Fungal Spore Richness and Abundance of Allergenic Taxa: Comparing a Portable Impactor and Passive Trap Indoors and Outdoors in an Urban Setting.

Fungal spores are common airborne allergens, and fungal richness has been implicated in allergic disease. Amplicon sequencing of environmental DNA from air samples is a promising method to estimate fungal spore richness with semi-quantification of hundreds of taxa and can be combined with quantitative PCR to derive abundance estimates. However, it remains unclear how the choice of air sampling method influences these estimates. This study compared active sampling with a portable impactor and passive sampling with a passive trap over different durations to estimate fungal spore richness and the abundance of allergenic taxa. Air sampling was conducted indoors and outdoors at 12 residences, including repeated measurements with a portable impactor and passive traps with 1-day and 7-day durations. ITS2 amplicon sequence data were transformed to spore equivalents estimated by quantitative PCR, repeated active samples were combined, and abundance-based rarefaction was performed to standardize sample coverage for estimation of genus-level richness and spore abundance. Rarefied fungal richness was similar between methods indoors but higher for passive traps with a 7-day duration outdoors. Rarefied abundance of allergenic genera was similar between methods but some genera had lower abundance for passive traps with a 1-day duration, which differed indoors and outdoors indicating stochasticity in the collection of spores on collocated samplers. This study found that similar estimates of fungal spore richness and abundance of allergenic taxa can be obtained using a portable impactor or a passive trap within one day and that increased passive sample duration provides limited additional information.

Fungal Spore Richness in School Classrooms is Related to Surrounding Forest in a Season-Dependent Manner.

Airborne fungal spores are important aeroallergens that are remarkably diverse in terms of taxonomic richness. Indoor fungal richness is dominated by outdoor fungi and is geographically patterned, but the influence of natural landscape is unclear. We aimed to elucidate the relationship between indoor fungal spore richness and natural landscape by examining the amount of surrounding forest cover. Passive sampling of airborne fungal spores was conducted in 24 schools in Taiwan during hot and cool seasons, and amplicon sequencing was used to study fungal spore (genus) richness targeting the internal transcribed spacer 2 (ITS2) region. In total, 693 fungal genera were identified, 12 of which were ubiquitous. Despite overall similarity of fungal spore richness between seasons, Basidiomycota and Ascomycota richness increased during the hot and cool seasons, respectively. Fungal spore richness in schools had a strong positive correlation with the amount of surrounding forest cover during the cool season, but not during the hot season. Fungal assemblages in schools were more similar during the hot season due to the increased ubiquity of Agaricomycetes genera. These observations indicate dispersal limitation at the kilometer scale during the cool season and increased long-distance dispersal during the hot season. Several allergenic fungi were commonly identified in schools, including some previously overlooked by conventional methods, which may be targeted as sensitizing agents in future investigations into atopic conditions. More generally, the relative importance of fungal spore richness in the development, chronicity, and severity of atopic conditions in children requires investigation.

Surveillance of Rice Blast Resistance Effectiveness and Emerging Virulent Isolates in Taiwan.

Rice blast caused by Magnaporthe oryzae is a dangerous threat to rice production and food security worldwide. Breeding and proper deployment of resistant varieties are effective and environmentally friendly strategies to manage this notorious disease. However, a highly dynamic and quickly evolved rice blast pathogen population in the field has made disease control with resistance germplasms more challenging. Therefore, continued monitoring of pathogen dynamics and application of effective resistance varieties are critical tasks to prolong or sustain field resistance. Here, we report a team project that involved evaluation of rice blast resistance genes and surveillance of M. oryzae field populations in Taiwan. A set of International Rice Research Institute-bred blast-resistant lines (IRBLs) carrying single blast resistance genes was utilized to monitor the field effectiveness of rice blast resistance. Resistance genes such as Ptr (formerly Pita2) and Pi9 exhibited the best and most durable resistance against the rice blast fungus population in Taiwan. Interestingly, line IRBLb-B harboring the Pib gene with good field protection has recently shown susceptible lesions in some locations. To dissect the genotypic features of virulent isolates against the Pib resistance gene, M. oryzae isolates were collected and analyzed. Screening of the AvrPib locus revealed that the majority of field isolates still maintained the wild-type AvrPib status but eight virulent genotypes were found. Pot3 insertion appeared to be a major way to disrupt the AvrPib avirulence function. Interestingly, a novel AvrPib double-allele genotype among virulent isolates was first identified. Pot2 repetitive element-based polymerase chain reaction (rep-PCR) fingerprinting analysis indicated that mutation events may occur independently among different lineages in different geographic locations of Taiwan. This study provides our surveillance experience of rice blast disease and serves as the foundation to sustain rice production.

