Nitrogen-doped carbon quantum dot regulates cell proliferation and differentiation by endoplasmic reticulum stress.

Quantum dots have diverse biomedical applications, from constructing biological infrastructures like medical imaging to advancing pharmaceutical research. However, concerns about human health arise due to the toxic potential of quantum dots based on heavy metals. Therefore, research on quantum dots has predominantly focused on oxidative stress, cell death, and other broader bodily toxicities. This study investigated the toxicity and cellular responses of mouse embryonic stem cells (mESCs) and mouse adult stem cells (mASCs) to nitrogen-doped carbon quantum dots (NCQDs) made of non-metallic materials. Cells were exposed to NCQDs, and we utilized a fluorescent ubiquitination-based cell system to verify whether NCQDs induce cytotoxicity. Furthermore, we validated the differentiation-inducing impact of NCQDs by utilizing embryonic stem cells equipped with the Oct4 enhancer-GFP reporter system. By analyzing gene expression including Crebzf, Chop, and ATF6, we also observed that NCQDs robustly elicited endoplasmic reticulum (ER) stress. We confirmed that NCQDs induced cytotoxicity and abnormal differentiation. Interestingly, we also confirmed that low concentrations of NCQDs stimulated cell proliferation in both mESCs and mASCs. In conclusion, NCQDs modulate cell death, proliferation, and differentiation in a concentration-dependent manner. Indiscriminate biological applications of NCQDs have the potential to cause cancer development by affecting normal cell division or to fail to induce normal differentiation by affecting embryonic development during pregnancy. Therefore, we propose that future biomedical applications of NCQDs necessitate comprehensive and diverse biological studies.

Rotator Cuff Tear Size: Could It Be Influenced by the Presence of One or More Diseases Capable of Altering the Peripheral Microcirculation?

Background: To date, it is not well known which systemic pathologies most frequently afflict patients with rotator cuff tear (RCT) and whether the coexistence of two or more pathologies can affect the lesion size. Therefore, we analyzed our database relative to a large group of patients who recently underwent rotator cuff repair. Methods: A total of 527 patients with full-thickness RCT were enrolled. For each patient, we checked the presence of at least one of diabetes, venous system diseases, cardiovascular diseases, hypercholesterolemia, blood hypertension, thyroid diseases, and a smoking habit. Patients were subdivided according to risk factors into five groups, representing those who had zero, one, two, three, and four or more risk factors, respectively. Statistical analysis was performed. Results: In total, 37% of our patients had no risk factors; 28% had one risk factor (arterial hypertension, smoking habit, and hypercholesterolemia were the most frequent); 23% had two risk factors (the hypertension/hypercholesterolemia association was the most frequent); and 8% suffered from three pathologies (the diabetes/arterial hypertension/hypercholesterolemia association was the most frequent). Comparing the cuff tear severity in patients without and with at least one risk factor, we observed that tear size increased in those with at least one risk factor. Conclusions: A total of 63% of patients with rotator cuff tears were either smokers and/or had at least one pathology capable of altering the peripheral microcirculation. Hypertension and hypercholesterolemia were the most frequent. Tear severity significantly increased with the presence of at least one risk factor.

Nursing care services to address unmet supportive care needs among cancer survivors: a systematic review.

BACKGROUND: The increasing population of cancer survivors poses a significant challenge for healthcare systems globally, necessitating comprehensive post-treatment care to address diverse physical, psychological, and social needs. OBJECTIVE: This systematic review aims to synthesize and critically evaluate the current evidence concerning the unmet needs for nursing services among cancer survivors, spanning various dimensions of survivorship care. METHODS: A systematic search was conducted across major databases, including PubMed, CINAHL, and PsycINFO, to identify relevant studies investigating the unmet needs and health-related quality-of-life (HRQOL) of nursing services led by nurses among cancer survivors. The final search update was conducted in June 2024. Unmet needs dimensions were categorized by the biopsychosocial-spiritual framework. RESULTS: Of the 9503 records searched, 18 studies were included. This review revealed mixed findings in the domains of unmet needs and interventions aimed at addressing them. While nurse-led interventions showed promise in addressing physical and daily living needs, outcomes related to psychological and emotional needs varied across studies. Additionally, nurse-led interventions were effective in addressing patient-clinician communication and health system/information needs, although statistical significance was not consistently observed. HRQOL assessments using general and cancer-specific measures yielded mixed findings. CONCLUSIONS: Despite limitations of the risk of bias of included studies and weak study designs for evaluating nurse-led intervention effects for cancer survivors, the findings highlight the potential of nursing practice to significantly contribute to improving unmet needs of physical, psychological, and social perspectives and ultimately improving their HRQOL. However, the impact on the spiritual needs of nursing care services is limited by the low number of studies. IMPLICATIONS FOR CANCER SURVIVORS: By providing comprehensive support and management, nursing practice can enhance post-treatment outcomes and HRQOL for cancer survivors, contributing to more patient-centered and effective care delivery. More rigorous research considering a biopsychosocial-spiritual perspective to help cancer survivors improve HRQOL is needed.

