Bacterial lacZ Research Articles

Gene-Directed Enzyme Prodrug Therapy by Dendrimer-Like Mesoporous Silica Nanoparticles against Tumor Cells.

We report herein a gene-directed enzyme prodrug therapy (GDEPT) system using gated mesoporous silica nanoparticles (MSNs) in an attempt to combine the reduction of side effects characteristic of GDEPT with improved pharmacokinetics promoted by gated MSNs. The system consists of the transfection of cancer cells with a plasmid controlled by the cytomegalovirus promoter, which promotes β-galactosidase (β-gal) expression from the bacterial gene lacZ (CMV-lacZ). Moreover, dendrimer-like mesoporous silica nanoparticles (DMSNs) are loaded with the prodrug doxorubicin modified with a galactose unit through a self-immolative group (DOXO-Gal) and modified with a disulfide-containing polyethyleneglycol gatekeeper. Once in tumor cells, the reducing environment induces disulfide bond rupture in the gatekeeper with the subsequent DOXO-Gal delivery, which is enzymatically converted by β-gal into the cytotoxic doxorubicin drug, causing cell death. The combined treatment of the pair enzyme/DMSNs-prodrug are more effective in killing cells than the free prodrug DOXO-Gal alone in cells transfected with β-gal.

Mutagenicity of N-hydroxy-4-aminobiphenyl in human TP53 knock-in (Hupki) mouse embryo fibroblasts.

TP53 harbors somatic mutations in more than half of human tumors with some showing characteristic mutation spectra that have been linked to environmental exposures. In bladder cancer, a unique distribution of mutations amongst several codons of TP53 has been hypothesized to be caused by environmental carcinogens including 4-aminobiphenyl (4-ABP). 4-ABP undergoes metabolic activation to N-hydroxy-4-aminobiphenyl (N-OH-4-ABP) and forms pre-mutagenic adducts in DNA, of which N-(deoxyguanosin-8-yl)-4-ABP (dG-C8-4-ABP) is the major one. Human TP53 knock-in mouse embryo fibroblasts (HUFs) are a useful model to study the influence of environmental carcinogens on TP53-mutagenesis. By performing the HUF immortalization assay (HIMA) TP53-mutant HUFs are generated and mutations can be identified by sequencing. Here we studied the induction of mutations in human TP53 after treatment of primary HUFs with N-OH-4-ABP. In addition, mutagenicity in the bacterial lacZ reporter gene and the formation of dG-C8-4-ABP, measured by 32 P-postlabelling analysis, were determined in N-OH-4-ABP-treated primary HUFs. A total of 6% TP53-mutants were identified after treatment with 40 μM N-OH-4-ABP for 24 hr (n = 150) with G>C/C>G transversion being the main mutation type. The mutation spectrum found in the TP53 gene of immortalized N-OH-4-ABP-treated HUFs was unlike the one found in human bladder cancer. DNA adduct formation (~40 adducts/108 nucleotides) was detected after 24 hr treatment with 40 μM N-OH-4-ABP, but lacZ mutagenicity was not observed. Adduct levels decreased substantially (sixfold) after a 24 hr recovery period indicating that primary HUFs can efficiently repair the dG-C8-4-ABP adduct possibly before mutations are fixed. In conclusion, the observed difference in the N-OH-4-ABP-induced TP53 mutation spectrum to that observed in human bladder tumors do not support a role of 4-ABP in human bladder cancer development.

Analysis of Gene Expression Using lacZ Reporter Mouse Lines.

Reporter mice transgenically expressing the bacterial (E. coli) lacZ gene encoding β-galactosidase (β-gal, EC 3.2.1.23) are a versatile and extensively used tool to study gene expression and cell lineage patterns. Enzymatic activity of the β-gal reporter can be effectively visualized at cellular resolution either histochemically using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) or by immunofluorescent detection using a β-gal-specific antibody. Here, we summarize protocols for the localization of β-gal expressing cells in whole embryos or organs as well as in histological tissue sections of lacZ reporter mice and discuss their limitations and common pitfalls.

