Local application of zoledronate inhibits early bone resorption and promotes bone formation.

Nonunion resulting from early bone resorption is common after bone transplantation surgery. In these patients, instability or osteoporosis causes hyperactive catabolism relative to anabolism, leading to graft resorption instead of fusion. Systemic zoledronate administration inhibits osteoclastogenesis and is widely used to prevent osteoporosis; however, evidence on local zoledronate application is controversial due to osteoblast cytotoxicity, uncontrolled dosing regimens, and local release methods. We investigated the effects of zolendronate on osteoclastogenesis and osteogenesis and explored the corresponding signaling pathways. In vitro cytotoxicity and differentiation of MC3T3E1 cells, rat bone marrow stromal cells (BMSCs) and preosteoclasts (RAW264.7 cells) were evaluated with different zolendronate concentrations. In vivo bone regeneration ability was tested by transplanting different concentrations of zolendronate with ß-tricalcium phosphate (TCP) bone substitute into rat femoral critical-sized bone defects. In vitro, zolendronate concentrations below 2.5 × 10-7 M did not compromise viability in the three cell lines and did not promote osteogenic differentiation in MC3T3E1 cells and BMSCs. In RAW264.7 cells, zoledronate inhibited extracellular regulated protein kinases and c-Jun n-terminal kinase signaling, downregulating c-Fos and NFATc1 expression, with reduced expression of fusion-related dendritic cell­specific transmembrane protein and osteoclast-specific Ctsk and tartrate-resistant acid phosphatase (. In vivo, histological staining revealed increased osteoid formation and neovascularization and reduced fibrotic tissue with 500 µM and 2000 µM zolendronate. More osteoclasts were found in the normal saline group after 6 weeks, and sequential osteoclast formation occurred after zoledronate treatment, indicating inhibition of bone resorption during early callus formation without inhibition of late-stage bone remodeling. In vivo, soaking ß-TCP artificial bone with 500 µM or 2000 µM zoledronate is a promising approach for bone regeneration, with potential applications in bone transplantation.

The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development.

Aims: Therapeutic agents that prevent chondrocyte loss, extracellular matrix (ECM) degradation, and osteoarthritis (OA) progression are required. The expression level of epidermal growth factor (EGF)-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) in damaged human cartilage is significantly higher than in undamaged cartilage. However, the effect of EDIL3 on cartilage is still unknown. Methods: We used human cartilage plugs (ex vivo) and mice with spontaneous OA (in vivo) to explore whether EDIL3 has a chondroprotective effect by altering OA-related indicators. Results: EDIL3 protein prevented chondrocyte clustering and maintained chondrocyte number and SOX9 expression in the human cartilage plug. Administration of EDIL3 protein prevented OA progression in STR/ort mice by maintaining the number of chondrocytes in the hyaline cartilage and the number of matrix-producing chondrocytes (MPCs). It reduced the degradation of aggrecan, the expression of matrix metalloproteinase (MMP)-13, the Osteoarthritis Research Society International (OARSI) score, and bone remodelling. It increased the porosity of the subchondral bone plate. Administration of an EDIL3 antibody increased the number of matrix-non-producing chondrocytes (MNCs) in cartilage and exacerbated the serum concentrations of OA-related pro-inflammatory cytokines, including monocyte chemotactic protein-3 (MCP-3), RANTES, interleukin (IL)-17A, IL-22, and GROα. Administration of ß1 and ß3 integrin agonists (CD98 protein) increased the expression of SOX9 in OA mice. Hence, EDIL3 might activate ß1 and ß3 integrins for chondroprotection. EDIL3 may also protect cartilage by attenuating the expression of IL-1ß-enhanced phosphokinase proteins in chondrocytes, especially glycogen synthase kinase 3 alpha/beta (GSK-3α/ß) and phospholipase C gamma 1 (PLC-γ1). Conclusion: EDIL3 has a role in maintaining the cartilage ECM and inhibiting the development of OA, making it a potential therapeutic drug for OA.

