Clinical performance of the TrueMark™ MSI assay for microsatellite instability detection in a Chinese colorectal cancer cohort.

BACKGROUND: Microsatellite instability (MSI) and mismatch repair (MMR) detection is valuable in assessing prognosis and treatment options. However, the conventional detection methods such as immunohistochemistry (IHC) are limited by not fully consistent results as well as a long turnaround time. TrueMark™ MSI Assay is a novel solution for MSI analysis, but lack of research support in the Chinese colorectal cancer (CRC) patients. MATERIALS AND METHODS: 60 dMMR and 60 pMMR CRC samples identified by IHC were collected and their MSI status were detected using TrueMark™ MSI assay with an expanded panel of 13 markers. The overall performance and diagnostic concordance between TrueMark™ MSI test and MMR IHC analysis were assessed and analyzed. RESULTS: According to the TrueMark™ test, 55 out of the 120 (45.8 %) CRCs were identified as MSI-high (MSI-H) with an instability at ≥ 4/13 markers. Compared with the MMR IHC analysis, an overall percent agreement of 94.2 % and a Kappa of 0.883 were achieved. For the seven inconsistent samples, tumor mutation burden analysis was performed and the results supported the diagnosis by TrueMark™ test. To confirm the robustness of the above findings, a validation was performed in an independent cohort comprising 51 consecutive CRCs. Furthermore, an optimized panel composed of NR-21, NR-24, NR-27, ABI-16, ABI-17 and ABI-20B was developed by multivariate logistic regression model, and showed 100 % agreement with the 13-marker panel for MSI detection in both the derivation and validation sets. CONCLUSION: TrueMark™ MSI provides a fast, reliable and highly automated solution to MSI detection in Chinese CRC patients, and the new 6-marker panel we established shows promise deserving further evaluation.

Sufu limits sepsis-induced lung inflammation via regulating phase separation of TRAF6.

Rationale: Sepsis is a potentially life-threatening condition caused by the body's response to a severe infection. Although the identification of multiple pathways involved in inflammation, tissue damage and aberrant healing during sepsis, there remain unmet needs for the development of new therapeutic strategies essential to prevent the reoccurrence of infection and organ injuries. Methods: Expression of Suppressor of Fused (Sufu) was evaluated by qRT-PCR, western blotting, and immunofluorescence in murine lung and peritoneal macrophages. The significance of Sufu expression in prognosis was assessed by Kaplan-Meier survival analysis. The GFP-TRAF6-expressing stable cell line (GFP-TRAF6 Blue cells) were constructed to evaluate phase separation of TRAF6. Phase separation of TRAF6 and the roles of Sufu in repressing TRAF6 droplet aggregation were analyzed by co-immunoprecipitation, immunofluorescence, Native-PAGE, FRAP and in vitro assays using purified proteins. The effects of Sufu on sepsis-induced lung inflammation were evaluated by cell function assays, LPS-induced septic shock model and polymicrobial sepsis-CLP mice model. Results: We found that Sufu expression is reduced in early response to lipopolysaccharide (LPS)-induced acute inflammation in murine lung and peritoneal macrophages. Deletion of Sufu aggravated LPS-induced and CLP (cecal ligation puncture)-induced lung injury and lethality in mice, and augmented LPS-induced proinflammatory gene expression in cultured macrophages. In addition, we identified the role of Sufu as a negative regulator of the Toll-Like Receptor (TLR)-triggered inflammatory response. We further demonstrated that Sufu directly interacts with TRAF6, thereby preventing oligomerization and autoubiquitination of TRAF6. Importantly, TRAF6 underwent phase separation during LPS-induced inflammation, which is essential for subsequent ubiquitination activation and NF-κB activity. Sufu inhibits the phase-separated TRAF6 droplet formation, preventing NF-κB activation upon LPS stimulation. In a septic shock model, TRAF6 depletion rescued the augmented inflammatory phenotype in mice with myeloid cell-specific deletion of Sufu. Conclusions: These findings implicated Sufu as an important inhibitor of TRAF6 in sepsis and suggest that therapeutics targeting Sufu-TRAF6 may greatly benefit the treatment of sepsis.

