Utilizing the allyl-terminated copolymer methoxy(poly(ethylene glycol))-block-poly(jasmine lactone) in the development of amorphous solid dispersions: A comparative study of functionalized and non-functionalized polymer.

Molecular interactions are crucial to stabilize amorphous drugs in amorphous solid dispersions (ASDs). Most polymers, however, have only a limited ability to form strong molecular interactions with drugs. Polymers tailored to fit the physicochemical properties of the drug molecule to be incorporated, for instance by allowing the incorporation of specific functional groups, would be highly sought-for in this regard. For this purpose, the novel allyl-terminated polymer methoxy(polyethylene glycol)-block-poly(jasmine lactone) (mPEG-b-PJL) has been synthesized and functionalized to potentially enhance specific drug-polymer interactions. This study investigated the use of mPEG-b-PJL in ASDs, using carvedilol (CAR), a weakly basic model drug. The findings revealed that the acidic functionalized form of the polymer (mPEG-b-PJL-COOH) indeed established stronger molecular interactions with CAR compared to its non-functionalized counterpart mPEG-b-PJL. Evaluations on polymer effectiveness in forming ASDs demonstrated that mPEG-b-PJL-COOH outperformed its non-functionalized counterpart in miscibility, drug loading ability, and stability, inferred from reduced molecular mobility. However, dissolution tests indicated that ASDs with mPEG-b-PJL-COOH did not significantly improve the dissolution behaviour compared to amorphous CAR alone, despite potential solubility enhancement through micelle formation. Overall, this study confirms the potential of functionalized polymers in ASD formulations, while the challenge of improving dissolution performance in these ASDs remains an area of further development.

A cyclic peptide-based PROTAC induces intracellular degradation of palmitoyltransferase and potently decreases PD-L1 expression in human cervical cancer cells.

Introduction: Our previous research has found that degradation of palmitoyltransferase in tumor cells using a linear peptide PROTAC leads to a significant decrease in PD-L1 expression in tumors. However, this degradation is not a sustained and efficient process. Therefore, we designed a cyclic peptide PROTAC to achieve this efficient anti-PD-L1 effect. Methods: We designed and synthesized an improvement in linear peptide PROTAC targeting palmitoyltransferase DHHC3, and used disulfide bonds to stabilize the continuous N- and C-termini of the peptides to maintain their structure. Cellular and molecular biology techniques were used to test the effect of this cyclic peptide on PD-L1. Results: In human cervical cancer cells, our cyclic peptide PROTAC can significantly downregulate palmitoyl transferase DHHC3 and PD-L1 expressions. This targeted degradation effect is enhanced with increasing doses and treatment duration, with a DC50 value much lower than that of linear peptides. Additionally, flow cytometry analysis of fluorescence intensity shows an increase in the amount of cyclic peptide entering the cell membrane with prolonged treatment time and higher concentrations. The Cellular Thermal Shift Assay (CETSA) method used in this study indicates effective binding between our novel cyclic peptide and DHHC3 protein, leading to a change in the thermal stability of the latter. The degradation of PD-L1 can be effectively blocked by the proteasome inhibitor MG132. Results from clone formation experiments illustrate that our cyclic peptide can enhance the proliferative inhibition effect of cisplatin on the C33A cell line. Furthermore, in the T cell-C33A co-culture system, cyclic peptides target the degradation of PD-L1, thereby blocking the interaction between PD-L1 and PD-1, and promoting the secretion of IFN-γ and TNF-α in the co-culture system supernatant. Conclusion: Our results demonstrate that a disulfide-bridged cyclic peptide PROTAC targeting palmitoyltransferase can provide a stable and improved anti-PD-L1 activity in human tumor cells.

Biological function research of Fusarium oxysporum f. sp. cubense inducible banana long noncoding RNA Malnc2310 in Arabidopsis.

