Exact reaction coordinates for flap opening in HIV-1 protease.

The primary goal of protein science is to understand how proteins function, which requires understanding the functional dynamics responsible for transitions between different functional structures of a protein. A central concept is the exact reaction coordinates that can determine the value of committor for any protein configuration, which provide the optimal description of functional dynamics. Despite intensive efforts, identifying the exact reaction coordinates (RCs) in complex molecules remains a formidable challenge. Using the recently developed generalized work functional, we report the discovery of the exact RCs for an important functional process-the flap opening of HIV-1 protease. Our results show that this process has six RCs, each one is a linear combination of ~240 backbone dihedrals, providing the precise definition of collectivity and cooperativity in the functional dynamics of a protein. Applying bias potentials along each RC can accelerate flap opening by [Formula: see text] to [Formula: see text] folds. The success in identifying the RCs of a protein with 198 residues represents a significant progress beyond that of the alanine dipeptide, currently the only other complex molecule for which the exact RCs for its conformational changes are known. Our results suggest that the generalized work functional (GWF) might be the fundamental operator of mechanics that controls protein dynamics.

Cornichon protein CNIH4 is not essential for mice gametogenesis and fertility.

BACKGROUND: Cornichon is a functionally conserved transmembrane protein family that generally acts as a cargo-sorting receptor and cycles between the ER and the Golgi. Four Cornichon family members (CNIH1-4) have been identified. The key residues responsible for CNIH1-3 to bind to AMPA receptors are not conserved in CNIH4. Additionally, the function of CNIH1-3 in GPCR signaling is less established, while more established in case of CNIH4 protein that interact with GPCR and control their exportation. Many GPCRs are known for their essential roles in male and female gonad development. But whether CNIH4 plays a role in gametogenesis remains unknown. DESIGN: Mice carrying the Cnih4 knockout allele (Cnih4tm1a-/-) were generated by insertion of a LacZ reporter and a polyadenylation site after exon 1. Western blot, Immunofluorescence, computer-aided sperm analysis and other methods were used in the functional analysis. RESULTS: We identified that both Cnih4tm1a-/- male and female mice have normal fertility. Though, the sperm count, morphology, and motility of Cnih4tm1a-/- mice were slightly impaired compared to those of wild-type mice, the testes to body weight ratio and testicular histology were similar to those in control mice. Histological examination of Cnih4tm1a-/- ovaries detected follicles from primordial to antral stages and the numbers of follicles at each stage were also comparable to wild-type controls. Normal fertility was noticed after six-month fertility tests. That was likely due to the compensatory role of Chin3, which significantly upregulated in the Cnih4tm1a-/- mice to preserve the fertility role. CONCLUSION: Despite CNIH4 showing enriched expression in mouse germ cells, our genetic knockout studies demonstrated that CNIH4 is not essential for gametogenesis and fertility in mice although with a slight reduction in count, motility and morphology of sperm in male mice.

OsGADD45a1: a multifaceted regulator of rice architecture, grain yield, and blast resistance.

KEY MESSAGE: The homolog gene of the Growth Arrest and DNA Damage-inducible 45 (GADD45) in rice functions in the regulation of plant architecture, grain yield, and blast resistance. The Growth Arrest and DNA Damage-inducible 45 (GADD45) family proteins, well-established stress sensors and tumor suppressors in mammals, serve as pivotal regulators of genotoxic stress responses and tumorigenesis. In contrast, the homolog and role of GADD45 in plants have remained unclear. Herein, using forward genetics, we identified an activation tagging mutant AC13 exhibited dwarf characteristics resulting from the loss-of-function of the rice GADD45α homolog, denoted as OsGADD45a1. osgadd45a1 mutants displayed reduced plant height, shortened panicle length, and decreased grain yield compared to the wild-type Kitaake. Conversely, no obvious differences in plant height, panicle length, or grain yield were observed between wild-type and OsGADD45a1 overexpression plants. OsGADD45a1 displayed relatively high expression in germinated seeds and panicles, with localization in both the nucleus and cytoplasm. RNA-sequencing analysis suggested a potential role for OsGADD45a1 in the regulation of photosynthesis, and binding partner identification indicates OsGADD45a1 interacts with OsRML1 to regulate rice growth. Intriguingly, our study unveiled a novel role for OsGADD45a1 in rice blast resistance, as osgadd45a1 mutant showed enhanced resistance to Magnaporthe oryzae, and the expression of OsGADD45a1 was diminished upon blast fungus treatment. The involvement of OsGADD45a1 in rice blast fungus resistance presents a groundbreaking finding. In summary, our results shed light on the multifaceted role of OsGADD45a1 in rice, encompassing biotic stress response and the modulation of several agricultural traits, including plant height, panicle length, and grain yield.

