METTL8 links mt-tRNA m3C modification to the HIF1α/RTK/Akt axis to sustain GBM stemness and tumorigenicity.

Epitranscriptomic RNA modifications are crucial for the maintenance of glioma stem cells (GSCs), the most malignant cells in glioblastoma (GBM). 3-methylcytosine (m3C) is a new epitranscriptomic mark on RNAs and METTL8 represents an m3C writer that is dysregulated in cancer. Although METTL8 has an established function in mitochondrial tRNA (mt-tRNA) m3C modification, alternative splicing of METTL8 can also generate isoforms that localize to the nucleolus where they may regulate R-loop formation. The molecular basis for METTL8 dysregulation in GBM, and which METTL8 isoform(s) may influence GBM cell fate and malignancy remain elusive. Here, we investigated the role of METTL8 in regulating GBM stemness and tumorigenicity. In GSC, METTL8 is exclusively localized to the mitochondrial matrix where it installs m3C on mt-tRNAThr/Ser(UCN) for mitochondrial translation and respiration. High expression of METTL8 in GBM is attributed to histone variant H2AZ-mediated chromatin accessibility of HIF1α and portends inferior glioma patient outcome. METTL8 depletion impairs the ability of GSC to self-renew and differentiate, thus retarding tumor growth in an intracranial GBM xenograft model. Interestingly, METTL8 depletion decreases protein levels of HIF1α, which serves as a transcription factor for several receptor tyrosine kinase (RTK) genes, in GSC. Accordingly, METTL8 loss inactivates the RTK/Akt axis leading to heightened sensitivity to Akt inhibitor treatment. These mechanistic findings, along with the intimate link between METTL8 levels and the HIF1α/RTK/Akt axis in glioma patients, guided us to propose a HIF1α/Akt inhibitor combination which potently compromises GSC proliferation/self-renewal in vitro. Thus, METTL8 represents a new GBM dependency that is therapeutically targetable.

PHF2 regulates genome topology and DNA replication in neural stem cells via cohesin.

Cohesin plays a crucial role in the organization of topologically-associated domains (TADs), which influence gene expression and DNA replication timing. Whether epigenetic regulators may affect TADs via cohesin to mediate DNA replication remains elusive. Here, we discover that the histone demethylase PHF2 associates with RAD21, a core subunit of cohesin, to regulate DNA replication in mouse neural stem cells (NSC). PHF2 loss impairs DNA replication due to the activation of dormant replication origins in NSC. Notably, the PHF2/RAD21 co-bound genomic regions are characterized by CTCF enrichment and epigenomic features that resemble efficient, active replication origins, and can act as boundaries to separate adjacent domains. Accordingly, PHF2 loss weakens TADs and chromatin loops at the co-bound loci due to reduced RAD21 occupancy. The observed topological and DNA replication defects in PHF2 KO NSC support a cohesin-dependent mechanism. Furthermore, we demonstrate that the PHF2/RAD21 complex exerts little effect on gene regulation, and that PHF2's histone-demethylase activity is dispensable for normal DNA replication and proliferation of NSC. We propose that PHF2 may serve as a topological accessory to cohesin for cohesin localization to TADs and chromatin loops, where cohesin represses dormant replication origins directly or indirectly, to sustain DNA replication in NSC.

A Prospective Multi-Centered Registry-Based Observational Study for Patients With Cancer: Design and Rationale for Korean Medicine Cancer Registry (KMCARE).