Identification of MaWRKY40 and MaDLO1 as Effective Marker Genes for Tracking the Salicylic Acid-Mediated Immune Response in Bananas.

Bananas are among the world's most important cash and staple crops but are threatened by various devastating pathogens. The phytohormone salicylic acid (SA) plays a key role in the regulation of plant immune response. Tracking the expression of SA-responsive marker genes under pathogen infection is important in pathogenesis elucidation. However, the common SA-responsive marker genes are not consistently induced in different banana cultivars or different organs. Here, we conducted transcriptome analysis for SA response of a banana cultivar, 'Pei-Chiao' (Cavendish, AAA genome), and identified three genes, MaWRKY40, MaWRKY70, and Downy Mildew Resistant 6 (DMR6)-Like Oxygenase 1 (MaDLO1) that are robustly induced upon SA treatment in both the leaves and roots. Consistent induction of these three genes by SA treatment was also detected in both the leaves and roots of bananas belonging to different genome types such as 'Tai-Chiao No. 7' (Cavendish, AAA genome), 'Pisang Awak' (ABB genome), and 'Lady Finger' (AA genome). Furthermore, the biotrophic pathogen cucumber mosaic virus elicited the expression of MaWRKY40 and MaDLO1 in infected leaves of susceptible cultivars. The hemibiotrophic fungal pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) also consistently induced the expression of MaWRKY40 and MaDLO1 in the infected roots of the F. oxysporum f. sp. cubense TR4-resistant cultivar. These results indicate that MaWRKY40 and MaDLO1 can be used as reliable SA-responsive marker genes for the study of plant immunity in banana. Revealing SA-responsive marker genes provides a stepping stone for further studies in banana resistance to pathogens.

Marker-Assisted Development and Evaluation of Monogenic Lines of Rice cv. Kaohsiung 145 Carrying Blast Resistance Genes.

Rice blast is a serious threat to global rice production. Large-scale and long-term cultivation of rice varieties with a single blast resistance gene usually leads to breakdown of resistance. To effectively control rice blast in Taiwan, marker-assisted backcrossing was conducted to develop monogenic lines carrying different blast resistance genes in the genetic background of an elite japonica rice cultivar, Kaohsiung 145 (KH145). Eleven International Rice Research Institute (IRRI)-bred blast-resistant lines (IRBLs) showing broad-spectrum resistance to local Pyricularia oryzae isolates were used as resistance donors. Sequencing analysis revealed that the recurrent parent, KH145, does not carry known resistance alleles at the target Pi2/9, Pik, Pita, and Ptr loci. For each IRBL × KH145 cross, we screened 21 to 370 (average of 108) plants per generation from the BC1F1 to BC3F1/BC4F1 generation. A total of 1,499 BC3F2/BC4F2 lines carrying homozygous resistance alleles were selected and self-crossed for four to six successive generations. The derived lines were also evaluated for background genotype using genotyping by sequencing, for blast resistance under artificial inoculation and natural infection conditions, and for agronomic performance in multiple field trials. In Chiayi and Taitung blast nurseries in 2018 to 2020, Pi2, Pi9, and Ptr conferred high resistance, Pi20 and Pik-h moderate resistance, and Pi1, Pi7, Pik-p, and Pik susceptibility to leaf blast; only Pi2, Pi9, and Ptr conferred effective resistance against panicle blast. The monogenic lines showed agronomic traits, yield, and grain quality similar to those of KH145, suggesting the potential of growing a mixture of lines to achieve durable resistance in the field.

The smut fungus Ustilago esculenta has a bipolar mating system with three idiomorphs larger than 500 kb.