Development of a predictive nomogram for 28-day mortality risk in non-traumatic or post-traumatic subarachnoid hemorrhage patients.

OBJECTIVE: Subarachnoid hemorrhage (SAH) is associated with high rates of mortality and permanent disability. At present, there are few definite clinical tools to predict prognosis in SAH patients. The current study aims to develop and assess a predictive nomogram model for estimating the 28-day mortality risk in both non-traumatic or post-traumatic SAH patients. METHODS: The MIMIC-III database was searched to select patients with SAH based on ICD-9 codes. Patients were separated into non-traumatic and post-traumatic SAH groups. Using LASSO regression analysis, we identified independent risk factors associated with 28-day mortality and incorporated them into nomogram models. The performance of each nomogram was assessed by calculating various metrics, including the area under the curve (AUC), net reclassification improvement (NRI), integrated discrimination improvement (IDI), and decision curve analysis (DCA). RESULTS: The study included 999 patients with SAH, with 631 in the non-traumatic group and 368 in the post-traumatic group. Logistic regression analysis revealed critical independent risk factors for 28-day mortality in non-traumatic SAH patients, including gender, age, glucose, platelet, sodium, BUN, WBC, PTT, urine output, SpO2, and heart rate and age, glucose, PTT, urine output, and body temperature for post-traumatic SAH patients. The prognostic nomograms outperformed the commonly used SAPSII and APSIII systems, as evidenced by superior AUC, NRI, IDI, and DCA results. CONCLUSION: The study identified independent risk factors associated with the 28-day mortality risk and developed predictive nomogram models for both non-traumatic and post-traumatic SAH patients. The nomogram holds promise in guiding prognosis improvement strategies for patients with SAH.

A novel NPHP4 homozygous missense variant identified in infertile brothers with multiple morphological abnormalities of the sperm flagella.

PURPOSE: Asthenozoospermia is an important cause of male infertility, and the most serious type is characterized by multiple morphological abnormalities of the sperm flagella (MMAF). However, the precise etiology of MMAF remains unknown. In the current study, we recruited a consanguineous Pakistani family with two infertile brothers suffering from primary infertility due to MMAF without obvious signs of PCD. METHODS: We performed whole-exome sequencing on DNAs of the patients, their parents, and a fertile brother and identified the homozygous missense variant (c.1490C > G (p.P497R) in NPHP4 as the candidate mutation for male infertility in this family. RESULTS: Sanger sequencing confirmed that this mutation recessively co-segregated with the MMAF in this family. In silico analysis revealed that the mutation site is conserved across different species, and the identified mutation also causes abnormalities in the structure and hydrophobic interactions of the NPHP4 protein. Different bioinformatics tools predict that NPHP4p.P497R mutation is pathogenic. Furthermore, Papanicolaou staining and scanning electron microscopy of sperm revealed that affected individuals displayed typical MMAF phenotype with a high percentage of coiled, bent, short, absent, and/or irregular flagella. Transmission electron microscopy images of the patient's spermatozoa revealed significant anomalies in the sperm flagella with the absence of a central pair of microtubules (9 + 0) in every section scored. CONCLUSIONS: Taken together, these results show that the homozygous missense mutation in NPHP4 is associated with MMAF.

Novel frameshift mutation in STK33 is associated with asthenozoospermia and multiple morphological abnormalities of the flagella.

Serine/threonine kinases domain-containing proteins are known to play important functions in sperm flagella and male fertility. However, the roles of these proteins in human reproduction remain poorly understood and whether their variants are associated with human asthenozoospermia have not been reported. Here, we recruited a Pakistani family having four infertile patients diagnosed with idiopathic asthenozoospermia without any ciliary-related symptoms. Whole-exome sequencing identified a novel homozygous frameshift mutation (c.1235del, p.T412Kfs*14) in serine/threonine kinase 33 (STK33), which displays a highly conserved and predominant expression in testis in humans. This variant led to a dramatic reduction of STK33 messenger RNA (mRNA) in the patients. Patients homozygous for the STK33 variant presented reduced sperm motility, frequent morphological abnormalities of sperm flagella and completely disorganized flagellar ultrastructures, which are typical for multiple morphological abnormalities of the flagella (MMAF) phenotypes. Overall, these findings present evidence establishing that STK33 is an MMAF-related gene and provide new insights for understanding the role of serine/threonine kinases domain-containing proteins in human male reproduction.

