Molecular Analysis of Drought Tolerance in Guava Based on In Vitro PEG Evaluation

Forty landraces of guava were subjected to drought tolerance evaluation based on in vitro polyethylene glycol (PEG) treatment. Nodal stem segments with lateral buds were used as explant. Five concentrations (i.e. 0, 4, 6, 8 and 10 %) of PEG were tested, among which the optimum concentration for screening was determined as 8 %. Analysis of variance showed highly significant variation among the tested landraces, concentrations of PEG and the interaction between them in the percentage of response (%R), number of shoot per explant (NSE) and drought susceptibility index. The average percentage of reduction due to PEG treatment was 48.30 and 52.57 % for the %R and NSE, respectively. Heritability and genetic advance were increased due to drought stress for %R, while they were decreased for NSE, indicating that %R was more related to drought stress than NSE. The molecular analysis of the highest and lowest responsive landraces was performed using sequence related amplified polymorphism (SRAP) and inter-simple sequence repeats (ISSR). Both markers were effective in discriminating the tested landraces and completely separated them into two clusters related to %R under PEG. ISSR showed a higher percentage of polymorphism, polymorphic information content and diversity index compared with SRAP. However, SRAP was more effective than ISSR in showing a higher primer resolving power and a number of unique specific bands for drought tolerance and susceptibility. Drought in vitro evaluation method established here is effective, inexpensive and manageable in genotype screening for drought tolerance in guava and could be used in other woody plant species.     

Tunable Photophysical Behavior of a Novel Fluorescent Probe by Silver/Gold and Core–Shell NPs

Plasmonics - The photophysical and photochemical behaviors of a novel fluorescent probe, namely 5-(4-(dimethylamino)phenyl)-1-(thiophen-2-yl)penta-2,4-dien-1-one (DMATP), were tuned by solvent...     

Theory and Experimental Validation of a Spatio-temporal Model of Chemotherapy Transport to Enhance Tumor Cell Kill

It has been hypothesized that continuously releasing drug molecules into the tumor over an extended period of time may significantly improve the chemotherapeutic efficacy by overcoming physical transport limitations of conventional bolus drug treatment. In this paper, we present a generalized space- and time-dependent mathematical model of drug transport and drug-cell interactions to quantitatively formulate this hypothesis. Model parameters describe: perfusion and tissue architecture (blood volume fraction and blood vessel radius); diffusion penetration distance of drug (i.e., a function of tissue compactness and drug uptake rates by tumor cells); and cell death rates (as function of history of drug uptake). We performed preliminary testing and validation of the mathematical model using in vivo experiments with different drug delivery methods on a breast cancer mouse model. Experimental data demonstrated a 3-fold increase in response using nano-vectored drug vs. free drug delivery, in excellent quantitative agreement with the model predictions. Our model results implicate that therapeutically targeting blood volume fraction, e.g., through vascular normalization, would achieve a better outcome due to enhanced drug delivery.

Author Summary

Cancer treatment efficacy can be significantly enhanced through the elution of drug from nano-carriers that can temporarily stay in the tumor vasculature. Here we present a relatively simple yet powerful mathematical model that accounts for both spatial and temporal heterogeneities of drug dosing to help explain, examine, and prove this concept. We find that the delivery of systemic chemotherapy through a certain form of nano-carriers would have enhanced tumor kill by a factor of 2 to 4 over the standard therapy that the patients actually received. We also find that targeting blood volume fraction (a parameter of the model) through vascular normalization can achieve more effective drug delivery and tumor kill. More importantly, this model only requires a limited number of parameters which can all be readily assessed from standard clinical diagnostic measurements (e.g., histopathology and CT). This addresses an important challenge in current translational research and justifies further development of the model towards clinical translation.     

Cytotoxic Oleanane‐Type Saponins from the Leaves of Albizia anthelmintica Brongn.

