PCR bias Toward the wild-type k-ras and p53 sequences: Implications for PCR detection of mutations and cancer diagnosis

PCR-based cancer diagnosis requires detection of rare mutations in k- ras, p53 or other genes. The assumption has been that mutant and wild-type sequences amplify with near equal efficiency, so that they are eventually present in proportions representative of the starting material. Work on factor IX suggests that this assumption is invalid for one case of near- sequence identity. To test the generality of this phenomenon and its relevance to cancer diagnosis, primers distant from point mutations in p53 and k-ras were used to amplify wild-type and mutant sequences from these genes. A substantial bias against PCR amplification of mutants was observed for two regions of the p53 gene and one region of k-ras. For k-ras and p53, bias was observed when the wild-type and mutant sequences were amplified separately or when mixed in equal proportions before PCR. Bias was present with proofreading and non-proofreading polymerase. Mutant and wild-type segments of the factor V, cystic fibrosis transmembrane conductance regulator and prothrombin genes were amplified and did not exhibit PCR bias. Therefore, the assumption of equal PCR efficiency for point mutant and wild-type sequences is invalid in several systems. Quantitative or diagnostic PCR will require validation for each locus, and enrichment strategies may be needed to optimize detection of mutants.

The relationship between Bayesian motor unit number estimation and histological measurements of motor neurons in wild-type and SOD1 G93A mice

Objective: To assess the relationship between Bayesian MUNE and histological motor neuron counts in wild-type mice and in an animal model of ALS. Methods: We performed Bayesian MUNE paired with histological counts of motor neurons in the lumbar spinal cord of wild-type mice and transgenic SOD1 G93A mice that show progressive weakness over time. We evaluated the number of acetylcholine endplates that were innervated by a presynaptic nerve. Results: In wild-type mice, the motor unit number in the gastrocnemius muscle estimated by Bayesian MUNE was approximately half the number of motor neurons in the region of the spinal cord that contains the cell bodies of the motor neurons supplying the hindlimb crural flexor muscles. In SOD1 G93A mice, motor neuron numbers declined over time. This was associated with motor endplate denervation at the end-stage of disease. Conclusion: The number of motor neurons in the spinal cord of wild-type mice is proportional to the number of motor units estimated by Bayesian MUNE. In SOD1 G93A mice, there is a lower number of estimated motor units compared to the number of spinal cord motor neurons at the end-stage of disease, and this is associated with disruption of the neuromuscular junction. Significance: Our finding that the Bayesian MUNE method gives estimates of motor unit numbers that are proportional to the numbers of motor neurons in the spinal cord supports the clinical use of Bayesian MUNE in monitoring motor unit loss in ALS patients. © 2012 International Federation of Clinical Neurophysiology.

No genetic bottleneck in Plasmodium falciparum wild-type Pfcrt alleles reemerging in Hainan Island, China, following high-level chloroquine resistance

Chloroquine-resistant Plasmodium falciparum was highly prevalent in Hainan, China, in the 1970s. Twenty-five years after cessation of chloroquine therapy, the prevalence of P. falciparum wild-type Pfcrt alleles has risen to 36% (95% confidence interval, 22.1 to 52.4%). The diverse origins of wild-type alleles indicate that there was no genetic bottleneck caused by high chloroquine resistance.

Characterization of the antibody response in birds following infection with wild-type and attenuated strains of Eimeria tenella and Eimeria necatrix.

Live vaccines containing attenuated parasite strains are increasingly used to control chicken coccidiosis. In this paper antibody responses elicited by infections with wild-type and attenuated strains of Eimeria tenella and E.necatrix were characterized by immunoblotting and ELISA with homologous and heterologous antisera. Few differences between antisera from birds infected with wild and attenuated strains of E. tenella were evident in immunoblots conducted with merozoite antigen preparations from both E. tenella strains, however the reactivity of sera raised in birds infected with the wild-type strain was noticeably more intense. In ELISAs conducted with merozoite antigen preparations, antisera from birds infected with the wild-type strains of E. tenella and E. necatrix consistently produced a significantly higher (P < 0.05) antibody response than antisera from birds infected with the attenuated strains. Likewise, avidity ELISAs conducted with the E. tenella strains demonstrated that antibodies in birds infected with the wild-type strain were of significantly higher avidity (P < 0.05) than antibodies in birds infected with the attenuated strain. The differences in the antibody responses are probably due to changes in the attenuated strain as a result of selection for precocious development and the less severe tissue damage and inflammation of the intestine resulting from infection with the attenuated strain.

