Tubulin Research Articles

Gene expression profiling of the masticatory muscle tendons and Achilles tendons under tensile strain in the Japanese macaque Macaca fuscata.

Both Achilles and masticatory muscle tendons are large load-bearing structures, and excessive mechanical loading leads to hypertrophic changes in these tendons. In the maxillofacial region, hyperplasia of the masticatory muscle tendons and aponeurosis affect muscle extensibility resulting in limited mouth opening. Although gene expression profiles of Achilles and patellar tendons under mechanical strain are well investigated in rodents, the gene expression profile of the masticatory muscle tendons remains unexplored. Herein, we examined the gene expression pattern of masticatory muscle tendons and compared it with that of Achilles tendons under tensile strain conditions in the Japanese macaque Macaca fuscata. Primary tenocytes isolated from the masticatory muscle tendons (temporal tendon and masseter aponeurosis) and Achilles tendons were mechanically loaded using the tensile force and gene expression was analyzed using the next-generation sequencing. In tendons exposed to tensile strain, we identified 1076 differentially expressed genes with a false discovery rate (FDR) < 10-10. To identify genes that are differentially expressed in temporal tendon and masseter aponeurosis, an FDR of < 10-10 was used, whereas the FDR for Achilles tendons was set at > 0.05. Results showed that 147 genes are differentially expressed between temporal tendons and masseter aponeurosis, out of which, 125 human orthologs were identified using the Ensemble database. Eight of these orthologs were related to tendons and among them the expression of the glycoprotein nmb and sphingosine kinase 1 was increased in temporal tendons and masseter aponeurosis following exposure to tensile strain. Moreover, the expression of tubulin beta 3 class III, which promotes cell cycle progression, and septin 9, which promotes cytoskeletal rearrangements, were decreased in stretched Achilles tendon cells and their expression was increased in stretched masseter aponeurosis and temporal tendon cells. In conclusion, cyclic strain differentially affects gene expression in Achilles tendons and tendons of the masticatory muscles.

Phaeotubakialithocarpicola gen. et sp. nov. (Tubakiaceae, Diaporthales) from leaf spots in China.

Tubakiaceae represents a distinct lineage of Diaporthales, including its type genus Tubakia and nine additional known genera. Tubakiaceous species are commonly known as endophytes in leaves and twigs of many tree species, but can also be plant pathogens causing conspicuous leaf symptoms. In the present study, isolates were obtained from diseased leaves of Lithocarpusglaber collected in Guangdong Province, China. The identification was conducted based on morphology and phylogeny of combined loci of 28S nrRNA gene (LSU), internal transcribed spacer regions and intervening 5.8S nrRNA gene (ITS) of the nrDNA operon, translation elongation factor 1-alpha (tef1) and beta tubulin (tub2). As a result, a distinct clade in Tubakiaceae was revealed named Phaeotubakialithocarpicola gen. et sp. nov., which was distinguished from the other tubakiaceous taxa by its dark brown conidiogenous cells and conidia.

Identification and Interaction Analysis of Molecular Markers in Pancreatic Ductal Adenocarcinoma by Bioinformatics and Next-Generation Sequencing Data Analysis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common cancers worldwide. Intense efforts have been made to elucidate the molecular pathogenesis, but the molecular mechanisms of PDAC are still not well understood. The purpose of this study is to further explore the molecular mechanism of PDAC through integrated bioinformatics analysis. To identify the candidate genes in the carcinogenesis and progression of PDAC, next-generation sequencing (NGS) data set GSE133684 was downloaded from Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified, and Gene Ontology (GO) and pathway enrichment analyses were performed. The protein-protein interaction network (PPI) was constructed and the module analysis was performed using Integrated Interactions Database (IID) interactome database and Cytoscape. Subsequently, miRNA-DEG regulatory network and TF-DEG regulatory network were constructed using miRNet database, NetworkAnalyst database, and Cytoscape software. The expression levels of hub genes were validated based on Kaplan-Meier analysis, expression analysis, stage analysis, mutation analysis, protein expression analysis, immune infiltration analysis, and receiver operating characteristic (ROC) curve analysis. A total of 463 DEGs were identified, consisting of 232 upregulated genes and 233 downregulated genes. The enriched GO terms and pathways of the DEGs include vesicle organization, secretory vesicle, protein dimerization activity, lymphocyte activation, cell surface, transferase activity, transferring phosphorus-containing groups, hemostasis, and adaptive immune system. Four hub genes (namely, cathepsin B [CCNB1], four-and-a-half LIM domains 2 (FHL2), major histocompatibility complex, class II, DP alpha 1 (HLA-DPA1) and tubulin beta 1 class VI (TUBB1)) were obtained via taking interaction of different analysis results. On the whole, the findings of this investigation enhance our understanding of the potential molecular mechanisms of PDAC and provide potential targets for further investigation.

