Development of a multiplex microsatellite marker set for the study of the solitary red mason bee, Osmia bicornis (Megachilidae)

Background Solitary bees, such as the red mason bee (Osmia bicornis), provide important ecosystem services including pollination. In the face of global declines of pollinator abundance, such haplodiploid Hymenopterans have a compounded extinction risk due to the potential for limited genetic diversity. In order to assess the genetic diversity of Osmia bicornis populations, we developed microsatellite markers and characterised them in two populations. Methods and results Microsatellite sequences were mined from the recently published Osmia bicornis genome, which was assembled from DNA extracted from a single male bee originating from the United Kingdom. Sequences were identified that contained dinucleotide, trinucleotide, and tetranucleotide repeat regions. Seventeen polymorphic microsatellite markers were designed and tested, sixteen of which were developed into four multiplex PCR sets to facilitate cheap, fast and efficient genotyping and were characterised in unrelated females from Germany (n = 19) and England (n = 14). Conclusions The microsatellite markers are highly informative, with a combined exclusion probability of 0.997 (first parent), which will enable studies of genetic structure and diversity to inform conservation efforts in this bee.

Tetranucleotide and Low Microsatellite Instability Are Inversely Associated with the CpG Island Methylator Phenotype in Colorectal Cancer

MSH3 gene or protein deficiency or loss-of-function in colorectal cancer can cause a DNA mismatch repair defect known as “elevated microsatellite alterations at selected tetranucleotide repeats” (EMAST). A high percentage of MSI-H tumors exhibit EMAST, while MSI-L is also linked with EMAST. However, the distribution of CpG island methylator phenotype (CIMP) within the EMAST spectrum is not known. Five tetranucleotide repeat and five MSI markers were used to classify 100 sporadic colorectal tumours for EMAST, MSI-H and MSI-L according to the number of unstable markers detected. Promoter methylation was determined using methylation-specific PCR for MSH3, MCC, CDKN2A (p16) and five CIMP marker genes. EMAST was found in 55% of sporadic colorectal carcinomas. Carcinomas with only one positive marker (EMAST-1/5, 26%) were associated with advanced tumour stage, increased lymph node metastasis, MSI-L and lack of CIMP-H. EMAST-2/5 (16%) carcinomas displayed some methylation but MSI was rare. Carcinomas with ≥3 positive EMAST markers (13%) were more likely to have a proximal colon location and be MSI-H and CIMP-H. Our study suggests that EMAST/MSI-L is a valuable prognostic and predictive marker for colorectal carcinomas that do not display the high methylation phenotype CIMP-H.

Oscillating bacterial expression states generate herd immunity to viral infection

Hypermutable loci are widespread in bacteria as mechanisms for rapid generation of phenotypic diversity, enabling individual populations to survive fluctuating, often antagonistic, selection pressures. As observed for adaptive immunity, hypermutation may facilitate survival of multiple, spatially-separated bacterial populations. We developed an ‘oscillating prey assay’ to examine bacteriophage (phage) spread through populations ofHaemophilus influenzaewhose phage receptor gene,lic2A, is switched ‘ON’ and ‘OFF’ by mutations in a hypermutable tetranucleotide repeat tract. Phage extinction was frequently observed when the proportion of phage-resistant sub-populations exceeded 34%.In silicomodelling indicated that phage extinction was interdependent on phage loss during transfer between populations and the frequency of resistant populations. In a fixed-area oscillating prey assay, heterogeneity in phage resistance was observed to generate vast differences in phage densities across multiple bacterial populations resulting in protective quarantining of some populations from phage attack. We conclude that phase-variable hypermutable loci produce bacterial ‘herd immunity’ with resistant intermediary-populations acting as a barricade to reduce the viral load faced by phage-sensitive sub-populations. This paradigm of meta-population protection is applicable to evolution of hypermutable loci in multiple bacteria-phage and host-pathogen interactions.ImportanceHerd immunity is a survival strategy wherein populations are protected against invading pathogens by resistant individuals within the population acting as a barrier to spread of the infectious agent. Although, this concept is normally only applied to higher eukaryotes, prokaryotic organisms also face invasion by infectious agents, such as bacterial viruses, bacteriophage (phage). Here we use novel experimental approaches and mathematical modelling, to show that bacteria exhibit a form of herd immunity through stochastically generated resistant variants acting as barricades to phage predation of sensitive cells. With hypermutable loci found in many prokaryotic systems, this phenomenon may be widely applicable to phage-bacteria interactions and could even impact phage-driven evolution in bacteria.

