Search

The use of electronic cigarettes (E-cig) is popular because of the perception that they are less addictive and safer compared to the traditional cigarettes. Nevertheless, there are still harmful effects associated with chemicals emitted from E-cig. Identifying the sources of the emitted compounds can be challenging because of the differences in the design of E-cig devices and the variability in the composition of E-cig liquids used in these devices. In this study, the emission profiles from impurity-free E-liquids containing only propylene glycol and glycerol in various percentage ratios along with two commercially available E-liquids were evaluated using gas chromatography-mass spectrometry (GC-MS). This study approach allows the elucidation of the transformation pathways of the major emitted compounds without the confounding effects of existing impurities or flavor ingredients added to E-liquids. Analysis of the vapor phases of E-cig emissions detected toxicants such as acetaldehyde, acrolein, benzaldehyde, as well as benzene, toluene, ethylbenzene, and xylene (BTEX) compounds. The amount of glycerol in the E-liquids has a major effect on the concentration of these hazardous compounds emitted because the concentration of these chemicals increased with increasing glycerol percentage in the E-liquid. Acetaldehyde and acrolein increased by 175-fold and 28-fold, respectively, when the glycerol composition was increased from 0 to 80%. Benzaldehyde, naphthalene, diphenyl ether, and glycerol along with menthol and nicotine that were present in the commercial E-liquids were also detected in the aerosol condensates. The cascade impactor data on the distribution of the nicotine and menthol in different size fractions from >2.5 to <2.5 μm allow the estimates of the extent of toxicant deposition in different parts of the pulmonary system including the oropharynx region, the trachea as well as inside the alveoli and bronchioles. In summary, users of E-cig are exposed to harmful chemicals even if the E-liquids contain only propylene glycol and glycerol without flavorings, nicotine, or impurities. Furthermore, this study shows that E-liquids containing higher percentages of glycerol will produce higher levels of toxicants compared to E-liquids with similar percentages of propylene glycol. This finding has important implications to E-cigarette vendors and manufacturers, consumers, and regulatory agencies., Competing Interests: The authors declare no competing financial interest.

This study evaluates the pre-treatment of cellulose from kenaf plant to yield sugar precursors for the production of ethanol or butanol for use as biofuel additives. In order to convert the crystalline cellulosic form to the amorphous form that can undergo enzymatic hydrolysis of the glycosidic bond to yield sugars, kenaf pulp samples were subjected to two different pre-treatment processes. In the acid pre-treatment, the pulp samples were treated with 37.5% hydrochloric acid in the presence of FeCl(3) at 50 °C or 90 °C whereas in the alkaline method, the pulp samples were treated with 25% sodium hydroxide at room temperature and with 2% or 5% sodium hydroxide at 50 °C. Microwave-assisted NaOH-treatment of the cellulose was also investigated and demonstrated to be capable of producing high glucose yield without adverse environmental impact by circumventing the use of large amounts of concentrated acids i.e., 83-85% phosphoric acid employed in most digestion processes. The treated samples were digested with the cellulase enzyme from Trichoderma reesei. The amount of glucose produced was quantified using the Quantichrom(™) glucose bioassay for assessing the efficiency of glucose production for each of the treatment processes. The microwave-assisted alkaline pre-treatment processes conducted at 50 °C were found to be the most effective in the conversion of the crystalline cellulose to the amorphous form based on the significantly higher yields of sugar produced by enzymatic hydrolysis compared to the untreated sample.

The Saccharomyces cerevisiae wine yeast strains 71B-1122 and K1-V1116 were used to derive strains that could tolerate and produce higher ethanol yields. Respiratory-deficient mutants resistant to 500 microg/mL lycorine were isolated. Two mutants, 71B-1122 YEBr L3 and K1-V1116 YEBr L4, were shown to achieve about 10% and 18% improvement in their glucose-to-ethanol conversion efficiency compared to their respective parent strains. The K1-V1116 YEBr L4 in particular can tolerate an ethanol yield of 18.8 +/- 0.8% at 3.5 weeks of fermentation and continued to consume most of the sugar until less than 1% glucose was left.

The nature and properties of a variety of plasmids are described that facilitate the construction of baculovirus vectors for expression of one or more heterologous genes. The plasmids are designed for use with Autographa californica nuclear polyhedrosis virus, AcMNPV, as a vector for protein production in insect cells and/or insect larvae. Several plasmids described here facilitate the simultaneous insertion and expression of two different genes. Some vector systems allow high and equal levels of transcription of both genes while others employ two different promoters for differential transcription. Four of the plasmids described here are designed for expression of both the viral polyhedrin-encoding gene and a heterologous gene. Such recombinants form polyhedral occlusion bodies which serve as visible markers of recombination and facilitate oral infection of insect larvae for mass-scale protein production. A synthetic promoter with a unique sequence can be used at a variety of sites in the viral genome and avoids sequence duplication. A series of plasmids are also described that supply an N terminus with an efficient translational initiation signal and convenient multiple cloning sites in the three different translational reading frames. The modular nature of all the constructs allows the use of other promoters with different temporal regulation to be utilized in the construction of additional plasmids for customized expression work.

