A novel long non-coding RNA MIR4500HG003 promotes tumor metastasis through miR-483-3p-MMP9 axis in triple-negative breast cancer.

Breast cancer (BC) is the most common cancer and the leading cause of cancer-related deaths in women worldwide. The 5-year survival rate is over 90% in BC patients, but once BC cells metastasis into distal organs, it is dramatically decreasing to less than 30%. Especially, triple-negative breast cancer (TNBC) patients usually lead to poor prognosis and survival because of metastasis. Understanding the underline mechanisms of TNBC metastasis is a critical issue. Non-coding RNAs, including of lncRNAs and microRNAs, are non-protein-coding transcripts and have been reported as important regulators in TNBC metastasis. However, the underline mechanisms for non-coding RNAs regulating TNBC metastasis remain largely unclear. Here, we found that lncRNA MIR4500HG003 was highly expressed in highly metastatic MDA-MB-231 TNBC cells and overexpression of MIR4500HG003 enhanced metastasis ability in vitro and in vivo and promoted MMP9 expression. Furthermore, we found MIR4500HG003 physically interacted with miR-483-3p and reporter assay showed miR-483-3p attenuated MMP9 expression. Importantly, endogenous high expressions of MIR4500HG003 were correlated with tumor recurrence in TNBC patients with tumor metastasis. Taken together, our findings suggested that MIR4500HG003 promotes metastasis of TNBC through miR-483-3p-MMP9 signaling axis and may be used as potential prognostic marker for TNBC patients.

A molecular simulation study of the clinical G409V mutant in PINK1 associated with early-onset Parkinson's disease.

The serine/threonine kinase PINK1 is responsible for phosphorylating a ubiquitin (Ub)-like domain in an E3 Ub ligase Parkin protein and a Parkin-bound Ub. PINK1 works as a mitochondrial quality control by phosphorylating and activating the E3 ubiquitin ligase Parkin. Recent medicinal study has reported that mutations of Parkin and PINK1 cause defects in mitophagy and induce early-onset Parkinson's disease (EOPD). In this study, we conducted molecular dynamics simulations to investigate the structural discrepancy caused by a clinical G409V mutation in PINK1 kinase domain's A-loop. The Ub phosphorylation begins with PINK1 D362 deprotonating the hydroxyl group of the substrate Ub's S65' and PINK1's A-loop is responsible for coordinating S65'. On contrary to G409 offering structural plasticity, the replaced, bulky V409 interferes with the alignment of D362-S65', seriously hampering Ub phosphorylation, leading to the accumulation of damaged mitochondria, and ultimately EOPD. In this study, we predicted the hPINK1WT-UbWT binding mode and detected the structural impact brought by G409V replacement. It is expected the concluded remarks to be beneficial for developing cures to alleviate structural interference and restore PINK1 function.

Novel metal peroxide nanoboxes restrain Clostridioides difficile infection beyond the bactericidal and sporicidal activity.

Clostridioides difficile spores are considered as the major source responsible for the development of C. difficile infection (CDI), which is associated with an increased risk of death in patients and has become an important issue in infection control of nosocomial infections. Current treatment against CDI still relies on antibiotics, which also damage normal flora and increase the risk of CDI recurrence. Therefore, alternative therapies that are more effective against C. difficile bacteria and spores are urgently needed. Here, we designed an oxidation process using H2O2 containing PBS solution to generate Cl- and peroxide molecules that further process Ag and Au ions to form nanoboxes with Ag-Au peroxide coat covering Au shell and AgCl core (AgAu-based nanoboxes). The AgAu-based nanoboxes efficiently disrupted the membrane structure of bacteria/spores of C. difficile after 30-45 min exposure to the highly reactive Ag/Au peroxide surface of the nano structures. The Au-enclosed AgCl provided sustained suppression of the growth of 2 × 107 pathogenic Escherichia coli for up to 19 days. In a fecal bench ex vivo test and in vivo CDI murine model, biocompatibility and therapeutic efficacy of the AuAg nanoboxes to attenuate CDI was demonstrated by restoring the gut microbiota and colon mucosal structure. The treatment successfully rescued the CDI mice from death and prevented their recurrence mediated by vancomycin treatment. The significant outcomes indicated that the new peroxide-derived AgAu-based nanoboxes possess great potential for future translation into clinical application as a new alternative therapeutic strategy against CDI.

Polyphenol-assisted assembly of Au-deposited polylactic acid microneedles for SERS sensing and antibacterial photodynamic therapy.