Zizania latifolia Turcz., which is mainly distributed in Asia, has had a long cultivation history as a cereal and vegetable crop. On infection with the smut fungus Ustilago esculenta, Z. latifolia becomes an edible vegetable, water bamboo. Two main cultivars, with a green shell and red shell, are cultivated for commercial production in Taiwan. Previous studies indicated that cultivars of Z. latifolia may be related to the infected U. esculenta isolates. However, related research is limited. The infection process of the corn smut fungus Ustilago maydis is coupled with sexual development and under control of the mating type locus. Thus, we aimed to use the knowledge of U. maydis to reveal the mating system of U. esculenta. We collected water bamboo samples and isolated 145 U. esculenta strains from Taiwan's major production areas. By using PCR and idiomorph screening among meiotic offspring and field isolates, we identified three idiomorphs of the mating type locus and found no sequence recombination between them. Whole-genome sequencing (Illumina and PacBio) suggested that the mating system of U. esculenta was bipolar. Mating type locus 1 (MAT-1) was 552,895 bp and contained 44% repeated sequences. Sequence comparison revealed that U. esculenta MAT-1 shared high gene synteny with Sporisorium reilianum and many repeats with Ustilago hordei MAT-1. These results can be utilized to further explore the genomic diversity of U. esculenta isolates and their application for water bamboo breeding.

The Blue-Light Photoreceptor Sfwc-1 Gene Regulates the Phototropic Response and Fruiting-Body Development in the Homothallic Ascomycete Sordaria fimicola.

Sordaria fimicola, a coprophilous ascomycete, is a homothallic fungus that can undergo sexual differentiation with cellular and morphological changes followed by multicellular tissue development to complete its sexual cycle. In this study, we identified and characterized the blue-light photoreceptor gene in S. fimicola The S. fimicola white collar-1 photoreceptor (SfWC-1) contains light-oxygen-voltage-sensing (LOV), Per-Arnt-Sim (PAS), and other conserved domains and is homologous to the WC-1 blue-light photoreceptor of Neurospora crassa The LOV domain of Sfwc-1 was deleted by homologous recombination using Agrobacterium-mediated protoplast transformation. The Sfwc-1(Δlov) mutant showed normal vegetative growth but produced less carotenoid pigment under illumination. The mutant showed delayed and less-pronounced fruiting-body formation, was defective in phototropism of the perithecial beaks, and lacked the fruiting-body zonation pattern compared with the wild type under the illumination condition. Gene expression analyses supported the light-induced functions of the Sfwc-1 gene in the physiology and developmental process of perithecial formation in S. fimicola Moreover, green fluorescent protein (GFP)-tagged SfWC-1 fluorescence signals were transiently strong upon light induction and prominently located inside the nuclei of living hyphae. Our studies focused on the putative blue-light photoreceptor in a model ascomycete and contribute to a better understanding of the photoregulatory functions and networks mediated by the evolutionarily conserved blue-light photoreceptors across diverse fungal phyla.IMPORTANCESordaria sp. has been a model for study of fruiting-body differentiation in fungi. Several environmental factors, including light, affect cellular and morphological changes during multicellular tissue development. Here, we created a light-oxygen-voltage-sensing (LOV) domain-deleted Sfwc-1 mutant to study blue-light photoresponses in Sordaria fimicola Phototropism and rhythmic zonation of perithecia were defective in the Sfwc-1(Δlov) mutant. Moreover, fruiting-body development in the mutant was reduced and also significantly delayed. Gene expression analysis and subcellular localization study further revealed the light-induced differential gene expression and cellular responses upon light stimulation in S. fimicola.

Dynamic Insertion of Pot3 in AvrPib Prevailing in a Field Rice Blast Population in the Philippines Led to the High Virulence Frequency Against the Resistance Gene Pib in Rice.

