The Lactulose Breath Test Can Predict Refractory Gastroesophageal Reflux Disease by Measuring Bacterial Overgrowth in the Small Intestine.

OBJECTIVE: The diagnosis of RGERD in patients typically involves 24-hour esophageal pH monitoring, but due to its invasiveness and low patient compliance, new screening methods are needed. In this study, a lactulose breath test (LBT) was conducted to detect the growth of small intestine bacteria (SIBO) and explore the potential relationship between LBT and RGERD to identify a new treatment method for RGERD. METHODS: A total of 178 patients with gastroesophageal reflux were enrolled from June 2020 to December 2022 in the Gastroenterology Department, Building 3, the First Affiliated Hospital of Kunming Medical University; these patients included 96 patients with nonrefractory GERD (NRGERD) and 82 patients with RGERD. The Gerd Q score, reflux symptom index (RSI) score, gastroscopy results, clinical symptoms, and other related indicators were collected. Statistical methods were used to analyze the gathered data. RESULTS: The incidence of acid reflux and heartburn in patients with RGERD was significantly greater than that in patients with NRGERD (67.10% vs. 42.70%, P<0.01 and 65.00% vs. 34.40%, P<0.01). The CH4 values of patients with RGERD were significantly greater than those of patients with NRGERD at each time point, and there was a correlation between the CH4 values at 60 min and RGERD (P<0.05). For patients with RGERD, the incidence of abdominal pain, acid regurgitation, and heartburn was greater in the CH4-positive group than in the CH4-negative group (61.90% vs. 57.50%, 69.05% vs. 65.00%, 69.05% vs. 57.50%, P>0.05). The incidence of nausea was also greater in the CH4-positive group than in the CH4-negative group (61.90% vs. 35.00%, P<0.05). CONCLUSION: Increased CH4 levels are correlated with RGERD. In addition, patients with RGERD may develop SIBO after long-term use of PPIs, and interventions involving SIBO could provide new ideas for the treatment of RGERD.

Photoactivatable Nanobody Conjugate Dimerizer Temporally Resolves Tiam1-Rac1 Signaling Axis.

The precise spatiotemporal dynamics of protein activities play a crucial role in cell signaling pathways. To control cellular functions in a spatiotemporal manner, a powerful method called photoactivatable chemically induced dimerization (pCID) is used. In this study, photoactivatable nanobody conjugate inducers of dimerization (PANCIDs) is introduced, which combine pCID with nanobody technology. A PANCID consists of a nanobody module that directly binds to an antigenic target, a photocaged small molecule ligand, and a cyclic decaarginine (cR10 *) cell-penetrating peptide (CPP) for efficient nonendocytic intracellular delivery. Therefore, PANCID photodimerizers also benefit from nanobodies, such as their high affinities (in the nm or pm range), specificities, and ability to modulate endogenous proteins. Additionally it is demonstrated that the nanobody moiety can be easily replaced with alternative ones, expanding the potential applications. By using PANCIDs, the dynamics of the Tiam1-Rac1 signaling cascade is investigated and made an interesting finding. It is found that Rac1 and Tiam1 exhibit distinct behaviors in this axis, acting as time-resolved "molecular oscillators" that transit between different functions in the signaling cascade when activated either slowly or rapidly.

ATNC: Versatile Nanobody Chimeras for Autophagic Degradation of Intracellular Unligandable and Undruggable Proteins.

Targeted protein degradation (TPD) through the autophagy pathway displays broad substrate scope and is gaining increasing interest in biology and medicine. However, current approaches using small-molecule degraders have limitations due to the lack of versatility, modularity, and ease of implementation and are restricted to addressing only ligandable proteins. Herein, we report a nonsmall molecule-based autophagy-targeting nanobody chimera (ATNC), or phagobody, for selective degradation of intracellular targets, which overcomes these limitations. The core of an ATNC features a nanobody for recruiting proteins as well as an autophagic pathway-directing module. ATNC turns out to be a general, modular, and versatile degradation platform. We show that ATNC can be versatilely implemented in different ways including expressed ATNC intrabodies for ease of use, chemically induced proximity (CIP)-operated logic-gated conditional and tunable degradation, and cyclic cell-penetrating peptide-tethered cell-permeable phagobodies that selectively degrade the undruggable therapeutically relevant HE4 protein, resulting in effective suppression of ovarian cancer cell proliferation and migration. Overall, ATNC represents a general, modular, and versatile targeted degradation platform that degrades unligandable proteins and offers therapeutic potential.

Potential inhibitors for blocking the interaction of the coronavirus SARS-CoV-2 spike protein and its host cell receptor ACE2.

