modifier play an important role in improving the functionalities of co-processed products. Generally, polymeric modifiers exist in an amorphous semi-crystalline state. The temperature at which the polymer changes from the glassy state to rubbery state is called glass transition temperature (Tg), which is the major thermodynamic property of polymeric modifiers. When the temperature is below the Tg, the polymer modifiers present greater resistance to deformation, and when the temperature is above the Tg, they exhibit decreased resistance to deformation. Therefore, the degree of elastic deformation is mainly determined by their Tg. The higher level of elastic deformation indicates less permanent inter-particulate bonding and shows poor compactibility. Thus, the use of an appropriate polymeric surface modifier can effectively improve the tabletability of co-processed products by co-processing technology.Some polymers absorb water and cause swelling in particles, which results in breaking. Some polymer modifiers  exert the swelling effect when dissolved in the water and can significantly improve the disintegration time of the drug.Surface modifiers offer significant advantages and can be used to produce a wider range of APIs/excipients with excellent properties for direct compaction . As a reIn this review, some frequently used modifiers and their binary and ternary composites were summarized. Additionally, they were analyzed in terms of their applications in direct compaction and modification mechanisms. Moreover, the use of suitable modification technology (based upon recent studies) indicates that modifiers could be used to effectively prepare functional composite particles and improve the direct compaction properties of APIs. In addition, this review also provides some potential guidance for formulation scientists involved in the development of tablet dosage forms. In this article, we have summarized the most commonly used pharmaceutical excipients for tablet formulation. Small amounts of excipients can be employed as modifiers through appropriate co-processing techniques thereby enhancing the DC properties of drug powders. As a result, it will provide a novel alternative to conventional tablet formulations, which contain a high proportion of excipients to enhance DC properties.


Given that COVID-19 continues to spread worldwide, attempts to restrain the virus and to prevent the effects that critically ill patients with COVID-19 have on healthcare systems, has become a public health priority. This ecological study aimed to investigate the correlation between the Human Development Index (HDI) and the epidemiological indicators of COVID-19, including the cumulative incidence rate of cases, the cumulative incidence rate of death, performed COVID-19 tests per million, recovery rate, and case fatality rate.In this ecological study, a data set was provided, which included the epidemiologic indices of COVID-19, HDI, and its components for each country. Correlation coefficients were used to determine linear correlation. Also, the scatter plots of the HDI for the studied countries based on the epidemiologic indices of COVID-19 were drawn.p < 0.001). HDI and two of its components, including literacy and Gross National Income (GNI) components had negative correlation with case fatality rate (CFR). Also, HDI and two of its components, including literacy and life expectancy components had negative correlation with recovery rate.This study showed that HDI and its components had positive correlation with a cumulative incidence rate of cases, the cumulative incidence rate of death, and performed COVID-19 tests (Our study showed that the HDI and its components can affect the epidemiological status of COVID-19. As HDI increased, the cumulative incidence rate of cases, cumulative incidence rate of death, and COVID-19 tests increased as well. As HDI increased, CFR and recovery rate decreased as well. Although the HDI is higher in high-income countries, these countries may have also better reporting and surveillance systems. COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), originated in Wuhan, China, in December 2019 and has spread worldwide  and has https://www.worldometers.info/ for the period from the date of the first reported case until November 30th, 2020. In this study, the relationship between COVID-19 indices, including cumulative incidence rate of cases, cumulative incidence rate of death, cumulative tests performed, recovery rate, case fatality rate, and HDI, were investigated. Data on HDI and its components, including literacy, life expectancy, and Gross National Income (GNI) in 2019, were also collected from human development reports [This survey is an ecological study, so all studied variables are aggregate variables. To collect the variables in the study, a data set was provided, which included the information of each country based on the cumulative cases, cumulative deaths, case fatality rate, recovery rate, the number of performed COVID-19 tests, HDI, and its components. Information about COVID-19 for each country was retrieved from  reports . The HDI reports . Life ex reports . Also, tr = 0.00– < 0.20), week (r = 0.20– < 0.40), moderate (r = 0.40– < 0.60), strong (r = 0.60– < 0.70), and very week (r = 0.80–1.00) [2) was calculate. The proportion of variance in the dependent variable which is described by the independent variables in the sample depicts by R2 [P-values less than 0.05 were considered as statistically significant.Scatter plots of HDI for the studied countries were drawn based on cumulative incidence rate of cases, cumulative incidence rate of death, tests performed, recovery rate, and case fatality rate of COVID-19. Furthermore, Spearman correlation coefficient was also used to verify the correlation between HDI and indicators related to COVID-19. The correlation coefficient was categorized as very weak (80–1.00) . Also, Rts by R2 . Which ap < 0.001). As HDI and three components increases, the cumulative incidence rate of cases, the cumulative incidence rate of death, and performed COVID-19 tests, will increase. Also, the case fatality rate had negative correlation with HDI and two of its components, including literacy and GNI (P < 0.05). With increasing HDI, literacy, and GNI, the case fatality rate will decrease. The correlation between HDI and two of its components, including literacy and life expectancy with the recovery rate was also negative; with increased HDI, literacy, and life expectancy, recovery rate decreased (P < 0.05). In countries with a population of 10 million and more, a significant positive correlation was observed between HDI and its components with the cumulative incidence rate of cases, the cumulative incidence rate of death, and performed COVID-19 tests (p < 0.001). However, HDI and its dimensions were not correlated with recovery rate and case fatality rate.This ecological study showed that among all countries surveyed, Montenegro  had the highest cumulative incidence rate of COVID-19, while Tanzania (8.42 per million) had the lowest cumulative incidence rate of COVID-19. Also, it revealed that Belgium (1425.15 per million) had the highest cumulative incidence rate of death due to COVID-19 when Burundi (0.08 per million) had the lowest cumulative incidence rate of death due to COVID-19. Luxembourg  was the country with the highest number of performed COVID-19 tests per million among studied countries. The lowest number of COVID-19 tests per million belonged to Yemen (560.05 per million). The highest recovery rate was in Timor-Leste (100%) when the lowest rate was in Belgium (6.48%). In addition, the highest case fatality rate of COVID-19 was in Yemen (28.34%) while the lowest case fatality rate of COVID-19 was in Singapore (0.05%). Also, the highest HDI was in Norway and Switzerland (0.95), and the lowest in Niger (0.38). At birth, the highest life expectancy was in Japan (84.63 years) and the lowest in Chad (54.24 years). The highest mean years of schooling were in