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Abstract Background Variability in the health effects of dietary fiber might arise from inter-individual differences in the gut microbiota’s ability to ferment these substrates into beneficial metabolites. Our understanding of what drives this individuality is vastly incomplete and will require an ecological perspective as microbiomes function as complex inter-connected communities. Here, we performed a parallel two-arm, exploratory randomized controlled trial in 31 adults with overweight and class-I obesity to characterize the effects of long-chain, complex arabinoxylan (n = 15) at high supplementation doses (female: 25 g/day; male: 35 g/day) on gut microbiota composition and short-chain fatty acid production as compared to microcrystalline cellulose (n = 16, non-fermentable control), and integrated the findings using an ecological framework. Results Arabinoxylan resulted in a global shift in fecal bacterial community composition, reduced α-diversity, and the promotion of specific taxa, including operational taxonomic units related to Bifidobacterium longum, Blautia obeum, and Prevotella copri. Arabinoxylan further increased fecal propionate concentrations (p = 0.012, Friedman’s test), an effect that showed two distinct groupings of temporal responses in participants. The two groups showed differences in compositional shifts of the microbiota (p ≤ 0.025, PERMANOVA), and multiple linear regression (MLR) analyses revealed that the propionate response was predictable through shifts and, to a lesser degree, baseline composition of the microbiota. Principal components (PCs) derived from community data were better predictors in MLR models as compared to single taxa, indicating that arabinoxylan fermentation is the result of multi-species interactions within microbiomes. Conclusion This study showed that long-chain arabinoxylan modulates both microbiota composition and the output of health-relevant SCFAs, providing information for a more targeted application of this fiber. Variation in propionate production was linked to both compositional shifts and baseline composition, with PCs derived from shifts of the global microbial community showing the strongest associations. These findings constitute a proof-of-concept for the merit of an ecological framework that considers features of the wider gut microbial community for the prediction of metabolic outcomes of dietary fiber fermentation. This provides a basis to personalize the use of dietary fiber in nutritional application and to stratify human populations by relevant gut microbiota features to account for the inconsistent health effects in human intervention studies. Trial registration Clinicaltrials.gov, NCT02322112 , registered on July 3, 2015. Video Abstract

Varicocele is one of the main reasons for male infertility the exact aetiology of which remains unclear. Methylenetetrahydrofolate reductase ( MTHFR) is important for DNA synthesis and methylation, which has a key role during spermatogenesis. Numerous literature suggests that the MTHFR polymorphism may be genetic risk factors for male infertility. In this study, we evaluated C677 T and A1298 C MTHFR gene polymorphism frequency in patients with varicocele and normal men. A total of 107 varicocele patients and 109 fertile healthy individuals were included. Genotyping of the MTHFR gene in C677 T and A1298 C base pairs carried out by using real-time PCR technique and afterwards, the statistical analysis accomplished. There is a statistical difference for the frequency of 1298 AA genotype in patients with varicocele compared with normal controls ( P = 0.0051, OR = 2.2750). Instead, subsequently, 1298/ A allel frequency in patient group was significantly higher in comparison with control group ( P = 0.0174). According to our results, 1298 AA genotype in MTHFR gene raises the risk of varicocele approximately 2.3 times more compared with men carrying other genotypes. The results show that genetic factors have an important role in the molecular basis of varicocele. [ABSTRACT FROM AUTHOR]

Titanium dioxide (TiO2) nanoparticles (NPs) are commonly used materials present in many consumables for which most people are exposed to. The biological hazards of the NPs on human health have been demonstrated previously. In this study, we aimed to assess the cytotoxicity potency of TiO2 NPs on the primary human amniotic fluid cells. The cells derived from amniotic fluid were treated with different dosages of TiO2 NPs for some periods. Cell adhesion status was assessed using a light microscopic observation. Cell proliferation and cell death rates were determined using trypan blue staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Also, mitotic index was determined using fluorescence in situ hybridization with chromosome 8 centromer-specific DNA probe. Disrupted cell adhesion, decreased proliferation, and increased mortality rates were detected in the cells that were treated with TiO2 NPs depending on the dosage (p < 0.001). Also, reduced mitotic index was determined in the cells depending on the time and TiO2 dosage when compared with the controls (p < 0.0001). These results showed that TiO2 NPs have high cytotoxicity for amniotic fluid-derived cells. Therefore, different products containing TiO2 NPs should be used with care, especially for pregnant women. [ABSTRACT FROM PUBLISHER]