Long noncoding RNAs (lncRNAs) participate in plant biological processes under biotic and abiotic stresses. However, little is known about the function and regulation mechanism of lncRNAs related to the pathogen at a molecular level. A banana lncRNA, Malnc2310, is a Fusarium oxysporum f. sp. cubense inducible lncRNA in roots. In this study, we demonstrate the nuclear localization of Malnc2310 by fluorescence in situ hybridization and it can bind to several proteins that are related to flavonoid pathway, pathogen response and programmed cell death. Overexpression of Malnc2310 increases susceptibility to Fusarium crude extract (Fu), salinity, and cold in transgenic Arabidopsis. In addition, Malnc2310 transgenic Arabidopsis accumulated more anthocyanins under Fusarium crude extract and cold treatments that are related to upregulation of these genes involved in anthocyanin biosynthesis. Based on our findings, we propose that Malnc2310 may participate in flavonoid metabolism in plants under stress. Furthermore, phenylalanine ammonia lyase (PAL) protein expression was enhanced in Malnc2310 overexpressed transgenic Arabidopsis, and Malnc2310 may participate in PAL regulation by binding to it. This study provides new insights into the role of Malnc2310 in mediating plant stress adaptation.

Potential Anti-Alzheimer Properties of Mogrosides in Vitamin B12-Deficient Caenorhabditis elegans.

Vitamin B12 deficiency can lead to oxidative stress, which is known to be involved in neurodegenerative diseases such as Alzheimer's disease (AD). Mogrosides are plant-derived triterpene glycosides that exhibit anti-inflammatory and antioxidant activity in animal cell lines and mouse models. Since amyloid-ß toxicity is known to cause oxidative stress and damage to brain cells, we hypothesized that mogrosides may have a protective effect against AD. In this study, we investigated the potential anti-AD effect of mogrosides in vitamin B12-deficient wild-type N2 and in transgenic CL2355 Caenorhabditis elegans expressing amyloid-ß peptide. Our data indicated that mogrosides have a beneficial effect on the lifespan and egg-laying rate of N2 and vitamin B12-deficient N2 worms. Additionally, the results revealed that mogrosides can effectively delay the paralysis of CL2355 worms as determined by serotonin sensitivity assay. Our analysis showed that mogrosides increase the expression of oxidative protective genes in N2 worms fed with vitamin B12-deficient OP50 bacterium. We conclude that mogrosides may exert preventative rather than curative effects that counteract the detrimental vitamin B12-deficient environment in N2 and CL2355 C. elegans by modulating oxidation-related gene expression.

Destabilization of Indomethacin-Paracetamol Co-Amorphous Systems by Mechanical Stress.

Using co-amorphous systems (CAMS) has shown promise in addressing the challenges associated with poorly water-soluble drugs. Quench-cooling is a commonly used CAMS preparation method, often followed by grinding or milling to achieve a fine powder that is suitable for subsequent characterization or further down-stream manufacturing. However, the impact of mechanical stress applied to CAMS has received little attention. In this study, the influence of mechanical stress on indomethacin-paracetamol CAMS was investigated. The investigation involved thermal analysis and solid-state characterization across various CAMS mixing ratios and levels of mechanical stress. The study revealed a negative effect of mechanical stress on stability, particularly on the excess components in CAMS. Higher levels of mechanical stress were observed to induce phase separation or recrystallization. Notably, samples at the optimal mixing ratio demonstrated greater resistance to the destabilization caused by mechanical stress. These results showed the significance of careful consideration of processing methods during formulation and the significance of optimizing mixing ratios in CAMS.

Ethylene insensitive 2 (EIN2) as a potential target gene to enhance Fusarium head blight disease resistance.

Fusarium head blight (FHB) caused by Fusarium graminearum (Fg) severely affects cereal crops, especially wheat and barley. FHB results in significant yield loss, reduces grain quality and contaminates grains with mycotoxin. The development of FHB-resistant cereal cultivars can be expedited through CRISPR gene editing. The Arabidopsis ethylene insensitive 2 (AtEIN2) plays a key role in ethylene signaling pathway and is critical for monitoring plant growth and defense responses. RNAi down-regulation of the wheat homolog TaEIN2 has been shown to enhance wheat FHB resistance. Here we generated site-specific mutations in AtEIN2 by CRISPR-editing. Detached inflorescence infection assays revealed that AtEIN2 knock-out (KO) mutants displayed enhanced Fg resistance and substantially reduced Fg spore production in planta. Gene expression profiling of defense genes revealed that impairment of AtEIN2 resulted in down-regulation of the ethylene signaling pathway while the salicylic acid signaling pathway was unaffected. Complementation of AtEIN2-KO plants with a barley orthologue, HvEIN2, restored Fg susceptibility, indicating that HvEIN2 is functionally equivalent to its Arabidopsis counterpart and, hence, may have a similar role in conditioning barley Fg susceptibility. These results provide insight into the defense role of EIN2 and a molecular and functional foundation for manipulating HvEIN2 to enhance FHB resistance in barley.