S1 nuclease protection and primer extension analyses were used to determine the 5' end of the Autographa californica nuclear polyhedrosis virus 603 open reading frame (ORF) transcript upstream of and on the opposite strand to the polyhedrin gene. These analyses suggested that the 5' end of the 603 ORF was located near the initiation site for polyhedrin gene transcription. Primer extension products of reverse transcription of 603 RNA were dependent on the presence of the TAAG sequence, an essential polyhedrin promoter element. The results could be interpreted as indicating bidirectional transcription from the TAAG element. However, the data could also be due to an artefact of antisense transcripts and RNA duplex formation in this region. Since the bidirectional transcriptional model is not consistent with other mapping data and Northern blot analysis, we conclude that the presence of antisense RNA can result in mapping artefacts and that caution must be taken in interpreting data where overlapping sense and antisense RNAs are present.

Transposon Tn5 mutagenesis of the Escherichia coli chromosome was used to isolate 21 independent insertion mutations conferring an altered colony color phenotype on MacConkey-glycerol plates. The polymerase chain reaction was used to map 16 of these Tn5 insertions within the glpFK region at 88 min. The most polar Tn5 insertion was shown by nucleotide sequencing to be in the proposed glpF open reading frame. The data suggest that the glpF and glpK genes are in an operon with a bent DNA segment (BENT-6) involved in transcriptional regulation of this operon.

A late 3.2-kilobase (kb) RNA initiated approximately 2 kb downstream of the 3' end of the Autographa californica nuclear polyhedrosis virus polyhedrin-coding sequence and traversed the polyhedrin gene in an antisense direction. This RNA was sense RNA for the two open reading frames flanking the polyhedrin gene. A mutant virus, vXpoly, which differs from wild-type virus only at the essential RNA initiation site in the polyhedrin promoter, exhibited higher levels of the 3.2-kb RNA than did wild-type virus during the polyhedrin transcriptional phase. Thus, transcription of the polyhedrin gene down regulates the levels of this 3.2-kb RNA.

The nucleotide sequence of the yeast mitochondrial olil gene has been obtained in a series of mit- mutants with mutations in this gene, which codes for subunit 9 of of the mitochondrial ATPase complex. Subunit 9 is the proteolipid, 76 amino acids in length, necessary for the proton translocation function of the membrane Fo-sector. These mutants were classified on the basis of their rescue by a petite strain shown here to retain the entire wild-type olil gene. The mutation in one mit- strain removes a positively charged residue (Arg39----Met) which is likely to be located in a segment of subunit 9 that protrudes from the inner mitochondrial membrane. In a second mit- mutant, a negatively charged residue replaces a conserved glycine residue (Gly18----Asp) in a glycine-rich segment of the protein that is most likely embedded within the membrane. Other mit- mutations result in frameshifts with predicted products 7, 65 and 68 amino acid residues long. In each mit- mutant, there is the loss of one or more of the amino acid residues that are highly conserved among diverse species. The location and nature of specific changes pinpoint amino acid residues in subunit 9 essential to the activity of the mitochondrial ATPase complex.

The nucleotide sequence of the oli1 gene encoding mitochondrial ATPase subunit 9 (76 amino acids) has been determined for five oligomycin-resistant mutants of Saccharomyces cerevisiae. Three of the mutations affect amino acids in the vicinity of the glutamic acid residue 59 at which dicylohexyl carbodiimide binds. Two other mutations lead to substitution of amino acid 23, which would lie very close to residue 59 in the folded hairpin conformation that this protein is thought to adopt in the inner mitochondrial membrane. The apposition of residues 23 and those adjacent to residue 59, lying respectively in the two hydrophobic membrane-spanning arms of subunit 9, is considered to constitute an oligomycin-binding domain. By consideration of the amino acid substitutions in those mutants cross-resistant to venturicidin, a domain of resistance for venturicidin is defined to lie within the oligomycin-binding domain, also centered on residues 23 and 59. These data also clarify the genetic recombination behaviour of alleles previously defined to form part of the oli3 locus (mutants characterized by resistance to both oligomycin and venturicidin) together with alleles defined to form part of the oli1 locus (mutants not cross-resistant to venturicidin). The oli1 and oli3 loci can now be seen to form two overlapping extended groups within the oli1 gene, with sequenced oli3 mutations being as far apart as 125 nucleotides within the subunit 9 coding region of 231 nucleotides.

