ALS-linked KIF5A ΔExon27 mutant causes neuronal toxicity through gain-of-function.

Mutations in the human kinesin family member 5A (KIF5A) gene were recently identified as a genetic cause of amyotrophic lateral sclerosis (ALS). Several KIF5A ALS variants cause exon 27 skipping and are predicted to produce motor proteins with an altered C-terminal tail (referred to as ΔExon27). However, the underlying pathogenic mechanism is still unknown. Here, we confirm the expression of KIF5A mutant proteins in patient iPSC-derived motor neurons. We perform a comprehensive analysis of ΔExon27 at the single-molecule, cellular, and organism levels. Our results show that ΔExon27 is prone to form cytoplasmic aggregates and is neurotoxic. The mutation relieves motor autoinhibition and increases motor self-association, leading to drastically enhanced processivity on microtubules. Finally, ectopic expression of ΔExon27 in Drosophila melanogaster causes wing defects, motor impairment, paralysis, and premature death. Our results suggest gain-of-function as an underlying disease mechanism in KIF5A-associated ALS.

Diffusion quantum Monte Carlo method on diradicals using single- and multi-determinant-Jastrow trial wavefunctions and different orbitals.

In this work, the diffusion quantum Monte Carlo (DMC) method is employed to calculate the energies of singlet and triplet states for a series of organic diradicals and diatomic diradicals with π2 configuration. Single-determinant-Jastrow (SDJ) trial wavefunctions for triplet states, two-determinant-Jastrow (2DJ) trial wavefunctions for the singlet states, and multi-determinant-Jastrow (MDJ) trial wavefunctions are employed in DMC calculations using restricted open-shell B3LYP (ROB3LYP) orbitals, complete-active-space self-consistent field (CASSCF) orbitals, state-average CASSCF orbitals, or frozen-CASSCF orbitals. Our results show that DMC energies using either SDJ/2DJ or MDJ with ROB3LYP orbitals are close to or lower than those with the other orbitals for organic diradicals, while they are not very sensitive to the employed orbitals for diatomic diradicals. Furthermore, using MDJ can reduce DMC energies to some extent for most of the investigated organic diradicals and some diatomic diradicals. The importance of MDJ on DMC energies can be estimated based on the percentage of main determinants in the CASCI wavefunction. On the other hand, singlet-triplet gaps can be calculated reasonably with DMC using MDJ with a mean absolute error of less than 2 kcal/mol with all these orbitals. CASCI wavefunctions using density functional theory orbitals are preferred in constructing MDJ trial wavefunctions in practical DMC calculations since it is easier to obtain such wavefunctions than CASSCF methods.

Optical Control of Mitosis with a Photoswitchable Eg5 Inhibitor.

Eg5 is a kinesin motor protein that is responsible for bipolar spindle formation and plays a crucial role during mitosis. Loss of Eg5 function leads to the formation of monopolar spindles, followed by mitotic arrest, and subsequent cell death. Several cell-permeable small molecules have been reported to inhibit Eg5 and some have been evaluated as anticancer agents. We now describe the design, synthesis, and biological evaluation of photoswitchable variants with five different pharmacophores. Our lead compound Azo-EMD is a cell permeable azobenzene that inhibits Eg5 more potently in its light-induced cis form. This activity decreased the velocity of Eg5 in single-molecule assays, promoted formation of monopolar spindles, and led to mitotic arrest in a light dependent way.

Recoding the Cancer Epigenome by Intervening in Metabolism and Iron Homeostasis with Mitochondria-Targeted Rhenium(I) Complexes.

The development and malignancy of cancer cells are closely related to the changes of the epigenome. In this work, a mitochondria-targeted rhenium(I) complex (DFX-Re3), integrating the clinical iron chelating agent deferasirox (DFX), has been designed. By relocating iron to the mitochondria and changing the key metabolic species related to epigenetic modifications, DFX-Re3 can elevate the methylation levels of histone, DNA, and RNA. As a consequence, DFX-Re3 affects the events related to apoptosis, RNA polymerases, and T-cell receptor signaling pathways. Finally, it is shown that DFX-Re3 induces immunogenic apoptotic cell death and exhibits potent antitumor activity in vivo. This study provides a new approach for the design of novel epigenetic drugs that can recode the cancer epigenome by intervening in mitochondrial metabolism and iron homeostasis.

Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains.

