A three-step screening procedure to identify the mode of phloem loading in intact leaves

A three-step screening method was developed to identify the mode of phloem loading in intact leaves. Phloem loading of ¹⁴CO₂-derived photosynthate was challenged by p-chloromercuribenzenesulfonic acid (PCMBS) in leaves of dicotyledons with either a symplasmic (type 1, with intermediary cells as companion cells) or apoplasmic (type 2b, with transfer cells as companion cells) minor-vein configuration. Firstly, photosynthate export as the result of phloem loading was measured by collection of phloem exudate from the petiole. The PCMBS had virtually no effect on photosynthate export in representatives of type-1 families (Lamiaceae, Lythraceae, Onagraceae, Saxifragaceae). In contrast, photosynthate export was strongly reduced by PCMBS in representatives of type-2b families (Asteraceae, Balsaminaceae, Dipsacaceae, Linaceae, Tropaeolaceae, Valerianaceae) and type-2b members of polytypical families (Fabaceae, Scrophulariaceae). Secondly, densitometric measurements of leaf autoradiographs demonstrated that the contrast between the mesophyll and the lower-order veins was hardly affected by PCMBS treatment in type-1 species, whereas PCMBS strongly reduced the contrast in type-2b species. Thirdly, separate ¹⁴C-radioassays of vein and mesophyll tissues confirmed this observation. The three-step procedure thus revealed a strong and consistent reduction of phloem loading by PCMBS in type-2b species which was absent in type-1 species. In conclusion, phloem loading in type-2b species occurs via the apoplast and type-1 species execute an alternative — most likely symplasmic — mode of phloem loading.Abbreviations PCMBS p-chloromercuribenzenesulfonic acid - SE/CC-complex sieve element/companion cell complex We gratefully acknowledge the expert help of Dr. Maarten Terlou, Department of Image Processing and Design, University of Utrecht, in carrying out the densitometric measurements.     

Probing binding mechanism of interleukin-6 and olokizumab: in silico design of potential lead antibodies for autoimmune and inflammatory diseases

Computer-aided antibody engineering has been successful in the design of new biologics for disease diagnosis and therapeutic interventions. Interleukin-6 (IL-6), a well-recognized drug target for various autoimmune and inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, and psoriasis, was investigated in silico to design potential lead antibodies. Here, crystal structure of IL-6 along with monoclonal antibody olokizumab was explored to predict antigen–antibody (Ag???Ab)-interacting residues using DiscoTope, Paratome, and PyMOL. Tyr56, Tyr103 in heavy chain and Gly30, Ile31 in light chain of olokizumab were mutated with residues Ser, Thr, Tyr, Trp, and Phe. A set of 899 mutant macromolecules were designed, and binding affinity of these macromolecules to IL-6 was evaluated through Ag???Ab docking (ZDOCK, ClusPro, and Rosetta server), binding free-energy calculations using Molecular Mechanics/Poisson Boltzman Surface Area (MM/PBSA) method, and interaction energy estimation. In comparison to olokizumab, eight newly designed theoretical antibodies demonstrated better result in all assessments. Therefore, these newly designed macromolecules were proposed as potential lead antibodies to serve as a therapeutics option for IL-6-mediated diseases.     

Actin pegs and ultrastructure of presumed sensory receptors of Beroë (Ctenophora)

Summary We have investigated the actin content and ultrastructure of two kinds of presumed sensory projections on the lip epidermis of beroid ctenophores. Transmission electron microscopy showed that conical pegs contain a large bundle of densely packed, parallel microfilaments. Rhodamine-phalloidin brightly stained the pegs, confirming that they contain filamentous actin. Epidermal cells with actin pegs also bear a single long cilium with an onion-root structure, previously described as arising from a different type of cell. The actin peg and onion-root cilium project side-by-side, defining a polarized axis of the cell which is shared by neighboring cells. The onion-root body is surrounded by a flattened membranes sac which lies immediately below the plasma membrane. The perimeter of the membrane sac is encircled by aggregates of dense material. An extra layer of dense material is found along the side of the membrane sac facing the peg; this material often makes direct contact with the adjacent actin filament bundle. Cells with actin pegs and onion-root cilia synapse onto adjacent neurites and secretory gland cells, indicating that one or both types of projections are sensory elements. Since the feeding responses of beroids are reported to depend on chemical and tactile stimuli to the lips, the cells bearing pegs and cilia may function as both mechanoreceptors and chemoreceptors, that is, as double sensory receptors.     

