Role of C9orf72 hexanucleotide repeat expansions in ALS/FTD pathogenesis.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive neurological disorders that share neurodegenerative pathways and features. The most prevalent genetic causes of ALS/FTD is the GGGGCC hexanucleotide repeat expansions in the first intron region of the chromosome 9 open reading frame 72 (C9orf72) gene. In this review, we comprehensively summarize the accumulating evidences elucidating the pathogenic mechanism associated with hexanucleotide repeat expansions in ALS/FTD. These mechanisms encompass the structural polymorphism of DNA and transcribed RNA, the formation of RNA foci via phase separation, and the cytoplasmic accumulation and toxicities of dipeptide-repeat proteins. Additionally, the formation of G-quadruplex structures significantly impairs the expression and normal function of the C9orf72 protein. We also discuss the sequestration of specific RNA binding proteins by GGGGCC RNA, which further contributes to the toxicity of C9orf72 hexanucleotide repeat expansions. The deeper understanding of the pathogenic mechanism of hexanucleotide repeat expansions in ALS/FTD provides multiple potential drug targets for these devastating diseases.

The N-terminal region of Cdc6 specifically recognizes human DNA G-quadruplex.

Guanine (G)-rich nucleic acid sequences can form diverse G-quadruplex structures located in functionally significant genome regions, exerting regulatory control over essential biological processes, including DNA replication in vivo. During the initiation of DNA replication, Cdc6 is recruited by the origin recognition complex (ORC) to target specific chromosomal DNA sequences. This study reveals that human Cdc6 interacts with G-quadruplex structure through a distinct region within the N-terminal intrinsically disordered region (IDR), encompassing residues 7-20. The binding region assumes a hook-type conformation, as elucidated by the NMR solution structure in complex with htel21T18. Significantly, mutagenesis and in vivo investigations confirm the highly specific nature of Cdc6's recognition of G-quadruplex. This research enhances our understanding of the fundamental mechanism governing the interaction between G-quadruplex and the N-terminal IDR region of Cdc6, shedding light on the intricate regulation of DNA replication processes.

Electronic Janus lattice and kagome-like bands in coloring-triangular MoTe2 monolayers.

Polymorphic structures of transition metal dichalcogenides (TMDs) host exotic electronic states, like charge density wave and superconductivity. However, the number of these structures is limited by crystal symmetries, which poses a challenge to achieving tailored lattices and properties both theoretically and experimentally. Here, we report a coloring-triangle (CT) latticed MoTe2 monolayer, termed CT-MoTe2, constructed by controllably introducing uniform and ordered mirror-twin-boundaries into a pristine monolayer via molecular beam epitaxy. Low-temperature scanning tunneling microscopy and spectroscopy (STM/STS) together with theoretical calculations reveal that the monolayer has an electronic Janus lattice, i.e., an energy-dependent atomic-lattice and a Te pseudo-sublattice, and shares the identical geometry with the Mo5Te8 layer. Dirac-like and flat electronic bands inherently existing in the CT lattice are identified by two broad and two prominent peaks in STS spectra, respectively, and verified with density-functional-theory calculations. Two types of intrinsic domain boundaries were observed, one of which maintains the electronic-Janus-lattice feature, implying potential applications as an energy-tunable electron-tunneling barrier in future functional devices.

Inhibition of VEGF binding to neuropilin-2 enhances chemosensitivity and inhibits metastasis in triple-negative breast cancer.

Although blocking the binding of vascular endothelial growth factor (VEGF) to neuropilin-2 (NRP2) on tumor cells is a potential strategy to treat aggressive carcinomas, a lack of effective reagents that can be used clinically has hampered this potential therapy. Here, we describe the generation of a fully humanized, high-affinity monoclonal antibody (aNRP2-10) that specifically inhibits the binding of VEGF to NRP2, conferring antitumor activity without causing toxicity. Using triple-negative breast cancer as a model, we demonstrated that aNRP2-10 could be used to isolate cancer stem cells (CSCs) from heterogeneous tumor populations and inhibit CSC function and epithelial-to-mesenchymal transition. aNRP2-10 sensitized cell lines, organoids, and xenografts to chemotherapy and inhibited metastasis by promoting the differentiation of CSCs to a state that is more responsive to chemotherapy and less prone to metastasis. These data provide justification for the initiation of clinical trials designed to improve the response of patients with aggressive tumors to chemotherapy using this monoclonal antibody.

