Microtubule Structure Research Articles

Microtubule-Rab5 mutual-influential system screening based on gene-regulatory networks map in Rab5 RNAi eye-degeneration model

Rabs are involved in neuronal development and protrusion formation. Existing studies support the notion that manipulation or mutation of Rab genes could lead to functional changes in neurons. However, whether Rabs gene-manipulation induced Drosophila eye-degeneration remains unknown. By down-regulating Rab5, but not Rab7, we first constructed a compound eye injury model in Drosophila. As the distribution, content, and even maturation of Rab5-positive endosomes are influenced by cytoskeletal proteins, like actin or tubulin-related proteins, the existence of a bidirectional regulatory relationship between Rab5 and the cytoskeleton remains unclear and worth researching. Through complete transcriptome sequencing combined immunofluorescence testing, we confirmed that down-regulation of Rab5 affected the increase of α-Tub84B (alternatively named TubA84B) but not γ-tubulin. Based on Weighted Gene Co-Expression Network Analysis (WGCNA) and multi-tissue screening verification, this study proposes that the apoptosis-related factors–Rab5–TubA84B have conserved regulatory functions with cooperative expression. Gene manipulation confirmed that apoptotic factors, especially rpr, strongly regulate Rab5, and may ultimately influence microtubule structure through complex routes, including the Rab5 variance and the intracellular configuration ratio of α-Tubulin to Glyceraldehyde-3-phosphate dehydrogenase.

The Impact of a Catalytic Site Mutation on the Shape and Mechanics of Tubulin Protofilaments

Alpha-beta heterodimers of tubulin proteins serve as the building blocks of microtubules, which are key biopolymers forming one of the principal systems of the cellular cytoskeleton. A detailed study of these building blocks, as well as their alterations caused by point mutations, contributes to a deeper understanding of physiological and pathological processes related to the cytoskeleton. This study presents an analysis of the impact of the E254N point mutation in the catalytic site of α-tubulin on the bending conformations of human recombinant tubulin tetramers using molecular dynamics methods. The models were constructed based on high-resolution cryo-electron microscopy data, allowing the reconstruction of three-dimensional structures of both wild-type and mutant tetramers. The simulations revealed that the primary difference between wild-type and mutant tubulin lies in the equilibrium bending direction of the protofilaments, while the bending amplitude, twisting, and associated stiffness remain largely unchanged. We propose that the observed differences in bending directions may be related to variations in protofilament tilts within microtubules, which aligns with previously published cryo-electron microscopy data. These findings provide valuable insights into the principles underlying the formation of the polymeric structure of microtubules based on the properties of their individual building blocks.

Highly-enhanced gas-sensing performance of metal-doped In2O3 microtubes from acceptor doping and double surface adsorption

The different valent metal-doping is a feasible and convenient way to adjust the microstructures and electron concentration of In2O3 gas sensors. In this paper, the different valence metals (Zn2+, Sb3+, Zr4+ and Nb5+) are doped into MIL-68 (In) metal–organic frameworks (MOFs) by solvothermal method, and then In2O3 and metal-doped In2O3 microtubes are obtained by pyrolysis MIL-68 MOFs. All samples exhibit the similar microtubular structures, indicating oxygen adsorption on both inner and outer surface. The average grain size of metal-doped In2O3 microtubes decreases a little while the specific surface area increases greatly. Metal-doping greatly affects the formaldehyde gas-sensing performance, and Zn2+-doped In2O3 sensor presents the highest response value (188.56), shortest response/recovery times and excellent selectivity to formaldehyde gas at 210 ℃. Compared the microstructural and gas-sensing parameters of In2O3 sensor, the specific surface area and oxygen vacancies of metal-doped In2O3 sensors enhance the surface O-. Moreover, acceptor Zn2+-doping directly extracts electrons from conduction band of Zn2+-doped In2O3 sensor, which greatly increases the resistance in air and the thickness of electron deletion layer for Zn2+-doped In2O3 sensor.

Mlf mediates proteotoxic response via formation of cellular foci for protein folding and degradation in Giardia.

