Functional Identification and Regulatory Active Site Screening of the DfDXS Gene of Dryopteris fragrans.

Dryopteris fragrans (L.) Schott has anti-inflammatory and antioxidant properties, and terpenoids are important components of its active constituents. The methyl-D-erythritol 4-phosphate (MEP) pathway is one of the major pathways for the synthesis of terpene precursors in plants, and 1-deoxy-D-xylulose-5-phosphate synthase (DXS) is the first rate-limiting enzyme in this pathway. DXS has been shown to be associated with increased stress tolerance in plants. In this experiment, two DXS genes were extracted from the D. fragrans transcriptome and named DfDXS1 and DfDXS2. Based on phylogenetic tree and conserved motif analyses, DXS was shown to be highly conserved evolutionarily and its localization to chloroplasts was determined by subcellular localization. Prokaryotic expression results showed that the number and growth status of recombinant colonies were better than the control under 400 mM NaCl salt stress and 800 mM mannitol-simulated drought stress. In addition, the DfDXS1 and DfDXS2 transgenic tobacco plants showed improved resistance to drought and salt stress. DfDXS1 and DfDXS2 responded strongly to methyl jasmonate (MeJA) and PEG-mimicked drought stress following exogenous hormone and abiotic stress treatments of D. fragrans. The transcriptional active sites were investigated by dual luciferase and GUS staining assays, and the results showed that the STRE element (AGGGG), the ABRE element (ACGTGGC), and the MYC element (CATTTG) were the important transcriptional active sites in the promoters of the two DXS genes, which were closely associated with hormone response and abiotic stress. These results suggest that the DfDXS gene of D. fragrans plays an important role in hormone signaling and response to stress. This study provides a reference for analyzing the molecular mechanisms of stress tolerance in D. fragrans.

Stem cell-like circulating tumor cells identified by Pep@MNP and their clinical significance in pancreatic cancer metastasis.

Objective: The circulating tumor cells (CTCs) could be captured by the peptide functionalized magnetic nanoparticles (Pep@MNP) detection system in pancreatic ductal adenocarcinoma (PDAC). CTCs and the CXCR4 expression were detected to explore their clinical significance. The CXCR4+ CTCs, this is highly metastatic-prone stem cell-like subsets of CTCs (HM-CTCs), were found to be associated with the early recurrence and metastasis of PDAC. Methods: CTCs were captured by Pep@MNP. CTCs were identified via immunofluorescence with CD45, cytokeratin antibodies, and the CXCR4 positive CTCs were assigned to be HM-CTCs. Results: The over-expression of CXCR4 could promote the migration of pancreatic cancer cell in vitro and in vivo. In peripheral blood (PB), CTCs were detected positive in 79.0% of all patients (49/62, 9 (0-71)/2mL), among which 63.3% patients (31/49, 3 (0-23)/2mL) were HM-CTCs positive. In portal vein blood (PVB), CTCs were positive in 77.5% of patients (31/40, 10 (0-40)/2mL), and 67.7% of which (21/31, 4 (0-15)/2mL) were HM-CTCs positive CTCs enumeration could be used as diagnostic biomarker of pancreatic cancer (AUC = 0.862), and the combination of CTCs positive and CA19-9 increase shows improved diagnostic accuracy (AUC = 0.963). in addition, PVB HM-CTCs were more accurate to predict the early recurrence and liver metastasis than PB HM-CTCs (AUC 0.825 vs. 0.787 and 0.827 vs. 0.809, respectively). Conclusions: The CTCs identified by Pep@MNP detection system could be used as diagnostic and prognostic biomarkers of PDAC patients. We identified and defined the CXCR4 over-expressed CTC subpopulation as highly metastatic-prone CTCs, which was proved to identify patients who were prone to suffering from early recurrence and metastasis.

Novel strategy of S. mutans gcrR gene over-expression plus antibacterial dimethylaminohexadecyl methacrylate suppresses biofilm acids and reduces dental caries in rats.