Germany (14.15 years) and the lowest in Burkina Faso (1.64 years). The highest GNI was in Qatar  when the lowest was in Burundi (753.9 dollars per capita). Table 2 was observed in the cumulative incidence rate of cases (R2 = 0.349) and the performed COVID-19 tests (R2 = 0.298). HDI explain about 35% and 30% of the variance in the cumulative incidence rate of cases and the performed COVID-19 tests, respectively. In countries with a population of 10 million or more, the highest R2 was for the tests performed (R2 = 0.530), followed by the cumulative incidence rate of cases (R2 = 0.436). In countries with a population of 10 million or more HDI explain about 53% and 44% of the variance in the performed COVID-19 tests and cumulative incidence rate of cases, respectively.Figure COVID-19 is a global health crisis in our time and the biggest challenge that the world is encountering . In this2 was observed in the cumulative incidence rate of cases and the performed COVID-19 tests. HDI explain about 35%, 30%, and 24% of the variance in the cumulative incidence rate of cases, the performed COVID-19 tests, and cumulative incidence rate of death, respectively. In countries with a population of 10 million or more HDI explain about 53%, 44%, and 37% of the variance in the performed COVID-19 tests, cumulative incidence rate of cases, and cumulative incidence rate of death, respectively. Therefore, in addition to the HDI, other factors may be related to these indices.Our study showed that the higher the HDI, the higher the cumulative incidence rate of the cases, the cumulative incidence rate of death, and performed tests. This correlation may be due to the strong infrastructure in these countries, these countries with higher HDI have been able to record more laboratory tests due to higher capacity. The high incidence in nations with a higher HDI may also be linked to the strength of an excellent health care system for early identification and detection of asymptomatic and subclinical kinds of illness, as well as more effective screening program implementation in these countries. Poor access to diagnostic equipment decreases the frequency of infection in nations with a low HDI. Furthermore, these nations' illness reporting systems are of poor quality, resulting in low disease reporting . It has Also, there was a negative correlation between literacy and life expectancy with recovery rate in our study. Life expectancy at birth is one of the components of HDI. A rise in the old population will occur as life expectancy increases. According to prior research, the elderly has the greatest prevalence and mortality of Covid-19. When it comes to the link between age and the occurrence and mortality of this illness, it can be noted that older individuals are more susceptible to severe instances of the disease since their immune systems are weaker against the disease –30. ThisSo, in general, the positive spatial correlations between the incidence of COVID-19 and the human development measured by HDI are unprecedented. Generally, epidemiological studies of infectious diseases have found an indirect indicator of health in HDI because the highest value of this index is related to life expectancy, income, and education . This seEcological studies have a history of use in public health issues and diseases , 40. In One of the strengths of the present study is that for the first time, the relationship between HDI and COVID-19 epidemiological indices including, mortality rate, recovery, especially new cases, and the number of tests, was examined in 157 countries. But the limitation of this study ecologic fallacy and using data at the aggregate level due to its nature.Our study showed that the HDI and its components can affect the epidemiological status of COVID-19 disease. As HDI increased, the cumulative incidence rate of cases, cumulative incidence rate of death, and COVID-19 tests increased as well. As HDI increased, CFR and recovery rate decreased as well. Although the HDI is higher in high-income countries, these countries may have also better reporting and surveillance systems. It seems that global assistance to countries with less HDI can effectively reduce the pandemic crisis in these countries and other regions. As in respiratory epidemics, borders cannot be a factor in disease prevention, if international cooperation is not practiced, we will not move towards disease control.


Hemophilia is an inherited recessive hemorrhagic disorder of the X-linked type, suffered by the male sex. Adults with hemophilia are coping with numerous diagnostics, associated comorbidities, pain, and difficult gait by arthropathy in ankles and feet. Physical pains contribute to depression in patients with hemophilia.Methods: The sample consisted of 100 participants (median age 42.50 ± 30). Patients with hemophilia were recruited from Spanish Hemophiliac Associations (n = 50) and healthy subjects (n = 50) from a Clinic Podiatry Practices .The study aimed to assess the impact of depression in adult patient with hemophilia and to compare it with healthy matched-paired controls. p < 0.001) was presented in the variation of the BDI scores between both groups. Patients with hemophilia presented worse results with a BDI = 7.50 ± 11.25 points compared to healthy subjects with BDI = 2.50 ± 5 points. In the BDI categories, statistically significant differences (p = 0.004) were found in greater BDI categories in the Hemophilia group compared with healthy subjects. Moderate and severe depression categories were only shown in patients with hemophilia.Results and categories of the Spanish-translated version of the Beck Depression Inventory (BDI) were gathered. A clear statistically significant difference (Greater depression scores and range status were observed in patients with hemophilia compared to non-patients with hemophilia. Patients with hemophilia are at increased risk of depressiveness. Hemophilia is an inherited recessive hemorrhagic disorder of the X-linked type, suffered by the male sex. There are principally 2 forms of Hemophilia: Hemophilia A, the most usual type caused by a missing clotting factor VIII , whereas Hemophilia B is produced by the absence of factor IX   intra-articular hemorrhage . The most frequent site of hemorrhage is the ankle, succeeded by the elbow and the knee . FrequenThis chronic illness is avowed as a significant public health problem, due to the negative impact posing for these patients, your families and health and/or economic burden, with consequent available resources, financial, medical practice, and socioeconomic implications affecting the healthcare , 7.On the order hand, various investigations have indicated the existence of a relationship between psychological disorders and different musculoskeletal variables , with depression and anxiety being regularly implicated as emotional factors in the context –10.However, at present, there are insufficient studies in Spain that examine the seriousness of depression taking into account its affective, behavioral, and cognitive dimensions and anxiety in patients with hemophilia. In this context, hemophilia is one general pathology in which older patients with hemophilia are frequently faced with numerous diagnostics, associated comorbidities, and pain due to arthropathy affectations .Despite the importance of this chronic disease, both due to its prevalence and its influence on the daily activities of living, there are insufficient studies in Spain that have measured the influence of anxiety and depression in these patients. The study aimed to assess the impact of depression in adult patients with hemophilia and also to compare healthy subjects with healthy matched-paired controls.n = 50) and male salubrious subjects . The participant selection and inclusion were: (1) male gender (2) older than 18, (3) persons with Hemophilia diagnosed by a specialist physician and not having neurological or psychological problems (case group), and persons without hemophilia and health problems (control group). The