Effects of doxofylline as an adjuvant on severe exacerbation and long-term prognosis for COPD with different clinical subtypes.

OBJECTIVE: This study aimed to investigate the effectiveness of doxofylline as an adjuvant in reducing severe exacerbation for different clinical subtypes of chronic obstructive pulmonary disease (COPD). METHODS: The clinical trial was an open-label non-randomized clinical trial that enrolled patients with COPD. The patients were divided into two groups (doxofylline group[DG] and non-doxofylline group[NDG]) according to whether the adjuvant was used. Based on the proportion of inflammatory cells present, the patients were divided into neutrophilic, eosinophilic, and mixed granulocytic subtypes. The rates of severe acute exacerbation, use of glucocorticoids, and clinical symptoms were followed up in the first month, the third month, and the sixth month after discharge. RESULTS: A total of 155 participants were included in the study. The average age of the participants was 71.2 ± 10.1 years, 52.3% of the patients were male, and 29.7% of the participants had extremely severe cases of COPD. In the third month after discharge the numbers of patients exhibiting severe exacerbation among the neutrophilic subtype were 5 (6.6%) in the DG versus 17 (22.4%) in the NDG (incidence rate ratio[IRR] = 0.4 [95% CI: 0.2-0.9] P = 0.024). In the sixth month after discharge, the numbers were 3 (3.9%) versus 13 (17.1%; IRR = 0.3 [95%; CI: 0.1-0.9], P = 0.045), and those for the eosinophilic subtype were 0 (0.0%) versus 4 (14.8%), P = 0.02. In the eosinophilic subtype, the results for forced expiratory volume in the first second and maximal mid-expiratory flow were significantly higher in the DG. The mean neutrophil and eosinophil levels were significantly lower than in the NDG among the neutrophilic subtype, and the neutrophil percentage was lower than in the NDG among the eosinophilic subtype. At the six-month follow-up, the dose adjustment rates of the neutrophilic and eosinophilic subtypes showed a significant difference (P< 0.05). CONCLUSIONS: As an adjuvant drug, doxofylline has a good therapeutic effect on patients with the neutrophilic and eosinophilic clinical subtypes of COPD. It can reduce the incidence of severe exacerbation, the use of glucocorticoids, and inflammatory reactions in the long term (when used for a minimum of 3 months).

Characterization and Advancement of an Evaluation Method for the Treatment of Spontaneous Osteoarthritis in STR/ort Mice: GRGDS Peptides as a Potential Treatment for Osteoarthritis.

STR/ort mice spontaneously exhibit the typical osteoarthritis (OA) phenotype. However, studies describing the relationship between cartilage histology, epiphyseal trabecular bone, and age are lacking. We aimed to evaluate the typical OA markers and quantify the subchondral bone trabecular parameters in STR/ort male mice at different weeks of age. We then developed an evaluation model for OA treatment. We graded the knee cartilage damage using the Osteoarthritis Research Society International (OARSI) score in STR/ort male mice with or without GRGDS treatment. We measured the levels of typical OA markers, including aggrecan fragments, matrix metallopeptidase-13 (MMP-13), collagen type X alpha 1 chain (COL10A1), and SRY-box transcription factor 9 (Sox9), and quantified epiphyseal trabecular parameters. Compared to the young age group, elderly mice showed an increased OARSI score, decreased chondrocyte columns of the growth plate, elevated expression of OA markers (aggrecan fragments, MMP13, and COL10A1), and decreased expression of Sox9 at the articular cartilage region in elderly STR/ort mice. Aging also significantly enhanced the subchondral bone remodeling and microstructure change in the tibial plateau. Moreover, GRGDS treatment mitigated these subchondral abnormalities. Our study presents suitable evaluation methods to characterize and measure the efficacy of cartilage damage treatments in STR/ort mice with spontaneous OA.

Characterization of the phenotypes of methicillin- and vancomycin-susceptible Staphylococcus argenteus after vancomycin passages.