OBJECTIVE: Streptococcus mutans (S. mutans) is a major contributor to dental caries, with its ability to synthesize extracellular polysaccharides (EPS) and biofilms. The gcrR gene is a regulator of EPS synthesis and biofilm formation. The objectives of this study were to investigate a novel strategy of combining gcrR gene over-expression with dimethylaminohexadecyl methacrylate (DMAHDM), and to determine their in vivo efficacy in reducing caries in rats for the first time. METHODS: Two types of S. mutans were tested: Parent S. mutans; and gcrR gene over-expressed S. mutans (gcrR OE S. mutans). Bacterial minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were measured with DMAHDM and chlorhexidine (CHX). Biofilm biomass, polysaccharide, lactic acid production, live/dead staining, colony-forming units (CFUs), and metabolic activity (MTT) were evaluated. A Sprague-Dawley rat model was used with parent S. mutans and gcrR OE S. mutans colonization to determine caries-inhibition in vivo. RESULTS: Drug-susceptibility of gcrR OE S. mutans to DMAHDM or CHX was 2-fold higher than that of parent S. mutans. DMAHDM reduced biofilm CFU by 3-4 logs. Importantly, the combined gcrR OE S. mutans+ DMAHDM dual strategy reduced biofilm CFU by 5 logs. In the rat model, the parent S. mutans group had a higher cariogenicity in dentinal (Dm) and extensive dentinal (Dx) regions. The DMAHDM + gcrR OE group reduced the Dm and Dx caries to only 20 % and 0 %, those of parent S. mutans + PBS control group (p < 0.05). The total caries severity of gcrR OE + DMAHDM group was decreased to 51 % that of parent S. mutans control (p < 0.05). SIGNIFICANCE: The strategy of combining S. mutans gcrR over-expression with antibacterial monomer reducing biofilm acids by 97 %, and reduced in vivo total caries in rats by 48 %. The gcrR over-expression + DMAHDM strategy is promising for a wide range of dental applications to inhibit caries and protect tooth structures.

Reprogramming Chromosome Ends by Functional Histone Acetylation.

Cancers harness embryonic programs to evade aging and promote survival. Normally, sequences at chromosome ends called telomeres shorten with cell division, serving as a countdown clock to limit cell replication. Therefore, a crucial aspect of cancerous transformation is avoiding replicative aging by activation of telomere repair programs. Mouse embryonic stem cells (mESCs) activate a transient expression of the gene Zscan4, which correlates with chromatin de-condensation and telomere extension. Head and neck squamous cell carcinoma (HNSCC) cancers reactivate ZSCAN4, which in turn regulates the phenotype of cancer stem cells (CSCs). Our study reveals a new role for human ZSCAN4 in facilitating functional histone H3 acetylation at telomere chromatin. Next-generation sequencing indicates ZSCAN4 enrichment at telomere chromatin. These changes correlate with ZSCAN4-induced histone H3 acetylation and telomere elongation, while CRISPR/Cas9 knockout of ZSCAN4 leads to reduced H3 acetylation and telomere shortening. Our study elucidates the intricate involvement of ZSCAN4 and its significant contribution to telomere chromatin remodeling. These findings suggest that ZSCAN4 induction serves as a novel link between 'stemness' and telomere maintenance. Targeting ZSCAN4 may offer new therapeutic approaches to effectively limit or enhance the replicative lifespan of stem cells and cancer cells.

Recombinant protein synthesis and isolation of human interferon alpha-2 in cyanobacteria.

