Construction of a murine model of latent infection and reactivation induced by Talaromyces marneffei.

OBJECTIVE: To establish a murine model of Talaromyces marneffei (T. marneffei) latent infection and reactivation, providing a foundation for exploring the molecular mechanisms underlying disease relapse. METHODS: BALB/c mice were tail vein injected with T. marneffei at 0 days post-infection (dpi) and treated with cyclophosphamide (CTX) intraperitoneally every four days, starting from 21 dpi or 42 dpi. Mice were observed for body weight changes, liver and spleen indices, histological characteristics of liver and spleen, fungal load detection in liver and spleen, and Mp1p qualitation in liver and spleen to assess T. marneffei infection severity. RESULTS: T. marneffei-infected mice exhibited a trend of initial weight loss followed by recovery and a subsequent decrease in weight after CTX injection throughout the observation period. Liver and spleen indices, as well as tissue damage, significantly increased during infection but later returned to normal levels, with a gradual rise observed after immunosuppression. Fungal load analysis revealed positive T. marneffei cultures in the liver and spleen at 7 dpi and 14 dpi, followed by negative T. marneffei cultures from 21 dpi until day 21 post-immunosuppression (42 dpi or 63 dpi); however, the spleen remained T. marneffei-cultured negative, consistent with the trend observed in Mp1p detection results. CONCLUSION: A latent infection and reactivation model of T. marneffei in mice was successfully established, with the liver likely serving as a key site for latent T. marneffei.

Application of deep learning algorithm in the recognition of cryptococcosis and talaromycosis skin lesions.

BACKGROUND: Cryptococcosis and talaromycosis are known as 'neglected epidemics' due to their high case fatality rates and low concern. Clinically, the skin lesions of the two fungal diseases are similar and easily misdiagnosed. Therefore, this study aims to develop an algorithm to identify cryptococcosis/talaromycosis skin lesions. METHODS: Skin images of tararomiasis and cryptococcosis were collected from published articles and augmented using the Python Imaging Library (PIL). Then, five deep artificial intelligence models, VGG19, MobileNet, InceptionV3, Incept ResNetV2 and DenseNet201, were developed based on the collected datasets using transfer learning technology. Finally, the performance of the models was evaluated using sensitivity, specificity, F1 score, accuracy, AUC and ROC curve. RESULTS: In total, 159 articles (79 for cryptococcosis and 80 for talaromycosis), including 101 cryptococcosis skin lesion images and 133 talaromycosis skin lesion images, were collected for further mode construction. Five methods showed good performance for prediction but did not yield satisfactory results for all cases. Among them, DenseNet201 performed best in the validation set, followed by InceptionV3. However, InceptionV3 showed the highest sensitivity, accuracy, F1 score and AUC values in the training set, followed by DenseNet201. The specificity of DenseNet201 in the training set is better than that of InceptionV3. CONCLUSIONS: DenseNet201 and InceptionV3 are equivalent to the optimal model in these conditions and can be used in clinical settings as decision support tools for the identification and classification of skin lesions of cryptococcus/talaromycosis.

Roles of extracellular vesicles in periodontal homeostasis and their therapeutic potential.

Periodontal tissue is a highly dynamic and frequently stimulated area where homeostasis is easily destroyed, leading to proinflammatory periodontal diseases. Bacteria-bacteria and cell-bacteria interactions play pivotal roles in periodontal homeostasis and disease progression. Several reviews have comprehensively summarized the roles of bacteria and stem cells in periodontal homeostasis. However, they did not describe the roles of extracellular vesicles (EVs) from bacteria and cells. As communication mediators evolutionarily conserved from bacteria to eukaryotic cells, EVs secreted by bacteria or cells can mediate interactions between bacteria and their hosts, thereby offering great promise for the maintenance of periodontal homeostasis. This review offers an overview of EV biogenesis, the effects of EVs on periodontal homeostasis, and recent advances in EV-based periodontal regenerative strategies. Specifically, we document the pathogenic roles of bacteria-derived EVs (BEVs) in periodontal dyshomeostasis, focusing on plaque biofilm formation, immune evasion, inflammatory pathway activation and tissue destruction. Moreover, we summarize recent advancements in cell-derived EVs (CEVs) in periodontal homeostasis, emphasizing the multifunctional biological effects of CEVs on periodontal tissue regeneration. Finally, we discuss future challenges and practical perspectives for the clinical translation of EV-based therapies for periodontitis.