A recessive ACTL7A founder variant leads to male infertility due to acrosome detachment in Pakistani Pashtuns.

Male infertility affects more than 20 million men worldwide and is a major public health concern. Male infertility has a strong genetic basis, particularly for those unexplained cases. Here, through genetic analysis of three Pakistani families having eight infertile men with normal parameters in routine semen analysis, we identified a novel ACTL7A variant (c.149_150del, p.E50Afs*6), recessively co-segregating with infertility in these three families. This variant leads to the loss of ACTL7A proteins in spermatozoa from patients. Transmission EM analyses revealed acrosome detachment from nuclei in 98.9% spermatozoa of patients. Interestingly, this ACTL7A variant was frequently detected in our sequenced Pakistani Pashtuns with a minor allele frequency of ~0.021 and all the carriers shared a common haplotype of about 240 kb flanking ACTL7A, indicating that it is likely originated from a single founder. Our findings reveal that a founder ACTL7A pathogenic variant confers a high genetic susceptibility for male infertility with normal routine semen parameters but acrosomal ultrastructural defects in Pakistani Pashtun descendants, and highlight that variants not rare should also be considered when trying to identify disease-causing variants in ethnic groups with the tradition of intra-ethnic marriages.

Plasma membrane-localized H+-ATPase OsAHA3 functions in saline-alkaline stress tolerance in rice.

KEY MESSAGE: A novel function of plasma membrane-localized H+-ATPase, OsAHA3, was identified in rice, which is involved in saline-alkaline tolerance and specifically responds to high pH during saline-alkaline stress. Saline-alkaline stress causes serious damage to crop production on irrigated land. Plants suffer more severe damage under saline-alkaline stress than under salinity stress alone. Plasma membrane-localized proton (H+) pump (H+-ATPase) is an important enzyme that controls plant growth and development by catalyzing H+ efflux and enabling effective charge balance. Many studies about the role of plasma membrane H+-ATPases in saline-alkaline stress tolerance have been reported in Arabidopsis, especially on the AtAHA2 (Arabidopsis thaliana H+-ATPase 2) gene; however, whether and how plasma membrane H+-ATPases play a role in saline-alkaline stress tolerance in rice remain unknown. Here, using the activation-tagged rice mutant pool, we found that the plasma membrane-localized H+-ATPase OsAHA3 (Oryza sativa autoinhibited H+-ATPase 3) is involved in saline-alkaline stress tolerance. Activation-tagged line 29 (AC29) was identified as a loss-of-function mutant of OsAHA3 and showed more severe growth retardation under saline-alkaline stress with high pH than under salinity stress. Moreover, osaha3 loss-of-function mutants generated by CRISPR/Cas9 system exhibited saline-alkaline stress sensitive phenotypes; staining of leaves with nitrotetrazolium blue chloride (NBT) and diaminobenzidine (DAB) revealed more reactive oxygen species (ROS) accumulation in osaha3 mutants. OsAHA3-overexpressing plants showed increased saline-alkaline stress tolerance than wild-type plants. Tissue-specific expression analysis revealed high expression level of OsAHA3 in leaf, sheath, glume, and panicle. Overall, our results revealed a novel function of plasma membrane-localized H+-ATPase, OsAHA3, which is involved in saline-alkaline stress tolerance and specifically responds to high pH.

Autophagy receptor OsNBR1 modulates salt stress tolerance in rice.

