Efficiency of ligation-mediated PCR and TAIL-PCR methods for isolation of RbcS promoter sequences from green microalgae Ankistrodesmus convolutus

Isolation of promoter sequences from known gene sequences is a tedious task in genome-related research. An efficient method of obtaining the promoter sequences is necessary in order to successfully use targeted promoters for genetic manipulations. Here, efficiency and usefulness of two PCR-based methods, namely: ligation-mediated PCR and thermal asymmetric interlaced (TAIL) PCR, for isolation of promoter sequences of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) gene from green microalgae Ankistrodesmus convolutus (A. convolutus) were evaluated. The results showed that the amplification efficiency of TAIL-PCR was higher than that of the ligation-mediated PCR method, i.e. the amplified promoter fragments of 1.2 and 0.8 kb in length or promoter sequences of 813 and 606 bp (after eliminating the unreadable sequences). The use of TAIL-PCR described here presents a low cost and efficient strategy for the isolation of promoter sequences of known genes, especially in GC-rich regions, and species with little or no available genome information such as A. convolutus.     

The DNLZ/HEP zinc-binding subdomain is critical for regulation of the mitochondrial chaperone HSPA9

Human mitochondrial DNLZ/HEP regulates the catalytic activity and solubility of the mitochondrial hsp70 chaperone HSPA9. Here, we investigate the role that the DNLZ zinc-binding and C-terminal subdomains play in regulating HSPA9. We show that truncations lacking portions of the zinc-binding subdomain (ZBS) do not affect the solubility of HSPA9 or its ATPase domain, whereas those containing the ZBS and at least 10 residues following this subdomain enhance chaperone solubility. Binding measurements further show that DNLZ requires its ZBS to form a stable complex with the HSPA9 ATPase domain, and ATP hydrolysis measurements reveal that the ZBS is critical for full stimulation of HSPA9 catalytic activity. We also examined if DNLZ is active in vivo. We found that DNLZ partially complements the growth of Δzim17 Saccharomyces cerevisiae, and we discovered that a Zim17 truncation lacking a majority of the C-terminal subdomain strongly complements growth like full-length Zim17. These findings provide direct evidence that human DNLZ is a functional ortholog of Zim17. In addition, they implicate the pair of antiparallel β-strands that coordinate zinc in Zim17/DNLZ-type proteins as critical for binding and regulating hsp70 chaperones.     

Effects of Typical and Atypical Antipsychotic Drugs on Rat Brain Muscarinic Receptors

Quantitative in vitro autoradiography was used to examine changes in muscarinic M1/M4 and M2/M4 receptors (targeted with [³H]pirenzepine and [³H]AF-DX384 respectively), in rats treated with the typical (haloperidol) and atypical (clozapine and olanzapine) antipsychotic medications for a period of 36 days. Rats were sacrificed at either 2 h or 48 h after the last drug administration to examine immediate effects as well as the effects at 48 h after drug withdrawal. Haloperidol significantly increased [³H]pirenzepine binding in the dentate gyrus (37%) and in the CA1 region of the hippocampus (34%) in animals sacrificed 2 h after the last drug administration compared to controls. Similarly, clozapine significantly increased [³H]pirenzepine binding in dentate gyrus (29%) in rats sacrificed 2 h after the last drug administration compared to controls. Haloperidol decreased [³H]AF-DX384 binding in the basolateral nucleus of the amygdala (20%) in the rats sacrificed 48 h after the last drug administration compared to controls. These findings suggest that muscarinic receptors and limbic brain regions such as hippocampus and amygdala might represent common targets that mediate beneficial clinical effects of antipsychotic drugs.     

Carbon emissions in the field of land use,land use change,and forestry in the Vietnam mainland

The biennial report of Vietnam includes updated information on the greenhouse gas emissions for the base years which are specified by the United Nations Framework Convention on Climate Change for reporting on greenhouse gas emissions by member nations of the Intergovernmental Panel on Climate Change. The estimation of greenhouse gas emissions in general and carbon emissions in particular in the field of land use, land use change, and forestry using advanced technology to provide the input data was recommended. Remote sensing technology with transparency, multi-time characteristics, and wide coverage is useful in this area. An experiment on carbon emission estimation was carried out based on land cover change over ten years between 2002 and 2012. The results obtained by remote sensing data classification for the land cover categories achieved a reliability of 68% for the year 2002 and 67% for the year 2012. Data in relation to the land cover change, soil zoning, and ecological/climate zoning in the Vietnamese mainland, through the process of integrating, processing, and synthesizing data, and using the reported activity data and carbon emission coefficients, were input into the Agriculture and Land Use Greenhouse Gas Inventory Software for carbon emission estimations based on the quality control and quality assurance work.

     

Chemical Defense in Millipedes (Myriapoda,Diplopoda): Do Representatives of the Family Blaniulidae Belong to the ‘Quinone’ Clade?