Immune-Hot tumor features associated with recurrence in early-stage ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is a distinct histotype of ovarian cancer, which usually presages a worse prognosis upon recurrence. Identifying patients at risk for relapse is an unmet need to improve outcomes. A retrospective cohort analysis of 195 early-stage OCCC patients diagnosed between January 2011 and December 2019 at National Taiwan University Hospital was conducted to identify prognostic factors for recurrence, progression-free survival (PFS) and overall survival (OS). Molecular profiling of tumors was performed in a case-controlled cohort matched for adjuvant therapy for biomarker discovery. Multivariate Cox proportional hazard model revealed that paclitaxel-based chemotherapy was associated with better PFS than nonpaclitaxel chemotherapy (HR = 0.19, P = .006). The addition of bevacizumab was associated with better PFS, compared to no bevacizumab (HR = 0.09, P = .02). Neither showed significant improvement in OS. Recurrence is associated with an Immune-Hot tumor feature (P = .03), the CTLA-4-high subtype (P = .01) and increased infiltration of immune cells in general. The Immune-Hot feature (HR = 3.39, P = .005) and the CTLA-4-high subtype (HR = 2.13, P = .059) were associated with worse PFS. Immune-Hot tumor features could prognosticate recurrence in early-stage OCCC.

Identification of a rare Gli1+ progenitor cell population contributing to liver regeneration during chronic injury.

In adults, hepatocytes are mainly replenished from the existing progenitor pools of hepatocytes and cholangiocytes during chronic liver injury. However, it is unclear whether other cell types in addition to classical hepatocytes and cholangiocytes contribute to hepatocyte regeneration after chronic liver injuries. Here, we identified a new biphenotypic cell population that contributes to hepatocyte regeneration during chronic liver injuries. We found that a cell population expressed Gli1 and EpCAM (EpCAM+Gli1+), which was further characterized with both epithelial and mesenchymal identities by single-cell RNA sequencing. Genetic lineage tracing using dual recombinases revealed that Gli1+ nonhepatocyte cell population could generate hepatocytes after chronic liver injury. EpCAM+Gli1+ cells exhibited a greater capacity for organoid formation with functional hepatocytes in vitro and liver regeneration upon transplantation in vivo. Collectively, these findings demonstrate that EpCAM+Gli1+ cells can serve as a new source of liver progenitor cells and contribute to liver repair and regeneration.

To evaluate the serum cortisol, salivary cortisol, and serum interleukin-1 B level in patients of chronic periodontitis with smoking and stress and without smoking and stress.

ABSTRACT: The role of cognitive, social and biological factors in the etiology of chronic periodontitis has been reported.The aim of this study was to evaluate the salivary cortisol level and interleukin-1 B level in patients of Chronic periodontitis in smokers and stress and nonsmokers without stress.The design of study randomized, prospective, double-blinded, and prospective study.The total sample size was comprised of 600 subjects between the ages of 20 and 50 years. The sample size was divided into 300 males and 300 females. Out of 600 subjects, 200 subjects comprised of subjects with chronic periodontitis with positive depression level with a history of smoking (Group I), 200 subjects comprised of subjects with chronic periodontitis without depression and without smoking (Group II), and 200 subjects who were taken as the control group comprised of healthy subjects without chronic periodontitis, without depression level, and no smoking history (Group III). Salivary cortisol levels were determined by enzyme-linked immunosorbent assay (ELISA).The result showed that there was a positive correlation between morning and evening salivary cortisol level in all the groups with correlation coefficient. There was significant higher value of salivary cortisol in Group I patients when compared with Group II and Group III. However, when the comparison of salivary cortisol levels was done between the Group II and Control group, the result showed nonsignificant P value.It is suggested that stress is positively correlated with the salivary cortisol levels in smokers and nonsmokers.

