Single-cell transcriptome profiling reveals neutrophil heterogeneity in homeostasis and infection.

The full neutrophil heterogeneity and differentiation landscape remains incompletely characterized. Here, we profiled >25,000 differentiating and mature mouse neutrophils using single-cell RNA sequencing to provide a comprehensive transcriptional landscape of neutrophil maturation, function and fate decision in their steady state and during bacterial infection. Eight neutrophil populations were defined by distinct molecular signatures. The three mature peripheral blood neutrophil subsets arise from distinct maturing bone marrow neutrophil subsets. Driven by both known and uncharacterized transcription factors, neutrophils gradually acquire microbicidal capability as they traverse the transcriptional landscape, representing an evolved mechanism for fine-tuned regulation of an effective but balanced neutrophil response. Bacterial infection reprograms the genetic architecture of neutrophil populations, alters dynamic transitions between subpopulations and primes neutrophils for augmented functionality without affecting overall heterogeneity. In summary, these data establish a reference model and general framework for studying neutrophil-related disease mechanisms, biomarkers and therapeutic targets at single-cell resolution.

Nonclinical Pharmacokinetics Study of OLX702A-075-16, N-Acetylgalactosamine Conjugated Asymmetric Small Interfering RNA (GalNAc-asiRNA).

In this study, the nonclinical pharmacokinetics of OLX702A-075-16, an RNA interference (RNAi) therapeutic currently in development, were investigated. OLX702A-075-16 is a novel N-acetylgalactosamine conjugated asymmetric small-interfering RNA (GalNAc-asiRNA) used for the treatment of an undisclosed liver disease. Its unique 16/21-mer asymmetric structure reduces nonspecific off-target effects without compromising efficacy. We investigated the plasma concentration, tissue distribution, metabolism, and renal excretion of OLX702A-075-16 following a subcutaneous administration in mice and rats. For bioanalysis, high-performance liquid chromatography with fluorescence detection (HPLC-FD) was used. The results showed rapid clearance from plasma (0.5 to 1.5 h of half-life) and predominant distribution to the liver, and/or kidney. Less than 1% of the liver concentration of OLX702A-075-16 was detected in the other tissues. Metabolite profiling using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) revealed that the intact duplex OLX702A-075-16 was the major compound in plasma. The GalNAc moiety was predominantly metabolized from the sense strand in the liver, with the unconjugated sense strand of OLX702A-075-16 accounting for more than 95% of the total exposure in the rat liver. Meanwhile, the antisense strand was metabolized by the sequential loss of nucleotides from the 3'-terminus by exonuclease, with the rat liver samples yielding the most diverse truncated forms of metabolites. Urinary excretion over 96 h was less than 1% of the administered dose in rats. High plasma protein binding of OLX702A-075-16 likely inhibited its clearance through renal filtration. Significance Statement This study presents the first comprehensive characterization of the in vivo pharmacokinetics of GalNAc-asiRNA. The pharmacokinetic insights gained from this research will aid in understanding toxicology and efficacy, optimizing delivery platforms, and improving the predictive power of preclinical species data for human applications.

Myeloid cell-derived reactive oxygen species externally regulate the proliferation of myeloid progenitors in emergency granulopoiesis.

The cellular mechanisms controlling infection-induced emergency granulopoiesis are poorly defined. Here we found that reactive oxygen species (ROS) concentrations in the bone marrow (BM) were elevated during acute infection in a phagocytic NADPH oxidase-dependent manner in myeloid cells. Gr1(+) myeloid cells were uniformly distributed in the BM, and all c-kit(+) progenitor cells were adjacent to Gr1(+) myeloid cells. Inflammation-induced ROS production in the BM played a critical role in myeloid progenitor expansion during emergency granulopoiesis. ROS elicited oxidation and deactivation of phosphatase and tensin homolog (PTEN), resulting in upregulation of PtdIns(3,4,5)P3 signaling in BM myeloid progenitors. We further revealed that BM myeloid cell-produced ROS stimulated proliferation of myeloid progenitors via a paracrine mechanism. Taken together, our results establish that phagocytic NADPH oxidase-mediated ROS production by BM myeloid cells plays a critical role in mediating emergency granulopoiesis during acute infection.