OBJECTIVES: Staphylococcus argenteus is generally more susceptible to antibiotic treatments than Staphylococcus aureus; however, the study showed that the daptomycin/vancomycin-resistant S. argenteus was isolated from a patient with repeated antibiotic treatments. In this study, the methicillin- and vancomycin-susceptible S. argenteus isolates were used to characterize the phenotypes of S. argenteus after vancomycin passages in vitro. METHODS: Eleven S. argenteus isolates were used for passaging under different concentrations of vancomycin. The minimal inhibitory concentration (MIC) of vancomycin was determined by the agar dilution assay, and the biofilm mass of the passaged variants was quantified by the crystal violet staining assay and observed under the confocal microscope. RESULTS: The MIC of vancomycin for eight of 11 S. argenteus isolates was increased from ≤2 µg/mL to ≤4-8 µg/mL after vancomycin passages. Two variants with the high-level vancomycin-intermediate (vancomycin MIC ≤8 µg/mL) phenotype were identified, and the parental strains of these variants did not have the heterogeneous vancomycin-intermediate population determined by the population profile analysis. Further, three S. argenteus isolates showed an increase in biofilm production and icaA transcription after the low-dose (2 µg/mL) vancomycin passages. CONCLUSIONS: S. argenteus is capable of acquiring a vancomycin-tolerant phenotype and/or converting to a strong biofilm producer after vancomycin passages, which could contribute to the decrease of their antibiotic susceptibility.

Serum Insufficiency Induces RANKL-Independent Osteoclast Formation during Developing Ischemic ONFH.

Blood supply interruption induces hypoxia and reduces serum provision to cause ischemia-induced osteonecrosis, including avascular osteonecrosis of the femoral head (ONFH). Oxygen deficiency (hypoxia) is known to induce different expression patterns in osteoblasts and osteoclasts, which have been extensively studied. However, the effects of serum insufficiency in nutrients, growth factors, and hormones on osteoblast and osteoclast activity in the damaged area and nearby regions remain poorly understood. In this study, the expression of osteoblast and osteoclast marker proteins was elucidated through in vitro and ex vivo studies. The results indicate that serum insufficiency accelerates the formation of monocyte-derived osteoclasts. The combined effect of serum insufficiency and hypoxia (mimicking ischemia) suppressed the activity of alkaline phosphatase and calcification in osteoblasts after the stimulation of osteogenic growth factors. Serum insufficiency increased the activity of tartrate-resistant acid phosphatase, expression of phosphorylated extracellular signal-regulated kinases, and production of reactive oxygen species in monocyte-derived osteoclasts in the absence of receptor activator of nuclear factor kappa-Β ligand stimulation. The findings indicate that changes in the expression of osteoblast and osteoclast markers in necrotic bone extracts were similar to those observed during an in vitro study. These results also suggest that serum insufficiency may be involved in the regulation of osteoclast formation in patients with ONFH.

Ibudilast Mitigates Delayed Bone Healing Caused by Lipopolysaccharide by Altering Osteoblast and Osteoclast Activity.

Bacterial infection in orthopedic surgery is challenging because cell wall components released after bactericidal treatment can alter osteoblast and osteoclast activity and impair fracture stability. However, the precise effects and mechanisms whereby cell wall components impair bone healing are unclear. In this study, we characterized the effects of lipopolysaccharide (LPS) on bone healing and osteoclast and osteoblast activity in vitro and in vivo and evaluated the effects of ibudilast, an antagonist of toll-like receptor 4 (TLR4), on LPS-induced changes. In particular, micro-computed tomography was used to reconstruct femoral morphology and analyze callus bone content in a femoral defect mouse model. In the sham-treated group, significant bone bridge and cancellous bone formation were observed after surgery, however, LPS treatment delayed bone bridge and cancellous bone formation. LPS inhibited osteogenic factor-induced MC3T3-E1 cell differentiation, alkaline phosphatase (ALP) levels, calcium deposition, and osteopontin secretion and increased the activity of osteoclast-associated molecules, including cathepsin K and tartrate-resistant acid phosphatase in vitro. Finally, ibudilast blocked the LPS-induced inhibition of osteoblast activation and activation of osteoclast in vitro and attenuated LPS-induced delayed callus bone formation in vivo. Our results provide a basis for the development of a novel strategy for the treatment of bone infection.