Myeloid leukemia factor 1 (Mlf1) was identified as a proto-oncoprotein that affects hematopoietic differentiation in humans. However, its cellular function remains elusive, spanning roles from cell cycle regulation to modulation of protein aggregate formation and participation in ciliogenesis. Given that structurally conserved homologs of Mlf1 can be found across the eukaryotic tree of life, we decided to characterize its cellular role underlying this phenotypic pleiotropy. Using a model of the unicellular eukaryote Giardia intestinalis, we demonstrate that its Mlf1 homolog (GiMlf) mainly localizes to two types of cytosolic foci: microtubular structures, where it interacts with Hsp40, and ubiquitin-rich, membraneless compartments, found adjacent to mitochondrion-related organelles known as mitosomes, containing the 26S proteasome regulatory subunit 4. Upon cellular stress, GiMlf either relocates to the affected compartment or disperses across the cytoplasm, subsequently accumulating into enlarged foci during the recovery phase. In vitro assays suggest that GiMlf can be recruited to membranes through its affinity for signaling phospholipids. Importantly, cytosolic foci diminish in the gimlf knockout strain, which exhibits extensive proteomic changes indicative of compromised proteostasis. Consistent with data from other cellular systems, we propose that Mlf acts in the response to proteotoxic stress by mediating the formation of function-specific foci for protein folding and degradation.

Utidelone plus pembrolizumab as the fourth-line combination treatment in non-small cell lung cancer with EGFR mutation: a case report.

Utidelone is an ebomycin derivative chemotherapeutic drug, which can promote tubulin polymerization and stabilize microtubule structure, so as to induce apoptosis. The drug is an innovative drug independently developed by China with independent intellectual property rights. Phase II clinical trials for advanced breast cancer are being approved by National Medical Products Administration for the treatment of advanced breast cancer. However, there is no report on the application in non-small cell lung cancer (NSCLC) patients with the epidermal growth factor receptor (EGFR) mutation. This case is a patient with EGFR mutant stage IV NSCLC who has progressed after third-line targeted therapy. The fourth line was treated with utidelone combined with pabolizumab. The patient had progressed after targeted therapy with oxitinib, ametinib, and vometinib. Due to the patient's physical reasons, the traditional platinum drugs were not suitable, so the patient was treated with utidelone combined with pabolizumab. The curative effect was evaluated as SD after two cycles and progesterone receptor after four cycles. At present, it is still in the maintenance of reduction of utidelone combined with pabolizumab, and the tumor continues to shrink. Although peripheral neurotoxicity occurred during treatment, it improved after symptomatic treatment. The treatment of EGFR mutant stage IV NSCLC with utidelone combined with pabolizumab has good effect and mild adverse reactions.

Thermal Shrinking of Biopolymeric Hydrogels for High Resolution 3D Printing of Kidney Tubules

AbstractThe effective replication of microtubular structures in tissue engineering remains a great challenge. In this study, the temperature‐responsive characteristics of poly(N‐isopropylacrylamide) (pNIPAM) to create intricate, high‐resolution tubular structures through a shrinking mechanism is investigated by exploring 2 thermosensitive hydrogels–gelatin methacryloyl (gelMA) and silk fibroin methacryloyl (silkMA)–combined with pNIPAM. Systematic investigations revealed precise control of shrinking behavior at elevated temperatures (33–37 °C) as a function of polymer concentration. The hydrogel sizes reduce by ≈15% from room temperature (RT) to 33 °C and ≈40% from RT to 37 °C for both hydrogel types. The shrinking affects the mechanical properties, increasing the compressive modulus by ≈2.8‐fold for gelMA‐pNIPAM gels and ≈5.1‐fold for silkMA‐pNIPAM gels at 37 °C. Combined with volumetric printing, these materials achieve resolution enhancements of ≈20% for positive features and ≈70% for negative features, enabling the creation of complex, high‐resolution structures within seconds, with open channels (≈50 µm). GelMA‐pNIPAM hydrogels show better cell compatibility compared to silkMA‐pNIPAM hydrogels, promoting cell adhesion and viability. This study demonstrates the thermosensitive hydrogels' capability to engineer intricate, high‐resolution tubular structures with volumetric printing–an efficient route to fabricate microenvironments mimicking native tissues with potential for developing relevant in vitro models.