KEY MESSAGE: Autophagy receptor OsNBR1 modulates salt stress tolerance by affecting ROS accumulation in rice. The NBR1 (next to BRCA1 gene 1), as important selective receptors, whose functions have been reported in animals and plants. Although the function of NBR1 responses to abiotic stress has mostly been investigated in Arabidopsis thaliana, the role of NBR1 under salt stress conditions remains unclear in rice (Oryza sativa). In this study, by screening the previously generated activation-tagged line, we identified a mutant, activation tagging 10 (AC10), which exhibited salt stress-sensitive phenotypes. TAIL-PCR (thermal asymmetric interlaced PCR) showed that the AC10 line carried a loss-of-function mutation in the OsNBR1 gene. OsNBR1 was found to be a positive regulator of salt stress tolerance and was localized in aggregates. A loss-of-function mutation in OsNBR1 increased salt stress sensitivity, whereas overexpression of OsNBR1 enhanced salt stress resistance. The osnbr1 mutants showed higher ROS (reactive oxygen species) production, whereas the OsNBR1 overexpression (OsNBR1OE) lines showed lower ROS production, than Kitaake plants under normal and salt stress conditions. Furthermore, RNA-seq analysis revealed that expression of OsRBOH9 (respiratory burst oxidase homologue) was increased in osnbr1 mutants, resulting in increased ROS accumulation in osnbr1 mutants. Together our results established that OsNBR1 responds to salt stress by influencing accumulation of ROS rather than by regulating transport of Na+ and K+ in rice.

Plasma membrane-localized Hsp40/DNAJ chaperone protein facilitates OsSUVH7-OsBAG4-OsMYB106 transcriptional complex formation for OsHKT1;5 activation.

The salinization of irrigated land affects agricultural productivity. HIGH-AFFINITY POTASSIUM (K+ ) TRANSPORTER 1;5 (OsHKT1;5)-dependent sodium (Na+ ) transport is a key salt tolerance mechanism during rice growth and development. Using a previously generated high-throughput activation tagging-based T-DNA insertion mutant pool, we isolated a mutant exhibiting salt stress-sensitive phenotype, caused by a reduction in OsHKT1;5 transcripts. The salt stress-sensitive phenotype of this mutant results from the loss of function of OsDNAJ15, which encodes plasma membrane-localized heat shock protein 40 (Hsp40). osdnaj15 loss-of-function mutants show decreased plant height, increased leaf angle, and reduced grain number caused by shorter panicle length and fewer branches. On the other h'and, OsDNAJ15-overexpression plants showed salt stress-tolerant phenotypes. Intriguingly, salt stress facilitates the nuclear relocation of OsDNAJ15 so that it can interact with OsBAG4, and OsDNAJ15 and OsBAG4 synergistically facilitate the DNA-binding activity of OsMYB106 to positively regulate the expression of OsHKT1;5. Overall, our results reveal a novel function of plasma membrane-localized Hsp40 protein in modulating, alongside chaperon regulator OsBAG4, transcriptional regulation under salinity stress tolerance.

The changing pattern of common respiratory and enteric viruses among outpatient children in Shanghai, China: Two years of the COVID-19 pandemic.

Nonpharmaceutical interventions (NPIs) taken to combat the coronavirus disease 2019 (COVID-19) pandemic have not only decreased the spread of severe acute respiratory syndrome coronavirus 2 but also have had an impact on the prevalence of other common viruses. This study aimed to investigate the long-term impact of NPIs on common respiratory and enteric viruses among children in Shanghai, China, as NPIs were relaxed after June 2020. The laboratory results and clinical data of outpatient children with acute respiratory tract infections (ARTI) and acute gastroenteritis (AGE) were analyzed and compared between the post-COVID-19 period (from June 2020 to January 2022) and pre-COVID-19 period (from June 2018 to January 2020). A total of 107 453 patients were enrolled from June 2018 to January 2022, including 43 190 patients with ARTI and 64 263 patients with AGE. The positive rates of most viruses decreased during the post-COVID-19 period, with the greatest decrease for influenza A (-0.94%), followed by adenoviruses (AdV) (-61.54%), rotaviruses (-48.17%), and influenza B (-40%). However, the positive rates of respiratory syncytial virus (RSV) and enteric AdV increased during the post-COVID-19 period as the NPIs were relaxed. Besides this, in the summer of 2021, an unexpected out-of-season resurgence of RSV activity was observed, and the resurgence was more prominent among children older than 5 years. The effectiveness of the current relaxed NPIs in control of common respiratory and enteric viruses was variable. Relaxation of NPIs might lead to the resurgence of common viruses.

Mechanism for the rare fluctuation that powers protein conformational change.