Comprehensive analysis of predominant pathogenic bacteria and viruses in seafood products.

Given the growing global demand for seafood, it is imperative to conduct a comprehensive study on the prevalence and persistence patterns of pathogenic bacteria and viruses associated with specific seafood varieties. This assessment thoroughly examines the safety of seafood products, considering the diverse processing methods employed in the industry. The importance of understanding the behavior of foodborne pathogens, such as Salmonella typhimurium, Vibrio parahaemolyticus, Clostridium botulinum, Listeria monocytogenes, human norovirus, and hepatitis A virus, is emphasized by recent cases of gastroenteritis outbreaks linked to contaminated seafood. This analysis examines outbreaks linked to seafood in the United States and globally, with a particular emphasis on the health concerns posed by pathogenic bacteria and viruses to consumers. Ensuring the safety of seafood is crucial since it directly relates to consumer preferences on sustainability, food safety, provenance, and availability. The review focuses on assessing the frequency, growth, and durability of infections that arise during the processing of seafood. It utilizes next-generation sequencing to identify the bacteria responsible for these illnesses. Additionally, it analyzes methods for preventing and intervening of infections while also considering the forthcoming challenges in ensuring the microbiological safety of seafood products. This evaluation emphasizes the significance of the seafood processing industry in promptly responding to evolving consumer preferences by offering current information on seafood hazards and future consumption patterns. To ensure the continuous safety and sustainable future of seafood products, it is crucial to identify and address possible threats.

Association between toothbrushing and cardiovascular risk factors: a cross-sectional study using Korean National Health and Nutrition Examination Survey 2015-2017.

BACKGROUND: Previous studies have suggested that frequent toothbrushing is associated with a lower risk of future cardiovascular events. We sought to investigate further the relationship between toothbrushing, cardiovascular risk factors, and lifestyle behaviours. METHODS: We analysed a cross-sectional survey including 13,761 adults aged 30 years or older without a history of cardiovascular diseases from the Korean National Health and Nutritional Examination Survey. Conventional cardiovascular risk factors (blood pressure, lipid profiles, and fasting glucose), and inflammatory markers (high-sensitivity C-reactive protein [hsCRP], and white blood cell counts [WBC]) were investigated in relation to the frequency of toothbrushing. RESULTS: The estimated 10-year atherosclerotic cardiovascular disease (ASCVD) risk, calculated using the pooled cohort equations was 13.7%, 9.1%, and 7.3% for participants who reported toothbrushing 0-1, 2, and ≥ 3 times a day, respectively. Both conventional risk factors and inflammatory markers were significantly associated with frequent toothbrushing. However, after adjusting potential confounding factors such as age, sex, comorbidities, and lifestyle behaviours, only inflammatory markers were remained as significant factors. CONCLUSIONS: Oral hygiene behaviours are closely linked to cardiovascular risk factors. This study suggests that reduced systemic inflammatory burden may explain the benefit of improved oral hygiene in terms of cardiovascular risk.

Comparative effectiveness of peroneus longus tendon (PLT) autografts versus hamstring tendon (HT) autografts in anterior cruciate ligament reconstruction: a comprehensive systematic review and meta analysis.

