Expedient Synthesis and Characterization of π-Extended Luciferins.

Bioluminescence imaging enables the sensitive tracking of cell populations and the visualization of biological processes in living systems. Bioluminescent luciferase/luciferin pairs with far-red and near-infrared emission benefit from the reduced competitive absorption by blood and tissue while also facilitating multiplexing strategies. Luciferins with extended π-systems, such as AkaLumine and recently reported CouLuc-1 and -3, can be used for bioluminescence imaging in this long wavelength regime. Existing synthetic routes to AkaLumine and similar π-extended compounds require a multistep sequence to install the thiazoline heterocycle. Here we detail the development of a two-step strategy for accessing these molecules via a Horner-Wadsworth-Emmons reaction and cysteine condensation sequence from readily available aldehyde starting materials. We detail an improved synthesis of AkaLumine, as well as the corresponding two-carbon homologues, Tri- and Tetra-AkaLumine. We then extended this approach to prepare coumarin- and naphthalene-derived luciferins. These putative luciferins were tested against a panel of luciferases to identify capable emitters. Of these, an easily prepared naphthalene derivative exhibits photon emission on par with that of the broadly used Akaluc/AkaLumine pair with similar emission maxima. Overall, this chemistry provides efficient access to several bioluminescent probes for a variety of imaging applications.

Impact of GSTT1 and GSTM1 Polymorphisms in the Susceptibility to Philadelphia Negative Chronic Myeloid Leukaemia.

BACKGROUND: Our research aimed to clarify the role of genetic polymorphisms in GST (T1 and M1) in the development of Ph-ve CML. MATERIALS AND METHODS: We report on a case-control study with 126 participants, divided into 26 patients with Ph-ve CML (57.7% male, 42.3% female) and 100 healthy volunteers (51% male, 49% female) with no medical history of cancer as a control population. All Ph-ve CML patients were diagnosed according to standard hematologic and cytogenetic criteria based on CBC, confirmed by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) to determine the presence or absence of the BCRABL gene, followed by bone marrow (BM) examination. RESULTS: Of the 26 studied cases, 50% had the GSTT1 null genotype against 21% of the control group, a statistically significant difference (CI= 1.519 - 9.317; p-value= 0.004). The GSTM1 null genotype was detected in 23.1% of cases and 35% of controls, a difference not statistically significant (OR= 0.557; CI= 0.205-1.515; p-value= 0.252). The distribution of GSTT1 and GSTM1 polymorphisms was also examined according to gender, age and ethnic grouping; these findings revealed no statistically significant differences. CONCLUSION: Our study reveals a strong correlation between GSTT1 polymorphism and Ph-ve CML, whereas the data for GSTM1 polymorphisms indicates no role in the initial development of the disease. More studies are required to further clarify these and other genes' roles in disease development.

Impact of Methionine Synthase Reductase Polymorphisms in Chronic Myeloid Leukemia Patients.

Introduction： Metabolism methionine and of folate play a vital function in cellular methylation reactions, DNA synthesis and epigenetic process.However, polymorphisms of methionine have received much attention in recent medical genetics research. Objectives: To ascertain whether the common polymorphisms of the MTRR (Methionine Synthase Reductase) A66G gene could play a role in affecting susceptibility to Chronic Myeloid Leukemia (CML) in Sudanese individuals. Methods: In a case-controlled study, we extracted and analyzed DNA from 200 CML patients and 100 healthy control subjects by the PCR-RFLP method. Results: We found no significant difference in age orgender between the patient group and controls. The MTRR A66G genotypes were distributed based on the Hardy-Weinberg equilibrium (p > 0.05). The variation of MTRR A66G was less significantly frequent in cases with CML (68.35%) than in controls (87%) (OR = 0.146, 95% CI = 0.162−0.662, p < 0.002). Additionally, AG and GG genotypes and G allele were reducing the CML risk (Odds ratio [OR] = 0.365; 95% CI [0.179−0.746]; p = 0.006; OR = 0.292; 95% CI [0.145−0.590]; p = 0.001 and OR = 0.146; 95% CI [0.162−0.662]; p = 0.002 and OR = 2.0; 95% CI [1.3853−2.817]; respectively, (p = 0.000)). Conclusions: Our data demonstrated that heterozygous and homozygous mutant genotypes of MTRR polymorphisms were associated with decreased risk of developing CML in the Sudanese population.

The Prognostic Value of Estrogen Receptor ß Isoform With Correlation of Estrogen Receptor α Among Sudanese Breast Cancer Patients.