MPK-09, a small molecule inspired from bioactive styryllactone restores the wild-type function of mutant p53

In the search for more efficacious and less toxic cancer drugs, the tumor suppressor p53 protein has long been a desirable therapeutic target. In the recent past, few independent studies have demonstrated that the antitumor activity of wild-type p53 can be restored in cancer cells harboring mutant form of p53 using small molecule activators. In this study, we describe a novel small molecule MPK-09, which is selective and highly potent against allele specific p53 mutations mainly, R175H, R249S, R273H, R273C, and E285K. Except E285K, all other mutations tested are among the six ``hot spot'' p53 mutations reported in majority of human cancer. Furthermore, our study conclusively demonstrates that the apoptotic activity of the small molecule MPK-09 against cancer cells harboring R273C and E285K mutations is due to restoration of the wild-type conformation to the corresponding mutant form of p53.

Novel Functions of (p)ppGpp and Cyclic di-GMP in Mycobacterial Physiology Revealed by Phenotype Microarray Analysis of Wild-Type and Isogenic Strains of Mycobacterium smegmatis

The bacterial second messengers (p)ppGpp and bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulate important functions, such as transcription, virulence, biofilm formation, and quorum sensing. In mycobacteria, they regulate long-term survival during starvation, pathogenicity, and dormancy. Recently, a Pseudomonas aeruginosa strain lacking (p) ppGpp was shown to be sensitive to multiple classes of antibiotics and defective in biofilm formation. We were interested to find out whether Mycobacterium smegmatis strains lacking the gene for either (p)ppGpp synthesis (Delta rel(Msm)) or c-di-GMP synthesis (Delta dcpA) would display similar phenotypes. We used phenotype microarray technology to compare the growth of the wild-type and the knockout strains in the presence of several antibiotics. Surprisingly, the Delta rel(Msm) and Delta dcpA strains showed enhanced survival in the presence of many antibiotics, but they were defective in biofilm formation. These strains also displayed altered surface properties, like impaired sliding motility, rough colony morphology, and increased aggregation in liquid cultures. Biofilm formation and surface properties are associated with the presence of glycopeptidolipids (GPLs) in the cell walls of M. smegmatis. Thin-layer chromatography analysis of various cell wall fractions revealed that the levels of GPLs and polar lipids were reduced in the knockout strains. As a result, the cell walls of the knockout strains were significantly more hydrophobic than those of the wild type and the complemented strains. We hypothesize that reduced levels of GPLs and polar lipids may contribute to the antibiotic resistance shown by the knockout strains. Altogether, our data suggest that (p)ppGpp and c-di-GMP may be involved in the metabolism of glycopeptidolipids and polar lipids in M. smegmatis.

Curcumin Pyrazole and its derivative (N-(3-Nitrophenylpyrazole) Curcumin inhibit aggregation, disrupt fibrils and modulate toxicity of Wild type and Mutant alpha-Synuclein

Accumulating evidence suggests that deposition of neurotoxic a-synuclein aggregates in the brain during the development of neurodegenerative diseases like Parkinson's disease can be curbed by anti-aggregation strategies that either disrupt or eliminate toxic aggregates. Curcumin, a dietary polyphenol exhibits anti-amyloid activity but the use of this polyphenol is limited owing to its instability. As chemical modifications in curcumin confiscate this limitation, such efforts are intensively performed to discover molecules with similar but enhanced stability and superior properties. This study focuses on the inhibitory effect of two stable analogs of curcumin viz. curcumin pyrazole and curcumin isoxazole and their derivatives against a-synuclein aggregation, fibrillization and toxicity. Employing biochemical, biophysical and cell based assays we discovered that curcumin pyrazole (3) and its derivative N-(3-Nitrophenylpyrazole) curcumin (15) exhibit remarkable potency in not only arresting fibrillization and disrupting preformed fibrils but also preventing formation of A11 conformation in the protein that imparts toxic effects. Compounds 3 and 15 also decreased neurotoxicity associated with fast aggregating A53T mutant form of a-synuclein. These two analogues of curcumin described here may therefore be useful therapeutic inhibitors for the treatment of a-synuclein amyloidosis and toxicity in Parkinson's disease and other synucleinopathies.