BACKGROUND: The outbreak of SARS-CoV-2 continues to pose a serious threat to human health and social. The ongoing pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made a serious threat to public health and economic stability worldwide. Given the urgency of the situation, researchers are attempting to repurpose existing drugs for treating COVID-19. METHODS: We first established an anti-coronavirus drug screening platform based on the Homogeneous Time Resolved Fluorescence (HTRF) technology and the interaction between the coronavirus spike protein and its host receptor ACE2. Two compound libraries of 2,864 molecules were screened with this platform. Selected candidate compounds were validated by SARS-CoV-2_S pseudotyped lentivirus and ACE2-overexpressing cell system. Molecular docking was used to analyze the interaction between S protein and compounds. RESULTS: We identified three potential anti-coronavirus compounds: tannic acid (TA), TS-1276 (anthraquinone), and TS-984 (9-Methoxycanthin-6-one). Our in vitro validation experiments indicated that TS-984 strongly inhibits the interaction of the coronavirus S protein and the human cell ACE2 receptor. Additionally, tannic acid showed moderate inhibitory effect on the interaction of S protein and ACE2. CONCLUSION: This platform is a rapid, sensitive, specific, and high throughput system, and available for screening large compound libraries. TS-984 is a potent blocker of the interaction between the S-protein and ACE2, which might have the potential to be developed into an effective anti-coronavirus drug.

Structure of Epstein-Barr virus tegument protein complex BBRF2-BSRF1 reveals its potential role in viral envelopment.

Epstein-Barr virus (EBV) is a γ-herpesvirus associated with the occurrence of several human malignancies. BBRF2 and BSRF1 are two EBV tegument proteins that have been suggested to form a hetero-complex and mediate viral envelopment, but the molecular basis of their interaction and the functional mechanism of this complex remains unknown. Here, we present crystal structures of BBRF2 alone and in complex with BSRF1. BBRF2 has a compact globular architecture featuring a central ß-sheet that is surrounded by 10 helices, it represents a novel fold distinct from other known protein structures. The central portion of BSRF1 folds into two tightly associated antiparallel α-helices, forming a composite four-helix bundle with two α-helices from BBRF2 via a massive hydrophobic network. In vitro, a BSRF1-derived peptide binds to BBRF2 and reduces the number of viral genome copies in EBV-positive cells. Exogenous BBRF2 and BSRF1 co-localize at the Golgi apparatus. Furthermore, BBRF2 binds capsid and capsid-associated proteins, whereas BSRF1 associates with glycoproteins. These findings indicate that the BBRF2-BSRF1 complex tethers EBV nucleocapsids to the glycoprotein-enriched Golgi membrane, facilitating secondary envelopment.

SOX1 promotes differentiation of nasopharyngeal carcinoma cells by activating retinoid metabolic pathway.

Undifferentiation is a key feature of nasopharyngeal carcinoma (NPC), which presents as a unique opportunity for intervention by differentiation therapy. In this study, we found that SOX1 inhibited proliferation, promoted differentiation, and induced senescence of NPC cells, which depended on its transcriptional function. RNA-Seq-profiling analysis showed that multiple undifferentiated markers of keratin family, including KRT5, KRT13, and KRT19, were reduced in SOX1 overexpressed NPC cells. Interestingly, gene ontology (GO) analysis revealed genes in SOX1 overexpressed cells were enriched in extracellular functions. The data of LC/MS untargeted metabolomics showed that the content of retinoids in SOX1 overexpressed cells and culture medium was both higher than that in the control group. Subsequently, we screened mRNA level of genes in retinoic acid (RA) signaling or metabolic pathway and found that the expression of UDP-glucuronosyltransferases was significantly decreased. Furtherly, UGT2B7 could rescue the differentiation induced by SOX1 overexpression. Inhibition of UGTs by demethylzeylasteral (T-96) could mimic SOX1 to promote the differentiation of NPC cells. Thus, we described a mechanism by which SOX1 regulated the differentiation of NPC cells by activating retinoid metabolic pathway, providing a potential target for differentiation therapy of NPC.

MicroRNA-494-3p Promotes Cell Growth, Migration, and Invasion of Nasopharyngeal Carcinoma by Targeting Sox7.