Two estrogen receptor isoforms (ERα and ERß) have been characterized with variable and sometimes contrasting responses to estrogens, partially explained by different receptor signaling pathways in estrogen-sensitive tissues. This is a retrospective, descriptive, cross-sectional study, aiming to evaluate the expression pattern of ERß, employing immunohistochemical techniques using specific monoclonal antibody for ERß, to correlate its expression with that of ERα in a Sudanese population. Two-hundred and fifty formalin-fixed paraffin-wax-embedded breast tissue blocks were used in this study. Of these, 200 were taken from breast cancer patients ascertained as study cases, and the remaining 50 were noninvolved surgical margin considered as normal breast tissue. Receptor expression was demonstrated using immunohistochemical techniques. The immune expression of ERß was detected in 57.5% of breast cancers. It was differentially expressed in breast tissues encompassing normal, noninvasive, as well as invasive carcinoma (P = .02). There was no evidence of a significant relationship between ERß and ERα expression. Among the ERα-negative tumor, 60.4% expressed ERß. The expression of ERß among this subgroup was significantly associated with good clinicopathological parameters such as negative Her2/neu, lower grade, and negative lymph node metastasis (P = .002). This study concludes that ERß was commonly expressed among Sudanese patients with breast cancer, either co-expressed with ERα or expressed alone. In the ERα-negative subgroup, it was associated with better tumor outcomes suggesting ERß should be included in the diagnostic protocol as an independent marker for favorable prognosis.

Evaluation of Genetic Polymorphisms of N-acetyltransferase 2 and Relation with Chronic Myeloid Leukemia.

OBJECTIVES: The N-Acetyltransferase 2 (NAT2) gene encodes a key enzyme involved in xenobiotic metabolism, which contributes to the detoxification of numerous cancer therapy-induced products. However, the NAT2 genotype/phenotype is not fully understood and few studies have reported its relationship with CML. The aim of this study was to determine whether its polymorphisms (C481T, G590A, 803A>G and 857G>A) have a role in chronic myeloid leukemia susceptibility (CML) in Sudanese population. METHODS: We performed a case- control study. DNA from 200 CML patients and 100 controls was analyzed for the NAT2 polymorphisms using PCR-RFLP assay. RESULTS: The study showed NAT2 polymorphisms 803AG are associated with CML protection by a factor of 2.3, (OR = 0.044, 95% CI: 0.020-0.095, p = 0. 000). The study indicated that the heterozygous (GA) and mutant (AA) variants of the G857A genotype also offer protection, (OR = 0.002, 95% CI: 0.002-0.019, p = 0. 000) and (OR = 0.018, 95% CI: 0.002-0.133, p = 0. 000), respectively. CONCLUSION: There was no significant difference in CML diagnosis among Sudanese cases with the 481C→T and 590G→A polymorphisms. But patients with the compound NAT2 genotypes 481CT/803 AG, 590AG/ 803AG, 590AG/ 803GG, 590AA/ 803AG and 590GG/ 803AG were found to have a reduced risk. The current study demonstrates that polymorphisms of NAT2 A803G and G857A might also act as protective factors against developing the disease.

Structural Insights into How Protein Environments Tune the Spectroscopic Properties of a Noncanonical Amino Acid Fluorophore.

Genetically encoded fluorescent noncanonical amino acids (fNCAAs) could be used to develop novel fluorescent sensors of protein function. Previous efforts toward this goal have been limited by the lack of extensive physicochemical and structural characterizations of protein-based sensors containing fNCAAs. Here, we report the steady-state spectroscopic properties and first structural analyses of an fNCAA-containing Fab fragment of the 5c8 antibody, which binds human CD40L. A previously reported 5c8 variant in which the light chain residue IleL98 is replaced with the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) exhibits a 1.7-fold increase in fluorescence upon antigen binding. Determination and comparison of the apparent pKas of 7-HCAA in the unbound and bound forms indicate that the observed increase in fluorescence is not the result of perturbations in pKa. Crystal structures of the fNCAA-containing Fab in the apo and bound forms reveal interactions between the 7-HCAA side chain and surrounding residues that are disrupted upon antigen binding. This structural characterization not only provides insight into the manner in which protein environments can modulate the fluorescence properties of 7-HCAA but also could serve as a starting point for the rational design of new fluorescent protein-based reporters of protein function.

CYP1A1 and CYP2D6 Polymorphisms and Susceptibility to Chronic Myelocytic Leukaemia.

