Inhibiting Cycloxygenase and Ornithine Decarboxylase by Diclofenac and Alpha-Difluoromethylornithine Blocks Cutaneous SCCs by Targeting Akt-ERK Axis

Non-melanoma skin cancer (NMSC) is the most common type of skin cancer in Caucasian populations. Its increasing incidence has been a major public health concern. Elevated expressions of ODC and COX-2 are associated with both murine and human NMSCs. Inhibition of these molecular targets singly employing their respective small molecule inhibitors showed limited success. Here, we show that combined blockade of ODC and COX-2 using their potent inhibitors, DFMO and diclofenac respectively abrogates growth of A431 epidermal xenograft tumors in nu/nu mice by more than 90%. The tumor growth inhibition was associated with a diminution in the proliferation and enhancement in apoptosis. The proliferation markers such as PCNA and cyclin D1 were reduced. TUNEL-positive apoptotic cells and cleaved caspase-3 were increased in the residual tumors. These agents also manifested direct target-unrelated effects. Reduced expression of phosphorylated MAPKAP-2, ERK, and Akt (ser⁴⁷³ & thr³⁰⁸) were noticed. The mechanism by which combined inhibition of ODC/COX attenuated tumor growth and invasion involved reduction in EMT. Akt activation by ODC+COX-2 over-expression was the key player in this regard as Akt inhibition manifested effects similar to those observed by the combined inhibition of ODC+COX-2 whereas forced over-expression of Akt resisted against DFMO+diclofenac treatment. These data suggest that ODC+COX-2 over-expression together leads to pathogenesis of aggressive and invasive cutaneous carcinomas by activating Akt signaling pathway, which through augmenting EMT contributes to tumor invasion.     

A high-throughput method to deliver targeted optogenetic stimulation to moving C. elegans populations

We present a high-throughput optogenetic illumination system capable of simultaneous closed-loop light delivery to specified targets in populations of moving Caenorhabditis elegans. The instrument addresses three technical challenges: It delivers targeted illumination to specified regions of the animal’s body such as its head or tail; it automatically delivers stimuli triggered upon the animal’s behavior; and it achieves high throughput by targeting many animals simultaneously. The instrument was used to optogenetically probe the animal’s behavioral response to competing mechanosensory stimuli in the the anterior and posterior gentle touch receptor neurons. Responses to more than 43,418 stimulus events from a range of anterior–posterior intensity combinations were measured. The animal’s probability of sprinting forward in response to a mechanosensory stimulus depended on both the anterior and posterior stimulation intensity, while the probability of reversing depended primarily on the anterior stimulation intensity. We also probed the animal’s response to mechanosensory stimulation during the onset of turning, a relatively rare behavioral event, by delivering stimuli automatically when the animal began to turn. Using this closed-loop approach, over 9,700 stimulus events were delivered during turning onset at a rate of 9.2 events per worm hour, a greater than 25-fold increase in throughput compared to previous investigations. These measurements validate with greater statistical power previous findings that turning acts to gate mechanosensory evoked reversals. Compared to previous approaches, the current system offers targeted optogenetic stimulation to specific body regions or behaviors with many fold increases in throughput to better constrain quantitative models of sensorimotor processing.

This study resents a new targeted illumination method for the nematode Caenorhabditis elegans, allowing delivery of optogenetic stimulation to specific body parts of many animals at once, automatically triggered by the animals’ behavior.     

Interference of N-hydroxysuccinimide with bicinchoninic acid protein assay

We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu²⁺ in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time–response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample.     

Genetic Variations of PIP4K2A Confer Vulnerability to Poor Antipsychotic Response in Severely Ill Schizophrenia Patients