Lipoteichoic Acid Accelerates Bone Healing by Enhancing Osteoblast Differentiation and Inhibiting Osteoclast Activation in a Mouse Model of Femoral Defects.

Lipoteichoic acid (LTA) is a cell wall component of Gram-positive bacteria. Limited data suggest that LTA is beneficial for bone regeneration in vitro. Thus, we used a mouse model of femoral defects to explore the effects of LTA on bone healing in vivo. Micro-computed tomography analysis and double-fluorochrome labeling were utilized to examine whether LTA can accelerate dynamic bone formation in vivo. The effects of LTA on osteoblastogenesis and osteoclastogenesis were also studied in vitro. LTA treatment induced prompt bone bridge formation, rapid endochondral ossification, and accelerated healing of fractures in mice with femoral bone defects. In vitro, LTA directly enhanced indicators of osteogenic factor-induced MC3T3-E1 cell differentiation, including alkaline phosphatase activity, calcium deposition and osteopontin expression. LTA also inhibited osteoclast activation induced by receptor activator of nuclear factor-kappa B ligand. We identified six molecules that may be associated with LTA-accelerated bone healing: monocyte chemoattractant protein 1, chemokine (C-X-C motif) ligand 1, cystatin C, growth/differentiation factor 15, endostatin and neutrophil gelatinase-associated lipocalin. Finally, double-fluorochrome, dynamic-labeling data indicated that LTA significantly enhanced bone-formation rates in vivo. In conclusion, our findings suggest that LTA has promising bone-regeneration properties.

Synovial Fluid Interleukin-16 Contributes to Osteoclast Activation and Bone Loss through the JNK/NFATc1 Signaling Cascade in Patients with Periprosthetic Joint Infection.

Because of lipopolysaccharide (LPS)-mediated effects on osteoclast differentiation and bone loss, periprosthetic joint infection (PJI) caused by Gram-negative bacteria increases the risk of aseptic loosening after reimplantation. Synovial fluid interleukin-16 (IL-16) expression was higher in patients with PJI than in patients without joint infection. Thus, we explored the effects of IL-16 on bone. We investigated whether IL-16 modulates osteoclast or osteoblast differentiation in vitro. An LPS-induced bone loss mice model was used to explore the possible advantages of IL-16 inhibition for the prevention of bone loss. IL-16 directly activated p38 and c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK) signaling and increased osteoclast activation markers, including tartrate-resistant acid phosphatase (TRAP), cathepsin K, and nuclear factor of activated T cells 1 (NFATc1). IL-16 directly caused monocytes to differentiate into TRAP-positive osteoclast-like cells through NFATc1 activation dependent on JNK/MAPK signaling. Moreover, IL-16 did not alter alkaline phosphatase activity or calcium deposition during osteoblastic differentiation. Finally, IL-16 inhibition prevented LPS-induced trabecular bone loss and osteoclast activation in vivo. IL-16 directly increased osteoclast activation through the JNK/NFATc1 pathway. IL-16 inhibition could represent a new strategy for treating infection-associated bone loss.

Rapid analysis of bacterial composition in prosthetic joint infection by 16S rRNA metagenomic sequencing.