The Chitinous Skeleton of Ianthella basta Marine Demosponge as a Renewable Scaffold-Based Carrier of Antiseptics

The chitinous skeleton of the marine demosponge Ianthella basta exhibits a unique network-like 3D architecture, excellent capillary properties, and chemical inertness, making it highly suitable for interdisciplinary research, especially in biomedical applications. This study investigates the potential of renewable I. basta chitinous scaffolds for drug delivery and wound dressing. The scaffolds, characterized by a microtubular structure, were impregnated with selected commercially available antiseptics, including solutions with hydrophilic and hydrophobic properties. Evaluations against selected clinical strains of bacteria, as well as fungi, demonstrated significant zones of growth inhibition with antiseptics such as brilliant green, gentian violet, decamethoxine, and polyhexanide. Notably, the antibacterial properties of these antiseptic-treated chitin matrices persisted for over 72 h, effectively inhibiting microbial growth in fresh cultures. These findings highlight the considerable potential of I. basta chitin scaffolds as sustainable, innovative biomaterials for controlled drug release and wound dressing applications.

Impaired Intestinal Barrier Function in Immune Thrombocytopenic Purpura Patients Measured by Serum Zonulin

Abstract Background Besides its involvement in the digestion and resorption of nutrients, one task of the intestine is to act as a barrier regulating the passage of antigens and macromolecules from the environment into the organism. This barrier is to a major extent formed by the intestinal epithelia and its lateral tight junctions whichclose off the paracellular interstices. The tight junctions consist of transmembrane barrier proteins such as occludin and claudins, which are bound via proteins of the intracellular cytoskeleton with actin and microtubular structures. The function of the intestinal barrier (IB) can be modulated by a variety of stimuli. These include nutrients such as gluten, toxins or bacteria or their respective metabolic products ab- sorbed with the nutrition which induce a raised secretion of zonulin through the cells of the lamina propria. Objective To evaluate serum Zonulin level, as a marker of increased intestinal permeability, in ITP patient compared to healthy subjects. Methods This Study was conducted between October 2022 and June 2023 on 60 patients diagnosed with ITP divided in to 2 sub-groups 30 patients on steroid therapy for a duration longer than 3 months and 30 steroid naïve patients togetherwith 30 apparently healthy individuals as a control group. Patients were recruitedfrom Hematology department and outpatient clinic, Ain Shams University Hospitals. Results There was highly statistically significant difference between chronic patients and newly diagnosed patients regarding PLT and Serum Zonulin p < 0.001. There was highly statistically significant difference between recently diagnosed patients and healthy controls regarding PLT p < 0.001, there was statistically significant difference regarding ALT and Serum Zonulin, (p value were 0.03 and 0.01) respectively. There was a statistically significant correlation among all study participants between Serum Zonulin and Creatinine, p value = .004. There was no statistically significant correlation among all study participants between Serum Zonulin and TLC, HB, PLT and ALT, (p value were 0.736, 0.227, 070 and 0.619) respectively. There was highly statistically significant correlation between serum zonulin level and PLT in chronic patients, p value <0.001. Conclusion There was highly statistically significant difference between three study groups regarding PLT and Serum Zonulin. There was a statistically significant correlation among all study participants between Serum Zonulin and Creatinine. There was highly statistically significant correlation between serum zonulin level and PLT in chronic patients.

Spastin accumulation and motor neuron defects caused by a novel SPAST splice site mutation

BackgroundHereditary spastic paraplegia (HSP) is a rare genetically heterogeneous neurodegenerative disorder. The most common type of HSP is caused by pathogenic variants in the SPAST gene. Various hypotheses regarding the pathogenic mechanisms of HSP-SPAST have been proposed. However, a single hypothesis may not be sufficient to explain HSP-SPAST.ObjectiveTo determine the causative gene of autosomal dominant HSP-SPAST in a pure pedigree and to study its underlying pathogenic mechanism.MethodsA four-generation Chinese family was investigated. Genetic testing was performed for the causative gene, and a splice site variant was identified. In vivo and in vitro experiments were conducted separately. Western blotting and immunofluorescence were performed after transient transfection of cells with the wild-type (WT) or mutated plasmid. The developmental expression pattern of zebrafish spasts was assessed via whole-mount in situ hybridization. The designed guide RNA (gRNA) and an antisense oligo spast-MO were microinjected into Tg(hb9:GFP) zebrafish embryos, spinal cord motor neurons were observed, and a swimming behavioral analysis was conducted.ResultsA novel heterozygous intron variant, c.1004 + 5G > A, was identified in a pure HSP-SPAST pedigree and shown to cosegregate with the disease phenotypes. This intron splice site variant skipped exon 6, causing a frameshift mutation that resulted in a premature termination codon. In vitro, the truncated protein was evenly distributed throughout the cytoplasm, formed filamentous accumulations around the nucleus, and colocalized with microtubules. Truncated proteins diffusing in the cytoplasm appeared denser. No abnormal microtubule structures were observed, and the expression levels of α-tubulin remained unchanged. In vivo, zebrafish larvae with this mutation displayed axon pathfinding defects, impaired outgrowth, and axon loss. Furthermore, spast-MO larvae exhibited unusual behavioral preferences and increased acceleration.ConclusionThe adverse effects of premature stop codon mutations in SPAST result in insufficient levels of functional protein, and the potential toxicity arising from the intracellular accumulation of spastin serves as a contributing factor to HSP-SPAST.