Meteorological factors, governmental responses and COVID-19: Evidence from four European countries.

With the global lockdown, meteorological factors are highly discussed for COVID-19 transmission. In this study, national-specific and region-specific data sets from Germany, Italy, Spain and the United Kingdom were used to explore the effect of temperature, absolute humidity and diurnal temperature range (DTR) on COVID-19 transmission. From February 1st to November 1st, a 7-day COVID-19 case doubling time (Td), meteorological factors with cumulative 14-day-lagged, government response index and other factors were fitted in the distributed lag nonlinear models. The overall relative risk (RR) of the 10th and the 25th percentiles temperature compared to the median were 0.0074 (95% CI: 0.0023, 0.0237) and 0.1220 (95% CI: 0.0667, 0.2232), respectively. The pooled RR of lower (10th, 25th) and extremely high (90th) absolute humidity were 0.3266 (95% CI: 0.1379, 0.7734), 0.6018 (95% CI: 0.4693, 0.7718) and 0.3438 (95% CI: 0.2254, 0.5242), respectively. While the DTR did not have a significant effect on Td. The total cumulative effect of temperature (10th) and absolute humidity (10th, 90th) on Td increased with the change of lag days. Similarly, a decline in temperature and absolute humidity at cumulative 14-day-lagged corresponded to the lower RR on Td in pooled region-specific effects. In summary, the government responses are important factors in alleviating the spread of COVID-19. After controlling that, our results indicate that both the cold and the dry environment also likely facilitate the COVID-19 transmission.

Suppression of histone deacetylases by SAHA relieves bone cancer pain in rats via inhibiting activation of glial cells in spinal dorsal horn and dorsal root ganglia.

BACKGROUND: Robust activation of glial cells has been reported to occur particularly during the pathogenesis of bone cancer pain (BCP). Researchers from our group and others have shown that histone deacetylases (HDACs) play a significant role in modulating glia-mediated immune responses; however, it still remains unclear whether HDACs are involved in the activation of glial cells during the development of BCP. METHODS: BCP model was established by intra-tibia tumor cell inoculation (TCI). The expression levels and distribution sites of histone deacetylases (HDACs) in the spinal dorsal horn and dorsal root ganglia were evaluated by Western blot and immunofluorescent staining, respectively. Suberoylanilide hydroxamic acid (SAHA), a clinically used HDAC inhibitor, was then intraperitoneally and intrathecally injected to rescue the increased expression levels of HDAC1 and HDAC2. The analgesic effects of SAHA administration on BCP were then evaluated by measuring the paw withdrawal thresholds (PWTs). The effects of SAHA on activation of glial cells and expression of proinflammatory cytokines (TNF-α, IL-1ß, and IL-6) in the spinal dorsal horn and dorsal root ganglia of TCI rats were further evaluated by immunofluorescent staining and Western blot analysis. Subsequently, the effects of SAHA administration on tumor growth and cancer cell-induced bone destruction were analyzed by hematoxylin and eosin (HE) staining and micro-CT scanning. RESULTS: TCI caused rapid and long-lasting increased expression of HDAC1/HDAC2 in glial cells of the spinal dorsal horn and dorsal root ganglia. Inhibiting HDACs by SAHA not only reversed TCI-induced upregulation of HDACs but also inhibited the activation of glial cells in the spinal dorsal horn and dorsal root ganglia, and relieved TCI-induced mechanical allodynia. Further, we found that SAHA administration could not prevent cancer infiltration or bone destruction in the tibia, which indicated that the analgesic effects of SAHA were not due to its anti-tumor effects. Moreover, we found that SAHA administration could inhibit GSK3ß activity in the spinal dorsal horn and dorsal root ganglia, which might contributed to the relief of BCP. CONCLUSION: Our findings suggest that HDAC1 and HDAC2 are involved in the glia-mediated neuroinflammation in the spinal dorsal horn and dorsal root ganglia underlying the pathogenesis of BCP, which indicated that inhibiting HDACs by SAHA might be a potential strategy for pain relief of BCP.