The blue-eyed man: A case of Waardenburg syndrome type 1 associated with mania and autistic spectrum disorder

Waardenburg syndrome (WS) is a rare genetic disorder characterised by varying degrees of sensorineural deafness, dystopia canthorum, musculokeletal defects, pigmentation anomalies such as bright blue iris, greying hair and in some cases intestinal pathology.A 21-year-old Chinese gentleman, diagnosed with WS type 1 (Figs. 1 and 2) at the age of two, presented at the emergency unit with manic symptoms for the past one month such as irritability, grandiosity, flight of ideas and reduced need for sleep. With regards to social integration, he had features suggestive of autism spectrum disorder (ASD). He often played by himself and was fixated on particular toys. He was eventually admitted to the psychiatric ward for acute management of mania. He was stabilised on olanzapine 10 mg BD and sodium valproate 600 mg BD. His sodium valproate was cross-titrated with lithium in the ward and his manic features gradually subsided. He was discharged well after 2 weeks of admission with lithium 300 mg BD and olanzapine 10 mg BD. WS type 1 has been localised to the locus 2q35 and researchers have identified that a tetranucleotide repeat marker on 2q35 is strongly associated with recurrent mood symptoms.In conclusion, it is important to note that individuals with WS may be at higher risk to develop ASD and mood disorders.Disclosure of interestThe authors have not supplied their declaration of competing interest.

Isolation and identification of a new set of microsatellite loci from Ucides cordatus genome

A new set of microsatellite loci (simple sequence repeats, SSRs) from the overexploited mangrove crab Ucides cordatus is described in this study. Microsatellite isolation used a highly simplified and inexpensive protocol based on (i) multiple enzyme digestion/ligation; (ii) mixed biotin-labeled probes and streptavidin-coated magnetic bead hybridization capture strategy, and (iii) a double-repeat-enrichment procedure. A genomic library, double-enriched for inserts containing tetranucleotide repeat motifs [(GACA)6, (GATA)7, (GGAT)5 and (GTAT)5], was constructed to increase the chance of recovering SSR-containing sequences within DNA fragments. Amplified enriched DNA was cloned and transformed into competent E. coli. Then, positive clones were identified by 'white/blue plaque selection?. One hundred and five colonies were PCR-screened for sequencing, and 72 of these were found to have unique SSR inserts. Microsatellite motifs contained more than five repeats, and most loci were found to have perfect tandem repeats (51.4%), of which 94.4% were dinucleotide and 5.5% trinucleotide. Only 20% of all loci were compound and 28.6% were imperfect repeats containing di-, tri- and/or tetranucleotides. The high frequency of perfect repeat motifs after enrichment is additional evidence of the importance of adopting this procedure for the isolation of SSR. The novel 34 SSRs described in this study are expected to be highly polymorphic and, therefore, useful in population/stocks discrimination of this valuable mangrove species throughout its range, currently subjected to excessive fishing efforts.

Association between a Tetranucleotide Repeat Polymorphism of SPAG16 Gene and Cataract in Male Children

Purpose. Studies involving genotyping of STR markers at 2q34 have repeatedly found the region to host the disease haplotype for pediatric cataract. Present study investigated the association of D2S2944 marker, in sperm associated antigen 16 (SPAG16) gene and rs2289917 polymorphism, in γ-crystallin B gene, with childhood cataract. Methods. 97 pediatric cataract cases and 110 children with no ocular defects were examined for tetranucleotide repeat marker/SNP using PCR-SSLP/RFLP techniques. Polymorphisms were assessed for association using contingency tables and linkage disequilibrium among alleles of the markers was estimated. Energy-optimization program predicted the secondary structure models of repeats of D2S2944. Results. Seven alleles of D2S2944, with 9–15 “GATA” repeats, were observed. Frequency of the longer allele of D2S2944, ≥(GATA)13 repeats, was 0.73 in cases and 0.56 in controls (P=0.0123). Male children bearing ≥(GATA)13 repeats showed >3-fold higher risk for cataract (CI95% = 1.43–7.00, P=0.0043, Pc=0.0086) as compared to female children (OR=1.19, CI95% = 0.49–2.92, P=0.70). Cases with haplotype—≥(GATA)13 of D2S2944 and “C” allele rs2289917—have a higher risk for pediatric cataract (OR=2.952, CI95% = 1.595~5.463, P=0.000453). >(GATA)13 repeats formed energetically more favorable stem-loop structure. Conclusion. Intragenic microsatellite repeat expansion in SPAG16 gene increases predisposition to pediatric cataract by probably interfering posttranscriptional events and affecting the expression of adjacent lens transparency gene/s in a gender bias manner.