A Novel Targeted Microtubules Transformable Nanopeptide System Yields Strong Anti-Prostate Cancer Effects by Suppressing Nuclear Translocation of Androgen Receptors.

The extended use of androgen deprivation therapy (ADT) may often lead to the progression from castration-sensitive prostate cancer (CSPC) to castration-resistant prostate cancer (CRPC) in prostate cancer. To address this, it is essential to inhibit the nuclear translocation of the androgen receptor (AR) as part of an effective disease-modifying strategy. Microtubules play a central role in facilitating AR nuclear translocation, highlighting their importance as a therapeutic target. In this regard, a designated as the targeted microtubules transformable nanopeptide system (MTN) is developed. This system is designed to disrupt microtubule structure and function through dual-targeting of prostate-specific membrane antigen (PSMA) and β-tubulin. Initially, MTN targets prostate cells via PSMA and then specifically binds to β-tubulin within microtubules, leading to the formation of nanofibers. These nanofibers subsequently induce the polymerization of microtubules, thereby disrupting AR transport. Notably, MTN exhibits efficient and prolonged suppression of prostate cancer across the spectrum from CSPC to CRPC, with a highly favorable safety profile in normal cells. These findings highlight the potential of MTN as a novel and promising approach for comprehensive prostate cancer therapy throughout its entire progression.

Structures of Native Doublet Microtubules from Trichomonas vaginalis Reveal Parasite-Specific Proteins as Potential Drug Targets.

Doublet microtubules (DMTs) are flagellar components required for the protist Trichomonas vaginalis (Tv) to swim through the human genitourinary tract to cause trichomoniasis, the most common non-viral sexually transmitted disease. Lack of DMT structures has prevented structure-guided drug design to manage Tv infection. Here, we determined the cryo-EM structure of native Tv-DMTs, identifying 29 unique proteins, including 18 microtubule inner proteins and 9 microtubule outer proteins. While the A-tubule is simplistic compared to DMTs of other organisms, the B-tubule features specialized, parasite-specific proteins, such as TvFAP40 and TvFAP35 that form filaments near the inner and outer junctions, respectively, to stabilize DMTs and enable Tv locomotion. Notably, a small molecule, assigned as IP6, is coordinated within a pocket of TvFAP40 and has characteristics of a drug molecule. This first atomic model of the Tv-DMT highlights the diversity of eukaryotic motility machinery and provides a structural framework to inform rational design of therapeutics.

Compression-dependent microtubule reinforcement enables cells to navigate confined environments.

Cells migrating through complex three-dimensional environments experience considerable physical challenges, including tensile stress and compression. To move, cells need to resist these forces while also squeezing the large nucleus through confined spaces. This requires highly coordinated cortical contractility. Microtubules can both resist compressive forces and sequester key actomyosin regulators to ensure appropriate activation of contractile forces. Yet, how these two roles are integrated to achieve nuclear transmigration in three dimensions is largely unknown. Here, we demonstrate that compression triggers reinforcement of a dedicated microtubule structure at the rear of the nucleus by the mechanoresponsive recruitment of cytoplasmic linker-associated proteins, which dynamically strengthens and repairs the lattice. These reinforced microtubules form the mechanostat: an adaptive feedback mechanism that allows the cell to both withstand compressive force and spatiotemporally organize contractility signalling pathways. The microtubule mechanostat facilitates nuclear positioning and coordinates force production to enable the cell to pass through constrictions. Disruption of the mechanostat imbalances cortical contractility, stalling migration and ultimately resulting in catastrophic cell rupture. Our findings reveal a role for microtubules as cellular sensors that detect and respond to compressive forces, enabling movement and ensuring survival in mechanically demanding environments.

