Systematic Study on Genetic and Epimutational Profile of a Cohort of Amsterdam Criteria-Defined Lynch Syndrome in Singapore

Background

Germline defects of mismatch repair (MMR) genes underlie Lynch Syndrome (LS). We aimed to gain comprehensive genetic and epigenetic profiles of LS families in Singapore, which will facilitate efficient molecular diagnosis of LS in Singapore and the region.

Methods

Fifty nine unrelated families were studied. Mutations in exons, splice-site junctions and promoters of five MMR genes were scanned by high resolution melting assay followed by DNA sequencing, large fragment deletions/duplications and promoter methylation in MLH1, MSH2, MSH6 and PMS2 were evaluated by multiplex ligation-dependent probe amplification. Tumor microsatellite instability (MSI) was assessed with five mononucleotide markers and immunohistochemical staining (IHC) was also performed.

Results

Pathogenic defects, all confined to MLH1 and MSH2, were identified in 17 out of 59 (28.8%) families. The mutational spectrum was highly heterogeneous and 28 novel variants were identified. One recurrent mutation in MLH1 (c.793C>T) was also observed. 92.9% sensitivity for indication of germline mutations conferred by IHC surpassed 64.3% sensitivity by MSI. Furthermore, 15.6% patients with MSS tumors harbored pathogenic mutations.

Conclusions

Among major ethnic groups in Singapore, all pathogenic germline defects were confined to MLH1 and MSH2. Caution should be applied when the Amsterdam criteria and consensus microsatellite marker panel recommended in the revised Bethesda guidelines are applied to the local context. We recommend a screening strategy for the local LS by starting with tumor IHC and the hotspot mutation testing at MLH1 c.793C>T followed by comprehensive mutation scanning in MLH1 and MSH2 prior to proceeding to other MMR genes.     

Genomic Stability of Lyophilized Sheep Somatic Cells before and after Nuclear Transfer

The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.     

Comparison of Anti-HBV Activity of β-D- and β-L-ddA-5′Monophosphate Prodrugs and Effectiveness in Combination with Lamivudine

Abstract

We have investigated the effects of several β-D-ddA 5′-monophospate (β-D-ddAMP), and their corresponding β-L-enantiomers prodrugs against HBV replication. All ddAMP prodrugs inhibited HBV replication in a dose-dependent manner.     

Microencapsulation in Na-alginate and in vitro development of sheep blastomeres]

Embryos at 4 cell stage obtained from Sarda ewes superovulated with FSHp (Sigma) were micromanipulated in order to obtain single blastomeres (1/4 E). The 1/4 E have been located randomly in two groups. In the first (Group A n. 30) the 1/4 E have been put back in empty zonae pellucidae; in the second (Group B n. 21) they have been microencapsulated in sodium alginate (1.1%) by dropping cell-alginate solution in a 1.5% CaCl2. Each capsule (1 mm diameter) contained four 1/4 E. The blastomeres have been co-cultured for 5 days in CZB medium on oviductal cell monolayer in a humidified incubator (5% CO2, 95% air, 38.5 degrees C). No differences were found between the groups reaching blastocyst stage after the end of the culture period (A 50%-B 47%).     

Use of a protein-free medium for the in vitro development of sheep embryos]

The employment of protein-free medium for the culture of ovine embryos collected at the 1-2 cell stage from superovulated ewes was investigated. For this purpose sheep zygotes were randomly allocated in four treatment groups: T1) CZB medium + bovine serum albumin (BSA) on sheep oviductal monolayer (SOM), T2) CZB + polyvinyl alcohol (PVA) + SOM, T3) CZB + PVA + SOM supplemented with inositol (I) and serine (S), T4) TCM 199 + 10% fetal calf serum + SOM. Standard culture conditions were 2 ml of medium in 35 mm Petri dishes, under 5% CO2 in air at 39 degrees C. The percentages of morulae and blastocysts were recorded after 4 and 7 days of culture. After 4 days of culture there was no significant difference (P > 0.05) in the percentage of morulae between embryos cultured in T1 (86%), T2 (85%), T3 (88.8%), and T4 (87.5%). After 7 days the percentages of blastocysts were T1 (70%), T2 (50%), T3 (55.5%) and T4 (46.8%). These data suggest that a protein-free medium, CZB + PVA and CZB + PVA + I + S, can support ovine preimplantation embryo development in vitro; however CZB medium supplemented with BSA enhances development to blastocyst.     

Influence of shifts over an 80-year period in forest composition on soil properties

Background and aims

Forest composition in North America has undergone important changes since the European settlement. The effects of such alterations on soil properties remain largely unknown. This study aims to understand the long-term effects of shifts in forest composition on soil properties.

Methods

Using data from 130 plots measured over an eighty-year period, the relationships between stand composition (both current and past), parent material and current soil chemical properties were studied with redundancy analyses.

