Bone-targeted lipoplex-loaded three-dimensional bioprinting bilayer scaffold enhanced bone regeneration.

Clinical bone-morphogenetic protein 2 (BMP2) treatment for bone regeneration, often resulting in complications like soft tissue inflammation and ectopic ossification due to high dosages and non-specific delivery systems, necessitates research into improved biomaterials for better BMP2 stability and retention. To tackle this challenge, we introduced a groundbreaking bone-targeted, lipoplex-loaded, three-dimensional bioprinted bilayer scaffold, termed the polycaprolactone-bioink-nanoparticle (PBN) scaffold, aimed at boosting bone regeneration. We encapsulated BMP2 within the fibroin nanoparticle based lipoplex (Fibroplex) and functionalized it with DSS6 for bone tissue-specific targeting. 3D printing technology enables customized, porous PCL scaffolds for bone healing and soft tissue growth, with a two-step bioprinting process creating a cellular lattice structure and a bioink grid using gelatin-alginate hydrogel and DSS6-Fibroplex, shown to support effective nutrient exchange and cell growth at specific pore sizes. The PBN scaffold is predicted through in silico analysis to exhibit biased BMP2 release between bone and soft tissue, a finding validated by in vitro osteogenic differentiation assays. The PBN scaffold was evaluated for critical calvarial defects, focusing on sustained BMP2 delivery, prevention of soft tissue cell infiltration and controlled fiber membrane pore size in vivo. The PBN scaffold demonstrated a more than eight times longer BMP2 release time than that of the collagen sponge, promoting osteogenic differentiation and bone regeneration in a calvarial defect animal. Our findings suggest that the PBN scaffold enhanced the local concentration of BMP2 in bone defects through sustained release and improved the spatial arrangement of bone formation, thereby reducing the risk of heterotopic ossification.

Comparative analysis of different surgical approaches for recurrent inguinal hernia: a single-center observational study.

Purpose: Managing recurrent inguinal hernias is complex, and choosing the right surgical approach (laparoscopic vs. open) is vital for patient outcomes. This study compared the outcomes of using the same vs. different surgical approaches for initial and subsequent hernia repairs. Methods: We retrospectively analyzed patients who underwent recurrent inguinal hernia repair at Seoul National University Bundang Hospital between January 2014 and May 2023. Patients were divided into the "concordant" and "discordant" groups, comprising patients who underwent same and different approaches in both surgeries, respectively. Preoperative baseline characteristics, index surgery data, postoperative outcomes, and recurrence rates were analyzed and compared. Results: In total, 131 patients were enrolled; the concordant and discordant groups comprised 31 (open, n = 19; laparoscopic, n = 12) and 100 patients (open to laparoscopic, n = 68; laparoscopic to open, n = 32), respectively. No significant differences were observed in the mean operation time (50.5 ± 21.7 minutes vs. 50.2 ± 20.0 minutes, P = 0.979), complication rates (6.5% vs. 14.0%, P = 0.356), or 36-month cumulative recurrence rates (9.8% vs. 9.8%; P = 0.865). The mean postoperative hospital stay was significantly shorter in the discordant than in the concordant group (1.8 ± 0.7 vs. 1.4 ± 0.6, P = 0.003). Conclusion: Most recurrent inguinal hernia repairs were performed using the discordant surgical approach. Overall, concordance in the surgical approach did not significantly affect postoperative outcomes. Therefore, the selection of the surgical approach based on the patient's condition and surgeon's preference may be advisable.

Foci-Xpress: Automated and Fast Nuclear Foci Counting Tool.