Complex evolutionary patterns within the tubulin gene family of ciliates, unicellular eukaryotes with diverse microtubular structures

BackgroundTubulins are major components of the eukaryotic cytoskeletons that are crucial in many cellular processes. Ciliated protists comprise one of the oldest eukaryotic lineages possessing cilia over their cell surface and assembling many diverse microtubular structures. As such, ciliates are excellent model organisms to clarify the origin and evolution of tubulins in the early stages of eukaryote evolution. Nonetheless, the evolutionary history of the tubulin subfamilies within and among ciliate classes is unclear.ResultsWe analyzed the evolutionary pattern of ciliate tubulin gene family based on genomes/transcriptomes of 60 species covering 10 ciliate classes. Results showed: (1) Six tubulin subfamilies (α_Tub, β_Tub, γ_Tub, δ_Tub, ε_Tub, and ζ_Tub) originated from the last eukaryotic common ancestor (LECA) were observed within ciliates. Among them, α_Tub, β_Tub, and γ_Tub were present in all ciliate species, while δ_Tub, ε_Tub, and ζ_Tub might be independently lost in some species. (2) The evolutionary history of the tubulin subfamilies varied. Evolutionary history of ciliate γ_Tub, δ_Tub, ε_Tub, and ζ_Tub showed a certain degree of consistency with the phylogeny of species after the divergence of ciliate classes, while the evolutionary history of ciliate α_Tub and β_Tub varied among different classes. (3) Ciliate α- and β-tubulin isoforms could be classified into an “ancestral group” present in LECA and a “divergent group” containing only ciliate sequences. Alveolata-specific expansion events probably occurred within the “ancestral group” of α_Tub and β_Tub. The “divergent group” might be important for ciliate morphological differentiation and wide environmental adaptability. (4) Expansion events of the tubulin gene family appeared to be consistent with whole genome duplication (WGD) events in some degree. More Paramecium-specific tubulin expansions were detected than Tetrahymena-specific ones. Compared to other Paramecium species, the Paramecium aurelia complex underwent a more recent WGD which might have experienced more tubulin expansion events.ConclusionsEvolutionary history among different tubulin gene subfamilies seemed to vary within ciliated protists. And the complex evolutionary patterns of tubulins among different ciliate classes might drive functional diversification. Our investigation provided meaningful information for understanding the evolution of tubulin gene family in the early stages of eukaryote evolution.

Effects of Acute High-Altitude Exposure on Morphology and Function of Retinal Ganglion Cell in Mice.

High altitude retinopathy (HAR) is a retinal functional disorder caused by inadequate adaptation after exposure to high altitude. However, the cellular and molecular mechanisms underlying retinal dysfunction remain elusive. Retinal ganglion cell (RGC) injury is the most important pathological basis for most retinal and optic nerve diseases. Studies focusing on RGC injury after high-altitude exposure (HAE) are scanty. Therefore, the present study sought to explore both functional and morphological alterations of RGCs after HAE. A mouse model of acute hypobaric hypoxia was established by mimicking the conditions of a high altitude of 5000 m. After HAE for 2, 4, 6, 10, 24, and 72hours, the functional and morphological alterations of RGCs were assessed using retinal hematoxylin and eosin (H&E) sections, retinal whole mounts, transmission electron microscopy (TEM), and the photopic negative response (PhNR) of the electroretinogram. Compared with the control group, the thickness of the ganglion cell layer and retinal nerve fiber layer increased significantly, RGC loss remained significant, and the amplitudes of a-wave, b-wave, and PhNR were significantly reduced after HAE. In addition, RGCs and their axons exhibited an abnormal ultrastructure after HAE, including nuclear membrane abnormalities, uneven distribution of chromatin in the nucleus, decreased cytoplasmic electron density, widening and vacuolization of the gap between axons, loosening and disorder of myelin sheath structure, widening of the gap between myelin sheath and axon membrane, decreased axoplasmic density, unclear microtubule and nerve fiber structure, and abnormal mitochondrial structure (mostly swollen, with widened membrane gaps and reduced cristae and vacuolization). The study findings confirm that the morphology and function of RGCs are damaged after HAE. These findings lay the foundation for further study of the specific molecular mechanisms of HAR and promote the effective prevention.