Current Approaches for Predicting a Lack of Response to Anti-EGFR Therapy in KRAS Wild-Type Patients

Targeting epidermal growth factor receptor (EGFR) has been one of the most effective colorectal cancer strategies. Anti-EGFR antibodies function by binding to the extracellular domain of EGFR, preventing its activation, and ultimately providing clinical benefit. KRAS mutations in codons 12 and 13 are recognized prognostic and predictive biomarkers that should be analyzed at the clinic prior to the administration of anti-EGFR therapy. However, still an important fraction of KRAS wild-type patients do not respond to the treatment. The identification of additional genetic determinants of primary or secondary resistance to EGFR targeted therapy for further improving the selection of patients is urgent. Herein, we review the latest published literature highlighting the most important genes that may predict resistance to anti-EGFR monoclonal antibodies in colorectal cancer patients. According to the available findings, the evaluation of BRAF, NRAS, PIK3CA, and PTEN status could be the right strategy to select patients who are likely to respond to anti-EGFR therapies. In the future, the combination of those biomarkers will help establish consensus that can be introduced into clinical practice.

Identification of genes differentially expressed in wild type and Purkinje cell degeneration mice

Purkinje cell degeneration (pcd) mice are characterized by death of virtually all cerebellar Purkinje cells by postnatal day 30. In this study, we used DNA microarray analysis to investigate differences in gene expression between the brains of wild type and pcd mice on postnatal day 20, before the appearance of clear-cut phenotypic abnormalities. We identified 300 differentially expressed genes, most of which were involved in metabolic and physiological processes. Among the differentially expressed genes were several calcium binding proteins including calbindin -28k, paravalbumin, matrix gamma-carboxygluta mate protein and synaptotagamins 1 and 13, suggesting the involvement of abnormal Ca2+ signaling in the pcd phenotype.

Research on the ultrafast fluorescence property of thylakoid membranes of the wild-type and mutant rice

A high yielding rice variety mutant (Oryza sativa L., Zhenhui 249) with low chlorophyll b (Chl b) has been discovered in natural fields. It has a quality character controlled by a pair of recessive genes (nuclear gene). The partial loss of Chl b in content affects the efficiency of light harvest in a light harvest complex (LHC), thus producing the difference of the exciting energy transfer and the efficiency of photochemistry conversion between the mutant and wild-type rice in photosynthetic unit. The efficiency of utilizing light energy is higher in the mutant than that in the wildtype rice relatively. For further discussion of the above-mentioned difference and learning about the mechanism of the increase in the photochemical efficiency of the mutant, the pico-second resolution fluorescence spectrum measurement with delay-frame-scanning single photon counting technique is adopted. Thylakoid membranes of the mutant and the wild-type rice are excited by an Ar+ laser with a pulse width of 120 ps, repetition rate of 4 MHz and wavelength of 514 nm. Compared with the time and spectrum property of exciting fluorescence, conclusions of those ultrafast dynamic experiments are: 1) The speeds of the exciting energy transferred in photo-system I are faster than that in photo-system II in both samples. 2) The speeds of the exciting energy transfer of mutant sample are faster than those of the wildtype. This might be one of the major reasons why the efficiency of photosynthesis is higher in mutant than that in the wild-type rice.

Adenovirus-mediated wild-type P53 Transfer radiosensitizes H1299 cells to subclinical-dose carbon-ion irradiation through the restoration of p53 function

To determine whether adenovirus-mediated wild-type p53 transfer after radiotherapy could radiosensitize non-small-cell lung cancer (NSCLC) cells to subclinical-dose carbon-ion beam (C-beam), H1299 cells were exposed to a C-beam or -ray and then infected with 5 MOI of AdCMV-p53 or GFP (C-beam or -ray with p53 or GFP).Cell cycle was detected by flow cytometric analysis. The apoptosis was examined by a fluorescent microscope with DAPI staining. DNA fragmentation was monitored by the TUNEL assay. P53 mRNA was detected by reverse-transcriptase polymerase chain reaction. The expression of p53, MDM2, and p21 was monitored by Western blot. Survival fractions were determined by colony-forming assay. The percentages of G1-phase cells in C-beam with p53 increased by 8.2%–16.0%, 5.2%–7.0%, and 5.8%–18.9%, respectively, compared with C-beam only, -ray with p53, or p53 only. The accumulation of G2-phase cells in C-beam with p53 increased by 5.7%–8.9% and 8.8%–14.8%, compared with those in -ray with p53 or p53 only, respectively. The percentage of apoptosis for C-beam with p53 increased by 7.4%–19.1%, 5.8%–11.7%, and 5.2%–19.2%, respectively, compared with C-beam only, -ray with p53, or p53 only. The level of p53 mRNA in C-beam with p53 was significantly higher than that in p53 only. The expression level of p53 and p21 in C-beam with p53 was significantly higher than that in both C-beam with GFP and p53 only. The survival fractions for C-beam with p53 were significantly less than those for the other groups (p 0.05). The data suggested that AdCMV-p53 transfer could more efficiently radiosensitize H1299 cells to subclinical-dose C-beam irradiation through the restoration of p53 function.