exclusion rule were: (1) people with depression, (2) antecedents of previous hematologic disease, (3) neurological problems, (4) a history of surgery and/or orthopedic problems, (5) loss of independence or self-sufficiency in daily routines, (6) refusal to approve the consent form, and (7) cognitive impairment of any etiology.We performed an observational, cross-sectional, descriptive, and case-control research in agreement with Strengthening the Reporting of Observational Studies in Epidemiology Statement (STROBE) guidelines. Using a consecutive sampling method, a total sample of 100 subjects was selected from the Hemophiliac Associations of Málaga, Aragon, and Cantabria (Spain) and a University Clinic  between December 2019 and February 2020. Eligible participants were male subjects with hemophilia type A or B diagnosed by a hematologist  demographic variables ; (2) characteristics of comorbid problems ; (3) foot problems. In addition, exact health questions associated with hemophilia, such as (4) type and (5) clinical grade of hemophilia, were recorded. Next, participants completed the BDI questionnaire . This inThis study got a positive report issued by the Ethical Committee from the University of Extremadura . Ethical principles for medical research on humans to the Helsinki Declaration  were respected . The inf* Power software  was used to calculate the sample size, using the differences between 2 groups of independent samples, based on a preliminary investigation (n = 32) with 2 groups (mean ± SD) and on the scores of the Beck Depression Inventory (BDI), 16 patients with hemophilia  and 16 sane paired participants . Also, for sample size calculation were used: 2-tailed test, the effect size of 0.78, the alpha error probability of 0.01, the power (probability of 1-beta error) of 0.90, and 1 allocation ratio (N2/N1). Therefore, a final sample size of 100 subjects, 50 persons with Hemophilia and 50 healthy matched-paired persons, was resolved with an actual power of 0.902.The G P < 0.05 and a 95% confidence interval (CI).Statistical analyses were performed using the SPSS 24.0 . For all analyses, Statistical significance was established at p-value minor than 0.05) and median and interquartile range (IR), as well as maximum and minimum (range) values, were utilized to describe the total sample. Mann–Whitney U-tests were used to obtain if the differences between both groups were statistically significant.Regarding the quantitative variable, the normality was evaluated by the Kolmogorov-Smirnov test. All data were distributed as non-parametric data .n = 50), and salubrious matched-paired participants  presented an age division from 18 to 80 years old. Furthermore, the participants with hemophilia revealed two types of this chronic illness, type A (90%) showed clinical grades of mild (18%), moderate (14%), and serious (68%), while type B is at 10%. Also, the patients with hemophilia stated they had suffered foot pain (86%) and ankle arthropathy (76%). Statistically significant differences were not shown (p > 0.01) between the two groups for descriptive data  was presented for the division of the BDI punctuations between both groups (p = 0.004) in greater BDI categories in a hemophilic group compared with healthy subjects. It is worthy to note that moderate and severe depression categories were only shown in patients with hemophilia  and type B (349), where one-third of children and two-thirds of adults were shown to have musculoskeletal bleeding, in this anatomical region .To the researchers' knowledge, this study may be considered one of the few investigations in patients with hemophilia using the BDI questionnaire  to measuThe results of our study confirm that people with hemophilia presented a “mild depression status” 24%), contrasted to healthy group without hemophilia (2%). Consequently, this research establishes that in patients with hemophilia, exist a relation with BDI depression results. Instead, some studies associate pain in different musculoskeletal conditions with worse physical and psychological factors , 19–21. %, contraFurthermore, it should be taken into account in patients with hemophilia that probability of depression is increased by the factor of suffering from chronic disease and chronic joint pain , 28. ThuIn addition to this, we found that hemophiliac majority subjects could suffer from overweight, being the median BMI was 25.45 in patients with hemophilia group. According to Wilding et al., the incidence of overweight and obesity in persons with hemophilia in Europe and USA is presently comparable to the universal people at 31%, and there has been an important increment in pediatric hemophilic population . In entiContrasting our results with implications of other studies was not possible due to the contrast in methodological and criteria variations.Further, this investigation had some limitations. First, a future study with bigger sample sizes, as well as longitudinal evaluation of results, would facilitate in determining definitive conclusions. Second, though appropriate validity and reliability have been created for the BDI questionnaire, their reliability has not yet been recognized expressly for patients with hemophilia and should be considered for future studies. Other limitation of the present study is heterogeneous characteristic s of the sample (subjects who lives in different locations of Spain). Ultimately, it would become of advantage to study patients with hemophilia and with depression in terms of factually calculated results, for example, a clinical and radiological evaluations of articulation state.Greater depression scores and ranges status was observed in patients with hemophilia compared to patients without hemophilia. Patients with hemophilia are at increased risk of depressiveness. Thus, individuals from this group should be monitored for potential depressive symptoms. Because hemophilia is a rare disease that requires a multidisciplinary approach, to promote physical and psychological wellness, patients should be attended in comprehensive care of hemophilia treatment center, including psychological evaluation and care.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University of Extremadura . The patients/participants provided their written informed consent to participate in this study.AJ-C, PP-L, RBV, ML-I, EN-F, MS-A, CC-L, and DL-L: conceptualization, formal analysis, methodology, supervision, writing—original draft, and writing—review and editing. AJ-C: data curation. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Acridine orange staining showed that the degree of apoptosis in zebrafish hearts was significantly increased under 6-BA, and the expression of cell-cycle-related genes was also changed. In addition, HPA axis assays revealed abnormally expressed mRNA levels of genes and significantly increased cortisol contents, which was also consistent with the observed anxiety behavior in zebrafish at 3 dpf. Transcriptional abnormalities of pro- and anti-inflammatory factors in immune signaling pathways were also detected in qPCR experiments. Collectively, we found that 6-BA induced cardiotoxicity in zebrafish, which may be related to altered HPA axis activity and the onset of inflammatory responses under 6-BA treatment.6-BA is a common plant growth regulator, but its safety has not been conclusive. The heart is one of the most important organs of living organisms, and the cardiogenesis process of zebrafish is similar to that of humans. Therefore, based on wild-type and transgenic zebrafish, we explored the development of zebrafish heart under 6-BA exposure and its mechanism. We found that 6-BA affected larval cardiogenesis, inducing defective expression of key genes for cardiac development ( The environment is closely related to human production and life, and environmental pollution has become one of the hot topics in recent years. Therefore, pesticides and their residues that are highly related to human diet are also increasingly aroused by people’s warnings. 