Project description:BackgroundLactose synthesis rate is an important factor in milk production and quality in mammals. Understanding the lactose synthesis mechanism is crucial for the improvement of milk quantity and quality. However, research on the temporal gene changes regarding lactose synthesis during the whole lactation is still limited. The objective of this study was to determine gene expression profiles related to lactose synthesis in sows during lactation, and further identify the critical steps or key factors in the lactose synthesis pathway.MethodsTo determine the temporal change of factors related to lactose synthesis in sows, milk from eight multiparous Yorkshire sows (parity 3 to 6) was collected at 0 h, 2 h, 6 h, 12 h, 24 h, day 2, 3, 4, 7, 14, and 21 after birth of the first piglet. Lactose content, prolactin and progesterone concentration, and gene or protein expression related to lactose synthesis were measured.ResultsThe lactose yield increased gradually from D2 to D21 and reached a maximum at D14 (3-fold from D2) during lactation (P < 0.05). A similar trend was observed in IGF-1 and insulin concentrations in milk, both of which were greatest at D3 with a subsequent decrease during middle to late lactation. Conversely, milk prolactin and progesterone concentrations moderately decreased with the progression of lactation. The mRNA or protein expressions related to glucose transportation (GLUT1), glucose-galactose interconversion (HK1 and UGP2), UDP-galactose transportation (SLC35A2), and lactose synthetase (LALBA and B4GALT1) in the lactose synthesis pathway were significantly upregulated during early to middle lactation and plateaued by late lactation (P < 0.05).ConclusionsThese novel findings suggest that the increased lactose synthesis in lactation was related to the coordinated upregulation of genes or enzymes in the lactose synthesis pathway, and glucose transportation (GLUT1) and lactose synthetase (LALBA and B4GALT1) might be the critical steps in the lactose synthesis pathway of sows during lactation.

Project description:Streptococcus mutans, the primary etiological agent of human dental caries, has developed multiple mechanisms to colonize and form biofilms on the tooth surface. The brpA gene codes for a predicted surface-associated protein with apparent roles in biofilm formation, autolysis, and cell division. In this study, we used two models to further characterize the biofilm-forming characteristics of a BrpA-deficient mutant, strain TW14. Compared to those of the parent strain, UA159, TW14 formed long chains and sparse microcolonies on hydroxylapatite disks but failed to accumulate and form three-dimensional biofilms when grown on glucose as the carbohydrate source. The biofilm formation defect was also readily apparent by confocal laser scanning microscopy when flow cells were used to grow biofilms. When subjected to acid killing at pH 2.8 for 45 min, the survival rate of strain TW14 was more than 1 log lower than that of the wild-type strain. TW14 was at least 3 logs more susceptible to killing by 0.2% hydrogen peroxide than was UA159. The expression of more than 200 genes was found by microarray analysis to be altered in cells lacking BrpA (P < 0.01). These results suggest that the loss of BrpA can dramatically influence the transcriptome and significantly affects the regulation of acid and oxidative stress tolerance and biofilm formation in S. mutans, which are key virulence attributes of the organism. RNA extraction. S. mutans strains were grown in 50 ml of BHI broth and harvested at an OD600 of [congruent with]0.5 by centrifugation at 3,800 × g at 4°C for 5 min. The pellets were quickly resuspended and treated with RNAprotect (QIAGEN, Inc., CA) by using the procedures recommended by the supplier. The treated cells were collected by centrifugation at 3,800 × g at 4°C for 10 min and stored at −80°C. RNA extractions were performed by using hot phenol as previously described (33). To remove all DNA, the purified RNAs were treated with DNaseI (Ambion, Inc., Austin, TX) and retrieved with the RNeasy purification kit (QIAGEN, Inc., CA). To prepare a reference RNA, S. mutans UA159 was grown in 3 liters of BHI until mid-exponential phase (OD600 [congruent with] 0.5) and total RNA was extracted as described above. The purified RNA was then aliquoted and stored at −80°C until use. cDNA synthesis, Cy dye coupling and array hybridization. Array analysis was performed by using the whole-genome S. mutans microarrays that were obtained from PFGRC at TIGR. The S. mutans genome array consisted of 70-mer oligonucleotides representing 1,960 open reading frames from strain UA159. The full 70-mer complement was printed four times on the surface of the slides. Experiments, including cDNA synthesis, Cy dye coupling, hybridization, and washing, were performed by using protocols from the PFGRC with minor modifications. Briefly, cDNA synthesis was carried out in a total volume of 45 μl by mixing 10 μg total RNA with 3 μg random hexamers (Invitrogen Life Technologies, CA), 9 μl 5× first-strand buffer, 4.5 μl 0.1 M dithiothreitol, 2 μl 12.5 mM dNTP/aa-UTP with a 1.5- to-1 ratio of aa-dUTP to dTTP, and 3 μl Superscript III reverse transcriptase. This mixture was incubated at 42°C for 16 h. Following the completion of cDNA synthesis, RNA was hydrolyzed with NaOH and the aminoallyl-labeled cDNA samples were purified by using the QIAGEN QIAquick PCR purification kit, followed by drying in a speed vacuum. The samples were then resuspended in 5 μl 0.1 M NaCO3 and incubated with 5 μl of Cy dye (resuspended as recommended by the manufacturer) for 2 h in the dark. For all experiments, the reference RNA samples were coupled to Cy5 and both wild-type and the BrpA-deficient mutant RNA samples were coupled to Cy3. Uncoupled Cy dyes were removed by the QIAquick PCR purification kit, and the coupled samples were eluted in 100 μl elution buffer. Each Cy3-labeled experimental sample was mixed with a Cy5-labeled reference sample, and the mixtures were allowed to dry in a speed vacuum. To hybridize, the samples were resuspended in 60 μl hybridization buffer and heated at 90°C for 10 min. Following a quick centrifugation, the Cy dye-coupled probes were applied to microarray slides and incubated at 42°C in a water bath for 17 h. Slides were then washed and scanned by using an Axon GenePix 4000B scanner (Axon Instruments, Foster City, CA). Array data normalization and statistical analysis. Data were collected from at least four separate array slides, which contained four copies of the genome, with RNA isolated from four independent experiments. The raw data were loaded into the TIGR Spotfinder program and further normalized with the TIGR microarray data analysis system using LOWESS iterative log mean centering parameters with the default settings, followed by in-slide replicate analysis. Spots that were missing or labeled as “bad” during the upstream processes in 50% of the slides were cut off in the output files. The ratios of channel A over channel B were then converted to log2 values and analyzed with BRB array tools (version 3.01, developed by Richard Simon and Amy Peng Lam, National Cancer Institute, Bethesda, MD). A pairwise Student's t test was used to analyze the mean log ratios of the BrpA-deficient strain and the wild type, and genes that were differentially expressed with the significance level of a P value of <0.001 were selected.

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