BK channels of five different subunit combinations underlie the de novo KCNMA1 G375R channelopathy.

The molecular basis of a severe developmental and neurological disorder associated with a de novo G375R variant of the tetrameric BK channel is unknown. Here, we address this question by recording from single BK channels expressed to mimic a G375R mutation heterozygous with a WT allele. Five different types of functional BK channels were expressed: 3% were consistent with WT, 12% with homotetrameric mutant, and 85% with three different types of hybrid (heterotetrameric) channels assembled from both mutant and WT subunits. All channel types except WT showed a marked gain-of-function in voltage activation and a smaller decrease-of-function in single-channel conductance, with both changes in function becoming more pronounced as the number of mutant subunits per tetrameric channel increased. The net cellular response from the five different types of channels comprising the molecular phenotype was a shift of -120 mV in the voltage required to activate half of the maximal current through BK channels, giving a net gain-of-function. The WT and homotetrameric mutant channels in the molecular phenotype were consistent with genetic codominance as each displayed properties of a channel arising from only one of the two alleles. The three types of hybrid channels in the molecular phenotype were consistent with partial dominance as their properties were intermediate between those of mutant and WT channels. A model in which BK channels randomly assemble from mutant and WT subunits, with each subunit contributing increments of activation and conductance, approximated the molecular phenotype of the heterozygous G375R mutation.

An allosteric modulator activates BK channels by perturbing coupling between Ca2+ binding and pore opening.

BK type Ca2+-activated K+ channels activate in response to both voltage and Ca2+. The membrane-spanning voltage sensor domain (VSD) activation and Ca2+ binding to the cytosolic tail domain (CTD) open the pore across the membrane, but the mechanisms that couple VSD activation and Ca2+ binding to pore opening  are not clear. Here we show that a compound, BC5, identified from in silico screening, interacts with the CTD-VSD interface and specifically modulates the Ca2+ dependent activation mechanism. BC5 activates the channel in the absence of Ca2+ binding but Ca2+ binding inhibits BC5 effects. Thus, BC5 perturbs a pathway that couples Ca2+ binding to pore opening to allosterically affect both, which is further supported by atomistic simulations and mutagenesis. The results suggest that the CTD-VSD interaction makes a major contribution to the mechanism of Ca2+ dependent activation and is an important site for allosteric agonists to modulate BK channel activation.

Hydrogen Spillover-Enhanced Heterogeneously Catalyzed Hydrodeoxygenation for Biomass Upgrading.

Hydrodeoxygenation (HDO) is regarded as a promising technology for biomass upgrading to obtain sustainable and competitive chemicals and fuels. In fact, biomass HDO over heterogeneous solid catalysts is often accompanied by the phenomenon of hydrogen spillover, which further affects the catalytic performance. Thus, it is necessary to gain in-depth understand the promoting effect of hydrogen spillover in the biomass HDO process to obtain desired conversion and selectivity. This Review summarized the extensive research on hydrogen spillover in biomass refining and discussed in detail the regulation mechanism of hydrogen spillover in biomass HDO process, mainly by regulating different active center sites on catalyst supports, such as metal sites, acid sites, surface functional groups, and defective sites, which exhibit independent and synergistic characteristics promoting catalyst activity, selectivity, and stability. Finally, the prospective of hydrogen spillover in biomass HDO applications was critically evaluated, and the key technical challenges in developing "hydrogen-free" HDO and upgrading biofuels were highlighted. The presentation of hydrogen spillover-enhanced catalytic biomass HDO in this Review will hopefully provide insight and guidance for further development of efficient catalysts and preparation of high-value chemicals in the future.

Crystal structure of parallel G-quadruplex formed by the two-repeat ALS- and FTD-related GGGGCC sequence.

The hexanucleotide repeat expansion, GGGGCC (G4C2), within the first intron of the C9orf72 gene is known to be the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 repeat expansions, either DNA or RNA, are able to form G-quadruplexes which induce toxicity leading to ALS/FTD. Herein, we report a novel crystal structure of d(G4C2)2 that self-associates to form an eight-layer parallel tetrameric G-quadruplex. Two d(G4C2)2 associate together as a parallel dimeric G-quadruplex which folds into a tetramer via 5'-to-5' arrangements. Each dimer consists of four G-tetrads connected by two CC propeller loops. Especially, the 3'-end cytosines protrude out and form C·C+•C·C+/ C·C•C·C+ quadruple base pair or C•C·C+ triple base pair stacking on the dimeric block. Our work sheds light on the G-quadruplexes adopted by d(G4C2) and yields the invaluable structural details for the development of small molecules to tackle neurodegenerative diseases, ALS and FTD.