Most functional processes of biomolecules are rare events. Key to a rare event is the rare fluctuation that enables the energy activation process that precedes and powers crossing of the activation barrier. However, the physical nature of this rare fluctuation and how it enables energy activation and subsequently barrier crossing are unknown. We developed a novel metric, the reaction capacity pC, that rigorously defines the beginning and parameterizes the progress of energy activation. This enabled us to identify the rare fluctuation as a special phase-space condition that is necessary and sufficient for initiating systematic energy flow from the non-reaction coordinates into the reaction coordinates. The energy activation of a prototype biomolecular isomerization reaction is dominated by kinetic energy transferring into and accumulating in the reaction coordinates, administered by inertial forces alone. This mechanism for energy activation is fundamentally different from the mechanism suggested by Kramers theory.

Novel loss-of-function variants in DNAH17 cause multiple morphological abnormalities of the sperm flagella in humans and mice.

Multiple morphological abnormalities of the flagella (MMAF) is a genetically heterogeneous disorder leading to male infertility. Recent studies have revealed that DNAH17 variants are associated with MMAF, yet there is no functional evidence in support of their pathnogenicity. Here, we recruited two consanguineous families of Pakistani and Chinese origins, respectively, diagnosed with MMAF. Whole-exome sequencing identified novel homozygous DNAH17 variants, which led to loss of DNAH17 proteins, in the patients. Transmission electron microscope analyses revealed completely disorganized axonemal structure as the predominant anomaly and increased frequencies of missings of microtubule doublet(s) 4-7 in sperm flagella of patients. Similar to those found in patients, Dnah17-/- mice also displayed MMAF phenotype along with completely disorganized axonemal structures. Clusters of disorganized microtubules and outer dense fibers were observed in developing spermatids, indicating impaired sperm flagellar assembly. Besides, we also noticed many elongating spermatids with a deformed nuclear shape and abnormal step 16 spermatids that failed to spermiate, which subsequently underwent apoptosis in Dnah17-null mice. These findings present direct evidence establishing that DNAH17 is a MMAF-related gene in humans and mice, extend the clinical interpretations of DNAH17 variants, and highlight an essential and complex role of DNAH17 in spermatogenesis.

Kinetic energy flows in activated dynamics of biomolecules.

Protein conformational changes are activated processes essential for protein functions. Activation in a protein differs from activation in a small molecule in that it involves directed and systematic energy flows through preferred channels encoded in the protein structure. Understanding the nature of these energy flow channels and how energy flows through them during activation is critical for understanding protein conformational changes. We recently [W. Li and A. Ma, J. Chem. Phys. 144, 114103 (2016)] developed a rigorous statistical mechanical framework for understanding potential energy flows. Here, we complete this theoretical framework with a rigorous theory for kinetic energy flows: potential and kinetic energies interconvert when impressed forces oppose inertial forces, whereas kinetic energy transfers directly from one coordinate to another when inertial forces oppose each other. This theory is applied to analyzing a prototypic system for biomolecular conformational dynamics: the isomerization of an alanine dipeptide. Among the two essential energy flow channels for this process, dihedral ϕ confronts the activation barrier, whereas dihedral θ1 receives energy from potential energy flows. Intriguingly, θ1 helps ϕ to cross the activation barrier by transferring to ϕ via direct kinetic energy flow all the energy it received-an increase in θ̇1 caused by potential energy flow converts into an increase in ϕ̇. As a compensation, θ1 receives kinetic energy from bond angle α via a direct mechanism and bond angle ß via an indirect mechanism.

A new Augmin subunit, Msd1, demonstrates the importance of mitotic spindle-templated microtubule nucleation in the absence of functioning centrosomes.

The Drosophila Augmin complex localizes gamma-tubulin to the microtubules of the mitotic spindle, regulating the density of spindle microtubules in tissue culture cells. Here, we identify the microtubule-associated protein Msd1 as a new component of the Augmin complex and demonstrate directly that it is required for nucleation of microtubules from within the mitotic spindle. Although Msd1 is necessary for embryonic syncytial mitoses, flies possessing a mutation in msd1 are viable. Importantly, however, in the absence of centrosomes, microtubule nucleation from within the spindle becomes essential. Thus, the Augmin complex has a crucial role in the development of the fly.

A benchmark for reaction coordinates in the transition path ensemble.