Literature suggests that disease severity and neurotransmitter signaling pathway genes can accurately identify antipsychotic response in schizophrenia patients. However, putative role of signaling molecules has not been tested in schizophrenia patients based on severity of illness, despite its biological plausibility. In the present study we investigated the possible association of polymorphisms from five candidate genes RGS4, SLC6A3, PIP4K2A, BDNF, PI4KA with response to antipsychotic in variably ill schizophrenia patients. Thus in present study, a total 53 SNPs on the basis of previous reports and functional grounds were examined for their association with antipsychotic response in 423 schizophrenia patients segregated into low and high severity groups. Additionally, haplotype, diplotype, multivariate logistic regression and multifactor-dimensionality reduction (MDR) analyses were performed. Furthermore, observed associations were investigated in atypical monotherapy (n = 355) and risperidone (n = 260) treated subgroups. All associations were estimated as odds ratio (OR) and 95% confidence interval (CI) and test for multiple corrections was applied. Single locus analysis showed significant association of nine variants from SLC6A3, PIP4K2A and BDNF genes with incomplete antipsychotic response in schizophrenia patients with high severity. We identified significant association of six marker diplotype ATTGCT/ATTGCT (rs746203-rs10828317-rs7094131-rs2296624-rs11013052-rs1409396) of PIP4K2A gene in incomplete responders (corrected p-value = 0.001; adjusted-OR = 3.19, 95%-CI = 1.46–6.98) with high severity. These associations were further observed in atypical monotherapy and risperidone sub-groups. MDR approach identified gene-gene interaction among BDNF_rs7103411-BDNF_rs1491851-SLC6A3_rs40184 in severely ill incomplete responders (OR = 7.91, 95%-CI = 4.08–15.36). While RGS4_rs2842026-SLC6A3_rs2975226 interacted synergistically in incomplete responders with low severity (OR = 4.09, 95%-CI = 2.09–8.02). Our findings provide strong evidence that diplotype ATTGCT/ATTGCT of PIP4K2A gene conferred approximately three-times higher incomplete responsiveness towards antipsychotics in severely ill patients. These results are consistent with the known role of phosphatidyl-inositol-signaling elements in antipsychotic action and outcome. Findings have implication for future molecular genetic studies as well as personalized medicine. However more work is warranted to elucidate underlying causal biological pathway.     

Hexafluoroisopropanol-induced helix–sheet transition of stem bromelain: Correlation to function

Stem bromelain is a proteolytic phytoprotein with a variety of therapeutic effects. Understanding its structural properties could provide insight into the mechanisms underlying its clinical utility. Stem bromelain was evaluated for its conformational and folding properties at the pH conditions it encounters when administered orally. It exists as a partially folded intermediate at pH 2.0. The conformational changes to this intermediate state were evaluated using fluorinated alcohols known to induce changes similar to those seen in vivo. Studies using circular dichroism, fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid and mass spectrometry indicate that treatment with 10–30% hexafluoroisopropanol induces the partially folded intermediate to adopt much of the native protein's secondary structure, but only a rudimentary tertiary structure, characteristic of the molten globule state. Addition of slightly higher concentrations of hexafluoroisopropanol caused transformation from an α-helix to a β-sheet and induced formation of a compact nonnative structure. This nonnative form was more inhibitory of cell survival than either the native or the partially folded intermediate forms, as measured by enhanced suppression of proliferative cues (e.g., extracellular-signal-regulated kinase) and initiation of apoptotic events. The nonnative form also showed better antitumorigenic properties, as evaluated using an induced two-stage mouse skin papilloma model. In contrast, the nonnative state showed only a fraction of the proteolytic activity of the native form. This study demonstrates that hexafluoroisopropanol can induce a conformational change in stem bromelain to a form with potentially useful therapeutic properties different from those of the native protein.     

A Genome-Wide Association Study of the Maize Hypersensitive Defense Response Identifies Genes That Cluster in Related Pathways

Much remains unknown of molecular events controlling the plant hypersensitive defense response (HR), a rapid localized cell death that limits pathogen spread and is mediated by resistance (R-) genes. Genetic control of the HR is hard to quantify due to its microscopic and rapid nature. Natural modifiers of the ectopic HR phenotype induced by an aberrant auto-active R-gene (Rp1-D21), were mapped in a population of 3,381 recombinant inbred lines from the maize nested association mapping population. Joint linkage analysis was conducted to identify 32 additive but no epistatic quantitative trait loci (QTL) using a linkage map based on more than 7000 single nucleotide polymorphisms (SNPs). Genome-wide association (GWA) analysis of 26.5 million SNPs was conducted after adjusting for background QTL. GWA identified associated SNPs that colocalized with 44 candidate genes. Thirty-six of these genes colocalized within 23 of the 32 QTL identified by joint linkage analysis. The candidate genes included genes predicted to be in involved programmed cell death, defense response, ubiquitination, redox homeostasis, autophagy, calcium signalling, lignin biosynthesis and cell wall modification. Twelve of the candidate genes showed significant differential expression between isogenic lines differing for the presence of Rp1-D21. Low but significant correlations between HR-related traits and several previously-measured disease resistance traits suggested that the genetic control of these traits was substantially, though not entirely, independent. This study provides the first system-wide analysis of natural variation that modulates the HR response in plants.