Diagnosis and functional prediction of microbial markers in tumor tissues of sporadic colorectal cancer patients associated with the MLH1 protein phenotype

ObjectiveMost patients with sporadic colorectal cancer (SCRC) develop microsatellite instability because of defects in mismatch repair (MMR). Moreover, the gut microbiome plays a vital role in the pathogenesis of SCRC. In this study, we assessed the microbial composition and diversity of SCRC tumors with varying MutL protein homolog 1 (MLH1) status, and the effects of functional genes related to bacterial markers and clinical diagnostic prediction.MethodsThe tumor microbial diversity and composition were profiled using high-throughput sequencing of the 16S ribosomal RNA (rRNA) gene V4 region. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) software and BugBase tool were used to predict the functional roles of the microbiome. We aimed to construct a high-accuracy model to detect and evaluate the area under the receiver operating characteristic curve with candidate biomarkers.ResultsThe study included 23 patients with negative/defective MLH1 (DM group) and 22 patients with positive/intact MLH1 (IM group). Estimation of alpha diversity indices showed that the Shannon index (p = 0.049) was significantly higher in the DM group than in the controls, while the Simpson index (p = 0.025) was significantly lower. At the genus level, we observed a significant difference in beta diversity in the DM group versus the IM group. Moreover, the abundance of Lachnoclostridium spp. and Coprococcus spp. was significantly more enriched in the DM group than in the IM group (q < 0.01 vs. q < 0.001). When predicting metagenomes, there were 18 Kyoto Encyclopedia of Genes and Genomes pathways and one BugBase function difference in both groups (all q < 0.05). On the basis of the model of diagnostic prediction, we built a simplified optimal model through stepwise selection, consisting of the top two bacterial candidate markers (area under the curve = 0.93).ConclusionIn conclusion, the genera Lachnoclostridium and Coprococcus as key species may be crucial biomarkers for non-invasive diagnostic prediction of DM in patients with SCRC in the future

The Scorpion Toxin Analogue BmKTX-D33H as a Potential Kv1.3 Channel-Selective Immunomodulator for Autoimmune Diseases

The Kv1.3 channel-acting scorpion toxins usually adopt the conserved anti-parallel β-sheet domain as the binding interface, but it remains challenging to discover some highly selective Kv1.3 channel-acting toxins. In this work, we investigated the pharmacological profile of the Kv1.3 channel-acting BmKTX-D33H, a structural analogue of the BmKTX scorpion toxin. Interestingly, BmKTX-D33H, with its conserved anti-parallel β-sheet domain as a Kv1.3 channel-interacting interface, exhibited more than 1000-fold selectivity towards the Kv1.3 channel as compared to other K+ channels (including Kv1.1, Kv1.2, Kv1.7, Kv11.1, KCa2.2, KCa2.3, and KCa3.1). As expected, BmKTX-D33H was found to inhibit the cytokine production and proliferation of both Jurkat cells and human T cells in vitro. It also significantly improved the delayed-type hypersensitivity (DTH) responses, an autoreactive T cell-mediated inflammation in rats. Amino acid sequence alignment and structural analysis strongly suggest that the “evolutionary” Gly11 residue of BmKTX-D33H interacts with the turret domain of Kv1 channels; it appears to be a pivotal amino acid residue with regard to the selectivity of BmKTX-D33H towards the Kv1.3 channel (in comparison with the highly homologous scorpion toxins). Together, our data indicate that BmKTX-D33H is a Kv1.3 channel–specific blocker. Finally, the remarkable selectivity of BmKTX-D33H highlights the great potential of evolutionary-guided peptide drug design in future studies

hERG Potassium Channel Blockage by Scorpion Toxin BmKKx2 Enhances Erythroid Differentiation of Human Leukemia Cells K562

<div><p>Background</p><p>The hERG potassium channel can modulate the proliferation of the chronic myelogenous leukemic K562 cells, and its role in the erythroid differentiation of K562 cells still remains unclear.</p> <p>Principal Findings</p><p>The hERG potassium channel blockage by a new 36-residue scorpion toxin BmKKx2, a potent hERG channel blocker with IC₅₀ of 6.7±1.7 nM, enhanced the erythroid differentiation of K562 cells. The mean values of GPA (CD235a) fluorescence intensity in the group of K562 cells pretreated by the toxin for 24 h and followed by cytosine arabinoside (Ara-C) treatment for 72 h were about 2-fold stronger than those of K562 cells induced by Ara-C alone. Such unique role of hERG potassium channel was also supported by the evidence that the effect of the toxin BmKKx2 on cell differentiation was nullified in hERG-deficient cell lines. During the K562 cell differentiation, BmKKx2 could also suppress the expression of hERG channels at both mRNA and protein levels. Besides the function of differentiation enhancement, BmKKx2 was also found to promote the differentiation-dependent apoptosis during the differentiation process of K562 cells. In addition, the blockage of hERG potassium channel by toxin BmKKx2 was able to decrease the intracellular Ca²⁺ concentration during the K562 cell differentiation, providing an insight into the mechanism of hERG potassium channel regulating this cellular process. </p> <p>Conclusions/Significance</p><p>Our results revealed scorpion toxin BmKKx2 could enhance the erythroid differentiation of leukemic K562 cells via inhibiting hERG potassium channel currents. These findings would not only accelerate the functional research of hERG channel in different leukemic cells, but also present the prospects of natural scorpion toxins as anti-leukemic drugs.</p> </div