The Magnaporthe oryzae avirulence gene AvrPib is required for the resistance mediated by its cognate resistance gene Pib, which has been intensively used in indica rice breeding programs in many Asian countries. However, the sequence diversity of AvrPib among geographically distinct M. oryzae populations was recently shown to be increasing. Here, we selected a field population consisting of 248 rice blast isolates collected from a disease hotspot in Philippine for the analysis of AvrPib haplotypes and their pathogenicity against Pib. We found that all of the isolates were virulent to Pib and each of them contained an insertion of Pot3 transposon in AvrPib. Moreover, Pot3 insertion was detected in different genomic positions, resulting in three different AvrPib haplotypes, designated avrPib-H1 to H3. We further conducted a genome-wide Pot2 fingerprinting analysis by repetitive element palindromic polymerase chain reaction (PCR) and identified seven different lineages out of 47 representative isolates. The isolates belonging to the same lineage often had the same AvrPib haplotype. In contrast, the isolates having the same AvrPib haplotypes did not always belong to the same lineages. Both mating types MAT1-1 and MAT1-2 were identified in the population in Bohol and the latter appeared dominant. On the host side, we found that 32 of 52 released rice varieties in the Philippines contained Pib diagnosed by PCR gene-specific primers and DNA sequencing of gene amplicons, suggesting that it was widely incorporated in different rice varieties. Our study highlights the genetic dynamics of rice blast population at both the AvrPib locus and the genome-wide levels, providing insight into the mechanisms of the mutations in AvrPib leading to the breakdown of Pib-mediated resistance in rice.

Discovery of An Orally Effective Factor IX-Transferrin Fusion Protein for Hemophilia B.

Hemophilia B is a severe blood clotting disorder caused by the deficiency of factor IX (FIX). FIX is not bioavailable when given orally due to poor stability and permeability in the gastrointestinal tract. The feasibility of fusing FIX with transferrin (Tf) to enhance the oral bioavailability of FIX is explored. Seven recombinant fusion proteins (rFIX-Tf) with different linkers were constructed and expressed in HEK293 cells and characterized by in vitro transcytosis and transferrin receptor (TfR) binding assay in Caco-2 cells and a one-stage clotting assay. The in vivo efficacy study was performed using a tail-bleeding model in hemophilia B mice. Fusion proteins rFIX-Tf/G2 and rFIX-Tf/SVSQ were most permeable and showed a specific binding ability to TfR in Caco-2 cells. Both proteins retained FIX activity in clotting generation. The in vivo efficacy study showed that both proteins by intravenous injection significantly reduced blood loss. Most significantly, rFIX-Tf/G2 demonstrated anti-bleeding activity when administered orally. Our results showed that the fusion protein technique with Tf could be potentially used for oral delivery of FIX and the linker between FIX and Tf in the fusion protein is crucial. rFIX-Tf/G2 appears to be the most promising fusion protein as potential oral therapeutics for hemophilia B.

Characterization and Oral Delivery of Proinsulin-Transferrin Fusion Protein Expressed Using ExpressTec.

Proinsulin-transferrin fusion protein (ProINS-Tf) has been designed and successfully expressed from the mammalian HEK293 cells (HEK-ProINS-Tf). It was found that HEK-ProINS-Tf could be converted into an activated form in the liver. Furthermore, HEK-ProINS-Tf was demonstrated as an extra-long acting insulin analogue with liver-specific insulin action in streptozotocin (STZ)-induced type 1 diabetic mice. However, due to the low production yield from transfected HEK293 cells, there are other interesting features, including the oral bioavailability, which have not been fully explored and characterized. To improve the protein production yield, an alternative protein expression system, ExpressTec using transgenic rice (Oryza sativa L.), was used. The intact and active rice-derived ProINS-Tf (ExpressTec-ProINS-Tf) was successfully expressed from the transgenic rice expression system. Our results suggested that, although the insulin-like bioactivity of ExpressTec-ProINS-Tf was slightly lower in vitro, its potency of in vivo blood glucose control was considerably stronger than that of HEK-ProINS-Tf. The oral delivery studies in type 1 diabetic mice demonstrated a prolonged control of blood glucose to near-normal levels after oral administration of ExpressTec-ProINS-Tf. Results in this report suggest that ExpressTec-ProINS-Tf is a promising insulin analog with advantages including low cost, prolonged and liver targeting effects, and most importantly, oral bioactivity.

Characterization of Polyelectrolyte Complex Formation Between Anionic and Cationic Poly(amino acids) and Their Potential Applications in pH-Dependent Drug Delivery.