The hamstring tendon (HT) autograft is currently the most widely utilised autograft option for anterior cruciate ligament (ACL) reconstruction. However, recent studies endorse the peroneus longus tendon (PLT) autograft as a viable alternative. To evaluate this, we systematically reviewed randomised controlled trials (RCTs) to compare the efficacy of PLT against HT autografts. Our search encompassed Cochrane, Embase, OVID, PubMed, and Scopus databases for RCTs comparing outcomes of PLT and HT autografts in ACL reconstruction. Primary outcomes included Lysholm and International Knee Documentation Committee (IKDC) scores, while secondary outcomes involved American Orthopaedic Foot and Ankle Society (AOFAS) scores, graft diameters and donor-site complications. Statistical analysis was performed using Review Manager 5.4 (Cochrane Collaboration) and heterogeneity was assessed with I2 statistics. 683 patients from 6 RCTs were included, with 338 (49.5%) patients treated with PLT autografts. Follow-up ranged from 12 to 30 months. Despite lower preoperative Lysholm scores in the PLT group, no significant differences were observed at 6 and 12 months. Although preoperative and 6-month IKDC scores were lower in the PLT group, no significant differences were found at 12 and 24 months. AOFAS scores showed no significant preoperative difference, but slightly lower scores were noted in the PLT group at 12 or 24 months. There was no significant difference in graft diameter, while donor-site complications were fewer in the PLT group. In summary, the PLT autograft is a promising and non-inferior alternative to the HT autograft, demonstrating equivalent outcomes in patient-reported knee and ankle metrics, comparable graft diameters and fewer donor-site complications.

Weissella koreensis and Pediococcus pentosaceus bacterial ghosts induce inflammatory responses as immunostimulants.

In this study, bacterial ghosts (BGs) were generated from Weissella koreensis LKS42 (WKorGs) and Pediococcus pentosacues KA94 (PPGs) by chemically inducing lysis using substances such as hydrochloric acid (HCl), sulfuric acid (H2SO4), nitric acid (HNO3), acetic acid (CH3COOH), sodium hydroxide (NaOH), potassium hydroxide (KOH), sodium carbonate (Na2CO3), n-butanol, and C6H8O7. HCl-induced WKorGs and PPGs exhibited complete removal of DNA and displayed transverse membrane dissolution tunnel structures under scanning electron microscopy (SEM). Cell viability assays showed high viability of RAW 264.7 cells exposed to HCl-induced WKorGs and PPGs. Additionally, treatment with HCl-induced WKorGs and PPGs elevated mRNA levels of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, iNOS) and the anti-inflammatory cytokine IL-10 in RAW 264.7 cells. These findings suggest that HCl-induced WKorGs and PPGs have the potential to be used as inactivated bacterial immunostimulants, highlighting their promising applications in immunization and immunotherapy.

House dust mite-induced Akt-ERK1/2-C/EBP beta pathway triggers CCL20-mediated inflammation and epithelial-mesenchymal transition for airway remodeling.

House dust mite (HDM) allergens cause inflammatory responses and chronic allergic diseases such as bronchial asthma and atopic dermatitis. Here, we investigate the mechanism by which HDM induces C-C chemokine ligand 20 (CCL20) expression to promote chronic inflammation and airway remodeling in an HDM-induced bronchial asthma mouse model. We showed that HDM increased CCL20 levels via the Akt-ERK1/2-C/EBPß pathway. To investigate the role of CCL20 in chronic airway inflammation and remodeling, we made a mouse model of CCL20-induced bronchial asthma. Treatment of anti-CCL20Ab in this mouse model showed the reduced airway hyper-responsiveness and inflammatory cell infiltration into peribronchial region by neutralizing CCL20. In addition, CCL20 induced the Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation through NLRP3 deubiquitination and transcriptional upregulation in BEAS-2B cells. As expected, anti-CCL20Ab markedly suppressed NLRP3 activation induced by CCL20. Moreover, HDM-induced CCL20 leads to epithelial-mesenchymal transition in the lung epithelium which appears to be an important regulator of airway remodeling in allergic asthma. We also found that anti-CCL20Ab attenuates airway inflammation and remodeling in an HDM-induced mouse model of bronchial asthma. Taken together, our results suggest that HDM-induced CCL20 is required for chronic inflammation that contributes airway remodeling in a mouse model of asthma.