3D SERS microneedles with self-assembled AuNPs were fabricated with tannic acid (chemical glue and reductant) on polylactic acid microneedles for in-depth chemical and biomolecular analysis, with LOD values below 200 ppb for small molecules and 102 CFU cm-2 for bacteria. The MB/Au-microneedles were used for photodynamic therapy with SERS-monitored photosensitizer degradation.

An order-to-disorder structural switch regulates HIF-1 transcription through S247 phosphorylation in the HIF1α PAS-B domain.

Hypoxia-inducible factor 1 (HIF-1), a transcriptional activator that mediates cellular responses to hypoxic stress, is essential for tumor progression. It is a heterodimer comprising HIF1α and HIF1ß, with multiple interfaces among their PAS-A, PAS-B, and bHLH domains. HIF1ß is also known as aryl hydrocarbon receptor nuclear translocator (ARNT). Casein kinase 1δ-dependent phosphorylation of the solvent-front residue S247 on the HIF1α PAS-B domain interrupts HIF1α-ARNT complex formation and reduces HIF-1 transcription activity. However, S247 is involved in neither HIF1α-ARNT complex formation nor stabilization of the relative orientation between the HIF1α PAS-A and PAS-B domains. To uncover the underlying allosteric mechanism, we conducted Gaussian accelerated molecular dynamics simulations and identified two distinct conformations of the pS247-carrying HIF1α PAS-B domain: H291-in and H291-out. The H291-in structure can associate with the HIF1α PAS-A domain and form a V-shaped pouch to accommodate the ARNT PAS-A domain, but it cannot associate with the ARNT PAS-B domain. By contrast, the H291-out structure can bind to the ARNT PAS-B domain, but its association with the HIF1α PAS-A domain leads to an unsuitable relative orientation to accommodate the ARNT PAS-A domain. Both conformations were also collected in parallel simulations of the unphosphorylated PAS-B domain. Both structures manage to associate with the ARNT PAS-B and HIF1α PAS-A domains; thus, they are adequate for HIF1α-ARNT complex formation. The domain-domain contact pattern in a phosphorylated variant is shuffled by an order-to-disorder structural switch, triggered by the newly formed K251-pS247 interaction.

A Novel Phenotype of the Factor 5 Gene Mutation (Homozygote Met1736Val and Heterozygote Asp68His) Is Associated With Moderate Factor V Deficiency.

Background: Factor V (FV) deficiency is a rare disease, with a low incidence rate in Asia. Therefore, the F5 mutation in the Taiwanese population is poorly understood. Methods: A Chinese family with FV deficiency was included, and the patient and his family members underwent mutation analysis. Then, patients from Keelung City (Taiwan) were screened for F5 polymorphism; the Chang Gung Human Database was used to determine single-nucleotide variants in the non-FV-deficient patient population. Results: Eight mutation sites on the F5 gene locus, including exon 16 homozygote Met1736Val and seven heterozygous mutations, including Asp68His, were found. Moreover, Met1736Val was found to be the dominant mutation in people living in the Taiwan community, and this result was compared with the records of the Chang Gung Human Database. The above-mentioned polymorphisms may result in a variable incidence of FV deficiency in Keelung City, thereby facilitating carrier diagnosis and prenatal diagnosis in most FV-deficient families. Conclusion: The homozygote Met1736Val and the co-inheritance of the Asp68His F5 gene are unique and worthy of screening in FV-deficient patients.

In Situ Formation of Au-Glycopolymer Nanoparticles for Surface-Enhanced Raman Scattering-Based Biosensing and Single-Cell Immunity.

Successful synthesis of glyconanoparticles has attracted much attention due to their various biointeractive capabilities, but it is still a challenge to understand different single-cell responses to exogenous particles among cell populations. Herein, we designed polyaniline-containing galactosylated gold nanoparticles (Au@PGlyco NPs) via in situ polymerization of ortho-nitrophenyl-ß-galactoside assisted by Au nucleation. The nanogold-carrying polyaniline block produced electromagnetic enhancement in surface-enhanced Raman scattering (SERS). The underlying polymerization mechanism of ortho-nitrophenyl compounds via the formation of Au nanoparticles was investigated. Depending on how the galactoside moiety reacted with ß-galactosidase derived from bacteria, the Au@PGlyco NPs-mediated SERS biosensor could detect low amounts of bacteria (∼1 × 102 CFU/mL). In addition, a high accumulation of Au@PGlyco NPs mediated the immune response of tumor-associated M2 macrophages to the immunogenic M1 macrophage transition, which was elicited by reactive oxygen levels biostimulation using single-cell SERS-combined fluorescence imaging. Our study suggested that Au@PGlyco NPs may serve as a biosensing platform with the labeling capacity on galactose-binding receptors expressed cell and immune regulation.

