Association of gene expression with syringyl to guaiacyl ratio in sugarcane lignin.

KEY MESSAGE: A transcriptome analysis reveals the transcripts and alleles differentially expressed in sugarcane genotypes with contrasting lignin composition. Sugarcane bagasse is a highly abundant resource that may be used as a feedstock for the production of biofuels and bioproducts in order to meet increasing demands for renewable replacements for fossil carbon. However, lignin imparts rigidity to the cell wall that impedes the efficient breakdown of the biomass into fermentable sugars. Altering the ratio of the lignin units, syringyl (S) and guaiacyl (G), which comprise the native lignin polymer in sugarcane, may facilitate the processing of bagasse. This study aimed to identify genes and markers associated with S/G ratio in order to accelerate the development of sugarcane bioenergy varieties with modified lignin composition. The transcriptome sequences of 12 sugarcane genotypes that contrasted for S/G ratio were compared and there were 2019 transcripts identified as differentially expressed (DE) between the high and low S/G ratio groups. These included transcripts encoding possible monolignol biosynthetic pathway enzymes, transporters, dirigent proteins and transcriptional and post-translational regulators. Furthermore, the frequencies of single nucleotide polymorphisms (SNPs) were compared between the low and high S/G ratio groups to identify specific alleles expressed with the phenotype. There were 2063 SNP loci across 787 unique transcripts that showed group-specific expression. Overall, the DE transcripts and SNP alleles identified in this study may be valuable for breeding sugarcane varieties with altered S/G ratio that may provide desirable bioenergy traits.

The functionality of African-specific variants in the TGFB1 regulatory region and their potential role in HIVAN.

BACKGROUND: Transcription of transforming growth factor beta-1 (TGF-ß1) is regulated by a polymorphic promoter region containing African-specific single nucleotide polymorphisms (SNPs). Some of these SNPs have higher frequencies among Southern Africans compared to other African populations and their functionality has only been partially studied. Due to the high prevalence of HIV-associated nephropathy (HIVAN) in Africans we hypothesized that functional African TGFB1-promoter SNPs may contribute to HIVAN pathogenesis. METHODS: The functionality of the TGFB1 -1347 C>T variant and African-specific variants (-1287 G>A, -1154 C>T, -387 C>T and -14 G>A) were examined by measuring reporter gene expression in kidney and fibroblast cell lines co-transfected with TGFB1-promoter constructs and an HIV-Tat expression vector. TGF-ß1 immunohistochemical staining was performed on kidney biopsies with HIVAN (n = 18) and compared to control biopsies without HIVAN or tubulointerstitial disease (n = 12) using semi-quantitative and digital image analysis. HIVAN cases were genotyped for TGFB1 -1347 and -387 SNP variants. RESULTS: TGFB1-promoter haplotypes containing the African -387 T-allele resulted in ~ five-fold repression of TGFB1-promoter activity compared to -387 C haplotypes (p ≤ 0.024). HIV-Tat upregulated TGFB1-promoter activity for haplotypes containing -1347 T and -387 T in transfected renal cells (≈ 1.6-fold; p ≤ 0.030) and fibroblasts (≈ 1.3-fold; p ≤ 0.016). The renal interstitium from HIVAN biopsies, compared to HIV-positive and -negative controls, differed in the semi-quantitative TGF-ß1 staining and digital optical density analyses. The TGFB1 -1347 and -387 genotypes in HIVAN cases were similar to population controls. CONCLUSION: African-specific haplotypes lower TGFB1-promoter activity and expression levels and HIV-Tat upregulates TGFB1 promoter activity irrespective of the haplotype.

Clinico-pathological features of repeat renal biopsies in patients with lupus nephritis at Groote Schuur Hospital, Cape Town.

Background Repeat renal biopsies in patients with lupus nephritis are usually done to guide treatment or to establish disease chronicity. Their value is not clear from available literature. There are also no available data in Africa to guide clinicians. Methods This was a retrospective study of patients undergoing a repeat renal biopsy between January 2003 and December 2014 from a single centre in Cape Town, South Africa. Relevant demographic, clinical and histological records of patients with repeat renal biopsies were documented. Comparison of data from first and second renal biopsy was performed. Results Forty-four patients had at least two biopsies done during the study period. Most patients were females (81.8%). The mean biopsy interval was 2.8 ± 1.8 (range 0.38-9.4) years. Proteinuria was the main indication for the repeat biopsy (36.1%). The glomerular filtration rate and proteinuria worsened between the two biopsies ( p = 0.001 and 0.019, respectively) suggesting disease progression. Most patients (65.4%) with a non-proliferative class of lupus nephritis at first biopsy progressed into a proliferative class, whereas patients with initial proliferative lupus nephritis at first biopsy (77.8%) remained as proliferative at repeat biopsy. Treatment was changed in 85% of patients at second biopsy. Conclusion Repeat renal biopsies in patients with lupus nephritis presents a useful means of assessing disease progression and provides guidance regarding modification of treatment. More studies are, however, required to evaluate the value of repeat biopsies and perhaps the need for protocol renal biopsies in patients with lupus nephritis.