Synechocystis sp. PCC 6803 (Synechocystis) is a unicellular photosynthetic microorganism that has been used as a model for photo-biochemical research. It comprises a potential cell factory for the generation of valuable bioactive compounds, therapeutic proteins, and possibly biofuels. Fusion constructs of recombinant proteins with the CpcA α-subunit or CpcB ß-subunit of phycocyanin in Synechocystis have enabled true over-expression of several isoprenoid pathway enzymes and biopharmaceutical proteins to levels of 10-20 % of the total cellular protein. The present work employed the human interferon α-2 protein, as a study case of over-expression and downstream processing. It advanced the state of the art in the fusion constructs for protein overexpression technology by developing the bioresource for target protein separation from the fusion construct and isolation in substantially enriched or pure form. The work brings the cyanobacterial cell factory concept closer to meaningful commercial application for the photosynthetic production of useful recombinant proteins.

Anthelmintic efficacy of an organic fraction from Guazuma ulmifolia leaves and evaluation of reactive oxidative stress on Haemonchus contortus.

This study describes the anthelmintic efficacy of an organic fraction (EtOAc-F) from Guazuma ulmifolia leaves and the evaluation of its reactive oxidative stress on Haemonchus contortus. The first step was to assess the anthelmintic effect of EtOAc-F at 0.0, 3.5, 7.0 and 14 mg kg of body weight (BW) in gerbil's (Meriones unguiculatus) artificially infected with H. contortus infective larvae (L3). The second step was to evaluate the preliminary toxicity after oral administration of the EtOAc-F in gerbils. Finally, the third step was to determine the relative expression of biomarkers such as glutathione (GPx), catalase (CAT), and superoxide dismutase (SOD) against H. contortus L3 post-exposition to EtOAc-F. Additionally, the less-polar compounds of EtOAc-F were identified by gas mass spectrophotometry (GC-MS). The highest anthelmintic efficacy (97.34%) of the organic fraction was found in the gerbils treated with the 14 mg/kg of BW. Histopathological analysis did not reveal changes in tissues. The relative expression reflects overexpression of GPx (p<0.05, fold change: 14.35) and over expression of SOD (p≤0.05, fold change: 0.18) in H. contortus L3 exposed to 97.44 mg/mL of EtOAc-F compared with negative control. The GC-MS analysis revealed the presence of 4-hydroxybenzaldehyde (1), leucoanthocyanidin derivative (2), coniferyl alcohol (3), ferulic acid methyl ester acetate (4), 2,3,4-trimethoxycinnamic acid (5) and epiyangambin (6) as major compounds. According to these results, the EtOAc-F from G. ulmifolia leaves exhibit anthelmintic effect and increased the stress biomarkers on H. contortus.

Functional characterization of a catalase gene PtCat associated with sclerotia formation in Pleurotus tuber-regium.

Pleurotus tuber-regium (Fr.) Sing. can evade oxygen by forming sclerotia under oxidative stress, consequently averting the development of hyperoxidative state, during which the expression level of catalase gene (PtCat) is significantly up-regulated. To investigate the relationship between the catalase gene and sclerotia formation, over-expression and interference strains of the PtCat gene were obtained by Agrobacterium tumefaciens-mediated transformation for phenotypic analysis. In the absence of hydrogen peroxide (H2O2) stress, a minor difference was observed in the mycelial growth rate and the activity of antioxidant enzymes between the over-expression and interference strains. However, when exposed to 1-2 mM H2O2, the colony diameter of the over-expression strain was approximately 2-3× that of the interference strain after 8 days of culturing. The catalase activity of the over-expression strain increased by 1000 U/g under 2 mM H2O2 stress, while the interference strain increased by only 250 U/g. After one month of cultivation, the interference strain formed an oval sclerotium measuring 3.5 cm on the long axis and 2 cm on the short axis, while the over-expression strain did not form sclerotia. Therefore, it is concluded that catalase activity regulates the formation of sclerotia in P. tuber-regium.

Insights on the enhancement of chilling tolerance in Rice through over-expression and knock-out studies of OsRBCS3.