Squalene epoxidase promotes colorectal cancer cell proliferation through accumulating calcitriol and activating CYP24A1-mediated MAPK signaling.

BACKGROUND: Colorectal cancer (CRC) is one of the most malignant tumors with high incidence, yet its molecular mechanism is not fully understood, hindering the development of targeted therapy. Metabolic abnormalities are a hallmark of cancer. Targeting dysregulated metabolic features has become an important direction for modern anticancer therapy. In this study, we aimed to identify a new metabolic enzyme that promotes proliferation of CRC and to examine the related molecular mechanisms. METHODS: We performed RNA sequencing and tissue microarray analyses of human CRC samples to identify new genes involved in CRC. Squalene epoxidase (SQLE) was identified to be highly upregulated in CRC patients. The regulatory function of SQLE in CRC progression and the therapeutic effect of SQLE inhibitors were determined by measuring CRC cell viability, colony and organoid formation, intracellular cholesterol concentration and xenograft tumor growth. The molecular mechanism of SQLE function was explored by combining transcriptome and untargeted metabolomics analysis. Western blotting and real-time PCR were used to assess MAPK signaling activation by SQLE. RESULTS: SQLE-related control of cholesterol biosynthesis was highly upregulated in CRC patients and associated with poor prognosis. SQLE promoted CRC growth in vitro and in vivo. Inhibition of SQLE reduced the levels of calcitriol (active form of vitamin D3) and CYP24A1, followed by an increase in intracellular Ca2+ concentration. Subsequently, MAPK signaling was suppressed, resulting in the inhibition of CRC cell growth. Consistently, terbinafine, an SQLE inhibitor, suppressed CRC cell proliferation and organoid and xenograft tumor growth. CONCLUSIONS: Our findings demonstrate that SQLE promotes CRC through the accumulation of calcitriol and stimulation of CYP24A1-mediated MAPK signaling, highlighting SQLE as a potential therapeutic target for CRC treatment.

LINC00176 facilitates CD4+T cell adhesion in systemic lupus erythematosus via the WNT5a signaling pathway by regulating WIF1.

Accruing research shows the implications of long non-coding RNAs (lncRNAs) in the progression of various autoimmune diseases including systemic lupus erythematosus (SLE). The present study aimed to identify the expression pattern of LINC00176 in SLE and to explore its effects on CD4+T cell adhesion in this context. The biological functions of LINC00176, WIF1 and WNT5a on CD4+T cells in SLE were evaluated via gain- and loss-of-function experiments, following delivery of pcDNA3-LINC00176, siRNA-LINC00176, pcDNA3-WIF1 and WNT-sFRP5 (an inhibitor for the WNT5a signaling pathway). High LINC00176 expression was evident in the CD4+T cells of SLE patients. Additionally, WIF1 was identified as a potential target gene of LINC00176, and was negatively regulated by LINC00176. The overexpression of LINC00176 could promote proliferation and adhesion of CD4+T cells in SLE. Such alternations were reversed following up-regulation of WIF1 or inhibition of the WNT5a signaling pathway. Taken together, the key findings of our study highlight the ability of LINC00176 to potentially promote the proliferation and adhesion of CD4+T cells in SLE by down-regulating WIF1 and activating the WNT5a signaling pathway, providing new insight and a theoretical basis for translation in SLE therapy.

SLAP reagents for the photocatalytic synthesis of C3/C5-substituted, N-unprotected selenomorpholines and 1,4-selenazepanes.

Herein, we disclose the first set of unique selenium-containing SLAP (SiLicon Amine Protocol) reagents for the direct synthesis of C3/C5-substituted selenomorpholines and 1,4-selenazepanes from diverse (hetero)aldehydes under mild photocatalytic conditions. Enantiomerically pure 1,2-amino alcohol/α-amino acid versions of these heterocycles were also synthesized. Further, we have shown the late-stage modification of certain biologically active agents using the developed seleno-SLAP reagents.

lncRNA Signature for Predicting Cerebral Vasospasm in Patients with SAH: Implications for Precision Neurosurgery.