Abstract: The effect of simultaneous alloying with Ca and rare earth (RE) elements on the age hardening kinetics of AZ91 was studied through the fitting of the Johnson–Mehl–Avrami (JMA) equation. The results showed that the addition of both Ca and RE elements not only suppress discontinuous precipitation of the Mg17Al12 phase during the age hardening process, but also decrease the alloy hardness. Fitting the JMA equation to the experimental data indicated that the phase transformation during age hardening of an alloy variant containing both Ca and RE (at 170°C and 190°C) and standard AZ91 (at 170°C) takes place by the nucleation of precipitates on dislocations. In contrast, the precipitation during age hardening of AZ91 at 190°C occurs via nucleation at grain boundaries. Although it was observed that the creep strength of age hardened specimens are lower than that of the as cast specimens, but age hardening treatment has lower deleterious influence on the creep resistance of the alloy containing Ca and RE in comparison with conventional AZ91. This may be ascribed to the decreased precipitation rate resulting from the addition of both Ca and RE elements. [Copyright &y& Elsevier]

Overexpression of HER2 occurs in 25% of breast cancer. Targeting HER2 has proven to be an effective therapeutic strategy for HER2-positive breast cancer. While trastuzumab is the most commonly used HER2 targeting agent, which has significantly improved outcomes, the overall response rate is low. To develop novel therapies to boost trastuzumab efficacy, it is critical to identify the mechanisms underlying trastuzumab action and resistance. We recently showed that the inhibition of breast cancer cell growth by trastuzumab is not through the inhibition of HER2 canonical signaling. Here we report the identification of a novel non-canonical HER2 signaling pathway and its interference by trastuzumab. We showed that HER2 signaled through a non-canonical pathway, regulated intramembrane proteolysis (RIP). In this pathway, HER2 is first cleaved by metalloprotease ADAM10 to produce an extracellular domain (ECD) that is released and the p95HER2 that contains the transmembrane domain (TM) and intracellular domain (ICD). p95HER2, if further cleaved by an intramembrane protease, γ-secretase, produced a soluble ICD p75HER2 with nuclear localization signal (NLS). p75HER2 is phosphorylated and translocated to the nucleus. Nuclear p75HER2 promotes cell proliferation. Trastuzumab targets this non-canonical HER2 pathway via inhibition of the proteolytic cleavage of HER2 by both ADAM10 and γ-secretase. However, p75HER2 pathway also confers resistance to trastuzumab once aberrantly activated. Combination of trastuzumab with ADAM10 and γ-secretase inhibitors completely blocks p75HER2 production in both BT474 and SKBR3 cells. We concluded that HER2 signals through the RIP signaling pathway that promotes cell proliferation and is targeted by trastuzumab. The aberrant HER2 RIP signaling confers resistance to trastuzumab that could be overcome by the application of inhibitors to ADAM10 and γ-secretase.

Cilia are hairlike protrusions that project from the surface of eukaryotic cells and play key roles in cell signaling and motility. Ciliary motility is regulated by the conserved nexin-dynein regulatory complex (N-DRC), which links adjacent doublet microtubules and regulates and coordinates the activity of outer doublet complexes. Despite its critical role in cilia motility, the assembly and molecular basis of the regulatory mechanism are poorly understood. Here, using cryo-electron microscopy in conjunction with biochemical cross-linking and integrative modeling, we localize 12 DRC subunits in the N-DRC structure of Tetrahymena thermophila. We also find that the CCDC96/113 complex is in close contact with the DRC9/10 in the linker region. In addition, we reveal that the N-DRC is associated with a network of coiled-coil proteins that most likely mediates N-DRC regulatory activity., (© 2023. Springer Nature Limited.)