Chilling stress is an important environmental factor that affects rice (Oryza sativa L.) growth and yield, and the booting stage is the most sensitive stage of rice to chilling stress. In this study, we focused on OsRBCS3, a rice gene related to chilling tolerance at the booting stage, which encodes the key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit in photosynthesis. The aim of this study was to elucidate the role and mechanism of OsRBCS3 in rice chilling tolerance at the booting stage. The expression levels of OsRBCS3 under chilling stress were compared in two japonica rice cultivars with different chilling tolerances: Kongyu131 (KY131) and Longjing11 (LJ11). A positive correlation was found between OsRBCS3 expression and chilling tolerance. Over-expression (OE) and knock-out (KO) lines of OsRBCS3 were constructed using over-expression and CRISPR/Cas9 technology, respectively, and their chilling tolerance was evaluated at the seedling and booting stages. The results showed that OE lines exhibited higher chilling tolerance than wild-type (WT) lines at both seedling and booting stages, while KO lines showed lower chilling tolerance than WT lines. Furthermore, the antioxidant enzyme activities, malondialdehyde (MDA) content and Rubisco activity of four rice lines under chilling stress were measured, and it was found that OE lines had stronger antioxidant and photosynthetic capacities, while KO lines had the opposite effects. This study validated that OsRBCS3 plays an important role in rice chilling tolerance at the booting stage, providing new molecular tools and a theoretical basis for rice chilling tolerance breeding.

Response characterization and target site mechanism study in glyphosate-resistant populations of Lolium multiflorum L. from Brazil.

Italian ryegrass (Lolium multiflorum L.) is an invasive species widely spread in croplands worldwide. The intensive use of glyphosate has resulted in the selection of resistance to this herbicide in Italian ryegrass. This work characterized the response to glyphosate of Italian ryegrass populations from the South and Southwest regions of Paraná, Brazil. A total of 44 Italian ryegrass populations were collected in farming areas, and were classified for glyphosate resistance with 75% of populations resistant to gloyphosate. Of these, 3 resistant (VT05AR, MR20AR and RN01AR) and three susceptible (VT07AS, MR05AS and RN01AS) of these populations were selected to determine the resistance level and the involvement of the target site mechanisms for glyphosate resistance. Susceptible populations GR50 ranged from 165.66 to 218.17 g.e.a. ha-1 and resistant populations from 569.37 to 925.94, providing RI ranging from 2.88 and 4.70. No mutation in EPSPS was observed in the populations, however, in two (MR20AR and RN02AR) of the three resistant populations, an increase in the number of copies of the EPSPs gene (11 to 57×) was detected. The number of copies showed a positive correlation with the gene expression (R2 = 0.86) and with the GR50 of the populations (R2 = 0.81). The increase in EPSPS gene copies contributes to glyphosate resistance in Italian ryegrass populations from Brazil.

Expression Patterns and Functional Analysis of Three SmTAT Genes Encoding Tyrosine Aminotransferases in Salvia miltiorrhiza.

Tyrosine aminotransferase (TAT, E.C. 2.6.1.5) is a pyridoxal phosphate-dependent aminotransferase that is widely found in living organisms. It catalyzes the transfer of the amino group on tyrosine to α-ketoglutarate to produce 4-hydroxyphenylpyruvic acid (4-HPP) and is the first enzyme for tyrosine degradation. Three SmTATs have been identified in the genome of Salvia miltiorrhiza (a model medicinal plant), but their information is very limited. Here, the expression profiles of the three SmTAT genes (SmTAT1, SmTAT2, and SmTAT3) were studied. All three genes expressed in different tissues and responded to methyl jasmonate stimuli. SmTAT proteins are localized in the cytoplasm. The recombinant SmTATs were subjected to in vitro biochemical properties. All three recombinant enzymes had TAT activities and SmTAT1 had the highest catalytic activity for tyrosine, followed by SmTAT3. Also, SmTAT1 preferred the direction of tyrosine deamination to 4-HPP, while SmTAT2 preferred transamination of 4-HPP to tyrosine. In parallel, transient overexpression of SmTATs in tobacco leaves revealed that all three SmTAT proteins catalyzed tyrosine to 4-HPP in vivo, with SmTAT1 exhibiting the highest enzymatic activity. Overall, our results lay a foundation for the production of tyrosine-derived secondary metabolites via metabolic engineering or synthetic biology in the future.