M2 Macrophages Enhance the Cementoblastic Differentiation of Periodontal Ligament Stem Cells via the Akt and JNK Pathways.

Although macrophage (Mφ) polarization has been demonstrated to play crucial roles in cellular osteogenesis across the cascade of events in periodontal regeneration, how polarized Mφ phenotypes influence the cementoblastic differentiation of periodontal ligament stem cells (PDLSCs) remains unknown. In the present study, human monocyte leukemic cells (THP-1) were induced into M0, M1, and M2 subsets, and the influences of these polarized Mφs on the cementoblastic differentiation of PDLSCs were assessed in both conditioned medium-based and Transwell-based coculture systems. Furthermore, the potential pathways and cyto-/chemokines involved in Mφ-mediated cementoblastic differentiation were screened and identified. In both systems, M2 subsets increased cementoblastic differentiation-related gene/protein expression levels in cocultured PDLSCs, induced more PDLSCs to differentiate into polygonal and square cells, and enhanced alkaline phosphatase activity in PDLSCs. Furthermore, Akt and c-Jun N-terminal Kinase (JNK) signaling was identified as a potential pathway involved in M2 Mφ-enhanced PDLSC cementoblastic differentiation, and cyto-/chemokines (interleukin (IL)-10 and vascular endothelial growth factor [VEGF]) secreted by M2 Mφs were found to be key players that promoted cell cementoblastic differentiation by activating Akt signaling. Our data indicate for the first time that Mφs are key modulators during PDLSC cementoblastic differentiation and are hence very important for the regeneration of multiple periodontal tissues, including the cementum. Although the Akt and JNK pathways are involved in M2 Mφ-enhanced cementoblastic differentiation, only the Akt pathway can be activated via a cyto-/chemokine-associated mechanism, suggesting that players other than cyto-/chemokines also participate in the M2-mediated cementoblastic differentiation of PDLSCs. Stem Cells 2019;37:1567-1580.

Association of air pollution with outpatient visits for respiratory diseases of children in an ex-heavily polluted Northwestern city, China.

BACKGROUND: A great number of studies have confirmed that children are a particularly vulnerable population to air pollution. METHODS: In the present study, 332,337 outpatient visits of 15 hospitals for respiratory diseases among children (0-13 years), as well as the simultaneous meteorological and air pollution data, were obtained from 2014 to 2016 in Lanzhou, China. The generalized additive model was used to examine the effects of air pollutants on children's respiratory outpatient visits, including the stratified analysis of age, gender and season. RESULTS: We found that PM2.5, NO2 and SO2 were significantly associated with the increased total respiratory outpatient visits. The increments of total respiratory outpatient visits were the highest in lag 05 for NO2 and SO2, a 10 µg/m3 increase in NO2 and SO2 was associated with a 2.50% (95% CI: 1.54, 3.48%) and 3.50% (95% CI: 1.51, 5.53%) increase in total respiratory outpatient visits, respectively. Those associations remained stable in two-pollutant models. Through stratification analysis, all air pollutants other than PM10 were significantly positive associated with the outpatients of bronchitis and upper respiratory tract infection. Besides, both NO2 and SO2 were positively related to the pneumonia outpatient visits. PM2.5 and SO2 were significantly related to the outpatient visits of other respiratory diseases, while only NO2 was positively associated with the asthma outpatients. We found these associations were stronger in girls than in boys, particularly in younger (0-3 years) children. Interestingly, season stratification analysis indicated that these associations were stronger in the cold season than in the transition or the hot season for PM10, PM2.5 and SO2. CONCLUSIONS: Our results indicate that the air pollution exposure may account for the increased risk of outpatient visits for respiratory diseases among children in Lanzhou, particularly for younger children and in the cold season.