OBJECTIVES: Prosthetic joint infection (PJI) is the most common cause of arthroplasty failure. However, infection is often difficult to detect by conventional bacterial cultures, for which false-negative rates are 23% to 35%. In contrast, 16S rRNA metagenomics has been shown to quantitatively detect unculturable, unsuspected, and unviable pathogens. In this study, we investigated the use of 16S rRNA metagenomics for detection of bacterial pathogens in synovial fluid (SF) from patients with hip or knee PJI. METHODS: We analyzed the bacterial composition of 22 SF samples collected from 11 patients with PJIs (first- and second-stage surgery). The V3 and V4 region of bacteria was assessed by comparing the taxonomic distribution of the 16S rDNA amplicons with microbiome sequencing analysis. We also compared the results of bacterial detection from different methods including 16S metagenomics, traditional cultures, and targeted Sanger sequencing. RESULTS: Polymicrobial infections were not only detected, but also characterized at different timepoints corresponding to first- and second-stage exchange arthroplasty. Similar taxonomic distributions were obtained by matching sequence data against SILVA, Greengenes, and The National Center for Biotechnology Information (NCBI). All bacteria isolated from the traditional culture could be further identified by 16S metagenomics and targeted Sanger sequencing. CONCLUSION: The data highlight 16S rRNA metagenomics as a suitable and promising method to detect and identify infecting bacteria, most of which may be uncultivable. Importantly, the method dramatically reduces turnaround time to two days rather than approximately one week for conventional cultures.Cite this article: M-F. Chen, C-H. Chang, C. Chiang-Ni, P-H. Hsieh, H-N. Shih, S. W. N. Ueng, Y. Chang. Rapid analysis of bacterial composition in prosthetic joint infection by 16S rRNA metagenomic sequencing. Bone Joint Res 2019;8:367-377. DOI: 10.1302/2046-3758.88.BJR-2019-0003.R2.

Periprosthetic Joint Infection Caused by Gram-Positive Versus Gram-Negative Bacteria: Lipopolysaccharide, but not Lipoteichoic Acid, Exerts Adverse Osteoclast-Mediated Effects on the Bone.

Periprosthetic joint infection (PJI)-the most common cause of knee arthroplasty failure-may result from Gram-positive (GP) or Gram-negative (GN) bacterial infections. The question as to whether PJI due to GP or GN bacteria can lead to different rates of aseptic loosening after reimplantation remains open. We have investigated this issue through a retrospective review of clinical records obtained from 320 patients with bacterial PJI. The results revealed that, compared with GP infections, GN infections were associated with an increased risk of aseptic loosening. In animal studies, mice underwent intrafemoral injection of lipopolysaccharide (LPS) from GN bacteria or lipoteichoic acid (LTA) from GP bacteria. We demonstrate that LPS-but not LTA-reduced both the number of trabeculae and the bone mineral density in mice. In addition, LPS-treated mice exhibited a reduced body weight, higher serum osteocalcin levels, and an increased number of osteoclasts. LPS accelerated monocyte differentiation into osteoclast-like cells, whereas LTA did not. Finally, ibudilast-a toll-like receptor (TLR)-4 antagonist-was found to inhibit LPS-induced bone loss and osteoclast activation in mice. Taken together, our data indicate that PJI caused by GN bacteria portends a higher risk of aseptic loosening after reimplantation, mainly because of LPS-mediated effects on osteoclast differentiation.

SEPT14 Mutations and Teratozoospermia: Genetic Effects on Sperm Head Morphology and DNA Integrity.

The main objective of this study was to evaluate the potential genetic effects of SEPT14 on male infertility through sequencing the SEPT14 coding region. To address this research gap, 254 men with sperm abnormalities and 116 normozoospermic men were recruited, and the whole-coding regions of SEPT14 were sequenced. Two heterozygous mutations, p.Ala123Thr (3/254 vs. 0/116) and p.Ile333Thr (3/254 vs. 0/116), were identified in these cases. A high percentage of defective sperm heads was found in sperm with mutated SEPT14. Both mutations are highly evolutionarily conserved among vertebrates. The results of a fine morphological and chromatin structural analysis indicated severely malformed sperm heads with abnormal chromatin packaging through transmission electron microscopy and Toluidine blue staining. Compared with controls, high DNA fragmentation was demonstrated in sperm from cases carrying SEPT14 mutations using the comet assay. In addition, these two mutations in SEPT14 affected its polymerization ability in vitro. These data revels that the two SEPT14 missense mutations impaired sperm head morphology and induced DNA damage. Our study suggests that genetic variant of SEPT14 is one of the effects for human sperm formation and male fertility.