Mahabalella pernambucana, a new hyphomycete from Brazil with notes on its phylogenetic positioning

A new species of Mahabalella B. Sutton & S. D. Patil was isolated from decaying leaves of an unidentified plant, during a survey in the Charles Darwin Ecological Refuge in Igarassu, Pernambuco, Brazil, in June 2018. The new species is morphologically distinguishable from the other Mahabalella species by the size of their conidia, conidiogenous cells and setae. In this study, the morphological descriptions, illustrations and cultural characteristics for Mahabalella pernambucana are provided, as well as a key to species of this genus. Total DNA from the isolate was extracted, and four genes were partially sequenced (nLSU, ITS cluster, TEF1 alpha, and beta tubulin). Based on the nLSU analysis, the phylogenetic position of the new species was determined within the Sordariomycetes, Ascomycota, as a sister genus to Neotracylla, in the Tracyllaceae family.

Selection of Stable Reference Genes for Quantitative Real-Time PCR on Herbaceous Peony (Paeonia lactiflora Pall.) in Response to Drought Stress

Herbaceous peony (Paeonia lactiflora Pall.), as a high-end cut flower in the international market, has high ornamental and medicinal values. But in Northern China, drought is a major environmental factor influencing the growth and development of P. lactiflora. Quantitative real-time polymerase chain reaction (qRT-PCR) can evaluate gene expression levels under different stress conditions, and stable internal reference is the key for qRT-PCR. At present, there is no systematic screening of internal reference for correcting gene expressions of P. lactiflora in response to drought stress. In this study, 10 candidate genes [ubiquitin (UBQ2), UBQ1, elongation factor 1-α (EF-1α), Histidine (His), eukaryotic initiation factor (eIF), tubulin (TUB), actin (ACT), UBQ3, ACT2, RNA polymerase II (RNA Pol II)] were chosen, and 4 analysis methods were used to compare the stabilities for these 10 genes coping with drought stress. Due to the difference of operation methods, the results of different analysis were distinct, and the final comprehensive analysis indicated that EF-1α was a relatively stable internal reference gene for P. lactiflora under drought stress. Also, UBQ1 and UBQ2 were the best reference gene combination according to GeNorm analysis. This study will lay a foundation for screening the key genes of P. lactiflora in response to drought stress.

Effects of dedifferentiated fat cells on neurogenic differentiation and cell proliferation in neuroblastoma cells.

Mesenchymal stem cells (MSCs) can induce differentiation of neuroblastoma (NB) cells. Properties of dedifferentiated fat cells (DFATs) are similar to those of MSCs. Here, we investigated whether DFATs can induce NB cell differentiation and suppress cell proliferation. DFATs were obtained from mature adipocytes isolated from adipose tissue from a ceiling culture. NB cells were cultured in a medium with or without DFATs and, subsequently, cultured in a DFAT-conditioned medium (CM) with or without phosphatidylinositol 3-kinase (PI3K) inhibitor. The neurite lengths were measured, and mRNA expression levels of the neurofilament (NF) and tubulin beta III (TUBβ3) were assessed using quantitative real-time RT-PCR. Cell viability was assessed using the WST-1 assay. NB cells cultured with DFATs caused elongation of the neurites and upregulated the expression of NF and Tubβ3. NB cells cultured in DFAT-CM demonstrated increased cell viability. NB cells cultured with DFAT-CM and PI3K inhibitors suppressed cell viability. NB cells cultured with DFAT-CM and PI3K inhibitor demonstrated increased neurite length and expression, and upregulation of Tubβ3. The combined use of DFAT-CM and PI3K inhibitors suppresses the proliferation of NB cells and induces their differentiation. Thus, DFAT may offer new insights into therapeutic approaches in neuroblastoma.

