Blood-Based Marker For Aggressive Breast Cancer Early Detection

Dr. Srinidi Mohan gives an overview presentation of his research, focused on nitric oxide metabolism, and its role in breast cancer biomarker development.https://dune.une.edu/biomed_facpres/1003/thumbnail.jp

Functional Role Of Recombinant Cysteine Protease On Spodoptera Frugiperda Peritrophic Matrix

Fall armyworm larvae (FAW), which are serious pests in the southern United States, show retarded growth when they feed on insect-resistant maize inbreds Mp704 and Mp708. These maize genotypes are not only resistant to FAW, but to a number of other lepidopteran pests. In these genotypes, a unique, extracellular, 33-KDa cysteine protease (Mir1-CP) rapidly accumulates in the whorl in response to insect feeding. Initial morphological studies on larvae feeding on resistant maize plants over-expressing the cysteine protease showed severe damage in insect?s first line of defense, the peritrophic matrix (PM). But it is not known whether the cysteine protease has unprecedented effect on insect defense mechanisms. This study focuses on understanding the functional involvement of the cysteine protease (Mir1-CP) in a plant-herbivore defense mechanism. I used purified, recombinant 33-KDa cysteine protease (Mir1-CP) and its two mutated forms (Mut1 and Mut2) to determine their effects on the permeability of PMs from fall armyworm and other lepidopteran larvae. The purified Mir1-CP was also used to determine its minimal effective dosage on lepidopteran larval growth as well as to qualitatively determine their direct morphological effects on PM and gut regions of fall armyworm larvae. In vitro permeability studies demonstrated that the recombinant Mir1-CP directly permeabilized the PM and requires both cysteine at the active site and the terminal 25 amino acids to achieve complete permeabilization. Dose response study suggested that physiologically relevant concentrations of Mir1-CP in the maize whorl would be effective in controlling a broad range of lepidopteran pests. The study also suggested that stacking Mir1-CP and Bt-toxin (Bt-CryIIA) genes in transgenic plants could broaden the normal range of both Mir1-CP and Bt-toxin. Morphological studies using three different microscopic techniques showed damaged PM in larvae fed on Mir1-CP diet. These results suggest that by directly permeabilizing and damaging the PM, the Mir1-CP provides critical defense in host plants against lepidopteran pests

A Naturally Occurring Plant Cysteine Protease Possesses Remarkable Toxicity against Insect Pests and Synergizes Bacillus thuringiensis Toxin

When caterpillars feed on maize (Zea maize L.) lines with native resistance to several Lepidopteran pests, a defensive cysteine protease, Mir1-CP, rapidly accumulates at the wound site. Mir1-CP has been shown to inhibit caterpillar growth in vivo by attacking and permeabilizing the insect's peritrophic matrix (PM), a structure that surrounds the food bolus, assists in digestion and protects the midgut from microbes and toxins. PM permeabilization weakens the caterpillar defenses by facilitating the movement of other insecticidal proteins in the diet to the midgut microvilli and thereby enhancing their toxicity. To directly determine the toxicity of Mir1-CP, the purified recombinant enzyme was directly tested against four economically significant Lepidopteran pests in bioassays. Mir1-CP LC50 values were 1.8, 3.6, 0.6, and 8.0 ppm for corn earworm, tobacco budworm, fall armyworm and southwestern corn borer, respectively. These values were the same order of magnitude as those determined for the Bacillus thuringiensis toxin Bt-CryIIA. In addition to being directly toxic to the larvae, 60 ppb Mir1-CP synergized sublethal concentrations of Bt-CryIIA in all four species. Permeabilization of the PM by Mir1-CP probably provides ready access to Bt-binding sites on the midgut microvilli and increases its activity. Consequently, Mir1-CP could be used for controlling caterpillar pests in maize using non-transgenic approaches and potentially could be used in other crops either singly or in combination with Bt-toxins

Mechanism of Cellular Oxidation Stress Induced by Asymmetric Dimethylarginine

The mechanism by which asymmetric dimethylarginine (ADMA) induces vascular oxidative stress is not well understood. In this study, we utilized human umbilical vein endothelial cells (HUVEC) to examine the roles of ADMA cellular transport and the uncoupling of endothelial nitric oxide synthase (eNOS) in contributing to this phenomenon. Dihydroethidium (DHE) fluorescence was used as an index of oxidative stress. Whole cells and their isolated membrane fractions exhibited measureable increased DHE fluorescence at ADMA concentrations greater than 10 µM. ADMA-induced DHE fluorescence was inhibited by co-incubation with L-lysine, tetrahydrobiopterin (BH<sub>4</sub>), or L-nitroarginine methyl ester (L-NAME). Oxidative stress induced in these cells by angiotensin II (Ang II) were unaffected by the same concentrations of L-lysine, L-NAME and BH<sub>4</sub>. ADMA-induced reduction in cellular nitrite or nitrite/nitrate production was reversed in the presence of increasing concentrations of BH<sub>4</sub>. These results suggest that ADMA-induced DHE fluorescence involves the participation of both the cationic transport system in the cellular membrane and eNOS instead of the Ang II-NADPH oxidase pathway