Role of NOX1 and NOX5 in protein kinase C/reactive oxygen species­mediated MMP­9 activation and invasion in MCF­7 breast cancer cells.

NADPH oxidases (NOXs) are a family of membrane proteins responsible for intracellular reactive oxygen species (ROS) generation by facilitating electron transfer across biological membranes. Despite the established activation of NOXs by protein kinase C (PKC), the precise mechanism through which PKC triggers NOX activation during breast cancer invasion remains unclear. The present study aimed to investigate the role of NOX1 and NOX5 in the invasion of MCF­7 human breast cancer cells. The expression and activity of NOXs and matrix metalloprotease (MMP)­9 were assessed by reverse transcription­quantitative PCR and western blotting, and the activity of MMP­9 was monitored using zymography. Cellular invasion was assessed using the Matrigel invasion assay, whereas ROS levels were quantified using a FACSCalibur flow cytometer. The findings suggested that NOX1 and NOX5 serve crucial roles in 12­O­tetradecanoylphorbol­13­acetate (TPA)­induced MMP­9 expression and invasion of MCF­7 cells. Furthermore, a connection was established between PKC and the NOX1 and 5/ROS signaling pathways in mediating TPA­induced MMP­9 expression and cellular invasion. Notably, NOX inhibitors (diphenyleneiodonium chloride and apocynin) significantly attenuated TPA­induced MMP­9 expression and invasion in MCF­7 cells. NOX1­ and NOX5­specific small interfering RNAs attenuated TPA­induced MMP­9 expression and cellular invasion. In addition, knockdown of NOX1 and NOX5 suppressed TPA­induced ROS levels. Furthermore, a PKC inhibitor (GF109203X) suppressed TPA­induced intracellular ROS levels, MMP­9 expression and NOX activity in MCF­7 cells. Therefore, NOX1 and NOX5 may serve crucial roles in TPA­induced MMP­9 expression and invasion of MCF­7 breast cancer cells. Furthermore, the present study indicated that TPA­induced MMP­9 expression and cellular invasion were mediated through PKC, thus linking the NOX1 and 5/ROS signaling pathways. These findings offer novel insights into the potential mechanisms underlying their anti­invasive effects in breast cancer.

Subconjunctival aflibercept inhibits corneal angiogenesis and VEGFR-3+CD11b+ cells.

PURPOSE: This study aimed to investigate the effects of subconjunctival injection of aflibercept, a soluble protein decoy for VEGFR-1 and VEGFR-2, on corneal angiogenesis and VEGFR-expressing CD11b+ cells in a mouse model of suture-induced corneal neovascularization. METHODS: Corneal neovascularization was induced in BALB/c mice by placing three sutures on the cornea. Immediately after surgery, either 200 µg aflibercept (5 µL) or an equal volume of phosphate-buffered saline (PBS) was administered into the subconjunctival space. Seven days after later, corneal new vessels were quantified through clinical examination and measurement of the CD31-stained area in corneal flat mounts. The levels of pro-angiogenic and inflammatory markers in the cornea were evaluated using RT-qPCR. The percentages of VEGFR-2+CD11b+ cells and VEGFR-3+CD11b+ cells were analyzed in the cornea, blood, and draining cervical lymph nodes (DLNs) using flow cytometry. RESULTS: Subconjunctival injection of aflibercept significantly reduced the growth of corneal new vessels compared to subconjunctival PBS injection. The mRNA levels of Cd31, vascular growth factors (Vegfc and Angpt1), and pro-angiogenic/inflammatory markers (Tek/Tie2, Mrc1, Mrc2, and Il6) in the cornea were downregulated by subconjunctival aflibercept. Also, the percentage of VEGFR-3+CD11b+ cells in the cornea, blood, and DLNs was decreased by aflibercept, whereas that of VEGFR-2+CD11b+ cells was unaffected. CONCLUSION: Subconjunctival aflibercept administration inhibits inflammatory angiogenesis in the cornea and reduces the numbers of cornea-infiltrating and circulating VEGFR-3+CD11b+ cells.