Dentists' attitudes toward patient-centered care and its predictors: a cross-sectional study in South Korea.

BACKGROUND: Patient-centered care (PCC) has been one of medical practice's most frequently discussed principles. However, attitudes toward PCC among dentists remain underexplored. This study focuses on examining dentists' patient-centered attitudes and investigating their predictors. METHODS: The Patient-Practitioner Orientation Scale which consists of Sharing and Caring subscales was used to assess patient-centered attitudes. The statistical analysis included 217 dentists from South Korea. Hierarchical linear regression analysis was performed to examine the predictors such as sociodemographic aspects, academic factors, work-related factors, and empathy. RESULTS: A patient-centered attitude of Caring subscale (M = 4.29, SD = 0.56) emerged, but the provider-centered attitude was higher in Sharing subscale (M = 3.40, SD = 0.48). Work year, academic track, and empathy were associated significantly with an overall caring aspect of patient-centered attitude, while the gender effect remained insignificant. Empathy had a critical and significant impact on the patient-centered attitude. CONCLUSIONS: Efforts to enhance patient-centeredness in Sharing are needed; post-graduate education and transition to a more patient-centered health system are recommended. Moreover, empathy still matters as it was found to be a significant predictor of patient-centered attitudes. The findings of this study support the need for efforts to enhance patient-centered attitudes among dentists, which will help generate discussion on improving the curriculum of post-graduate education and health system reform.

Robot-Assisted Parathyroidectomy Using Indocyanine Green (ICG) Fluorescence in Primary Hyperparathyroidism.

Background and Objectives: Surgical treatment for primary hyperparathyroidism (PHPT) has evolved from bilateral exploration through a long transcervical incision to focused parathyroidectomy with a minimal incision above the pathologic gland. Recently, endoscopic or robot-assisted parathyroid surgery without direct neck incision has been introduced. The aim of this study was to investigate the effectiveness of indocyanine green (ICG) fluorescence as a new method for the visual identification of abnormal hyperfunctioning parathyroid glands in robot-assisted parathyroidectomy using FireflyTM technology. We also aimed to conduct a comparative analysis between robot-assisted parathyroidectomy and conventional focused parathyroidectomy in order to identify clinical differences between the two surgical approaches. Materials and Methods: A total of 37 patients with PHPT underwent parathyroidectomy at a single university hospital between September 2018 and December 2022. Thirty-one patients underwent open focused parathyroidectomy (open group), and six patients underwent robot-assisted parathyroidectomy (robot group). Pre-operative localization via parathyroid SPECT-CT and an intraoperative parathyroid hormone (IOPTH) assay were used to successfully remove the pathologic parathyroid in both groups. ICG was administered only in the robot group. Results: Pathologic parathyroid showed a persistent fluorescence pattern under near-infrared vision. After the removal of the fluorescent parathyroid gland, IOPTH was normalized in all six patients in the robot group. However, the open group showed shorter hospital stays (1.8 ± 1.2 vs. 3.0 ± 0.0 days, p < 0.001) and shorter operation times (91.1 ± 69.1 vs. 152.5 ± 23.6 min, p = 0.001) than the robot group. After 6 months of surgery, PTH, calcium, and ionized calcium levels were all normalized without significant differences between the groups. Conclusions: Robot-assisted parathyroidectomy using ICG is helpful for the visual identification of the pathologic parathyroid gland. The advantage of robot parathyroidectomy is a better cosmetic outcome. However, it still does not show better clinical outcomes than conventional open focused parathyroidectomy.

Pathologic angiogenesis in the bone marrow of humanized sickle cell mice is reversed by blood transfusion.

