Invasive aspergillosis due to cryptic Aspergillus species: A prospective study from a single centre in India.

BACKGROUND: & objective: The existence of visually identical cryptic Aspergillus species that can be distinguished only by molecular techniques is becoming more widely acknowledged. For the majority of antifungal drugs, these are known to exhibit a greater minimal inhibitory concentration in vitro. For the purpose of receiving the proper care, it is crucial to identify these species at right time. Our aim in this work is to identify and describe the Aspergillus species that are cryptic from all of the clinical samples. METHODS: Routine samples from inpatients and outpatients received in department of Microbiology, All India Institute of Medical Sciences, New Delhi, showing growth of Aspergillus species were included in this study. Phenotypic and Matrix Assisted Laser Desorption Ionisation - Time of Flight identified isolates were analysed for cryptic species, by PCR and ITS/ß - tubulin sequencing. In accordance with CLSI recommendations, antifungal susceptibility testing was conducted using micro broth dilution. RESULTS: Of the 94 isolates, 54 A. fumigatus, 34 A. flavus, 3 A. nidulans, 2 A. terreus, and 1 A. niger were morphologically identified. MALDI-TOF misidentified 2 A. nidulans isolates and 1 A, stellatus isolate. The ß - tubulin sequence analysis revealed that 2 isolates (2.08 %) were cryptic, one was A. stellatus and another one was A. tubingensis.

The first molecular detection of benzimidazole resistance in Haemonchus contortus from sheep in Hejing and Minfeng counties of Southern Xinjiang.

To understand the benzimidazole (BZ) resistance of Haemonchus contortus in Southern Xinjiang, three single nucleotide polymorphisms (SNPs) designated as F167Y, E198A, and F200Y, in the isotype-1 ß-tubulin gene which are associated with BZ resistance, were investigated for H. contortus populations from sheep in Hejing and Minfeng counties of Southern Xinjiang. In brief, a total of 190 H. contortus adults were collected from 52 out of 70 slaughtered sheep in city abattoirs across two regions in Southern Xinjiang. The species identity of each adult worm was confirmed by PCR amplification of ITS-2 using H. contortus-specific primers targeting the ITS-2. The samples were then investigated for BZ-related SNPs at locus 167, 198, and 200, by PCR-sequencing of the isotype-1 ß-tubulin gene. The results showed that only E198A and F200Y mutations were detected in the investigated H. contortus populations. The E198A mutation (homozygous and heterozygote resistant: found in 40% and 30% of sequenced samples from Minfeng and Hejing counties, respectively) was predominant compared with the F200Y mutation (homozygous and heterozygote resistant: found in 14% and 13.3% of sequenced samples from Minfeng and Hejing counties, respectively). The results indicate a high prevalence of BZ resistance in H. contortus populations from certain areas of Southern Xinjiang. Our findings provide valuable information for the prevention and control of H. contortus in areas with similar conditions.

Toxic Advanced Glycation End-Products Inhibit Axonal Elongation Mediated by ß-Tubulin Aggregation in Mice Optic Nerves.

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-ß-tubulin aggregation and tau phosphorylation in neural cultures. In this study, we further illustrated GA-induced suppression of optic nerve axonal elongation via abnormal ß-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.

Surveillance of Ancylostoma caninum in naturally infected dogs in Quebec, Canada, and assessment of benzimidazole anthelmintics reveal a variable efficacy with the presence of a resistant isolate in imported dogs.