Selection and Validation of Suitable Reference Genes for Gene Expression Studies in Callerya speciosa (Champ. ex Benth.) Schot under Different Experimental Conditions

The accuracy of reverse transcription quantitative real-time PCR (RT-qPCR) strongly depends on the stability of reference gene. It is essential to select a suitable reference gene in order to obtain reliable RT-qPCR results when gene expression profiles were evaluated. Callerya speciosa, a traditional Chinese medicine, has a long cultivation history in south China. However, suitable reference genes in C. speciosa have not yet been investigated for accurate gene expression quantification under different experimental conditions. In this study, eight candidate reference genes, including GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 60S (60S ribosomal protein L34), ACTIN (actin), TUA2 (tubulin alpha chain), TUB1 (tubulin beta-1 chain), TIF5 (eukaryotic translation initiation factor 5A), UBQ (polyubiquitin), and EF2 (elongation factor 2), were selected from the transcriptome databases, and their expression stability under six different experimental conditions (developmental stages, tissues, MeJA treatment, GA3 treatment, CPPU treatment, and PP333 treatment) was evaluated using ΔCT, geNorm, NormFinder, BestKeeper, and RefFinder programs. The results showed that GAPDH was the optimal reference gene for all different experimental conditions, whereas ACTIN showed the best stability under most of the hormone treatments. TUA2 and EF2 were the two least suitable ones as reference genes in C. speciosa. The expression pattern of CsMYB36, a gene associated with the regulation of isoflavonoid biosynthesis in C. speciosa, verified the accuracy of our experimental results. These results provided a theoretical basis for subsequent research on the regulation of functional gene expression in C. speciosa and other Callerya species in the future.

Phenotype-based screening rediscovered benzopyran-embedded microtubule inhibitors as anti-neuroinflammatory agents by modulating the tubulin–p65 interaction

Neuroinflammation is one of the critical processes implicated in central nervous system (CNS) diseases. Therefore, alleviating neuroinflammation has been highlighted as a therapeutic strategy for treating CNS disorders. However, the complexity of neuroinflammatory processes and poor drug transport to the brain are considerable hurdles to the efficient control of neuroinflammation using small-molecule therapeutics. Thus, there is a significant demand for new chemical entities (NCEs) targeting neuroinflammation. Herein, we rediscovered benzopyran-embedded tubulin inhibitor 1 as an anti-neuroinflammatory agent via phenotype-based screening. A competitive photoaffinity labeling study revealed that compound 1 binds to tubulin at the colchicine-binding site. Structure–activity relationship analysis of 1’s analogs identified SB26019 as a lead compound with enhanced anti-neuroinflammatory efficacy. Mechanistic studies revealed that upregulation of the tubulin monomer was critical for the anti-neuroinflammatory activity of SB26019. We serendipitously found that the tubulin monomer recruits p65, inhibiting its translocation from the cytosol to the nucleus and blocking NF-κB-mediated inflammatory pathways. Further in vivo validation using a neuroinflammation mouse model demonstrated that SB26019 suppressed microglial activation by downregulating lba-1 and proinflammatory cytokines. Intraperitoneal administration of SB26019 showed its therapeutic potential as an NCE for successful anti-neuroinflammatory regulation. Along with the recent growing demands on tubulin modulators for treating various inflammatory diseases, our results suggest that colchicine-binding site-specific modulation of tubulins can be a potential strategy for preventing neuroinflammation and treating CNS diseases.

Discovery of novel whitefly vector proteins that interact with a virus capsid component mediating virion retention and transmission

Specificity and efficiency of plant virus transmission depend largely on protein-protein interactions of vectors and viruses. Cucurbit chlorotic yellows virus (CCYV), transmitted specifically by tobacco whitefly, Bemisia tabaci, in a semi-persistent manner, has caused serious damage on cucurbit and vegetable crops around the world. However, the molecular mechanism of interaction during CCYV retention and transmission are still lacking. CCYV was proven to bind particularly to the whitefly foregut, and here, we confirmed that the minor coat protein (CPm) of CCYV is participated in the interaction with the vector. In order to identify proteins of B. tabaci that interact directly with CPm of CCYV, the immunoprecipitation (IP) assay and DUALmembrane cDNA library screening technology were applied. The cytochrome c oxidase subunit 5A (COX), tubulin beta chain (TUB) and keratin, type I cytoskeletal 9-like (KRT) of B. tabaci shown strong interactions with CPm and are closely associated with the retention within the vector and transmission of CCYV. These findings on whitefly protein-CCYV CPm interactions are crucial for a much better understanding the mechanism of semi-persistent plant virus transmission by insect vectors, as well as for implement new strategies for effective management of plant viruses and their vector insects.

Decreased CSTB, RAGE, and Axl Receptor Are Associated with Zika Infection in the Human Placenta.