BACKGROUND: Cancer is one of the leading causes of death in most countries with an expected increased burden on healthcare systems. Since integrative medical treatments are not collected within the scope of existing cancer registries, the establishment of the Korean Medicine Cancer Registry (KMCARE), gathering integrative therapies, including conservative care and Korean medicine, is warranted. METHODS: A prospective observational study based on the registry will be conducted in 5 Korean medical hospitals. A total of 650 eligible participants undergoing Korean medicine treatments within 1 month of a diagnosis of lung, colorectal, stomach, or breast cancer are anticipated to be enrolled in the registry. Data collected in the KMCARE can be classified into patient information, received treatments, and outcomes. The primary outcome is the Functional Assessment of Cancer Therapy-General Questionnaire score at 3 months. Secondary outcomes include the MD Anderson Symptom Inventory-Core and the Body Constitution Questionnaire at 3 and 6 months. After 6 months of follow-up periods, survival surveillance will be continued for additional 18 months. Descriptive and statistical analysis of primary and secondary outcomes, baseline data, safety, survival, and prognostic factors will be performed. DISCUSSION: This is the first prospective, multi-centered, registry-based observational study of cancer patients in Korean medicine hospitals, which could reveal the current status of cancer patients receiving integrative cancer therapies, and provide better insight into the role of Korean medicine in palliative care for patients with cancer. TRIAL REGISTRATION: Clinical Research Information Service (CRIS), KCT0007447.

CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex to induce mitotic checkpoint in glioma.

MAD2 is a spindle assembly checkpoint protein that participates in the formation of mitotic checkpoint complex, which blocks mitotic progression. RNF8, an established DNA damage response protein, has been implicated in mitotic checkpoint regulation but its exact role remains poorly understood. Here, RNF8 proximity proteomics uncovered a role of RNF8-MAD2 in generating the mitotic checkpoint signal. Specifically, RNF8 competes with a small pool of p31comet for binding to the closed conformer of MAD2 via its RING domain, while CAMK2D serves as a molecular scaffold to concentrate the RNF8-MAD2 complex via transient/weak interactions between its p-Thr287 and RNF8's FHA domain. Accordingly, RNF8 overexpression impairs glioma stem cell (GSC) mitotic progression in a FHA- and RING-dependent manner. Importantly, low RNF8 expression correlates with inferior glioma outcome and RNF8 overexpression impedes GSC tumorigenicity. Last, we identify PLK1 inhibitor that mimics RNF8 overexpression using a chemical biology approach, and demonstrate a PLK1/HSP90 inhibitor combination that synergistically reduces GSC proliferation and stemness. Thus, our study has unveiled a previously unrecognized CAMK2D-RNF8-MAD2 complex in regulating mitotic checkpoint with relevance to gliomas, which is therapeutically targetable.

Traditional herbal medicine for anorexia in patients with cancer: a systematic review and meta-analysis of randomized controlled trials.

Background: The purpose of this systematic review and meta-analysis was to evaluate the efficacy and safety of traditional herbal medicine (THM) for improving anorexia in patients with cancer. Methods: We searched for randomized controlled trials (RCTs) that evaluated orally administered THM for cancer-related anorexia using 10 databases from the inception to 1 August 2021. The primary outcome was an improvement in anorexia, measured with the total effective rate (TER) or visual analog scale (VAS). The secondary outcomes were the changes in body weight, the Karnofsky performance scale, acylated ghrelin, and adverse events. We used the Cochrane risk of bias assessment tool and the Grading of Recommendations Assessment, Development, and Evaluation method to assess the quality of the studies and the quality of the evidence. Results: A total of 26 RCTs were included, of which 23 were subjected to quantitative analysis. THM showed a significant improvement in anorexia measured with the TER [risk ratio (RR) 1.12, 95% confidence intervals (CI) 1.04-1.20] than appetite stimulants with moderate quality evidence and in the Karnofsky performance scale (RR 1.38, 95% CI 1.12-1.70) with low quality evidence but not in body weight gain (RR 0.98, 95% CI 0.80-1.20). THM showed a significant improvement in anorexia measured with the TER (RR 1.74, 95% CI 1.23-2.48) compared with usual care with low-quality evidence but did not significantly improve the VAS score (mean difference 0.72, 95% CI 0.00-1.43) or the level of acylated ghrelin (mean difference 0.94, 95% CI 1.08-2.97). There were no serious adverse events. Conclusion: This review suggests that THM may be considered a safe alternative therapeutic option for improving anorexia in patients with cancer. Nonetheless, more rigorous RCTs are needed due to methodological limitations. Systematic Review Registration: https://www.crd.york.ac.uk/prospero, identifier CRD42021276508.

Impact of traditional East Asian medicine as an add-on therapy on survival and recurrence after surgery for breast cancer: A systematic review and meta-analysis.