Ancylostoma caninum is a widely prevalent parasitic nematode in dogs across the world. There has been a notable increase in reports of anthelmintic resistance in A. caninum within the United States of America in recent years, which has led us to investigate the potential of this scenario in Canada. The study objectives were to assess the prevalence of A. caninum in two different groups, including a colony of rescued dogs in Canada and three imported Greyhound dogs from USA, and to evaluate the efficacy of two benzimidazole (BZ) anthelmintics against A. caninum, complemented with a molecular genetic analysis adapted to low prevalence. Fecal samples were collected at pre- and post-treatment with fenbendazole for the native shelters-origin group, and a combination of anthelmintic formulations, including the pro-BZ febantel for the USA-origin group. The coprology analyses found several genera of internal parasites. Canine ancylostomiasis was the most prevalent parasitosis with 30.77% in the native group and 100% in the USA group, but with overall low average of A. caninum eggs per gram. Through the fecal egg count reduction test (FECRT), applying a cut-off at 90% as baseline of egg reduction for successful efficacy, BZ showed variable efficacy. Furthermore, molecular analysis confirmed the presence of A. caninum in both groups of dogs and found differences in the genetics linked to BZ resistance on the A. caninum ß-tubulin isotype 1 gene. In the isolate from the native group, both codons 167 and 200 were homozygous without the presence of single nucleotide polymorphism (SNP). In contrast, the selected isolate from the USA group, showed a homozygous allele at position 200 and a heterozygous SNP at position 167. The latter was congruent with the low efficacy in FECRT and agrees with the recent findings of USA A. caninum isolate resistant phenotype to the BZ anthelmintics. The limitations of the study include an overall low eggs-per-gram in both canine groups, and the shortage of additional fecal samples from the USA group, restraining the molecular analysis only to one out of the three Greyhounds. This study provided some insights on the efficacy of BZs against A. caninum and revealed the presence of BZ resistant isolates in imported dogs in Quebec, Canada. All this information should be considered, for choosing the best strategy in the control of A. caninum using anthelmintic drugs.

New Insights on Tools for Detecting ß-Tubulin Polymorphisms in Trichuris trichiura Using rhAmpTM SNP Genotyping.

Soil-transmitted helminth (STH) infections, commonly treated with benzimidazoles, are linked to resistance through single nucleotide polymorphisms (SNPs) at position 167, 198, or 200 in the ß-tubulin isotype 1 gene. The aim of this study was to establish a novel genotyping assay characterized by its rapidity and specificity. This assay was designed to detect the presence of SNPs within the partial ß-tubulin gene of Trichuris trichiura. This was achieved through the biallelic discrimination at codons 167, 198, and 200 by employing the competitive binding of two allele-specific forward primers. The specificity and reliability of this assay were subsequently confirmed using Trichuris samples isolated from captive primates. Furthermore, a molecular study was conducted to substantiate the utility of the ß-tubulin gene as a molecular marker. The assays showed high sensitivity and specificity when applied to field samples. Nevertheless, none of the SNPs within the ß-tubulin gene were detected in any of the adult worms or eggs from the analyzed populations. All specimens consistently displayed an SS genotype. The examination of the ß-tubulin gene further validated the established close relationships between the T. trichiura clade and Trichuris suis clade. This reaffirms its utility as a marker for phylogenetic analysis.

Evaluation of tubulin ß-3 and 5 hydroxy-methyl cytosine as diagnostic and prognostic markers in malignant melanoma.

Tubulin ß-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-ß-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 ß-3 staining. However, in most nevi, tubulin ß-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 ß-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-ß-3 staining was predictive for poor prognosis. As single markers, tubulin-ß-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-ß-3 staining may have prognostic value.

Worldwide absence of canonical benzimidazole resistance-associated mutations within ß-tubulin genes from Ascaris.

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 ß-tubulins and block their function, and several benzimidazole resistance-associated mutations have been described in the ß-tubulins of ruminant nematodes. Recent research on ascarids has shown that these canonical benzimidazole resistance-associated mutations are likely not present in the ß-tubulins of Ascaris, Ascaridia or Parascaris, even in phenotypically resistant populations. METHODS: To further determine the putative absence of key ß-tubulin polymorphisms, we screened two ß-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 ß-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 ß-tubulin isotype A and 20 haplotypes of isotype B. Single nucleotide polymorphisms were seen at 14 and 37 positions for ß-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 ß-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 ß-tubulin mutations within Ascaris, but alternative modes of anthelminthic resistance may emerge necessitating continued genetic scrutiny alongside monitoring of drug efficacy.

Moderate Physical Activity Increases the Expression of ADNP in Rat Brain.

