Synthesis and spectroscopic studies of functionalized graphene quantum dots with diverse fluorescence characteristics.

In this research, we report a facile method for synthesizing a series of carboxyl functionalized graphene quantum dots (GQDs) using graphite flakes (300 meshes) as raw material. These highly luminescent GQDs emitted blue, light blue, green, yellow, and red light (400-700 nm intensity peaks) under ultraviolet irradiation conditions, while exhibiting quantum yields in the range of 50-70%. The products were comprehensively characterized using ultraviolet-visible, photoluminescence, infrared, Raman, and dynamic light scattering spectroscopies. The GQDs were found to remain highly stable against photobleaching when stored over a prolonged period of more than three months. The proposed method for the synthesis of high quality, multicolor GQDs can be utilized to extend the application of these nanoparticles to molecular biotechnology and bioengineering; for example, the immobilization of cancer markers on their surface. As such, carboxylic acid groups present on the surface of these GQDs help create complexes for in vivo sensing applications.

Prevalence of genetic variants associated with cardiovascular disease risk and drug response in the Southern Indian population of Kerala.

BACKGROUND AND AIM: This study reports the prevalence of five clinically significant variants associated with increased risk of cardiovascular disorders, and variable responses of individuals to commonly prescribed cardiovascular drugs in a South Indian population from the state of Kerala. MATERIALS AND METHODS: Genomic DNA isolated from 100 out-patient samples from Kerala were sequenced to examine the frequency of clinically relevant polymorphisms in the genes MYBPC3 (cardiomyopathy), SLCO1B1 (statin-induced myopathy), CYP2C9, VKORC1 (response to warfarin) and CYP2C19 (response to clopidogrel). RESULTS: Our analyses revealed the frequency of a 25 bp deletion variant of MYBPC3 associated with risk of cardiomyopathy was 7%, and the SLCO1B1 "C" allele associated with risk for statin-induced myopathy was 15% in this sample group. Among the other variants associated with dose-induced toxicity of warfarin, VKORC1 (c.1639G>A), was detected at 22%, while CYP2C9*3 and CYP2C9*2 alleles were present at a frequency of 15% and 3% respectively. Significantly, the tested sample population showed high prevalence (66%) of CYP2C19*2 variant, which determines response to clopidogrel therapy. CONCLUSIONS: We have identified that certain variants associated with cardiovascular disease and related drug response in the five genes, especially those in VKORC1, CYP2C19 and MYBPC3, are highly prevalent in the Kerala population, with almost 2 times higher prevalence of CYP2C19*2 variant compared with other regions in the country. Since the variants chosen in this study have relevance in disease phenotype and/or drug response, and are detected at a higher frequency, this study is likely to encourage clinicians to perform genetic testing before prescribing therapy.

Sequencing and analysis of a South Asian-Indian personal genome.

BACKGROUND: With over 1.3 billion people, India is estimated to contain three times more genetic diversity than does Europe. Next-generation sequencing technologies have facilitated the understanding of diversity by enabling whole genome sequencing at greater speed and lower cost. While genomes from people of European and Asian descent have been sequenced, only recently has a single male genome from the Indian subcontinent been published at sufficient depth and coverage. In this study we have sequenced and analyzed the genome of a South Asian Indian female (SAIF) from the Indian state of Kerala. RESULTS: We identified over 3.4 million SNPs in this genome including over 89,873 private variations. Comparison of the SAIF genome with several published personal genomes revealed that this individual shared ~50% of the SNPs with each of these genomes. Analysis of the SAIF mitochondrial genome showed that it was closely related to the U1 haplogroup which has been previously observed in Kerala. We assessed the SAIF genome for SNPs with health and disease consequences and found that the individual was at a higher risk for multiple sclerosis and a few other diseases. In analyzing SNPs that modulate drug response, we found a variation that predicts a favorable response to metformin, a drug used to treat diabetes. SNPs predictive of adverse reaction to warfarin indicated that the SAIF individual is not at risk for bleeding if treated with typical doses of warfarin. In addition, we report the presence of several additional SNPs of medical relevance. CONCLUSIONS: This is the first study to report the complete whole genome sequence of a female from the state of Kerala in India. The availability of this complete genome and variants will further aid studies aimed at understanding genetic diversity, identifying clinically relevant changes and assessing disease burden in the Indian population.

The splicing-factor related protein SFPQ/PSF interacts with RAD51D and is necessary for homology-directed repair and sister chromatid cohesion.