Heterologous Expression of Platycodon grandiflorus PgF3'5'H Modifies Flower Color Pigmentation in Tobacco.

Flavonoid-3',5'-hydroxylase (F3'5'H) is the key enzyme for the biosynthesis of delphinidin-based anthocyanins, which are generally required for purple or blue flowers. Previously, we isolated a full-length cDNA of PgF3'5'H from Platycodon grandiflorus, which shared the highest homology with Campanula medium F3'5'H. In this study, PgF3'5'H was subcloned into a plant over-expression vector and transformed into tobacco via Agrobacterium tumefaciens to investigate its catalytic function. Positive transgenic tobacco T0 plants were obtained by hygromycin resistance screening and PCR detection. PgF3'5'H showed a higher expression level in all PgF3'5'H transgenic tobacco plants than in control plants. Under the drive of the cauliflower mosaic virus (CaMV) 35S promoter, the over-expressed PgF3'5'H produced dihydromyricetin (DHM) and some new anthocyanin pigments (including delphinidin, petunidin, peonidin, and malvidin derivatives), and increased dihydrokaempferol (DHK), taxifolin, tridactyl, cyanidin derivatives, and pelargonidin derivatives in PgF3'5'H transgenic tobacco plants by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, resulting in a dramatic color alteration from light pink to magenta. These results indicate that PgF3'5'H products have F3'5'H enzyme activity. In addition, PgF3'5'H transfer alters flavonoid pigment synthesis and accumulation in tobacco. Thus, PgF3'5'H may be considered a candidate gene for gene engineering to enhance anthocyanin accumulation and the molecular breeding project for blue flowers.

Genome-Wide Identification of GSTs Gene Family and Functional Analysis of BraGSTF2 of Winter Rapeseed (Brassica rapa L.) under Cold Stress.

The largest gene families in plants were found to be Glutathione transferases (GSTs), which played significant roles in regulating plant growth, development, and stress response. Within the GSTs gene family, members were found to play a crucial role in the low-temperature response process of plants. A comprehensive study identified a total of 70 BraGSTs genes. Cluster analysis results demonstrated that the BraGSTs in Brassica rapa (B. rapa) could be categorized into eight sub-families and were unevenly distributed across ten chromosomes. The 39 BraGSTs genes were found to be organized into 15 tandem gene clusters, with the promoters containing multiple cis-elements associated with low-temperature response. Cold stress was observed to stimulate the expression of 15 genes, with the BraGSTF2 gene exhibiting the highest level of expression, suggesting its significant involvement in winter B. rapa's response to low-temperature stress. Subcellular localization analysis of the BraGSTF2 protein indicated its potential expression in both the cell membrane and nucleus. The analysis of stress resistance in BraGSTF2 transgenic Arabidopsis thaliana lines demonstrated that the over-expression of this gene resulted in significantly elevated levels of SOD, POD activity, and SP content compared to the wild type following exposure to low temperatures. These levels reached their peak after 24 h of treatment. Conversely, the MDA content was lower in the transgenic plants compared to the wild-type (WT) Arabidopsis (Arabidopsis thaliana L.). Additionally, the survival rate of BraGSTF2 transgenic Arabidopsis was higher than that of the WT Arabidopsis thaliana, suggesting that the BraGSTF2 gene may play a crucial role in enhancing the cold stress tolerance of winter B. rapa. This study lays a foundation for further research on the role of the BraGSTs gene in the molecular regulation of cold resistance in winter B. rapa.

