Adding value to cocoa (Theobroma cacao L.) germplasm information with domestication history and admixture mapping

A sound understanding of crop history can provide the basis for deriving novel genetic information through admixture mapping. We confirmed this, by using characterization data from an international collection of cocoa, collected 25 years ago, and from a contemporary plantation. We focus on the trees derived from three centuries of admixture between Meso-American Criollo and South American Forastero genomes. In both cacao sets of individuals, linkage disequilibrium extended over long genetic distances along chromosome regions, as expected in populations derived from recent admixture. Based on loose genome scans, genomic regions involved in useful traits were identified. Fifteen genomic regions involved in seed and fruit weight variation were highlighted. They correspond to ten previously identified QTLs and five novel ones. Admixture mapping can help to add value to genetic resources and thus, help to encourage investment in their conservation. Maria Marcano and Tatiana Pugh contributed equally to this work.     

Phase I evaluation of intranasal trivalent inactivated influenza vaccine with nontoxigenic Escherichia coli enterotoxin and novel biovector as mucosal adjuvants, using adult volunteers

Trivalent influenza virus A/Duck/Singapore (H5N3), A/Panama (H3N2), and B/Guandong vaccine preparations were used in a randomized, controlled, dose-ranging phase I study. The vaccines were prepared from highly purified hemagglutinin and neuraminidase from influenza viruses propagated in embryonated chicken eggs and inactivated with formaldehyde. We assigned 100 participants to six vaccine groups, as follows. Three intranasally vaccinated groups received 7.5-microg doses of hemagglutinin from each virus strain with either 3, 10, or 30 microg of heat-labile Escherichia coli enterotoxin (LTK63) and 990 microg of a supramolecular biovector; one intranasally vaccinated group was given 7.5-microg doses of hemagglutinin with 30 microg of LTK63 without the biovector; and another intranasally vaccinated group received saline solution as a placebo. The final group received an intramuscular vaccine containing 15 microg hemagglutinin from each strain with MF59 adjuvant. The immunogenicity of two intranasal doses, delivered by syringe as drops into both nostrils with an interval of 1 week between, was compared with that of two inoculations by intramuscular delivery 3 weeks apart. The intramuscular and intranasal vaccine formulations were both immunogenic but stimulated different limbs of the immune system. The largest increase in circulating antibodies occurred in response to intramuscular vaccination; the largest mucosal immunoglobulin A (IgA) response occurred in response to mucosal vaccination. Current licensing criteria for influenza vaccines in the European Union were satisfied by serum hemagglutination inhibition responses to A/Panama and B/Guandong hemagglutinins given with MF59 adjuvant by injection and to B/Guandong hemagglutinin given intranasally with the highest dose of LTK63 and the biovector. Geometric mean serum antibody titers by hemagglutination inhibition and microneutralization were significantly higher for each virus strain at 3 and 6 weeks in recipients of the intramuscular vaccine than in recipients of the intranasal vaccine. The immunogenicity of the intranasally delivered experimental vaccine varied by influenza virus strain. Mucosal IgA responses to A/Duck/Singapore (H5N3), A/Panama (H3N2), and B/Guandong were highest in participants given 30 microg LTK63 with the biovector, occurring in 7/15 (47%; P=0.0103), 8/15 (53%; P=0.0362), and 14/15 (93%; P=0.0033) participants, respectively, compared to the placebo group. The addition of the biovector to the vaccine given with 30 microg LTK63 enhanced mucosal IgA responses to A/Duck/Singapore (H5N3) (P=0.0491) and B/Guandong (P=0.0028) but not to A/Panama (H3N2). All vaccines were well tolerated.     

Soluble adenylyl cyclase (sAC) is indispensable for sperm function and fertilization