Airborne particulate matter, population mobility and COVID-19: a multi-city study in China.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is an emerging infectious disease, which has caused numerous deaths and health problems worldwide. This study aims to examine the effects of airborne particulate matter (PM) pollution and population mobility on COVID-19 across China. METHODS: We obtained daily confirmed cases of COVID-19, air particulate matter (PM2.5, PM10), weather parameters such as ambient temperature (AT) and absolute humidity (AH), and population mobility scale index (MSI) in 63 cities of China on a daily basis (excluding Wuhan) from January 01 to March 02, 2020. Then, the Generalized additive models (GAM) with a quasi-Poisson distribution were fitted to estimate the effects of PM10, PM2.5 and MSI on daily confirmed COVID-19 cases. RESULTS: We found each 1 unit increase in daily MSI was significantly positively associated with daily confirmed cases of COVID-19 in all lag days and the strongest estimated RR (1.21, 95% CIs:1.14 ~ 1.28) was observed at lag 014. In PM analysis, we found each 10 µg/m3 increase in the concentration of PM10 and PM2.5 was positively associated with the confirmed cases of COVID-19, and the estimated strongest RRs (both at lag 7) were 1.05 (95% CIs: 1.04, 1.07) and 1.06 (95% CIs: 1.04, 1.07), respectively. A similar trend was also found in all cumulative lag periods (from lag 01 to lag 014). The strongest effects for both PM10 and PM2.5 were at lag 014, and the RRs of each 10 µg/m3 increase were 1.18 (95% CIs:1.14, 1.22) and 1.23 (95% CIs:1.18, 1.29), respectively. CONCLUSIONS: Population mobility and airborne particulate matter may be associated with an increased risk of COVID-19 transmission.

A colorimetric probe for detection of Cu2+ by the naked eye and application in test paper.

A novel colorimetric probe RP1 was synthesized using rhodamine derivatives and heterocyclic compounds for the purpose of detecting Cu2+ . RP1 showed good selectivity, high sensitivity and affinity toward Cu2+ over other competing ions in CH3 OH-H2 O (1/1, v/v) solution. Absorbance intensity showed a good linear fit between probe R1 and Cu2+ over the concentration range 1-8 µM and the association constant was also calculated to be 1.145 × 105 M-1 . The sensing mechanism was deduced using Job's plot, Fourier transform infrared spectroscopy, and density functional theory studies. In addition, the colorimetric experiment indicated that probe RP1 could be made into test paper to detect Cu2+ with a colour change from colourless to pink.

The effects of conditioned media generated by polarized macrophages on the cellular behaviours of bone marrow mesenchymal stem cells.

Macrophages (Mφs) are involved in a variety of physiological and pathological events including wound healing and tissue regeneration, in which they play both positive and negative roles depending on their polarization state. In this study, we investigated the cellular behaviours of bone marrow mesenchymal stem cells (BMMSCs) after incubation in different conditioned media (CMs) generated by unpolarized Mφs (M0) or polarized Mφs (M1 and M2). Mφ polarization was induced by stimulation with various cytokines, and CMs were obtained from in vitro Mφ cultures termed CM0, CM1 and CM2 based on each Mφ phenotype. We found that CM1 supported the proliferation and adipogenic differentiation of BMMSCs, whereas CM0 had a remarkable effect on cell osteogenic differentiation. To a certain degree, CM2 also facilitated BMMSC osteogenesis; in particular, cells incubated with CM2 exhibited an enhanced capacity to form robust stem cell sheets. Although incubation with CM1 also increased production of extracellular matrix components, such as fibronectin, COL-1 and integrin ß1during sheet induction, the sheets generated by CM2-incubated cells were thicker than those generated by CM1-incubated cells (P < 0.001). Our data suggest that each Mφ phenotype has a unique effect on BMMSCs. Fine-tuning Mφ polarization following transplantation may serve as an effective method to modulate the therapeutic potential of BMMSCs.

Administration of signalling molecules dictates stem cell homing for in situ regeneration.