Light-harvesting complex gene regulation by a MYB-family transcription factor in the marine diatom, Phaeodactylum tricornutum.

Unicellular photoautotrophs adapt to variations in light intensity by changing the abundance of light harvest pigment-protein complexes (LHCs) on time scales of hours to days. This process requires a feedback signal between the plastid (where light intensity is sensed) to the nucleus (where the genes for LHCs are encoded). The signals must include heretofore unidentified transcription factors that modify the expression level of the LHCs. Analysis of the nuclear genome of the model diatom Phaeodactylum tricornutum revealed that all the lhc genes have potential binding sites for transcription factors belonging to the MYB-family proteins. Functional studies involving antisense RNA interference of a hypothetical protein with a MYB DNA-binding domain were performed. The resultant strains with altered photosynthetic and physiological characteristics lost their ability to acclimate to changes in irradiance; i.e., cellular chlorophyll content became independent of growth irradiance. Our results strongly suggest that the inter-organellar signaling cascade was disrupted, and the cell could no longer communicate the environmental signal from the plastid to the nucleus. Here, we identify, for the first time, an LHC Regulating Myb (LRM) transcription factor, which we propose is involved in lhc gene regulation and photoacclimation mechanisms in response to changes in light intensity.

Identification of two Eleusine indica (goosegrass) biotypes of cool-season turfgrass resistant to dithiopyr.

BACKGROUND: Turfgrass managers reported poor Eleusine indica control following applications of the mitosis-inhibiting herbicide dithiopyr in cool-season turfgrass. Field, glasshouse, and laboratory experiments were conducted to understand the response of these biotypes to dithiopyr and prodiamine. RESULTS: In field experiments at two locations with putative dithiopyr-resistant E. indica, preemergence applications of dithiopyr provided no E. indica control. Single applications of the protoporphyrinogen oxidase (PPO)-inhibitor, oxadiazon, provided > 85% control at these locations. When subjected to agar-based bioassays, root growth of putative resistant biotypes planted with 0.01 mmol L-1 dithiopyr was slightly reduced (< 25%) whereas roots were completely inhibited in the susceptible biotype. Glasshouse whole plant rate-response experiments found that the cytochrome P450 inhibitor, piperonyl butoxide (PBO), did not increase the sensitivity of these putative resistant biotypes to dithiopyr. Sequencing of α-tubulin 1 (TUA1) revealed a Leu-136-Phe substitution in both dithiopyr-resistant populations. CONCLUSION: Eleusine indica biotypes with resistance to dithiopyr are present in cool-season turfgrass systems in the United States. Resistance is possibly related to a single nucleotide polymorphism (SNP) of an α-tubulin gene. If turfgrass managers suspect resistance to dithiopyr, oxadiazon can still be an effective alternative for preemergence control. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

CRISPR-Editing of Sweet Basil (Ocimum basilicum L.) Homoserine Kinase Gene for Improved Downy Mildew Disease Resistance.

Sweet basil (Ocimum basilicum L.) downy mildew disease (DM) caused by Peronospora belbahrii is a worldwide threat to the basil industry due to the lack of natural genetic resistance in sweet basil germplasm collections. In this study, we used CRISPR-gene editing to modify the sweet basil DM susceptibility gene homoserine kinase (ObHSK). Gene-edited plants challenged with P. belbahrii displayed a significantly reduced susceptibility to DM, based on phenotypic disease indices and on in planta pathogen load. These results suggest that ObHSK plays a role in conditioning DM susceptibility, similar to that observed for the AtHSK gene in Arabidopsis. These results demonstrate the utility of CRISPR-gene editing in enhancing DM resistance and contributing to sweet basil breeding programs.