Zika virus (ZIKV) compromises placental integrity, infecting the fetus. However, the mechanisms associated with ZIKV penetration into the placenta leading to fetal infection are unknown. Cystatin B (CSTB), the receptor for advanced glycation end products (RAGE), and tyrosine-protein kinase receptor UFO (AXL) have been implicated in ZIKV infection and inflammation. This work investigates CSTB, RAGE, and AXL receptor expression and activation pathways in ZIKV-infected placental tissues at term. The hypothesis is that there is overexpression of CSTB and increased inflammation affecting RAGE and AXL receptor expression in ZIKV-infected placentas. Pathological analyses of 22 placentas were performed to determine changes caused by ZIKV infection. Quantitative proteomics, immunofluorescence, and western blot were performed to analyze proteins and pathways affected by ZIKV infection in frozen placentas. The pathological analysis confirmed decreased size of capillaries, hyperplasia of Hofbauer cells, disruption in the trophoblast layer, cell agglutination, and ZIKV localization to the trophoblast layer. In addition, there was a significant decrease in CSTB, RAGE, and AXL expression and upregulation of caspase 1, tubulin beta, and heat shock protein 27. Modulation of these proteins and activation of inflammasome and pyroptosis pathways suggest targets for modulation of ZIKV infection in the placenta.

Identification of TUBB8 Variants in 5 Primary Infertile Women with Multiple Phenotypes in Oocytes and Early Embryos

Tubulin beta 8 class VIII (TUBB8) is a β-tubulin isotype that is specifically expressed in human oocytes and early embryos. It has been identified as a disease-causing gene in primary female infertility by affecting oocyte maturation arrest. This study investigated the genetic cause of female infertility in five patients from four families. Five women with primary infertility were recruited. Medical-exome sequencing and Sanger sequencing were performed on the patients, and their family members to identify candidate genes that explained infertility. Additionally, the morphology of oocytes and zygotes from the patients and controls were assessed. We observed recurrent oocytes MI arrest, oocytes abnormal fertilization, uncleaved embryos, and embryo transfer failure in the patients. Heterozygous missense variants in TUBB8, c.538G > A (p.V180M), c.527C > G (p.S176W), c.124C > G (p.L42V), and c.628A > C (p.I210L), were verified in four unrelated families. This study expanded the mutational spectrum of TUBB8 by identifying three novel heterozygous missense variants. Screening for TUBB8 mutation demonstrated the diagnostic utility of female infertility.

Taxol and β-tubulins from endophytic fungi isolated from the Himalayan Yew, Taxus wallichiana Zucc.

Paclitaxel, better known as the anticancer drug Taxol®, has been isolated from several plant species and has been shown to be produced by fungi, actinomycetes, and even bacteria isolated from marine macroalgae. Given its cytostatic effect, studies conducted in the 1990's showed that paclitaxel was toxic to many pathogenic fungi and oomycetes. Further studies led to the idea that the differences in paclitaxel sensitivity exhibited by different fungi were due to differences in the β-tubulin protein sequence. With the recent isolation of endophytic fungi from the leaves and bark of the Himalayan Yew, Taxus wallichiana Zucc., and the availability of genomes from paclitaxel-producing fungi, we decided to further explore the idea that endophytic fungi isolated from Yews should be well-adapted to their environment by encoding β-tubulin proteins that are insensitive to paclitaxel. Our results found evidence of episodic positive/diversifying selection at 10 sites (default p-value threshold of 0.1) in the β-tubulin sequences, corresponding to codon positions 33, 55, 172, 218, 279, 335, 359, 362, 379, and 406. Four of these positions (i.e., 172, 279, 359, and 362) have been implicated in the binding of paclitaxel by β-tubulin or formed part of the binding pocket. As expected, all the fungal endophytes grew in different media regardless of the paclitaxel concentration tested. Furthermore, our results also showed that Taxomyces andreanae CBS 279.92, the first fungus shown to produce paclitaxel, is a Basidiomycete fungus as the two beta tubulins encoded by the fungus clustered together with other Basidiomycete fungi.

Homology Modeling, Molecular Docking, Molecular Dynamic Simulation, and Drug-Likeness of the Modified Alpha-Mangostin against the β-Tubulin Protein of Acanthamoeba Keratitis.