Subarachnoid hemorrhage (SAH) patients' surgery is performed to prevent extravasation of blood into the subarachnoid space. Cerebral vasospasm (CVS; narrowing of cerebral arteries) occurs following SAH and represents a major cause of associated mortality and morbidity. To improve postsurgery care of SAH patients and their prognosis, the ability to predict CVS onset is critical. We report a long noncoding RNA (lncRNA) signature to distinguish SAH patients with CVS from SAH patients without CVS. Cerebrospinal fluid (CSF) was obtained from SAH patients without CVS (n = 10) and SAH patients with CVS (n = 10). lncRNAs ZFAS1 and MALAT1 were significantly upregulated (p < 0.05), whereas lncRNAs LINC00261 and LINC01619 were significantly downregulated in SAH patients with CVS (p < 0.05) compared to SAH patients without CVS. We applied this lncRNA signature to retrospectively predict CVS in SAH patients (n = 38 for SAH patients without CVS, and n = 27 for SAH patients with CVS). The 4-lncRNA signature was found to be predictive in >40% of samples and the 2-lncRNA comprising MALAT1 and LINC01619 accurately predicted CVS in ∼90% cases. These results are initial steps toward personalized management of SAH patients in clinics and provide novel CSF biomarkers that can substantially improve the clinical management of SAH patients.

Visualized nutrition education and dietary behavioral change: A systematic review and meta-analysis.

Effectiveness of using visual approaches in health education and its influential factors were still in debate. This study aimed to asess the effects of visualized nutrition education on dietary knowledge and behavioral changes, and factors influencing them. A comprehensive search of PubMed, EMBASE, Scopus, and Cochrane Library was conducted. Eligible studies were trials assessed effects of visualized nutrition education on dietary knowledge or behavior changes, compared with non-visualized or no education group. Fourteen studies (n = 7,259) were qualitatively analyzed and 7 of them were included in the meta-analysis. We found a higher fiber intake in both short term (1.59 g/1000 kcals, 95% CI 0.90-2.27) and long term (1.36 g/1000 kcals, 95% CI 0.64-2.09). A marginal advantage was shown in short-term fruits and vegetables consumption (F&V consumption) (standardized mean difference [SMD] = 0.08, 95% CI -0.00 to 0.16). The education effects were more pronounced when education was delivered in series (SMDF&V consumption = 0.09, 95% CI 0.00-0.17), avoiding loss-framing (SMDFat intake = 0.31, 95% CI 0.10-0.51) and video modeling (SMDF&V consumption = 0.23, 95% CI 0.03-0.43), with short length plus cultural adaptation. Visualized nutrition education was overall promising in improving dietary behaviors. Delivering in series, short in length, with cultural adaptation were features tended to enlarge the benefits of visualized education while loss-framing and video modeling might be avoided.

Fsh-Pc-Sce complex mediates active transcription of Cubitus interruptus (Ci).

The Hedgehog (Hh) signaling pathway plays important roles in both embryonic development and adult tissue homeostasis. Such biological functions are mediated by the transcription factor Cubitus interruptus (Ci). Yet the transcriptional regulation of the effector Ci itself is poorly investigated. Through an RNAi-based genetic screen, we identified that female sterile (1) homeotic (Fsh), a transcription co-activator, directly activates Ci transcription. Biochemistry assays demonstrated physical interactions among Fsh, Sex combs extra (Sce), and Polycomb (Pc). Functional assays further showed that both Pc and Sce are required for Ci expression, which is not likely mediated by the derepression of Engrailed (En), a repressor of Ci, in Pc or Sce mutant cells. Finally, we provide evidence showing that Pc/Sce facilitates the binding of Fsh at Ci locus and that the physical interaction between Fsh and Pc is essential for Fsh-mediated Ci transcription. Taken together, we not only uncover that Ci is transcriptionally regulated by Fsh-Pc-Sce complex but also provide evidence for the coordination between Fsh and PcG proteins in transcriptional regulation.

UbcD1 regulates Hedgehog signaling by directly modulating Ci ubiquitination and processing.