HER2 receptor tyrosine kinase (encoded by the ERBB2 gene) is overexpressed in approximately 25% of all breast cancer tumors (HER2-positive breast cancers). Resistance to HER2-targeting therapies is partially due to the loss of HER2 expression in tumor cells during treatment. However, little is known about the exact mechanism of HER2 downregulation in HER2-positive tumor cells. Here, by analyzing publicly available genomic data we investigate the hypothesis that epithelial-mesenchymal transition (EMT) abrogates HER2 expression by epigenetic silencing of the ERBB2 gene as a mechanism of acquired resistance to HER2-targeted therapies. As result, HER2 expression was found to be positively and negatively correlated with the expression of epithelial and mesenchymal phenotype marker genes, respectively. The ERBB2 chromatin of HER2-high epithelial-like breast cancer cells and HER2-low mesenchymal-like cells were found to be open/active and closed/inactive, respectively. Decreased HER2 expression was correlated with increased EMT phenotype, inactivated chromatin and lower response to lapatinib. We also found that induction of EMT in the HER2-positive breast cancer cell line BT474 resulted in downregulated HER2 expression and reduced trastuzumab binding. Our results suggest that ERBB2 gene silencing by epigenetic regulation during EMT may be a mechanism of de novo resistance of HER2-positive breast cancer cells to trastuzumab and lapatinib.

Background: Hypertension has been identified as the most common comorbidity in coronavirus disease 2019 (COVID-19) patients, and has been suggested as a risk factor for COVID-19 disease outcomes. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host human cells via binding to host cell angiotensin-converting enzyme 2 (ACE2) receptors. Inhibition of ACE2 has been proposed as a potential therapeutic approach to block SARS-CoV-2 contagion. However, some experts suggest that ACE2 inhibition could worsen the infection. Here, we aimed to study the effect of ACE2 inhibition on the SARS-CoV-2 spike protein binding to ACE2., Method: Crystallographic structures of the SARS-CoV-2 spike protein, the spike receptor-binding domain, native ACE2, and the ACE2 complexed with MLN-4760 were used as the study model structures. The spike proteins were docked to the ACE2 structures and the dynamics of the complexes, ligand-protein, and protein-protein interactions were studied by molecular dynamics simulation for 100 ns., Results: Our result showed that inhibition of ACE2 by MLN-4760 increased the affinity of the SARS-CoV-2 spike protein binding to ACE2. Results also revealed that spike protein binding to the ACE2 inhibited by MLN-4760 restored the enzymatic active conformation of the ACE2 from closed/inactive to open/active conformation by removing MLN-4760 binding from the ligand-binding pocket of ACE2., Conclusion: We conclude that using ACE2 inhibitors can increase the risk of SARS-CoV-2 infection and worsen COVID-19 disease outcome. We also found that the SARS-CoV-2 can abrogate the function of ACE2 inhibitors and rescue the enzymatic activity of ACE2. Therefore, ACE2 inhibition is not a useful treatment against COVID-19 infection., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Pertuzumab (Perjeta) is an anti-HER2 monoclonal antibody that is used for treatment of HER2-positive breast cancers in combination with trastuzumab (Herceptin) and docetaxel and showed promising clinical outcomes. Pertuzumab is suggested to block heterodimerization of HER2 with EGFR and HER3 that abolishes canonical function of HER2. However, evidence on the exact mode of action of pertuzumab in homodimerization of HER2 are limited. In this study, we investigated the effect of pertuzumab and its combination with trastuzumab on HER2 homodimerization, phosphorylation and whole gene expression profile in Chinese hamster ovary (CHO) cells stably overexpressing human HER2 (CHO-K6). CHO-K6 cells were treated with pertuzumab, trastuzumab, and their combination, and then HER2 homodimerization and phosphorylation at seven pY sites were investigated. The effects of the monoclonal antibodies on whole gene expression and the expression of cell cycle stages, apoptosis, autophagy, and necrosis were studied by cDNA microarray. Results showed that pertuzumab had no significant effect on HER2 homodimerization, however, trastuzumab increased HER2 homodimerization. Interestingly, pertuzumab increased HER2 phosphorylation at Y1127, Y1139, and Y1196 residues, while trastuzumab increased HER2 phosphorylation at Y1196. More surprisingly, combination of pertuzumab and trastuzumab blocked the phosphorylation of Y1005 and Y1127 of HER2. Our results also showed that pertuzumab, but not trastuzumab, abrogated the effect of HER2 overexpression on cell cycle in particular G1/S transition, G2/M transition, and M phase, whereas trastuzumab abolished the inhibitory effect of HER2 on apoptosis. Our findings confirm that pertuzumab is unable to inhibit HER2 homodimerization but induces HER2 phosphorylation at some pY sites that abolishes HER2 effects on cell cycle progress. These data suggest that the clinical effects of pertuzumab may mostly through the inhibition of HER2 heterodimers, rather than HER2 homodimers and that pertuzumab binding to HER2 may inhibit non-canonical HER2 activation and function in non-HER-mediated and dimerization-independent pathway(s)., Competing Interests: The authors declare no conflict of interest.