In the nucleus, distinct, discrete spots or regions called "foci" have been identified, each harboring a specific molecular function. Accurate and efficient quantification of these foci is essential for understanding cellular dynamics and signaling pathways. In this study, we present an innovative automated image analysis method designed to precisely quantify subcellular foci within the cell nucleus. Manual foci counting methods can be tedious and time-consuming. To address these challenges, we developed an open-source software that automatically counts the number of foci from the indicated image files. We compared the foci counting efficiency, velocity, accuracy, and convenience of Foci-Xpress with those of other conventional methods in foci-induced models. We can adjust the brightness of foci to establish a threshold. The Foci-Xpress method was significantly faster than other conventional methods. Its accuracy was similar to that of conventional methods. The most significant strength of Foci-Xpress is automation, which eliminates the need for analyzing equipment while counting. This enhanced throughput facilitates comprehensive statistical analyses and supports robust conclusions from experiments. Furthermore, automation completely rules out biases caused by researchers, such as manual errors or daily variations. Thus, Foci-Xpress is a convincing, convenient, and easily accessible focus-counting tool for cell biologists.

Different oncological features of colorectal cancer codon-specific KRAS mutations: Not codon 13 but codon 12 have prognostic value.

BACKGROUND: Approximately 40% of colorectal cancer (CRC) cases are linked to Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations. KRAS mutations are associated with poor CRC prognosis, especially KRAS codon 12 mutation, which is associated with metastasis and poorer survival. However, the clinicopathological characteristics and prognosis of KRAS codon 13 mutation in CRC remain unclear. AIM: To evaluate the clinicopathological characteristics and prognostic value of codon-specific KRAS mutations, especially in codon 13. METHODS: This retrospective, single-center, observational cohort study included patients who underwent surgery for stage I-III CRC between January 2009 and December 2019. Patients with KRAS mutation status confirmed by molecular pathology reports were included. The relationships between clinicopathological characteristics and individual codon-specific KRAS mutations were analyzed. Survival data were analyzed to identify codon-specific KRAS mutations as recurrence-related factors using the Cox proportional hazards regression model. RESULTS: Among the 2203 patients, the incidence of KRAS codons 12, 13, and 61 mutations was 27.7%, 9.1%, and 1.3%, respectively. Both KARS codons 12 and 13 mutations showed a tendency to be associated with clinical characteristics, but only codon 12 was associated with pathological features, such as stage of primary tumor (T stage), lymph node involvement (N stage), vascular invasion, perineural invasion, tumor size, and microsatellite instability. KRAS codon 13 mutation showed no associations (77.2% vs 85.3%, P = 0.159), whereas codon 12 was associated with a lower 5-year recurrence-free survival rate (78.9% vs 75.5%, P = 0.025). In multivariable analysis, along with T and N stages and vascular and perineural invasion, only codon 12 (hazard ratio: 1.399; 95% confidence interval: 1.034-1.894; P = 0.030) among KRAS mutations was an independent risk factor for recurrence. CONCLUSION: This study provides evidence that KRAS codon 13 mutation is less likely to serve as a prognostic biomarker than codon 12 mutation for CRC in a large-scale cohort.

Electroconvulsive therapy and seizure: a double-edged sword?

Electroconvulsive therapy (ECT) is a widely used therapeutic option of drug-refractory psychiatric disorders. ECT treats psychiatric symptoms by inducing brief controlled seizures through electrical stimulation, but ECT does not generally cause prolonged seizures or epilepsy. However, several studies have reported cases of prolonged seizures after ECT. This review aimed to determine the mechanism of epileptogenesis with neurobiological changes after ECT. Contrary to epileptogenesis by ECT, several cases have reported that ECT was successfully applied for treatment of refractory status epilepticus. In addition, ECT might be applied to hyperkinetic movement and psychiatric symptoms of encephalitis. We also investigated the anticonvulsant mechanism of ECT and how it controls encephalitis symptoms.

Peptidylarginine deiminase 2 plays a key role in osteogenesis by enhancing RUNX2 stability through citrullination.