The Hh pathway controls many morphogenetic processes in metazoans and plays important roles in numerous pathologies and in cancer. Hh signaling is mediated by the activity of the Gli/Ci family of transcription factors. Several studies in Drosophila have shown that ubiquitination by the ubiquitin E3 ligases Slimb and Rdx(Hib) plays a crucial role in controlling Ci stability dependent on the levels of Hh signals. If Hh levels are low, Slimb adds K11- and K48-linked poly-ubiquitin chains on Ci resulting in partial degradation. Ubiquitin E2 enzymes are pivotal in determining the topologies of ubiquitin chains. However, which E2 enzymes participate in the selective ubiquitination-degradation of Ci remains elusive. Here, we find that the E2 enzyme UbcD1 negatively regulates Hh signaling activity in Drosophila wing disks. Genetic and biochemical analyses in wing disks and in cultured cells reveal that UbcD1 directly controls Ci stability. Interestingly, UbcD1 is found to be selectively involved in Slimb-mediated Ci degradation. Finally, we show that the homologs of UbcD1 play a conserved role in modulating Hh signaling in vertebrates.

Repression of Abd-B by Polycomb is critical for cell identity maintenance in adult Drosophila testis.

Hox genes play a fundamental role in regulating animal development. However, less is known about their functions on homeostasis maintenance in adult stem cells. Here, we report that the repression of an important axial Hox gene, Abdominal-B (Abd-B), in cyst stem cells (CySCs) is essential for the homeostasis and cell identity maintenance in the adult Drosophila testis. Derepression of Abd-B in CySCs disrupts the proper self-renewal of both germline stem cells (GSCs) and CySCs, and leads to an excessive expansion of early stage somatic cells, which originate from both lineages. We further demonstrate that canonical Polycomb (Pc) and functional pathway of Polycomb group (PcG) proteins are responsible for maintaining the germline cell identity non-autonomously via repressing Abd-B in CySCs in the adult Drosophila testis.

NRF2 Plays a Critical Role in Both Self and EGCG Protection against Diabetic Testicular Damage.

Activation of nuclear factor erythroid 2-related factor 2 (NRF2) has been found to ameliorate diabetic testicular damage (DTD) in rodents. However, it was unclear whether NRF2 is required for these approaches in DTD. Epigallocatechin gallate (EGCG) is a potent activator of NRF2 and has shown beneficial effects on multiple diabetic complications. However, the effect of EGCG has not been studied in DTD. The present study aims to explore the role of NRF2 in both self and EGCG protection against DTD. Therefore, streptozotocin-induced diabetic C57BL/6 wild type (WT) and Nrf2 knockout (KO) mice were treated in the presence or absence of EGCG, for 24 weeks. The Nrf2 KO mice exhibited more significant diabetes-induced loss in testicular weight and spermatozoa count, and increase in testicular apoptotic cell death, as compared with the WT mice. EGCG activated NRF2 expression and function, preserved testicular weight and spermatozoa count, and attenuated testicular apoptotic cell death, endoplasmic reticulum stress, inflammation, and oxidative damage in the WT diabetic mice, but not the Nrf2 KO diabetic mice. The present study demonstrated for the first time that NRF2 plays a critical role in both self and EGCG protection against DTD.

SUMO regulates somatic cyst stem cell maintenance and directly targets the Hedgehog pathway in adult Drosophila testis.

SUMO (Small ubiquitin-related modifier) modification (SUMOylation) is a highly dynamic post-translational modification (PTM) that plays important roles in tissue development and disease progression. However, its function in adult stem cell maintenance is largely unknown. Here, we report the function of SUMOylation in somatic cyst stem cell (CySC) self-renewal in adult Drosophila testis. The SUMO pathway cell-autonomously regulates CySC maintenance. Reduction of SUMOylation promotes premature differentiation of CySCs and impedes the proliferation of CySCs, which leads to a reduction in the number of CySCs. Consistent with this, CySC clones carrying a mutation of the SUMO-conjugating enzyme are rapidly lost. Furthermore, inhibition of the SUMO pathway phenocopies disruption of the Hedgehog (Hh) pathway, and can block the proliferation of CySCs induced by Hh activation. Importantly, the SUMO pathway directly regulates the SUMOylation of Hh pathway transcription factor Cubitus interruptus (Ci), which is required for promoting CySC proliferation. Thus, we conclude that SUMO directly targets the Hh pathway and regulates CySC maintenance in adult Drosophila testis.