Background: Breast cancer is the most common cancer in women. Patients with cancer increasingly incorporate complementary and alternative medicines, including traditional East Asian medicine (TEAM), for cancer prevention and treatment. This review aimed to determine the effectiveness and safety of TEAM for survival and recurrence after surgery in patients with breast cancer. Methods: We searched nine electronic databases up to 25 August 2022, for randomized controlled trials (RCTs) of TEAM to prevent the recurrence of breast cancer in female patients after mastectomy or breast-conserving surgery. The primary outcome was 5-year disease-free survival (DFS), and secondary outcomes were 5-year overall survival, locoregional and distant recurrence rates, and toxicity. This study adhered to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) was used to evaluate the quality of evidence. Results: From 368 citations, data from nine studies reporting on a total of 1240 patients were included in the systematic review, and eight studies were deemed suitable for the meta-analysis. TEAM combined with adjuvant chemotherapy showed a significant improvement in DFS (odds ratio [OR] 0.42%, 95% confidence interval [CI] 0.28 to 0.61, p < 0.00001) and overall survival (OR 0.44%, 95% CI 0.27 to 0.73, p = 0.001) compared to adjuvant chemotherapy alone. The reduction in the rate of total recurrence was favorable for TEAM combined with adjuvant chemotherapy compared to adjuvant chemotherapy alone (Risk ratio 0.49%, 95% CI 0.35 to 0.70; p < 0.0001). TEAM after adjuvant chemotherapy showed a significant advantage in DFS compared to no TEAM (OR 0.61%, 95% CI 0.41 to 0.92, p = 0.02). No severe adverse events related to TEAM were reported. The overall certainty of the evidence for DFS, overall survival, and the total recurrence rate were moderate when postoperative breast cancer patients used TEAM combined with adjuvant chemotherapy. Conclusion: Moderate-quality evidence suggests TEAM as an add-on therapy to adjuvant chemotherapy. TEAM may have the potential to improve long-term survival and prevent postoperative recurrence in patients with breast cancer. In future, more rigorous RCTs should be conducted to confirm these findings.

E2F and STAT3 provide transcriptional synergy for histone variant H2AZ activation to sustain glioblastoma chromatin accessibility and tumorigenicity.

The histone variant H2AZ is overexpressed in diverse cancer types where it facilitates the accessibility of transcriptional regulators to the promoters of cell cycle genes. However, the molecular basis for its dysregulation in cancer remains unknown. Here, we report that glioblastomas (GBM) and glioma stem cells (GSCs) preferentially overexpress H2AZ for their proliferation, stemness and tumorigenicity. Chromatin accessibility analysis of H2AZ2 depleted GSC revealed that E2F1 occupies the enhancer region within H2AZ2 gene promoter, thereby activating H2AZ2 transcription. Exploration of other H2AZ2 transcriptional activators using a customized "anti-H2AZ2" query signature for connectivity map analysis identified STAT3. Co-targeting E2F and STAT3 synergistically reduced the levels of H2AZ, histone 3 lysine 27 acetylation (H3K27ac) and cell cycle gene transcription, indicating that E2F1 and STAT3 synergize to activate H2AZ gene transcription in GSCs. Remarkably, an E2F/STAT3 inhibitor combination durably suppresses GSC tumorigenicity in an orthotopic GBM xenograft model. In glioma patients, high STAT3 signaling is associated with high E2F1 and H2AZ2 expression. Thus, GBM has uniquely opted the use of E2F1- and STAT3-containing "enhanceosomes" that integrate multiple signaling pathways to achieve H2AZ gene activation, supporting a translational path for the E2F/STAT3 inhibitor combination to be applied in GBM treatment.

Deciphering Nanoparticle Trafficking into Glioblastomas Uncovers an Augmented Antitumor Effect of Metronomic Chemotherapy.