The molecular mechanism of a reaction is embedded in its transition path ensemble, the complete collection of reactive trajectories. Utilizing the information in the transition path ensemble alone, we developed a novel metric, which we termed the emergent potential energy, for distinguishing reaction coordinates from the bath modes. The emergent potential energy can be understood as the average energy cost for making a displacement of a coordinate in the transition path ensemble. Where displacing a bath mode invokes essentially no cost, it costs significantly to move the reaction coordinate. Based on some general assumptions of the behaviors of reaction and bath coordinates in the transition path ensemble, we proved theoretically with statistical mechanics that the emergent potential energy could serve as a benchmark of reaction coordinates and demonstrated its effectiveness by applying it to a prototypical system of biomolecular dynamics. Using the emergent potential energy as guidance, we developed a committor-free and intuition-independent method for identifying reaction coordinates in complex systems. We expect this method to be applicable to a wide range of reaction processes in complex biomolecular systems.

Reaction mechanism and reaction coordinates from the viewpoint of energy flow.

Reaction coordinates are of central importance for correct understanding of reaction dynamics in complex systems, but their counter-intuitive nature made it a daunting challenge to identify them. Starting from an energetic view of a reaction process as stochastic energy flows biased towards preferred channels, which we deemed the reaction coordinates, we developed a rigorous scheme for decomposing energy changes of a system, both potential and kinetic, into pairwise components. The pairwise energy flows between different coordinates provide a concrete statistical mechanical language for depicting reaction mechanisms. Application of this scheme to the C7eq → C7ax transition of the alanine dipeptide in vacuum revealed novel and intriguing mechanisms that eluded previous investigations of this well studied prototype system for biomolecular conformational dynamics. Using a cost function developed from the energy decomposition components by proper averaging over the transition path ensemble, we were able to identify signatures of the reaction coordinates of this system without requiring any input from human intuition.

Some studies on generalized coordinate sets for polyatomic molecules.

Generalized coordinates are widely used in various analyses of the trajectories of polyatomic molecules from molecular dynamics simulations, such as normal mode analysis and force distribution analysis. Here, we presented detailed discussions on the properties of some specific sets of generalized coordinates, which separate translational, rotational, and vibrational motions of a molecule from one another once the trajectories of dynamical systems are known. Efficient methods were suggested for estimating the transformation matrix between generalized and Cartesian coordinates. Some properties of the well-known BAT coordinates (bond length, angle, and torsional coordinates) were discussed as well.

Reducing the cost of evaluating the committor by a fitting procedure.

Correct identification of reaction coordinates in complex systems is essential for understanding the mechanisms of their reaction dynamics. Existing methods for identifying reaction coordinates typically require knowledge of the committor--the probability of a given configuration to reach the product basin. The high computational cost of evaluating committors has limited applications of methods for identifying reaction coordinates. We proposed a fitting procedure that can reduce the cost of evaluating committors by an order of magnitude or more. The method only requires evaluating the committors of a few configurations in a transition path by the standard and costly shooting procedure. The committors of the other configurations are then estimated with great accuracy by a sigmoid function derived from fitting the few numerically evaluated committors. The method has been systematically tested on a model system of a Brownian particle moving in a one-dimensional double-well potential, and a small biomolecular system--the isomerization of alanine dipeptide in vacuum and in explicit water.

Nanoclay-Modified Hyaluronic Acid Microspheres for Bone Induction by Sustained rhBMP-2 Delivery.

Microspheres (MSs) are ideal candidates as biological scaffolds loading with growth factors or cells for bone tissue engineering to repair irregular alveolar bone defects by minimally invasive injection. However, the high initial burst release of growth factor and low cell attachment limit the application of microspheres. The modification of microspheres often needs expensive experiments facility or complex chemical reactions, which is difficult to achieve and may bring other problems. In this study, a sol-grade nanoclay, laponite XLS is used to modify the surface of MSs to enhance its affinity to either positively or negatively charged proteins and cells without changing the interior structure of the MSs. Recombinant human bone morphogenetic protein-2 (rhBMP-2) is used as a representation of growth factor to check the osteoinduction ability of laponite XLS-modified MSs. By modification, the protein sustained release, cell loading, and osteoinduction ability of MSs are improved. Modified by 1% laponite XLS, the MSs can not only promote osteogenic differentiation of MC3T3-E1 cells by themselves, but also enhance the effect of the rhBMP-2 below the effective dose. Collectively, the study provides an easy and viable method to modify the biological behavior of microspheres for bone tissue regeneration.