Regulatable assembly of synthetic microtubule architectures using engineered microtubule-associated protein-IDR condensates

Microtubule filaments are assembled into higher-order structures using microtubule-associated proteins. However, synthetic MAPs that direct the formation of new structures are challenging to design, as nanoscale biochemical activities must be organized across micron length-scales. Here, we develop modular MAP-IDR condensates (synMAPs) that enable inducible assembly of higher-order microtubule structures for synthetic exploration invitro and in mammalian cells. synMAPs harness a small microtubule-binding domain from oligodendrocytes (TPPP) whose activity we show can be rewired by interaction with unrelated condensate-forming IDR sequences. This combination is sufficient to allow synMAPs to self-organize multivalent structures that bind and bridge microtubules into higher-order architectures. By regulating the connection between the microtubule-binding domain and condensate-forming components of a synMAP, the formation of these structures can be triggered by small molecules or cell-signaling inputs. We systematically test a panel of synMAP circuit designs to define how the assembly of these synthetic microtubule structures can be controlled at the nanoscale (via microtubule-binding affinity) and microscale (via condensate formation). synMAPs thus provide a modular starting point for the design of higher-order microtubule systems and an experimental testbed for exploring condensate-directed mechanisms of higher-order microtubule assembly from the bottom-up.

P-076 Role of N-DRC complex in male fertility

Abstract Study question There are many causes of human infertility, what’s the role of Nexin-dynein regulatory complex (N-DRC) in male fertility? Summary answer Many gene mutants in N-DRC components have been identified in infertile men, which affect the assembly of N-DRC complex and cause defects of sperm. What is known already human infertility affects 10–15% of couples, asthenozoospermia accounting for 18% of infertile men and is a common male infertility phenotype. Nexin-dynein regulatory complex (N-DRC) is a large protein complex regulating device in the flagellum and connects adjacent doublets of microtubules (DMTs). Some N-DRC components were reported to be associated with human disease, such as Primary ciliary dyskinesia (PCD). In recent years, more and more studies reported mutants in subunits of N-DRC are related to male infertility. For example, DRC1, DRC5 and DRC3 mutant were identified in patients displayed multiple morphological abnormalities of the flagella (MMAF) and obstructive azoospermia. Study design, size, duration We searched articles about N-DRC and male infertility in pumped and select research studies and the tidied up the gene mutants, gene knock out mouse modals and IVF outcomes of different N-DRC components in infertile men. Besides, we summarized the parameters of semen and ultrastructure of sperm flagella in these patients. Participants/materials, setting, methods We searched articles about N-DRC and male infertility in pumped and select research studies and the tidied up the gene mutants, gene knock out mouse modals and IVF outcomes of different N-DRC components in infertile men. Main results and the role of chance N-DRC is a highly conserved structural complex located in the axoneme of the flagellum and cilium, containing at least 11 different subunits (DRC1∼DRC11). Patients who were diagnosed as MMAF and male infertility carrying homozygous DRC1 mutants. TEM analyses of the patients’ sperm revealed the flagellar axoneme structure is disordered. A frameshift mutant of DRC5 was identified in an infertile male with asthenozoospermia, but the patient’s problem of infertility could be successfully solved by vitro Fertilization (IVF). A recent study identified two bi-allelic DRC3 frameshift variants in two unrelated patients, one patient displayed MMAF and the other present obstructive azoospermia. The sperm flagella function of DRC5 deletion mice was abrogated but flagellum ultrastructure remained normal and the mice behaved as asthenozoospermia and sterile. DRC3, DRC7 and DRC9 knockout mice exhibit MMAF and male infertility, and in DRC7 KO mice, the axoneme of sperm is disorganized and it was difficult to detect the ‘9 + 2’ microtubule structure. DRC1 mutant mice on the C57BL/6 background died before puberty, KO DRC6 in mice neither has no obvious effects on sperm motility function nor destroy the “9 + 2” arrangement of microtubules in cilia, and male mice was fertile. Limitations, reasons for caution More samples of MMAF and asthenozoospermia men to sequence N-DRC gene, and track their assisted reproductive outcomes. Wider implications of the findings The conclusions in our study uncover certain mechanism of N-DRC deficient in MMAF and asthenozoospermia phenotype, which could provide a new basis for diagnosis of male infertility. Trial registration number not applicable

Expanding the Structural Diversity of Tubulin-Targeting Agents: Development of Highly Potent Benzimidazoles for Treating Fungal Diseases.