Acanthamoeba species are capable of causing amoebic keratitis (AK). As a monotherapy, alpha-mangostin is effective for the treatment of AK; however, its bioavailability is quite poor. Moreover, the efficacy of therapy is contingent on the parasite and virulent strains. To improve readiness against AK, it is necessary to find other derivatives with accurate target identification. Beta-tubulin (BT) has been used as a target for anti-Acanthamoeba (A. keratitis). In this work, therefore, a model of the BT protein of A. keratitis was constructed by homology modeling utilizing the amino acid sequence from NCBI (GenBank: JQ417907.1). Ramachandran Plot was responsible for validating the protein PDB. The verified BT PDB was used for docking with the specified ligand. Based on an improved docking score compared to alpha-mangostin (AM), two modified compounds were identified: 1,6-dihydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (C1) and 1,6-dihydroxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (C2). In addition, molecular dynamics simulations were conducted to analyze the interaction characteristics of the two bound BT–new compound complexes. During simulations, the TRP9, ARG50, VAL52, and GLN122 residues of BT-C1 that align to the identical residues in BT-AM generate consistent hydrogen bond interactions with 0–3 and 0–2. However, the BT-C2 complex has a different binding site, TYR 258, ILE 281, and SER 302, and can form more hydrogen bonds in the range 0–4. Therefore, this study reveals that C1 and C2 inhibit BT as an additive or synergistic effect; however, further in vitro and in vivo studies are needed.

P096 Comprehensive analysis of Trichophyton mentagrophytes/interdigitale complex from human and animal origin

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesTaxonomic delineation of etiologic agents responsible for recalcitrant dermatophytosis causing epidemic in India is still debated. The organism responsible for this epidemic was previously designated as Trichophyton indotineae, a clonal offshoot of T. mentagrophytes.To evaluate the accurate identity of the agent causing the epidemic, we performed multigene sequence analysis of Trichophyton species isolated from human and animal origin.MethodsThe clinical isolates of T. mentagrophytes/interdigitale complex (n = 213) from PGIMER, and six isolates of animal origin (canine) from IVRI were included in the study. Seven genomic loci including internal transcribed spacer (ITS) region (n = 219), translational elongation factor (TEF 1-α) (n = 40), partial beta tubulin (BT) (n = 40), large ribosomal subunit (LSU) (n = 34), calmodulin (CAL) (n = 29), high mobility group transcription factor (HMG) (n = 17), and α-box (n = 17) were amplified by polymerase chain reaction (PCR). Phylogenetic tree was constructed by neighbor-joining method using ours and sequences retrieved from NCBI database. Trichophyton quinckeanum was used as the outgroup due to its high divergence. The identity labeling was validated as per the phylogenetic analysis of ITS gene in the present study [ITS gene (n = 374), TEF1-α (n = 184), BT (n = 52), LSU (n = 50), CAL (n = 69) and HMG transcription factor (n = 102)].ResultsPhylogenetic analysis of ITS, TEF 1-α, LSU gene revealed that except for one isolate (PGI-IVRI B24-A) of animal origin belonged to ITS genotype III and rest all isolates [human (n = 213) and animal origin (n = 5)] belonged to the same cluster, i.e., T. mentagrophytes ITS genotype VIII. The phylogenetic tree constructed based on the BT gene (n = 39) also clustered all our isolates together and isolates belonging to T. mentagrophytes ITS genotype IV, VIII, and T. interdigitale and one isolate (PGI-IVRI B24-A) formed a separate cluster and did not coalesce with other genotypes. Whereas CAL gene clustered all our isolates (n = 28) together except PGI-IVRI B24-A isolate which grouped with T. mentagrophytes type VII. Further HMG analysis reveals that all the isolated Trichophyton species from humans (n = 11) and animals (n = 5) contained HMG transcription factors but lacked α-box genes. One animal isolate (PGI-IVRI B24-A) consisted of both HMG transcription factor and α-box genes. The accurate identity of the isolates was based on our phylogenetic analysis (ITS gene). Based on the analysis of the 374 ITS sequences deposited in the GenBank database, 107 sequences were mislabeled, ie, T. mentagrophytes was named T. interdigitale or vice versa. Similarly, 184, 52, 50, 69, and 102 sequences of TEF1-α, BT, LSU, CAL, and HMG transcription factor deposited in the GenBank have 6, 23, 25, 3, and 7 sequences were mislabeled, respectively. Further, for the identical isolates (n = 6), we found multiple accession numbers with different labels.ConclusionIn this study, for the first time we isolated ‘Indian ITS genotype VIII’ from animal. Isolation of T. mentagrophytes type III from animal but none among humans indicates its niche among animals. Adding to the confusion, outdated/inaccurate naming for these dermatophytes in the public database has created confusion in using appropriate species designation.

First Report ofPyricularia oryzae Causing Blast onWild Rice (Oryza rufipogon) in China.

