Advanced glycation end-products (AGEs) form through non-enzymatic glycation of various proteins. Optic nerve degeneration is a frequent complication of diabetes, and retinal AGE accumulation is strongly linked to the development of diabetic retinopathy. Type 2 diabetes mellitus is a major risk factor for Alzheimer\'s disease (AD), with patients often exhibiting optic axon degeneration in the nerve fiber layer. Notably, a gap exists in our understanding of how AGEs contribute to neuronal degeneration in the optic nerve within the context of both diabetes and AD. Our previous work demonstrated that glyceraldehyde (GA)-derived toxic advanced glycation end-products (TAGE) disrupt neurite outgrowth through TAGE-β-tubulin aggregation and tau phosphorylation in neural cultures. In this study, we further illustrated GA-induced suppression of optic nerve axonal elongation via abnormal β-tubulin aggregation in mouse retinas. Elucidating this optic nerve degeneration mechanism holds promise for bridging the knowledge gap regarding vision loss associated with diabetes mellitus and AD.

Tubulin β-3 staining pattern and staining intensity of 5-hydroxymethyl cytosine (5-hmC) are potential diagnostic and prognostic markers in melanocytic lesions that need further evaluation. Melanocytic nevi and primary cutaneous melanomas were immunohistochemically stained for tubulin-β-3 and 5-hmC. Immunoreactivity and staining patterns were correlated with Breslow-thickness, clinical and pathological characteristics, and progression-free survival. Melanocytes showed positive tubulin β-3 staining. However, in most nevi, tubulin β-3 staining appeared as a gradient with intense cytoplasmic staining in cells of the superficial part of the lesion that faded to weak staining in the deep dermal part, while no gradient was found in deep penetrating nevi and melanomas. In 53 % of the melanomas, areas with loss of tubulin β-3 staining were found. 5-hmC staining intensity was significantly higher in melanocytic nevi compared to melanomas. Breslow thickness in combination with low 5-hmC score and loss of tubulin-β-3 staining was predictive for poor prognosis. As single markers, tubulin-β-3 and 5-hmC can be useful to distinguish between melanocytic nevi and melanoma, but staining variability limits the use of 5-hmC. In melanomas measuring >1.5 mm, combination of low 5-hmC score and loss of tubulin-β-3 staining may have prognostic value.

BACKGROUND: The giant roundworm Ascaris is an intestinal nematode, causing ascariasis by infecting humans and pigs worldwide. Recent estimates suggest that Ascaris infects over half a billion people, with chronic infections leading to reduced growth and cognitive ability. Ascariasis affects innumerable pigs worldwide and is known to reduce production yields via decreased growth and condemnation of livers. The predominant anthelminthic drugs used to treat ascariasis are the benzimidazoles. Benzimidazoles interact with β-tubulins and block their function, and several benzimidazole resistance-associated mutations have been described in the β-tubulins of ruminant nematodes. Recent research on ascarids has shown that these canonical benzimidazole resistance-associated mutations are likely not present in the β-tubulins of Ascaris, Ascaridia or Parascaris, even in phenotypically resistant populations.
METHODS: To further determine the putative absence of key β-tubulin polymorphisms, we screened two β-tubulin isotypes of Ascaris, highly expressed in adult worms. Using adult and egg samples of Ascaris obtained from pigs and humans worldwide, we performed deep amplicon sequencing to look for canonical resistance-associated mutations in Ascaris β-tubulins. Subsequently, we examined these data in closer detail to study the population dynamics of Ascaris and genetic diversity within the two isotypes and tested whether genotypes appeared to partition across human and pig hosts.
RESULTS: In the 187 isolates, 69 genotypes were found, made up of eight haplotypes of β-tubulin isotype A and 20 haplotypes of isotype B. Single nucleotide polymorphisms were seen at 14 and 37 positions for β-tubulin isotype A and isotype B, respectively. No evidence of any canonical benzimidazole resistance-associated mutations was found in either human- or pig-derived Ascaris isolates. There was, however, a difference in the genetic diversity of each isotype and distribution of β-tubulin genotypes between human- and pig-derived Ascaris. Statistical tests of population differentiation show significant differences (p < 0.001) between pig- and human-derived worms; however, more diversity was seen between worms from different populations than worms from different hosts.
CONCLUSIONS: Our work suggests an absence of canonical β-tubulin mutations within Ascaris, but alternative modes of anthelminthic resistance may emerge necessitating continued genetic scrutiny alongside monitoring of drug efficacy.

Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. Our previous work established that gonadotropin-releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope-derived LβT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry (MS) to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterized the temporal dynamics of this modification. Our results show that actin and tubulin are citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. The data show that 10 nM GnRHa induces the citrullination of β-actin, with elevated levels occurring at 10 min. The level of β-actin citrullination is reduced in the presence of the pan-PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa-induced actin reorganization in dispersed murine gonadotrope cells. GnRHa induces the citrullination of β-tubulin, with elevated levels occurring at 30 min, and this response is attenuated in the presence of PAD inhibition. To examine the functional consequence of β-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LβT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that the MT average lifetime increases following 30 min of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa-induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells.

Type 2 diabetes mellitus (T2DM) is a risk factor for Alzheimer\'s Disease (AD). However, the detailed mechanism underlying T2DM-related AD remains unknown. In DM, many types of advanced glycation end-products (AGEs) are formed and accumulated. In our previous study, we demonstrated that Glyceraldehyde (GA)-derived Toxic Advanced Glycation End-products (Toxic AGEs, TAGE) strongly showed cytotoxicity against neurons and induced similar alterations to those observed in AD. Further, GA induced dysfunctional neurite outgrowth via TAGE-β-- tubulin aggregation, which resulted in the TAGE-dependent abnormal aggregation of β-tubulin and tau phosphorylation. Herein, we provide a perspective on the possibility that T2DM increases the probability of AD onset and accelerates its progression.

Tetrahymenosis is caused by the ciliated protozoan Tetrahymena and is responsible for serious economic losses to the aquaculture industry worldwide. However, information regarding the molecular mechanism leading to tetrahymenosis is limited. In previous transcriptome sequencing work, it was found that one of the two β-tubulin genes in T. pyriformis was significantly expressed in infected fish, we speculated that β-tubulin is involved in T. pyriformis infecting fish. Herein, the potential biological function of the β-tubulin gene in Tetrahymena species when establishing infection in guppies was investigated by cloning the full-length cDNA of this T. pyriformis β-tubulin (BTU1) gene. The full-length cDNA of T. pyriformis BTU1 gene was 1873 bp, and the ORF occupied 1134 bp, whereas 5\' UTR 434 bp, and 3\' UTR 305 bp whose poly (A) tail contained 12 bases. The predicted protein encoded by T. pyriformis BTU1 gene had a calculated molecular weight of 42.26 kDa and pI of 4.48. Moreover, secondary structure analysis and tertiary structure prediction of BTU1 protein were also conducted. In addition, morphology, infraciliature, phylogeny, and histopathology of T. pyriformis isolated from guppies from a fish market in Harbin were also investigated. Furthermore, qRT-PCR analysis and experimental infection assays indicated that the expression of BTU1 gene resulted in efficient cell proliferation during infection. Collectively, our data revealed that BTU1 is a key gene involved in T. pyriformis infection in guppies, and the findings discussed herein provide valuable insights for future studies on tetrahymenosis.

The mechanisms of action of ligands competing for the Colchicine Binding Site (CBS) of the α,β-Tubulin are non-standard compared to the commonly witnessed ligand-induced inhibition of proteins. This is because their potencies are not solely judged by the binding affinity itself, but also by their capacity to bias the conformational states of the dimer. Regarding the latter requirement, it is observed that ligands competing for the same pocket that binds colchicine exhibit divergence in potential clinical outcomes. Molecular dynamics-based ∼5.2 µs sampling of α,β-Tubulin complexed with four different ligands has revealed that each ligand has its customized way of influencing the complex. Primarily, it is the proportion of twisting and/or bending characteristic of modes of the intrinsic dynamics which is revealed to be \'fundamental\' to tune this variation in the mechanism. The milder influence of \'bending\' makes a ligand (TUB092), better classifiable under the group of vascular disrupting agents (VDAs), which are phenotypically effective on cytoskeletons; whereas a stronger impact of \'bending\' makes the classical ligand Colchicine (COL) a better Anti-Mitotic agent (AMA). Two other ligands BAL27862 (2RR) and Nocodazole (NZO) fall in the intermediate zone as they fail to explicitly induce bending modes. Random Forest Classification method and K-means Clustering is applied to reveal the efficiency of Machine Learning methods in classifying the Tubulin conformations according to their ligand-specific perturbations and to highlight the significance of specific amino acid residues, mostly positioned in the α-β and β-β interfaces involved in the mechanism. These key residues responsible to yield discriminative actions of the ligands are likely to be highly useful in future endeavours to design more precise inhibitors.