We previously demonstrated that male mice deficient in the soluble adenylyl cyclase (sAC) are sterile and produce spermatozoa with deficits in progressive motility and are unable to fertilize zona-intact eggs. Here, analyses of sAC(-/-) spermatozoa provide additional insights into the functions linked to cAMP signaling. Adenylyl cyclase activity and cAMP content are greatly diminished in crude preparations of sAC(-/-) spermatozoa and are undetectable after sperm purification. HCO(3)(-) is unable to rapidly accelerate the flagellar beat or facilitate evoked Ca(2+) entry into sAC(-/-) spermatozoa. Moreover, the delayed HCO(3)(-)-dependent increases in protein tyrosine phosphorylation and hyperactivated motility, which occur late in capacitation of wild-type spermatozoa, do not develop in sAC(-/-) spermatozoa. However, sAC(-/-) sperm fertilize zona-free oocytes, indicating that gamete fusion does not require sAC. Although ATP levels are significantly reduced in sAC(-/-) sperm, cAMP-AM ester increases flagellar beat frequency, progressive motility, and alters the pattern of tyrosine phosphorylated proteins. These results indicate that sAC and cAMP coordinate cellular energy balance in wild-type sperm and that the ATP generating machinery is not operating normally in sAC(-/-) spermatozoa. These findings demonstrate that sAC plays a critical role in cAMP signaling in spermatozoa and that defective cAMP production prevents engagement of multiple components of capacitation resulting in male infertility.     

Genomic analysis of Pseudomonas aeruginosa phages LKD16 and LKA1: establishment of the phiKMV subgroup within the T7 supergroup

Lytic Pseudomonas aeruginosa phages LKD16 and LKA1 were locally isolated and morphologically classified as Podoviridae. While LKD16 adsorbs weakly to its host, LKA1 shows efficient adsorption (ka = 3.9 x 10(-9) ml min(-1)). LKA1, however, displays a narrow host range on clinical P. aeruginosa strains compared to LKD16. Genome analysis of LKD16 (43,200 bp) and LKA1 (41,593 bp) revealed that both phages have linear double-stranded DNA genomes with direct terminal repeats of 428 and 298 bp and encode 54 and 56 genes, respectively. The majority of the predicted structural proteins were experimentally confirmed as part of the phage particle using mass spectrometry. Phage LKD16 is closely related to bacteriophage phiKMV (83% overall DNA homology), allowing a more thoughtful gene annotation of both genomes. In contrast, LKA1 is more distantly related, lacking significant DNA homology and showing protein similarity to phiKMV in 48% of its gene products. The early region of the LKA1 genome has diverged strongly from phiKMV and LKD16, and intriguing differences in tail fiber genes of LKD16 and LKA1 likely reflect the observed discrepancy in infection-related properties. Nonetheless, general genome organization is clearly conserved among phiKMV, LKD16, and LKA1. The three phages carry a single-subunit RNA polymerase gene adjacent to the structural genome region, a feature which distinguishes them from other members of the T7 supergroup. Therefore, we propose that phiKMV represents an independent and widespread group of lytic P. aeruginosa phages within the T7 supergroup.     

Completion total thyroidectomy in children with differentiated thyroid cancer

INTRODUCTION: The optimal surgical treatment of children with differentiated thyroid cancer remains an important point of discussion. Especially the need for completion operation is questioned in young patients. Our objective was to examine the rate of residual neoplastic disease after non radical initial operation. MATERIAL AND METHODS: From the 235 children diagnosed with differentiated thyroid cancer, 131 (56%) needed completion operation due to incomplete primary surgery. Completion operation involved thyroid bed, lymph nodes or both respectively in 91 (39%), 13 (6%) and 27 (11%) cases. Risk factors responsible for residual disease were evaluated by means of logistic regression analysis. RESULTS: Residual disease was detected in 46 (35%) of reoperated children (25% in thyroid bed and 85% in lymph node of lateral neck compartment). Sex and age did not influence the risk of residual disease in thyroid bed or lymph nodes. Papillary type of cancer and multifocality increased risk of residual disease in thyroid bed respectively by the factor of 15 (95% CI: 2-125) and 2.3 (95% CI: 1.2-4.4). Infiltration of thyroid capsule did not correlate with the risk of residual disease. Lymph node metastases in primary operation increased risk of residual disease by the factor of 16 (95% CI: 1.2-245). Histopathology, multifocality of primary tumour or infiltration of lymph node capsule did not influence the risk of residual disease in lymph nodes of lateral neck compartment. CONCLUSIONS: In children with differentiated thyroid cancer residual disease is diagnosed in about 1/3 of non radically operated cases. This high incidence justifies completion operations. The risk of residual disease is significantly increased in papillary thyroid cancer, multifocal tumours and cases with lymph node metastases.     