Mercuration of thiacalix[4]arenes in the cone and 1,3-alternate conformations.

The first mercuration in the thiacalixarene series using thiacalix[4]arenes immobilized in the cone or 1,3-alternate conformations gave a mixture of two monomercurated regioisomers (meta and para) in approx. 4 : 1 and 2 : 1 ratios, respectively. The organomercurial intermediates show unusual solid-state behaviour, as evidenced by the formation of η(6) complexes, and can be easily transformed into halogen-substituted derivatives, so far inaccessible in thiacalixarene chemistry. This paves the way towards the synthesis of inherently chiral thiacalixarene-based receptors with an unusual substitution pattern.

Investigating the threshold for early renal allograft biopsy: A South African single-centre perspective.

BACKGROUND: The most common clinical indication for renal biopsy in the early post-transplant period is early graft dysfunction (EGD), which may present either as delayed graft function (DGF) or acute graft dysfunction. Even though it is a valuable diagnostic tool, renal allograft biopsy is not without risk of major complications. Recent studies have suggested that, with modern immunosuppressive induction regimens and more accurate ways to determine high immunological risk transplants, early acute rejection (AR) is uncommon and routine biopsy for EGD does not result in a change in management. OBJECTIVES: To describe the histological findings and complications of renal allograft biopsies for EGD in our setting, and to determine whether our current threshold for biopsy is appropriate. METHODS: This study was a retrospective audit that included all patients who underwent renal allograft biopsy within the first 30 days of transplantation at Groote Schuur Hospital, Cape Town, South Africa, from 1 June 2010 to 30 June 2018. The indication for biopsy was any patient who showed significant EGD, characterised by acute graft dysfunction or DGF with dialysis dependence. RESULTS: During the study period, 330 patients underwent renal transplantation, of whom 105 (32%) had an early biopsy and were included in the study. The median age of recipients was 39 (range 17 - 62) years, with 65% males and 35% females. The majority of donors were deceased donations after brain death (70%), with an overall median cold ischaemic time of 9 hours (interquartile range (IQR) 4 - 16). The average number of human leukocyte antigen mismatches was 5 (IQR 4 - 7). A donor-specific antibody was recorded for 18% of recipients and a panel-reactive antibody score of >30% was recorded for 21%. The median duration after transplant for biopsy was 8 (IQR 6 - 10) days. During the first month of EGD, AR was diagnosed in 42% of patients who underwent biopsies. In 21% of these patients, there was acute cellular rejection, in 16% antibody-mediated rejection, and in 5% both of these. Acute tubular necrosis was the primary finding in 32%, with acute interstitial nephritis in 8%, and acute calcineurin toxicity in 4% of cases. A significant biopsy-related complication was recorded in 3 patients: 1 small-bowel perforation repaired via laparotomy, and 2 vascular injuries successfully embolised by interventional radiology. CONCLUSIONS: Considering the relative safety and high rate of detection of AR, a liberal approach to renal biopsy for EGD remains justifiable in our setting.

Antimicrobial efficacy and irrigating potential of irrigation solutions using different activation methods.