Nanoparticles have been explored in glioblastomas as they can traverse the blood-brain barrier and target glioblastoma selectively. However, direct observation of nanoparticle trafficking into glioblastoma cells and their underlying intracellular fate after systemic administration remains uncharacterized. Here, based on high-resolution transmission electron microscopy experiments of an intracranial glioblastoma model, it is shown that ligand-modified nanoparticles can traverse the blood-brain barrier, endocytose into the lysosomes of glioblastoma cells, and undergo endolysosomal escape upon photochemical ionization. Moreover, an optimal dose of metronomic chemotherapy using dual-drug-loaded nanocarriers can induce an augmented antitumor effect directly on tumors, which has not been recognized in previous studies. Metronomic chemotherapy enhances antitumor effects 3.5-fold compared with the standard chemotherapy regimen using the same accumulative dose in vivo. This study provides a conceptual framework that can be used to develop metronomic nanoparticle regimens as a safe and viable therapeutic strategy for treating glioblastomas and other advanced-stage solid tumors.

Traditional Herbal Medicine for Insomnia in Patients With Cancer: A Systematic Review and Meta-Analysis.

Background: Insomnia is one of the most prevalent cancer-related symptoms and has a severe impact on the quality of life. This study aimed to evaluate the efficacy and safety of traditional herbal medicine (THM) for improving sleep quality in patients with cancer. Methods: Randomized controlled trials (RCTs) evaluating orally administered THM in a cancer population with insomnia were searched using nine electronic databases up to November 30, 2020. The outcome measurements were sleep quality measured by validated questionnaire such as the Pittsburgh Sleep Quality Index (PSQI), total effective rate, and adverse effects. The included studies were appraised using the Cochrane risk of bias tool and meta-analyzed. The quality of evidence was assessed using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) method. Results: Fourteen RCTs were included in the systematic review, and 10 RCTs were analyzed quantitatively. Compared to hypnotics, THM showed a significant improvement in sleep quality by reducing the PSQI score [mean difference (MD) -2.25, 95% confidence interval (CI) -3.46 to -1.05, I 2 = 84%] and increasing the total effective rate [risk ratio (RR) 1.26, 95% CI 1.07 to 1.48, I 2 = 70%] with low quality of evidence. Compared to placebo, THM also reduced the PSQI score significantly (MD -2.56, 95% CI -3.81 to -1.31, I 2 = 91%) with moderate quality of evidence. The most frequently used herbs were Ziziphus jujuba Mill. No serious adverse events were observed. Conclusion: This review suggests that THM may be an effective therapeutic option for insomnia in patients with cancer. However, considering the limited methodological qualities and inconsistent results of the included trials, further rigorous RCTs are required. Systematic Review Registration: [https://www.crd.york.ac.uk/prospero], PROSPERO 2021 [CRD42021265070].

Traditional Herbal Medicine, Sipjeondaebo-Tang, for Cancer-Related Fatigue: A Randomized, Placebo-Controlled, Preliminary Study.

PURPOSE: Sipjeondaebo-tang (SDT) is a widely used traditional herbal medicine for relieving fatigue. This randomized, placebo-controlled, preliminary study evaluated SDT for cancer-related fatigue, which is the most common symptom experienced by patients with cancer. PATIENTS AND METHODS: Patients with a Brief Fatigue Inventory (BFI) score of at least 4 were randomly assigned in a double-blinded manner to receive SDT (3 g 3 times daily) or placebo orally for 3 weeks. The BFI was the primary outcome measure and secondary outcome measures included the Hospital Anxiety and Depression Scale (HADS), the European Organization for Research and Treatment of Cancer-Quality of Life Questionnaire (EORTC QLQ-C30), immunoregulatory tests, and safety. RESULTS: A total of 50 participants were randomly assigned and 48 patients completed the trial. Based on intention-to-treat analysis, fatigue, which was the primary outcome, was improved in both arms compared with the baseline, and was significantly better in the SDT group than in the placebo group at week 3 (3.56 ± 1.18 vs 4.63 ± 1.83, P = .019). Secondary outcomes, including anxiety, depression, and immunoregulatory tests, did not improve significantly in either group. However, quality of life measured using the EORTC QLQ-C30 improved in both arms compared with the baseline, and the global health subscale was significantly better in the SDT group than in the placebo group (P = .02). No significant toxicities were observed. CONCLUSION: SDT may improve cancer-related fatigue and quality of life in patients with cancer. A further randomized clinical trial with large sample size is warranted.