Combined Effect of the Preparation Method and Compression on the Physical Stability and Dissolution Behavior of Melt-Quenched Amorphous Celecoxib.

In an earlier investigation, amorphous celecoxib was shown to be sensitive to compression-induced destabilization. This was established by evaluating the physical stability of uncompressed/compressed phases in the supercooled state (Be̅rzins . Mol. Pharmaceutics, 2019, 16(8), 3678-3686). In this study, we investigated the ramifications of compression-induced destabilization in the glassy state as well as the impact of compression on the dissolution behavior. Slow and fast melt-quenched celecoxib disks were compressed with a range of compression pressures (125-500 MPa) and dwell times (0-60 s). These were then monitored for crystallization using low-frequency Raman spectroscopy when kept under dry (∼20 °C; <5% RH) and humid (∼20 °C; 97% RH) storage conditions. Faster crystallization was observed from the samples, which were compressed using more severe compression parameters. Furthermore, crystallization was also affected by the cooling rate used to form the amorphous phases; slow melt-quenched samples exhibited higher sensitivity to compression-induced destabilization. The behavior of the melt-quench disks, subjected to different compression conditions, was continuously monitored during dissolution using low-frequency Raman and UV/vis for the solid-state form and dissolution properties, respectively. Surprisingly the compressed samples exhibited higher apparent dissolution (i.e., higher area under the dissolution curve and initial celecoxib concentration in solution) than the uncompressed samples; however, this is attributed to biaxial fracturing throughout the compressed compacts yielding a greater effective surface area. Differences between the slow and fast melt quenched samples showed some trends similar to those observed for their storage stability.

A Multivariate Approach for the Determination of the Optimal Mixing Ratio of the Non-Strong Interacting Co-Amorphous System Carvedilol-Tryptophan.

Converting crystalline compounds into co-amorphous systems is an effective way to improve the solubility of poorly water-soluble drugs. It is, however, of critical importance for the physical stability of co-amorphous systems to find the optimal mixing ratio of the drug with the co-former. In this study, a novel approach for this challenge is presented, exemplified with the co-amorphous system carvedilol-tryptophan (CAR-TRP). Following X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) of the ball-milled samples to confirm their amorphous form, Fourier-transform infrared spectroscopy (FTIR) and principal component analysis (PCA) were applied to investigate intermolecular interactions. A clear deviation from a purely additive spectrum of CAR and TRP was visualized in the PCA score plot, with a maximum at around 30% drug (mol/mol). This deviation was attributed to hydrogen bonds of CAR with TRP ether groups. The sample containing 30% drug (mol/mol) was also the most stable sample during a stability test. Using the combination of FTIR with PCA is an effective approach to investigate the optimal mixing ratio of non-strong interacting co-amorphous systems.

Parafoveal retinal massage combined with autologous blood cover in the management of giant, persistent or recurrent macular holes.

AIM: To assess the efficacy and safety of parafoveal retinal massage combined with autologous whole blood cover in the treatment of refractory macular holes (MHs) and present the surgical procedure. METHODS: Patients with giant (minimum diameter ≥800 µm), recurrent or persistent MHs who underwent PPV combined with parafoveal retinal massage and autologous whole blood cover using C3F8 as tamponade agent from February 2018 to May 2019 were enrolled in this retrospective study. After surgery, all patients were informed to maintain a prone position for at least 7d. Preoperative and postoperative best-corrected visual acuities (BCVAs) were compared and MH closure rate was measured as the main outcome. RESULTS: A total of 13 MH patients consisted of 6 giant MHs, 4 persistent holes and 3 recurrent holes (5 men and 8 women; average age was 56.40±11.72y) were enrolled in this study. MH closure was achieved in 11 eyes by this modified surgical technique while 2 eyes failed. Revitrectomy with autologous neurosensory retinal patch transplantations was applied for those 2 patients and then both holes were closed. No intraoperative complications were observed. BCVA improved from 1.73 logMAR to 0.74 logMAR at 6mo postoperation. There was significant difference in BCVA before versus after the surgery (P<0.05). There were no adverse events occurred during the follow-up period. CONCLUSION: With easier surgical procedure, parafoveal retinal massage combined with autologous whole blood cover is an effective addition to the surgical options for the management of refractory MHs.