The objective of this in vitro study was to establish the antimicrobial efficacy and the effect of different activation methods on the smear layer at the coronal level of straight root canals of four different root canal irrigation solutions. The four irrigation solutions were 3.5% sodium hypochlorite liquid (NaOCl), 2% sodium hypochlorite gel, chlorhexidine gluconate liquid and a mixture of 100 mg doxycycline capsules with 2 ml sterile water. ANTIMICROBIAL EFFECTS: The surfaces of four agar plates were inoculated with Enterococcus faecalis and divided into four equal quadrants. Ten microlitres of each test solution was dispensed onto the four filter paper disks on each agar plate. The antibacterial activity of materials was apparent from circular clear inhibition zones forming around the filtration paper. The diameters of these inhibition zones were measured using a micrometer gauge. EFFECT ON SMEAR LAYER: Access cavities were prepared on fifty, extracted, single rooted, human teeth and the root canals prepared with rotary files. The teeth were randomly divided into five groups (n = 10) and each group irrigated with a different irrigation solution. Different activation methods were used in the coronal portion of each root canal. The solutions were activated in the canals using one of the following methods: a 30 gauge needle (Control), a sonic scaler tip, and a rotary brush. After sampling, the roots of the treated teeth were fractured and prepared for Scanning Electron Microscopy (SEM) according to standard methods. The one-way ANOVA test was used to determine whether there were any statistical significant differences between the different groups. The average zones of inhibition for 3.5% NaOCl, 2% NaOCl, 2.5% chlorhexidine and doxycycline were 2.7mm, 2.0 mm, 11.2 mm and 12.4 mm respectively. Sterile water, 3.5% NaOCl and 2% NaOCl had no significant effect on the smear layer. However, when chlorhexidine and doxycycline solutions were activated with a rotary brush, 90 and 80 per cent of the observed surfaces were free of smear layer respectively. Doxycyline and 2.5% chlorhexidine demonstrated the highest antimicrobial activity against Enterococcus faecalis and removed most of the smear layer when the solutions were activated with a rotary brush.

Relationship between sugarcane culm and leaf biomass composition and saccharification efficiency.

BACKGROUND: Lignocellulosic biomass is recognized as a promising renewable feedstock for the production of biofuels. However, current methods for converting biomass into fermentable sugars are considered too expensive and inefficient due to the recalcitrance of the secondary cell wall. Biomass composition can be modified to create varieties that are efficiently broken down to release cell wall sugars. This study focused on identifying the key biomass components influencing plant cell wall recalcitrance that can be targeted for selection in sugarcane, an important and abundant source of biomass. RESULTS: Biomass composition and the amount of glucan converted into glucose after saccharification were measured in leaf and culm tissues from seven sugarcane genotypes varying in fiber composition after no pretreatment and dilute acid, hydrothermal and ionic liquid pretreatments. In extractives-free sugarcane leaf and culm tissue, glucan, xylan, acid-insoluble lignin (AIL) and acid-soluble lignin (ASL) ranged from 20 to 32%, 15% to 21%, 14% to 20% and 2% to 4%, respectively. The ratio of syringyl (S) to guaiacyl (G) content in the lignin ranged from 1.5 to 2.2 in the culm and from 0.65 to 1.1 in the leaf. Hydrothermal and dilute acid pretreatments predominantly reduced xylan content, while the ionic liquid (IL) pretreatment targeted AIL reduction. The amount of glucan converted into glucose after 26 h of pre-saccharification was highest after IL pretreatment (42% in culm and 63.5% in leaf) compared to the other pretreatments. Additionally, glucan conversion in leaf tissues was approximately 1.5-fold of that in culm tissues. Percent glucan conversion varied between genotypes but there was no genotype that was superior to all others across the pretreatment groups. Path analysis revealed that S/G ratio, AIL and xylan had the strongest negative associations with percent glucan conversion, while ASL and glucan content had strong positive influences. CONCLUSION: To improve saccharification efficiency of lignocellulosic biomass, breeders should focus on reducing S/G ratio, xylan and AIL content and increasing ASL and glucan content. This will be key for the development of sugarcane varieties for bioenergy uses.

Antimicrobial activity of medicinal plants against oral microorganisms.

Ethanol extracts of eight plant species used traditionally in South Africa for the treatment of oral diseases were investigated for in vitro antimicrobial activity against oral pathogens namely Actinobacillus actinomycetemcomitans, Actinomyces naeslundii, Actinomyces israelii, Candida albicans, Porphyromonus gingivalis, Privotella intermedia and Streptococcus mutans using the disk diffusion method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ethanol extracts were determined against these microorganisms using micro dilution. The cytotoxicity and therapeutic index (TI) of selected active extracts were also determined. Out of eight plants, six (Annona senegalensis, Englerophytum magalismontanum, Dicerocarym senecioides, Euclea divinorum, Euclea natalensis, Solanum panduriforme and Parinari curatellifolia) exhibited MIC values ranging from 25.0 mg/ml to 0.8 mg/ml. Gram negative bacteria were found to be more resistant to the plant extracts than Gram positive bacteria, except for Euclea natalensis which inhibited all three Gram negative bacteria tested in this study. All plant extracts showed moderate cytotoxicity on the Vero cell line. The fifty percent inhibitory concentration (IC(50)) of all plants tested range from 92.3 to 285.1 microg/ml.