Using a Human Papillomavirus Model to Study DNA Replication and Repair of Wild Type and Damaged DNA Templates in Mammalian Cells

Human papillomaviruses have 8kbp DNA episomal genomes that replicate autonomously from host DNA. During initial infection, the virus increases its copy number to 20–50 copies per cell, causing torsional stress on the replicating DNA. This activates the DNA damage response (DDR) and HPV replicates its genome, at least in part, using homologous recombination. An active DDR is on throughout the HPV life cycle. Two viral proteins are required for replication of the viral genome; E2 binds to 12bp palindromic sequences around the A/T rich origin of replication and recruits the viral helicase E1 via a protein–protein interaction. E1 forms a di-hexameric complex that replicates the viral genome in association with host factors. Transient replication assays following transfection with E1–E2 expression plasmids, along with an origin containing plasmid, allow monitoring of E1-E2 replication activity. Incorporating a bacterial lacZ gene into the origin plasmid allows for the determination of replication fidelity. Here we describe how we exploited this system to investigate replication and repair in mammalian cells, including using damaged DNA templates. We propose that this system has the potential to enhance the understanding of cellular components involved in DNA replication and repair.

New insights of polyamine metabolism in testicular physiology: A role of ornithine decarboxylase antizyme inhibitor 2 (AZIN2) in the modulation of testosterone levels and sperm motility.

The specific role of polyamines in the testis physiology is not fully understood. Antizymes (OAZs) and antizyme inhibitors (AZINs) are modulators of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis and polyamine uptake. Although the three known OAZs are expressed in the testis, only OAZ3 is testis specific and has been proven to have an essential role in male fertility. Regarding the two existing AZINs, AZIN2 is the most abundantly expressed member in this gonad. Whereas previous studies suggested that AZIN2 might participate in mouse spermatogenesis, immunohistological analysis of human testicular sections revealed that AZIN2 is also detected in the steroidogenic Leydig cells but not in the germinal epithelium. In the present study, we found a close ontogenic similarity in the mRNA levels of OAZs and AZINs between mice and rats, but an opposite expression pattern of ODC activity. Further analysis of AZIN2 and OAZ3 in the testis of mice with different alterations in spermatogenesis and fertility, induced either genetically or pharmacologically, corroborated that both AZIN2 and OAZ3 are mainly expressed in the haploid germinal cells. Finally, by using transgenic mice with a truncated Azin2 gene fused to the bacterial lacZ gene, we studied the expression of Azin2 in testes, epididymides and spermatozoa. AZIN2 was detected in spermatids and spermatozoa, as well as in Leydig cells, and in epithelial epidydimal cells. Azin2 knock-out male mice were fertile; however, they showed marked decreases in testicular putrescine and plasma and testicular testosterone levels, and a dramatic reduction in the sperm motility. These results suggest an important role for AZIN2 in testicular cells by modulating polyamine concentrations, testosterone synthesis and sperm function. Overall, our data corroborate the relevance of polyamine regulation in testis functions, where both AZIN2 and OAZ3 play fundamental roles.

New reporter gene assays for detecting natural and synthetic molting hormone agonists using yeasts expressing ecdysone receptors of various insects.

Synthetic nonsteroidal ecdysone agonists, a class of insect growth regulators (IGRs), target the ecdysone receptor (EcR), which forms a heterodimer with ultraspiracle (USP) to transactivate ecdysone response genes. These compounds have high binding affinities to the EcR–USP complexes of certain insects and their toxicity is selective for certain taxonomic orders. In the present study, we developed reporter gene assay (RGA) systems to detect molting hormone (ecdysone) activity by introducing EcR–USP cDNA and a bacterial lacZ reporter gene into yeast. EcR and USP were derived from the insect species of three different taxonomic orders: Drosophila melanogaster (Diptera), Chilo suppressalis (Lepidoptera), and Leptinotarsa decemlineata (Coleoptera). Transcriptional coactivator taiman (Tai) cDNA cloned from D. melanogaster was also used in this RGA system. This yeast RGA system responded to various EcR ligands in a dose‐dependent and ecdysteroid‐specific manner. Furthermore, the insect order‐selective ligand activities of synthetic nonsteroidal ecdysone agonists were linearly related to their binding activities, which were measured against in vitro translated EcR–USP complexes. Our newly established yeast RGA is useful for screening new molting hormone agonists that work selectively on target insects.

A pilot study for construction of a new cadmium-sensing yeast strain carrying a reporter plasmid with the JLP1 promoter.