6-Benzylaminopurine (6-BA), a major plant growth regulator, plays a major role in plant growth promotion and fruit and vegetable preservation . There aDanio rerio), as a vertebrate, has a high degree of genome homology with humans, which makes it an invaluable model for developmental, neurological, and toxicological studies [Many studies on acute toxicology are based on zebrafish models. The zebrafish  axis is a body system that involves the hypothalamus, pituitary, and adrenal glands and is activated in response to environmental stress . SpecifiIn this experiment, zebrafish embryos were used as the research object to study the toxicological effects of 6-BA exposure through acute exposure treatment. The cardiac morphology, cardiac development and function, and cell apoptosis in the early embryonic development of zebrafish were counted, and the levels of inflammation-related factors and HPA activity-related genes and hormones were also detected. In addition, thigmotaxis behavior, as a behavioral indicator of early zebrafish development and a cascade of altered HPA activity, was further explored. These results were used to assess the effects of acute 6-BA exposure on early cardiac development in aquatic vertebrates.It was observed that at 48 hpf, the hearts of control larvae circled into an S shape, with partially overlapping atria and ventricles. In contrast, some zebrafish exposed to 6-BA exhibited cardiac developmental malformations, such as increased pericardial edema and linearization of the relative position of the atrium and ventricle, which also resulted in a significant increase in the BA–SV distance in larvae A. Based hbae3 and hbbe1, with significant differences compared with the control group , the embryo hatches from the chorion and reaches the larval stage at about 3 dpf, when most of the internal organs have matured . The heaAmhc and vmhc are the earliest marker genes to label the atrium and ventricle of the heart [Myl7, also known as cmlc2, is expressed in the atrium and ventricle, and is closely related to various physiological processes, such as cardiomyocyte proliferation, cardiac contraction, and myofibril assembly [myl7, amhc, and vmhc expression was observed, which may be related to the various cardiac defects observed as well as pericardial edema and subsequent cell death [bmp4, notch1b, and another marker expressed in the myocardium, tbx2b, were downregulated upon 6-BA exposure, suggesting that 6-BA interferes with the molecular conditions required for atrioventricular valve formation in AVC. Developmental defects caused by inappropriate AVC formation have devastating consequences in affected individuals [Cardiogenesis is a complex process involving cell specification and differentiation as well as tissue morphogenesis and remodeling . Spatialhe heart ,18, respassembly . Signifill death . Differell death . We founividuals . We specividuals . The redbax, bcl2, caspase3, p53) was induced to change. The balance between pro- and antiapoptotic protein regulators is a critical point in determining whether cells undergo apoptosis. Combined with the staining results, the above data indicated that 6-BA induced apoptosis in zebrafish. When DNA damage or other exogenous stress stimuli occur, the cell cycle repairs it to the normal stage of genome replication. However, dysregulation of multiple cell cycle regulators, including the cyclin/cdk complex, induces cell cycle arrest and cellular senescence, leading to apoptosis [ccnd1, ccne1, cdk2, cdk6 was blocked. The increased expression of the cell cycle activation genes ccnd1 and cdk2 and the mitogenic gene c-myc in the 6-BA exposed group at 20 mg/L was likely due to the compensatory response of the organism after the effect of 6-BA on the cell cycle of zebrafish larvae. These data suggest that 6-BA treatment induces apoptosis during early stages of zebrafish development, which may be affected by cell cycle dysregulation, ultimately impairing cardiac development.Apoptosis is an important and irreversible damage in biological, physiological, and pathological processes , and apopoptosis . Resultsstat3 is activated by the combination of IL-4 and IL-4r [stat3 gene level was downregulated in zebrafish under 6-BA exposure, and transcription of the stat3-targeted mitogenic genes c-myc and gata3 was also induced by 6-BA. Changes in downstream genes of cytokines further prove that 6-BA induces inflammatory responses, which may have negative effects on cardiogenesis. Inflammatory responses can disrupt the activities of key factors in cardiac development, leading to the failure of early cardiac formation [Altered phenotype and function of immune cells underlie adverse cardiovascular remodeling . Studiesnd IL-4r , and is ormation , so the crha, crhb, pomca, pomcb, nr3c1) in zebrafish, suggesting that the HPA axis activity of larvae was altered by 6-BA induction. The HPA axis is an important aspect of the neuroendocrine system as it controls stress responses and regulates many physical activities [The HPA axis is the main system by which animals respond to changes in their external environment and stress, and functions in a variety of physiological processes, and the HPA axis in zebrafish and humans is highly similar . GR is ativities . Numeroutivities . The restivities . These btivities ,39. Thustivities . There itivities . Our obsIn conclusion, we found that acute 6-BA exposure induced cardiotoxicity in zebrafish. The experimental results showed that 6-BA caused defects in cardiac phenotype, hindered cardiac morphogenesis, and resulted in abnormal cardiac circularization and weakened cardiac function. In addition, the degree of apoptosis in zebrafish hearts was significantly increased under 6-BA treatment compared with controls, and the expression of cell-cycle-related genes was induced to decrease. At the same time, we also found that the landmark cytokines of inflammatory response were changed by 6-BA induction, and the mRNA levels of HPA-axis-related genes and cortisol content were increased under 6-BA treatment, which may contribute to 6-BA-induced cardiotoxicity. In general, the experimental results indicate that the potential toxic effects of 6-BA cannot be ignored, and the biological effects of 6-BA need to be further studied.2, and 0.33 mM MgSO4, pH 7.4), and transferred to 6-well plates for culture at 28.5 °C.Wild-type AB strains and transgenic zebrafish Tg(myl7:GFP) were purchased from the Chinese Zebrafish Resource Center and were reared in a standard recirculating aquaculture system :10 (dark)). Zebrafish feed on freshly hatched brine shrimp twice a day. The night before the experiment, male and female zebrafish were placed in the mating box at a ratio of 1:1, separated by a transparent partition. After the partitions were removed the next morning, the males chased the females for mating. Fifteen minutes after spawning, embryos were collected in clean petri dishes, washed with fresh zebrafish water  and dimethyl sulfoxide (purity ≥ 99.8%) were purchased from Beijing Coolaber Company  and Shanghai Aladdin Company , respectively. The 6-BA powder was weighed and dissolved in dimethyl sulfoxide (DMSO) solvent to make a 1 mg/mL 6-BA stock solution and stored at −20 °C protected from light. At 2 hpf, fresh system water was used to dilute the stock solutions to obtain the desired final concentrations: 10 and 20 mg/L of 6-BA solutions; system water with a DMSO ratio of 0.05% was used as the control. Subsequently, they were added to the corresponding well plates for drug exposure.The zebrafish experimental techniques and related operations involved have been approved by the Committee for Animal Experimentation of the College of Life Science at Nankai University (no. 2008), and were carried out in strict accordance with the requirements of the NIH Guide for the Care and Use of Laboratory Animals .According to the previous method ,42, zebrZebrafish developed to 72 hpf were anesthetized and mounted on grooved slides to allow clear visualization of larval