Molecular dynamics study of enhanced autophosphorylation by S904F mutation of the RET kinase domain.

The aberrant kinase activity of RET (rearranged during transfection), a transmembrane tyrosine kinase, is associated with human cancer. A point mutation caused by the replacement of solvent-front hydrophilic S904, located on the activation loop (A-loop), with a bulky hydrophobic phenylalanine residue can induce resistance to the type I kinase inhibitor vandetanib. A possible mechanism of this drug resistance is the release of a cis-autoinhibited conformation of RET for autophosphorylation, which activates RET kinase. Because the association between S904F mutation and enhanced autophosphorylation is unclear, we conducted molecular modeling analysis to compare unphosphorylated apo wild-type and S904F mutant structures. The structural compactness of the A-loop promoted ATP binding. When the A-loop is extended, the αC helix moves toward the glycine-rich loop, resulting in the protrusion of F735. The extruded F735 connects with E734 and R912 and constrains the ATP pocket entrance. Contrarily, a contracted A-loop pulls the αC helix away from the glycine-rich loop, burying F734 and making the ATP pocket accessible. The mutated F904 stabilizes the contracted A-loop and releases the autoinhibited conformation of RET, thereby facilitating autophosphorylation. We also simulated two ATP-bound systems. The binding free energies of ATP, estimated through the molecular mechanics with a generalized Born and surface area solvation approach, revealed that the S904F mutant was bound more tightly than was the wild type with the ATP. The findings support the premise of autophosphorylation promotion in the S904F mutant.

The differences in nurses' willingness to discuss palliative care with patients and their family members.

BACKGROUND: This study aimed to evaluate the differences in nurses' willingness to discuss palliative care with terminally ill patients and their family members. METHODS: The participants were randomly recruited from registered staff nurses ≥20 years of age who were responsible for clinical inpatient care in a tertiary hospital in northern Taiwan. A semi-structured questionnaire was administered to evaluate nurses' experiences of discussing do-not-resuscitate (DNR) decisions and their willingness to discuss palliative care with terminal patients and their family members. The differences in nurses' experiences regarding DNR and willingness to discuss palliative care with terminally ill patients and their family members were compared using the Chi-square test. Logistic regressions were used to analyze factors associated with nurses' willingness to discuss palliative care with patients and their families. RESULTS: More participants had experienced initiating discussions about DNR with patients' families than with patients (72.2% vs 61.9%, p < 0.001). Unadjusted logistic regression analysis showed that the experiences of actively initiating DNR discussions with patients were a significant factor associated with palliative care discussion with patients (odds ratio [OR] = 2.91, 95% confidence interval [CI]: 1.09-7.79). On the other hand, the experiences of actively initiating DNR discussions with patients and with patients' families were significant factors associated with palliative care discussion with patients' families (OR = 3.84, 95% CI: 1.22-12.06 and OR = 3.60, 95% CI: 1.19-10.90, respectively). After adjusting for covariates, no significant factors were found to be independently associated with nurses' willingness to discuss palliative care with patients and their family members. CONCLUSION: There are significant differences in nurses' willingness to discuss palliative care with patients and their family members. Further research is needed to evaluate factors associated with nurses' willingness to discuss palliative care with patients and their families to facilitate these discussions and protect patients' autonomy.

Loss of function in SAGA deubiquitinating module caused by Sgf73 H93A mutation: A molecular dynamics study.

The Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitinating module (DUBm), comprising Ubp8, Sgf11, Sus1, and Sgf73, functions as a deubiquitinase. Data from recent biomolecular experiments have indicated that the H93A mutation in Sgf73 abrogates the enzyme function without interfering with module formation. Interestingly Sgf73 H93 residue is neither involved in the active site nor near the ubiquitin substrate binding site but is capable of influencing the active site through an allosteric mechanism. In this study, molecular dynamics simulations were used to analyze the structural discrepancy between wild-type and H93A mutant of the SAGA DUBm to reveal the interprotein communication pathway linking the mutation site to the catalytic site. We concluded that H93A mutation directly impairs Sgf73's zinc finger motif and further induces a downward movement of Sgf11's N-terminal long α-helix. The structural influence gradually propagates toward Sgf11's C-terminal end and results in the rearrangement of the catalytic triad of Ubp8 protein. The repositioned catalytic Cys-His-Asn triad could no longer execute the deubiquitinating function.