Area Socioeconomic Status is Associated with Refusal of Recommended Surgery in Patients with Metastatic Bone and Joint Disease.

BACKGROUND: This study sought to identify associations between the Yost Index, a geocoded area neighborhood socioeconomic status (nSES) score, and race/ethnicity with patient refusal of recommended surgery for metastatic bone disease. METHODS: Patients with metastatic bone disease were extracted from the Surveillance, Epidemiology, and End Results database. The Yost Index was geocoded using factor analysis and categorized into quintiles using census tract-level American Community Service (ACS) 5-year estimates and seven nSES measures. Multivariable logistic regression models calculated odds ratios (ORs) of refusal of recommended surgery and 95% confidence intervals (CIs), adjusting for clinical covariates. RESULTS: A total of 138,257 patients were included, of which 14,943 (10.8%) were recommended for surgical resection. Patients in the lowest nSES quintile had 57% higher odds of refusing surgical treatment than those in the highest quintile (aOR = 1.57, 95% CI 1.30-1.91, p < 0.001). Patients in the lowest nSES quintile also had a 31.2% higher age-adjusted incidence rate of not being recommended for surgery compared with those in the highest quintile (186.4 vs. 142.1 per 1 million, p < 0.001). Black patients had 34% higher odds of refusing treatment compared with White patients (aOR = 1.34, 95% CI 1.14-1.58, p = 0.003). Advanced age, unmarried status, and patients with aggressive cancer subtypes were associated with higher odds of refusing surgery (p < 0.001). CONCLUSIONS: nSES and race/ethnicity are independent predictors of a patient refusing surgery for metastatic cancer to bone, even after adjusting for various clinical covariates. Effective strategies for addressing these inequalities and improving the access and quality of care of patients with a lower nSES and minority backgrounds are needed.

Radiation dose estimation with multiple artificial neural networks in dicentric chromosome assay.

PURPOSE: The dicentric chromosome assay (DCA), often referred to as the 'gold standard' in radiation dose estimation, exhibits significant challenges as a consequence of its labor-intensive nature and dependency on expert knowledge. Existing automated technologies face limitations in accurately identifying dicentric chromosomes (DCs), resulting in decreased precision for radiation dose estimation. Furthermore, in the process of identifying DCs through automatic or semi-automatic methods, the resulting distribution could demonstrate under-dispersion or over-dispersion, which results in significant deviations from the Poisson distribution. In response to these issues, we developed an algorithm that employs deep learning to automatically identify chromosomes and perform fully automatic and accurate estimation of diverse radiation doses, adhering to a Poisson distribution. MATERIALS AND METHODS: The dataset utilized for the dose estimation algorithm was generated from 30 healthy donors, with samples created across seven doses, ranging from 0 to 4 Gy. The procedure encompasses several steps: extracting images for dose estimation, counting chromosomes, and detecting DC and fragments. To accomplish these tasks, we utilize a diverse array of artificial neural networks (ANNs). The identification of DCs was accomplished using a detection mechanism that integrates both deep learning-based object detection and classification methods. Based on these detection results, dose-response curves were constructed. A dose estimation was carried out by combining a regression-based ANN with the Monte-Carlo method. RESULTS: In the process of extracting images for dose analysis and identifying DCs, an under-dispersion tendency was observed. To rectify the discrepancy, classification ANN was employed to identify the results of DC detection. This approach led to satisfaction of Poisson distribution criteria by 32 out of the initial pool of 35 data points. In the subsequent stage, dose-response curves were constructed using data from 25 donors. Data provided by the remaining five donors served in performing dose estimations, which were subsequently calibrated by incorporating a regression-based ANN. Of the 23 points, 22 fell within their respective confidence intervals at p < .05 (95%), except for those associated with doses at levels below 0.5 Gy, where accurate calculation was obstructed by numerical issues. The accuracy of dose estimation has been improved for all radiation levels, with the exception of 1 Gy. CONCLUSIONS: This study successfully demonstrates a high-precision dose estimation method across a general range up to 4 Gy through fully automated detection of DCs, adhering strictly to Poisson distribution. Incorporating multiple ANNs confirms the ability to perform fully automated radiation dose estimation. This approach is particularly advantageous in scenarios such as large-scale radiological incidents, improving operational efficiency and speeding up procedures while maintaining consistency in assessments. Moreover, it reduces potential human error and enhances the reliability of results.

Does Epidural Corticosteroid Application During Spinal Surgery Reduce Postoperative Pain?: An Adjunct to Multimodal Analgesia.