cardiac structures. In order to count the heart rate of zebrafish under drug treatment, a 20 s video was taken for heartbeat count and converted to beats per minute. Approximately 12–15 larvae were photographed per group. In addition, to calculate the diastolic and systolic velocities of the heart, the 10 s video recordings of the heartbeats of the larvae were taken according to the previous method . The vidTo assess the effect of 6-BA on cardiac hemoglobin activity, zebrafish were stained with o-dianisidine solution. Amounts 0.03 g of o-dianisidine and 0.041 g of sodium acetate were accurately weighed and placed in a 50 mL volumetric flask. After adding 1.1 mL of 35% hydrogen peroxide, the remaining volume was filled with 40% ethanol. A total of 15 zebrafish developed to 72 hpf were randomly selected from each group, stained with freshly prepared o-dianisidine solution in the dark for 15 min, and then quickly washed with DMSO solution three times. The larvae were placed side by side on a grooved glass slide, and the heart position was imaged under a stereo microscope. ImageJ was used to perform statistics on staining results for quantitative analysis. In addition, we performed video filming to analyze the blood flow rate of larvae under drug exposure, as described in the At 72 hpf, larvae were stained with 2.5 μg/mL of acridine orange (AO) solution for 30 min at room temperature in the dark, then washed three times with system water for 5 min each. The treated zebrafish were anesthetized with 0.168 g/L tricaine and observed under a fluorescence microscope , and whole-body and local cardiac images were collected. The cardiac fluorescence intensity of larval fish after acridine orange staining was quantitatively analyzed using Image J software  to indicate the degree of cardiac apoptosis in zebrafish larvae exposed to 6-BA.w:v) of PBS (pH 7.4) was added for thorough homogenization. Centrifuge was at 2000–3000 rpm for about 20 min. The supernatant was carefully collected and aliquoted, one for testing and the rest frozen for later use. The experimental operation was carried out according to the instructions of the Fish Cortisol ELISA . Four replicates were set up for each sample. After the measurement, the actual concentration of the sample was calculated. The experiment was repeated three times using different batches of embryos.A total of 60 larvae that developed to 72 hpf were randomly selected from each group and placed in 1.5 mL centrifuge tubes. The weights were accurately weighed, and nine times the volume (myl7: EGFP) background, as previously described [Atrioventricular canal (AVC) marker genes, apoptosis-related genes, and cell cycle genes were isolated from control and different concentrations of 6-BA-exposed fish hearts in a Tg  and Fisher’s LSD test, and the nonparametric Kruskal–Wallis test and Dunn’s multiple comparisons test were used to analyze data that did not conform to a normal distribution. SPSS 20  and GraphPad Prism 6.0  were also used for data analysis and graphing. All data are presented as mean ± standard error (SEM).


Micropterus salmoides). Seven isoproteic and isolipidic experimental diets were formulated , containing 0, 3, 6, 9, 12, 15, and 18% non-starch polysaccharides (NSPs) , respectively. Dietary inclusion of NSPs below 9% showed no negative impacts on fish growth and feed utilization efficiency, whereas dietary NSPs inclusion level above 9% decreased weight gain rate, specific growth rate, protein efficiency, protein deposition rate, apparent digestibility of dry matter and protein, and were accompanied by a reduction in intestinal protease, Na+/K+-ATPase and alkaline phosphatase activity and an increase in feed intake and feed coefficient. The activity of lipase was significantly decreased when dietary inclusion of 15 and 18% NSPs. Moreover, the lipid deposition rate and the apparent digestibility of lipids were significantly decreased since dietary inclusion of 9% NSPs. Dietary inclusion of NSPs above 12% significantly up-regulated intestinal GLP-2 gene’s expression, and was accompanied by significant changes in hindgut morphology, including increases in villus length and width, muscularis thickness and number of goblet cell, as well as a decrease in crypt depth. Additionally, dietary inclusion of NSPs above 3% significantly increased intestinal length index, and the viserosomatic index was significantly increased when dietary NSPs exceeded 15%. The linear regression analysis based on weight gain rate and feed coefficient showed that the appropriate dietary NSPs level of juvenile largemouth bass should not above 5.51%. In conclusion, high dietary NSPs adversely affects digestive enzyme activity and intestinal morphology, which in turn reduced the apparent digestibility of dietary nutrients and growth of juvenile largemouth bass.An 8-weeks feeding trial was carried out to evaluate the effects of different levels of dietary non-starch polysaccharide on the growth, apparent nutrient digestibility, intestinal development, and morphology of largemouth bass ( Non-starch polysaccharides (NSPs) are the main components of plant cell walls, including cellulose, hemicellulose, and pectin, and are highly abundant in plant-based raw materials . The NSPRachycentron canadum), gilthead sea bream (Sparus aurata), zebrafish (Danio rerio), and Jian carp (Cyprinus carpio var. jian)  –22. The r. jian) . The altr. jian) , 9, 24, r. jian) , 25, whiThe juvenile largemouth bass used in this study was approved by the Animal Research and Ethics Committee of Guangdong Ocean University, China. Additionally, all experiments were performed in strict compliance with its guidelines.Seven isoproteic and isolipidic experimental diets were formulated , containing 0, 3, 6, 9, 12, 15, and 18% NSPs , respectively. Dietary NSPs were formulated with purified cellulose, β-glucan, mannan, arabinoxylan, and pectin in proportions as described in a previous study . During the breeding period, the water quality was kept fresh, the water temperature was maintained at 28–31°C, the dissolved oxygen was more than 6.0 mg/L, and the ammonia nitrogen was less than 0.02 mg/L. The feeding experiment lasted for 8 weeks, and the experimental fish were fed twice daily until they were apparently satiated (07:00 and 17:00). The feed consumption and the number of test fish mortalities in each cage were recorded.The digestibility trial was performed during the feeding trial. Yttrium trioxide  was used as an indicator in the experimental diets. After the test fish were acclimated to the test diet for 2 weeks, the collection of feces in each cage was started. The feces at the bottom of the cages were collected with a 200-mesh brail every day after the test fish had been fed for 5–7 h. The formed feces were then selected, dried at 65°C for 6 h, and stored at –20°C for subsequent analysis. Fecal collection was continued for 6 weeks to ensure that the volume of fecal samples met the assay requirements (dry matter >10 g).The test fish were fasted 24 h at the end of the feeding trial, and then the fish in each net cage were weighed and counted to calculate the growth index. Before sampling, the test fish were anesthetized with 100 mg/L of MS-222 . Three fish were randomly selected from each cage and frozen at −20°C for whole-body composition analysis. Four fish were randomly selected from each cage, and the total length of the fish was measured, weighed, and then dissected; the visceral mass, liver and intestine were sequentially weighed, and the length of the intestine was measured. Specifically, intestinal weights were weighed after removal of mesenteric fat. Another two fish were randomly selected from each cage for dissection, and the intestines were taken out and placed in EP tubes with RNA letter, snap-frozen in liquid nitrogen and then stored at −80°C for subsequent analysis.N × 6.25); crude lipid by the Soxhlet extraction method; crude ash by burning in a muffle furnace at 550°C for 16 h. The determination of yttrium content in feed and