Cadmium contamination still occurs in some parts of the world, and its concentrations in the environment are monitored in most countries due to its adverse effects on human health. We herein established yeast (Saccharomyces cerevisiae) reporter assay strains carrying plasmids with the yeast JLP1, SEO1, and CUP1 promoters connected to the bacterial lacZ reporter gene. The strain carrying the high-copy number pESC-JLP1-lacZ reporter plasmid was more responsive to cadmium than strains with other reporter plasmids. This JLP1-lacZ reporter assay strain will be useful for monitoring cadmium contamination in environmental water and soil as a first screening tool preceding official instrumental analyses, because the assay is rapid, easy to handle, and has the ability to process a large number of samples at a low cost.

Beware of your Cre‐Ation: lacZ expression impairs neuronal integrity and hippocampus‐dependent memory

Expression of the lacZ-sequence is a widely used reporter-tool to assess the transgenic and/or transfection efficacy of a target gene in mice. Once activated, lacZ is permanently expressed. However, protein accumulation is one of the hallmarks of neurodegenerative diseases. Furthermore, the protein product of the bacterial lacZ gene is ß-galactosidase, an analog to the mammalian senescence-associated ß-galactosidase, a molecular marker for aging. Therefore we studied the behavioral, structural and molecular consequences of lacZ expression in distinct neuronal sub-populations. lacZ expression in cortical glutamatergic neurons resulted in severe impairments in hippocampus-dependent memory accompanied by marked structural alterations throughout the CNS. In contrast, GFP expression or the expression of the ChR2/YFP fusion product in the same cell populations did not result in either cognitive or structural deficits. GABAergic lacZ expression caused significantly decreased hyper-arousal and mild cognitive deficits. Attenuated structural and behavioral consequences of lacZ expression could also be induced in adulthood, and lacZ transfection in neuronal cell cultures significantly decreased their viability. Our findings provide a strong caveat against the use of lacZ reporter mice for phenotyping studies and point to a particular sensitivity of the hippocampus formation to detrimental consequences of lacZ expression. © 2016 Wiley Periodicals, Inc.

Drinking water quality and public health in Southwestern Saudi Arabia: The need for a national monitoring program.

Aim of the Study:The aim was to investigate the bacteriological quality of drinking water, and explore the factors involved in the knowledge of the public about the quality of drinking water in Najran region, Saudi Arabia.Study Design:A cross-sectional descriptive study.Materials and Methods:A total of 160 water samples were collected. Total coliforms, fecal coliform, and fecal streptococci were counted using Most Probable Number method. The bacterial genes lacZ and uidA specific to total coliforms and Escherichia coli, respectively, were detected using multiplex polymerase chain reaction. An interview was conducted with 1200 residents using a questionnaire.Results:Total coliforms were detected in 8 (20%) of 40 samples from wells, 13 (32.5%) of 40 samples from tankers, and 55 (68.8%) of 80 samples from roof tanks. Twenty (25%) and 8 (10%) samples from roof tanks were positive for E. coli and Streptococcus faecalis, respectively. Of the 1200 residents participating in the study, 10%, 45.5%, and 44.5% claimed that they depended on municipal water, bottled water, and well water, respectively. The majority (95.5%) reported the use of roof water tanks as a source of water supply in their homes. Most people (80%) believed that drinking water transmitted diseases. However, only 25% of them participated in educational programs on the effect of polluted water on health.Conclusions:Our results could help health authorities consider a proper regular monitoring program and a sustainable continuous assessment of the quality of well water. In addition, this study highlights the importance of the awareness and educational programs for residents on the effect of polluted water on public health.

Systemic RNAi delivery to the muscles of ROSA26 mice reduces lacZ expression.

RNAi has potential for therapeutically downregulating the expression of dominantly inherited genes in a variety of human genetic disorders. Here we used the ROSA26 mouse, which constitutively expresses the bacterial lacZ gene in tissues body wide, as a model to test the ability to downregulate gene expression in striated muscles. Recombinant adeno-associated viral vectors (rAAVs) were generated that express short hairpin RNAs (shRNAs) able to target the lacZ mRNA. Systemic delivery of these rAAV6 vectors led to a decrease of β-galactosidase expression of 30–50-fold in the striated muscles of ROSA26 mice. However, high doses of vectors expressing 21 nucleotide shRNA sequences were associated with significant toxicity in both liver and cardiac muscle. This toxicity was reduced in cardiac muscle using lower vector doses. Furthermore, improved knockdown in the absence of toxicity was obtained by using a shorter (19 nucleotide) shRNA guide sequence. These results support the possibility of using rAAV vectors to deliver RNAi sequences systemically to treat dominantly inherited disorders of striated muscle.