Enhancing plasmid transformation efficiency and enabling CRISPR-Cas9/Cpf1-based genome editing in Clostridium tyrobutyricum

Clostridium tyrobutyricum ATCC 25755 is known as a natural hyper-butyrate producer with great potentials as an excellent platform to be engineered for valuable biochemical production from renewable resources. However, limited transformation efficiency and the lack of genetic manipulation tools have hampered the broader applications of this micro-organism. In this study, the effects of Type I restriction-modification system and native plasmid on conjugation efficiency of C. tyrobutyricum were investigated through gene deletion. The deletion of Type I restriction endonuclease resulted in a 3.7-fold increase in conjugation efficiency, while the additional elimination of the native plasmid further enhanced conjugation efficiency to 6.05 ± 0.75 × 10³ CFU/ml-donor, which was 15.3-fold higher than the wild-type strain. Fermentation results indicated that the deletion of those two genetic elements did not significantly influence the end-products production in the resultant mutant ΔRMIΔNP. Thanks to the increased conjugation efficiency, the CRISPR-Cas9/Cpf1 systems, which previously could not be implemented in C. tyrobutyricum, were successfully employed for genome editing in ΔRMIΔNP with an efficiency of 12.5–25%. Altogether, approaches we developed herein offer valuable guidance for establishing efficient DNA transformation methods in nonmodel micro-organisms. The ΔRMIΔNP mutant can serve as a great chassis to be engineered for diverse valuable biofuel and biochemical production.     

Acute effects of Amomum villosum Lour. fruit extract on postprandial glycemia and insulin secretion: A single-blind,placebo-controlled,crossover study in healthy subjects

BackgroundAmomum villosum Lour., (Zingiberaceae) an herbaceous plant in the ginger family, has been used to treat various diseases. In a single-blind, randomized, crossover study, we assessed the postprandial blood insulin and blood glucose responses in healthy subjects (n = 40) after the Amomum villosum water extract (AVE) (5 g/person) or a placebo (5 g/person) consumption.MethodsDuring each treatment course, the healthy subject consumed a regular late afternoon meal, followed by fasting for 12 h, and arrived at the clinical study center the next morning. Blood insulin and blood glucose levels were assessed at 0, 30, 60, 90, and 120 min after AVE consumption. Between each treatment, the subjects accomplished one week of a washout period.ResultsThe AVE intake demonstrated a significant (67.26%) decline in postprandial blood glucose AUC_{0–120 min} (incremental area under the curve from 0 to 120 min) versus the placebo (P = 0.011). Furthermore, AVE reduced postprandial blood insulin AUC_{0–120 min} by 59.95% compared to the placebo group (P < 0.003), supporting the blood glucose results.ConclusionThis study revealed that AVE consumption significantly reduced postprandial insulin and glucose levels in healthy individuals, due in part to inhibition of α-glucosidase, and glucose transport.     

Evaluation of cetuximab as a candidate for targeted α-particle radiation therapy of HER1-positive disseminated intraperitoneal disease

Although the epidermal growth factor receptor (EGFR), also known as HER1, has been studied for over a decade, it continues to be a molecule of great interest and focus of investigators for development of targeted therapies. The marketed monoclonal antibody cetuximab binds to HER1, and thus might serve as the basis for creation of imaging or therapies that target this receptor. The potential of cetuximab as a vehicle for the delivery of α-particle radiation was investigated in an intraperitoneal tumor mouse model. The effective working dose of 10 μCi of ²¹²Pb-cetuximab was determined from a dose (10–50 μCi) escalation study. Toxicity, as indicated by the lack of animal weight loss, was not evident at the 10 μCi dose of ²¹²Pb-cetuximab. A subsequent study demonstrated ²¹²Pb-cetuximab had a therapeutic efficacy similar to that of ²¹²Pb-trastuzumab (p = 0.588). Gemcitabine given 24 h prior to ²¹²Pb-cetuximab increased the median survival from 174 d to 283 d, but carboplatin suppressed the effectiveness of ²¹²Pb-cetuximab. Notably, concurrent treatment of tumor-bearing mice with ²¹²Pb-labeled cetuximab and trastuzumab provided therapeutic benefit that was greater than either antibody alone. In conclusion, cetuximab proved to be an effective vehicle for targeting HER1-expressing tumors with α-radiation for the treatment of disseminated intraperitoneal disease. These studies provide further evidence that the multimodality therapy regimens may have greater efficacy and benefit in the treatment of cancer patients.     

Furazan and furoxan sulfonamides are strong α-carbonic anhydrase inhibitors and potential antiglaucoma agents

A series of furazan and furoxan sulfonamides were prepared and studied for their ability to inhibit human carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, hCA II, hCA IX, and hCA XII. The simple methyl substituted products 3–5 were potent inhibitors. Differing structural modifications of these leads had differing effects on potency and selectivity. In particular, products in which the sulfonamide group is separated from the hetero ring by a phenylene bridge retained high potency only on the hCA XII isoform. The sulfonamides 3–5 exerted intraocular pressure (IOP) lowering effects in vivo in hypertensive rabbits more efficiently than dorzolamide. Some other products (39–42), although less effective in vitro hCA II/XII inhibitors, also effectively lowered IOP in two different animal models of glaucoma.