Ex vivo-expanded stem cells have long been a cornerstone of biotherapeutics and have attracted increasing attention for treating intractable diseases and improving tissue regeneration. However, using exogenous cellular materials to develop restorative treatments for large numbers of patients has become a major concern for both economic and safety reasons. Advances in cell biological research over the past two decades have expanded the potential for using endogenous stem cells during wound healing processes, and in particular, recent insight into stem cell movement and homing has prompted regenerative research and therapy based on recruiting endogenous cells. Inspired by the natural healing process, artificial administration of specific chemokines as signals systemically or at the injury site, typically using biomaterials as vehicles, is a state-of-the-art strategy that potentiates stem cell homing and recreates an anti-inflammatory and immunomodulatory microenvironment to enhance in situ tissue regeneration. However, pharmacologically coaxing endogenous stem cells to act as therapeutics in the field of biomedicine remains in the early stages; its efficacy is limited by the lack of innovative methodologies for chemokine presentation and release. This review describes how to direct the homing of endogenous stem cells via the administration of specific signals, with a particular emphasis on targeted signalling molecules that regulate this homing process, to enhance in situ tissue regeneration. We also provide an outlook on and critical considerations for future investigations to enhance stem cell recruitment and harness the reparative potential of these recruited cells as a clinically relevant cell therapy.

The analgesic effects of triptolide in the bone cancer pain rats via inhibiting the upregulation of HDACs in spinal glial cells.

BACKGROUND: Bone cancer pain (BCP) severely compromises the quality of life, while current treatments are still unsatisfactory. Here, we tested the antinociceptive effects of triptolide (T10), a substance with considerable anti-tumor efficacies on BCP, and investigated the underlying mechanisms targeting the spinal dorsal horn (SDH). METHODS: Intratibial inoculation of Walker 256 mammary gland carcinoma cells was used to establish a BCP model in rats. T10 was intrathecally injected, and mechanical allodynia was tested by measuring the paw withdrawal thresholds (PWTs). In mechanism study, the activation of microglia, astrocytes, and the mitogen-activated protein kinase (MAPK) pathways in the SDH were evaluated by immunofluorescence staining or Western blot analysis of Iba-1, GFAP, p-ERK, p-p38, and p-JNK. The expression and cellular localization of histone deacetylases (HDACs) 1 and 2 were also detected to investigate molecular mechanism. RESULTS: Intrathecal injection of T10 inhibited the bone cancer-induced mechanical allodynia with an ED50 of 5.874 µg/kg. This effect was still observed 6 days after drug withdrawal. Bone cancer caused significantly increased expression of HDAC1 in spinal microglia and neurons, with HDAC2 markedly increased in spinal astrocytes, which were accompanied by the upregulation of MAPK pathways and the activation of microglia and astrocytes in the SDH. T10 reversed the increase of HDACs, especially those in glial cells, and inhibited the glial activation. CONCLUSIONS: Our results suggest that the upregulation of HDACs contributes to the pathological activation of spinal glial cells and the chronic pain caused by bone cancer, while T10 help to relieve BCP possibly via inhibiting the upregulation of HDACs in the glial cells in the SDH and then blocking the neuroinflammation induced by glial activation.

The role and mechanisms of PD-L1 in immune evasion during Talaromyces marneffei infection.

Talaromycosis, caused by Talaromyces marneffei (T. marneffei), is a systemic fungal disease that involves dissemination throughout the body. The ability of T. marneffei to evade the immune system is considered a crucial factor in its persistent infection, although the specific mechanisms are not yet fully understood. This study aims to investigate the molecular mechanisms underlying the occurrence of latent T. marneffei infection and immune evasion. The gene expression profile analysis in T. marneffei-infected mouse revealed that Pd-l1 exhibited the highest correlation strength with other hub genes, with a median of 0.60 (IQR: 0.50-0.69). T. marneffei infection upregulated the expression of PD-1 and PD-L1 in PBMCs from HIV patients, which was also observed in the T. marneffei-infected mouse and macrophage models. Treatment with a PD-L1 inhibitor significantly reduced fungal burden in the liver and spleen tissues of infected mice and in the kupffer-CTLL-2 co-culture system. PD-L1 inhibitor treatment increased CTLL-2 cell proliferation and downregulated the expression of PD-1, SHP-2, and p-SHP-2, indicating the activation of T cell viability and T cell receptor signaling pathway. Additionally, treatment with a PI3K inhibitor downregulated PD-L1 in T. marneffei-infected kupffer cells. Similar results were observed with treatment using the T. marneffei cell wall virulence factor ß-glucan. Overall, T. marneffei infection upregulated PD-L1 expression in HIV / T. marneffei patients, mice, and kupffer cells. Treatment with a PD-L1 inhibitor significantly reduced fungal burden, while activating T cell activity and proliferation, thereby promoting fungal clearance. Furthermore, the PI3K signaling pathway may be involved in the regulation of PD-L1 by T. marneffei.