Nisin controlled homologous Over-expression of an exopolysaccharide biosynthetic glycosyltransferase gene for enhanced EPS production in Lactobacillus plantarum BR2.

Glycosyltransferases synthesize a variety of exopolysaccharides (EPS) with different properties by altering the type of glycosidic linkage, degree of branching, length, mass, and conformation of the polymers. The genome analysis of an EPS-producing, Lactobacillus plantarum BR2 (Accession No: MN176402) showed twelve glycosyltransferase genes, and the gene BR2gtf (1116 bp), annotated as an EPS biosynthetic glycosyltransferase was cloned into the pNZ8148 vector. The recombinant pNZ8148 vector along with pNZ9530, a regulatory plasmid, were electroporated to L. plantarum BR2 for the over-expression of gtf gene under a nisin-controlled expression system and the glycosyltransferase activity of the recombinant and the wild-type strains were analysed. The recombinant strain showed 54.4% increase in EPS production with the maximum EPS production of 23.2 ± 0.5 g/L in a 5 L bioreactor study after 72 h of fermentation. This study shows an effective molecular strategy possibly to be adopted in lactic acid bacteria to enhance exopolysaccharide production.

Genome-Wide Identification of LBD Genes in Foxtail Millet (Setaria italica) and Functional Characterization of SiLBD21.

Plant-specific lateral organ boundaries domain (LBD) proteins play important roles in plant growth and development. Foxtail millet (Setaria italica) is one new C4 model crop. However, the functions of foxtail millet LBD genes are unknown. In this study, a genome-wide identification of foxtail millet LBD genes and a systematical analysis were conducted. A total of 33 SiLBD genes were identified. They are unevenly distributed on nine chromosomes. Among these SiLBD genes, six segmental duplication pairs were detected. The thirty-three encoded SiLBD proteins could be classified into two classes and seven clades. Members in the same clade have similar gene structure and motif composition. Forty-seven kinds of cis-elements were found in the putative promoters, and they are related to development/growth, hormone, and abiotic stress response, respectively. Meanwhile, the expression pattern was investigated. Most SiLBD genes are expressed in different tissues, while several genes are mainly expressed in one or two kinds of tissues. In addition, most SiLBD genes respond to different abiotic stresses. Furthermore, the function of SiLBD21, which is mainly expressed in roots, was characterized by ectopic expression in Arabidopsis and rice. Compared to controls, transgenic plants generated shorter primary roots and more lateral roots, indicating the function of SiLBD21 in root development. Overall, our study laid the foundation for further functional elucidation of SiLBD genes.

Cyanobacterial phycobilisomes as a platform for the stable production of heterologous enzymes and other proteins.

Efforts to stably over-express recombinant proteins in cyanobacteria are hindered due to cellular proteasome activity that efficiently degrades foreign proteins. Recent work from this lab showed that a variety of exogenous genes from plants, humans, and bacteria can be successfully and stably over-expressed in cyanobacteria, as fusion constructs with the abundant ß-subunit of phycocyanin (the cpcB gene product) in quantities up to 10-15% of the total cell protein. The CpcB*P fusion proteins did not simply accumulate in a soluble free-floating form in the cell but, rather, they assembled as functional (α,ß*P)3CpcG1 heterohexameric light-harvesting phycocyanin antenna discs, where α is the CpcA α-subunit of phycocyanin, ß*P is the CpcB*P fusion protein, the asterisk denoting fusion, and CpcG1 is the 28.9 kDa phycocyanin disc linker polypeptide (Hidalgo Martinez et al., 2022). The present work showed that the CpcA α-subunit of phycocyanin and the CpcG1 28.9 kDa phycocyanin disc linker polypeptide can also successfully serve as leading sequences in functional heterohexameric (α*P,ß)3CpcG1 and (α,ß)3CpcG1*P fusion constructs that permit stable recombinant protein over-expression and accumulation. These were shown to form a residual light-harvesting antenna and to contribute to photosystem-II photochemistry in the cyanobacterial cells. The work suggested that cyanobacterial cells need phycocyanin for light absorption, photosynthesis, and survival and, therefore, may tolerate the presence of heterologous recombinant proteins, when the latter are in a fusion construct configuration with essential cellular proteins, e.g., phycocyanin, thus allowing their substantial and stable accumulation.