Wild rice (Oryza rufipogon) is an excellent genetic resource for rice breeding programs. In June 2019, typical symptoms of blast on the leaves of wild rice cv. 'Haihong-12' were observed in a 3.3-ha field in Zhanjiang (20.93° N, 109.79° E), China. The symptoms included fusiform lesions with yellowish halo at the age of lesion, grayish-white color at the center, brown and elongated central veins at both ends of lesion, and grayish-white mold layer formed on the back of lesion under humid weather conditions. Disease incidence was more than 10%. Thirty diseased leaves were collected, and infected tissues were cut into 2 × 2 mm pieces, surface disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s and rinsed three times with sterile water. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 °C in darkness for 3 days. Three single-spore isolates (Pos-1, Pos-2, and Pos-3) were obtained using the method described by Jia (Jia 2009) and were subjected to further morphological and molecular identification. Colonies on PDA were light grey, with cottony mycelium. Conidiophores were solitary, erect, straight or curved, septate, and pale brown and measured 68 to 125×3 to 4 µm. Conidiogenous cells were sympodial and denticulate. Conidia were pale brown, pyriform, and 18.2 to 42.4×5.1 to 8.5 µm (n=30) in size, with two septa. Appressoria were spherical and had the size ranging 4.3 to 6.5×4.7 to 6.5 µm (n = 20). These morphological features agreed with the previous description of Pyricularia oryzae Cavara (Klaubauf et al. 2014). For molecular identification, the colony PCR method with MightyAmp DNA Polymerase (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS), calmodulin (CAL), actin (ACT), -tubulin (TUB) loci of the isolates using primer pairs ITS1/ITS4, CL1C/CL2C, ACT-512F/ACT-783R, and T1/βt2b, respectively (O'Donnell et al. 1997; Weir et al. 2012; White et al. 1990). Analysis of ITS (acc. nos. MW042176 to MW042178), ACT (MW091444 to MW091446), CAL (MW091447 to MW091449), and TUB (MW091441 to MW091443) sequences revealed 100% identity with the corresponding ITS (MH859782), ACT (MH589787), CAL (MH589663), and TUB (MH589547) sequences of P. oryzae in GenBank. A phylogenetic tree was generated based on the ITS sequences using maximum likelihood method that clustered Isolates Pos-1, Pos-2, and Pos-3 with known P. oryzae. Thus, the isolates were identified asP. oryzae. Pathogenicity tests were performed using Isolate Pos-1 in a greenhouse at 24to 30°C with 80% relative humidity. Individual rice plants (cv. 'Haihong-12') with three leaves were grown in 10 pots, with 50 plants per pot (40 × 60 cm). Five pots were spray inoculated with a spore suspension (105 spores/ml) until runoff from leaves, and the remaining five pots were sprayed with sterile water to serve as the controls. The test was conducted three times. Disease symptoms were observed on 10% of leaves at 10 days after inoculation, but the control plants remained healthy. The fungus was re-isolated from the diseased plants and morphologically identified as P. oryzae. Thus, this is the first report of P. oryzae causing blast on O. rufipogon in China. The results provide the information that can be used by rice breeders and fungal geneticists for further studies.

Transcriptomics of circulating neutrophils in dairy cows with subclinical hypocalcemia.

Hypocalcemia is closely associated with inflammatory diseases in dairy cows. Recent research has underscored the key role of calcium in the adaptations of the innate immune system during this period. The main objective in the present study was to compare the transcriptome profiles and analyze differences in the expression of neutrophil (PMNL) immune function-related genes and calcium binding-related genes in hypocalcemic cows. At 2 days postpartum, a concentration >2.10 mmol Ca2+/L was used to classify cows as controls (CON), and a concentration <2.00 mmol Ca2+/L used to classify cows as low-calcium (LCAL) (n = 8 in each group). A routine medical examination was conducted by the attending veterinarian to ensure there were no other complications and that the blood β-hydroxybutyrate was <1.2 mmol/L. Blood was collected from the tail vein (20 mL) to isolate PMNL, and 5 cows in each group were used for RNA sequencing and statistical analysis of gene expression differences. Transcriptome RNA-seq sequencing analysis was via omicsstudio using the R package edgeR. GO and KEGG enrichment analysis were used for bioinformatics. The remaining 3 cows in each group were used for validation of RNA sequencing data via quantitative PCR, which confirmed the observed responses. Compared with CON, 158 genes in LCAL were significantly up-regulated and 296 genes were down-regulated. The downregulation of Interleukin-12 (CXCL12), Tubulin beta chain (TUBB1), L1 cell adhesion molecule (L1CAM), and Myeloperoxidase (MPO) indicated a decrease in immune function of PMNL in LCAL cows. The decreased expression of calcium-binding pathway-related genes in PMNL of LCAL cows indicated a decrease in immune function of PMNL likely related to calcium ions. For example, cartilage acid protein 1 (CRTAC1) and calcium/calmodulin-dependent kinase 4 (CAMK4) were significantly reduced in LCAL cows. The upregulation of Cyclin dependent kinase inhibitor 1A (CDKN1A), Perforin 1 (PRF1), and Homeodomain interacting protein kinase 3 (HIPK3) indicated that LCAL led to greater cell apoptosis and senescence. Overall, the analyses indicated that the reduction in PMNL immune function during hypocalcemia is associated with downregulation of intracellular Ca2+ related genes and upregulation of genes controlling apoptosis and senescence. Together, these alterations contribute to an immunosuppressive state during the transition period.