Structural basis of DNA replication origin recognition by human Orc6 protein binding with DNA.

The six-subunit origin recognition complex (ORC), a DNA replication initiator, defines the localization of the origins of replication in eukaryotes. The Orc6 subunit is the smallest and the least conserved among ORC subunits. It is required for DNA replication and essential for viability in all species. Orc6 in metazoans carries a structural homology with transcription factor TFIIB and can bind DNA on its own. Here, we report a solution structure of the full-length human Orc6 (HsOrc6) alone and in a complex with DNA. We further showed that human Orc6 is composed of three independent domains: N-terminal, middle and C-terminal (HsOrc6-N, HsOrc6-M and HsOrc6-C). We also identified a distinct DNA-binding domain of human Orc6, named as HsOrc6-DBD. The detailed analysis of the structure revealed novel amino acid clusters important for the interaction with DNA. Alterations of these amino acids abolish DNA-binding ability of Orc6 and result in reduced levels of DNA replication. We propose that Orc6 is a DNA-binding subunit of human/metazoan ORC and may play roles in targeting, positioning and assembling the functional ORC at the origins.

Coupling of Ca2+ and voltage activation in BK channels through the αB helix/voltage sensor interface.

Large-conductance Ca2+ and voltage-activated K+ (BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is composed of a voltage sensor domain (VSD), a central pore-gate domain, and a large cytoplasmic domain (CTD) that contains the Ca2+ sensors. While it is known that BK channels are activated by voltage and Ca2+, and that voltage and Ca2+ activations interact, less is known about the mechanisms involved. We explore here these mechanisms by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the Horrigan, Cui, and Aldrich model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca2+ activation for both Ca2+ bowl and RCK1 Ca2+ sites, suggesting that both high-affinity Ca2+ sites transduce their effect, at least in part, through the αB helix. Mg2+ activation also decreased. The crystal structure of the CTD with proline mutation L390P showed a flattening of the first helical turn in the αB helix compared to wild type, without other notable differences in the CTD, indicating that structural changes from the mutation were confined to the αB helix. These findings indicate that an intact αB helix/VSD interface is required for effective coupling of Ca2+ binding and voltage depolarization to pore opening and that shared Ca2+ and voltage transduction pathways involving the αB helix may be involved.

The crystal structure of an antiparallel chair-type G-quadruplex formed by Bromo-substituted human telomeric DNA.

Human telomeric guanine-rich DNA, which could adopt different G-quadruplex structures, plays important roles in protecting the cell from recombination and degradation. Although many of these structures were determined, the chair-type G-quadruplex structure remains elusive. Here, we present a crystal structure of the G-quadruplex composed of the human telomeric sequence d[GGGTTAGG8GTTAGGGTTAGG20G] with two dG to 8Br-dG substitutions at positions 8 and 20 with syn conformation in the K+ solution. It forms a novel three-layer chair-type G-quadruplex with two linking trinucleotide loops. Particularly, T5 and T17 are coplanar with two water molecules stacking on the G-tetrad layer in a sandwich-like mode through a coordinating K+ ion and an A6•A18 base pair. While a twisted Hoogsteen A12•T10 base pair caps on the top of G-tetrad core. The three linking TTA loops are edgewise and each DNA strand has two antiparallel adjacent strands. Our findings contribute to a deeper understanding and highlight the unique roles of loop and water molecule in the folding of the G-quadruplex.

A chair-type G-quadruplex structure formed by a human telomeric variant DNA in K+ solution.