Novel Role for miR-1290 in Host Species Specificity of Influenza A Virus.

The role of microRNA (miRNA) in influenza A virus (IAV) host species specificity is not well understood as yet. Here, we show that a host miRNA, miR-1290, is induced through the extracellular signal-regulated kinase (ERK) pathway upon IAV infection and is associated with increased viral titers in human cells and ferret animal models. miR-1290 was observed to target and reduce expression of the host vimentin gene. Vimentin binds with the PB2 subunit of influenza A virus ribonucleoprotein (vRNP), and knockdown of vimentin expression significantly increased vRNP nuclear retention and viral polymerase activity. Interestingly, miR-1290 was not detected in either chicken cells or mouse animal models, and the 3' UTR of the chicken vimentin gene contains no binding site for miR-1290. These findings point to a host species-specific mechanism by which IAV upregulates miR-1290 to disrupt vimentin expression and retain vRNP in the nucleus, thereby enhancing viral polymerase activity and viral replication.

Testis-Specific SEPT12 Expression Affects SUN Protein Localization and is Involved in Mammalian Spermiogenesis.

Male infertility is observed in approximately 50% of all couples with infertility. Intracytoplasmic sperm injection (ICSI), a conventional artificial reproductive technique for treating male infertility, may fail because of a severe low sperm count, immotile sperm, immature sperm, and sperm with structural defects and DNA damage. Our previous studies have revealed that mutations in the septin (SEPT)-coding gene SEPT12 cause teratozoospermia and severe oligozoospermia. These spermatozoa exhibit morphological defects in the head and tail, premature chromosomal condensation, and nuclear damage. Sperm from Sept12 knockout mice also cause the developmental arrest of preimplantation embryos generated through in vitro fertilization and ICSI. Furthermore, we found that SEPT12 interacts with SPAG4, a spermatid nuclear membrane protein that is also named SUN4. Loss of the Spag4 allele in mice also disrupts the integration nuclear envelope and reveals sperm head defects. However, whether SEPT12 affects SPAG4 during mammalian spermiogenesis remains unclear. We thus conducted this study to explore this question. First, we found that SPAG4 and SEPT12 exhibited similar localizations in the postacrosomal region of elongating spermatids and at the neck of mature sperm through isolated murine male germ cells. Second, SEPT12 expression altered the nuclear membrane localization of SPAG4, as observed through confocal microscopy, in a human testicular cancer cell line. Third, SEPT12 expression also altered the localizations of nuclear membrane proteins: LAMINA/C in the cells. This effect was specifically due to the expression of SEPT12 and not that of SEPT1, SEPT6, SEPT7, or SEPT11. Based on these results, we suggest that SEPT12 is among the moderators of SPAG4/LAMIN complexes and is involved in the morphological formation of sperm during mammalian spermiogenesis.

TBC1D21 Potentially Interacts with and Regulates Rap1 during Murine Spermatogenesis.

Few papers have focused on small guanosine triphosphate (GTP)-binding proteins and their regulation during spermatogenesis. TBC1D21 genes (also known as male germ cell RAB GTPase-activating protein MGCRABGAP) are related to sterility, as determined through cDNA microarray testing of human testicular tissues exhibiting spermatogenic defects. TBC1D21 is a protein specifically expressed in the testes that exhibits specific localizations of elongating and elongated spermatids during mammalian spermiogenesis. Furthermore, through co-immunoprecipitation (co-IP) and nano liquid chromatography⁻tandem mass spectrometry (nano LC⁻MS/MS), Rap1 has been recognized as a potential TBC1D21 interactor. This study determined the possible roles of Rap1 and TBC1D21 during mammalian spermiogenesis. First, the binding ability between Rap1 and TBC1D21 was verified using co-IP. Second, the stronger signals of Rap1 expressed in elongating and elongated murine spermatids extracted from testicular sections, namely spermatogonia, spermatocytes, and round spermatids, were compared. Third, Rap1 and TBC1D21 exhibited similar localizations at postacrosomal regions of spermatids and at the midpieces of mature sperms, through isolated male germ cells. Fourth, the results of an activating Rap1 pull-down assay indicated that TBC1D21 overexpression inactivates Rap1 activity in cell models. In conclusion, TBC1D21 may interact with and potentially regulate Rap1 during murine spermatogenesis.