Polyelectrolyte complexes (PECs) are self-assembling nano-sized constructs that offer several advantages over traditional nanoparticle carriers including controllable size, biodegradability, biocompatibility, and lack of toxicity, making them particularly appealing as tools for drug delivery. Here, we discuss potential application of PECs for drug delivery to the slightly acidic tumor microenvironment, a pH in the range of 6.5-7.0. Poly(l-glutamic acid) (En), poly(l-lysine) (Kn), and a copolymer composed of histidine-glutamic acid repeats ((HE)n) were studied for their ability to form PECs, which were analyzed for size, polydispersity, and pH sensitivity. PECs showed concentration dependent size variation at residue lengths of E51/K55 and E135/K127, however, no complexes were observed when E22 or K21 were used, even in combination with the longer chains. (HE)20/K55 PECs could encapsulate daunomycin, were stable from pH 7.4-6.5, and dissociated completely between pH 6.5-6.0. Conversely, the E51-dauno/K55 PEC dissociated between pH 4.0 and 3.0. These values for pH-dependent particle dissociation are consistent with the pKa's of the ionizable groups in each formulation and indicate that the specific pH-sensitivity of (HE)20-dauno/K55 PECs is mediated by incorporation of histidine. This response within a pH range that is physiologically relevant to the acidic tumors suggests a potential application of these PECs in pH-dependent drug delivery.

Proinsulin-Transferrin Fusion Protein Exhibits a Prolonged and Selective Effect on the Control of Hepatic Glucose Production in an Experimental Model of Type 1 Diabetes.

An ideal basal insulin (INS) replacement therapy requires the distribution or action of exogenous INS to more closely mimic physiological INS in terms of its preferential hepatic action. In this paper, we introduce a novel strategy to exert liver-specific INS action by hepatic activation of INS's precursor, proinsulin (ProINS). We demonstrated the conversion of human ProINS-transferrin (Tf) fusion protein, ProINS-Tf, into an active and immuno-reactive form of INS-Tf in the liver via the slow Tf receptor mediated recycling pathway. ProINS-Tf displayed prolonged basal blood glucose lowering effects for up to 40 h in streptozotocin-induced type 1 diabetic mice following a single subcutaneous injection. The effect of ProINS-Tf on blood glucose levels was observed predominantly under fasting conditions, with little effect under free-feeding conditions. In addition, both the pyruvate tolerance assay in normal mice and the Akt-phosphorylation assay in H-4-II-E hepatoma cells indicated that the hepatic-activated ProINS-Tf possessed a much longer effect on the control of hepatic glucose production than INS. These results indicated that ProINS-Tf may serve as an effective and safe hepatoselective INS analog to reduce the frequency of INS injections as well as avert severe hypoglycemia episodes and other side effects frequently encountered with long-acting INS therapeutics due to their peripheral action.

Selective Intracellular Delivery of Recombinant Arginine Deiminase (ADI) Using pH-Sensitive Cell Penetrating Peptides To Overcome ADI Resistance in Hypoxic Breast Cancer Cells.

Arginine depletion strategies, such as pegylated recombinant arginine deiminase (ADI-PEG20), offer a promising anticancer treatment. Many tumor cells have suppressed expression of a key enzyme, argininosuccinate synthetase 1 (ASS1), which converts citrulline to arginine. These tumor cells become arginine auxotrophic, as they can no longer synthesize endogenous arginine intracellularly from citrulline, and are therefore sensitive to arginine depletion therapy. However, since ADI-PEG20 only depletes extracellular arginine due to low internalization, ASS1-expressing cells are not susceptible to treatment since they can synthesize arginine intracellularly. Recent studies have found that several factors influence ASS1 expression. In this study, we evaluated the effect of hypoxia, frequently encountered in many solid tumors, on ASS1 expression and its relationship to ADI-resistance in human MDA-MB-231 breast cancer cells. It was found that MDA-MB-231 cells developed ADI resistance in hypoxic conditions with increased ASS1 expression. To restore ADI sensitivity as well as achieve tumor-selective delivery under hypoxia, we constructed a pH-sensitive cell penetrating peptide (CPP)-based delivery system to carry ADI inside cells to deplete both intra- and extracellular arginine. The delivery system was designed to activate the CPP-mediated internalization only at the mildly acidic pH (6.5-7) associated with the microenvironment of hypoxic tumors, thus achieving better selectivity toward tumor cells. The pH sensitivity of the CPP HBHAc was controlled by recombinant fusion to a histidine-glutamine (HE) oligopeptide, generating HBHAc-HE-ADI. The tumor distribution of HBHAc-HE-ADI was comparable to ADI-PEG20 in a mouse xenograft model of human breast cancer cells in vivo. In addition, HBHAc-HE-ADI showed increased in vitro cellular uptake in cells incubated in a mildly acidic pH (hypoxic conditions) compared to normal pH (normoxic conditions), which correlated with pH-sensitive in vitro cytotoxicity in hypoxic MDA-MB-231 and human prostate cancer PC3 cells. Together, we conclude that the HBHAc-HE-based peptide delivery offers a useful means to overcome hypoxia-induced resistance to ADI in breast cancer cells, and to target the mildly acidic tumor microenvironment.

