Cytosolic monoterpene biosynthesis is supported by plastid‐generated geranyl diphosphate substrate in transgenic tomato fruits

Geranyl diphosphate (GPP), the precursor of most monoterpenes, is synthesized in plastids from dimethylallyl diphosphate and isopentenyl diphosphate by GPP synthases (GPPSs). In heterodimeric GPPSs, a non‐catalytic small subunit (GPPS‐SSU) interacts with a catalytic large subunit, such as geranylgeranyl diphosphate synthase, and determines its product specificity. Here, snapdragon (Antirrhinum majus) GPPS‐SSU was over‐expressed in tomato fruits under the control of the fruit ripening‐specific polygalacturonase promoter to divert the metabolic flux from carotenoid formation towards GPP and monoterpene biosynthesis. Transgenic tomato fruits produced monoterpenes, including geraniol, geranial, neral, citronellol and citronellal, while exhibiting reduced carotenoid content. Co‐expression of the Ocimum basilicum geraniol synthase (GES) gene with snapdragon GPPS‐SSU led to a more than threefold increase in monoterpene formation in tomato fruits relative to the parental GES line, indicating that the produced GPP can be used by plastidic monoterpene synthases. Co‐expression of snapdragon GPPS‐SSU with the O. basilicum α–zingiberene synthase (ZIS) gene encoding a cytosolic terpene synthase that has been shown to possess both sesqui‐ and monoterpene synthase activities resulted in increased levels of ZIS‐derived monoterpene products compared to fruits expressing ZIS alone. These results suggest that re‐direction of the metabolic flux towards GPP in plastids also increases the cytosolic pool of GPP available for monoterpene synthesis in this compartment via GPP export from plastids.     

New embedding medium for sectioning undecalcified bone

Methylmethacrylate (MMA) is the most commonly used embedding medium for sectioning undecalcified bone; however, a number of problems exist with its use in a research laboratory. MMA requires a long infiltration time and temperature control, and it reacts with many polymers. We used Kleer Set resin? as an alternative embedding medium for sectioning undecalcified bone specimens. Fluorochrome labeled bone specimens were sectioned transversely using a ground section technique and longitudinally on a sledge macrotome. The slides were viewed using both transmitted light and epifluorescence microscopy. High quality sections were obtained using Kleer Set resin? for both sectioning techniques. We have shown that this new embedding medium is simpler, safer, quicker to use and does not interfere with visualization of fluorochromes.     

Analysis of factors lowering sensitivity of interferon-γ release assay for tuberculosis

Background

Imperfect sensitivity of interferon-γ release assay (IGRA) is a potential problem to detect tuberculosis. We made a thorough investigation of the factors that can lead to false negativity of IGRA.

Methods

We recruited 543 patients with new smear-positive pulmonary tuberculosis in Hanoi, Viet Nam. At diagnosis, peripheral blood was collected and IGRA (QuantiFERON-TB Gold In-Tube) was performed. Clinical and epidemiological information of the host and pathogen was collected. The test sensitivity was calculated and factors negatively influencing IGRA results were evaluated using a logistic regression model in 504 patients with culture-confirmed pulmonary tuberculosis.

Results

The overall sensitivity of IGRA was 92.3% (95% CI, 89.6%–94.4%). The proportions of IGRA-negative and -indeterminate results were 4.8% (95% CI, 3.1%–7.0%) and 3.0% (95% CI, 1.7%–4.9%). Age increased by year, body mass index <16.0, HIV co-infection and the increased number of HLA-DRB1*0701 allele that patients bear showed significant associations with IGRA negativity (OR = 1.04 [95% CI, 1.01–1.07], 5.42 [1.48–19.79], 6.38 [1.78–22.92] and 5.09 [2.31–11.22], respectively). HIV co-infection and the same HLA allele were also associated with indeterminate results (OR = 99.59 [95% CI, 15.58–625.61] and 4.25 [1.27–14.16]).

Conclusions

Aging, emaciation, HIV co-infection and HLA genotype affected IGRA results. Assessment of these factors might contribute to a better understanding of the assay.     

Thermodynamic parameters of the binding of an oligo-alpha-thymidylate to its complementary oligo-beta-adenylate sequences

The temperature dependence of the formation of a double-stranded complex between an oligo-alpha-thymidylate and its complementary sequence, beta-riboadenylate or beta-deoxyriboadenylate, was studied using circular dichroism and compared to the duplexes formed using an oligo-beta-thymidylate. Analysis of the melting profiles allowed us to determine the thermodynamic parameters (delta H, delta S and delta G) associated with the formation of the various duplexes. The order of the stability was found: alpha-dT:beta-dA approximately alpha-dt:beta-rA greater than beta-dT:beta-dA greater than beta-dT:beta-rA.     