Peptidylarginine deiminase (PADI) 2 catalyzes the post-translational conversion of peptidyl-arginine to peptidyl-citrulline in a process called citrullination. However, the precise functions of PADI2 in bone formation and homeostasis remain unknown. In this study, our objective was to elucidate the function and regulatory mechanisms of PADI2 in bone formation employing global and osteoblast-specific Padi2 knockout mice. Our findings demonstrate that Padi2 deficiency leads to the loss of bone mass and results in a cleidocranial dysplasia (CCD) phenotype with delayed calvarial ossification and clavicular hypoplasia, due to impaired osteoblast differentiation. Mechanistically, Padi2 depletion significantly reduces RUNX2 levels, as PADI2-dependent stabilization of RUNX2 protected it from ubiquitin-proteasomal degradation. Furthermore, we discovered that PADI2 binds to RUNX2 and citrullinates it, and identified ten PADI2-induced citrullination sites on RUNX2 through high-resolution LC-MS/MS analysis. Among these ten citrullination sites, the R381 mutation in mouse RUNX2 isoform 1 considerably reduces RUNX2 levels, underscoring the critical role of citrullination at this residue in maintaining RUNX2 protein stability. In conclusion, these results indicate that PADI2 plays a distinct role in bone formation and osteoblast differentiation by safeguarding RUNX2 against proteasomal degradation. In addition, we demonstrate that the loss-of-function of PADI2 is associated with CCD, thereby providing a new target for the treatment of bone diseases.

Nicotinamide enhances osteoblast differentiation through activation of the mitochondrial antioxidant defense system.

Although the normal physiological level of oxidative stress is beneficial for maintaining bone homeostasis, imbalance between reactive oxygen species (ROS) production and antioxidant defense can cause various bone diseases. The purpose of this study was to determine whether nicotinamide (NAM), an NAD+ precursor, can support the maintenance of bone homeostasis by regulating osteoblasts. Here, we found that NAM enhances osteoblast differentiation and mitochondrial metabolism. NAM increases the expression of antioxidant enzymes, which is due to increased FOXO3A transcriptional activity via SIRT3 activation. NAM has not only a preventive effect against weak and chronic oxidative stress but also a therapeutic effect against strong and acute exposure to H2O2 in osteoblast differentiation. Collectively, the results indicate that NAM increases mitochondrial biogenesis and antioxidant enzyme expression through activation of the SIRT3-FOXO3A axis, which consequently enhances osteoblast differentiation. These results suggest that NAM could be a potential preventive or therapeutic agent for bone diseases caused by ROS.

Effects of CYP2C19 genetic polymorphisms on the pharmacokinetics of lacosamide in Korean patients with epilepsy.

OBJECTIVE: Many pharmacokinetic studies of lacosamide (LCM) have been reported, but no large-scale clinical study has been conducted on genetic polymorphisms that affect the metabolism of LCM. Therefore, we designed a pharmacogenetic study of LCM to explore the effect of genetic polymorphisms on serum LCM concentration. We evaluated the pharmacodynamic characteristics of LCM, including clinical efficacy and toxicity. METHODS: Adult patients with epilepsy who received LCM at Seoul National University Hospital were enrolled. Blood samples were obtained from 115 patients taking LCM for more than 1 month with unchanged doses and were used to analyze the serum LCM concentration, the concentration/dose (C/D) ratio and the single nucleotide polymorphisms (SNPs) of the cytochrome P450 (CYP)2C9 and CYP2C19 genes. In addition, clinical information-including efficacy, toxicity, and concomitant drugs-was collected. RESULTS: The serum LCM concentration showed a linear correlation with the daily dose (r = .66, p < .001). In genetic analysis, 43 patients (38.7%) were extensive metabolizers (EMs), 51 (45.9%) were intermediate metabolizers (IMs), and 17 (15.3%) were poor metabolizers (PMs). In the group comparison, mean serum concentrations and the C/D ratio showed significant differences between the three groups (p = .01 and p < .001, respectively). The C/D ratios of IM (27.78) and PM (35.6) were 13% and 39% higher than those of EM (25.58), respectively. In the pharmacodynamic subgroup analysis, patients in the ineffective LCM group had significantly lower serum concentrations (6.39 ± 3.25 vs. 8.44 ± 3.68 µg/ml, p = .024), whereas patients with adverse events had higher serum concentrations than those without adverse events (11.03 ± 4.32 vs. 7.4 ± 3.1 µg/ml, p < .001). Based on this, we suggest a reference range for LCM in the Korean population (6-9 µg/ml). SIGNIFICANCE: Genetic polymorphisms of the CYP2C19 gene affect the serum LCM concentration. Because efficacy and toxicity are apparently related to serum LCM levels, the genetic phenotype of CYP2C19 should be considered when prescribing LCM for patients with epilepsy.