Activity-dependent neuroprotective protein (ADNP) is a neuroprotective protein essential for embryonic development, proper brain development, and neuronal plasticity. Its mutation causes the autism-like ADNP syndrome (also called the Helsmoortel-Van der Aa syndrome), characterized by neural developmental disorders and motor dysfunctions. Similar to the ADNP syndrome, the ADNP haploinsufficient mouse shows low synapse density, leading to motor and cognitive ability delays. Moderate physical activity (PA) has several neuroprotective and cognitive benefits, promoting neuronal survival, differentiation, neurogenesis, and plasticity. Until now, no study has investigated the effect of moderate exercise on ADNP expression and distribution in the rat brain. The aim of the current investigation was to study the effects of moderate exercise on the ADNP expression and neuronal activation measured by the microtubule protein ß-Tubulin III. In pursuit of this objective, twenty-four rats were selected and evenly distributed into two categories: sedentary control rats and rats exposed to moderate physical activity on a treadmill over a span of 12 weeks. Our results showed that moderate PA increases the expression of ADNP and ß-Tubulin III in the dentate gyrus (DG) hippocampal region and cerebellum. Moreover, we found a co-localization of ADNP and ß-Tubulin III in both DG and cerebellum, suggesting a direct association of ADNP with adult neuronal activation induced by moderate PA.

GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells.

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 LßT2 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 ß-actin, with elevated levels occurring at 10 min. The level of ß-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 ß-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 ß-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 LßT2 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.

The prognostic and predictive role of class III ß-Tubulin and hENT1 expression in patients with advanced pancreatic ductal adenocarcinoma.

BACKGROUND: FOLFIRINOX and gemcitabine-nabpaclitaxel (GnP) are standard first-line treatment regimens for advanced pancreatic ductal adenocarcinoma (PDAC). However, currently, there is a lack of predictive biomarkers to aid in the treatment selection. We aimed to explore the prognostic and predictive value of class III ß-Tubulin (TUBB3) and human equilibrative nucleoside transporter 1 (hENT1) expression, which have previously been shown to be associated with taxane and gemcitabine resistance in advanced PDAC. METHODS: We conducted a retrospective analysis of 106 patients with advanced PDAC treated with GnP and/or FOLFIRINOX at our institution. TUBB3 and hENT1 immunohistochemical staining was performed on tumor specimens and subsequently evaluated based on the intensity and percentage of expression. RESULTS: In patients who received the GnP regimen, a high combined score (TUBB3low/hENT1high) was associated with a higher DCR and longer PFS compared to those with intermediate (TUBB3high/hENT1high or TUBB3low/hENT1low) and low score (TUBB3high/hENT1low). In the multivariate analysis, a high combined score was an independent predictor of higher DCR (OR:11.96; 95 % CI:2.61-54.82; p = 0.001) and longer PFS (HR:0.33; 95%CI:0.18-0.60; p < 0.001). However, there was no difference in response rates or PFS based on TUBB3 and hENT1 expression among patients receiving the FOLFIRINOX regimen. CONCLUSION: Our findings indicate that tumor TUBB3 and hENT1 expression may predict the efficacy of the GnP regimen, and low TUBB3 and high hENT1 expression (TUBB3low/hENT1high) are associated with a higher DCR and longer PFS in patients treated with GnP. Evaluating TUBB3 and hENT1 jointly can identify the patients most (as well as least) likely to benefit from GnP chemotherapy.

Identification of a Tetrahymena species infecting guppies, pathology, and expression of beta-tubulin during infection.

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 ß-tubulin genes in T. pyriformis was significantly expressed in infected fish, we speculated that ß-tubulin is involved in T. pyriformis infecting fish. Herein, the potential biological function of the ß-tubulin gene in Tetrahymena species when establishing infection in guppies was investigated by cloning the full-length cDNA of this T. pyriformis ß-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.

Functional Roles of Two ß-Tubulin Isotypes in Regulation of Sensitivity of Colletotrichum fructicola to Carbendazim.