Scorpion toxin BmKKx2 primary structure and its pharmacological effect on the hERG channel.

<p>(A) the sequence alignments between scorpion toxins BmKKx2 and BeKm-1 [11,19]. (B) The pharmacological effect of BmKKx2 on hERG channels. The hERG channels were transfected in HEK 293 cells, and their current traces were shown in the absence (control) or presence 10 nM BmKKx2. (C) Dose-dependence curve of BmKKx2 on hERG channels expressed in HEK 293 cells. Symbols and associated error bars represent means ± SD for several cells (n=5). (D) The current trace of hERG potassium channels in absence of BmKKx2 in wild type K562 cells; (E) The pharmacological effect of BmKKx2 on hERG channels in wild type K562 cells. Current traces were shown in the absence (control) or presence 1 μM BmKKx2 .</p

BmKKx2 binding causing the Ca²⁺ concentration decrease during the erythroid differentiation of K562 cells.

<p>(A) Intracellular Ca²⁺ was stained by Fluo-8, and the Ca²⁺ concentration was measured by flow cytometric analysis. The mean values were mean ± SD from three independent experiments. **<i>p</i><0.01 (Student’s <i>t</i>-test). (B) Flow cytometric analysis of Fluo-8 fluorescence intensity in K562 cells with or without BmKKx2 after Ara-C induced for 24 h.</p

Advanced Structural Analysis of Innovative Steel–Glass Structures with Respect to the Architectural Design

This paper provides a comprehensive analysis of a steel–glass spindle torus structure based on the prototype of the Jewel Changi Airport, Singapore. Instead of studying a common cuboid building, the research in this paper focuses on a spindle torus shape structure which incorporates tremendous, curved members. Hence, the advanced modeling and structural analysis of this structure provides valuable information about an irregularly shaped building. Meanwhile, the modeling and analysis process of this innovative structure also gives rise to some practical design recommendations for both architects and engineers. In this paper, both global structure stability and local member buckling behavior were studied. With the use of commercial finite element software, Strand7 (R2.4.6) and ABAQUS (6.14), a series of numerical simulations were conducted. In terms of the behavior of the global structure, both numerical spindle torus models incorporating straight and curved steel members were tested under different load combinations specified in Australian building standards. A significant difference was observed between the results of the two models; therefore, research on the individual curved members was undertaken. Regarding the local member buckling behavior, the effective length factor for curved members with braced and sway boundaries conditions was investigated in Strand7. Moreover, the interaction curves of curved beams with different L/R ratios were compared with perfectly straight members in Australian building standards. ABAQUS can provide more precise predictions of local buckling behavior; therefore, the elastic local buckling behavior of the perimeter beams on different levels was investigated using ABAQUS. Additionally, the impacts of boundary conditions and L/R ratios on the beam buckling behavior are discussed

Induction of apoptosis by BmKKx2 during the erythroid differentiation of K562 cells.

<p>(A-B) K562 cells were stained with annexin V-FITC and PI and analyzed by flow cytometry. K562 cells were treated with BmKKx2, and BSA was used as the control for 48 h (A). K562 cells were treated with Ara-C (1 μM) and Ara-C (1 μM)+ BmKKx2 for 48 h (B). Flow cytometry data show representative results from one of three independent experiments. (C) Apoptotic cells were stained with annexin V-FITC and PI analyzed by flow cytometry. Values were mean ± SD from three experiments. *<i>p</i><0.05 (Student’s <i>t</i>-test).</p

No effect of BmKKx2 on the erythroid differentiation in the hERG channel-deficient K562 cells.

<p>(A) hERG channel expression in hERG-silenced and control cells shown by quantitative real-time PCR. (B) hERG channel expression shown by western blotting analysis. HSC70 was used as the endogenous control. (C) BmKKx2 enhancing the differentiation in the lentiviral vector-infected control cells. (D) BmKKx2 with no effect on the differentiation of the hERG-silenced cells. K562 cells in (C) and (D) were treated with Ara-C for 48 h. The right panel of (C) and (D) showed the mean values of GPA fluorescence from three independent samples. **<i>p</i><0.01 (Student’s <i>t</i>-test).</p