Neuropsychiatric symptoms and seizure related with serum cytokine in epilepsy patients.

Neuroinflammation contributes to epileptogenesis and ictogenesis. Various signals of neuroinflammation lead to neuronal hyper-excitability. Since an interplay between epilepsy, psychiatric comorbidities and neuroinflammation has been suggested, we explored psychiatric symptoms in epilepsy patients, and the relationship with neuroinflammation. We screened epilepsy patients who were admitted for video-EEG monitoring between July 2019 and December 2020. Enrolled patients were asked to respond to neuropsychiatric questionnaires (Hospital Anxiety and Depression Scale (HADS) and Neuropsychiatric Inventory-Questionnaire (NPI-Q)) on admission. Serum cytokines (IL-1ß, IL-2, IL-6, IFN-γ, CCL2, and CCL5) were measured by ELISA on admission, and within 6 h after a seizure. We enrolled 134 patients, and 32 patients (23.9%) had seizures during monitoring. Cytokine levels did not change after seizures, but IL-2 and IL-6 increased in cases of generalized tonic-clonic seizures. The HADS-A score was lower in Q4 of CCL5 (p-value = 0.016) and anxiety was also less common in Q4 of CCL5 (p-value = 0.042). NPI-Q question 4 (depression) severity was higher in CCL2 (p-value = 0.024). This suggested that psychiatric symptoms may also be related to inflammatory processes in epilepsy patients. Further large, standardized studies are necessary to underpin the inflammatory mechanisms in epilepsy and psychiatric symptoms.

ROS-induced PADI2 downregulation accelerates cellular senescence via the stimulation of SASP production and NFκB activation.

Cellular senescence is closely related to tissue aging including bone. Bone homeostasis is maintained by the tight balance between bone-forming osteoblasts and bone-resorbing osteoclasts, but it undergoes deregulation with age, causing age-associated osteoporosis, a main cause of which is osteoblast dysfunction. Oxidative stress caused by the accumulation of reactive oxygen species (ROS) in bone tissues with aging can accelerate osteoblast senescence and dysfunction. However, the regulatory mechanism that controls the ROS-induced senescence of osteoblasts is poorly understood. Here, we identified Peptidyl arginine deiminase 2 (PADI2), a post-translational modifying enzyme, as a regulator of ROS-accelerated senescence of osteoblasts via RNA-sequencing and further functional validations. PADI2 downregulation by treatment with H2O2 or its siRNA promoted cellular senescence and suppressed osteoblast differentiation. CCL2, 5, and 7 known as the elements of the senescence-associated secretory phenotype (SASP) which is a secretome including proinflammatory cytokines and chemokines emitted by senescent cells and a representative feature of senescence, were upregulated by H2O2 treatment or Padi2 knockdown. Furthermore, blocking these SASP factors with neutralizing antibodies or siRNAs alleviated the senescence and dysfunction of osteoblasts induced by H2O2 treatment or Padi2 knockdown. The elevated production of these SASP factors was mediated by the activation of NFκB signaling pathway. The inhibition of NFκB using the pharmacological inhibitor or siRNA effectively relieved H2O2 treatment- or Padi2 knockdown-induced senescence and osteoblast dysfunction. Together, our study for the first time uncover the role of PADI2 in ROS-accelerated cellular senescence of osteoblasts and provide new mechanistic and therapeutic insights into excessive ROS-promoted cellular senescence and aging-related bone diseases.