BACKGROUND: CYP1A1 and CYP2D6 are both xenobiotic metabolizing enzymes belonging to the CYP450 enzyme family. Polymorphisms in these genes vary between individuals, resulting in dissimilar patterns of susceptibility to the effects of carcinogenic substances and drugs. OBJECTIVE: In a prospective study, the influence of CYP1A1*2C and CYP2D6*4 gene polymorphisms on the susceptibility to chronic myelocytic leukaemia (CML) were investigated. METHODS: Prevalence of CYP1A1*2C and CYP2D6*4 was detected in blood specimens from three hundred participants - two hundred patients and a hundred healthy individuals as a control group, using PCR-RFLP. RESULTS: CYP1A1 Ile/Val and Val/Val genotype frequency in our study population was 82% & 15% in CML patients and 55% & 8% in controls, respectively. This suggests that carriers had an elevated risk (OR=18.38, 95% CI=7.364-45.913, p value; =0.000 and OR=23.125,95 % CI=7.228-73.980, p value=0.000, respectively). Individuals carrying the CYP2D6 heterozygous genotype (IM) were notably fewer in number within the CML group at 43.5%, as opposed to 93% of the controls. This suggests that the IM genotype may have a prophylactic function in lowering CML risk (OR=0.036, 95% CI=0.005-0.271, p value =0.001). In spite of the distribution of the homozygous mutant (PM) genotype being higher in cases with CML (87% as opposed to 6% in the control), this difference was deemed non-significant (OR=0.558, 95% CI=0.064-4.845, p value =0.597). CONCLUSION: These findings indicate that polymorphic CYP1A1 and CYP2D6 genes affect the susceptibility to CML.

Genetic Polymorphism of GSTP1, GSTM1 and GSTT1 Genes and Susceptibility to Chronic Myeloid Leukaemia.

BACKGROUND: The development of cancer results from an imbalance between exposure to carcinogens and the capacity of various enzyme systems engaged in activation or in the detoxification of xenobiotics. The aim of the present study is to investigate the association of GSTP1, GSTM1 and GSTT1 gene polymorphisms in susceptibility to Chronic Myeloid Leukaemia (CML). METHODS: A total of 200 CML patients and 100 controls were enrolled in a case-control study with GSTM1 and GSTT1 analysis with PCR and GSTP1 analysis with PCR-RFLP. RESULTS: The GSTT1 null genotype was significantly higher among CML patients suggesting that this genotype is associated with an increased risk of CML. It was found in 42% of cases as compared with 21% of the controls, (OR =2.78, 95% CI: 1.59 - 4.85; p-value =0.000). The presence of the GSTT1 genotype may thus be considered a protective factor for CML. The frequency of individuals carrying GSTM1 null genotype was slightly higher in the control group but this difference was not statistically significant. The GSTM1 null genotype was present in 35% of control cases and 34% of the CML patients, (OR=0.975, 95%CI: 0.58-1.58;p-value=0.863). Individuals with a combined GSTM1 null/GSTT1null genotype had an estimated 2.85-fold increased risk of CML, but no associated risk between GSTP1 Ile 105 Val polymorphism and CML was found (OR=1.99, 95% CI: 0.40 - 9.32; p-value = 0.417). CONCLUSIONS: No association between GSTP1 and GSTM1 with susceptibility to CML was found. GSTT1 genotype may be a protective factor for CML, while the null genotype shows association with developing CML.
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Omega 3 (n-3) fatty acids down-regulate nuclear factor-kappa B (NF-κB) gene and blood cell adhesion molecule expression in patients with homozygous sickle cell disease.

Chronic inflammation and reduced blood levels of omega-3 fatty acids (n-3) are known characteristics of sickle cell disease (SCD).The anti-inflammatory properties of n-3 fatty acids are well recognized. Omega-3 treated (n = 24), hydroxyurea (HU) treated (n = 18), and n-3 untreated (n=21) homozygous SCD patients (HbSS) and healthy (HbAA) controls (n = 25) matched for age (5-16 years), gender and socioeconomic status were studied. According to age (5-10) or (11-16) years, two or three capsules containing 277.8 mg docosahexaenoic (DHA) and 39.0mg eicosapentaenoic (EPA) or high oleic acid placebo (41%) were assigned to n-3 treated and n-3 untreated groups, respectively. Hydroxyurea treated group was on dosage more than 20 mg/kg/day. The effect of supplementation on systemic and blood cell markers of inflammation was investigated. The n-3 treated group had higher levels of DHA and EPA (p < 0.001) and lower white blood cell count and monocyte integrin (p < 0.05) compared with the n-3 untreated. No difference was detected between the two groups regarding C-reactive protein, granulocytes integrin and selectin, plasma tumour necrosis factor-α and interleukin-10. The n-3 treated group had lowered nuclear factor-kappa B (NF-κB) gene expression compared to n-3 untreated and HU treated groups (p < 0.05). This study provides evidence that supplementation with n-3 fatty acids may ameliorate inflammation and blood cell adhesion in patients with SCD.