Quantitative proteomics identifies TUBB6 as a biomarker of muscle‐invasion and poor prognosis in bladder cancer

Muscle-invasive urothelial carcinoma (MIUC) of the bladder shows highly aggressive tumor behavior, which has prompted the quest for robust biomarkers predicting invasion. To discover such biomarkers, we first employed high-throughput proteomic method and analyzed tissue biopsy cohorts from patients with bladder urothelial carcinoma (BUC), stratifying them according to their pT stage. Candidate biomarkers were selected through bioinformatic analysis, followed by validation. The latter comprised 2D and 3D invasion and migration assays, also a selection of external public datasets to evaluate mRNA expression and an in-house patient-derived tissue microarray (TMA) cohort to evaluate protein expression with immunohistochemistry (IHC). Our multilayered platform-based analysis identified tubulin beta 6 class V (TUBB6) as a promising prognostic biomarker predicting MIUC of the bladder. The in vitro 2D and 3D migration and invasion assays consistently showed that inhibition of TUBB6 mRNA significantly reduced cell migration and invasion ability in two BUC cell lines with aggressive phenotype (TUBB6 migration, P=.0509 and P < .0001; invasion, P=.0002 and P=.0044; TGFBI migration, P=.0214 and P=.0026; invasion, P < .0001 and P=.0001; T24 and J82, respectively). Validation through multiple public datasets, including The Cancer Genome Atlas (TCGA) and selected GSE (Genomic Spatial Event) databases, confirmed TUBB6 as a potential biomarker predicting MIUC. Further protein-based validation with our TMA cohort revealed concordant results, highlighting the clinical implication of TUBB6 expression in BUC patients (overall survival: P < .001). We propose TUBB6 as a novel IHC biomarker to predict invasion and poor prognosis, also select the optimal treatment in BUC patients.

First Report of Neopestalotiopsis cubana Causing leaf blight on Ixora chinensis in Malaysia.

Ixora chinensis (family Rubiaceae), locally known as 'Bunga Jejarum', is widely grown as an ornamental shrub and as sources for phytochemicals with medicinal properties in Malaysia. In May 2021, irregular brown spots were found on the leaves of some 'Bunga Jejarum' in Universiti Malaysia Sabah (6°02'01.0"N 116°07'20.2"E) located in Sabah province. As the disease progressed, the spots enlarged and coalesced into large necrotic areas giving rise to drying of infected leaves. The disease severity was about 70% with 20% incidence. Five symptomatic leaves (5 x 5 mm) from five plants were excised and sterilized based on Khoo et al. (2022) before plated on five potato dextrose agar (PDA) and cultured at 25°C. After 5 days, white to pale honey and dense mycelia with lobate edge were observed on all PDA plates. Globose, black conidiomata semi-immersed on PDA were observed after a week. Two to four hyaline filamentous appendages 7.7 to 17.6 μm long attached to fusoid conidia (11.8 to 20.9 x 5.7 to 7.6 μm, n = 20), which consisted of a hyaline apical cell, basal cell, and three versicolored median cells. The upper two median cells were dark brown, while the lowest median cell was pale brown. The isolate of the causal pathogen was characterized molecularly. Genomic DNA of isolate UMS01 was extracted based on Khoo et al. (2021) and Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS), tubulin (TUB) and translation elongation factor 1-α (TEF) region was performed based on Khoo et al. (2022) using primers ITS1/ITS4 (White et al. 1990), T1/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1997) and EF1-728/EF2 (O'Donnell et al. 1998; Carbone and Kohn, 1999), respectively. PCR products with positive amplicons were sent to Apical Scientific Sdn. Bhd. for sequencing. The isolate's sequences were deposited in GenBank as OM320626 (ITS), OM339539 (TUB) and OM339540 (TEF). They were 99% to 100% identical to ITS(KM199347) (545 out of 545 bp), TUB (KM199438) (768 out of 769 bp) and TEF (KM199521) (480 out of 481 bp) of the type sequences (CBS 600.96). Phylogenetic analysis using the maximum likelihood method based on the combined ITS, TEF and TUB sequences placed the isolate UMS01 in the same clade as the isolate CBS 600.96 of Neopestalotiopsis cubana. Thus, the pathogen was identified as N. cubanabased on the morphological description from Pornsuriya et al. (2020), molecular data in Genbank database and multigene sequence analysis. To further confirm its pathogenicity, the first and second leaves of three 'Bunga Jejarum' plants were inoculated by pipetting 1 ml aliquots of a 1 × 106 conidia/ml spore suspension. Three additional 'Bunga Jejarum' plants were mock inoculated by pipetting 1 ml of sterile distilled water on similar age leaves. The plants were covered with plastic bags after inoculation for 48 h before placing them in a glasshouse under room temperature. The leaves were sprayed with water to keep the leaf surfaces moist along the experiment. The incubation and disease observation were performed based on Chai et al. (2017) and Iftikhar et al. (2022). After 7 days post-inoculation, all infected leaves exhibited the symptoms observed in the field, whereas the controls showed no symptoms. The same fungus was isolated from the diseased leaves and, thus confirmed Koch's postulates. The experiment was repeated two more times. The reisolated fungi were visually and genetically identical to the original isolate obtained from the field samples. To our knowledge, this is the first report of N. cubana causing leaf blight on 'Bunga Jejarum' in Malaysia, as well as the world. Our finding has broadened the distribution and host range of N. cubana, indicating that it poses potential damage to the medicinal plant Bunga Jejarum in Malaysia.