Human epidermal growth factor receptor (HER) 2 (HER2) is overexpressed in 20⁻30% of breast cancers. HER2 is a preferred target for treating HER2-positive breast cancer. Trastuzumab and pertuzumab are two HER2-targeted monoclonal antibodies approved by the Food and Drug Administration (FDA) to use as adjuvant therapy in combination with docetaxel to treat metastatic HER2-positive breast cancer. Adding the monoclonal antibodies to treatment regimen has changed the paradigm for treatment of HER2-positive breast cancer. Despite improving outcomes, the percentage of the patients who benefit from the treatment is still low. Continued research and development of novel agents and strategies of drug combinations is needed. A thorough understanding of the molecular mechanisms underlying the action and synergism of trastuzumab and pertuzumab is essential for moving forward to achieve high efficacy in treating HER2-positive breast cancer. This review examined and analyzed findings and hypotheses regarding the action and synergism of trastuzumab and pertuzumab and proposed a model of synergism based on available information.

Taxanes are a class of chemotherapeutic agents that inhibit cell division by disrupting the mitotic spindle through the stabilization of microtubules. Most breast cancer (BC) tumors show resistance against taxanes partially due to alterations in tubulin genes. In this project we investigated tubulin isoforms in BC to explore any correlation between tubulin alterations and taxane resistance. Genetic alteration and expression profiling of 28 tubulin isoforms in 6714 BC tumor samples from 4205 BC cases were analyzed. Protein-protein, drug-protein and alterations neighbor genes in tubulin pathways were examined in the tumor samples. To study correlation between promoter activity and expression of the tubulin isoforms in BC, we analyzed the ChIP-seq enrichment of active promoter histone mark H3K4me3 and mRNA expression profile of MCF-7, ZR-75-30, SKBR-3 and MDA-MB-231 cell lines. Potential correlation between tubulin alterations and taxane resistance, were investigated by studying the expression profile of taxane-sensitive and resistant BC tumors also the MDA-MB-231 cells acquired resistance to paclitaxel. All genomic data were obtained from public databases. Results showed that TUBD1 and TUBB3 were the most frequently amplified and deleted tubulin genes in the BC tumors respectively. The interaction analysis showed physical interactions of α-, β- and γ-tubulin isoforms with each other. The most of FDA-approved tubulin inhibitor drugs including taxanes target only β-tubulins. The analysis also revealed sex tubulin-interacting neighbor proteins including ENCCT3, NEK2, PFDN2, PTP4A3, SDCCAG8 and TBCE which were altered in at least 20% of the tumors. Three of them are tubulin-specific chaperons responsible for tubulin protein folding. Expression of tubulin genes in BC cell lines were correlated with H3K4me3 enrichment on their promoter chromatin. Analyzing expression profile of BC tumors and tumor-adjacent normal breast tissues showed upregulation of TUBA1A , TUBA1C , TUBB and TUBB3 and downregulation of TUBB2A , TUBB2B , TUBB6 , TUBB7P pseudogene, and TUBGCP2 in the tumor tissues compared to the normal breast tissues. Analyzing taxane-sensitive versus taxane-resistant tumors revealed that expression of TUBB3 and TUBB6 was significantly downregulated in the taxane-resistant tumors. Our results suggest that downregulation of tumor βIII- and βV-tubulins is correlated with taxane resistance in BC. Based on our results, we conclude that aberrant protein folding of tubulins due to mutation and/or dysfunction of tubulin-specific chaperons may be potential mechanisms of taxane resistance. Thus, we propose studying the molecular pathology of tubulin mutations and folding in BC and their impacts on taxane resistance.