BRCA 1 & 2 mutations in Sudanese secondary school girls with known breast cancer in their families.

OBJECTIVE: Breast cancer is a major cause of morbidity and mortality in women worldwide. In Sudan, it is the most commonly diagnosed cancer. This study assesses the prevalence of BRCA1 and BRCA2 mutations among female students with a family history of breast cancer, in secondary schools of Marawi Locality, Northern State, Sudan. METHODS: From a survey of 2370 students, 67 cases (47 with family history and 20 controls) were analyzed for BRCA1 and BRCA2 mutations with a single-stranded conformation polymorphism (SSCP) mutation detection method applied to peripheral blood. Eighteen subjects knew of first degree female relatives with breast cancer, 23 with second degree female family members affected and 6 with related male sufferers. Twenty randomly selected girls from the remainder of the survey population with no known family history were also tested. RESULTS: The breast cancer susceptibility genes BRCA1 and BRCA2 accounted, respectively, for 1.21% of responders or 51% of those claiming a family history. Mutations were found in 20% of the group selected with no family history. Only 2 BRCA 2 mutations were found, both in girls with no known afflicted relatives. Six girls knew of male relatives with breast cancer; five of these girls carried mutant BRCA 1. Most of the BRCA1- mutations located to exon 11 fragments 11.9 and 11.1. CONCLUSION: The study indicates a high prevalence of genetically associated breast cancer in the Marawi locality suggesting a need to focus on the two mutation sites in developing screening protocols for at least this area of Sudan.

Characterisation of CYP2C8, CYP2C9 and CYP2C19 polymorphisms in a Ghanaian population.

BACKGROUND: Genetic influences on drug efficacy and tolerability are now widely known. Pharmacogenetics has thus become an expanding field with great potential for improving drug efficacy and reducing toxicity. Many pharmacologically-relevant polymorphisms do show variability among different populations. Knowledge of allelic frequency distribution within specified populations can be useful in explaining therapeutic failures, identifying potential risk groups for adverse drug reactions (ADRs) and optimising doses for therapeutic efficacy. We sought to determine the prevalence of clinically relevant Cytochrome P450 (CYP) 2C8, CYP2C9, and CYP2C19 variants in Ghanaians. We compared the data with other ethnic groups and further investigated intra country differences within the Ghanaian population to determine its value to pharmacogenetics studies. METHODS: RFLP assays were used to genotype CYP2C8 (*2, *3, *4) variant alleles in 204 unrelated Ghanaians. CYP2C9*2 and CYP2C19 (*2 and *3) variants were determined by single-tube tetra-primer assays while CYP2C9 (*3, *4, *5 and *11) variants were assessed by direct sequencing. RESULTS: Allelic frequencies were obtained for CYP2C8*2 (17%), CYP2C8*3 (0%), CYP2C8*4 (0%), CYP2C9*2 (0%), CYP2C9*3 (0%), CYP2C9*4 (0%), CYP2C9*5 (0%), CYP2C9*11 (2%), CYP2C19*2 (6%) and CYP2C19*3 (0%). CONCLUSION: Allele frequency distributions for CYP2C8, CYP2C9 and CYP2C19 among the Ghanaian population are comparable to other African ethnic groups but significantly differ from Caucasian and Asian populations. Variant allele frequencies for CYP2C9 and CYP2C19 are reported for the first time among indigenous Ghanaian population.

Protein evolution with an expanded genetic code.

We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in V(H) CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded "synthetic" genetic code can confer a selective advantage in the directed evolution of proteins with specific properties.

An evaluation of the Psychache Scale on an offender population.

This study examined the generalizability of a self-report measure of psychache to an offender population. The factor structure, construct validity, and criterion validity of the Psychache Scale was assessed on 136 male prison inmates. The results showed the Psychache Scale has a single underlying factor structure and to be strongly associated with measures of depression and hopelessness and moderately associated with psychiatric symptoms and the criterion variable of a history of prior suicide attempts. The variables of depression, hopelessness, and psychiatric symptoms all contributed unique variance to psychache. Discussion centers on psychache's theoretical application to the prediction of suicide.