feces was performed by inductively coupled plasma mass spectrometry (Agilent Agilent 7500cx).The chemical composition analysis of the experimental diets, whole-body and feces refers to the standard method . The sam+/K+-ATPase activities were determined by commercial kits , and the sample processing and determination process were strictly in accordance with the kit instructions.Intestinal protein concentration and amylase, lipase, protease, creatine kinase (CK), alkaline phosphatase (AKP) and NaOne fish was randomly selected from each cage for dissection, and the hindgut (1 cm) was taken out and fixed with 4% formaldehyde. After standing for 24 h, the tissue samples were dehydrated in a series of graded ethanol, and embedded in paraffin after dehydration. After the paraffin was solidified, the intestinal paraffin was cut into slices with a thickness of 5 μm with a microtome, followed by hematoxylin-eosin staining, and encapsulated to make H&E sections. The prepared sections were observed with a Nikon Ni-U microscope imaging system , and the intestinal villus height and width, crypt depth, muscularis thickness, and number of goblet cells were counted according to the method described in a previous study .Another one fish was randomly selected from each cage in the control group, NSPS 6 and NSPS 18 groups for dissection, and the hindgut tissue was fixed with 2.5% glutaraldehyde. After 24 h of fixation, the tissues were transferred into phosphate-buffered saline containing 2% osmium tetroxide, and then the tissues were dehydrated with a series of graded ethanol. After dehydration, the tissue was embedded in epoxy resin 812 and then cut into ultrathin sections using an ultra-microtome  for uranyl acetate and lead citrate staining. Finally, the morphology of intestinal microvilli and intestinal epithelial cells was observed using a transmission electron microscope .® ND-2000, Thermo, USA) to detect the purity and concentration of total RNA. Subsequently, the total RNA was used as a template to synthesize complementary DNA (cDNA) with a reverse transcription kit , and stored at −20°C until use. The primer sequences used for fluorescence quantification are shown in ® Green Premix Pro Taq HS qPCR Kit II  reaction system. Each sample was assayed in four replicates, and melting curve analysis was performed after each reaction to check product specificity. The mRNA expression of all genes was calculated by the 2–ΔΔCT method, and the mRNA expression of the target gene was normalized with the Ef1α mRNA expression of the control group as the standard.Intestinal total RNA was extracted with Trizol Reagent kit , followed by 1.2% denatured agarose to detect the integrity of total RNA, and a spectrophotometer .All data were subjected to one-way analysis of variance (ANOVA) with SPSS (v22.0) software, and Turkey’s multiple range test was performed when the difference was significant. P > 0.05; Wt, WGR, PER, and PDR in the NSPs12, NSPs15, and NSPs18 groups were significantly lower than those in the control group, in contrast to the FCR and FI (P < 0.05). In addition, the LDR in the NSPs9, NSPs12, NSP15, and NSPs18 groups was significantly lower than that in the control group (P < 0.05). The linear regression analysis based on WGR and FCR showed that the appropriate dietary NSPs level of juvenile largemouth bass was 5.28–5.51% . The FCR in the NSPs3 group was significantly lower than that in the control group, and the OI in the treatment group was all significantly higher than that in the control group (P < 0.05). The HSI in the NSPs9 group and the VSI in the NSPs15 and NSPs18 groups were significantly higher than those in the control group (P < 0.05). The ILI in the NSPs3 and control groups were relatively equal (P > 0.05), while the ILI in the rest treatment groups was significantly higher than that in the control group (P < 0.05).Dietary inclusion of different levels of NSPs had no significant effect on the moisture and crude ash contents of juvenile largemouth bass .The apparent digestibility of dietary crude lipids in the NSPs9 group was significantly lower than that in the control group . Additionally, the intestinal lipase activity in the NSPs15 and NSPs18 groups was significantly lower than that in the control group (P < 0.05). The intestinal amylase activity in the treatment group was significantly higher than that in the control group (P < 0.05).Dietary inclusion of different levels of NSPs had no significant effect on the activity of intestinal CK in juvenile largemouth bass . The width of hindgut villus in the NSPs9, NSPs12, NSPs15, and NSPs18 groups was significantly higher than that in the control group, in contrast to the crypt depth (P < 0.05). The muscularis thickness of hindgut in the NSPs12, NSPs15, and NSPs18 groups was significantly higher than that in the control group (P < 0.05). Except for the NSPs3 group, the relative goblet cell numbers of hindgut villus in the rest treatment groups were significantly higher than that in the control group (P < 0.05). Intestinal epithelial cell death was observed in the NSPs18 group . Conversely, several studies have found that the dietary inclusion of high NSPs had no negative effects on the growth performance of rainbow trout (15% cellulose) and European sea bass   , 33. TheCtenopharyngodon idella)  , 35. The idella) –38. Mean idella) , 40. In organism , 42. ThuDietary protein and lipid need to be decomposed by protease and lipase before they can be absorbed and utilized by the intestinal tract. Consequently, the apparent digestibility of dietary protein and lipid in fish was linearly correlated with the activity of intestinal digestive enzymes . In thisThe effects of dietary NSPs on the activity of intestinal digestive enzymes may be based on the physicochemical properties of NSPs. For instance, some soluble NSPs  are naturally in viscous and can increase the viscosity of chyme and adhere to digestive enzymes, thereby inhibiting digestive enzymes ; meanwhiOreochromis niloticus L.) and calcium, magnesium, phosphorus (P) and Na in African catfish (Clarias gariepinus)  , 50. Simiepinus) ; and inciepinus) . These rt uptake , 53. Themembrane , and is membrane . Gal-GarFeed ingredients inevitably affect the development and morphology of the intestine, therefore, intestinal morphology is often used to assess the potential physiological effects of diet on the intestine . MoreoveMucin secreted by goblet cells is an important component of the immune barrier of the intestinal mucosa and plays an important role in the maintenance of the intestinal health of fish –68. TherStudies have revealed that IGF1 and GLP-2 play an important role in regulating nutrient metabolism and promoting cell proliferation and differentiation, and their expression levels can effectively reflect the development status of tissues –75. AddiIn summary, using weight gain rate and feed coefficient as evaluation indicators, the suitable level of NSPs for largemouth bass juveniles should not above 5.51%. Excessive dietary NSPs adversely affected digestive and absorptive enzyme activities and intestinal morphology, which in turn reduced the apparent digestibility of feed nutrients and the growth of juvenile largemouth bass. Overall, our data suggest that carnivorous fish have limited tolerance to dietary NSPs, and for juvenile largemouth bass, dietary NSPs should not exceed 5.51%.The original contributions presented in this study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.This animal study was reviewed and approved by Animal Research and Ethics Committee of Guangdong Ocean University.YL: conceptualization, formal analysis, data curation, writing—original draft, and writing—review and editing. JF, HH, and HZ: methodology, project administration, and data curation. YC and YZ: conceptualization, formal analysis, and data curation. WJ and WZ: project administration and supervision. JD and BT: investigation, methodology, and resources. All authors contributed to the article and approved the submitted version.