Results

Results indicated that the parent material remained the dominant factor explaining soil properties, followed by current tree species composition. No legacy effect of past forest composition was found, but shifts in forest composition explained part of the current soil properties. Specifically, an increase in balsam fir was related to higher C/N ratio in the O-horizon, while an increase in maple species was related to higher net nitrification in both the O and B-horizons, and higher extractable P in the B-horizon.

Conclusion

Our results suggest that increasing the maple component at the expense of conifers over several decades may enhance nutrient availability in the O-horizon.

     

Nitric oxide is inactivated by the bacterial pigment pyocyanin.

Pyocyanin is a phenazine pigment produced by the bacterium Pseudomonas aeruginosa and found in human lung secretions. Micromolar concentrations of pyocyanin inhibited the bioactivity of endothelium-derived relaxing factor (EDRF) generated from bovine pulmonary-artery endothelium in response to bradykinin. This inhibition was reversed by perfusing the EDRF-bioassay system with pyocyanin-free buffer for 15 min, but persisted in the presence of superoxide dismutase (20 units/ml). When nitric oxide, the major component of EDRF, was passed into an aqueous solution of pyocyanin in the absence of O2, a rapid colour change occurred from blue to pink; m.s. analysis of the products showed that the pyocyanin had been converted into a nitrosylated species.     

Development of (1-->3,1-->4)-beta-d-Glucan Endohydrolase Isoenzymes in Isolated Scutella and Aleurone Layers of Barley (Hordeum vulgare)

An immunological assay has been used to investigate the synthesis of (1→3,1→4)-β-glucanase (EC 3.2.1.73) isoenzymes from isolated barley aleurone layers and scutella. Enzyme release from both tissues is enhanced by 1 micromolar gibberellic acid and 10 millimolar Ca²⁺, although increases induced by gibberellic acid are observed only in the presence of Ca²⁺. Isoenzyme I is synthesized predominantly in the scutellum, while isoenzyme II is synthesized exclusively in the aleurone. A third, putative isoenzyme III has been detected in significant proportions in scutellar secretions and may also be secreted from aleurone layers. Both gibberellic acid and Ca²⁺ appear to preferentially enhance isoenzyme II secretion from the aleurone and isoenzyme III secretion from scutella. The patterns of isoenzyme secretion are suggestive of tissue-specific differences in expression of the genes which code for (1→3,1→4)-β-glucanase isoenzymes. Qualitatively similar results were obtained with barley cultivars harvested in Australia and North America.     

Genetic engineering of self-assembled protein hydrogel based on elastin-like sequences with metal binding functionality

Recombinant DNA methods have been exploited to enable the creation of protein-based block copolymers with programmable sequences, desired properties, and predictable three-dimensional structures. These advantages over conventional polymer counterparts facilitate the utility of this new class of biomaterials in a wide range of applications. In this project, we exploited the environmental application of protein-based block copolymers based on elastin-like protein (ELP) sequences. Triblock copolymers containing charged and hydrophobic segments were synthesized. Chain lengths of each segment were manipulated in order to maintain a gelation point below room temperature. Polyhistidine sequences were successfully incorporated into the hydrophilic segment without disruption of the self-assembled hydrogel formation. The microscopic structure was further investigated using laser confocal microscopy. The metal binding capability and capacity of resulting hydrogel were studied to demonstrate the functionality of polyhistidine and its environmental application for heavy metal removal. Reversibility of metal binding was demonstrated, indicating the cost-effectiveness of this hydrogel. Significantly, we envision that this versatile strategy of incorporating functional groups within a 3-D protein network provides new possibilities in creation of biomaterials with great control over structure-property relationships.     

Molecular analysis of a novel FMRFamide-related peptide gene (SOFaRP(2)) and its expression pattern in the brain of the European cuttlefish Sepia officinalis

FMRFamide-related peptides (FaRPs) are among several neurotransmitters known to regulate the chromatophore function in the European cuttlefish Sepia officinalis. Here we report the cloning and sequencing of a novel S. officinalis FaRP gene (SOFaRP(2)). The complete 835-base pair cDNA sequence of the SOFaRP(2) gene contains an open reading frame of 567 base pairs encoding 188 amino acids and four putative FaRPs, NSLFRFamide, GNLFRFamide, TIFRFamide and PHTPFRFamide. All except TIFRFamide cause chromatophore expansion when assayed in an in vitro chromatophore bioassay. To investigate the expression pattern of SOFaRP(2) gene in the cuttlefish brain, in situ hybridization was performed using a full length RNA probe. The SOFaRP(2) gene was expressed primarily in the posterior chromatophore, anterior chromatophore, lateral basal and optic lobes among other brain locations. The SOFaRP(2) gene appears to be expressed in all brain regions involved in chromatophore regulation. These data suggests that some or all of the four FaRPs encoded by SOFaRP(2) might be involved in controlling chromatophore activity in cuttlefish.