A systematic computational analysis of Mycobacterium tuberculosis H37Rv and human CD34+ genomic expression reveals crucial molecular entities involved in infection progression.

The co-evolution of Mycobacterium tuberculosis H37Rv along with its host systems enables the pathogenic bacterium to emerge as a multi-drug resistant form. This creates challenges for a more efficacious treatment strategy that can mitigate the infection. Working towards the same, our study followed a mathematical and statistical approach proposing that mycobacterial transcription factors regulating virulence and adaptation, host cell cytoplasmic component metabolism, oxidoreductase activity and respiratory ETC would be targets for antibiotics against Mycobacterium tuberculosis. Simultaneously, extending the statistical study on Mycobacterium-infected human cord blood CD34+ cells revealed that the human CD34+ genes, S100A8 and FGR (tyrosine-protein kinase, Src2), might be affected in the infection pathogenesis by Mycobacterium. Further, the deduced Mycobacterium-human gene interaction network proposed that mycobacterial coregulators Rv0452 (MarR family regulator) and Rv3862c (WhiB6) triggered genes controlling bacterial metabolism, which influences human immunological pathways involving TLR2 and CXCL8/MAPK8.Communicated by Ramaswamy H. Sarma.

Establishment of a novel 70K Mac-2 binding protein antibody through screening of fucosylation-related antibodies.

Mac-2 binding protein (Mac-2bp) is a serum glycoprotein that contains seven N-glycans, and Mac-2bp serum levels are increased in patients with several types of cancer and liver disease. Mac-2bp glycosylation isomer has been applied as a clinical biomarker of several diseases, including liver fibrosis. In the present study, we identified fucosylated Mac-2bp in the conditioned medium of cancer cells resistant to anticancer therapies using glycoproteomic analyses. Fucosylation is one of the most important types of glycosylation involved in carcinogenesis and cancer stemness. To establish a next-generation glycan antibody for fucosylated Mac-2bp, we used fucosylation-deficient HEK293T cells to prepare reference Mac-2bp antigens and performed antibody screening. Unexpectedly, the 19-8H mAb obtained with our screen recognized 70K Mac-2bp, which is C-terminus-truncated product, rather than specifically recognizing fucosylated Mac-2bp. We performed immunocytochemistry using our novel 19-8H mAb, which resulted in strong cell surface staining of anticancer drug-resistant cancer cells. Therefore, our novel 19-8H mAb represents a valuable tool for cancer biology research that can help elucidate the biological function of 70K Mac-2bp.

Integrated analysis of mRNA and microRNA expression pattern reveals differential transcriptome signatures in RIPK2 over-expressing chicken macrophages infected with avian pathogenic E. coli.

1. As RIPK2 (receptor interacting serine/threonine kinase 2) has been shown to to alleviate excessive inflammatory responses, the following study conducted a systematic and in-depth analysis of the mRNA-seq and miRNA-seq data from chicken macrophages with/without over-expression of RIPK2 (oeRIPK2) combined with/without avian pathogenic E. coli (APEC) infection to identify the miRNA-mRNA interaction network and potential signalling pathways involved.2. A total of 9,201 differentially expressed (DE) mRNAs and 300 DE miRNA were identified in both oeRIPK2+APEC vs. APEC and oeRIPK2 vs. the wild-type (WT). Moreover, 4,269 instances of co-expression between miRNAs and mRNAs were seen involving 1,652 DE mRNAs and 164 DE miRNAs.3. Functional analysis of the DE mRNAs in the miRNA-mRNA interaction network showed that 223 biological processes and five KEGG pathways were significantly enriched in the two comparisons. In total, 128 pairs of miRNA-mRNA interactions were involved in the identified MAPK signalling pathway and focal adhesion immune related pathways.4. Significantly, these screened miRNAs (gga-miR-222b-5p and gga-miR-214) and their target genes were highly correlated with APEC infection and RIPK2. These recognised key genes, miRNA and the overall miRNA-mRNA regulatory network, enables better understanding of the molecular mechanism of host response to APEC infection, especially related to RIPK2.