Sickle cell disease (SCD) is a monogenic red blood cell (RBC) disorder with high morbidity and mortality. Here, we report, for the first time, the impact of SCD on the bone marrow (BM) vascular niche, which is critical for hematopoiesis. In SCD mice, we find a disorganized and structurally abnormal BM vascular network of increased numbers of highly tortuous arterioles occupying the majority of the BM cavity, as well as fragmented sinusoidal vessels filled with aggregates of erythroid and myeloid cells. By in vivo imaging, sickle and control RBCs have significantly slow intravascular flow speeds in sickle cell BM but not in control BM. In sickle cell BM, we find increased reactive oxygen species production in expanded erythroblast populations and elevated levels of HIF-1α. The SCD BM exudate exhibits increased levels of proangiogenic growth factors and soluble vascular cell adhesion molecule-1. Transplantation of SCD mouse BM cells into wild-type mice recapitulates the SCD vascular phenotype. Our data provide a model of SCD BM, in which slow RBC flow and vaso-occlusions further diminish local oxygen availability in the physiologic hypoxic BM cavity. These events trigger a milieu that is conducive to aberrant vessel growth. The distorted neovascular network is completely reversed by a 6-week blood transfusion regimen targeting hemoglobin S to <30%, highlighting the plasticity of the vascular niche. A better insight into the BM microenvironments in SCD might provide opportunities to optimize approaches toward efficient and long-term hematopoietic engraftment in the context of curative therapies.

Inhibitory effects of atmospheric dielectric barrier discharge plasma against Bacillus cereus and Staphylococcus aureus on Gwamegi (semi-dried Cololabis saira).

AIMS: The current study was conducted to investigate the effects of atmospheric dielectric barrier discharge (DBD) plasma on the reduction of B. cereus and S. aureus, both potential hazardous bacteria on Gwamegi. METHODS AND RESULTS: DBD plasma devices (1.1 kV, 43 kHz, 5-60 min, N2 : 1.5 lpm for 5, 10, 20, 30 and 60 min) were used to investigate the reduction effect. In the B. cereus by DBD plasma treatment, the 5-60 min indicated a reduction of 0.2-1.2 log CFU/g. The reductions of S. aureus at the same duration time of DBD plasma were 0.1-1.1 log CFU/g. The D-values for B. cereus and S. aureus were 49.0 (R2  = 0.98) and 61.0 (R2  = 0.94) min, respectively. The pH values for 0-30 min (6.00-6.01) were not significantly different, but significant differences at 60 min (6.09). There were no significant sensorial differences in colour (6.4-5.2) and flavour (6.2-5.3), but showing significant differences in appearance (6.6-5.2), texture (6.3-5.1) and overall acceptability (6.5-5.5). CONCLUSIONS: This study indicates that the 60 min of DBD plasma treatment resulted in >1 log CFU/g of B. cereus and S. aureus without concomitant adverse changes in pH and most sensory properties in semi-dried Gwamegi. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel DBD plasma technology can be applied in semi-dried food production and distribution processes to enhance dried fishery food hygiene and safety.

A waterproof cellulose nanofibril sheet prepared by the deposition of an alkyl ketene dimer on a controlled porous structure.

This study prepared a waterproof cellulose nanofibril (CNF) sheet via the deposition of an alkyl ketene dimer (AKD) on the sheet's controlled porous structure. The porosity of the CNF sheet was controlled by drying under different conditions, which included hot-press drying (HD) and solvent-exchange drying (SD), and the effect on the hydrophobization and water-related barrier performance of the sheet were investigated. When the SD sheet was immersed in an AKD wax solution, the sheet exhibited super-hydrophobicity and a lower water vapor transmission rate, compared with the HD sheet. This indicated that the porous structure of the SD sheet enabled AKD to be adsorbed on both the surface and the inner surface and it filled in the pores of the sheet, thereby giving rise to excellent waterproofing properties. The performance of a hydrophobized SD sheet as a water barrier material was comparable to a linear low-density polyethylene film. This study confirms the possibility for AKD wax to be immersed in a porous CNF sheet and used as a potential barrier material in hydrogel packaging.