Guanine tracts of human telomeric DNA sequences are known to fold into eight different four-stranded structures that vary by the conformation of guanine nucleotides arranged in the stack of G-tetrads in their core and by different kinds and orders of connecting loops, called G-quadruplexes. Here, we present a novel G-quadruplex structure formed in K+ solution by a human telomeric variant d[(GGGTTA)2GGGTTTGGG], htel21T18. This variant DNA is located in the subtelomeric regions of human chromosomes 8, 11, 17, and 19 as well as in the DNase hypersensitive region and in the subcentromeric region of chromosome 5. Interestingly, single A18T substitution that makes htel21T18 different from the human telomeric sequence results in the formation of a three-layer chair-type G-quadruplex, a fold previously unknown among human telomeric repeats, with two loops interacting through the reverse Watson-Crick A6·T18 base pair. The loops are edgewise; glycosidic conformation of guanines is syn·anti·syn·anti around each tetrad, and each strand of the core has two antiparallel adjacent strands. Our results expand the repertoire of known G-quadruplex folding topologies and may provide a potential target for structure-based anticancer drug design.

Prognostic values of common clinical parameters in advanced pancreatic ductal adenocarcinoma: a large multicenter cohort study of ten years.

OBJECTIVE: We conducted a multicenter cohort study to investigate the prognostic value of some commonly-used laboratory indices in advanced pancreatic ductal adenocarcinoma (PDAC). METHODS: A multicenter cohort study was conducted from 2004 to 2013. The associations between laboratory indices and prognosis of advanced PDAC were examined. RESULTS: This cohort consisted of 553 females (36.2%) and 973 males (63.8%). Patients at cancer stage III and IV were 595 (39.0%) and 931 (61.0%), respectively. The median survival of stage III patients was 9.0 months, with 3-, 6-, and 12-month survival rates of 94.5%, 73.4%, and 28.5%, respectively. The median survival of stage IV patients was 5.4 months, with 3-, 6-, and 12-month survival rates of 79.3%, 42.9%, and 15.0%, respectively. In multivariate analyses, primary tumor diameter, low albumin, and elevated CA19-9 were associated with decreased survival for stage III patients. Age, smoking, primary tumor diameter, elevated ALT or AST, low albumin, and elevated CA19-9 were associated with decreased survival for stage IV patients. CONCLUSION: Elevated CA19-9 level, decreased albumin level, and tumor size were associated with worse survival in stage III patients. Meanwhile, advanced age, smoking, and ALT or AST level were negatively correlated to prognosis in stage IV patients.

Characterizations of distinct parallel and antiparallel G-quadruplexes formed by two-repeat ALS and FTD related GGGGCC sequence.

The large expansion of GGGGCC (G4C2) repeats of the C9orf72 gene have been found to lead to the pathogenesis of devastating neurological diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The structural polymorphisms of C9orf72 HRE DNA and RNA may cause aberrant transcription and contribute to the development of ALS and FTD. Here we showed that the two-repeat G4C2 DNA, d(G4C2)2, simultaneously formed parallel and antiparallel G-quadruplex conformations in the potassium solution. We separated different folds of d(G4C2)2 by anion exchange chromatography, followed with characterizations by circular dichroism and nuclear magnetic resonance spectroscopy. The parallel d(G4C2)2 G-quadruplex folded as a symmetric tetramer, while the antiparallel d(G4C2)2 adopted the topology of an asymmetric dimer. These folds are distinct from the antiparallel chair-type conformation we previously identified for the d(G4C2)4 G-quadruplex. Our findings have demonstrated the conformational heterogeneity of the C9orf72 HRE DNA, and provided new insights into the d(G4C2)n folding. Meanwhile, the purified d(G4C2)2 G-quadruplex samples are suitable for further three-dimensional structure characterizations, which are required for the structure-based design of small molecules targeting ALS and FTD related C9orf72 HRE.

Safety of Metformin Therapy in Patients with Type 2 Diabetes Living on an Oxygen-Deficient Plateau, Tibet, China.

BACKGROUND: In the general population, the absolute risk of lactic acidosis in patients treated with metformin appears to be low. However, in the Tibetan plateau, an extreme oxygen-deficient environment, there are no data available concerning the safety of metformin. The aim of our study is to assess the safety of metformin in people of the plateau area. METHODS: We conducted an observational cross-sectional study in Tibet. All the 166 subjects were divided into two groups: T2DM+metformin group and T2DM group. Clinical characteristics as well as lactate levels were measured in all subjects. The association between lactate, metformin use, FBG, HbA1c, eGFR, and other potential predictors was evaluated. RESULTS: A total of 166 subjects were enrolled in this study. Average age was 51.7±12.3 years, and the percentage of male participants was 67%. The median level of lactate was 1.89 (1.35-2.91) mmol/L in all the subjects. The mean (±SD) lactate concentration in patients treated with metformin, versus those who were not, was 2.35±1.42 vs 2.29±1.65 mmol/L, respectively (mean difference 0.06 mmol/L, 95% CI: -0.48-0.60, P=0.556). FBG was significantly higher in the high lactate group than in the low lactate group (12.1±4.1 vs. 10.5±4.0 mmol/L, P=0.018). Similarly, HbA1c level was also significantly higher in the high lactate group than in the low lactate group (12.3±2.6 vs. 11.0±3.0%, P=0.008). CONCLUSIONS: In the oxygen-deficient Tibetan plateau, lactate concentration among patients on metformin was not significantly different from patients without metformin. The level of lactate was significantly associated with FBG and HbA1c levels.