STUDY DESIGN: A prospective, randomized, placebo-controlled, double-blinded study. OBJECTIVE: To examine the effect of intraoperative epidural administration of Depo-Medrol on postoperative back pain and radiculitis symptoms in patients undergoing Transforaminal Lumbar Interbody Fusion (TLIF). SUMMARY OF BACKGROUND DATA: Postoperative pain is commonly experienced by patients undergoing spinal fusion surgery. Adequate management of intense pain is necessary to encourage early ambulation, increase patient satisfaction, and limit opioid consumption. Intraoperative steroid application has been shown to improve postoperative pain in patients undergoing lumbar decompression surgeries. There have been no studies examining the effect of epidural steroids on both back pain and radicular pain in patients undergoing TLIF. METHOD: In all, 151 patients underwent TLIF surgery using rh-BMP2 with 3 surgeons at a single institution. Of those, 116 remained in the study and were included in the final analysis. Based on a 1:1 randomization, a collagen sponge saturated with either Saline (1 cc) or Depo-Medrol (40 mg/1 cc) was placed at the annulotomy site on the TLIF level. Follow-up occurred on postoperative days 1, 2, 3, 7, and postoperative months 1, 2, and 3. Lumbar radiculopathy was measured by a modified symptom- and laterality-specific Visual Analog Scale (VAS) regarding the severity of back pain and common radiculopathy symptoms. RESULTS: The patients who received Depo-Medrol, compared with those who received saline, experienced significantly less back pain on postoperative days 1, 2, 3, and 7 ( P <0.05). There was no significant difference in back pain beyond day 7. Radiculopathy-related symptoms such as leg pain, numbness, tingling, stiffness, and weakness tended to be reduced in the steroid group at most time points. CONCLUSION: This study provides Level 1 evidence that intraoperative application of Depo-Medrol during a TLIF surgery with rh-BMP2 significantly reduces back pain for the first week after TLIF surgery. The use of epidural Depo-Medrol may be a useful adjunct to multimodal analgesia for pain relief in the postoperative period.

Effect of Porcine-Derived Absorbable Patch-Type Atelocollagen for Arthroscopic Rotator Cuff Repair: A Prospective Randomized Controlled Trial.

BACKGROUND: Even though arthroscopic rotator cuff repair is recognized as a standard treatment option, the risk of postoperative retear is a major concern. PURPOSE: To evaluate the effect of porcine-derived absorbable patch-type atelocollagen during arthroscopic rotator cuff repair. STUDY DESIGN: Randomized controlled trial; Level of evidence, 1. METHODS: A total of 64 patients with rotator cuff tears diagnosed on magnetic resonance imaging (MRI) were enrolled prospectively from November 2020 to December 2021. Both groups had repairs using the suture bridge technique. For the atelocollagen group, before securing the lateral anchors, we inserted porcine-derived absorbable patch-type atelocollagen between the footprint and the tendon. On postoperative day 2, the patients underwent MRI to confirm containment of the patch-type atelocollagen. At 6 months and 1 year postoperatively, the signal intensity of the repaired tendon was assessed using MRI. Patients were evaluated using the Constant score as the primary outcome, along with the visual analog scale for pain; range of motion; American Shoulder and Elbow Surgeons score; University of California, Los Angeles, score; and Korean Shoulder Score preoperatively and at 2, 3, 6, and 12 months postoperatively. RESULTS: No significant changes in the Constant score as primary outcome, pain or other functional scores, and range of motion were observed between the groups at 1 year postoperatively. The patch-type atelocollagen was confirmed to be contained by the time-zero MRI scan taken 2 days postoperatively. Among the 55 patients included in final analysis, 12 retear cases were recorded (21.8% retear rate). A significantly lower retear rate was found in the atelocollagen group, as 3 cases were observed in this group (10.3%) and 9 cases were observed in the conventional repair group (34.6%) (P = .048). CONCLUSION: The Constant score was not different between the groups. The retear rate after rotator cuff repair was significantly lower in the group that received porcine-derived absorbable patch-type atelocollagen compared with in the conventional group. REGISTRATION: KCT0005184 (Clinical Research Information Service [CRIS]; https://cris.nih.go.kr).

SIRT1 ISGylation accelerates tumor progression by unleashing SIRT1 from the inactive state to promote its deacetylase activity.