Murine CD8+ T cells but not macrophages express the vitamin D 1α-hydroxylase

The active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is synthesized by the 1α-hydroxylase, which is encoded by the Cyp27B1 gene. Using transgenic mice that have replaced the Cyp27B1 gene with the bacterial lacZ reporter gene (β-galactosidase), the inflammatory conditions that induce Cyp27B1 in the immune system were probed. A variety of stimuli including lipopolysaccharide, anti-CD3 or PMA/ionomycin were used to stimulate splenocytes and bone marrow derived macrophage in vitro. Only anti-CD3 stimulation resulted in a low induction of β-galactosidase activity in the spleen, indicating that T cells might be a source of Cyp27B1. In vivo, challenge with lipopolysaccharide, α-galactosylceramide, and Listeria monocytogenes failed to induce β-galactosidase activity outside of the kidneys. During more prolonged and severe inflammation there was staining in both the lungs and the gastrointestinal tract for β-galactosidase. Furthermore, wild-type reconstitution of the hematopoietic cell population in Cyp27B1 KO mice protected the mice from experimental colitis. T cell production of Cyp27B1 activity was shown to be from the CD8+ but not the CD4+ T cell population. CD8+ T cells expressed the reporter gene only after 48 h of stimulation. The data is consistent with a model where CD8+ T cells are activated to produce Cyp27B1 and 1,25(OH)2D3 that serves to turn off the local immune response.

P∆TubHA4C, a new versatile vector for constitutive expression in Drosophila

Several vectors for gene expression are available in Drosophila, a hub for genetics and genomics innovation. However, the vectors for ubiquitous expression have a complex structure, including coding exons, that makes in-frame cloning of cDNAs very complicated. In this report we describe a new Drosophila expression vector (p∆TubHA4C) for ubiquitous expression of coding sequences under the control of a minimal 0.9kb promoter of α1 tubulin (α1t). This plasmid was designed to include optimized multiple cloning sites (polylinker) to provide flexibility in cloning strategies. We also added the option of double labeling the expressed proteins with two C-terminal tags, the viral epitope hemagglutinin and a synthetic tetracysteine (4C) tag that binds small fluorescent compounds. This dual tag allows both in situ and biochemical detection of the desired protein. In particular, the new 4C tag technology combines easy fluorescent labeling with small arsenical compounds in live or fixed cells and tissues, while producing minimal alterations to the tagged protein due to its small size. To demonstrate the potent and ubiquitous expression under the control of the ∆Tub promoter, bacterial lacZ was expressed and monitored in cell culture and transgenic flies. We found that the modified 0.9kb ΔTub promoter induced similar expression levels to the intact 2.6kb α1t promoter, supporting the inclusion of all critical regulatory elements in the new and flexible ∆TubHA4C vector.

Development and characterization of a vector set with regulated promoters for systematic metabolic engineering in Saccharomyces cerevisiae

A set of vectors was constructed that enable combined and systematic testing of metabolic pathway genes in Saccharomyces cerevisiae. The vectors are available as CEN/ARS and 2 µ-based plasmids with a choice of three inducible promoters, P(GAL1) , P(CUP1) and P(ADH2) . These features offer control over the initiation and level of gene expression. In addition, the vectors can be used as templates to generate PCR fragments for targeted chromosomal integration of gene expression cassettes. Selection markers are flanked by loxP elements to allow efficient CreA-mediated marker removal and recycling after genomic integration. For each promoter, expression of a bacterial lacZ reporter gene was characterized from plasmid-based and integrated chromosomal cassettes, and compared to that of the glycolytic P(PGK1) promoter. Plasmid stabilities were also determined. The promoters showed distinct activity profiles useful for modulating expression of metabolic pathway genes. This series of plasmids with inducible promoters extends our previous vector set carrying the constitutive promoters P(PGK1) , P(TEF1) and P(HXT7-391) .