Background: Targeted therapy with trastuzumab has become a mainstay for HER2-positive breast cancer without a clear understanding of the mechanism of its action. While many mechanisms have been suggested for the action of trastuzumab, most of them are not substantiated by experimental data. It has been suggested that trastuzumab functions by inhibiting intracellular signaling initiated by HER2, however, the data are very controversial. A major issue is the different cellular background of various breast cancer cells lines used in these studies. Each breast cancer cell line has a unique expression profile of various HER receptors, which could significantly affect the effects of trastuzumab., Methods: To overcome this problem, in this research we adopted a cell model that allow us to specifically examine the effects of trastuzumab on a single HER receptor without the influence of other HER receptors. Three CHO cell lines stably expressing only human EGFR (CHO-EGFR), HER2 (CHO-K6), or HER3 (CHO-HER3) were used. Various methods including cytotoxicity assay, immunoblotting, indirect immunofluorescence, cross linking, and antibody-dependent cellular cytotoxicity (ADCC) were employed in this research., Results: We showed that trastuzumab did not bind EGFR and HER3, and thus did not affect the homodimerization and phosphorylation of EGFR and HER3. However, overexpression of HER2 in CHO cells, in the absence of other HER receptors, resulted in the homodimerization of HER2 and the phosphorylation of HER2 at all major pY residues. Trastuzumab bound to HER2 specifically and with high affinity. Trastuzumab inhibited neither the homodimerization of HER2, nor the phosphorylation of HER2 at most phosphotyrosine residues. Moreover, trastuzumab did not inhibit the phosphorylation of ERK and AKT in CHO-K6 cells, and did not inhibit the proliferation of CHO-K6 cells. However, trastuzumab induced strong ADCC in CHO-K6 cells., Conclusion: We concluded that, in the absence of other HER receptors, trastuzumab exerts its antitumor activity through the induction of ADCC, rather than the inhibition of HER2-homodimerization and phosphorylation.

HER2 receptor tyrosine kinase that is overexpressed in approximately 20% of all breast cancers (BCs) is a poor prognosis factor and a precious target for BC therapy. Trastuzumab is approved by FDA to specifically target HER2 for treating HER2+ BC. However, about 60% of patients with HER2+ breast tumor develop de novo resistance to trastuzumab, partially due to the loss of expression of HER2 extracellular domain on their tumor cells. This is due to shedding/cleavage of HER2 by metalloproteinases (ADAMs and MMPs). HER2 shedding results in the accumulation of intracellular carboxyl-terminal HER2 (p95HER2), which is a common phenomenon in trastuzumab-resistant tumors and is suggested as a predictive marker for trastuzumab resistance. Up-regulation of the metalloproteinases is a poor prognosis factor and is commonly seen in mesenchymal-like cancer stem cells that are risen during epithelial to mesenchymal transition (EMT) of tumor cells. HER2 cleavage during EMT can explain why secondary metastatic tumors with high percentage of mesenchymal-like cancer stem cells are mostly resistant to trastuzumab but still sensitive to lapatinib. Importantly, many studies report HER2 interaction with oncogenic/stemness signaling pathways including TGF-β/Smad, Wnt/β-catenin, Notch, JAK/STAT and Hedgehog. HER2 overexpression promotes EMT and the emergence of cancer stem cell properties in BC. Increased expression and activation of metalloproteinases during EMT leads to proteolytic cleavage and shedding of HER2 receptor, which downregulates HER2 extracellular domain and eventually increases trastuzumab resistance. Here, we review the hypothesis that a negative feedback loop between HER2 and stemness signaling drives resistance of BC to trastuzumab.