Pyroptotic macrophages induce disruption of glutamate metabolism in periodontal ligament stem cells contributing to their compromised osteogenic potential.

Macrophage pyroptosis is of key importance to host defence against pathogen infections and may participate in the progression and recovery of periodontitis. However, the role of pyroptotic macrophages in regulating periodontal ligament stem cells (PDLSCs), the main cell source for periodontium renewal, remains unclear. First, we found that macrophage pyroptosis were enriched in gingiva tissues from periodontitis patients compared with those of healthy people through immunofluorescence. Then the effects of pyroptotic macrophages on the PDLSC osteogenic differentiation were investigated in a conditioned medium (CM)-based coculture system in vitro. CM derived from pyroptotic macrophages inhibited the osteogenic differentiation-related gene and protein levels, ALP activity and mineralized nodule formation of PDLSCs. The osteogenic inhibition of CM was alleviated when pyroptosis was inhibited by VX765. Further, untargeted metabolomics showed that glutamate limitation may be the underlying mechanism. However, exogenous glutamate supplementation aggravated the CM-inhibited osteogenic differentiation of PDLSCs. Moreover, CM increased extracellular glutamate and decreased intracellular glutamate levels of PDLSCs, and enhanced the gene and protein expression levels of system xc - (a cystine/glutamate antiporter). After adding cystine to CM-based incubation, the compromised osteogenic potency of PDLSCs was rescued. Our data suggest that macrophage pyroptosis is related to the inflammatory lesions of periodontitis. Either pharmacological inhibition of macrophage pyroptosis or nutritional supplements to PDLSCs, can rescue the compromised osteogenic potency caused by pyroptotic macrophages.

Harnessing Mechanical Stress with Viscoelastic Biomaterials for Periodontal Ligament Regeneration.

The viscoelasticity of mechanically sensitive tissues such as periodontal ligaments (PDLs) is key in maintaining mechanical homeostasis. Unfortunately, PDLs easily lose viscoelasticity (e.g., stress relaxation) during periodontitis or dental trauma, which disrupt cell-extracellular matrix (ECM) interactions and accelerates tissue damage. Here, Pluronic F127 diacrylate (F127DA) hydrogels with PDL-matched stress relaxation rates and high elastic moduli are developed. The hydrogel viscoelasticity is modulated without chemical cross-linking by controlling precursor concentrations. Under cytomechanical loading, F127DA hydrogels with fast relaxation rates significantly improved the fibrogenic differentiation potential of PDL stem cells (PDLSCs), while cells cultured on F127DA hydrogels with various stress relaxation rates exhibited similar fibrogenic differentiation potentials with limited cell spreading and traction forces under static conditions. Mechanically, faster-relaxing F127DA hydrogels leveraged cytomechanical loading to activate PDLSC mechanotransduction by upregulating integrin-focal adhesion kinase pathway and thus cytoskeletal rearrangement, reinforcing cell-ECM interactions. In vivo experiments confirm that faster-relaxing F127DA hydrogels significantly promoted PDL repair and reduced abnormal healing (e.g., root resorption and ankyloses) in delayed replantation of avulsed teeth. This study firstly investigated how matrix nonlinear viscoelasticity influences the fibrogenesis of PDLSCs under mechanical stimuli, and it reveals the underlying mechanobiology, which suggests novel strategies for PDL regeneration.

Whole-Genome Analysis of Termite-Derived Bacillus velezensis BV-10 and Its Application in King Grass Silage.