Traditional herbal medicine combined with first-line platinum-based chemotherapy for advanced non-small-cell lung cancer: A PRISMA-compliant systematic review and meta-analysis.

BACKGROUND: Non-small-cell lung cancer (NSCLC) is a major health burden in many countries. This review aimed to evaluate the efficacy of traditional herbal medicine (THM) combined with first-line platinum-based chemotherapy (PBCT) for the treatment of advanced NSCLC. METHODS: From inception to April 2021, relevant studies were retrieved from 9 electronic databases. Randomized controlled trials (RCTs) comparing survival outcomes of THM + PBCT treatment with PBCT treatment in patients with advanced NSCLC were reviewed. The risk of bias was evaluated using the Cochrane Risk of Bias Tool. Overall survival, 1-year survival, progression-free survival or time to progression, tumor response rate, and adverse effects were analyzed. RESULTS: Sixteen RCTs comprising 1445 patients were included. The meta-analysis indicated that THM + PBCT treatment, compared to PBCT alone, could improve overall survival (median survival ratio = 1.24, 95% confidence intervals [CI] [1.11, 1.39], P < .001), progression-free survival/time to progression (median survival ratio = 1.22, 95% CI [1.09, 1.37], P < .001), and the 1-year survival rate (risk ratio [RR] = 1.56, 95% CI [1.31, 1.86], P < .001). THM + PBCT also led to a higher tumor response rate (RR = 1.39, 95% CI [1.22, 1.59], P < .001) and lower incidence of thrombocytopenia (RR = 0.72, 95% CI [0.56, 0.92], P = .009) and nausea/vomiting (RR = 0.35, 95% CI [0.21, 0.57], P < .001), while there was no significant effect observed on leukopenia (RR = 0.68, 95% CI [0.34, 1.36], P = .27). CONCLUSION: THM, when used in combination with PBCT, might increase survival and the tumor response rate while decreasing the side effects caused by chemotherapy in patients with advanced NSCLC. However, considering the limited methodological qualities of the included trials, more rigorous RCTs are needed.

Comparative Efficacy of Jaungo, A Traditional Herbal Ointment, and the Water-in-Oil Type Non-Steroidal Moisturizer for Radiation-Induced Dermatitis in Patients With Breast Cancer: A Study Protocol for a Prospective, Randomized, Single-Blinded, Pilot Study.

Background: Radiation-induced dermatitis (RID) is a common complication of radiation therapy (RT). Although it has a high prevalence and can even trigger the premature end of conventional cancer therapies, there is no standard management. This study aims to evaluate whether topical use of Jaungo (Shiunko), a traditional herbal ointment mainly composed of Lithospermi radix and Angelica sinensis, could reduce RID compared to the water-in-oil type non-steroidal moisturizer in patients with breast cancer. Methods: This is a prospective, single-blinded, randomized controlled pilot trial that investigates the effect of topical application of Jaungo for the prevention of RID in postoperative breast cancer patients scheduled for RT, in comparison with the non-steroidal moisturizer, with a random distribution of 50 patients across the two groups. RT will be administered for 5-7 weeks with a biological equivalent dose (BED10) of 60 Gy or more, and the interventions will be applied 3 times a day during RT duration. Participants will be assessed a total of nine times, including eight visits during the period of RT and one visit at a 2-week follow-up period after the end of treatment. The incidence and severity of RID, quality of life, skin reaction symptoms, and maximum pain related to RID will be measured. The incidence rate of grade 2 or higher RID using the Radiation Therapy Oncology Group (RTOG) in the two groups will be statistically compared as the primary outcome. The types and frequencies of adverse events will be also collected and evaluated. All assessments will be performed by independent radiology oncologists. Discussion: This trial is currently ongoing and is recruiting. This study will determine the preventive efficacy of Jaungo in RID with postoperative breast cancer patients and provide evidence in traditional Korean medicine clinical practice.

A chemical biology approach reveals a dependency of glioblastoma on biotin distribution.