Cytoplasmic dynein is a homodimeric microtubule (MT) motor protein responsible for most MT minus-end-directed motility. Dynein contains four AAA+ ATPases (AAA: ATPase associated with various cellular activities) per motor domain (AAA1-4). The main site of ATP hydrolysis, AAA1, is the only site considered by most dynein motility models. However, it remains unclear how ATPase activity and MT binding are coordinated within and between dynein's motor domains. Using optical tweezers, we characterize the MT-binding strength of recombinant dynein monomers as a function of mechanical tension and nucleotide state. Dynein responds anisotropically to tension, binding tighter to MTs when pulled toward the MT plus end. We provide evidence that this behavior results from an asymmetrical bond that acts as a slip bond under forward tension and a slip-ideal bond under backward tension. ATP weakens MT binding and reduces bond strength anisotropy, and unexpectedly, so does ADP. Using nucleotide binding and hydrolysis mutants, we show that, although ATP exerts its effects via binding AAA1, ADP effects are mediated by AAA3. Finally, we demonstrate "gating" of AAA1 function by AAA3. When tension is absent or applied via dynein's C terminus, ATP binding to AAA1 induces MT release only if AAA3 is in the posthydrolysis state. However, when tension is applied to the linker, ATP binding to AAA3 is sufficient to "open" the gate. These results elucidate the mechanisms of dynein-MT interactions, identify regulatory roles for AAA3, and help define the interplay between mechanical tension and nucleotide state in regulating dynein motility.

High activity of cubic PtRh alloys supported on graphene towards ethanol electrooxidation.

Cubic PtRh alloys supported on graphene (PtxRhy/GN) with different atomic ratio of Pt and Rh were directly synthesized for the first time using the modified polyol method with Br(-) for the shape-directing agents. The process didn't use surface-capping agents such as PVP that easily occupy the active sites of electrocatalysts and are difficult to remove. Graphene is the key factor for cubic shape besides Br(-) and keeping catalysts high-dispersed. The X-ray diffraction (XRD), scanning electron microscope (SEM) and transmission electron microscope (TEM) were used to characterize the structure and morphology of these electrocatalysts. The results showed that they were composed of homogeneous cubic PtRh alloys. Traditional electrochemical methods, such as cyclic voltammetry and chronoamperometry, were used to investigate the electrocatalytic properties of PtxRhy/GN towards ethanol electrooxidation. It can be seen that PtxRhy/GN with all atomic ratios exhibited high catalytic activity, and the most active one has a composition with Pt : Rh = 9 : 1 atomic ratio. Electrochemical in situ FTIR spectroscopy was used to evaluate the cleavage of C-C bond in ethanol at room temperature in acidic solutions, the results illustrated that Rh in an alloy can promote the split of C-C bond in ethanol, and the alloy catalyst with atomic ratio Pt : Rh = 1 : 1 showed obviously better performance for the C-C bond breaking in ethanol and higher selectivity for the enhanced activity of ethanol complete oxidation to CO2 than alloys with other ratios of Pt and Rh. The investigation indicates that high activity of PtxRhy/GN electrocatalyst towards ethanol oxidation is due to the specific shape of alloys and the synergistic effect of two metal elements as well as graphene support.

Structure and Function of Dynein's Non-Catalytic Subunits.

Dynein, an ancient microtubule-based motor protein, performs diverse cellular functions in nearly all eukaryotic cells, with the exception of land plants. It has evolved into three subfamilies-cytoplasmic dynein-1, cytoplasmic dynein-2, and axonemal dyneins-each differentiated by their cellular functions. These megadalton complexes consist of multiple subunits, with the heavy chain being the largest subunit that generates motion and force along microtubules by converting the chemical energy of ATP hydrolysis into mechanical work. Beyond this catalytic core, the functionality of dynein is significantly enhanced by numerous non-catalytic subunits. These subunits are integral to the complex, contributing to its stability, regulating its enzymatic activities, targeting it to specific cellular locations, and mediating its interactions with other cofactors. The diversity of non-catalytic subunits expands dynein's cellular roles, enabling it to perform critical tasks despite the conservation of its heavy chains. In this review, we discuss recent findings and insights regarding these non-catalytic subunits.

Kinesin-14 HSET and KlpA are non-processive microtubule motors with load-dependent power strokes.

Accurate chromosome segregation during cell division relies on coordinated actions of microtubule (MT)-based motor proteins in the mitotic spindle. Kinesin-14 motors play vital roles in spindle assembly and maintenance by crosslinking antiparallel MTs at the spindle midzone and anchoring spindle MTs' minus ends at the poles. In this study, we investigate the force generation and motility of the Kinesin-14 motors HSET and KlpA. Our findings reveal that both motors are non-processive, producing single load-dependent power strokes per MT encounter, with estimated load-free power strokes of ~30 and ~35 nm, respectively. Each homodimeric motor generates forces of ~0.5 pN, but when assembled in teams, they cooperate to generate forces of 1 pN or more. Notably, the cooperative activity among multiple motors leads to increased MT-sliding velocities. These results quantitatively elucidate the structure-function relationship of Kinesin-14 motors and underscore the significance of cooperative behavior in their cellular functions.