DNA double-stranded breaks (DSBs) are among the most severe forms of DNA damage and responsible for chromosomal translocations that may lead to gene fusions. The RAD51 family plays an integral role in preserving genome stability by homology directed repair of DSBs. From a proteomics screen, we recently identified SFPQ/PSF as an interacting partner with the RAD51 paralogs, RAD51D, RAD51C and XRCC2. Initially discovered as a potential RNA splicing factor, SFPQ was later shown to have homologous recombination and non-homologous end joining related activities and also to bind and modulate the function of RAD51. Here, we demonstrate that SFPQ interacts directly with RAD51D and that deficiency of both proteins confers a severe loss of cell viability, indicating a synthetic lethal relationship. Surprisingly, deficiency of SFPQ alone also leads to sister chromatid cohesion defects and chromosome instability. In addition, SFPQ was demonstrated to mediate homology directed DNA repair and DNA damage response resulting from DNA crosslinking agents, alkylating agents and camptothecin. Taken together, these data indicate that SFPQ association with the RAD51 protein complex is essential for homologous recombination repair of DNA damage and maintaining genome integrity.

RAD51D protects against MLH1-dependent cytotoxic responses to O(6)-methylguanine.

S(N)1-type methylating agents generate O(6)-methyl guanine (O(6)-meG), which is a potently mutagenic, toxic, and recombinogenic DNA adduct. Recognition of O(6)-meG:T mismatches by mismatch repair (MMR) causes sister chromatid exchanges, which are representative of homologous recombination (HR) events. Although the MMR-dependent mutagenicity and toxicity caused by O(6)-meG has been studied, the mechanisms of recombination induced by O(6)-meG are poorly understood. To explore the HR and MMR genetic interactions in mammals, we used the Rad51d and Mlh1 mouse models. Ablation of Mlh1 did not appreciably influence the developmental phenotypes conferred by the absence of Rad51d. Mouse embryonic fibroblasts (MEFs) deficient in Rad51d can only proliferate in p53-deficient background. Therefore, Rad51d(-/-)Mlh1(-/-)Trp53(-/-) MEFs with a combined deficiency of HR and MMR were generated and comparisons between MLH1 and RAD51D status were made. To our knowledge, these MEFs are the first mammalian model system for combined HR and MMR defects. Rad51d-deficient MEFs were 5.3-fold sensitive to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) compared to the Rad51d-proficient MEFs. A pronounced G2/M arrest in Rad51d-deficient cells was accompanied by an accumulation of gamma-H2AX and apoptosis. Mlh1-deficient MEFs were resistant to MNNG and showed no G2/M arrest or apoptosis at the doses used. Importantly, loss of Mlh1 alleviated sensitivity of Rad51d-deficient cells to MNNG, in addition to reducing gamma-H2AX, G2/M arrest and apoptosis. Collectively, the data support the hypothesis that MMR-dependent sensitization of HR-deficient cells is specific for O(6)-meG and suggest that HR resolves DNA intermediates created by MMR recognition of O(6)-meG:T. This study provides insight into recombinogenic mechanisms of carcinogenesis and chemotherapy resulting from O(6)-meG adducts.

The interaction profile of homologous recombination repair proteins RAD51C, RAD51D and XRCC2 as determined by proteomic analysis.

The RAD51 family of proteins is involved in homologous recombination (HR) DNA repair and maintaining chromosome integrity. To identify candidates that interact with HR proteins, the mouse RAD51C, RAD51D and XRCC2 proteins were purified using bacterial expression systems and each of them used to co-precipitate interacting partners from mouse embryonic fibroblast cellular extracts. Mass spectroscopic analysis was performed on protein bands obtained after 1-D SDS-PAGE of co-precipitation eluates from cell extracts of mitomycin C treated and untreated mouse embryonic fibroblasts. Profiling of the interacting proteins showed a clear bias toward nucleic acid binding and modification proteins. Interactions of four candidate proteins (SFPQ, NONO, MSH2 and mini chromosome maintenance protein 2) were confirmed by Western blot analysis of co-precipitation eluates and were also verified to form ex vivo complexes with RAD51D. Additional interacting proteins were associated with cell division, embryo development, protein and carbohydrate metabolism, cellular trafficking, protein synthesis, modification or folding, and cell structure or motility functions. Results from this study are an important step toward identifying interacting partners of the RAD51 paralogs and understanding the functional diversity of proteins that assist or regulate HR repair mechanisms.

Functional characterization and identification of mouse Rad51d splice variants.