Prompt diagnosis of nontuberculous Mycobacterial (NTM) vertebral osteomyelitis is challenging, yet necessary to prevent serious morbidity and mortality. Here, we report 3 cases of vertebral osteomyelitis caused by NTM with imaging findings.Case 1, a 58-year-old male patient, was admitted to our hospital because of the presence of a pulmonary mass for 6 months with cough and chest pain.Case 2, a 50-year-old male patient, had fever and cough for 3 years and was diagnosed with tuberculosis. Antituberculosis treatment was ineffective, accompanied by lymph node enlargement and osteosclerotic changes involving vertebral bodies.Case 3, a 66-year-old female patient, was admitted to our hospital with a mass on the top of her head for 1 month, which ruptured in the last 2 weeks.Mycobacterium (M.) avium.Case 1: Sputum culture revealed M. intracellulare.Case 2: The final culture results of the lymph node biopsy samples were M. gordon.Case 3: Culture results of the sputum and pus from the abscess were We found sclerosing lesions in the spine in all 3 NTM patients, which were easily misdiagnosed as metastatic tumors. In 2 cases, there was bone destruction in the ilium with limbic sclerosis, and there were abscesses near the ilium and in front of the sacrum in 1 case.Case 1 was transferred to other specialist hospital.Case 3 received surgical treatment for cranial lesions and abscess drainage.Case 2 and case 3 received targeted treatment for nontuberculous mycobacteria in our hospital.The condition of case 1 was unknown.Recovery of case 2 was uneventful because of prolonged illness; however, inflammation gradually improved overall.Case 3 had no recurrence following surgical treatment.In our 3 cases of NTM vertebral osteomyelitis, bone lesions were often misdiagnosed as bony metastases because of the presence of multiple sclerotic lesions. Diagnoses were challenging and delayed. It is important to consider osteomyelitis by NTM when disseminated osteosclerosis with or without osteolytic bone lesions is present in conjunction with continuous inflammatory symptoms and signs. Moreover, an open biopsy of the lesion should be performed for a definitive diagnosis. NTM is readily isolated from environmental sources, such as water, dust, and soil, and infections are presumed to occur via the respiratory or gastrointestinal tract.,2 In general, this mycobacterium is less pathogenic to humans than is Mycobacterium TB; however, if there is a susceptibility factor, local or systemic immune dysfunction of the host can lead to infectious lesions.–3 In addition, human-to-human transmission is rare.Nontuberculous Mycobacterium (NTM) refers to all NTM can invade human lungs, bones, joints, lymph nodes, skin, soft tissue, and other tissues and cause systemic disseminated diseases. In many low-burden TB countries, the incidence of NTM has surpassed that of TB. the diagnosis of NTM osteomyelitis is often delayed and misdiagnosed, which results in delayed treatment. Imaging reports of vertebral osteomyelitis with NTM are scarce. Here, we report imaging findings of 3 cases of vertebral osteomyelitis with NTM. We have summarized our imaging findings of NTM vertebral osteomyelitis, alongside typical clinical histories to facilitate prompt and accurate diagnoses in the future.Clinically, the diagnosis and differential diagnosis of spinal NTM disease are challenging because of the currently limited understanding of the condition. Improving our understanding of the imaging features of spinal NTM infection is valuable for accurate diagnoses and timely treatment of NTM. According to our experience and the literature,22.19/L, the percentage of neutrophile granulocyte was 82.4%, red blood cell count was 3.74 × 1012/L, hemoglobin level was 92 g/L, C-reactive-protein level was 156.4 mg/L, and erythrocyte sedimentation rate (ESR) was 108 mm/h. Serum carbohydrate antigen 199, carbohydrate antigen 125, alpha- fetoprotein, carcinoembryonic antigen, and prostate-specific antigen were negative.The patient was a 58-year-old man who was admitted to our hospital because of the presence of a pulmonary mass for 6 months and a clinical history of cough and chest pain for more than 10 days. The patient had a fever of 38°C during hospitalization. The patient had no specific underlying disease, had never undergone surgery, and had an unremarkable family history. Laboratory findings showed that white blood cell count was 14.2 × 10The patient underwent computed tomography (CT) scans of the chest and whole abdomen. The chest CT scan Fig. A showed On the CT scans of the chest and whole abdomen, abnormalities of the vertebral bodies were simultaneously observed. Multiple sclerotic lesions were found in the thoracic and lumbar vertebrae and the iliac bone with small circular high-density lesions and clumpy high-density shadows with blurred edges; moreover, rounded bone destruction could be seen on the lower edge of the T10 and T11 vertebral bodies, accompanied by a sclerotic margin that mimicked Schmorl node  were performed because of suspected malignancy. However, no evidence of tumors or other diseases was found.9/L, the percentage of neutrophile granulocyte was 83.5%, hemoglobin level was 94 g/L, C-reactive-protein level was 147.32 mg/L, procalcitonin was 0.33 ng/mL, and ESR was 51 mm/h.Laboratory finding showed that white blood cell count was 16.4 × 10The enhanced abdominal CT Fig. A showed MR of the left ankle showed multiple nodules or patchy hyperintensity on T2WI fat-saturation images of the left ankle and various bones of the left foot.M. intracellulare.Retroperitoneal lymph node biopsy showed positive acid-fast staining, and the final culture results of lymph node biopsy samples were After 5.5 months of treatment, the T1-weighted MR examination showed that the vertebral lesions had gradually changed to yellow bone marrow Fig. F, and th2.39/L, red blood cell count was 2.69 × 1012/L, the percentage of neutrophile granulocyte was 75.4%, hemoglobin level was 70 g/L, C-reactive-protein level was 112.7 mg/L, procalcitonin was 0.24 ng/mL, and ESR was 101 mm/h. On physical examination, a scalp defect of approximately 20 × 20 mm involving the skull was observed at the top of the patient's head, and purulent exudation was observed in the cavity. Skull CT and MRI  or causative factor (36.5%). In the review by Shinohara et al, about half of all NTM spondylitis cases occurred in healthy people. Blunt-force trauma was a risk factor for NTM spondylitis, and it was hypothesized that macrophages, which phagocytose NTM, migrate to the injury site for tissue restoration but release the pathogen, resulting in the development of vertebral osteomyelitis. Moral et al also reported a case of NTM spondylitis with normal immunity. Gray et al reported a case who underwent an extended period of corticosteroid therapy and proposed that chronic corticosteroid therapy poses a greater risk for the development of NTM vertebral osteomyelitis. These findings demonstrate that a diagnosis of NTM spondylitis should not be ignored because of a patient's normal immunity.It is generally acknowledged that people with immunodeficiency or long-term hormone use are susceptible to NTM infections. However, in our cases, except hepatitis B in case 3, there were no underlying diseases. Garcia