Neuroprotective Effect of Bcl-2 on Lipopolysaccharide-Induced Neuroinflammation in Cortical Neural Stem Cells.

Neuroinflammation is involved in the pathogenesis of neurodegenerative diseases due to increased levels of pro-inflammatory cytokines in the central nervous system (CNS). Chronic neuroinflammation induced by neurotoxic molecules accelerates neuronal damage. B-cell lymphoma 2 (Bcl-2) is generally accepted to be an important anti-apoptotic factor. However, the role of Bcl-2 in neuroprotection against neuroinflammation remains to be determined. The purpose of this study was to investigate the neuroprotective effect of Bcl-2 on lipopolysaccharide (LPS)-induced neuroinflammation in cortical neural stem cells (NSCs). LPS decreased mRNA and protein levels of Tuj-1, a neuron marker, and also suppressed neurite outgrowth, indicating that LPS results in inhibition of neuronal differentiation of NSCs. Furthermore, LPS treatment inhibited Bcl-2 expression during neuronal differentiation; inhibition of neuronal differentiation by LPS was rescued by Bcl-2 overexpression. LPS-induced pro-inflammatory cytokines, including interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α), were decreased by Bcl-2 overexpression. Conversely, Bcl-2 siRNA increased the LPS-induced levels of IL-6 and TNF-α, and decreased neuronal differentiation of NSCs, raising the possibility that Bcl-2 mediates neuronal differentiation by inhibiting the LPS-induced inflammatory response in NSC. These results suggest that Bcl-2 has a neuroprotective effect by inhibiting the LPS-induced inflammatory response in NSCs.

Nitration of protein phosphatase 2A increases via Epac1/PLCε/CaMKII/HDAC5/iNOS cascade in human endometrial stromal cell decidualization.

Decidualization of the endometrial stroma is an essential differentiation process for embryo implantation and maintenance of pregnancy. We previously reported that protein phosphatase 2A (PP2A) acts as a key mediator during cAMP-induced decidualization of human endometrial stromal cells (hESCs). However, the mechanism underlying its activation has remained obscure in hESCs. In the present study, we aimed to reveal the mechanism that induces the nitration of PP2A catalytic subunit (PP2Ac) during cAMP-induced decidualization of hESCs. First, cAMP-induced PP2Ac nitration was significantly repressed using L-NAME, an inhibitor of nitric oxide synthase (NOS). Among several NOS isoforms, only inducible NOS (iNOS) was highly expressed in hESCs, indicating that iNOS directly induces the nitration of PP2Ac. Second, cAMP-induced iNOS expression and PP2Ac nitration were decreased by treatment with TSA, an inhibitor of histone deacetylase 5 (HDAC5). cAMP-induced phosphorylation of CaMKII and HDAC5 was suppressed by treatment with U73122 (an inhibitor of phospholipase C) or transfection of PLCε siRNA. Finally, small G protein Rap1 and its guanine nucleotide exchange factor Epac1 were found to be involved in cAMP-induced PP2A activation. Taken together, our results suggest that PP2Ac nitration during cAMP-induced decidualization of hESCs is induced through the Epac1-Rap1-PLCε-CaMKII-HDAC5-iNOS signaling pathway.

Factors related to children's tooth-brushing at different ages: an application of the theory of planned behavior.

Tooth-brushing is one of the most important health behaviors to teach children considering potentially serious ramifications of poor dental health. However, children's tooth-brushing behavior is affected by various developmental factors. The aim of this cross-sectional study was to investigate factors related to the tooth-brushing behavior of children adopting the Theory of Planned Behavior. A preliminary elicitation study with 33 primary school students identified underlying beliefs related to tooth-brushing intentions. This data was analysed, synthesized, and incorporated into the development of survey items for three age-appropriate, closed-ended questionnaires administered to 709 primary public school students. Path analyses using structural equation modelling were used to discover the structural relationships among the direct and indirect determinants of tooth-brushing behaviors, and path estimates and the model fit were calculated. Social recognition, peer influence, self-motivating strategies, and cognitive aspects of tooth-brushing were found to become more influential in facilitating behavioral intention with age. In the path models, the tooth-brushing behavioral intention was significantly related to perceived behavioral control rather than behavioral beliefs and normative beliefs. Subjective norms were found to become more significant as children matured. This result suggests that education to improve children's tooth-brushing behavior needs be appropriate to the developmental stage of children.