A fast and sensitive alternative for β-galactosidase detection in mouse embryos

The bacterial lacZ gene is widely used as a reporter in a myriad of mouse transgenic experiments. β-Galactosidase, encoded by lacZ, is usually detected using X-gal in combination with ferric and ferrous ions. This assay produces a blue indole precipitate that is easy to detect visually. Here, we show that Salmon-gal in combination with tetrazolium salts provides a more sensitive and faster staining reaction than the traditional β-galactosidase assay in mouse embryos. Using a combination of Salmon-gal and tetranitroblue tetrazolium, we were able to visualize the activity of β-galactosidase in embryos at stages when the customary X-gal reaction failed to detect staining. Our studies provide an enhanced alternative for β-galactosidase detection in expression and cell fate studies that use lacZ-based transgenic mouse lines.

General steps in the production and establishment of transgenic chicken lines carrying a bacterial lacZ gene expressing β-galactosidase

Replication-defective retroviral vectors are efficient vehicles for the delivery of exogenous genes, and they may be used in the generation of transgenic animals. The replication-defective retroviral SNTZ vector carrying the lacZ gene with a nuclear localized signal was injected into the subgerminal cavity of freshly laid eggs. Subsequently, the eggs were allowed to hatch, and the chickens were screened for the lacZ gene by using the polymerase chain reaction. Eight of 15 male chickens that survived to sexual maturity contained the lacZ gene in their semen. Subsequently, these males were mated with wild-type female chickens. In this study, performance of the SNTZ vector carrying the Eschrichia coli lacZ gene to express a nuclear-localized β-galactosidase was injected into the subgerminal cavity of embryos collected from newly laid eggs was reviewed.

Thermal regulation of membrane lipid fluidity by a two‐component system in Bacillus subtilis

This article describes a simple and robust laboratory exercise on the regulation of membrane unsaturated fatty acid composition in bacteria by a decrease in growth temperature. We take advantage of the well characterized Des pathway of Bacillus subtilis, composed of a Δ5-desaturase (encoded by the des gene) and the canonical two-component system DesK-DesR, to study the transcriptional regulation of des during cold shock. Students analyze the expression of a reporter transcriptional fusion between the des promoter and the bacterial lacZ gene in a wild-type B. subtilis strain and in des or desK-desR mutants grown under different culture conditions. Measurements of β-galactosidase activity allow them to investigate how the Des pathway works and to assess the role of each component of this regulatory system.

Inflammation fails to induce the vitamin D3 1α-hydroxylase. (135.13)

Abstract The active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH) 2D3], is largely produced by a renally expressed enzyme 1α-hydroxylase, which is encoded by the Cyp27B1 gene. Using transgenic mice that have replaced the Cyp27B1 gene with the bacterial lacZ reporter gene (β-galactosidase), we wanted to investigate the conditions that induce extrarenal 1α-hydroxylase expression. Expression of the 1α-hydroxylase in extrarenal tissues including the immune system has been reported. We did not detect β-galactosidase expression in LPS-stimulated BMDM, splenocytes, and peritoneal cells. Moreover, in vivo challenges of LPS, α-galactosylceramide, and Listeria monocytogenes showed no detectable β-galactosidase expression in lung, liver, spleen, and blood. Cyp27B1 expression has been reported in the colon of both DSS-treated and control mice. Cyp27B1 KO mice were more susceptible to DSS colitis than WT mice and showed severe weight loss, inflammation, bleeding, and shrinkage of the colon. DSS treatment of Cyp27B1 KO mice resulted in spotty induction of β-galactosidase in the distal small intestine with a frequency of 3 positives for 22 tested and in the colon with a frequency of 6 positives for 22 tested. No β-galactosidase activity was detected in the intestines of untreated controls. The induction of Cyp27B1 expression was sporadic and not associated with the severity of disease. Our data does not support a role for the induction of Cyp27B1 following inflammation.