ISG15 is an interferon-stimulated ubiquitin-like protein (UBL) with multifaceted roles as a posttranslational modifier in ISG15 conjugation (ISGylation). However, the mechanistic consequences of ISGylation in cancer have not been fully elucidated, largely due to a lack of knowledge on the ISG15 target repertoire. Here, we identified SIRT1, a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase, as a new target for ISGylation. SIRT1 ISGylation impairs the association of SIRT1 with its negative regulator, deleted in breast cancer 1 (DBC1), which unleashes SIRT1 from its inactive state and leads to an increase in its deacetylase activity. Importantly, SIRT1 ISGylation promoted lung cancer progression and limited lung cancer cell sensitivity to DNA damage-based therapeutics in vivo and in vitro models. The levels of ISG15 mRNA and protein were significantly higher in lung cancer tissues than in adjacent normal tissues. Accordingly, elevated expression of SIRT1 and ISG15 was associated with poor prognosis in lung cancer patients, a finding that could be translated for lung cancer patient stratification and disease outcome evaluation. Taken together, our findings provide a mechanistic understanding of the regulatory effect of SIRT1 ISGylation on tumor progression and therapeutic efficacy in lung cancer.

Analytical Performance Evaluation of a Digital Real-Time PCR for Quantifying Major BCR::ABL1 Transcripts.

BACKGROUND: Accurate quantification of the BCR::ABL1 transcripts is essential for measurable residual disease (MRD) monitoring in chronic myeloid leukemia (CML) after tyrosine kinase inhibitor (TKI) treatment. This study evaluated the newly developed digital real-time PCR method, Dr. PCR, as an alternative reverse transcription-PCR (qRT-PCR) for MRD detection. METHODS: The performance of Dr. PCR was assessed using reference and clinical materials. Precision, linearity, and correlation with qRT-PCR were evaluated. MRD levels detected by Dr. PCR were compared with qRT-PCR, and practical advantages were investigated. RESULTS: Dr. PCR detected MRD up to 0.0032%IS (MR4.5) with excellent precision and linearity and showed a strong correlation with qRT-PCR results. Notably, Dr. PCR identified higher levels of MRD in 12.7% (29/229) of patients than qRT-PCR, including six cases of MR4, which is a critical level for TKI discontinuation. Dr. PCR also allowed for sufficient ABL1 copies in all cases, while qRT-PCR necessitated multiple repeat tests in 3.5% (8/229) of cases. CONCLUSION: Our study provides a body of evidence supporting the clinical application of Dr. PCR as a rapid and efficient method for assessing MRD in patients with CML under the current treatment regimen.

Association of Socioeconomic Status With Worse Overall Survival in Patients With Bone and Joint Cancer.

BACKGROUND: The effect of socioeconomic status (SES) on the outcomes of patients with metastatic cancer to bone has not been adequately studied. We analyzed the association between the Yost Index, a composite geocoded SES score, and overall survival among patients who underwent nonprimary surgical resection for bone metastases. METHODS: This population-based study used data from the National Cancer Institute's Surveillance, Epidemiology, and End Results database (2010 to 2018). We categorized bone and joint sites using International Classification of Disease-O-3 recodes. The Yost Index was geocoded using a factor analysis and categorized into quintiles using census tract-level American Community Service 5-year estimates and seven measures: median household income, median house value, median rent, percent below 150% of the poverty line, education index, percent working class, and percent unemployed. Multivariate Cox regression models were used to calculate adjusted hazard ratios of overall survival and 95% confidence intervals. RESULTS: A total of 138,158 patients were included. Patients with the lowest SES had 34% higher risk of mortality compared with those with the highest SES (adjusted hazard ratio of 1.34, 95% confidence interval: 1.32 to 1.37, P < 0.001). Among patients who underwent nonprimary surgery of the distant bone tumor (n = 11,984), the age-adjusted mortality rate was 31.3% higher in the lowest SES patients compared with the highest SES patients (9.9 versus 6.8 per 100,000, P < 0.001). Patients in the lowest SES group showed more racial heterogeneity (63.0% White, 33.5% Black, 3.1% AAPI) compared with the highest SES group (83.9% White, 4.0% Black, 11.8% AAPI, P < 0.001). Higher SES patients are more likely to be married (77.5% versus 59.0%, P < 0.0001) and to live in metropolitan areas (99.6% versus 73.6%, P < 0.0001) compared with lower SES patients. DISCUSSION: Our results may have implications for developing interventions to improve access and quality of care for patients from lower SES backgrounds, ultimately reducing disparities in orthopaedic surgery.

Initial experience with the enhanced recovery after surgery (ERAS) protocols in gynecologic surgery at an urban academic tertiary medical center.