Mass spectrometry study of a transferrin-based protein drug reveals the key role of protein aggregation for successful oral delivery.

A recently designed human growth hormone/transferrin fusion protein (GHT) remains one of the very few examples of a protein capable of eliciting measurable therapeutic response after oral administration. To better understand the underlying factors that resulted in this rare success of nonparenteral protein drug delivery, we analyzed proteolytic stability and receptor binding properties of this protein, the key factors in overcoming the primary barriers to successful oral delivery. Analysis of GHT by a combination of size exclusion chromatography and mass spectrometry revealed that a significant protein population exists in an oligomeric (GHTx) state in addition to the anticipated monomer (GHT1). These states of GHT were evaluated for their survivability in stomach-like conditions, as well as their ability to bind transferrin receptor (TfR). Our results reveal an exceptional stability of GHTx, as well as the preserved ability to bind TfR, a critical first step in crossing the epithelial-intestinal barrier through receptor-mediated transcytosis.

Interaction between cell-penetrating peptides and acid-sensitive anionic oligopeptides as a model for the design of targeted drug carriers.

Overcoming the nonspecific cellular uptake of cell-penetrating peptides (CPPs) is a major hurdle in their clinical application. Using pH as the activation switch, histidine-glutamic acid (HE) dipeptide repeats were fused to CPPs to trigger the membrane-penetrating activity at mildly acidic pH environments (i.e., pH 6.5 or below) while masking the internalization at neutral pH (i.e., pH 7.0 or above). In this study, a series of recombinant GST-fusion proteins containing an HE oligopeptide sequence (i.e., (HE)n with n = 8, 10, or 12) and a cationic CPP (i.e., YG(RG)6, YGR6G6, or Tat) were engineered for a pH-sensitive study comparing their cellular uptake and surface binding in cultured HeLa cells. Circular dichroism (CD) spectroscopy was performed to correlate differences between CPPs in secondary structure with the pH sensitivity. YGR6G6 with clustered arginine residues exhibited greater pH sensitivity in cellular uptake than YG(RG)6 with separated arginine residues. Increasing the stretch of HE repeats decreased cellular uptake and surface binding for both YG(RG)6 and YGR6G6. The ratio of cellular internalization at pH 7.5 vs 6.0 was not changed by the presence of serum. CD spectral data revealed that both (HE)10-Tat and (HE)10-YGR6G6 exhibited an unordered secondary structure, whereas (HE)10-YG(RG)6 adopted an antiparallel ß-sheet conformation. This ß-sheet conformation presumably stabilized the association of (HE)10 with YG(RG)6, leading to weakened pH sensitivity of (HE)10-YG(RG)6. On the other hand, the random-coiled structures, that is, (HE)10-YGR6G6 and (HE)10-Tat, both showed higher pH sensitivity as determined in cell experiments. The data presented in this study provide a basis for the future design of pH-sensitive HE-CPP carrier for targeted drug delivery.

Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativa L.).