H-ABC tubulinopathy revealed by label-free second harmonic generation microscopy

Hypomyelination with atrophy of the basal ganglia and cerebellum is a recently described tubulinopathy caused by a mutation in the tubulin beta 4a isoform, expressed in oligodendrocytes. The taiep rat is the only spontaneous tubulin beta 4a mutant available for the study of this pathology. We aimed to identify the effects of the tubulin mutation on freshly collected, unstained samples of the central white matter of taiep rats using second harmonic generation microscopy. Cytoskeletal differences between the central white matter of taiep rats and control animals were found. Nonlinear emissions from the processes and somata of oligodendrocytes in tubulin beta 4a mutant rats were consistently detected, in the shape of elongated structures and cell-like bodies, which were never detected in the controls. This signal represents the second harmonic trademark of the disease. The tissue was also fluorescently labeled and analyzed to corroborate the origin of the nonlinear signal. Besides enabling the description of structural and molecular aspects of H-ABC, our data open the door to the diagnostic use of nonlinear optics in the study of neurodegenerative diseases, with the additional advantage of a label-free approach that preserves tissue morphology and vitality.

Silencing LINC00665 inhibits cutaneous melanoma in vitro progression and induces apoptosis via the miR-339-3p/TUBB.

BackgroundLncRNAs are closely related to cutaneous melanoma (CM) tumorigenesis and metastasis, and it can affect the progression of CM by regulating cell proliferation, migration, invasion, apoptosis, and other cellular mechanisms. This study investigated the role of LINC00665 in CM.MethodsExpressions of LINC00665, miR‐339‐3p, and tubulin beta chain (TUBB) in CM cells were analyzed by qRT‐PCR and/or Western blot. The LINC00665/miR‐339‐3p/TUBB targeting network was predicted by bioinformatics tools, screened out by Venn diagrams and analyzed by Pearson's correlation coefficients, followed by validation via dual‐luciferase reporter assay and/or pull‐down assay. Transfection of siLINC00665 or miR‐339‐3p inhibitor/mimic was conducted with CM cells whose viability, proliferation, migration, invasion, cell cycle progression, and apoptosis were measured by CCK‐8 assay, colony formation assay, wound healing assay, Transwell assay, and flow cytometry. The associations of TUBB with tumor biological characteristics and other proteins were analyzed by CanserSEA and String, respectively.ResultsHigh‐expressed LINC00665 was detected in CM cells. Silencing LINC00665 decreased CM cell viability; inhibited colony formation, cell cycle progression, migration and invasion; enhanced apoptosis; and upregulated miR‐339‐3p. LINC00665 targeted miR‐339‐3p which targeted TUBB. MiR‐339‐3p upregulation induced effects similar to the LINC00665‐silencing‐induced effects and could downregulate TUBB, which was associated with malignant behaviors and related to other five proteins. MiR‐339‐3p downregulation induced the opposite effects of what miR‐339‐3p upregulation induced, and the miR‐339‐3p downregulation‐induced effects could be reversed by LINC00665 silencing.ConclusionSilencing LINC00665 inhibits in vitro CM progression and induces apoptosis via the miR‐339‐3p/TUBB axis.