A genetic variant of the sperm-specific SLO3 K+ channel has altered pH and Ca2+ sensitivities.

To fertilize an oocyte, sperm must first undergo capacitation in which the sperm plasma membrane becomes hyperpolarized via activation of potassium (K+) channels and resultant K+ efflux. Sperm-specific SLO3 K+ channels are responsible for these membrane potential changes critical for fertilization in mouse sperm, and they are only sensitive to pH i However, in human sperm, the major K+ conductance is both Ca2+- and pH i -sensitive. It has been debated whether Ca2+-sensitive SLO1 channels substitute for human SLO3 (hSLO3) in human sperm or whether human SLO3 channels have acquired Ca2+ sensitivity. Here we show that hSLO3 is rapidly evolving and reveal a natural structural variant with enhanced apparent Ca2+ and pH sensitivities. This variant allele (C382R) alters an amino acid side chain at a principal interface between the intramembrane-gated pore and the cytoplasmic gating ring of the channel. Because the gating ring contains sensors to intracellular factors such as pH and Ca2+, the effectiveness of transduction between the gating ring and the pore domain appears to be enhanced. Our results suggest that sperm-specific genes can evolve rapidly and that natural genetic variation may have led to a SLO3 variant that differs from wild type in both pH and intracellular Ca2+ sensitivities. Whether this physiological variation confers differences in fertility among males remains to be established.

Deletion of cytosolic gating ring decreases gate and voltage sensor coupling in BK channels.

Large conductance Ca2+-activated K+ channels (BK channels) gate open in response to both membrane voltage and intracellular Ca2+ The channel is formed by a central pore-gate domain (PGD), which spans the membrane, plus transmembrane voltage sensors and a cytoplasmic gating ring that acts as a Ca2+ sensor. How these voltage and Ca2+ sensors influence the common activation gate, and interact with each other, is unclear. A previous study showed that a BK channel core lacking the entire cytoplasmic gating ring (Core-MT) was devoid of Ca2+ activation but retained voltage sensitivity (Budelli et al. 2013. Proc. Natl. Acad. Sci. USA http://dx.doi.org/10.1073/pnas.1313433110). In this study, we measure voltage sensor activation and pore opening in this Core-MT channel over a wide range of voltages. We record gating currents and find that voltage sensor activation in this truncated channel is similar to WT but that the coupling between voltage sensor activation and gating of the pore is reduced. These results suggest that the gating ring, in addition to being the Ca2+ sensor, enhances the effective coupling between voltage sensors and the PGD. We also find that removal of the gating ring alters modulation of the channels by the BK channel's ß1 and ß2 subunits.

Topology of a G-quadruplex DNA formed by C9orf72 hexanucleotide repeats associated with ALS and FTD.

Abnormal expansions of an intronic hexanucleotide GGGGCC (G4C2) repeat of the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Previous studies suggested that the C9orf72 hexanucleotide repeat expansion (HRE), either as DNA or the transcribed RNA, can fold into G-quadruplexes with distinct structures. These structural polymorphisms lead to abortive transcripts and contribute to the pathogenesis of ALS and FTD. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, we analyzed the structures of C9orf72 HRE DNA with various G4C2 repeats. They exhibited diverse G-quadruplex folds in potassium ions. Furthermore, we determined the topology of a G-quadruplex formed by d(G4C2)4. It favors a monomeric fold and forms a chair-type G-quadruplex with a four-layer antiparallel G-tetra core and three edgewise loops, which is distinct from known structures of chair-type G-quadruplexes. Our findings highlight the conformational heterogeneity of C9orf72 HRE DNA, and may lay the necessary structural basis for designing small molecules for the modulation of ALS/FTD pathogenesis.