CDC42 Negatively Regulates Testis-Specific SEPT12 Polymerization.

Septin (SEPT) genes encode well-preserved polymerizing GTP-binding cytoskeletal proteins. The cellular functions of SEPTs consist of mitosis, cytoskeletal remodeling, cell polarity, and vesicle trafficking through interactions with various types of cytoskeletons. We discovered that mutated SEPTIN12 in different codons resulted in teratozoospermia or oligozoospermia. In mouse models with a defective Septin12 allele, sperm morphology was abnormal, sperm count decreased, and sperms were immotile. However, the regulators of SEPT12 are completely unknown. Some studies have indicated that CDC42 negatively regulates the polymerization of SEPT2/6/7 complexes in mammalian cell lines. In this study, we investigated whether CDC42 modulates SEPT12 polymerization and is involved in the terminal differentiation of male germ cells. First, through scanning electron microscopy analysis, we determined that the loss of Septin12 caused defective sperm heads. This indicated that Septin12 is critical for the formation of sperm heads. Second, CDC42 and SEPT12 were similarly localized in the perinuclear regions of the manchette at the head of elongating spermatids, neck region of elongated spermatids, and midpiece of mature spermatozoa. Third, wild-type CDC42 and CDC42Q61L (a constitutive-acting-mutant) substantially repressed SEPT12 polymerization, but CDC42T17N (a dominant-negative-acting mutant) did not, as evident through ectopic expression analysis. We concluded that CDC42 negatively regulates SEPT12 polymerization and is involved in terminal structure formation of sperm heads.

Epitope-associated and specificity-focused features of EV71-neutralizing antibody repertoires from plasmablasts of infected children.

Protective antibody levels are critical for protection from severe enterovirus 71 infection. However, little is known about the specificities and functional properties of the enterovirus 71-specific antibodies induced by natural infection in humans. Here we characterize 191 plasmablast-derived monoclonal antibodies from three enterovirus 71-infected children, each of whom shows a distinct serological response. Of the 84 enterovirus 71-specific antibodies, neutralizing antibodies that target the rims and floor of the capsid canyon exhibit broad and potent activities at the nanogram level against viruses isolated in 1998-2016. We also find a subset of infected children whose enterovirus 71-specific antibodies are focused on the 3- and 2-fold plateau epitopes localized at the margin of pentamers, and this type of antibody response is associated with lower serum titers against recently circulating strains. Our data provide new insights into the enterovirus 71-specific antibodies induced by natural infection at the serological and clonal levels.Enterovirus 71 is a leading cause of hand-foot-and-mouth disease and herpangina. Here, the authors characterize a large panel of plasmablast-derived IgG mAbs that target the capsid of EV71 to identify neutralizing antibodies induced by natural infection.

RAB10 Interacts with the Male Germ Cell-Specific GTPase-Activating Protein during Mammalian Spermiogenesis.