Background: The enhanced recovery after surgery (ERAS) protocols have been consistently associated with improved patient experience and surgical outcomes. Despite the release of ERAS Society guidelines specific to gynecologic oncology, the adoption of ERAS in gynecology on global level has been disappointingly low and some centers have shown minimal improvement in clinical outcomes after adopting ERAS. The aim of this study is to describe the development and early experience of ERAS protocols in gynecologic surgery at an urban academic tertiary medical center. Methods: This was an observational prospective cohort study. The target patient population included those with low comorbidities who were scheduled to undergo various types of gynecologic surgeries for both benign and malignant diseases between October 2020 and February 2021. Two attending surgeons implemented the protocols for their patients (ERAS cohort) while three attending surgeons maintained the conventional perioperative care for their patients (non-ERAS cohort). Baseline characteristics, surgical outcomes and patients' answers to a 12-question survey were compared. A case-matched comparative analysis was also performed between the ERAS cohort and the historical non-ERAS cohort (those who received the same types of surgical procedures from the two ERAS attending surgeons prior to the implementation of the protocols). Results: A total of 244 patients were evaluated (122 in the ERAS cohort vs. 122 in the non-ERAS cohort). The number of vials of opioid analgesia used during the first two postoperative days was significantly lower whereas the use of nonsteroidal anti-inflammatory drugs (NSAIDs) and acetaminophen was more frequent in the ERAS cohort group. The patients in the ERAS group reported less postoperative pain, feelings of hunger and thirst, and greater amount of exercise postoperatively. These benefits of the ERAS cohort were more pronounced in the patients who underwent laparotomic surgeries than those who underwent laparoscopic surgeries. The case-matched comparative analysis also showed similar results. The length of hospital stay did not differ between those who underwent the ERAS protocols and those who did not. Conclusions: The results of the study demonstrated the safety, clinical feasibility and benefits of the ERAS protocols for patients undergoing gynecologic surgeries for both benign and malignant indications.

Flipped C-Terminal Ends of APOA1 Promote ABCA1-Dependent Cholesterol Efflux by Small HDLs.

BACKGROUND: Cholesterol efflux capacity (CEC) predicts cardiovascular disease independently of high-density lipoprotein (HDL) cholesterol levels. Isolated small HDL particles are potent promoters of macrophage CEC by the ABCA1 (ATP-binding cassette transporter A1) pathway, but the underlying mechanisms are unclear. METHODS: We used model system studies of reconstituted HDL and plasma from control and lecithin-cholesterol acyltransferase (LCAT)-deficient subjects to investigate the relationships among the sizes of HDL particles, the structure of APOA1 (apolipoprotein A1) in the different particles, and the CECs of plasma and isolated HDLs. RESULTS: We quantified macrophage and ABCA1 CEC of 4 distinct sizes of reconstituted HDL. CEC increased as particle size decreased. Tandem mass spectrometric analysis of chemically cross-linked peptides and molecular dynamics simulations of APOA1, the major protein of HDL, indicated that the mobility of C-terminus of that protein was markedly higher and flipped off the surface in the smallest particles. To explore the physiological relevance of the model system studies, we isolated HDL from LCAT-deficient subjects, whose small HDLs (like reconstituted HDLs) are discoidal and composed of APOA1, cholesterol, and phospholipid. Despite their very low plasma levels of HDL particles, these subjects had normal CEC. In both the LCAT-deficient subjects and control subjects, the CEC of isolated extra-small HDL (a mixture of extra-small and small HDL by calibrated ion mobility analysis) was 3- to 5-fold greater than that of the larger sizes of isolated HDL. Incubating LCAT-deficient plasma and control plasma with human LCAT converted extra-small and small HDL particles into larger particles, and it markedly inhibited CEC. CONCLUSIONS: We present a mechanism for the enhanced CEC of small HDLs. In smaller particles, the C-termini of the 2 antiparallel molecules of APOA1 are "flipped" off the lipid surface of HDL. This extended conformation allows them to engage with ABCA1. In contrast, the C-termini of larger HDLs are unable to interact productively with ABCA1 because they form a helical bundle that strongly adheres to the lipid on the particle. Enhanced CEC, as seen with the smaller particles, predicts decreased cardiovascular disease risk. Thus, extra-small and small HDLs may be key mediators and indicators of the cardioprotective effects of HDL.

Comparison of three different lactic acid bacteria-fermented proteins on RAW 264.7 osteoclast and MC3T3-E1 osteoblast differentiation.