An Assessment of Mutagenic Effect of 3, 3', 4, 4', 5 Pentachlorobiphenyl (PCB126) in Muta Mouse Fetuses

PCBs are persistent environmental agents that induce multiple impairments in living beings. In this study we used a transgenic mouse model (Muta(TM) Mouse), carrying bacterial lacZ genes for mutation assays and for assessment of the genotoxic effect of PCB126 on fetal mice. Mothers of experimental groups were subjected to a single oral dose of PCB126 (125, 250 and 500 microg/kg) on the 10th day of pregnancy, respectively. Fetuses were autopsied on the 18th day of gestation. Cleft palate was observed in 2 out of 11 fetuses from 3 litters in 500 microg/kg treated group. Other external malformations were not observed. The DNA mutation frequencies (MF) of fetuses in each group were 1.15 +/- 0.24 x 10(-5), 0.90 +/- 0.20 x 10(-5) and 1.08 +/- 0.24 x 10(-5) in fetuses of 125, 250 and 500 microg/kg treated groups, respectively. The MF of controls was 0.81 +/- 0.22 x 10(-5). There were no significant differences among the groups. However, the MF of each treated group was a little highter than that of control group. Possible relationships between PCB and its mutagenic effects in the offspring of mice are discussed.

Differential regulation of human and mouse telomerase reverse transcriptase (TERT) promoter activity during testis development

In adult testis, telomerase activity is exclusively detectable during the early steps of spermatogenesis and is downregulated during differentiation to spermatozoa. Knowledge about telomerase activity during testis development from birth to adulthood is still scarce. Telomerase activity is regulated primarily via the transcriptional initiation of TERT expression which encodes its catalytic subunit. We used the hTERTp-lacZ transgenic mouse model that expresses the bacterial lacZ reporter gene under the control of an 8.0-kbp human TERT promoter fragment to analyze simultaneously endogenous mouse Tert gene expression as well as human TERT promoter activity during mouse testis development. We show that human TERT promoter activity increased during puberty and was highest in adult mouse testis whereas mouse Tert expression and telomerase activity were found to be high in testis from the earliest time point tested (6 days postpartum). Histological analysis revealed that beta-galactosidase expression, encoded by the lacZ reporter gene, is present in all seminiferous tubules in adult testis, but in a subset of tubules before puberty. We further analyzed the expression of SCF/ c-KIT, which was described to regulate spermatogonia proliferation and mouse Tert expression, in prepubertal and adult testis by immunohistochemistry. Whereas SCF and c-KIT were detectable in all seminiferous tubules, a spatial and preclusive expression pattern of human TERT promoter activity and activated c-KIT (p-c-KIT Tyr 567/569) was observed in prepubertal testes.

Whole Mount Enzyme Histochemistry as a Rapid Screen at Necropsy for Expression of β-Galactosidase (LacZ)–Bearing Transgenes: Considerations for Separating Specific LacZ Activity from Nonspecific (Endogenous) Galactosidase Activity

Whole mount enzyme histochemistry to localize lacZ-bearing transgenes (lacZ-WMH) also detects endogenous beta-galactosidases. The experiments reported here evaluated lacZ-WMH as a potential tool for transgene expression analysis during high-throughput rodent necropsies. A lacZ-WMH survey of organs from adult, wild-type, male and female mice (C57BL/6, FVB/N) and female rats (Sprague-Dawley) performed at the optimal pH (> or = 7.0) for bacterial lacZ yielded intense endogenous staining in the gonads, kidney, male accessory sex organs, salivary glands, submucosal glands in the duodenum, and thyroid. Substantial staining occurred in the adrenal cortex, lymph nodes, and linings of the gastrointestinal tract, the urinary bladder and uterus, and (for rat only) in the adenohypophysis, bone marrow, thymus, and trigeminal ganglia. Endogenous galactosidases were distributed similarly in sections of flash-frozen organs used for slide-based lacZ histochemistry (lacZ-SBH) at pH < or = 5.0 (optimal for eukaryotic enzymes). Cerebral neurons were labeled only by lacZ-SBH. At pH 7.4, endogenous but not specific lacZ activity was abolished for lacZ-SBH, while endogenous activity was not halted without reducing specific activity for lacZ-WMH. These data demonstrate that lacZ-WMH is feasible during rodent necropsies for many but not all organs if species-, strain-, and sex-specific divergence in endogenous galactosidase activity is considered and special fixation (3% paraformaldehyde for 3 hours at 4 degrees C) is used.