A positive role for polycomb in transcriptional regulation via H4K20me1.

The highly conserved polycomb group (PcG) proteins maintain heritable transcription repression of the genes essential for development from fly to mammals. However, sporadic reports imply a potential role of PcGs in positive regulation of gene transcription, although systematic investigation of such function and the underlying mechanism has rarely been reported. Here, we report a Pc-mediated, H3K27me3-dependent positive transcriptional regulation of Senseless (Sens), a key transcription factor required for development. Mechanistic studies show that Pc regulates Sens expression by promoting H4K20me1 at the Sens locus. Further bioinformatic analysis at genome-wide level indicates that the existence of H4K20me1 acts as a selective mark for positive transcriptional regulation by Pc/H3K27me3. Both the intensities and specific patterns of Pc and H3K27me3 are important for the fates of target gene transcription. Moreover, binding of transcription factor Broad (Br), which physically interacts with Pc and positively regulates the transcription of Sens, is observed in Pc(+)H3K27me3(+)H4K20me1(+) genes, but not in Pc(+)H3K27me3(+)H4K20me1(-) genes. Taken together, our study reveals that, coupling with the transcription factor Br, Pc positively regulates transcription of Pc(+)H3K27me3(+)H4K20me1(+) genes in developing Drosophila wing disc.

Antagonistic roles of Nibbler and Hen1 in modulating piRNA 3' ends in Drosophila.

In eukaryotes, aberrant expression of transposable elements (TEs) is detrimental to the host genome. Piwi-interacting RNAs (piRNAs) of ∼23 to 30 nucleotides bound to PIWI clade Argonaute proteins silence transposons in a manner that is strictly dependent on their sequence complementarity. Hence, a key goal in understanding piRNA pathways is to determine mechanisms that modulate piRNA sequences. Here, we identify a protein-protein interaction between the 3'-to-5' exoribonuclease Nibbler (Nbr) and Piwi that links Nbr activity with piRNA pathways. We show that there is a delicate balance in the interplay between Nbr and Hen1, a methyltransferase involved in 2'-O-methylation at the 3' terminal nucleotides of piRNAs, thus connecting two genes with opposing activities in the biogenesis of piRNA 3' ends. With age, piRNAs become shorter and fewer in number, which is coupled with the derepression of select TEs. We demonstrate that activities of Nbr and Hen1 inherently contribute to TE silencing and age-dependent profiles of piRNAs. We propose that antagonistic roles of Nbr and Hen1 define a mechanism to modulate piRNA 3' ends.

Gut-neuron interaction via Hh signaling regulates intestinal progenitor cell differentiation in Drosophila.

Intestinal homeostasis is maintained by intestinal stem cells (ISCs) and their progenies. A complex autonomic nervous system spreads over posterior intestine. However, whether and how neurons regulate posterior intestinal homeostasis is largely unknown. Here we report that neurons regulate Drosophila posterior intestinal homeostasis. Specifically, downregulation of neuronal Hedgehog (Hh) signaling inhibits the differentiation of ISCs toward enterocytes (ECs), whereas upregulated neuronal Hh signaling promotes such process. We demonstrate that, among multiple sources of Hh ligand, those secreted by ECs induces similar phenotypes as does neuronal Hh. In addition, intestinal JAK/STAT signaling responds to activated neuronal Hh signaling, suggesting that JAK/STAT signaling acts downstream of neuronal Hh signaling in intestine. Collectively, our results indicate that neuronal Hh signaling is essential for the determination of ISC fate.