Bacillus velezensis (B. velezensis) is a cellulose-degrading strain that has the potential as an additive in fermented feed. B. velezensis BV-10 was isolated and screened from the termite gut. We sequenced the whole genome of this new source of B. velezensis to reveal its potential for use in cellulose degradation. Whole-genome sequencing of B. velezensis BV-10 showed that it has a circular chromosome of 3929792 bp containing 3873 coding genes with a GC content of 45.51% and many genes related to cellulose, hemicellulose, and lignin degradation. King grass silage was inoculated with B. velezensis BV-10 and mixed with other feed additives to assess the effect of B. velezensis BV-10 on the fermentation quality of silage. Six treatment groups were established: the control, B. velezensis BV-10, molasses, cellulase, B. velezensis BV-10 plus molasses, and B. velezensis BV-10 plus cellulase groups. After 30 days of silage-fermentation testing, B. velezensis BV-10 was found to rapidly reduce the silage pH value and significantly reduce the acid-detergent fiber (ADF) content (p < 0.05). The addition of B. velezensis BV-10 plus molasses and cellulase in fermented feed significantly reduced the silage neutral-detergent fiber and ADF content and promoted organic-acid accumulation (p < 0.05). The above results demonstrate that B. velezensis BV-10 promotes the fermentation quality of silage and that this effect is greater when other silage-fermentation additives are included. In conclusion, genes involved in cellulose degradation in B. velezensis BV-10 were identified by whole-genome sequencing and further experiments explored the effects of B. velezensis BV-10 and different feed additives on the fermentation quality of king grass silage, revealing the potential of Bacillus velezensis as a new silage additive.

Biotin-Avidin System-Based Delivery Enhances the Therapeutic Performance of MSC-Derived Exosomes.

Exosomes (EXs) shed by mesenchymal stem cells (MSCs) are potent therapeutic agents that promote wound healing and regeneration, but when used alone in vivo, their therapeutic potency is diminished by rapid clearance and bioactivity loss. Inspired by the biotin-avidin interaction, we developed a simple yet versatile method for the immobilization of MSC-derived EXs (MSC-EXs) into hydrogels and achieved sustained release for regenerative purposes. First, biotin-modified gelatin methacryloyl (Bio-GelMA) was fabricated by grafting NHS-PEG12-biotin onto the amino groups of GelMA. Biotin-modified MSC-EXs (Bio-EXs) were then synthesized using an in situ self-assembling biotinylation strategy, which provided sufficient binding sites for MSC-EX delivery with little effect on their cargo composition. Thereafter, Bio-EXs were immobilized in Bio-GelMA via streptavidin to generate Bio-GelMA@Bio-EX hydrogels. An in vitro analysis demonstrated that Bio-EXs could be taken up by macrophages and exerted immunomodulatory effects similar to those of MSC-EXs, and Bio-GelMA@Bio-EX hydrogels provided sustained release of MSC-EXs for 7 days. After subcutaneous transplantation, a more constant retention of MSC-EXs in Bio-GelMA@Bio-EX hydrogels was observed for up to 28 days. When placed in an artificial periodontal multitissue defect, the functionalized hydrogels exhibited an optimized therapeutic performance to regrow complex periodontal tissues, including acellular cementum, periodontal ligaments (PDLs), and alveolar bone. In this context, Bio-GelMA@Bio-EX hydrogels exerted a robust immunomodulatory effect that promoted macrophage polarization toward an M2 phenotype. Our findings demonstrate that MSC-EXs delivered with the aid of the biotin-avidin system exhibit robust macrophage-modulating and repair-promoting functions and suggest a universal approach for the development of MSC-EX-functionalized biomaterials for advanced therapies.

NADPH-dependent ROS accumulation contributes to the impaired osteogenic differentiation of periodontal ligament stem cells under high glucose conditions.