Benzimidazoles, the representative pharmacophore of fungicides, have excellent antifungal potency, but their simple structure and single site of action have hindered their wider application in agriculture. In order to extend the structural diversity of tubulin-targeted benzimidazoles, novel benzimidazole derivatives were prepared by introducing the attractive pyrimidine pharmacophore. 2-((6-(4-(trifluoromethyl)phenoxy)pyrimidin-4-yl)thio)-1H-benzo[d]imidazole (A25) exhibited optimal antifungal activity against Sclerotinia sclerotiorum (S. s.), affording an excellent half-maximal effective concentration (EC50) of 0.158 μg/mL, which was higher than that of the reference agent carbendazim (EC50 = 0.594 μg/mL). Pot experiments revealed that compound A25 (200 μg/mL) had acceptable protective activity (84.7%) and curative activity (78.1%), which were comparable with that of carbendazim (protective activity: 90.8%; curative activity: 69.9%). Molecular docking displayed that multiple hydrogen bonds and π-π interactions could be formed between A25 and β-tubulin, resulting in a stronger bonding effect than carbendazim. Fluorescence imaging revealed that the structure of intracellular microtubules can be changed significantly after A25 treatment. Overall, these remarkable antifungal profiles of constructed novel benzimidazole derivatives could facilitate the application of novel microtubule-targeting agents.

P-154 Structural changes of the human sperm tail during the early post-fertilization

Abstract Study question What happens to the sperm tails that are no longer needed after the goal of fertilization has been achieved? Summary answer The sperm tail lost its fibrous sheath and changed to a coiled morphology with a whisker root-like structure at the tip during the early post-fertilization. What is known already The sperm tail has been thought to play only a role in allowing the head and neck to reach the oocyte. The flagellum is found in the mammalian sperm tail and consists of a middle, main, and terminal part. It also contains an axon composed of two central microtubules surrounded by nine peripheral microtubule doublets (9d + 2s microtubule pattern) surrounded by an accessory structure called the fibrous sheath (FS), but few studies have been conducted on the post-fertilization fate of the mammalian sperm tail. Study design, size, duration To investigate sperm tail structure from the pronuclear stage to the second cleavage, we used abnormally fertilized oocytes derived from either conventional in vitro fertilization (cIVF, n = 41) or intracytoplasmic sperm injection (ICSI, n = 11). In this study, abnormally fertilized oocytes were collected between March 2022 and November 2023 and subjected to immunofluorescence analysis and field emission scanning electron microscopy (FE-SEM) analysis. Participants/materials, setting, methods Under ethical approval, abnormally fertilized oocytes were donated by infertile couples undergoing cIVF or ICSI cycles at the Yamashita Shonan Yume Clinic in Japan. Informed consent was obtained from all participants. Immunofluorescence assay was performed to analyze sperm tail morphology using antibodies against H3K27me3, α-tubulin, and AKAP4. FE-SEM analysis was performed to detect the 9d + 2s structure characteristic of the sperm tail axon. Main results and the role of chance Tri-pronuclear zygotes derived from conventional in vitro fertilization (cIVF-derived 3PN) had two male pronuclei, whereas those derived from intracytoplasmic sperm injection (ICSI-derived 3PN) had one. Each zygote had the same number of coiled microtubule structures as the male pronucleus, and the 9d + 2s structure characteristic of the axon was identified, suggesting a sperm tail. No difference in sperm tail shape was observed between ICSI-derived 3PN and cIVF-derived 3PN. AKAP4, which is specifically localized in the FS associated with the principal piece of the flagellum, was observed in sperm tails attached to the zona pellucida but not in those in the oocyte, suggesting that the FS of the flagellum is lost before the pronuclear stage, leaving the microtubules of the axonemes exposed. The sperm tail was attached to one of the spindle poles during the first cleavage of abnormally fertilized oocytes, but no differences in the structure of the spindle pole with or without the sperm tail were observed. The tip of the sperm tail was branched like a “whisker root”, suggesting that the sperm tail was degraded from the end, but the degree of tip branching did not differ from the pronuclear stage to the second cleavage. Limitations, reasons for caution Normal fertilized oocytes were not used in this study because they are used for clinical treatment. The cell biological significance of sperm tail branching is currently unknown and requires further experimentation. Wider implications of the findings These findings shed light on the processes that occur during human fertilization, suggest new roles for the sperm tail, and may have implications for assisted fertilization approaches in the future. Trial registration number not applicable