Two new oleanane‐type saponins: β‐d ‐xylopyranosyl‐(1 → 4)‐6‐deoxy‐α‐l ‐mannopyranosyl‐(1 → 2)‐1‐O‐{(3β)‐28‐oxo‐3‐[(2‐O‐β‐d ‐xylopyranosyl‐β‐d ‐glucopyranosyl)oxy]olean‐12‐en‐28‐yl}‐β‐d ‐glucopyranose ( 1 ) and 1‐O‐[(3β)‐28‐oxo‐3‐{[β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐arabinopyranosyl‐(1 → 6)‐2‐acetamido‐2‐deoxy‐β‐d ‐glucopyranosyl]oxy}olean‐12‐en‐28‐yl]β‐d ‐glucopyranose ( 2 ), along with two known saponins: (3β)‐3‐[(β‐d ‐Glucopyranosyl‐(1 → 2)‐β‐d ‐glucopyranosyl)oxy]olean‐12‐en‐28‐oic acid ( 3 ) and (3β)‐3‐{[α‐l ‐arabinopyranosyl‐(1 → 6)‐[β‐d ‐glucopyranosyl‐(1 → 2)]‐β‐d ‐glucopyranosyl]oxy}olean‐12‐en‐28‐oic acid ( 4 ) were isolated from the acetone‐insoluble fraction obtained from the 80% aqueous MeOH extract of Albizia anthelmintica Brongn . leaves. Their structures were identified using different NMR experiments including: ¹H‐ and ¹³C‐NMR, HSQC, HMBC and ¹H,¹H‐COSY, together with HR‐ESI‐MS/MS, as well as by acid hydrolysis. The four isolated saponins and the fractions of the extract exhibited cytotoxic activity against HepG‐2 and HCT‐116 cell lines. Compound 2 showed the most potent cytotoxic activity among the other tested compounds against the HepG2 cell line with an IC₅₀ value of 3.60μm . Whereas, compound 1 showed the most potent cytotoxic effect with an IC₅₀ value of 4.75μm on HCT‐116 cells.     

Differential proteome and cellular adhesion analyses of the probiotic bacterium Lactobacillus acidophilus NCFM grown on raffinose – an emerging prebiotic

Whole cell and surface proteomes were analyzed together with adhesive properties of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) grown on the emerging prebiotic raffinose, exemplifying a synbiotic. Adhesion of NCFM to mucin and intestinal HT‐29 cells increased three‐fold after culture with raffinose versus glucose, as also visualized by scanning electron microscopy. Comparative proteomics using 2D‐DIGE showed 43 unique proteins to change in relative abundance in whole cell lysates from NCFM grown on raffinose compared to glucose. Furthermore, 14 unique proteins in 18 spots of the surface subproteome underwent changes identified by differential 2DE, including elongation factor G, thermostable pullulanase, and phosphate starvation inducible stress‐related protein increasing in a range of +2.1 ? +4.7 fold. By contrast five known moonlighting proteins decreased in relative abundance by up to ?2.4 fold. Enzymes involved in raffinose catabolism were elevated in the whole cell proteome; α‐galactosidase (+13.9 fold); sucrose phosphorylase (+5.4 fold) together with metabolic enzymes from the Leloir pathway for galactose utilization and the glycolysis; β‐galactosidase (+5.7 fold); galactose (+2.9/+3.1 fold) and fructose (+2.8 fold) kinases. The insights at the molecular and cellular levels contributed to the understanding of the interplay of a synbiotic composed of NCFM and raffinose with the host.     

Higher relatedness mitigates mortality in a nematode with lethal male fighting

According to kin selection theory, individuals show less aggression towards their relatives. Limited dispersal promotes interactions among relatives but also increases competition among them. The evolution of cooperation in viscous populations has been subject of mainly theoretical exploration. We investigated the influence of relatedness on aggression in males of entomopathogenic nematode Steinernema longicaudum that engage in lethal fighting. In a series of in vitro experiments, we found that both competitor male group size and relatedness influence male mortality rates. Higher relatedness led to progressively lower rates of male mortality. In experimentally infected insects, wherein large numbers of males and females interact, the proportion of dead and paralysed (= terminally injured) males was higher when infection was established by infective juveniles originating from a mixture of three lines than in those infected by a single line. The results collectively show that Steinernema longicaudum males recognize their kin and consequently male mortality rates are lower in groups consisting of more related males. Furthermore, this monotonic negative relationship between aggression and relatedness suggests that kin selection benefits are still substantial even under extreme competition. Our experiments also suggest that kin recognition in entomopathogenic nematodes has a genetic basis rather than being strictly based on environmental cues. We discuss our findings within the theoretical context of the evolution of altruistic/cooperative behaviour in structured populations.     

Association of fibroblast growth factor receptor 1 with the adaptor protein Grb14. Characterization of a new receptor binding partner

Using the cytoplasmic domain of fibroblast growth factor receptor 1 (FGFR1) as bait in a yeast two-hybrid screen, Grb14 was identified as a FGFR1 binding partner. A kinase-inactive mutant of FGFR1 failed to interact with Grb14, indicating that activation of FGFR1 is necessary for binding. Deletion of the C-tail or mutation of both C-tail tyrosine residues of FGFR1 to phenylalanine abolished binding, and deletion of the juxtamembrane domain of the receptor reduced binding, suggesting that Grb14 binds to FGFR1 at multiple sites. Co-immunoprecipitation and in vitro binding assays demonstrated that binding of Grb14 to FGFR1 in mammalian cells was dependent on receptor activation by fibroblast growth factor-2 (FGF-2). Deletion of the Src homology 2 (SH2) domain of Grb14 reduced but did not block binding to FGFR1 and eliminated dependence on receptor activation. The SH2 domain alone bound both FGFR1 and platelet-derived growth factor receptor, whereas full-length Grb14 bound only FGFR1, suggesting that regions upstream of the SH2 domain confer specificity for FGFR1. Grb14 was phosphorylated on serine and threonine residues in unstimulated cells, and treatment with FGF-2 enhanced this phosphorylation. Expression of exogenous Grb14 inhibited FGF-2-induced cell proliferation, whereas a point-mutated form of Grb14 incapable of binding to FGFR1 enhanced FGF-2-induced mitogenesis. These data demonstrate an interaction between activated FGFR1 and Grb14 and suggest a role for Grb14 in FGF signaling.     