Glioblastoma (GBM) is a uniformly lethal disease driven by glioma stem cells (GSCs). Here, we use a chemical biology approach to unveil previously unknown GBM dependencies. By studying sulconazole (SN) with anti-GSC properties, we find that SN disrupts biotin distribution to the carboxylases and histones. Transcriptomic and metabolomic analyses of SN-treated GSCs reveal metabolic alterations that are characteristic of biotin-deficient cells, including intracellular cholesterol depletion, impairment of oxidative phosphorylation, and energetic crisis. Furthermore, SN treatment reduces histone biotinylation, histone acetylation, and expression of superenhancer-associated GSC critical genes, which are also observed when biotin distribution is genetically disrupted by holocarboxylase synthetase (HLCS) depletion. HLCS silencing impaired GSC tumorigenicity in an orthotopic xenograft brain tumor model. In GBM, high HLCS expression robustly indicates a poor prognosis. Thus, the dependency of GBM on biotin distribution suggests that the rational cotargeting of biotin-dependent metabolism and epigenetic pathways may be explored for GSC eradication.

Efficacy of Chemotherapy Integrated With Traditional Korean Medicine in Patients With Metastatic Pancreatic Cancer: A Single-Center Retrospective Study.

BACKGROUND: This retrospective study investigated the efficacy and safety of chemotherapy (CTX) integrated with Traditional Korean Medicine (TKM) in patients with metastatic pancreatic cancer, in a single Korean center. METHODS: From January, 2014 to February, 2019, patients with metastatic pancreatic cancer who had received CTX were enrolled. Overall survival (OS), demographic characteristics, and adverse events were examined. Statistical analysis was utilized to evaluate the differences in characteristics and to compare the survival rates between the CTX group and CTX+TKM group. Kaplan-Meier curves were used to compare the differences in survival time. A Cox regression analysis was performed to determine the hazard ratio of the risk of mortality. RESULTS: A total 37 participants were included and visited a TKM hospital 7.4 ± 8.3 months after being diagnosed with metastatic pancreatic cancer. The median age of the participants was 62 years; 26 patients (70.3%) had an Eastern Cooperative Oncology Group (ECOG) performance status between 0 and 2, and 23 patients (62.2 %) had first-line CTX failure. The median OS of all patients was 3.8 months (95% CI, 3.1-4.6). The CTX + TKM group showed longer survival (4.1 months; 95% CI, 2.4- .8) than the CTX group (2.4 months, 95% CI 0.2-4.6) but this was not statistically significant (P = .217). Chemotherapy with TKM treatment for more than 30 days (CTX + TKM ≥ 30) significantly prolonged median OS (9.1 months; 95% CI, 3.6-14.5; P = .025) compared to chemotherapy alone. Cox hazard ratio analysis revealed that CTX + TKM ≥ 30 and prior chemotherapy were significantly independent prognostic factors for OS. The main herbs in the TKM treatment were Rhus verniciflua Stokes and Astragalus. Severe adverse events with respect to TKM treatment were not reported. CONCLUSIONS: TKM treatment integrated with chemotherapy may prolong OS in patients with metastatic pancreatic cancer compared to chemotherapy treatment alone. More rigorous prospective clinical trials are needed to confirm this result.

Gold Nanoparticles for Photothermal Cancer Therapy.

Gold is a multifunctional material that has been utilized in medicinal applications for centuries because it has been recognized for its bacteriostatic, anticorrosive, and antioxidative properties. Modern medicine makes routine, conventional use of gold and has even developed more advanced applications by taking advantage of its ability to be manufactured at the nanoscale and functionalized because of the presence of thiol and amine groups, allowing for the conjugation of various functional groups such as targeted antibodies or drug products. It has been shown that colloidal gold exhibits localized plasmon surface resonance (LPSR), meaning that gold nanoparticles can absorb light at specific wavelengths, resulting in photoacoustic and photothermal properties, making them potentially useful for hyperthermic cancer treatments and medical imaging applications. Modifying gold nanoparticle shape and size can change their LPSR photochemical activities, thereby also altering their photothermal and photoacoustic properties, allowing for the utilization of different wavelengths of light, such as light in the near-infrared spectrum. By manufacturing gold in a nanoscale format, it is possible to passively distribute the material through the body, where it can localize in tumors (which are characterized by leaky blood vessels) and be safely excreted through the urinary system. In this paper, we give a quick review of the structure, applications, recent advancements, and potential future directions for the utilization of gold nanoparticles in cancer therapeutics.