Raman Spectroscopy Reveals Photobiomodulation-Induced α-Helix to β-Sheet Transition in Tubulins: Potential Implications for Alzheimer's and Other Neurodegenerative Diseases.

In small clinical studies, the application of transcranial photobiomodulation (PBM), which typically delivers low-intensity near-infrared (NIR) to treat the brain, has led to some remarkable results in the treatment of dementia and several neurodegenerative diseases. However, despite the extensive literature detailing the mechanisms of action underlying PBM outcomes, the specific mechanisms affecting neurodegenerative diseases are not entirely clear. While large clinical trials are warranted to validate these findings, evidence of the mechanisms can explain and thus provide credible support for PBM as a potential treatment for these diseases. Tubulin and its polymerized state of microtubules have been known to play important roles in the pathology of Alzheimer's and other neurodegenerative diseases. Thus, we investigated the effects of PBM on these cellular structures in the quest for insights into the underlying therapeutic mechanisms. In this study, we employed a Raman spectroscopic analysis of the amide I band of polymerized samples of tubulin exposed to pulsed low-intensity NIR radiation (810 nm, 10 Hz, 22.5 J/cm2 dose). Peaks in the Raman fingerprint region (300-1900 cm-1)-in particular, in the amide I band (1600-1700 cm-1)-were used to quantify the percentage of protein secondary structures. Under this band, hidden signals of C=O stretching, belonging to different structures, are superimposed, producing a complex signal as a result. An accurate decomposition of the amide I band is therefore required for the reliable analysis of the conformation of proteins, which we achieved through a straightforward method employing a Voigt profile. This approach was validated through secondary structure analyses of unexposed control samples, for which comparisons with other values available in the literature could be conducted. Subsequently, using this validated method, we present novel findings of statistically significant alterations in the secondary structures of polymerized NIR-exposed tubulin, characterized by a notable decrease in α-helix content and a concurrent increase in β-sheets compared to the control samples. This PBM-induced α-helix to β-sheet transition connects to reduced microtubule stability and the introduction of dynamism to allow for the remodeling and, consequently, refreshing of microtubule structures. This newly discovered mechanism could have implications for reducing the risks associated with brain aging, including neurodegenerative diseases like Alzheimer's disease, through the introduction of an intervention following this transition.

Study on the mesoscopic dynamic effects of tumor treating fields on cell tubulin

Tumor treatment fields (TTFields) can effectively inhibit the proliferation of tumor cells, but its mechanism remains exclusive. The destruction of cellular microtubule structure caused by TTFields through electric field force is considered to be the main reason for inhibiting tumor cell proliferation. However, the validity of this hypothesis still lacks exploration at the mesoscopic level. Therefore, in this study, we built force models for tubulins subjected to TTFields, based on the physical and electrical properties of tubulin molecules. We theoretically analyzed and simulated the dynamic effects of electric field force and torque on tubulin monomer polymerization, as well as the alignment and orientation of α/β tubulin heterodimer, respectively. Research results indicate that the interference of electric field force induced by TTFields on tubulin monomer is notably weaker than the inherent electrostatic binding force among tubulin monomers. Additionally, the electric field torque generated by the TTFileds on α/β tubulin dimers is also difficult to affect their random alignment. Therefore, at the mesoscale, our study affirms that TTFields are improbable to destabilize cellular microtubule structures via electric field dynamics effects. These results challenge the traditional view that TTFields destroy the microtubule structure of cells through TTFields electric field force, and proposes a new approach that should pay more attention to the "non-mechanical" effects of TTFields in the study of TTFields mechanism. This study can provide reliable theoretical basis and inspire new research directions for revealing the mesoscopic bioelectrical mechanism of TTFields.