Dimerization of the epithelial growth factor (EGF) family of receptor tyrosine kinases is a crucial step for activation of these receptors. Different chemicals such as BS 3 and DSS have been introduced to covalently bind the interacting receptors and fix the dimers. Unique properties of BS 3 including higher water solubility and membrane impermeability make it suitable for assessing receptor-receptor interactions in live cells. In this protocol, we aim to explain a method to evaluate the dimerization of EGF receptors family using BS 3 as a cross-linker reagent.

Immunofluorescence staining is an effective method for visualizing ErbB family of receptor tyrosine kinases and to monitor their expression, phosphorylation, and localization in individual cells. However, immunofluorescence staining for membrane proteins is required preserving integrity of cell membrane intact by protecting the cells from organic solvents. Permeabilization of cells may increase nonspecific intracellular background signal as an inevitable result. This chapter describes procedures for the application of indirect immunofluorescence staining for ErbB receptors in adherent and suspended cells as well as in paraffin-embedded tissue sections.

Tunicamycin is an inhibitor of glycosylation that disturbs protein folding machinery in eukaryotic cells. Tunicamycin causes accumulation of unfolded proteins in cell endoplasmic reticulum (ER) and induces ER stress. ER stress is an essential mechanism for cellular homeostasis has role in cell death via reprogramming of protein processing, regulation of autophagy and apoptosis. In this study we show effect of tunicamycin on subpopulation and invasion of CD44+/CD24- MCF7 breast cancer stem cells. CD44+/CD24- cells were isolated from MCF7 cell line by fluorescence activated cell sorting (FACS) and treated with tunicamycin. ER stress was monitored by evaluation of X-box binding protein 1(XBP-1) mRNA splicing, cleaved activating transcription factor 6 (ATF6) nuclear translocation and CCAAT/enhancer-binding protein homologous protein (CHOP) expression. CD44+/CD24- subpopulation was analyzed using flow cytometry. Invasion was investigated by scratch assay, trypan blue staining, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation and in vitro migration assays. Increased level of spliced XBP-1, ATF6 nuclear translocation and CHOP protein expression were detected in CD44+/CD24- and original MCF7 cells treated with tunicamycin. Also, a significant decline in CD44+/CD24- cell subpopulation was determined in the cells treated with tunicamycin. The results also showed inhibited invasion, increased cell death, suppressed proliferation and reduced migration in the CD44+/CD24- and CD44+/CD24- rich MCF7 cell culture, under effect of tunicamycin. Our results indicate that CD44+/CD24- phenotype MCF7 cells are susceptible to tunicamycin. The results showed that tunicamycin-induced ER stress suppresses CD44+/CD24- phenotype cell subpopulation and in vitro invasion and accelerates tumorosphore formation. These results suggest that tunicamycin-induced ER stress inhibits CD44+/CD24- phenotype MCF7 breast cancer stem cells. We conclude that using ER-targeting chemicals like tunicamycin is an interesting approach to target breast cancer stem cells inside tumor., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Introduction: Breast cancer stem cell with CD44(hi)/CD24(lo) phonotype is described having stem cell properties and represented as the main driving factor in breast cancer initiation, growth, metastasis and low response to anti-cancer agents. Glucoseregulated proteins (GRPs) are heat shock protein family chaperons that are charged with regulation of protein machinery and modulation of endoplasmic reticulum homeostasis whose important roles in stem cell development and invasion of various cancers have been demonstrated. Here, we investigated the expression levels of GRP78 and GRP94 in CD44(hi)/CD24(lo) phenotype breast cancer stem cells (BCSCs)., Methods: MCF7, T-47D and MDA-MB-231 breast cancer cell lines were used. CD44(hi)/CD24(lo) phenotype cell population were analyzed and sorted by fluorescence-activated cell sorting (FACS). Transcriptional and translational expression of GRP78 and GRP94 were investigated by western blotting and quantitative real time PCR., Results: RESULTS showed different proportion of CD44(hi)/CD24(lo) phenotype cell population in their original bulk cells. The ranking of the cell lines in terms of CD44(hi)/CD24(lo) phenotype cell population was as MCF7