Correlating Pharmacy Student’s Stress Levels with their Stress Perception and Performance

To assess quantitative and qualitative levels of stress in PharmD courses, and correlate students’ stress levels with their academic performance in these courses. Quantitative stress levels in first and second-year students were monitored by evaluating salivary concentrations of cortisol (stress hormone). Student’s perception about their stress levels (qualitative) was assessed via Acute and Perceived Stress Scale surveys. The mean salivary cortisol concentrations during examinations were significantly higher than non-examination days. Surveys showed that student\u27s perception was different from cortisol levels regarding personal stress. There was a significant correlation between students stress and their academic performances

Dose response analysis for tobacco budworm larvae fed (a) Mir1-CP, (b) Bt-CryIIA, and (c) sub-lethal doses of Bt-CryIIA and Mir1-CP.

<p>The y-axis is the relative growth rate (RGR measured as Δmg/(mg_avgd) and vertical bars indicate one standard deviation. The least significant difference (LSD) at the 0.05 confidence level for the RGR values were as follows: Mir1-CP – 0.31, Bt-CryIIA – 0.44, sub-lethal doses of Bt-CryIIA and Mir1-CP combined – 0.57. Numbers (mean±SD) in the histogram bars are the average percentage mortality for each treatment. The least significant difference (LSD) at the 0.05 confidence level for mortality were as follows: Mir1-CP – 8.5, Bt-CryIIA – 7.4, sub-lethal doses of Bt-CryIIA and Mir1-CP combined – 15.83. Results are based on four independent bioassays. Concentrations are given as parts per million (ppm).</p

Dose response analysis for corn earworm larvae fed (a) Mir1-CP, (b) Bt-CryIIA, and (c) sub-lethal doses of Bt-CryIIA and Mir1-CP.

<p>The y-axis is the relative growth rate (RGR measured as Δmg/(mg_avgd) and vertical bars indicate one standard deviation. The least significant difference (LSD) at the 0.05 confidence level for the RGR values were as follows: Mir1-CP = 0.25, Bt-CryIIA = 0.3, sub-lethal doses of Bt-CryIIA and Mir1-CP = 0.42. Numbers (mean±SD) in the histogram bars are the average percentage mortality for each treatment. The least significant difference (LSD) at the 0.05 confidence level for mortality were as follows: Mir1-CP – 7.4, Bt-CryIIA – 9.5, sub-lethal doses of Bt-CryIIA and Mir1-CP combined – 15.94. Results are based on four independent bioassays. Concentrations are given as parts per million (ppm).</p

Relative growth rate (RGR) and percentage mortality of Lepidopteran larvae fed on Mir1-CP, Bt-CryIIA or Mir1-CP and Bt-CryIIA in combination.

<p>RGR = Δmg/(mg_avgd); SE = standard error. P values indicate that % mortality and RGR values significantly differ from additivity and show synergy at the 95% confidence level.</p

SDS-PAGE analysis of Bt-CryIIA protoxin and its processed form incubated with 0.06 ppm of Mir1-CP.

<p>Lane M – markers; lane 1 -processed Bt-CryIIA; lane 2 – unprocessed Bt-CryIIA protoxin; lane 3 - unprocessed Bt-CryIIA protoxin incubated with Mir1-CP for 8 hr; lane 4 – unprocessed Bt-CryIIA protoxin incubated with Mir1-CP for 24 hr; lane 5 – processed Bt-CryIIA toxin incubated with Mir1-CP for 8 hr. Numbers on the left margin refer to the molecular mass of the marker bands in kD. Arrow A indicates the position of the protoxin, arrow B indicates the position of the processed toxin and arrow C marks the position of Mir1-CP (molecular mass of 33 kD) added to the incubation.</p

Probit analysis of LC₅₀ values for several Lepidopteran species fed Mir1-CP and Bt-CryIIA. LC₅₀ values are in ppm and numbers in parenthesis represent the 95% confidence interval.

<p>Probit analysis of LC₅₀ values for several Lepidopteran species fed Mir1-CP and Bt-CryIIA. LC₅₀ values are in ppm and numbers in parenthesis represent the 95% confidence interval.</p