Osteoporosis is a state of bone weakening caused by an imbalance in osteoblast and osteoclast activity. In this study, the anti-osteoporotic effects of three proteins fermented by lactic acid bacteria (LAB) were assessed. Commercial proteins sodium caseinate (SC), whey protein isolate (WPI), and soy protein isolate (SPI) were fermented by LAB strains for 48 h. The fermented products (F-SC, F-WPI, and F-SPI, respectively) were used in an in vitro osteoclast and osteoblast-like cell model to assess their effects on bone health. Despite no difference in the results of TRAP staining of RANKL-induced osteoclastogenesis, F-WPI and F-SPI were effective in normalizing the altered gene expression of osteoclastogenesis markers such as TRAP, Nfatc1, RANK, and ATP6v0d. F-SPI was also effective in modulating osteoblasts by enhancing the expression of the osteoblastogenesis markers T1Col, Col2a, and OSX to levels higher than those in the SPI group, indicating that protein characteristics could be enhanced through bacterial fermentation. Moreover, these boosted effects of F-SPI may be involved with isoflavone-related metabolism during LAB-fermentation of SPI. These results demonstrate the potential of LAB-fermented proteins as dietary supplements to prevent bone loss. However, further understanding of its effects on balancing osteoblasts and osteoclasts and the underlying mechanisms is needed.

Plate prebending using a three-dimensional-printed model affords effective anatomical reduction in clavicular shaft fractures.

BACKGROUND: A precontoured plate rarely fits properly within the patient's clavicle and must be bent intraoperatively. This study aimed to determine whether anatomical reduction could be achieved using a plate bent before surgery. METHODS: This study included 87 consecutive patients with displaced mid-shaft clavicle fractures who underwent plate fixation and were followed-up for a minimum of 1 year. After exclusions, 39 consecutive patients underwent fixation with a precontoured plate bent intraoperatively (intraoperative bending group), and 28 underwent fixation with the plate bent preoperatively (preoperative bending group). Using free software and a three-dimensional (3D) printer, ipsilateral clavicle 3D-printed models were constructed. Using plain radiographs, the distance between the edge of the lateral inferior cortex and the medial inferior cortex was measured. The angle between the line connecting the inferior cortex edge and the line passing through the flat portion of the superior cortex of the distal clavicle was measured. RESULTS: Mean length differences between the ipsilateral and contralateral clavicle were smaller on both anteroposterior (AP; P=0.032) and axial images (P=0.029) in the preoperative bending group. The mean angular differences on both AP (P=0.045) and axial images (P=0.008) were smaller in the preoperative bending group. No significant differences were observed between the two groups in functional scores at the last follow-up. CONCLUSIONS: Smaller differences in length and angle between the ipsilateral and contralateral clavicle, indicative of reduction, were observed in the preoperative bending group. Using the precontoured technique with low expense, the operation was performed more effectively as reflected by a shorter operation time. Level of evidence: III.

A CRISPR activation screen identifies MUC-21 as critical for resistance to NK and T cell-mediated cytotoxicity.

BACKGROUND: Immunotherapy has significantly advanced cancer treatments, but many patients do not respond to it, partly due to immunosuppressive mechanisms used by tumor cells. These cells employ immunosuppressive ligands to evade detection and elimination by the immune system. Therefore, the discovery and characterization of novel immunosuppressive ligands that facilitate immune evasion are crucial for developing more potent anti-cancer therapies. METHODS: We conducted gain-of-function screens using a CRISPRa (CRISPR activation) library that covered the entire human transmembrane sub-genome to identify surface molecules capable of hindering NK-mediated cytotoxicity. The immunosuppressive role and mechanism of MUC21 were validated using NK and T cell mediated cytotoxicity assays. Bioinformatics tools were employed to assess the clinical implications of mucin-21 (MUC21) in cancer cell immunity. RESULTS: Our genetic screens revealed that MUC21 expression on cancer cell surfaces inhibits both the cytotoxic activity of NK cells and antibody-dependent cellular cytotoxicity, but not affecting complement-dependent cytotoxicity. Additionally, MUC21 expression hinders T cell activation by impeding antigen recognition, thereby diminishing the effectiveness of the immune checkpoint inhibitor, anti-PD-L1. Moreover, MUC21 expression suppress the antitumor function of both CAR-T cells and CAR-NK cells. Mechanistically, MUC21 facilitates immune evasion by creating steric hindrance, preventing interactions between cancer and immune cells. Bioinformatics analysis revealed elevated MUC21 expression in lung cancer, which correlated with reduced infiltration and activation of cytotoxic immune cells. Intriguingly, MUC21 expression was higher in non-small cell lung cancer (NSCLC) tumors that were non-responsive to anti-PD-(L)1 treatment compared to responsive tumors. CONCLUSIONS: These findings indicate that surface MUC21 serves as a potent immunosuppressive ligand, shielding cancer cells from NK and CD8+T cell attacks. This suggests that inhibiting MUC21 could be a promising strategy to improve cancer immunotherapy.