According to recent estimates, 2%-15% of couples are sterile, and approximately half of the infertility cases are attributed to male reproductive factors. However, the reasons remain undefined in approximately 25% of male infertility cases, and most infertility cases exhibit spermatogenic defects. Numerous genes involved in spermatogenesis still remain unknown. We previously identified Male Germ Cells Rab GTPase-Activating Proteins (MGCRABGAPs) through cDNA microarray analysis of human testicular tissues with spermatogenic defects. MGCRABGAP contains a conserved RABGAP catalytic domain, TBC (Tre2/Bub2/Cdc16). RABGAP family proteins regulate cellular function (e.g., cytoskeletal remodeling, vesicular trafficking, and cell migration) by inactivating RAB proteins. MGCRABGAP is a male germ cell-specific protein expressed in elongating and elongated spermatids during mammalian spermiogenesis. The purpose of this study was to identify proteins that interact with MGCRABGAP during mammalian spermiogenesis using a proteomic approach. We found that MGCRABGAP exhibited GTPase-activating bioability, and several MGCRABGAP interactors, possible substrates (e.g., RAB10, RAB5C, and RAP1), were identified using co-immunoprecipitation (co-IP) and nano liquid chromatography-mass spectrometry/mass spectrometry (nano LC-MS/MS). We confirmed the binding ability between RAB10 and MGCRABGAP via co-IP. Additionally, MGCRABGAP-RAB10 complexes were specifically colocalized in the manchette structure, a critical structure for the formation of spermatid heads, and were slightly expressed at the midpiece of mature spermatozoa. Based on these results, we propose that MGCRABGAP is involved in mammalian spermiogenesis by modulating RAB10.

Motor coordination and balance measurements reveal differential pathogenicity of currently spreading enterovirus 71 strains in human SCARB2 transgenic mice.

Enterovirus 71 (EV71) has caused large-scale epidemics with neurological complications in the Asia-Pacific region. The C4a and B5 strains are the two major genotypes circulating in many countries recently. This study used a new protocol, a motor coordination task, to assess the differential pathogenicity of C4a and B5 strains in human SCARB2 transgenic mice. We found that the pathogenicity of C4a viruses was more severe than that of B5 viruses. Moreover, we discovered that an increased level of monocyte chemoattractant protein-1 was positively correlated with severely deficient motor function. This study provides a new method for evaluating EV71 infection in mice and distinguishing the severity of the symptoms caused by different clinical strains, which would contribute to studies of pathogenesis and development of vaccines and antivirals in EV71 infections.

SEPT12-NDC1 Complexes Are Required for Mammalian Spermiogenesis.

Male factor infertility accounts for approximately 50 percent of infertile couples. The male factor-related causes of intracytoplasmic sperm injection failure include the absence of sperm, immotile sperm, immature sperm, abnormally structured sperm, and sperm with nuclear damage. Our knockout and knock-in mice models demonstrated that SEPTIN12 (SEPT12) is vital for the formation of sperm morphological characteristics during spermiogenesis. In the clinical aspect, mutated SEPT12 in men results in oligozoospermia or teratozoospermia or both. Sperm with mutated SEPT12 revealed abnormal head and tail structures, decreased chromosomal condensation, and nuclear damage. Furthermore, several nuclear or nuclear membrane-related proteins have been identified as SEPT12 interactors through the yeast 2-hybrid system, including NDC1 transmembrane nucleoporin (NDC1). NDC1 is a major nuclear pore protein, and is critical for nuclear pore complex assembly and nuclear morphology maintenance in mammalian cells. Mutated NDC1 cause gametogenesis defects and skeletal malformations in mice, which were detected spontaneously in the A/J strain. In this study, we characterized the functional effects of SEPT12-NDC1 complexes during mammalian spermiogenesis. In mature human spermatozoa, SEPT12 and NDC1 are majorly colocalized in the centrosome regions; however, NDC1 is only slightly co-expressed with SEPT12 at the annulus of the sperm tail. In addition, SEPT12 interacts with NDC1 in the male germ cell line through coimmunoprecipitation. During murine spermiogenesis, we observed that NDC1 was located at the nuclear membrane of spermatids and at the necks of mature spermatozoa. In male germ cell lines, NDC1 overexpression restricted the localization of SEPT12 to the nucleus and repressed the filament formation of SEPT12. In mice sperm with mutated SEPT12, NDC1 dispersed around the manchette region of the sperm head and annulus, compared with concentrating at the sperm neck of wild-type sperm. These results indicate that SEPT12-NDC1 complexes are involved in mammalian spermiogenesis.