BACKGROUND: The homologous recombination (HR) pathway is vital for maintaining genomic integrity through the restoration of double-stranded breaks and interstrand crosslinks. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) are essential for this process in vertebrates, and the RAD51D paralog is unique in that it participates in both HR repair and telomere maintenance. RAD51D is also known to directly interact with the RAD51C and XRCC2 proteins. Rad51d splice variants have been reported in mouse and human tissues, supportive of a role for alternative splicing in HR regulation. The present study evaluated the interaction of the Rad51d splice isoform products with RAD51C and XRCC2 and their expression patterns. RESULTS: Yeast-2-hybrid analysis was used to determine that the Mus musculus Rad51d splice variant product RAD51DDelta7b (deleted for residues 219 through 223) was capable of interacting with both RAD51C and XRCC2 and that RAD51D+int3 interacted with XRCC2. In addition, the linker region (residues 54 through 77) of RAD51D was identified as a region that potentially mediates binding with XRCC2. Cellular localization, detected by EGFP fusion proteins, demonstrated that each of the splice variant products tested was distributed throughout the cell similar to the full-length protein. However, none of the splice variants were capable of restoring resistance of Rad51d-deficient cell lines to mitomycin C. RT-PCR expression analysis revealed that Rad51dDelta3 (deleted for exon 3) and Rad51dDelta5 (deleted for exon 5)transcripts display tissue specific expression patterns with Rad51dDelta3 being detected in each tissue except ovary and Rad51dDelta5 not detected in mammary gland and testis. These expression studies also led to the identification of two additional Rad51d ubiquitously expressed transcripts, one deleted for both exon 9 and 10 and one deleted for only exon 10. CONCLUSION: These results suggest Rad51d alternative splice variants potentially modulate mechanisms of HR by sequestering either RAD51C or XRCC2.

Functional characterization of the RAD51D E233G genetic variant.

BACKGROUND AND OBJECTIVE: RAD51D, a paralog of the mammalian RAD51 gene, is an important component for DNA repair and telomere maintenance. A RAD51D variant, E233G, was initially identified as a potential susceptibility allele in high-risk, site-specific, familial breast cancer. We describe in this report, the effects of this amino acid change on RAD51D protein interaction and function. METHODS AND RESULTS: To examine the effect of the variant on cellular resistance to DNA damage, a complementation analysis by using Rad51d-deficient mouse embryonic fibroblasts was performed. Results indicated that the E233G variant actually increased the cellular resistance to the DNA-damaging agents, mitomycin C, cisplatin, methyl methane sulfonate, and ultraviolet light as well as to taxol. In addition, the E233G variant reduced the anaphase bridge index, a telomere dysfunction correlate, and conferred increased cellular proliferation, suggesting that the E to G substitution may affect telomere function. Yeast two-hybrid analyses demonstrated that interaction between RAD51C and RAD51D (E233G) was decreased by two fold, whereas normal levels of interaction between XRCC2 and the variant were maintained. Molecular modeling suggested that the glutamic acid-233 forms a salt bridge with lysine-23 in the N-terminal domain of RAD51D, and the glycine substitution may disrupt an interdomain interaction. CONCLUSION: Our findings suggest that the E233G variant affects RAD51D functions and protein interactions and increases cellular chemoresistance. This study is the first to analyze the functional effects of a clinically relevant RAD51D amino acid substitution. Further study of this variant will provide mechanistic insight into the role of RAD51D in cellular response to anticancer agents and as a molecular target for cancer therapy.

Cell cycle regulation in mammalian germ cells.

Meiosis is a unique form of cellular division by which a diploid cell produces genetically distinct haploid gametes. Initiation and regulation of mammalian meiosis differs between the sexes. In females, meiosis is initiated during embryo development and arrested shortly after birth during prophase I. In males, spermatogonial stem cells initiate meiosis at puberty and proceed through gametogenesis with no cell cycle arrest. Mouse genes required for early meiotic cell cycle events are being identified by comparative analysis with other eukaryotic systems, by virtue of gene knockout technology and by mouse mutagenesis screens for reproductive defects. This review focuses on mouse reproductive biology and describes the available mouse mutants with defects in the early meiotic cell cycle and prophase I regulatory events. These research tools will permit rapid advances in such medically relevant research areas as infertility, embryo lethality and developmental abnormalities.

The ATPase motif in RAD51D is required for resistance to DNA interstrand crosslinking agents and interaction with RAD51C.