The defensive secretions of two blaniulid millipedes, Nopoiulus kochii and Cibiniulus phlepsii, were characterized by GC‐FID and GC/MS analyses, which showed the presence of a complex mixture of benzoquinones, hydroquinones, and oleates. Altogether, 13 compounds were identified. The major compound in the secretions of both analyzed species was 2‐methyl‐1,4‐benzoquinone (toluquinone). The second major constituent in the N. kochii secretion was 2‐methyl‐3,4‐(methylenedioxy)phenol, while in that of C. phlepsii, it was 2‐methoxy‐3‐methyl‐1,4‐benzoquinone. The defensive secretion of N. kochii also showed a high content of hydroquinones (13.5%) in comparison to that of C. phlepsii (0.8%). Hexyl oleate and octyl oleate were detected for the first time in defensive millipede fluids. The chemical composition of the defensive secretions supports the chemotaxonomic position of the family Blaniulidae in the ‘quinone’ millipede clade.     

Further Amphoricarpolides from the Surface Extracts of Amphoricarpos Complex from Montenegro

Analysis of composition of sesquiterpene lactone fraction of leaf cuticular neutral lipids of Amphoricarpos complex from two different localities in north Montenegro, i.e., canyon of river Tara (A. autariatus ssp. autariatus) and mountain Zeletin (A. autariatus ssp. bertisceus) afforded sesquiterpene lactones with guaianolide skeletons (17 compounds), so called amphoricarpolides, typical for this genus. Nine of them, 9 – 17 , were new compounds, and their structures were elucidated by detailed analyses of IR, NMR, and MS data.     

Bacterial Load of Pneumococcal Serotypes Correlates with Their Prevalence and Multiple Serotypes Is Associated with Acute Respiratory Infections among Children Less Than 5 Years of Age

Background

Among pneumococcal serotypes, some serotypes are more prevalent in the nasopharynx than others; determining factors for higher prevalence remain to be fully explored. As non-vaccine serotypes have emerged after the introduction of 7-valent conjugate vaccines, study of serotype specific epidemiology is in need. When two or more serotypes co-colonize, they evolve rapidly to defend host''s immune responses; however, a clear association of co-colonization with a clinical outcome is lacking.

Methods

Children less than 5 years old who were admitted to hospital due to acute respiratory infections (ARI) (n = 595) and healthy children (n = 350) were recruited. Carriage of pneumococcus was determined by culture and lytA PCR in the nasopharyngeal samples. Serotype/serogroup detection and its quantification were done by the nanofluidic real time PCR system. Spearman''s correlation and logistic regression were used to examine a correlation of serotype/serogroup specific bacterial load with its prevalence and an association of co-colonization with ARI respectively.

Results

Serotype/serogroup specific bacterial load was correlated with its prevalence, both in ARI cases (Spearman''s rho = 0.44, n = 186; P<0.0001) and healthy children (Spearman''s rho = 0.41, n = 115; P<0.0001). The prevalence of multiple serotypes was more common in ARI cases than in healthy children (18.5% vs 7.1%; aOR 2.92, 95% CI: 1.27–6.71; P = 0.01). The dominant serotype in the co-colonization had a 2 log10 higher bacterial load than the subdominant serotype, both in ARI cases (P<0.001) and healthy children (P<0.05).

Conclusions

High bacterial load in the nasopharynx may help transmit pneumococci among hosts, and increase the chance of successful acquisition and colonization. Co-colonization of multiple serotypes of pneumococci is linked with ARI, which infers the interactions of multiple serotypes may increase their pathogenicity; however, they may compete for growth in number.     

Various methods for isolation of multipotent human periodontal ligament cells for regenerative medicine

Periodontal ligament (PDL) is a specialized connective tissue that connects cementum and alveolar bone to maintain and support the teeth in situ and preserve tissue homeostasis. Recent studies have revealed the existence of stem cells in human dental tissues including periodontal ligament that play an important role, not only in the maintenance of the periodontium but also in promoting periodontal regeneration. In this study, human periodontal ligament cells (hPDLCs) were isolated by outgrowth and enzymatic dissociation methods. Expression of surface markers on PDLCs as human mesenchymal stem cells (MSCs) was identified by flow cytometry. In addition, proliferation and differentiation capacity of cultured cells to osteoblasts, adipocytes were evaluated. As a result, we successfully cultured cells from the human periodontal ligament tissues. PDLCs express mesenchymal stem cell (MSC) markers such as CD44, CD73, and CD90 and do not express CD34, CD45, and HLA-DR. PDLCs also possess the multipotential to differentiate into various types of cells, such as osteoblast and adipocytes, in vitro. Therefore, these cells have high potential to serve as materials for tissue engineering, especially dental tissue engineering.