BACKGROUND: There is mounting evidence that microRNAs play an important role in nasopharyngeal carcinoma, which is widely prevalent in South China and is the most prevalent metastatic cancer among head and neck cancers. Recently, it has been shown that miR-494 is involved in the progression and prognosis of nasopharyngeal carcinoma. However, little is known about the function and mechanism of miR-494-3p in nasopharyngeal carcinoma. In the present study, we aimed to investigate the effects of miR-494-3p on the migration and invasion of nasopharyngeal carcinoma and to further explore the underlying mechanisms of these processes. METHODS: The expression levels of miR-494-3p and Sox7 in nasopharyngeal carcinoma specimens and nasopharyngeal carcinoma cell lines were measured using quantitative reverse transcription polymerase chain reaction. Luciferase reporter assay, quantitative reverse transcription polymerase chain reaction, and Western blotting were used to confirm whether Sox7 was a direct target of miR-494-3p. Additionally, the roles of miR-494-3p and Sox7 on cell proliferation, migration, and invasion of nasopharyngeal carcinoma were analyzed by Cell Counting Kit-8 (CCK-8) assay, wound healing assay, and Boyden chamber assay, respectively. RESULTS: Our study demonstrated that miR-494-3p was commonly upregulated in nasopharyngeal carcinoma specimens and nasopharyngeal carcinoma cell lines compared with nontumor nasopharyngeal epithelial tissue or nasopharyngeal cells (NP69). Moreover, miR-494-3p negatively regulated Sox7 at the posttranscriptional level by binding to a specific site in the Sox7 3'-untranslated region. In addition, synthetic miR-494-3p mimics significantly promoted proliferation, migration, and invasion of S18 and S26 nasopharyngeal carcinoma cells, while a synthetic miR-494-3p inhibitor resulted in suppressed nasopharyngeal carcinoma cell migration and invasion. CONCLUSION: miR-494-3p promotes nasopharyngeal carcinoma cell growth, migration, and invasion by directly targeting Sox7. Our results suggest that miR-494-3p might be a potential therapeutic target for nasopharyngeal carcinoma.

[Synergistic Antitumor Effect of Amorphigenin Combined with Cisplatin in Human Lung Adenocarcinoma A549/DDP Cells].

BACKGROUND: Amorphigenin, a rotenoid compouns, from seeds of Amorpha fruticosa, has been shown to possess anti-proliferation activities in several cancer cells. To explore the antitumor effects of amorphigenin on cisplatin-resistant human lung adenocarcinoma A549/DDP cells and explore the underlying mechanisms. METHODS: CCK-8 assay was used to measure the proliferation of A549/DDP cells; Colony formation assay was used to measure the colony formation of A549/DDP cells; Flow cytometry assay was used to detect the apoptosis rates; Western blot analysis was used to explore the expression of apoptosis-related proteins (caspase-3 protein, PARP protein) and lung resistance protein (LRP). RESULTS: Our results demonstrated that amorphigenin could inhibit the proliferation of A549/DDP cells with a inhibition concentration of 50% cell growth (IC50) at 48 h of (2.19±0.92) µmol/L. Amorphigenin could inhibit the colony formation ability and induce apoptosis of A549/DDP cells; Furthermore, amorphigenin combined with cisplatin showed synergistic proliferation-inhibitory effect and apoptosis-promoting effect in A549/DDP cells; reduced the expression of LRP of A549/DDP cells. CONCLUSIONS: Amorphigenin remarkably inhibits the proliferation and induces apoptosis in A549/DDP cells. Combination of amorphigenin with cisplatin had the synergistic inhibitory effect on A549/DDP cells by downregulating the expression of LRP.
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Clinical significance of microRNA-155 expression in hepatocellular carcinoma.

The present study aimed to evaluate the expression of microRNA-155 (miR-155) in hepatocellular carcinoma (HCC) and adjacent normal tissues, and assess its correlation with clinicopathological characteristics of this tumor type. miR-155 expression was detected in 40 HCC tissue samples and 40 samples of adjacent tumor-free tissue using fluorescent reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The association between miR-155 expression, clinicopathological features and 1-year relapse-free survival (RFS) in HCC and adjacent normal tissue samples was analyzed. RT-qPCR results revealed that, in 25 cases (62.5%), miR-155 expression levels were significantly increased in HCC tissues compared with the expression levels observed in pericarcinomatous tissues (P<0.05). miR-155 expression was observed to be significantly correlated with vessel invasion, Edmonson classification and clinical stage (P<0.05). However, miR-155 expression was not significantly correlated with gender, age, tumor size, tumor number, hepatitis B virus DNA copy number, cirrhosis or concentration of α-fetoprotein (P>0.05). A positive correlation was observed between late TNM classification of malignant tumor stage and 1-year RFS (P<0.05). Patients exhibiting high miR-155 expression levels were observed to exhibit a lower 1-year RFS than that of patients with reduced expression of miR-155 (48 vs. 73.3%), however this difference was not statistically significant (P=0.105). Additionally, correlations were observed between miR-155 expression and reduced differentiation, increased invasiveness and late stages of HCC. The current results demonstrated that miR-155 may be involved in the tumorigenesis of HCC and may be associated with clinical characteristics of HCC patients. Additional studies are required to clarify the mechanism of miR-155.