Antibacterial properties of five bonding agents.

PURPOSE: This study compared antibacterial properties of five bonding agents with that of a control, Chlorhexidine (2.5%). Products evaluated were the self-etch primers (-P) and adhesives (-A) of Clearfil SE Bond (SE-P; SE-A) [Kuraray Dental], Clearfil Protect Bond (PB-P; PB-A) [Kuraray Dental], Optibond Solo Self-etch (OS-P; OS-A) [Kerr] and the one-bottle products, self-etch Clearfil Tri-S Bond (3S) [Kuraray Dental] and total-etch Adper Scotchbond 1 XT (XT) [3M ESPE]. METHODS: Spread plates of three different bacteria (Streptococcus mutans, Lactobacillus paracasei and Actinomyces naeslundii) were prepared on Casein-peptone-Soymeal-peptone Agar (CASO-Agar). Controls, Primers, Adhesives, and Primer & Adhesive combinations were placed on standardized, sterilized filtration paper or composite disks and then placed on the inoculated agar and incubated at 37 degrees C for 48 hours. Inhibition zones were measured and data was statistically analyzed using the Student t-test. An additional test was performed by which growth inhibiting of 1/10 and 1/100 dilutions of the test suspensions were measured spectrophotometrically as turbidity at 600 nm and expressed as percentage growth (%). RESULTS: Compared to the controls, the only cured product which produced significant inhibition was Scotchbond 1 XT (XT), and that for Actinomyces naeslundii only. The primer of Clearfil Protect Bond (PB-P) showed statistically significant growth inhibition for all three test bacteria, the primer of SE Bond (SE-P) had significant inhibitive properties against Streptoccocus mutans and Actinomyces naeslundii and the primer of Optibond Solo Self-etch (OS-P) inhibited growth of Actinomyces naeslundii significantly. CONCLUSIONS: The primers of Clearfil Protect Bond, Clearfil SE Bond, Optibond Solo Self-etch and the product Adper Scotchbond 1 XT may be beneficial in eliminating remaining bacteria after cavity preparation, but further research on a possible long-term antibacterial benefit of these products needs to be undertaken.

Carbon Partitioning during Sucrose Accumulation in Sugarcane Internodal Tissue.

The temporal relationship between sucrose (Suc) accumulation and carbon partitioning was investigated in developing sugarcane internodes. Radiolabeling studies on tissue slices, which contained Suc concentrations ranging from 14 to 42% of the dry mass, indicated that maturation coincided with a redirection of carbon from water-insoluble matter, respiration, amino acids, organic acids, and phosphorylated intermediates into Suc. It is evident that a cycle of Suc synthesis and degradation exists in all of the internodes. The decreased allocation of carbon to respiration coincides with a decreased flux from the hexose pool. Both the glucose and fructose (Fru) concentrations significantly decrease during maturation. The phosphoenolpyruvate, Fru-6-phosphate (Fru-6-P), and Fru-2,6-bisphosphate (Fru-2, 6-P2) concentrations decrease between the young and older internodal tissue, whereas the inorganic phosphate concentration increases. The calculated mass-action ratios indicate that the ATP-dependent phosphofructokinase, pyruvate kinase, and Fru-1,6-bisphosphatase reactions are tightly regulated in all of the internodes, and no evidence was found that major changes in the regulation of any of these enzymes occur. The pyrophosphate-dependent phosphofructokinase reaction is in apparent equilibrium in all the internodes. Substrate availability might be one of the prime factors contributing to the observed decrease in respiration.

Induction of Pyrophosphate-Dependent Phosphofructokinase in Watermelon (Citrullus lanatus) Cotyledons Coincides with Insufficient Cytosolic D-Fructose-1,6-Bisphosphate 1-Phosphohydrolase to Sustain Gluconeogenesis.

During germination of Citrullus lanatus, pyrophosphate-dependent phosphofructokinase (PFP) activity is induced. The peak of PFP activity coincides with the maximum gluconeogenic flux and high fructose-2,6-bisphosphate (Fru-2,6-P2) concentrations. Determination of cytosolic fructose-1,6 bisphosphatase (FBPase) activity in crude extracts is unreliable because of the high PFP activity. The FBPase activity, after correction for the contaminating PFP, is only one-third of the PFP activity. Purified cytosolic FBPase is inhibited by Fru-2,6-P2. The low cytosolic FBPase activity and high Fru-2,6-P2 most probably result in inadequate in vivo activity to catalyze the observed gluconeogenic flux. The total PFP activity is sufficient to catalyze the required carbon flux.