Cryo-EM unveils kinesin KIF1A's processivity mechanism and the impact of its pathogenic variant P305L.

Mutations in the microtubule-associated motor protein KIF1A lead to severe neurological conditions known as KIF1A-associated neurological disorders (KAND). Despite insights into its molecular mechanism, high-resolution structures of KIF1A-microtubule complexes remain undefined. Here, we present 2.7-3.5 Å resolution structures of dimeric microtubule-bound KIF1A, including the pathogenic P305L mutant, across various nucleotide states. Our structures reveal that KIF1A binds microtubules in one- and two-heads-bound configurations, with both heads exhibiting distinct conformations with tight inter-head connection. Notably, KIF1A's class-specific loop 12 (K-loop) forms electrostatic interactions with the C-terminal tails of both α- and ß-tubulin. The P305L mutation does not disrupt these interactions but alters loop-12's conformation, impairing strong microtubule-binding. Structure-function analysis reveals the K-loop and head-head coordination as major determinants of KIF1A's superprocessive motility. Our findings advance the understanding of KIF1A's molecular mechanism and provide a basis for developing structure-guided therapeutics against KAND.

Measurements of the Force-Dependent Detachment Rates of Cytoplasmic Dynein from Microtubules.

Cytoplasmic dynein, the largest and most intricate cytoskeletal motor protein, powers the movement of numerous intracellular cargos toward the minus ends of microtubules (MT). Despite its essential roles in eukaryotic cells, dynein's molecular mechanism, the regulatory functions of its subunits and accessory proteins, and the consequences of human disease mutations on dynein force generation remain largely unclear. Recent work combining mutagenesis, single-molecule fluorescence, and optical tweezers-based force measurement have provided valuable insights into how dynein's multiple AAA+ ATPase domains regulate dynein's attachment to MTs. Here, we describe detailed protocols for the measurements of the force-dependent dynein-MT detachment rates. We provide updated and optimized protocols for the expression and purification of a tail-truncated single-headed Saccharomyces cerevisiae dynein, for polarity-marked MT polymerization, and for the non-covalent attachment of MTs to cover glass surfaces for the measurement of dynein-MT detachment forces.

Kinesin-14 HSET and KlpA are non-processive microtubule motors with load-dependent power strokes.

Accurate chromosome segregation during cell division relies on coordinated actions of microtubule (MT)-based motor proteins in the mitotic spindle. Kinesin-14 motors play vital roles in spindle assembly and maintenance by crosslinking antiparallel MTs at the spindle midzone and anchoring spindle MTs' minus ends at the poles. We investigate the force generation and motility of the Kinesin-14 motors HSET and KlpA, revealing that both motors function as non-processive motors under load, producing single power strokes per MT encounter. Each homodimeric motor generates forces of ∼0.5 pN, but when assembled in teams, they cooperate to generate forces of 1 pN or more. Importantly, cooperative activity among multiple motors leads to increased MT-sliding velocities. Our findings deepen our understanding of the structure-function relationship of Kinesin-14 motors and underscore the significance of cooperative behavior in their cellular functions.

Cryo-EM Unveils the Processivity Mechanism of Kinesin KIF1A and the Impact of its Pathogenic Variant P305L.

Mutations in the microtubule-associated motor protein KIF1A lead to severe neurological conditions known as KIF1A-associated neurological disorders (KAND). Despite insights into its molecular mechanism, high-resolution structures of KIF1A-microtubule complexes remain undefined. Here, we present 2.7-3.4 Å resolution structures of dimeric microtubule-bound KIF1A, including the pathogenic P305L mutant, across various nucleotide states. Our structures reveal that KIF1A binds microtubules in one- and two-heads-bound configurations, with both heads exhibiting distinct conformations with tight inter-head connection. Notably, KIF1A's class-specific loop 12 (K-loop) forms electrostatic interactions with the C-terminal tails of both α- and ß-tubulin. The P305L mutation does not disrupt these interactions but alters loop-12's conformation, impairing strong microtubule-binding. Structure-function analysis reveals the K-loop and head-head coordination as major determinants of KIF1A's superprocessive motility. Our findings advance the understanding of KIF1A's molecular mechanism and provide a basis for developing structure-guided therapeutics against KAND.