Coronavirus Disease Outbreak in Call Center, South Korea.

We describe the epidemiology of a coronavirus disease (COVID-19) outbreak in a call center in South Korea. We obtained information on demographic characteristics by using standardized epidemiologic investigation forms. We performed descriptive analyses and reported the results as frequencies and proportions for categoric variables. Of 1,143 persons who were tested for COVID-19, a total of 97 (8.5%, 95% CI 7.0%-10.3%) had confirmed cases. Of these, 94 were working in an 11th-floor call center with 216 employees, translating to an attack rate of 43.5% (95% CI 36.9%-50.4%). The household secondary attack rate among symptomatic case-patients was 16.2% (95% CI 11.6%- 22.0%). Of the 97 persons with confirmed COVID-19, only 4 (1.9%) remained asymptomatic within 14 days of quarantine, and none of their household contacts acquired secondary infections. Extensive contact tracing, testing all contacts, and early quarantine blocked further transmission and might be effective for containing rapid outbreaks in crowded work settings.

Contact Tracing during Coronavirus Disease Outbreak, South Korea, 2020.

We analyzed reports for 59,073 contacts of 5,706 coronavirus disease (COVID-19) index patients reported in South Korea during January 20-March 27, 2020. Of 10,592 household contacts, 11.8% had COVID-19. Of 48,481 nonhousehold contacts, 1.9% had COVID-19. Use of personal protective measures and social distancing reduces the likelihood of transmission.

Bcl-2 Overexpression Induces Neurite Outgrowth via the Bmp4/Tbx3/NeuroD1 Cascade in H19-7 Cells.

Bcl-2 is overexpressed in the nervous system during neural development and plays an important role in modulating cell survival. In addition to its anti-apoptotic function, it has been suggested previously that Bcl-2 might act as a mediator of neuronal differentiation. However, the mechanism by which Bcl-2 might influence neurogenesis is not sufficiently understood. In this study, we aimed to determine the non-apoptotic functions of Bcl-2 during neuronal differentiation. First, we used microarrays to analyze the whole-genome expression patterns of rat neural stem cells overexpressing Bcl-2 and found that Bcl-2 overexpression induced the expression of various neurogenic genes. Moreover, Bcl-2 overexpression increased the neurite length as well as expression of Bmp4, Tbx3, and proneural basic helix-loop-helix genes, such as NeuroD1, NeuroD2, and Mash1, in H19-7 rat hippocampal precursor cells. To determine the hierarchy of these molecules, we selectively depleted Bmp4, Tbx3, and NeuroD1 in Bcl-2-overexpressing cells. Bmp4 depletion suppressed the upregulation of Tbx3 and NeuroD1 as well as neurite outgrowth, which was induced by Bcl-2 overexpression. Although Tbx3 knockdown repressed Bcl-2-mediated neurite elaboration and downregulated NeuroD1 expression, it did not affect Bcl-2-induced Bmp4 expression. While the depletion of NeuroD1 had no effect on the expression of Bcl-2, Bmp4, or Tbx3, Bcl-2-mediated neurite outgrowth was suppressed. Taken together, these results demonstrate that Bcl-2 regulates neurite outgrowth through the Bmp4/Tbx3/NeuroD1 cascade in H19-7 cells, indicating that Bcl-2 may have a direct role in neuronal development in addition to its well-known anti-apoptotic function in response to environmental insults.