Activation-Induced Cytidine Deaminase-Initiated Off-Target DNA Breaks Are Detected and Resolved during S Phase

Activation-induced cytidine deaminase (AID) initiates DNA double-strand breaks (DSBs) in the IgH gene (Igh) to stimulate isotype class switch recombination (CSR), and widespread breaks in non-Igh (off-target) loci throughout the genome. Because the DSBs that initiate class switching occur during the G(1) phase of the cell cycle, and are repaired via end joining, CSR is considered a predominantly G(1) reaction. By contrast, AID-induced non-Igh DSBs are repaired by homologous recombination. Although little is known about the connection between the cell cycle and either induction or resolution of AID-mediated non-Igh DSBs, their repair by homologous recombination implicates post-G(1) phases. Coordination of DNA breakage and repair during the cell cycle is critical to promote normal class switching and prevent genomic instability. To understand how AID-mediated events are regulated through the cell cycle, we have investigated G(1)-to-S control in AID-dependent genome-wide DSBs. We find that AID-mediated off-target DSBs, like those induced in the Igh locus, are generated during G(1). These data suggest that AID-mediated DSBs can evade G(1)/S checkpoint activation and persist beyond G(1), becoming resolved during S phase. Interestingly, DSB resolution during S phase can promote not only non-Igh break repair, but also Ig CSR. Our results reveal novel cell cycle dynamics in response to AID-initiated DSBs, and suggest that the regulation of the repair of these DSBs through the cell cycle may ensure proper class switching while preventing AID-induced genomic instability.     

The Outcome of the Oxidations of Unusual Enediamide Motifs Is Governed by the Stabilities of the Intermediate Iminium Ions

We compare the results from the oxidation of two unusual “enediamide” motifs (3,4-dihydropyrazin-2(1H)-ones), where a double bond is flanked by two amides. In one case the oxidation led to a ring-opened product arising from the cleavage of the double bond, and in the other a rare cis-dioxygenated compound was obtained. Both products have been characterized by X-ray crystallography. The outcomes of the key reactions are rationalized based on calculated free energies of intermediates.     

Forced expression of MMP9 rescues the loss of angiogenesis and abrogates metastasis of pancreatic tumors triggered by the absence of host SPARC

Pancreatic adenocarcinoma is characterized by desmoplasia, local invasion, and metastasis. These features are regulated in part by MMP9 and SPARC. To explore the interaction of SPARC and MMP9 in cancer, we first established orthotopic pancreatic tumors in SPARC-null and wild-type mice with the murine pancreatic adenocarcinoma cell line, PAN02. MMP9 expression was higher in tumors from wild-type compared to SPARC-null mice. Coincident with lower MMP9 expression, tumors grown in SPARC-null mice were significantly larger, had decreased ECM deposition and reduced microvessel density compared to wild-type controls. In addition, metastasis was enhanced in the absence of host SPARC. Therefore, we next analyzed the orthotopic tumor growth of PAN02 cells transduced with MMP9 or a control empty vector. Forced expression of MMP9 by the PAN02 cells resulted in larger tumors in both wild-type and SPARC-null animals compared to empty vector controls and further diminished ECM deposition. Importantly, forced expression of MMP9 within the tumor reversed the decrease in angiogenesis and abrogated the metastatic potential displayed by control tumors grown in SPARC-null mice. Finally, contrary to the in vivo results, MMP9 increased cell migration in vitro, which was blocked by the addition of SPARC. These results suggest that SPARC and MMP9 interact to regulate many stages of tumor progression including ECM deposition, angiogenesis and metastasis.     

Upregulation of Cdc2 and cyclin A during apoptosis of endothelial cells induced by cleaved high-molecular-weight kininogen

We (8) reported that the cleaved high-molecular-weight kininogen (HKa) and its domain 5 (D5) inhibited angiogenesis. Further studies (15) revealed that D5 could inhibit cell proliferation and induce apoptosis of proliferating endothelial cells, which together may represent a critical part of antiangiogenic activity of HKa and D5. In the present study, we further examined the effect of HKa on cell cycle progression and cell viability. We report that HKa induced a significant upregulation of Cdc2 and cyclin A in proliferating endothelial cells, concurrent with a marked increase of Cdc2 activity. The increased expression of Cdc2 and cyclin A by HKa was not associated with an apparent change in cell cycle profiles of basic fibroblast growth factor-stimulated proliferating cells, but closely correlated with a marked increase of apoptosis, suggesting that the elevated Cdc2 activity is involved in HKa-induced apoptosis of proliferating endothelial cells. Our results support an emerging hypothesis that Cdc2 and cyclin A are important regulators for cell cycle as well as for apoptosis.     