BACKGROUND: Transferrin (TF) plays a critical physiological role in cellular iron delivery via the transferrin receptor (TFR)-mediated endocytosis pathway in nearly all eukaryotic organisms. Human serum TF (hTF) is extensively used as an iron-delivery vehicle in various mammalian cell cultures for production of therapeutic proteins, and is also being explored for use as a drug carrier to treat a number of diseases by employing its unique TFR-mediated endocytosis pathway. With the increasing concerns over the risk of transmission of infectious pathogenic agents of human plasma-derived TF, recombinant hTF is preferred to use for these applications. Here, we carry out comparative studies of the TFR binding, TFR-mediated endocytosis and cellular iron delivery of recombinant hTF from rice (rhTF), and evaluate its suitability for biopharmaceutical applications. RESULT: Through a TFR competition binding affinity assay with HeLa human cervic carcinoma cells (CCL-2) and Caco-2 human colon carcinoma cells (HTB-37), we show that rhTF competes similarly as hTF to bind TFR, and both the TFR binding capacity and dissociation constant of rhTF are comparable to that of hTF. The endocytosis assay confirms that rhTF behaves similarly as hTF in the slow accumulation in enterocyte-like Caco-2 cells and the rapid recycling pathway in HeLa cells. The pulse-chase assay of rhTF in Caco-2 and HeLa cells further illustrates that rice-derived rhTF possesses the similar endocytosis and intracellular processing compared to hTF. The cell culture assays show that rhTF is functionally similar to hTF in the delivery of iron to two diverse mammalian cell lines, HL-60 human promyelocytic leukemia cells (CCL-240) and murine hybridoma cells derived from a Sp2/0-Ag14 myeloma fusion partner (HB-72), for supporting their proliferation, differentiation, and physiological function of antibody production. CONCLUSION: The functional similarity between rice derived rhTF and native hTF in their cellular iron delivery, TFR binding, and TFR-mediated endocytosis and intracellular processing support that rice-derived rhTF can be used as a safe and animal-free alternative to serum hTF for bioprocessing and biopharmaceutical applications.

Comparison of cationic and amphipathic cell penetrating peptides for siRNA delivery and efficacy.

Cell penetrating peptides (CPPs) are short strands of arginine- and/or lysine-rich peptides (<30 amino acids) that use their cationic nature for efficient intracellular accumulation. CPPs have been used for small interfering RNA (siRNA) delivery by direct complexation with the siRNA anionic phosphate backbone. During this process, however, part of the CPP cationic charges are neutralized, and the resultant loss of free positive charges may substantially compromise CPP's internalization capabilities and eventually reduce siRNA delivery efficiency. The purpose of this study was to design a novel type of polyplex for siRNA delivery to overcome the CPP neutralization issue. This novel polyplex consists of three components: siRNA, 21mer oligolysine (K21) chemically modified to incorporate CPP conjugation sites (K21-PDP), and CPP delivery moiety. The siRNA was first neutralized by cationic charges of K21-PDP to form a polyplex. Then a cationic (hexaarginine, R6) or an amphipathic (model amphipathic peptide, MAP) CPP was conjugated to the polyplex. Agarose gel shift assays indicated that the siRNA could be released from the polyplex after K21-PDP degradation or polyplex dilution. Furthermore, the total intracellular internalization of these two CPP-polyplexes was studied. Compared with R6-polyplex, MAP-polyplex exhibited 170- and 600-fold greater uptake of fluorescently labeled siRNA at 1 and 6 h post-transfection, respectively. MAP-polyplex also exhibited comparable GFP silencing effects as Lipofectamine 2000 complex in Huh7.5 cells stably transfected to express GFP-light chain 3 protein, whereas R6-polyplex did not demonstrate significant silencing activity. Further studies indicated that the K21-PDP-siRNA polyplex formation and conjugation of MAP to the polyplex were essential for siRNA polyplex uptake and gene silencing. MAP-polyplex was also shown to be unaffected by the presence of 10% FBS during transfection. In addition, MAP-polyplex uptake was dependent on vesicle formation and fusion due to 70 and 54% loss of uptake at 4 and 16 °C, respectively, compared to incubation at 37 °C. Therefore, the amphipathic CPP is a more suitable carrier moiety for delivery of siRNA polyplex.

Mating differentiation in Cryptococcus neoformans is negatively regulated by the Crk1 protein kinase.