et al reported that the mean age of patients with vertebral osteomyelitis caused by NTM was 46.2 ± 18 years, and prevalence was higher in men (53.6%). Our 3 cases of NTM osteomyelitis of 2 males and 1 female aged 50 to 66 years are consistent with the review by Garcia et al.Garcia et al the most commonly reported agent in NTM vertebral osteomyelitis was M. avium, followed by M. xenopi. Kim et al reported 6 cases and reviewed 63 cases of vertebral osteomyelitis caused by NTM and found that the most common species, regardless of the presence of HIV co-infection, was the M. avium complex, followed by M. xenopi. The Runyon classification divides NTM into 4 groups according to colony pigment and growth rate: photochromogen, scotochromogen, non-photochromogen, and rapid grower. M. avium or M. avium-intracellulare complex (MAC), together with M. intracellulare, are classified into Runyon group III non-photochromogenic Mycobacterium. MAC, contains 2 types of Mycobacteria: M. avium and M. intracellulare. Although molecular techniques can identify MAC isolates as M. avium or M. intracellulare, they provide no prognostic or therapeutic advantage. In our report, 1 case had an M. avium infection, another had an M. intracellulare infection, and the third case had an M. Gordon infection. The source of infection in our report is consistent with the abovementioned studies.According to the report by Garcia et al, reported a case of multiple osteomyelitides due to M. avium infection, and CT revealed osteosclerotic lesions in the left femur and lumbar vertebrae, which was initially suspected by the radiologist as multiple malignant bone metastases. Liang et al summarized the CT findings of 21 patients with NTM spondylitis and reported the following CT features: involvement of more than 3 segments of the vertebral body, osteogenic bone destruction, and multi-segment polymorphic bone destruction without intervertebral disc involvement. This is largely consistent with the imaging findings of our cases. NTM causes osteogenic bone destruction, which may be related to bone repair; NTM causes nonspecific chronic inflammation and chronic stimulation of the bone, which leads to reactive osteosclerosis, and sclerosis prevents the vertebra from collapsing. The toxicity of NTM is low, the lesions caused by NTM are confined to the vertebral body, and the intervertebral disc is surrounded by the annulus fibrosus; therefore, it is difficult for NTM to break through the annulus fibrosus and affect the intervertebral disc.Table  did not specifically mention the low-density shadow in nodular sclerosis; however, it could be seen in the images provided in their report. We believe that small circular sclerotic foci are an early sign of infection, whereby the lesion initially appears as a small ring-like area of sclerosis. The gradual increase in lesion size leads to the small ring-like sclerotic lesion evolving into a larger cluster with a high density and an ill-defined margin. If the sclerotic lesion occurs in the anterior margin of the vertebral body, the anterior margin of the vertebral body appears highly irregular. With careful observation, the low-density dots could be seen inside most of the high-density lesions. Furthermore, these low-density speckles inside the sclerotic lesions did not have high signal on T2WI. This suggested that the necrotic material inside the large lesions were not pure water or pus; rather, they may have been the caseous material of TB.Liang et alIf the lesion occurred in the vertebral endplate or articular surface, we observed bone destruction, accompanied by a sclerotic edge, which suggested an indolent destruction process. In the case of vertebral endplate infection, initially, the edge of the vertebral body became irregular, followed by bone destruction of the irregular margin, which should be distinguished from vertebral endplate osteochondritis and Schmorl node. Vertebral endplate osteochondritis and Schmorl node started in the degenerative disc, and the disc signal was reduced and protruded into the adjacent endplate. However, we observed normal MRI signal in the disc in the NTM osteomyelitis. In addition, MRI showed that the signal of the diseased bone on T2WI was not excessive, which may be related to the sclerosis, and the enhancement was mild. osteomyelitis caused by NTM was found to resemble acute pyogenic infection but with slower progression. That is, NTM vertebral infection could result in bone destruction of the articular surface, accompanied by marginal sclerosis, and may be associated with abscess formation. This is consistent with the more severe manifestations in our cases . Moreover, Daphne et al found that additional features of spine infection may include the involvement of only a single end plate and noncontiguous levels. Indeed, we found the same patterns in cases 1 and 2. Liang et al compared the radiographic findings of NTM spondylitis with those of spinal TB. Because of the strong virulence of Mycobacterium TB, spinal TB is more likely to involve the 2 adjacent vertebrae and damage adjacent structures, such as intervertebral discs or paravertebral soft tissues; moreover, the paravertebral abscesses are likely to be more extensive. In contrast, NTM spondylitis rarely shows these symptoms.In the review by Daphne et al,In our cases, in addition to the vertebral and sacroiliac joints, other bones were also involved, such as the ilium. However, the lesions were predominantly sclerotic.Although the number of cases in our study was small, the imaging findings of the 3 cases had distinct features of NTM vertebral osteomyelitis that have not been described previously. Firstly, when the lesions were small, they presented as small annular or nodular sclerosis. Secondly, larger lesions presented as large sclerotic masses with ill-defined margins. Further progression of the lesions may result in destruction of the vertebral endplates or articular bone , and in more severe cases, abscess formation could be observed in the adjacent soft tissue. Such progression of infection could be observed simultaneously in 1 patient in whom the vertebral body lesions were multiple and disseminated, and the discs were not involved.In our 3 cases of NTM vertebral osteomyelitis, bone lesions were often misdiagnosed as bony metastases owing to the presence of multiple sclerotic lesions. The diagnoses were challenging and delayed, with the longest delay in diagnosis of 3 years in case 2. In addition, cases 1 and 2 were required to undergo excessive invasive tests in order to obtain an accurate diagnosis.Thus, accurate and prompt diagnosis and effective treatment of NTM infection of the vertebrae are crucial to prevent severe bone destruction and neurological complications. In conclusion, NTM vertebral infection should be considered when multiple lesions  with osteosclerosis and end plate destruction (or articular surface destruction) with sclerotic edges are observed alongside persistent inflammatory symptoms .Clinical data curation: Shu Juan Ji.Data curation: Peng Hu, Shu Juan Ji, Xiao Jing Yu, Yu Dong Lin.Imaging data curation: Xiao Jing Yu, Yu Dong Lin, Peng Hu.Methodology: Fei Zhou, Peng Hu.Writing – original draft: Xiao Jing Yu, Yu Dong Lin.Writing – review & editing: Chi Shing Zee.