Blood groups and protein polymorphisms in five goat breeds (Capra hircus)

Data on allele frequencies at six red cell blood group systems and three blood protein polymorphic loci in five goat breeds are reported. Two blood proteins, albumin and carbonic anhy-drase, were not found to be polymorphic. The B blood group system of goats, like its homologue in cattle and sheep, is highly complex. At least 44 B phenogroups (haplotypes) have been distinguished in this study. Based on the variation in allele frequencies between breeds, genetic distances were calculated. The distances estimated by four different methods were in close agreement with data from the history and geographic origins of the breeds examined.     

Sequence-targeted photosensitized reactions in nucleic acids by oligo-alpha-deoxynucleotides and oligo-beta-deoxynucleotides covalently linked to proflavin

Proflavin was covalently linked to the 3'-end or to the 5'-end of an octadeoxythymidylate. This oligonucleotide was synthesized with either the natural beta-anomer of thymidine or its synthetic alpha-anomer. A polymethylene chain was used to link one of the amino groups of proflavin to a terminal thiophosphate group of the oligonucleotide. A 27-mer oligodeoxynucleotide containing an octadeoxyadenylate sequence was used as a target for the proflavin-substituted octadeoxythymidylates. Upon irradiation with visible light, photo-cross-linking reactions induced the formation of branched species that migrated more slowly than the 27-mer on denaturing polyacrylamide gels. Piperidine treatment of the photo-cross-linked species induced strand breaks in the 27-mer. In addition, proflavin induced photosensitized reactions at guanine residues in the 27-mer sequence which were converted to strand breaks following piperidine treatment. Triple-helix formation by the oligothymidylates with their complementary oligodeoxyadenylate sequence at high salt concentration led to photo-cross-linking and cleavage reactions on both sides of the target sequence. These results show that it is possible to target photosensitized reactions to specific sequences on nucleic acids. This opens new possibilities for site-directed mutagenesis and the development of photoactive anti-messenger oligodeoxynucleotides.     

Prevalence and Risk Factors for Tuberculosis Infection among Hospital Workers in Hanoi,Viet Nam

Background

Transmission of tuberculosis (TB) to health care workers (HCWs) is a global issue. Although effective infection control measures are expected to reduce nosocomial TB, HCWs'' infection has not been assessed enough in TB high burden countries. We conducted a cross-sectional study to determine the prevalence of TB infection and its risk factors among HCWs in Hanoi, Viet Nam.

Methodology/Principal Findings

A total of 300 HCWs including all staff members in a municipal TB referral hospital received an interferon-gamma release assay (IGRA), QuantiFERON-TB Gold In-Tube^TM, followed by one- and two-step tuberculin skin test (TST) and a questionnaire-based interview. Agreement between the tests was evaluated by kappa statistics. Risk factors for TB infection were analyzed using a logistic regression model. Among the participants aged from 20 to 58 years (median = 40), prevalence of TB infection estimated by IGRA, one- and two-step TST was 47.3%, 61.1% and 66.3% respectively. Although the levels of overall agreement between IGRA and TST were moderate, the degree of agreement was low in the group with BCG history (kappa = 0.29). Working in TB hospital was associated with twofold increase in odds of TB infection estimated by IGRA. Increased age, low educational level and the high body mass index also demonstrated high odds ratios of IGRA positivity.

Conclusions/Significance

Prevalence of TB infection estimated by either IGRA or TST is high among HCWs in the hospital environment for TB care in Viet Nam and an infection control program should be reinforced. In communities with heterogeneous history of BCG vaccination, IGRA seems to estimate TB infection more accurately than any other criteria using TST.     

Capparis dongvanensis sp. nov. (Capparaceae) from Vietnam

Capparis dongvanensis (Capparaceae) is described as a new species and illustrated from Ha Giang Province of Vietnam. It is morphologically similar to C. lanceolaris and C. fengii, but differs in the number of flowers on peduncles, length of spine and petiole, apex of the leaf, type of inflorescence, petal color, cylindrical ovary without any beak, fruit shape and number of seeds per fruit. Diagnostic morphological characters along with pollen and seed SEM images of C. dongvanensis and allied species are provided. Its ecology, habitat and conservation status are also described.