Optimal Processing Conditions of Boswellia carteri Birdw. Using Response Surface Methodology.

BACKGROUND: Boswellia carteri Bridw. is being widely used for its anti-inflammatory properties, as well as for wound healing, antimicrobial, and immunomodulatory properties, and boswellic acids (BAs) are considered to be the main active constituents. OBJECTIVES: To investigate optimal conditions of stir-baking process for the resin of B. carteri with vinegar of using response surface methodology (RSM). MATERIALS AND METHODS: The concentration of acetic acid, heating temperature, and heating time were set as influential factors, and the yields of chemical compounds were the response values which were optimally designed by a Box-Behnken design. The amounts of 11-keto-ß-boswellic acid (KBA) and α-boswellic acid (αBA) in B. carteri resin were quantified using high-performance liquid chromatography analysis. RESULTS: Maximum amounts of KBA and αBA in B. carteri resin were obtained using 6% acetic acid for 10 min at 90°C in preliminary test. Two factor interactions, such as acetic acid concentration-heating temperature and heating temperature-heating time, were significantly observed by multiple regression analysis. Optimal processing conditions from RSM were 5.83% for acetic acid concentration, 9.56 min for heating time, and 89.87°C for heating temperature. Under the modified conditions, the experimental value of the response was 11.25 mg/g, which was similar to the predicted value. CONCLUSIONS: The results suggest that the optimal conditions for the stir-baking process of B. carteri resin were determined by RSM, which was reliable and applicable to practical processing of herbal medicine. SUMMARY: The resin of Boswellia carteri was macerated in aqueous acetic acid and heated using an oven for stir baking processThe interaction between heating temperature and heating time was the most significantOptimal conditions for processing B. carteri resin were determined as 5.83% acetic acid, 9.56 min for heating time, and 89.87°C for heating temperature. Abbreviations used: BAs: Boswellic acids; KBA: 11 keto ß boswellic acid; αBA: α boswellic acid; BBD: Box-Behnken design; RSM: Response surface method; HPLC: High performance liquid chromatography; LOD: Limits of determination; LOQ: Limits of quantification; RSD: Relative standard deviation; ANOVA: Analysis of variance.

A Drosophila Reporter for the Translational Activation of ATF4 Marks Stressed Cells during Development.

Eukaryotic cells have evolved signaling pathways that help to restore cellular homeostasis in response to various physiological or pathological conditions. ATF4 is a transcription factor whose mRNA translation is stimulated in response to stress-activated eIF2alpha kinases. Established conditions that activate eIF2alpha phosphorylation and ATF4 translation include excessive stress in the endoplasmic reticulum (ER) and amino acid deprivation. ATF4 is activated through a unique translational activation mechanism that involves multiple upstream open reading frames (uORFs) in the 5'-untranslated region (UTR), which is conserved from yeast to mammals. Taking advantage of this, we developed a translational activation reporter of ATF4 in Drosophila, in which the dsRed reporter coding sequence was placed downstream of the Drosophila ATF4 5' UTR. This reporter remained inactive in most tissues under normal conditions, but showed dsRed expression when starved, or when challenged with conditions that imposed ER stress. In normally developing flies, a small number of cell types showed reporter expression even without exogenous stress, which included the salivary gland, gut, the male reproductive organ, and the photoreceptor cells, suggestive of inherent stress during the normal development of these cell types. These results establish a new tool to study ATF4-mediated stress response in Drosophila development and disease.

Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis isolates from Korea and analysis of the correlation between the mutations and pyrazinamidase activity.