Detection and tracking of cytoplasmic G-quadruplexes in live cells.

A TTPP probe was developed to distinguish G-quadruplexes (G4s) from other nucleic acid topologies through longer fluorescence lifetimes and higher quantum yields. In fluorescence lifetime imaging microscopy, TTPP enabled the visualization of cytoplasmic G4s in live cells, and showed the potential to detect cell apoptosis and ferroptosis by tracking cytoplasmic G4s.

Controllable Preparation and Research Progress of Photosensitive Antibacterial Complex Hydrogels.

Hydrogels are materials consisting of a network of hydrophilic polymers. Due to their good biocompatibility and hydrophilicity, they are widely used in biomedicine, food safety, environmental protection, agriculture, and other fields. This paper summarizes the typical complex materials of photocatalysts, photosensitizers, and hydrogels, as week as their antibacterial activities and the basic mechanisms of photothermal and photodynamic effects. In addition, the application of hydrogel-based photoresponsive materials in microbial inactivation is discussed, including the challenges faced in their application. The advantages of photosensitive antibacterial complex hydrogels are highlighted, and their application and research progress in various fields are introduced in detail.

Association between lipocalin-2 and mild cognitive impairment or dementia: A systematic review and meta-analysis of population-based evidence.

BACKGROUND: The associations between lipocalin-2 (LCN2) with mild cognitive impairment (MCI) and dementia have gained growing interest. However, population-based studies have yielded inconsistent findings. Therefore, we conducted this essential systematic review and meta-analysis to analyze and summarize the existing population-based evidence. METHODS: PubMed, EMBASE, and Web of Science were systematically searched until Mar 18, 2022. Meta-analysis was performed to generate the standard mean difference (SMD) of peripheral blood and cerebrospinal fluid (CSF) LCN2. A qualitative review was performed to summarize the evidence from postmortem brain tissue studies. RESULTS: In peripheral blood, the overall pooled results showed no significant difference in LCN2 across Alzheimer's disease (AD), MCI and control groups. Further subgroup analysis revealed higher serum LCN2 levels in AD compared to controls (SMD =1.28 [0.44;2.13], p = 0.003), while the difference remained insignificant in plasma (SMD =0.04 [-0.82;0.90], p = 0.931). Besides, peripheral blood LCN2 were higher in AD when age difference between AD and controls ≥ 4 years (SMD =1.21 [0.37;2.06], p = 0.005). In CSF, no differences were found in LCN2 across groups of AD, MCI and controls. However, CSF LCN2 was higher in vascular dementia (VaD) compared to controls (SMD =1.02 [0.17;1.87], p = 0.018), as well as compared to AD (SMD =1.19 [0.58;1.80], p < 0.001). Qualitative analysis supported that LCN2 was increased in the brain tissue of AD-related areas, especially in astrocytes and microglia; while LCN2 increased in infarct-related brain areas and over-expressed in astrocytes and macrophages in mixed dementia (MD). CONCLUSION: The difference in peripheral blood LCN2 between AD and controls may be affected by the type of biofluid and age. No differences were found in CSF LCN2 across AD, MCI and controls groups. In contrast, CSF LCN2 was elevated in VaD patients. Moreover, LCN2 was increased in AD-related brain areas and cells in AD, while in infarcts-related brain areas and cells in MD.

Single-Molecule Studies on the Motion and Force Generation of the Kinesin-3 Motor KIF1A.

KIF1A is a neuron-specific member of the kinesin-3 family of microtubule (MT) plus-end-directed motor proteins. It powers the migration of nuclei in differentiating brain stem cells and the transport of synaptic precursors and dense core vesicles in axons. Its dysfunction causes severe neurodevelopmental and neurodegenerative diseases termed KIF1A-associated neurological disorders (KAND). KAND mutations span the entirety of the KIF1A protein sequence, of which the majority are located within the motor domain and are thus predicted to affect the motor's motility and force-generating properties. Unfortunately, the molecular etiologies of KAND remain poorly understood, in part because KIF1A's molecular mechanism remains unclear. Here, we describe detailed methods for how to express a tail-truncated dimeric KIF1A in E. coli cells and provide step-by-step protocols for performing single-molecule studies with total internal reflection fluorescence microscopy and optical tweezers assays, which, when combined with structure-function studies, help to decipher KIF1A's molecular mechanism.

Osmanthus-Loaded PVP/PVA Hydrogel Inhibits the Proliferation and Migration of Oral Squamous Cell Carcinoma Cells CAL-27.