Role of the human immunodeficiency virus type 1 envelope gene in viral fitness

A human host offers a variety of microenvironments to the infecting human immunodeficiency virus type 1 (HIV-1), resulting in various selective pressures, most of them directed against the envelope (env) gene. Therefore, it seems evident that the replicative capacity of the virus is largely related to viral entry. In this study we have used growth competition experiments and TaqMan real-time PCR detection to measure the fitness of subtype B HIV-1 primary isolates and autologous env-recombinant viruses in order to analyze the contribution of wild-type env sequences to overall HIV-1 fitness. A significant correlation was observed between fitness values obtained for wild-type HIV-1 isolates and those for the corresponding env-recombinant viruses (r = 0.93; P = 0.002). Our results suggest that the env gene, which is linked to a myriad of viral characteristics (e.g., entry into the host cell, transmission, coreceptor usage, and tropism), plays a major role in fitness of wild-type HIV-1. In addition, this new recombinant assay may be useful for measuring the contribution of HIV-1 env to fitness in viruses resistant to novel antiretroviral entry inhibitors.     

Population Genetic Structure of Endemic and Endangered Yellow Catfish, Horabagrus brachysoma, Using Allozyme Markers

The allozyme variation and population genetic structure of Horabagrus brachysoma in three natural populations from the southern part of the Western Ghats region, India, were investigated by polyacrylamide gel electrophoresis. Variations at 14 loci from 14 enzyme systems were analyzed. The allozyme analysis revealed a high level of genetic variation in this species, with an average of observed alleles per locus of 2.357 and observed heterozygosity of 0.178. The positive value of the fixation index (FIS=0.507) implied a significant deficiency of heterozygosity at the population level. The highly significant probability (P<0.0001) for the overall loci suggested that the three sample sets were not part of the same gene pool.     

In vitro development of microcorms and stigma like structures in saffron (Crocus sativus L.)

Saffron is an important spice derived from the stigmas of Crocus sativus, a species belonging to the family Iridaceae. Due to its triploid nature it is sterile and is not able to set seeds, so it is propagated only by corms. The natural propagation rate of most geophytes including saffron is relatively low. An in vitro multiplication technique like micropropagation has been used for the propagation of saffron. In the present study, various explants were cultured on different nutrient media supplemented with various concentrations of plant growth regulators to standardize the best media combination for obtaining optimum response with respect to corm production and development of Stigma Like Structures (SLS). Highest response (60 %) was observed with half ovaries on G-5 media supplemented with 27 μM NAA and 44.4 μM BA followed by 55 % on LS media with 27 μM NAA and 44.4 μM BA. Maximum size (1.3 g) of microcorms were obtained from apical buds on the LS media supplemented with 21.6 μM NAA and 22.2 μM. Stigma Like Structures were developed from half ovary explants both directly and indirectly. Maximum number (120 indirectly and 20 directly) and size (5.2 cm) of SLS were obtained in G-5 medium supplemented with 27 μM NAA and 44.4 μM BA followed by 100 indirectly and 20 directly and 4.5 cm long on LS medium supplemented with 27 μM NAA and 44.4 μM BA.     

Help Seeking Behavior of Women with Self-Discovered Breast Cancer Symptoms: A Meta-Ethnographic Synthesis of Patient Delay

Background and Objective

Patient delay makes a critical contribution to late diagnosis and poor survival in cases of breast cancer. Identifying the factors that influence patient delay could provide information for adopting strategies that shorten this delay. The aim of this meta-ethnography was to synthesize existing qualitative evidence in order to gain a new understanding of help seeking behavior in women with self-discovered breast cancer symptoms and to determine the factors that influence patient delay.

Methods

The design was a meta-ethnography approach. A systematic search of the articles was performed in different databases including Elsevier, PubMed, ProQuest and SCOPUS. Qualitative studies with a focus on help seeking behaviors in women with self-discovered breast cancer symptoms and patient delay, published in the English language between 1990 and 2013 were included. The quality appraisal of the articles was carried out using the Critical Appraisal Skills Programme qualitative research checklist and 13 articles met the inclusion criteria. The synthesis was conducted according to Noblit and Hare’s meta-ethnographic approach (1988), through reciprocal translational analysis and lines-of-argument.

Findings

The synthesis led to identification of eight repeated key concepts including: symptom detection, initial symptom interpretation, symptom monitoring, social interaction, emotional reaction, priority of medical help, appraisal of health services and personal-environmental factors. Symptom interpretation is identified as the important step of the help seeking process and which changed across the process through active monitoring of their symptoms, social interactions and emotional reactions. The perceived seriousness of the situation, priority to receive medical attention, perceived inaccessibility and unacceptability of the health care system influenced women’s decision-making about utilizing health services.

Conclusion

Help seeking processes are influenced by multiple factors. Educational programs aimed at correcting misunderstandings, erroneous social beliefs and improving self-awareness could provide key strategies to improve health policy which would reduce patient delay.