A Case Report of Varicella Zoster Meningitis as Co-Infection With Breakthrough COVID-19 in an Immunocompetent Patient.

There are several previous reports that infection or reactivation of varicella zoster virus (VZV) can occur after coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Herein, we report a rare case of VZV meningitis in breakthrough COVID-19. An 18-years-old male visited the emergency room, presenting with a headache and fever of up to 38.4°C for 5 days. He received the second dose of BNT162b2 mRNA SARS-CoV-2 vaccine 7 weeks prior to symptom onset. The symptoms persisted with headache, fever, and nausea. His cerebrospinal fluid (CSF) showed an elevated opening pressure of 27 cm H2O, 6/µL red blood cells, 234/µL white blood cells (polymorphonuclear leukocytes 3%, lymphocytes 83%, and other 14%), 43.9 mg/dL protein, and 59 mg/dL glucose, and CSF polymerase chain reaction (PCR) test was positive for VZV. Also, he was diagnosed with COVID-19 by reverse transcriptase-PCR examining upper and lower respiratory tract. We administered intravenous acyclovir for 12 days, and he was discharged without any neurologic complication.

Excessive osteoclast activation by osteoblast paracrine factor RANKL is a major cause of the abnormal long bone phenotype in Apert syndrome model mice.

The fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling pathway plays important roles in the development and growth of the skeleton. Apert syndrome caused by gain-of-function mutations of FGFR2 results in aberrant phenotypes of the skull, midface, and limbs. Although short limbs are representative features in patients with Apert syndrome, the causative mechanism for this limb defect has not been elucidated. Here we quantitatively confirmed decreases in the bone length, bone mineral density, and bone thickness in the Apert syndrome model of gene knock-in Fgfr2S252W/+ (EIIA-Fgfr2S252W/+ ) mice. Interestingly, despite these bone defects, histological analysis showed that the endochondral ossification process in the mutant mice was similar to that in wild-type mice. Tartrate-resistant acid phosphatase staining revealed that trabecular bone loss in mutant mice was associated with excessive osteoclast activity despite accelerated osteogenic differentiation. We investigated the osteoblast-osteoclast interaction and found that the increase in osteoclast activity was due to an increase in the Rankl level of osteoblasts in mutant mice and not enhanced osteoclastogenesis driven by the activation of FGFR2 signaling in bone marrow-derived macrophages. Consistently, Col1a1-Fgfr2S252W/+ mice, which had osteoblast-specific expression of Fgfr2 S252W, showed significant bone loss with a reduction of the bone length and excessive activity of osteoclasts was observed in the mutant mice. Taken together, the present study demonstrates that the imbalance in osteoblast and osteoclast coupling by abnormally increased Rankl expression in Fgfr2S252W/+ mutant osteoblasts is a major causative mechanism for bone loss and short long bones in Fgfr2S252W/+ mice.

Safety and efficacy of dietary supplement (gintonin-enriched fraction from ginseng) in subjective memory impairment: A randomized placebo-controlled trial.