The ivermectin is a potent nematocide and insecticide, which has low toxicity for humans and domestic animals, but due to low biotransformation, it can be dangerous for non-target organisms. The recent determination of ivermectin absorption and accumulation in tissues of higher plants and multiple shreds of evidence of its negative impact on plant physiology provide a basis for the search for ivermectin\'s molecular targets and mechanisms of action in plant cells. In this research, for the first time, the ivermectin effect on microtubules of Arabidopsis thaliana cells was studied. It was revealed that ivermectin (250 μg mL-1) disrupts the microtubule network, induces the loss of microtubule orientation, leads to microtubule curvature and shrinkage, and their longitudinal and cross-linked bundling in various cells of A. thaliana primary roots. Further, the previously proposed binding of ivermectin to the β1-tubulin taxane site was developed and confirmed using molecular dynamics simulations of ivermectin complexes with Haemonchus contortus and A. thaliana β1-tubulins. It was predicted that similar to other microtubule stabilizing agents ivermectin binding causes M-loop stabilization in both H. contortus and A. thaliana β-tubulin, which leads to the enhancement of lateral contacts between subunits of adjacent protofilaments preventing microtubule depolymerization.

Methyl benzimidazole carbamate (MBC) fungicides were once widely used for brown rot (Monilinia fructicola) control of peach (Prunus persica (L.) Batsch) in the southeastern US, but their use was substantially reduced and often eliminated due to widespread resistance. In this study, 233 M. fructicola isolates were collected from major peach production areas in Alabama, Georgia, and South Carolina, and sensitivity to thiophanate-methyl was examined. Isolates were also collected from one organic and two experimental peach orchards. A discriminatory dose of 1 μg/ml was used to distinguish sensitive (S) and moderately sensitive (S-LR) isolates from low resistant phenotypes, while 50 and 500 μg/ml thiophanate-methyl concentrations were used to determine high resistant (HR) phenotypes. Sequence analyses were performed to identify mutations in the β-tubulin target gene and detached fruit assays were performed to determine the efficacy of a commercial product against isolates representing each phenotype. Results indicated 55.7%, 63.5%, and 75.9% of isolates from Alabama, Georgia, and South Carolina, respectively, were S to thiophanate-methyl; 44.3%, 36.5%, and 21.4% were S-LR; no isolates were LR; and only 3 isolates (1.3%) from South Carolina were HR. No mutations in S or S-LR isolates were found, but HR isolates revealed the E198A mutation, an amino acid change of glutamic acid to alanine conferring high resistance. The high label rate of a commercial product containing thiophanate-methyl controlled brown rot caused by S and S-LR isolates in detached fruit studies but was ineffective against HR isolates. The combinations of thiophanate-methyl with azoxystrobin or isofetamid, when mixed together and applied in an experimental orchard 14 days preharvest, significantly reduced brown rot incidence on pre and postharvest commercially ripe fruit and efficacy was comparable to that of a grower standard fungicide. These results indicate that thiophanate-methyl may again be useful to peach growers in the southeastern US for brown rot and fungicide resistance management.

Aspergillus udagawae is a cryptic species of Aspergillus section Fumigati. Here, we report a case of canaliculitis with isolated A. udagawae. Fungal canaliculitis is a rare lacrimal disease, and its clinical features are poorly understood. The causative fungus was initially misclassified as Aspergillus thermomutatus by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) but was finally identified as A. udagawae by β-tubulin genetic analysis. The patient showed rapid improvement and did not experience relapse after drainage alone, without antifungal therapy. A. udagawae has low virulence, which may be related to the minimally invasive nature of the infection.