Colletotrichum fructicola is the major pathogen of anthracnose in tea-oil trees in China. Control of anthracnose in tea-oil trees mainly depends on the application of chemical fungicides such as carbendazim. However, the current sensitivity of C. fructicola isolates in tea-oil trees to carbendazim has not been reported. Here, we tested the sensitivity of 121 C. fructicola isolates collected from Guangdong, Guangxi, Guizhou, Hainan, Hunan, Jiangsu, and Jiangxi provinces in China to carbendazim. One hundred and ten isolates were sensitive to carbendazim, and 11 isolates were highly resistant to carbendazim. The growth rates, morphology, and pathogenicity of three resistant isolates were identical to those of three sensitive isolates, which indicates that these resistant isolates could form a resistant population under carbendazim application. These results suggest that carbendazim should not be the sole fungicide in control of anthracnose in tea-oil trees; other fungicides with different mechanisms of action or mixtures of fungicides could be considered. In addition, bioinformatics analysis identified two ß-tubulin isotypes in C. fructicola: Cfß1tub and Cfß2tub. E198A mutation was discovered in the Cfß2tub of three carbendazim-resistant isolates. We also investigated the functional roles of two ß-tubulin isotypes. CfΔß1tub exhibited slightly increased sensitivity to carbendazim and normal phenotypes. Surprisingly, CfΔß2tub was highly resistant to carbendazim and showed a seriously decreased growth rate, conidial production, pathogenicity, and abnormal hyphae morphology. Promoter replacement mutant CfΔß2-2×ß1 showed partly restored phenotypes, but it was still highly resistant to carbendazim, which suggests that Cfß1tub and Cfß2tub are functionally interchangeable to a certain degree.

Molecular Dynamics and Machine Learning reveal distinguishing mechanisms of Competitive Ligands to perturb α,ß-Tubulin.

The mechanisms of action of ligands competing for the Colchicine Binding Site (CBS) of the α,ß-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,ß-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-ß and ß-ß 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.

Ivermectin affects Arabidopsis thaliana microtubules through predicted binding site of ß-tubulin.

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 ß1-tubulin taxane site was developed and confirmed using molecular dynamics simulations of ivermectin complexes with Haemonchus contortus and A. thaliana ß1-tubulins. It was predicted that similar to other microtubule stabilizing agents ivermectin binding causes M-loop stabilization in both H. contortus and A. thaliana ß-tubulin, which leads to the enhancement of lateral contacts between subunits of adjacent protofilaments preventing microtubule depolymerization.

Low frequency of resistance to thiophanate-methyl in Monilinia fructicola populations from southeastern United States peach orchards.

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 ß-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.

Novel 8-Methoxycoumarin-3-Carboxamides with potent anticancer activity against liver cancer via targeting caspase-3/7 and ß-tubulin polymerization.

In the present study, we explored the potential of coumarin-based compounds, known for their potent anticancer properties, by designing and synthesizing a novel category of 8-methoxycoumarin-3-carboxamides. Our aim was to investigate their antiproliferative activity against liver cancer cells. Toward this, we developed a versatile synthetic approach to produce a series of 8-methoxycoumarin-3-carboxamide analogues with meticulous structural features. Assessment of their antiproliferative activity demonstrated their significant inhibitory effects on the growth of HepG2 cells, a widely studied liver cancer cell line. Among screened compounds, compound 5 exhibited the most potent antiproliferative activity among the screened compounds (IC50 = 0.9 µM), outperforming the anticancer drug staurosporine (IC50 = 8.4 µM), while showing minimal impact on normal cells. The flow cytometric analysis revealed that compound 5 induces cell cycle arrest during the G1/S phase and triggers apoptosis in HepG2 cells by increasing the percentage of cells arrested in the G2/M and pre-G1 phases. Annexin V-FITC/PI screening further supported the induction of apoptosis without significant necrosis. Further, compound 5 exhibited the ability to activate caspase3/7 protein and substantially inhibited ß-tubulin polymerization activity in HepG2 cells. Finally, molecular modelling analysis further affirmed the high binding affinity of compound 5 toward the active cavity of ß-tubulin protein, suggesting its mechanistic involvement. Collectively, our findings highlight the therapeutic potential of the presented class of coumarin analogues, especially compound 5, as promising candidates for the development of effective anti-hepatocellular carcinoma agents.

Isolation of Aspergillus udagawae in Canaliculitis: A Case Report.

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 ß-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.

Cystathionine ß-synthase affects organization of cytoskeleton and modulates carcinogenesis in colorectal carcinoma cells.