Conventional medical agents for oral squamous cell carcinoma (OSCC) with some adverse effects no longer meet the needs of the public. In this study, the prognosis-related hub genes of osmanthus-targeted therapy for OSCC were predicted and analyzed by network pharmacology and molecular docking. Osmanthus was extracted using the ethanol reflux method and osmanthus-loaded PVP/PVA (OF/PVP/PVA) hydrogel was prepared by electron beam radiation. The molecular structure, crystal structure and microscopic morphology of hydrogels were observed by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM), respectively. OSCC cells CAL-27 were cultured with OF/PVP/PVA hydrogel at different concentrations of extract to discover cell proliferation by MTT assay. The scratching test and JC-1 staining were used to observe the migration and mitochondrial membrane potential. Through experimental exploration, we found that a total of six prognosis-related targets were predicted, which are PYGL, AURKA, SQLE, etc., and osmanthus extract had good binding activity to AURKA. In vitro, except for proliferation inhibition, OF/PVP/PVA hydrogel prevented cell migration and changed the mitochondrial membrane potential of CAL-27 cells at a concentration equal to or greater than 50 µg/mL (p < 0.05). The addition of autophagy inhibitor chloroquine and 3-methyladenine weakened the migration inhibition of hydrogel (p < 0.05).

Osmanthus Fragrans Loaded NIPAAM Hydrogel Promotes Osteogenic Differentiation of MC3T3-E1.

There is an urgent need to find long-acting, natural osteogenesis-promoting drug systems. In this study, first the potential targets and mechanism of osmanthus fragrans (O. fragrans) extract in regulating osteogenic differentiation based on autophagy were analyzed by network pharmacology and molecular docking. Then, osmanthus fragrans was extracted using the ethanol reflux method and an osmanthus fragrans extract loaded Poly N-isopropylacrylamide (OF/NIPAAM) hydrogel was prepared by electron beam radiation. The chemical components of the osmanthus fragrans extract and the microstructure of OF/NIPAAM hydrogels were characterized by ultraviolet-visible spectrophotometry (UV-Vis) and X-ray diffraction (XRD), respectively. Mouse embryonic osteoblast precursor cells MC3T3-E1 were cultured with different concentrations of OF/NIPAAM hydrogel to discover cell proliferation activity by CCK-8 assay. Alkaline phosphatase (ALP) staining and alizarin red staining were used to observe the differentiation and calcification. Through experimental exploration, we found that a total of 11 targets were predicted, which are TP53, CASP3, SIRT1, etc., and osmanthus fragrans had good binding activity to TP53. In vitro, except for proliferation promotion, OF/NIPAAM hydrogel enhanced ALP activity and formation of mineralized nodules of MC3T3-E1 cells at a concentration equal to or less than 62.5 µg/mL (p < 0.05). The addition of autophagy inhibitor 3-methyladenine (3-MA) reduced ALP activity and mineralized nodule formation.

Doublecortin and JIP3 are neural-specific counteracting regulators of dynein-mediated retrograde trafficking.

Mutations in the microtubule (MT)-binding protein doublecortin (DCX) or in the MT-based molecular motor dynein result in lissencephaly. However, a functional link between DCX and dynein has not been defined. Here, we demonstrate that DCX negatively regulates dynein-mediated retrograde transport in neurons from Dcx-/y or Dcx-/y;Dclk1-/- mice by reducing dynein's association with MTs and disrupting the composition of the dynein motor complex. Previous work showed an increased binding of the adaptor protein C-Jun-amino-terminal kinase-interacting protein 3 (JIP3) to dynein in the absence of DCX. Using purified components, we demonstrate that JIP3 forms an active motor complex with dynein and its cofactor dynactin with two dyneins per complex. DCX competes with the binding of the second dynein, resulting in a velocity reduction of the complex. We conclude that DCX negatively regulates dynein-mediated retrograde transport through two critical interactions by regulating dynein binding to MTs and regulating the composition of the dynein motor complex.

Recent Advances in Smart Hydrogels Prepared by Ionizing Radiation Technology for Biomedical Applications.

Materials with excellent biocompatibility and targeting can be widely used in the biomedical field. Hydrogels are an excellent biomedical material, which are similar to living tissue and cannot affect the metabolic process of living organisms. Moreover, the three-dimensional network structure of hydrogel is conducive to the storage and slow release of drugs. Compared to the traditional hydrogel preparation technologies, ionizing radiation technology has high efficiency, is green, and has environmental protection. This technology can easily adjust mechanical properties, swelling, and so on. This review provides a classification of hydrogels and different preparation methods and highlights the advantages of ionizing radiation technology in smart hydrogels used for biomedical applications.