Connexin-26 gene analysis in hearing-impaired newborns

The efficacy and utility of the Connexin-26 (Cx-26) gene (also called GJB2) analysis from DNA isolated from Guthrie newborn screening cards is demonstrated. This analysis precisely defined a major cause of prelingual nonsyndromic deafness in those children requiring amplification in our study. Guthrie cards were obtained from 49 deaf children requiring amplification identified over the last 5 years by the Rhode Island Newborn Screening Program. Children with syndromes or other recognizable causes of hearing loss were excluded. DNA was extracted from the Guthrie cards and analyzed sequentially for the Cx-26 35delG mutation and then for the 167delT mutation followed by gene sequencing on remaining heterozygotes. Three of 42 children were 35delG homozygotes; 2/42 children were 35delG/167delT compound heterozygotes. One child was identified as being a 35delG heterozygote with no other mutation found by sequencing. Nine Guthrie cards yielded no amplification or uninterpretable results. Cx-26 mutations were identified as causing 11.9% of the deafness in the children studied. In conclusion, Cx-26 analysis is an important test that identifies a major cause of prelingual nonsyndromic deafness. Molecular analysis of hearing-impaired newborns will be important for genetic counseling in these families. Failures with Guthrie cards may make use of other collection methods preferable.     

Listeria monocytogenes Scott A: cell surface charge, hydrophobicity, and electron donor and acceptor characteristics under different environmental growth conditions

We determined the variations in the surface physicochemical properties of Listeria monocytogenes Scott A cells that occurred under various environmental conditions. The surface charges, the hydrophobicities, and the electron donor and acceptor characteristics of L. monocytogenes Scott A cells were compared after the organism was grown in different growth media and at different temperatures; to do this, we used microelectrophoresis and the microbial adhesion to solvents method. Supplementing the growth media with glucose or lactic acid affected the electrical, hydrophobic, and electron donor and acceptor properties of the cells, whereas the growth temperature (37, 20, 15, or 8 degrees C) primarily affected the electrical and electron donor and acceptor properties. The nonlinear effects of the growth temperature on the physicochemical properties of the cells were similar for cells cultivated in two different growth media, but bacteria cultivated in Trypticase soy broth supplemented with 6 g of yeast extract per liter (TSYE) were slightly more hydrophobic than cells cultivated in brain heart infusion medium (P < 0.05). Adhesion experiments conducted with L. monocytogenes Scott A cells cultivated in TSYE at 37, 20, 15, and 8 degrees C and then suspended in a sodium chloride solution (1.5 x 10(-1) or 1.5 x 10(-3) M NaCl) confirmed that the cell surface charge and the electron donor and acceptor properties of the cells had an influence on their attachment to stainless steel.     

Disruption of Cell–Substrate Adhesion Activates the Protein Tyrosine Kinase pp60

Treatment of confluent chicken embryo fibroblasts (CEFs) with trypsin results in a dose- and time-dependent increase in c-Src protein tyrosine kinase (PTK) activity. A similar, but less marked, increase in c-Src PTK activity occurs upon incubation of CEFs in calcium-free phosphate-buffered saline, which also causes a decrease in cell–substrate adhesion. The increase in c-Src PTK activity following disruption of cell–substrate adhesion correlates with a decrease in the phosphorylation of c-Src at the regulatory site, Tyr527. The phosphotyrosine phosphatase inhibitor phenylarsine oxide blocks the increase in c-Src PTK activity seen following treatment with trypsin and the morphological changes associated with the disruption of cell–substrate adhesion. In contrast, disruption of cell–substrate adhesion causes a decrease in FAK PTK activity that rapidly returns to control levels when the cells are plated on fibronection-coated dishes. Treatment of cells with cytochalasin D, which disrupts actin filaments but not cell–substrate adhesion, causes only a slight increase in c-Src PTK activity. Thus, these studies demonstrate a ligand-independent mechanism for the activation of c-Src that is consistent with its role in both cell adhesion and cell motility. Furthermore, these data suggest that similar to adhesion, loss of adhesion is not a passive process but can activate specific signaling pathways that may have significant effects on cellular function.