A temperature-conditional mit- mutant of Saccharomyces cerevisiae has been characterized; the mutant strain h45 cannot grow at 36 degrees C on nonfermentable substrates yet appears to be normal at 28 degrees C. The mutation in strain h45 maps genetically to the oli1 region of the mitochondrial DNA (mtDNA) genome, and prevents the synthesis at 36 degrees C of the oli1 gene product, subunit 9 of the mitochondrial ATPase complex. Since the level of oli1 mRNA in mutant h45 is close to normal at 36 degrees C, it is concluded that there is a specific block in translation of this mRNA at the non-permissive temperature. DNA sequence analysis of mtDNA from strain h45 reveals an additional T residue inserted 88 bp upstream of the oli1 coding region, in the A,T-rich sequence that is transcribed into the 5'-untranslated region of the oli1 mRNA. Sequence data on two revertants show that one returns to wild-type parental (J69-1B) mtDNA sequence, whilst the other contains an inserted A residue adjacent to the T inserted in the original h45 mutant. The results are discussed in terms of the stability of folds in RNA upstream of putative ribosome-binding sites in mitochondrial mRNA, and the potential action of nuclear-coded proteins that might be activators of the translation of specific mitochondrial mRNAs in yeast mitochondria.

A mitochondrial gene (denoted aap1) in Saccharomyces cerevisiae has been characterized by nucleotide sequence analysis of a region of mtDNA between the oxi3 and oli2 genes. The reading frame of the aap1 gene specifies a hydrophobic polypeptide containing 48 amino acids. The functional nature of this reading frame was established by sequence analysis of a series of mit- mutants and revertants. Evidence is presented that the aap1 gene codes for a mitochondrially synthesized polypeptide associated with the mitochondrial ATPase complex. This polypeptide (denoted subunit 8) is a proteolipid whose size has been previously assumed to be 10 kilodaltons based on its mobility on SDS-polyacrylamide gels, but the sequence of the aap1 gene predicts a molecular weight of 5,815 for this protein.

The effects of mutations within the 92-bp region immediately upstream from the translational initiation ATG of the polyhedrin gene of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) were determined by plasmid transient expression assays in the presence of wild-type (wt) AcMNPV DNA. Clustered point mutations were generated by substitution of 10-bp stretches of the polyhedrin promoter/leader region with a 10-bp HindIII linker. Three of these linker scan (LS) mutations in the region from nucleotides (nt) -62 to -84 (relative to the original polyhedrin ATG at +1, +2, +3) had no effect or a mild stimulatory effect on reporter gene expression. One mutation immediately upstream (nt -52 to -60) from the transcription start point (at nt -50) reduced expression four-fold. Three overlapping mutations affecting 8 bp from nt -44 to -51 (encompassing the transcriptional start point) reduced expression by 1000-fold. A 1000-fold reduction was also observed for a total deletion of nt -1 and -92. Five LS mutations between nt -1 and -43 each reduced expression two- to five-fold, whereas combining an LS mutation and a 9-bp deletion mutation in the leader reduced expression approx. nine-fold. Reversing the orientation of the reporter gene along with the wt 92-bp upstream polyhedrin promoter/leader sequences resulted in slightly higher expression levels than those observed for the normal orientation indicating that all the essential cis-acting promoter elements, with the possible exception of long-range enhancer sequences, are located downstream from nt -92. Sequences of the AcMNPVhr5 enhancer (homologous region No. 5 of AcMNPV) had only a minor effect on expression from the polyhedrin promoter in transient assays. The results show that 8 bp encompassing the transcriptional start point, a sequence which is conserved in all late AcMNPV promoters, is essential for polyhedrin gene expression. Additional nucleotides within the leader region are necessary for optimal expression.

A series of isonuclear oligomycin-resistant mutants of Saccharomyces cerevisiae carrying mutations in the mitochondrial oli1 gene has been studied. DNA sequence analysis of this gene has been used to define the amino acid substitutions in subunit 9 of the mitochondrial ATPase complex. A domain of amino acids involved in oligomycin resistance can be recognized which encompasses residues in each of the two hydrophobic portions of the subunit 9 polypeptide that are thought to span the inner mitochondrial membrane. Certain amino acid substitutions also confer cross-resistance to venturicidin: these residues define an inner domain for venturicidin resistance. The expression of venturicidin resistance resulting from one particular substitution is modulated by nuclear genetic factors.