To investigate the effect of natural pyrazinamidase (PncA) mutations on protein function, we analyzed expression and PncA activity of eight pncA point mutants identified in nineteen pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates. Among them, two mutants (Y99D and T135P) showed high expression level and solubility comparable to those of the wild-type PncA protein, two (K48E and G97D) displayed low expression level and solubility, and four (C14R, H51P, W68S, and A146V) were insoluble. Interestingly, when possible structural effects of these mutations were predicted by the CUPSAT program based on the proposed three-dimensional structure of M. tuberculosis PncA, only two highly soluble mutant proteins (Y99D and T135P) were predicted to be stabilizing and have favorable torsion angles. However, the others exhibiting either low solubility or precipitation were foreseen to be destabilizing and/or have unfavorable torsion angles, suggesting that the alterations could interfere with proper protein folding, thereby decreasing or depleting protein solubility. A PncA activity assay demonstrated that two mutants (G97D and T135P) showed virtually no activity, but two other mutants (K48E and Y99D) exhibited wild-type activity, indicating that the PncA residues (Cys14, His51, Trp68, Gly97, Thr135, and Ala146) may be important for PncA activity and/or proper protein folding.

Wnt modulators in the aqueous humor are associated with outer retinal damage severity in patients with neovascular age-related macular degeneration.

PURPOSE: To investigate the associations of the Wnt modulators Wnt inhibitory factor 1 (WIF-1) and Dickkopf 3 (DKK-3) in the aqueous humor with neovascular age-related macular degeneration (nAMD) and to determine their clinical implications. METHODS: Seventy-four nAMD patients initially treated with an intravitreal injection of ranibizumab (IVR) and 74 age- and sex-matched controls were studied. Aqueous humor WIF-1 and DKK-3 levels were measured by Western blotting and an ELISA before and 1 month after two consecutive IVRs (pre- and post-IVR). Visual acuity assessments and spectral domain optical coherence tomography were performed pre- and post-IVR. RESULTS: Western blotting showed increased WIF-1 and DKK-3 in 12 nAMD patients compared with 12 controls. The ELISA analysis demonstrated elevated WIF-1 (pre) and DKK-3 (pre) in 62 patients compared with 62 controls (54.7 vs. 23.0 and 114.3 vs. 93.0 ng/mL, respectively). In multivariate analyses, high WIF-1 (pre) levels were associated with increased disruption in the photoreceptor junction's inner and outer segments (IS/OS) (pre and post) and high WIF-1 (post) levels. Interestingly, WIF-1 (pre) levels were significantly higher in type 3 neovascularization (NV) patients than in type 1 or 2 NV (90.5 ± 36.7 vs. 48.3 ± 22.5 and 41.3 ± 28.8 ng/mL, respectively). However, choroidal thickness was not correlated with WIF-1 levels. CONCLUSIONS: We report, for the first time, the possibility of phenotypic, anatomic, and ocular proteomic correlations, demonstrating correlated WIF-1 and DKK-3 upregulation in nAMD patients' aqueous humor. Secreted WIF-1, reflecting the degree of retinal structure damage, may be a new biomarker for the retina's healthy and disease states.

Exosomal proteins in the aqueous humor as novel biomarkers in patients with neovascular age-related macular degeneration.

Age-related macular degeneration (AMD) describes the progressive degeneration of the retinal pigment epithelium (RPE), retina, and choriocapillaris and is the leading cause of blindness in people over 50. The molecular mechanisms underlying this multifactorial disease remain largely unknown. To uncover novel secretory biomarkers related to the pathogenesis of AMD, we adopted an integrated approach to compare the proteins identified in the conditioned medium (CM) of cultured RPE cells and the exosomes derived from CM and from the aqueous humor (AH) of AMD patients by LC-ESI-MS/MS. Finally, LC-MRM was performed on the AH from patients and controls, which revealed that cathepsin D, cytokeratin 8, and four other proteins increased in the AH of AMD patients. The present study has identified potential biomarkers and therapeutic targets for AMD treatment, such as proteins related to the autophagy-lysosomal pathway and epithelial-mesenchymal transition, and demonstrated a novel and effective approach to identifying AMD-associated proteins that might be secreted by RPE in vivo in the form of exosomes. The proteomics-based characterization of this multifactorial disease could help to match a particular marker to particular target-based therapy in AMD patients with various phenotypes.