Background: Cystathionine ß-synthase (CBS), one of three enzymes that endogenously produce hydrogen sulfide, is extensively studied for its relevance in the cells of various tumors. In our previous work, we observed that the immunofluorescence pattern of CBS is very similar to that of tubulin and actin. Therefore, we focused on the potential interaction of CBS with cytoskeletal proteins ß-actin and ß-tubulin and the functional relevance of the potential interaction of these proteins in colorectal carcinoma cell lines. Methods: To study the potential interaction of CBS with cytoskeletal proteins and its functional consequences, a CBS-knockout DLD1 (DLDx) cell line was established by using the CRISPR/Cas9 gene editing method. The interaction of the selected cytoskeletal protein with CBS was studied by immunoprecipitation, Western blot analysis, immunofluorescence, and proximity ligation assay. The functional consequences were studied by proliferation and migration assays and by generation of xenografts in SCID/bg mice. Results: We have found that CBS, an enzyme that endogenously produces H2S, binds to cytoskeletal ß-tubulin and, to a lesser extent, also to ß-actin in colorectal carcinoma-derived cells. When CBS was knocked out by the CRISPR/Cas9 technique (DLDx), we observed a de-arranged cytoskeleton compared to the unmodified DLD1 cell line. Treatment of these cells with a slow sulfide donor GYY4137 resulted in normal organization of the cytoskeleton, thus pointing to the role of CBS in microtubule dynamics. To evaluate the physiological importance of this observation, both DLD1 and DLDx cells were injected into SCID/bg mice, and the size and mass of the developed xenografts were evaluated. Significantly larger tumors developed from DLDx compared to the DLD1 cells, which correlated with the increased proliferation of these cells. Conclusions: Taken together, in colorectal cancer DLD1 cells, CBS binds to the cytoskeleton, modulates microtubule dynamics, and thus affects the proliferation and migration in the colorectal carcinoma stable cell line.

Protein Modelling and Molecular Docking Analysis of Fasciola hepatica ß-Tubulin's Interaction Sites, with Triclabendazole, Triclabendazole Sulphoxide and Triclabendazole Sulphone.

PURPOSE: Fasciola hepatica is a globally distributed trematode that causes significant economic losses. Triclabendazole is the primary pharmacological treatment for this parasite. However, the increasing resistance to triclabendazole limits its efficacy. Previous pharmacodynamics studies suggested that triclabendazole acts by interacting mainly with the ß monomer of tubulin. METHODS: We used a high-quality method to model the six isotypes of F. hepatica ß-tubulin in the absence of three-dimensional structures. Molecular dockings were conducted to evaluate the destabilization regions in the molecule against the ligands triclabendazole, triclabendazole sulphoxide and triclabendazole sulphone. RESULTS: The nucleotide binding site demonstrates higher affinity than the binding sites of colchicine, albendazole, the T7 loop and pßVII (p < 0.05). We suggest that the binding of the ligands to the polymerization site of ß-tubulin can lead a microtubule disruption. Furthermore, we found that triclabendazole sulphone exhibited significantly higher binding affinity than other ligands (p < 0.05) across all isotypes of ß-tubulin. CONCLUSIONS: Our investigation has yielded new insight on the mechanism of action of triclabendazole and its sulphometabolites on F. hepatica ß-tubulin through computational tools. These findings have significant implications for ongoing scientific research ongoing towards the discovery of novel therapeutics to treat F. hepatica infections.

Purification and evaluation of 2, 4-di-tert butylphenol (DTBP) as a biocontrol agent against phyto-pathogenic fungi.

A fungal strain, Marasmiellus sp (PUK64), isolated from the mangrove forests in Muthupet, Tamil Nadu, East coast of India, along with others were screened for the search of potent bioactive compounds. A phenolic compound, 2,4-di-tert-butylphenol (DTBP), was isolated from the most promising strain PUK64 and its chemical structure was ascertained. DTBP demonstrated remarkable antifungal activity against the phytopathogenic fungi Aspergillus oryzae, Curvularia lunata and Fusarium verticillioides. In an in-vitro experimental setup, DTBP suppressed the growth of all three fungi, among which F. verticillioides was found to be highly susceptible. This effect relates with the inhibition of spore germination and hyphal growth that we observed. DTBP showed high affinity with the F. verticillioides's ß-tubulin protein (determined by ligand-protein docking) as compared to the standard fungicide carbendazim (CBZ). Molecular docking and simulation studies of DTBP with target ß-tubulin further confirmed the potential of ß-tubulin binding in F. verticillioides. To our knowledge, this is the first report on DTBP-mediated biocontrol of phytopathogenic fungi, produced by Marasmiellus sp. PUK64 that can be potent inhibitor of ß-tubulin protein of F. verticillioides.