Cryptococcus neoformans is a heterothallic basidiomycete that grows vegetatively as yeast and filamentous hyphae are produced in the sexual state. Previous studies have shown that C. neoformans Cwc1 and Cwc2 are two central photoregulators which form a complex to inhibit the production of sexual filaments upon light treatment. To reveal the detailed regulatory mechanisms, a genome wide mutagenesis screen was conducted and components in the Cwc1/Cwc2 complex mediated pathway have been identified. In this study, one suppressor mutant, DJ22, is characterized and T-DNA is found to disrupt the C. neoformans CRK1 gene, a homologue of Saccharomyces cerevisiae IME2 and Ustilago maydis crk1. Ime2 is a meiosis-specific gene with the conserved Ser/Thr kinase domain and TXY dual phosphorylation site. Consistent with the findings of other suppressors in our screen, C. neoformans Crk1 plays a negative role in the mating process. Dikaryotic filaments, basidia, and basidiospores are produced earlier in the crk1 mutant crosses and mating efficiency is also increased. Artificial elevation of the CRK1 mRNA level inhibits mating. Interestingly, monokaryotic fruiting is defective both in the MATα crk1 mutant and CRK1 overexpression strains. Our studies demonstrate that C. neoformans CRK1 gene functions as a negative regulator in the mating differentiation.

Effects of receptor binding on plasma half-life of bifunctional transferrin fusion proteins.

In contrast to the wide applications of recombinant bifunctional fusion proteins in clinical usage, the systematic study for the pharmacokinetics (PK) of bifunctional fusion proteins is left blank. In this report, recombinant fusion proteins consisting of transferrin (Tf) and growth hormone (GH) or granulocyte colony-stimulating factor (G-CSF) have been constructed as a model for studying the PK of bifunctional fusion proteins. The results showed that the insertion of different linkers between the two protein domains altered the binding affinities of the fusion proteins to both domain receptors, and that the fusion proteins' plasma half-lives were greatly affected. A strong correlation between GH receptor binding affinity and plasma half-life of GH-Tf fusion proteins was observed. In addition, we demonstrated that the intracellular processing after receptor binding plays an important role in determining the half-life of fusion proteins. While the binding of the GH domain to the GH receptor will lead to endocytosis and lysosomal degradation in target cells, binding of the Tf domain to the Tf receptor may recycle the fusion protein and prolong its plasma half-life. To further confirm the effects of receptor binding on plasma half-life, G-CSF-Tf bifunctional fusion proteins with the same three linkers as GH-Tf were evaluated. While the 3 fusion proteins showed a similar G-CSF receptor binding affinity, the G-CSF-Tf fusion protein with the higher Tf receptor binding affinity exhibited longer plasma half-life. The linker insertion further demonstrated the involvement of Tf in recycling and prolonging plasma half-life. Based on our results, a model was developed to summarize the factors in determining the PK of bifunctional fusion proteins. Our findings are useful for predicting the plasma half-lives, as well as for improving the pharmacokinetic profiles of therapeutic bifunctional fusion proteins by applying linker technology.

Develop an efficient inoculation technique for Fusarium solani isolate "TJP-2178-10" pathogeny assessment in Phalaenopsis orchids.

BACKGROUND: Phalaenopsis is one of the important ornamental plants worldwide. It plays the most significant role in flower exportation in Taiwan. However, the yellow leaf disease caused by Fusarium spp. has reduced the orchid flower yield 10-50 % yearly. Varieties resistant to yellow leaf disease associated with Fusarium is urgently needed for orchid growers and breeders, and is the ultimate solution for the long-term goal. To achieve this, phenotyping is the first step and the most necessary information for further studies, such as resistance gene identification, quantitative trait loci identification, and genome-wide association study. RESULTS: The inoculation of Fusarium was performed in either abbreviated stem or detached leaf, and the pros and cons were compared. The former is the general method of phenotyping for estimating the tolerance to yellow leaf disease of Phalaenopsis, but it is time-consuming and spacy, and thus not suitable for the assessment of large numbers of samples. In contrast, the latter not only showed a similar trend of disease severity with time reduced to only one fourth of the former one but also less space needed. CONCLUSIONS: This solution allows a better phenotyping approach for the fast detection of yellow leaf disease associated with Fusarium in a large number of Phalaenopsis samples.