BACKGROUND: Gintonin inhibits ß-amyloid production, increases acetylcholine level in the brain, and promotes neurogenesis. We evaluated the efficacy of gintonin-enriched fraction (GEF) in improving the cognitive performance in subjective memory impairment. METHODS: In this 8-week, randomized, assessor and participant blinded, placebo-controlled study, participants with subjective memory impairment but preserved cognitive function (Korean Mini-Mental State Examination [K-MMSE] score ≥23) were assigned to GEF 300mg/day or placebo. K-MMSE, Korean versions of the Alzheimer's disease assessment scale, color-word stroop test (K-CWST), clinical dementia rating, and Beck depression inventory-II were evaluated along with the safety profiles. The primary outcome was set as the change in the K-MMSE. RESULTS: Seventy-six participants complete the study protocol. After 8 weeks, there was no inter-group difference in the primary or secondary outcome score changes. However, GEF group showed an improvement in the K-MMSE scores (P= 0.026), and in the number of correct answers in both word reading (P= 0.008) and color reading (P= 0.005) of K-CWST, although only the improvement in the K-CWST scores were higher than the minimum clinically important difference. The frequency of adverse events was comparable between the groups and all were of mild severity. CONCLUSION: GEF is safe but might not be effective in treating subjective memory impairment within the current study setting. However, GEF showed a trend of improving the global cognition and the frontal executive function. Further large-sized studies with longer follow-up period are warranted. CLINICAL TRIAL REGISTRATION: This clinical trial was registered at Clinical Research Information Service of Korea Centers for Disease Control and Prevention: KCT0004636.

Alerting network alteration in isolated rapid eye movement sleep behavior disorder patients with mild cognitive impairment.

OBJECTIVE: Mild cognitive impairment (MCI) was found in 30-50% of the isolated REM sleep behavior disorder (iRBD) patients. Furthermore, it is known that patients with Parkinson's disease have attention network defects. Given that iRBD is known to be the prodromal disease of α-synucleinopathies, our aim was to investigate whether there are attention network dysfunctions in iRBD patients following the presence of MCI. METHODS: 14 healthy controls, 48 iRBD patients, 24 with MCI and 24 without MCI, were included in this study. Attention network task (ANT) was used to assess alerting, orienting, and executive control networks. Event-related potentials (ERPs) and behavioral performances were recorded during the ANT. Parietal N1 and P3 components were analyzed to find effects of the three attention networks. RESULTS: IRBD patients without MCI showed neuropsychological, behavioral, and ERP results similar to those of healthy controls. On the other hand, iRBD patients with MCI showed a general decline in cognitive domains with no alerting effect (controls, p = 0.043; iRBD-noMCI, p = 0.014; iRBD-MCI, p = 0.130) while preserving orienting and executive control effect. Furthermore, iRBD patients with MCI had impairments in executive function and verbal memory domains, compared to iRBD patients without MCI. CONCLUSIONS: Our findings indicate that when cognition is reduced to MCI levels in iRBD patients, the attention network, especially the alerting component, is impaired. The attention network and cognition, on the other hand, can be preserved in iRBD patients due to the compensatory mechanism.

Probing TGF-ß1-induced cytoskeletal rearrangement by fluorescent-labeled silica nanoparticle uptake assay.

Cytoskeletal proteins are essential in maintaining cell morphology, proliferation, and viability as well as internalizing molecules in phagocytic and non-phagocytic cells. Orderly aligned cytoskeletons are disturbed by a range of biological processes, such as the epithelial-mesenchymal transition, which is observed in cancer metastasis. Although many biological methods have been developed to detect cytoskeletal rearrangement, simple and quantitative in vitro approaches are still in great demand. Herein, we applied a flow cytometry-based nanoparticle uptake assay to measure the degree of cytoskeletal rearrangement induced by transforming growth factor ß1 (TGF-ß1). For the assay, silica nanoparticles, selected for their high biocompatibility, were fluorescent-labeled to facilitate quantification with flow cytometry. Human keratinocyte HaCaT cells were treated with different concentrations of TGF-ß1 and then exposed to FITC-labeled silica nanoparticles. Increasing concentrations of TGF-ß1 induced gradual changes in cytoskeletal rearrangement, as confirmed by conventional assays. The level of nanoparticle uptake increased by TGF-ß1 treatment in a dose-dependent manner, indicating that our nanoparticle uptake assay can be used as a quick and non-destructive approach to measure cytoskeletal rearrangement.