Molecular Modeling for Structural Insights Concerning the Activation Mechanisms of F1174L and R1275Q Mutations on Anaplastic Lymphoma Kinase.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase involved in various cancers. In its basal state, the structure of ALK is in an autoinhibitory form stabilized by its A-loop, which runs from the N-lobe to the C-lobe of the kinase. Specifically, the A-loop adopts an inhibitory pose with its proximal A-loop helix (αAL-helix) to anchor the αC-helix orientation in an inactive form in the N-lobe; the distal portion of the A-loop is packed against the C-lobe to block the peptide substrate from binding. Upon phosphorylation of the first A-loop tyrosine (Y1278), the αAL-helix unfolds; the distal A-loop detaches from the C-lobe and reveals the P+1 pocket that accommodates the residues immediately after their phosphorylation, and ALK is activated accordingly. Recently, two neuroblastoma mutants, F1174L and R1275Q, have been determined to cause ALK activation without phosphorylation on Y1278. Notably, F1174 is located on the C-terminus of the αC-helix and away from the A-loop, whereas R1275 sits on the αAL-helix. In this molecular modeling study, we investigated the structural impacts of F1174L and R1275Q that lead to the gain-of-function event. Wild-type ALK and ALK with phosphorylated Y1278 were also modeled for comparison. Our modeling suggests that the replacement of F1174 with a smaller residue, namely leucine, moves the αC-helix and αAL-helix into closer contact and further distorts the distal portion of the A-loop. In wild-type ALK, R1275 assumes the dual role of maintaining the αAL-helix⁻αC-helix interaction in an inactive form and securing αAL-helix conformation through the D1276⁻R1275 interaction. Accordingly, mutating R1275 to a glutamine reorients the αC-helix to an active form and deforms the entire A-loop. In both F1174L and R1275Q mutants, the A-loop rearranges itself to expose the P+1 pocket, and kinase activity resumes.

Molecular modeling on HIF-2α-ARNT dimer destabilization caused by R171A and/or V192D mutations in HIF-2α.

Oxygen homeostasis in normal and tumor cells is mediated by hypoxia-inducible factors (HIFs), which are active as heterodimer complexes, such as HIF-2α-aryl hydrocarbon receptor nuclear translocator (ARNT) and HIF-1α-ARNT. A series of mutations on the interfaces between HIF-2α and ARNT and on the domain-domain interface within HIF-2α has been reported to exert varying effects on HIF-2α-ARNT dimerization. In the present study, molecular dynamic simulations were conducted to evaluate HIF-2α mutations, namely R171A, V192D, and R171A/V192D, which are not involved in the interaction with ARNT but impede HIF-2α-ARNT dimerization. Our results indicate that these mutations induct local conformation leading to a shortened (by V192D) or widened (by R171A and R171A/V192D) Y91-E346 separation distance, where E346 and Y91 are located on the HIF-2α and interact with ARNT according to electrostatic and geometrical shape complementarity, respectively.

Structural and Dynamic Characterization of Mutated Keap1 for Varied Affinity toward Nrf2: A Molecular Dynamics Simulation Study.

Keap1 is an adaptor protein that regulates Nrf2 in response to oxidative stress. Under basal conditions, Nrf2 is negatively regulated through ubiquitination by Keap1. However, upon exposure to oxidative stress, the ubiquitination of Nrf2 is inhibited, resulting in an increased steady-state level of Nrf2 in the nucleus and increased transcription of cytoprotective genes. A gene variant G364C and somatic mutation G430C on Keap1 have recently been reported to substantially impair the Keap1-Nrf2 interaction and to be associated with lung cancer. By contrast, alanine scanning experiments have shown that the mutations S363A, S508A, S555A, and S602A do not affect the ability of Keap1 to bind to Nrf2, regardless of the fact that G364 and G430 are not in contact with Nrf2 whereas the four serine residues are involved in the accommodation of Nrf2 with their hydroxy groups. In this study, molecular dynamics simulations were performed to investigate the structural and dynamic variances among wild-type (WT) Keap1 and the six mutants in unbound form. Principal component analysis of the collected MD trajectories was performed to provide dynamic diversity. Our dynamic and structural observations suggest that the G364C and G430C mutants possess a mobile D385 that moves toward R380, an anchor residue to accommodate an acidic residue in Nrf2, thereby hampering the Keap1-Nrf2 recognition of an electrostatic nature. By contrast, none of the four serine-to-alanine mutants alters the H-bond network formed by the serine backbone to its partner; accordingly, these mutants are almost as intact as the WT structurally and dynamically.