C1q/TNF-related Protein 9 Inhibits High Glucose-Induced Oxidative Stress and Apoptosis in Retinal Pigment Epithelial Cells Through the Activation of AMPK/Nrf2 Signaling Pathway.

Diabetic retinopathy (DR) is one of the common complications of diabetes mellitus. C1q/TNF-related protein 9 (CTRP9) has been demonstrated to be associated with the progression of diabetes and relative complications. However, its role in DR and underlying action of mechanism are not yet well understood. In the present study, human retinal pigment epithelial ARPE-19 cells were cultured under high concentration of glucose to simulate hyperglycemia condition in vitro. Our results showed that the expression of CTRP9 was significantly decreased in high glucose (HG)-stimulated ARPE-19 cells. CTRP9 overexpression improved HG-caused reduction in cell viability of ARPE-19 cells. CTRP9 overexpression significantly attenuated HG-induced oxidative stress, as proved by decreased levels of reactive oxygen species and malondialdehyde, and increased superoxide dismutase activity. Moreover, CTRP9 also prevented apoptosis in ARPE-19 cells in response to HG stimulation with decreased caspse-3 activity and bax expression, as well as increased bcl-2 expression. In contrast, knockdown of CTRP9 aggravated HG-induced oxidative stress and apoptosis. Furthermore, CTRP9 significantly induced the activation of AMPK/Nrf2 pathway in HG-induced ARPE-19 cells. Notably, inhibiting AMPK or Nrf2 blocked the protective effect of CTRP9 on ARPE-19 cells exposed to HG stimulation. Taken together, our findings suggested a protective effect of CTRP9 on HG-induced ARPE-19 cells and a putative mechanism involving the activation of AMPK/Nrf2 signaling pathway.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Ixeris denticulata in China.

Ixeris denticulata (Houtt.) Stebb is an annual herbaceous plant in the family of Asteraceae, which is native to Europe or central Asia. This plant is widely distributed in China and is commonly used for edible and medicinal purposes. In February 2019, typical symptoms of powdery mildew were observed on 70% of I. denticulata plants on the campus of Hainan University (20° 3' 25″ N; 110° 19' 4″ E) in Haikou, Hainan Province, China. White, superficial mycelia and conidia covered the leaf surfaces of affected plants, resulting in leaf curling, discoloration and defoliation. Hyphal appressoria were nipple-shaped, and solitary. Conidiophores were straight, cylindrical, 109 to 259 × 9 to 16 µm (n = 50), and produced 3 to 5 immature conidia in chains with a crenate outline. Foot cells were cylindrical, straight or sometimes constricted at the basal septum, 30 to 62 µm long (n = 100). Conidia were ellipsoid-ovoid to doliiform, 23 to 33 × 15 to 23 µm (n = 100) with a length/width ratio of 1.1 to 1.9, with well developed fibrosin bodies, and produced germ tubes from the lateral position. Based on these morphological characteristics, this pathogen was provisionally identified as Podosphaera xanthii (Braun and Cook 2012). The teleomorph was not detected. A specimen was deposited in the Hainan University Plant Pathology Herbarium as HNID-18. In order to confirm the identification, genomic DNA was extracted from mycelium and conidia collected from a single leaf using a fungal DNA kit (Omega Bio-Tek, USA). The rDNA internal transcribed spacer (ITS) region and 28S rDNA were amplified with the primer pairs ITS1 and ITS4 (White et al. 1990) and sequenced directly. The resulting 577-bp sequence was deposited in GenBank (Accession No. MT739423). The GenBank BLAST analysis of the ITS sequence showed 100% similarity with P. xanthii on Bidens sp. from Thailand (LC270780), as well as with P. xanthii from Eclipta prostrata (MT260063) and Cyanthillium cinereum (MN203658) from China. Additionally, the 28S rDNA region was amplified using the primer pairs NL1 and NL4 (O'Donnell 1993, Accession No. MT739424). The amplicon was sequenced in both directions and shared 100% similarity with P. xanthii (MK357436, LC371333 and MH137264). To fulfill Koch's postulates, five healthy potted plants of I. denticulata were inoculated by gently pressing a powdery mildew-infected leaf onto 15 young leaves. Five non-inoculated plants served as controls. All plants were maintained in a greenhouse at 24 to 30°C, 70% relative humidity and a 16-h photoperiod. After 7 days, inoculated leaves showed signs and symptoms of powdery mildew whereas no signs or symptoms were observed on the control plants. The fungus observed on the inoculated plants was identical morphologically to that on the originally infected leaves. Powdery mildew of I. chinensis caused by Golovinomyces sonchicola has been reported previously from Korea (Choi et al. 2014). Recently, P. xanthii was also shown to infect Ixeridium dentatum in Korea (Lee and Nguyen 2018). To our knowledge, this is the first record of P. xanthii infecting I. denticulata in China. We are concerned that the pathogen will cause severe damage and affect the yield and quality of the host, and even pose a threat to I. denticulata in the future.