Homologous recombination (HR) is a mechanism for repairing DNA interstrand crosslinks and double-strand breaks. In mammals, HR requires the activities of the RAD51 family (RAD51, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1), each of which contains conserved ATP binding sequences (Walker Motifs A and B). RAD51D is a DNA-stimulated ATPase that interacts directly with RAD51C and XRCC2. To test the hypothesis that ATP binding and hydrolysis by RAD51D are required for the repair of interstrand crosslinks, site-directed mutations in Walker Motif A were generated, and complementation studies were performed in Rad51d-deficient mouse embryonic fibroblasts. The K113R and K113A mutants demonstrated a respective 96 and 83% decrease in repair capacity relative to wild-type. Further examination of these mutants, by yeast two-hybrid analyses, revealed an 8-fold reduction in the ability to associate with RAD51C whereas interaction with XRCC2 was retained at a level similar to the S111T control. These cell-based studies are the first evidence that ATP binding and hydrolysis by RAD51D are required for efficient HR repair of DNA interstrand crosslinks.

Gene knockouts that cause female infertility: search for novel contraceptive targets.

The gene knockout technology has revolutionized the fertility/infertility field. It has revealed several essential previously undiscovered molecules, new insights and novel mechanisms involved in steps of the fertility cascade in females. Using database and literature search, knockouts of at least 83 genes were discovered that demonstrated an effect on fertility of female mice. These effects ranged from abnormality in reproductive structure, ovarian function, oocyte, fertilization, embryonic and fetal development, implantation and pregnancy to delivery. However, only a few of these knockout of genes such as encoding oocyte glycoprotein coat comprised of zona pellucida (ZP) 1, ZP2 and ZP3 and oocute plasma membrane specific proteins showed a specific and exclusive target infertility effect without concomitant effects on the non-reproductive organ system. These molecules will provide novel targets of contraception including contraceptive vaccine development.

Passive immunization for immunocontraception: lessons learned from infectious diseases.

Development of vaccine for contraception is an exciting proposition that could provide a valuable alternative to the presently available methods for birth control. Various targets such as gonadotropin releasing hormone (GnRH), follicle stimulating hormone (FSH), leutinizing hormone (LH), zona pellucida (ZP) antigens, sperm antigens, and human chorionic gonadotropin (hCG) are being explored for immunocontraception. Besides specific concerns associated with each contraceptive vaccine, the progress has been restricted by the variability of the immune response after active immunization, attain and maintain high antibody titers, time lag to achieve reasonably good antibody titers, and uncertainty regarding how long the bioeffective antibodies will remain in circulation. It is envisaged that these concerns may be taken care of by using the preformed antibodies in the passive immunization approach. The antibody therapies have been tried and found to be successful against various infectious diseases both in animals as well as humans. Some have become treatment modalities in the clinics. This manuscript will review the data available for the passive immunization of preformed polyclonal and murine/humanized/human monoclonal antibodies, their efficacy, mode of delivery, duration of the effects, and limitations, if any. The overall objective is to examine the feasibility and practicability of the passive immunization approach for immunocontraception.

Cloning and sequencing of a cDNA encoding for a protein specifically expressed in human testis, ovary and placenta.

Reproductive cell/tissue-specific antigens are attractive candidates for the development of a contraceptive vaccine. Using the differential display technology, a human cDNA fragment of 322-bp, designated as T17, was identified showing specific expression in the human testis. The T17 cDNA fragment was used as a probe to screen the human testis cDNA-lambda gt10 library. After screening, one positive clone of approximately 1.1 kb having T17 nucleotide (nt) sequence was obtained. The 5' and 3' termini of this cDNA clone were extended by using 5' and 3' RACE procedures that yielded a full-length cDNA, designated as the TOP gene. The TOP cDNA is 1,480-bp long and has an ORF of 463 aa with the first ATG Met start codon at nucleotide (nt) 64 and the stop codon TGA at nt 1452. The translated protein has a calculated molecular mass of 49.3 kD with isoelectric point of 12.17. The deduced amino acid (aa) has one potential N-glycosylation site, and several phosphorylation and myristoylation sites. Hydropathy plot generated from the deduced aa sequence showed it to be a surface protein. Comprehensive computer search in the database did not reveal any homology to any existing sequences both at the nt and aa levels. The TOP cDNA was found to be completely localized on the human chromosome 16 at the nt position 507833-506354, with the TOP ORF at the nt position 507770-506354. Northern blot analysis using three human Northern blots, indicated the specific expression of TOP gene in the human testis, ovary and placenta. RT-PCR-Southern blot analysis also confirmed tissue-specific expression of the TOP gene. The TOP cDNA may help us to gain insight into transcriptional control of the differentially expressed reproductive tissue-specific genes. It may also find clinical applications in the development of a contraceptive vaccine, and specific diagnosis and treatment of infertility in humans.