A xanthophyll-derived apocarotenoid regulates carotenogenesis in tomato chromoplasts.

Carotenoids possess important biological functions that make them essential components of the human diet. ß-Carotene and some other carotenoids have vitamin A activity while lutein and zeaxanthin, typically referred to as the macular pigments, are involved in good vision and in delaying the onset of age-related eye diseases. In order to create a zeaxanthin-producing tomato fruit, two transgenic lines, one with a high ß-carotene cyclase activity and the other with a high ß-carotene hydroxylase activity, have been genetically crossed. Ripe fruits from the resulting progeny contained significant levels of violaxanthin, antheraxanthin, and xanthophyll esters. However, their zeaxanthin content was not as high as expected, and the total level of carotenoids was only 25% of the carotenoids found in ripe fruits of the comparator line. Targeted transcript analysis and apocarotenoids determinations indicated that transcriptional regulation of the pathway or degradation of synthesized carotenoids were not responsible for the low carotenoid content of hybrid fruits which instead appeared to result from a substantial reduction of carotenoid biosynthesis. Notably, the content of an unidentified hydroxylated cyclic (C13) apocarotenoid was 13 times higher in the hybrid fruits than in the control fruits. Furthermore, a GC-MS-based metabolite profiling demonstrated a perturbation of carotenogenesis in ripening hybrid fruits compatible with a block of the pathway. Moreover, carotenoid profiling on leaf, fruit, and petal samples from a set of experimental lines carrying the hp3 mutation, in combination with the two transgenes, indicated that the carotenoid biosynthesis in petal and fruit chromoplasts could be regulated. Altogether the data were consistent with the hypothesis of the regulation of the carotenoid pathway in tomato chromoplasts through a mechanism of feedback inhibition mediated by a xanthophyll-derived apocarotenoid. This chromoplast-specific post-transcriptional mechanism was disclosed in transgenic fruits of HU hybrid owing to the abnormal production of zeaxanthin and antheraxanthin, the more probable precursors of the apocarotenoid signal. A model describing the regulation of carotenoid pathway in tomato chromoplasts is presented.

Clinicopathological pattern of oestrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 over-expression of epithelial ovarian carcinomas in Nigeria.

Background: Ovarian cancer is the leading cause of death from all gynaecological malignancies. Only few biomarkers of epithelial ovarian cancer (EOC) prognosis have been studied so far among Nigerian patients. Objective: To determine the pattern of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) expression in patients with EOC seen in Nigeria. Materials and Methods: This was a retrospective multicentre study of 102 cases of epithelial ovarian cancers. Relevant clinical information was obtained from hospital-based records in the 3 participating centres. Tissue microarrays were constructed using representative tumour tissue and the ER, PR and HER2 immunohistochemical staining was carried out at the University of Chicago, United States of America. Results: Serous carcinomas predominated (71% of cases). ER positivity was observed in 31.4%, PR positivity in 21.5% and HER2/neu in 16.7% of tumours. Fifty-two percent of tumours were triple negative. Serous tumours were significantly associated with ER positivity (p=0.001). Mean patient age for EOC was 52.6 ± 13.1 years. There were no statistically significant associations between hormone receptor status and histological grade, FIGO staging or survival. Conclusion: Serous tumours were significantly associated with ER expression while non-serous tumours tended to be triple negative.