Metformin Corrects Abnormal Circadian Rhythm and Kir4.1 Channels in Diabetes.

Purpose: Diabetic retinopathy (DR) is a leading cause of visual impairment. Müller cells in DR are dysfunctional due to downregulation of the inwardly rectifying potassium channel Kir4.1. Metformin, a commonly used oral antidiabetic drug, is known to elicit its action through 5' adenosine monophosphate-activated protein kinase (AMPK), a cellular metabolic regulator; however, its effect on Kir4.1 channels is unknown. For this study, we hypothesized that metformin treatment would correct circadian rhythm disruption and Kir4.1 channel dysfunction in db/db mice. Methods: Metformin was given orally to db/db mice. Wheel-running activity, retinal levels of Kir4.1, and AMPK phosphorylation were determined at study termination. In parallel, rat retinal Müller cell line (rMC-1) cells were treated using metformin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to assess the effect of AMPK activation on the Kir4.1 channel. Results: The wheel-running activity of the db/db mice was improved following the metformin treatment. The Kir4.1 level in Müller cells was corrected after metformin treatment. Metformin treatment led to an upregulation of clock regulatory genes such as melanopsin (Opn4) and aralkylamine N-acetyltransferase (Aanat). In rMC-1 cells, AMPK activation via AICAR and metformin resulted in increased Kir4.1 and intermediate core clock component Bmal-1 protein expression. The silencing of Prkaa1 (gene for AMPKα1) led to decreased Kir4.1 and Bmal-1 protein expression. Conclusions: Our findings demonstrate that metformin corrects abnormal circadian rhythm and Kir4.1 channels in db/db mouse a model of type 2 diabetes. Metformin could represent a critical pharmacological agent for preventing Müller cell dysfunction observed in human DR.

Validation of barley 2OGO gene as a functional orthologue of Arabidopsis DMR6 gene in Fusarium head blight susceptibility.

Fusarium head blight (FHB) caused by Fusarium graminearum (Fg) is a devastating disease of crops, especially wheat and barley, resulting in significant yield loss and reduced grain quality. Fg infection leads to the production of mycotoxins, whose consumption is toxic to humans and livestock. The Arabidopsis DMR6 gene encodes a putative 2-oxoglutarate Fe(II)-dependent oxygenase (2OGO) and has been identified as a susceptibility factor to downy mildew. We generated site-specific mutations in Arabidopsis At2OGO by CRISPR/Cas9 gene editing. The resulting At2OGO knock-out (KO) mutants display enhanced resistance to Fg in a detached inflorescence infection assay. Expression profiling of defense genes revealed that impairment of At2OGO function resulted in the upregulation of defense genes that are regulated by salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) pathways. Complementation of the At2OGO-KO lines with a barley (cv. Conlon) orthologue, Hv2OGO, restored susceptibility to Fg. This result indicates that the Hv2OGO gene is functionally equivalent to its Arabidopsis counterpart and, hence, may have a similar role in conditioning susceptibility to FHB in barley. These results provide a molecular basis for proposing 2OGO as a plant immunity suppressor in Arabidopsis and potentially in barley plants and establish a rationale and strategy for enhancing FHB resistance in barley.

Draft Genome Sequence of Streptomyces aureoverticillatus HN6, a Strain Antagonistic against Fusarium oxysporum f. sp. cubense Race 4.