Inhibitory effect of Sanguisorba hakusanensis Makino ethanol extract on atopic dermatitis-like responses in NC/Nga mice and human keratinocytes.

Atopic dermatitis (AD) is an allergic, inflammatory skin disease caused by immune dysregulation. In this study, we investigated anti-atopic and anti-inflammatory activities of Sanguisorba hakusanensis ethanol extract (SHE) both in vivo using NC/Nga mice and in vitro using human HaCaT keratinocytes. Oral administration of SHE suppressed several atopic symptoms associated with house dust mites (induced with Dermatophagoides farinae extract) in NC/Nga mice and decreased serum levels of inflammatory mediators such as immunoglobulin E, histamine, and inflammatory chemokines. Additionally, SHE treatment reduced the infiltration of immune cells such as mast cells and macrophages in AD skin lesions. In vitro, interferon-γ- and tumor necrosis factor-α-stimulated HaCaT cells exhibited increased expression of T helper 1 and 2 chemokines; their expression was inhibited by SHE treatment. The anti-inflammatory effects of SHE treatment involved blocking of the mitogen-activated protein kinase and signal transducer and activator of transcription 1 signaling pathways. In conclusion, SHE exerts potent anti-atopic and anti-inflammatory effects and should be considered for the clinical treatment of AD.

Diagnosis and management of acute colonic diverticulitis: results of a survey among Korean gastroenterologists.

BACKGROUND/AIMS: Some management strategies for acute colonic diverticulitis remain controversial in Korean real-world practice because their clinical features differ from those in the West. This study aimed to investigate the opinions of Korean physicians regarding the diagnosis and treatment of acute diverticulitis. METHODS: A web-based survey was conducted among gastroenterologists specializing on treating lower gastrointestinal disorders. The questionnaires concerned overall management strategies for colonic diverticulitis, including diagnosis, treatment, and follow-up. RESULTS: In total, 209 gastroenterologists responded to the survey. Less than one-fourth of the respondents (23.6%) answered that left-sided colonic diverticulitis is more likely to be complicated than right-sided colonic diverticulitis. Most respondents agreed that immunocompromised patients with diverticulitis have worse clinical outcomes than immunocompetent patients (71.3%). Computed tomography was the most preferred tool for diagnosing diverticulitis (93.9%). Approximately 89% of the respondents answered that they believed antibiotic treatment is necessary to treat acute uncomplicated diverticulitis. Most respondents (92.6%) agreed that emergency surgery is not required for diverticulitis with an abscess or microperforation without panperitonitis. Further, 94.7% of the respondents agreed that colon cancer screening is necessary in patients aged ≥ 50 years with diverticulitis after they have recovered from acute illness. Many respondents (71.4%) agreed that surgery for recurrent diverticulitis should be individualized. CONCLUSION: Opinions regarding management strategies for colonic diverticulitis among Korean gastroenterologists were well agreed upon in some areas but did not agree well in other areas. Evidence-based guidelines that meet the practical needs of the Korean population should be developed.

In vitro production of N-degron fused proteins and its application.

The N-degron pathway, first discovered several decades ago by Varshavsky's laboratory, controls the half-life of target proteins depending on their N-terminal residues. In vivo cell biology studies have established the physiological role of the N-degron pathway. However, in vitro studies such as biochemical assays and structural biology studies are relatively limited. The N-degron substrates cannot be obtained via simple protein expression. The N-degron residues are exposed via the proteolytic process from the translated nascent polypeptide chains. Thus, methods for the fusion expression with several cleavable tags and subsequent treatment with specific proteases to design the exposed N-degron signals have been introduced. Recently, we developed a unique fusion technique using microtubule-associated protein 1A/1B light chain 3B (LC3B), a key marker protein of autophagy, to obtain a high yield of the purified target proteins with variable N-terminal residues for various biochemical studies including enzymatic and binding assays, and crystallization of N-degron complex. This chapter describes the protocols that include the vector map designed for producing LC3B fused target proteins, methods for expression and purification of an example protein, p62/SQSMT1, using different N-terminal residues, and methods to obtain the purified ATG4B protease, which is used for processing LC3B tag and exposing the required N-terminal residues of the target protein.