A chromosome 8 gene-cluster polymorphism with low human beta-defensin 2 gene copy number predisposes to Crohn disease of the colon

Defensins are endogenous antimicrobial peptides that protect the intestinal mucosa against bacterial invasion. It has been suggested that deficient defensin expression may underlie the chronic inflammation of Crohn disease (CD). The DNA copy number of the beta-defensin gene cluster on chromosome 8p23.1 is highly polymorphic within the healthy population, which suggests that the defective beta-defensin induction in colonic CD could be due to low beta-defensin-gene copy number. Here, we tested this hypothesis, using genomewide DNA copy number profiling by array-based comparative genomic hybridization and quantitative polymerase-chain-reaction analysis of the human beta-defensin 2 (HBD-2) gene. We showed that healthy individuals, as well as patients with ulcerative colitis, have a median of 4 (range 2-10) HBD-2 gene copies per genome. In a surgical cohort with ileal or colonic CD and in a second large cohort with inflammatory bowel diseases, those with ileal resections/disease exhibited a normal median HBD-2 copy number of 4, whereas those with colonic CD had a median of only 3 copies per genome (P=.008 for the surgical cohort; P=.032 for the second cohort). Overall, the copy number distribution in colonic CD was shifted to lower numbers compared with controls (P=.002 for both the surgical cohort and the cohort with inflammatory bowel diseases). Individuals with < or = 3 copies have a significantly higher risk of developing colonic CD than did individuals with > or = 4 copies (odds ratio 3.06; 95% confidence interval 1.46-6.45). An HBD-2 gene copy number of < 4 was associated with diminished mucosal HBD-2 mRNA expression (P=.033). In conclusion, a lower HBD-2 gene copy number in the beta-defensin locus predisposes to colonic CD, most likely through diminished beta-defensin expression.     

The genetic code--40 years on

The genetic code discovered 40 years ago, consists of 64 triplets (codons) of nucleotides. The genetic code is almost universal. The same codons are assigned to the same amino acids and to the same START and STOP signals in the vast majority of genes in animals, plants, and microorganisms. Each codon encodes for one of the 20 amino acids used in the synthesis of proteins. That produces some redundancy in the code and most of the amino acids being encoded by more than one codon. The two cases have been found where selenocysteine or pyrrolysine, that are not one of the standard 20 is inserted by a tRNA into the growing polypeptide.     

First reported patient with human ERCC1 deficiency has cerebro-oculo-facio-skeletal syndrome with a mild defect in nucleotide excision repair and severe developmental failure

Nucleotide excision repair (NER) is a genome caretaker mechanism responsible for removing helix-distorting DNA lesions, most notably ultraviolet photodimers. Inherited defects in NER result in profound photosensitivity and the cancer-prone syndrome xeroderma pigmentosum (XP) or two progeroid syndromes: Cockayne and trichothiodystrophy syndromes. The heterodimer ERCC1-XPF is one of two endonucleases required for NER. Mutations in XPF are associated with mild XP and rarely with progeria. Mutations in ERCC1 have not been reported. Here, we describe the first case of human inherited ERCC1 deficiency. Patient cells showed moderate hypersensitivity to ultraviolet rays and mitomycin C, yet the clinical features were very severe and, unexpectedly, were compatible with a diagnosis of cerebro-oculo-facio-skeletal syndrome. This discovery represents a novel complementation group of patients with defective NER. Further, the clinical severity, coupled with a relatively mild repair defect, suggests novel functions for ERCC1.     

Temperature impacts the multiple attack action of amylases

The action pattern of several amylases was studied at 35, 50, and 70 degrees C using potato amylose, a soluble (Red Starch) and insoluble (cross-linked amylose) chromophoric substrate. With potato amylose as substrate, Bacillus stearothermophilus alpha-amylase (BStA) and porcine pancreatic alpha-amylase displayed a high degree of multiple attack (DMA, i.e., the number of bonds broken during the lifetime of an enzyme-substrate complex minus one), the fungal alpha-amylase from Aspergillus oryzae a low DMA, and the alpha-amylases from B. licheniformis, Thermoactinomyces vulgaris, B. amyloliquifaciens, and B. subtilis an intermediate DMA. These data are discussed in relation to structural properties of the enzymes. The level of multiple attack (LMA), based on the relation between the drop in iodine binding of amylose and the increase in total reducing value, proved to be a good alternative for DMA measurements. The LMA of the endo-amylases increased with temperature to a degree depending on the amylase. In contrast, BStA showed a decreased LMA when temperature was raised. Furthermore, different enzymes had different activities on Red Starch and cross-linked amylose. Hence, next to the temperature, the action pattern of alpha-amylases is influenced by structural parameters of the starch substrate.