Streptomyces aureoverticillatus HN6 was isolated in Hainan, China. It is highly inhibitory to Fusarium oxysporum f. sp. cubense race 4 (FOC4), which causes banana Fusarium wilt. The HN6 genome was fully sequenced and assembled. Bionformatic analysis shows that the HN6 genome contains at least 208 genes involved in antibiotic biosynthesis.

The MAPK substrate MASS proteins regulate stomatal development in Arabidopsis.

Stomata are specialized pores in the epidermis of the aerial parts of a plant, where stomatal guard cells close and open to regulate gas exchange with the atmosphere and restrict excessive water vapor from the plant. The production and patterning of the stomatal lineage cells in higher plants are influenced by the activities of the widely-used mitogen-activated protein kinase (MAPK) signaling components. The phenotype caused by the loss-of-function mutations suggested pivotal roles of the canonical MAPK pathway in the suppression of stomatal formation and regulation of stomatal patterning in Arabidopsis, whilst the cell type-specific manipulation of individual MAPK components revealed the existence of a positive impact on stomatal production. Among a large number of putative MAPK substrates in plants, the nuclear transcription factors SPEECHLESS (SPCH) and SCREAM (SCRM) are targets of MAPK 3 and 6 (MPK3/6) in the inhibition of stomatal formation. The polarity protein BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) is phosphorylated by MPK3/6 for localization and function in driving divisional asymmetries. Here, by functionally characterizing three MAPK SUBSTRATES IN THE STOMATAL LINEAGE (MASS) proteins, we establish that they are plasma membrane-associated, positive regulators of stomatal production. MPK6 can phosphorylate the MASS proteins in vitro and mutating the putative substrate sites interferes the subcellular partition and function of MASS in planta. Our fine-scale domain analyses identify critical subdomains of MASS2 required for specific subcellular localization and biological function, respectively. Furthermore, our data indicate that the MASS proteins may directly interact with the MAPKK Kinase YODA (YDA) at the plasma membrane. Thus, the deeply conserved MASS proteins are tightly connected with MAPK signaling in Arabidopsis to fine-tune stomatal production and patterning, providing a functional divergence of the YDA-MPK3/6 cascade in the regulation of plant developmental processes.

Lily steroidal glycoalkaloid promotes early inflammatory resolution in wounded human fibroblasts.

ETHNOPHARMACOLOGICAL RELEVANCE: The bulbs and flowers of plants from the Lilium genus have historically been used in Asian and Greco-Roman medicine to treat burns and promote skin healing. AIM OF THE STUDY: To evaluate a steroidal glycoalkaloid isolated from Easter lily bulbs for its potential wound healing promoting properties. MATERIALS AND METHODS: A lily-derived steroidal glycoalkaloid (LSGA), (22R, 25R)-spirosol-5-en-3ß-yl O-α-L-rhamnopyranosyl-(1→2)-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranoside, was isolated from Easter lily bulbs, and its structure was confirmed by LC-MS and NMR spectrometry. LSGA effects on wound scratch closure were evaluated in a primary human dermal fibroblast cell culture, and the changes in gene expression profiles were quantitated using an 84 wound-related gene qPCR microarray. RESULTS: LSGA promoted migration of dermal fibroblasts into the wounded area. The treatment was associated with a rapid upregulation of early inflammatory (CD40LG, CXCL11, IFNG, IL10, IL2 and IL4), cell growth (CSF3 and TNF) and remodeling (CTSG, F13A1, FGA, MMP and PLG) genes both in the wounded and unwounded cells treated with LSGA. A selective decrease in gene expression profiles associated with inflammatory (CXCL2 and CCL7) and remodeling (MMP7 and PLAT) phases was observed in wounded cells treated with LSGA, in contrast to the wounded cells (control). CONCLUSION: This study demonstrates that a glycoalkaloid present in lilies promoted fibroblast migration in vitro and affected inflammatory, remodeling and growth factor gene expression. The decreases in expression of key genes may impact the wound healing process, possibly contributing to an earlier end of the inflammatory response and shortening the early phases of model tissue reconstitution. The results of this preliminary investigation may provide a basis for the historical use of lily bulbs to promote dermal healing after injury.

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription.

A transcription factor (TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two σ factors, namely σ66 and σ28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.