Diabetes mellitus is an established risk factor for periodontal disease that can aggravate the severity of periodontal inflammation and accelerate periodontal destruction. The chronic high glucose condition is a hallmark of diabetes-related pathogenesis, and has been demonstrated to impair the osteogenic differentiation of periodontal ligament stem cells (PDLSCs), leading to delayed recovery of periodontal defects in diabetic patients. Reactive oxygen species (ROS) are small molecules that can influence cell fate determination and the direction of cell differentiation. Although excessive accumulation of ROS has been found to be associated with high glucose-induced cell damage, the underlying mechanisms remain unclear. Nicotinamide adenine dinucleotide phosphate (NADPH) is an important electron donor and functions as a critical ROS scavenger in antioxidant systems. It has been identified as a key mediator of various biological processes, including energy metabolism and cell differentiation. However, whether NADPH is involved in the dysregulation of ROS and further compromise of PDLSC osteogenic differentiation under high glucose conditions is still not known. In the present study, we found that PDLSCs incubated under high glucose conditions showed impaired osteogenic differentiation, excessive ROS accumulation and increased NADPH production. Furthermore, after inhibiting the synthesis of NADPH, the osteogenic differentiation of PDLSCs was significantly enhanced, accompanied by reduced cellular ROS accumulation. Our findings demonstrated the crucial role of NADPH in regulating cellular osteogenic differentiation under high glucose conditions and suggested a new target for rescuing high glucose-induced cell dysfunction and promoting tissue regeneration in the future.

Long non-coding RNA AC018926.2 regulates palmitic acid exposure-compromised osteogenic potential of periodontal ligament stem cells via the ITGA2/FAK/AKT pathway.

Although obesity has been proposed as a risk factor for periodontitis, the influence of excessive fat accumulation on the development of periodontitis and periodontal recovery from disease remains largely unknown. This study investigated the cellular response of periodontal ligament stem cells (PDLSCs) to elevated levels of a specific fatty acid, namely, palmitic acid (PA). The mechanism by which PA exposure compromises the osteogenic potential of cells was also explored. It was found that exposure of PDLSCs to abundant PA led to decreased cell osteogenic differentiation. Given that long non-coding RNAs (lncRNAs) play a key role in the stem cell response to adverse environmental stimuli, we screened the lncRNAs that were differentially expressed in PDLSCs following PA exposure using lncRNA microarray analysis, and AC018926.2 was identified as the lncRNA that was most sensitive to PA. Next, gain/loss-of-function studies illustrated that AC018926.2 was an important regulator in PA-mediated osteogenic differentiation of PDLSCs. Mechanistically, AC018926.2 upregulated integrin α2 (ITGA2) expression and therefore activated ITGA2/FAK/AKT signalling. Further functional studies revealed that inactivation of ITGA2/FAK/AKT signalling by silencing ITGA2 counteracted the pro-osteogenic effect induced by AC018926.2 overexpression. Moreover, the results of bioinformatics analysis and RNA immunoprecipitation assay suggested that AC018926.2 might transcriptionally regulate ITGA2 expression by binding to PARP1 protein. Our data suggest that AC018926.2 may serve as a therapeutic target for the management of periodontitis in obese patients.

A 3D-printed molybdenum-containing scaffold exerts dual pro-osteogenic and anti-osteoclastogenic effects to facilitate alveolar bone repair.

The positive regulation of bone-forming osteoblast activity and the negative feedback regulation of osteoclastic activity are equally important in strategies to achieve successful alveolar bone regeneration. Here, a molybdenum (Mo)-containing bioactive glass ceramic scaffold with solid-strut-packed structures (Mo-scaffold) was printed, and its ability to regulate pro-osteogenic and anti-osteoclastogenic cellular responses was evaluated in vitro and in vivo. We found that extracts derived from Mo-scaffold (Mo-extracts) strongly stimulated osteogenic differentiation of bone marrow mesenchymal stem cells and inhibited differentiation of osteoclast progenitors. The identified comodulatory effect was further demonstrated to arise from Mo ions in the Mo-extract, wherein Mo ions suppressed osteoclastic differentiation by scavenging reactive oxygen species (ROS) and inhibiting mitochondrial biogenesis in osteoclasts. Consistent with the in vitro findings, the Mo-scaffold was found to significantly promote osteoblast-mediated bone formation and inhibit osteoclast-mediated bone resorption throughout the bone healing process, leading to enhanced bone regeneration. In combination with our previous finding that Mo ions participate in material-mediated immunomodulation, this study offers the new insight that Mo ions facilitate bone repair by comodulating the balance between bone formation and resorption. Our findings suggest that Mo ions are multifunctional cellular modulators that can potentially be used in biomaterial design and bone tissue engineering.