Fluctuation of the volume of distribution of amikacin and its effect on once-daily dosage and clearance in a seriously ill patient.

OBJECTIVE: The main aim of the trial was to determine the extent to which the volume of distribution of amikacin fluctuates in a seriously ill patient receiving copious quantities of i.v. fluid over an extended term of treatment. The impact of the volume fluctuation on amikacin therapeutic peak concentrations was also assessed. DESIGN AND SETTING: The case report describes a young, previously healthy male adult admitted to the surgical ICU of a teaching hospital following trauma to the head and central nervous system. INTERVENTION: The patient received 1 g of amikacin once-daily i.v. for 35 consecutive days as part of an antimicrobial regimen. Blood samples were drawn for routine amikacin concentration determinations on 14 occasions, extending over the entire term of treatment, from which the required pharmacokinetic parameters were determined. RESULTS: The volume of distribution of amikacin varied extensively from 0.27 to 0.61 l/kg (normal range 0.27 +/- 0.06 1/kg) notwithstanding the fact that amikacin clearance remained satisfactorily high throughout the term of treatment. CONCLUSIONS: Once-daily therapeutic amikacin concentrations fluctuate extensively and rapidly in the seriously ill patient receiving copious quantities of i.v. fluids, despite competent renal function. The volume expansion seen in our patient is difficult to account for in terms of the extracellular fluid compartment only. RECOMMENDATIONS: (a) Once-daily regimen amikacin peak concentrations should be frequently monitored in the seriously ill patient; (b) once-daily amikacin regimens are best monitored using blood specimens drawn at 1 and 6-8 h post administration.

Partial purification and characterisation of sugarcane neutral invertase.

Sugarcane neutral invertase (SNI) has been partially purified from mature sugarcane stem tissue to remove any potential competing activity. The enzyme is non-glycosylated and exhibits catalytic activity as a monomer, dimer and tetramer, most of the activity elutes as a monomer of native M(r) ca 60 k. The enzyme displays typical hyperbolic saturation kinetics for Suc hydrolysis. It has a K(m) of 9.8 mM for Suc and a pH optimum of 7.2. An Arrhenius plot shows the energy of activation of the enzyme for Suc to be 62.5 kJ mol-1 below 30 degrees and -11.6 kJ mol-1 above 30 degrees. SNI is inhibited by its products, with Fru being a more effective inhibitor than Glc. SNI is significantly inhibited by HgCl2, AgNO3, ZnCl2, CuSO4 and CoCl2 but not by CaCl2, MgCl2 or MnCl2. SNI showed no significant hydrolysis of cellobiose or trehalose.

The introduction of an inverted repeat to the 5' untranslated leader sequence of a transgene strongly inhibits gene expression.

Stable secondary structures in naturally occurring 5' untranslated regions have been shown to down-regulate both transcription and translation. We introduced a synthetic GC-rich inverted repeat to the leader sequence of a transgene to determine its influence on gene expression. The addition of a 54-bp inverted repeat led to a more than 90% reduction in transient gene expression, while the addition of an inverted repeat of 42-bp reduced gene expression by 88%. Complete removal of the inverted repeat abolished this inhibiting effect. This dramatic decrease in transgene expression is probably due to the formation of stable stem-loop structures in the 5' untranslated region of the reporter gene. The secondary structure energy of the putative stem-loop structures in the 54-bp and 42-bp repeats are -64.6 and -40.3 kcal mol-1, respectively. In comparison, the most stable stem-loop structure in the control construct's leader has a free energy of -15.4 kcal mol-1. This has important implications for the design of expression vectors where the recombination of multiple cloning sites and other 5' leader sequences can lead to the introduction of inverted repeats that has the potential of forming stable stem-loop structures and resulting in a significant decrease in gene expression.

Laboratory analysis of fluoride containing toothpaste available in South Africa.

Since the introduction of fluoridated toothpaste more than 30 years ago, it has become by far the most widespread source of fluoride in the world and after fluoridated water the most effective at reducing dental caries. Both the total soluble and the soluble ionic fluoride present in toothpaste are important in determining the effectiveness of the paste as it is important in remineralising demineralised lesions, a characteristic of fluoride which has proven to be the most important in preventing dental caries. The purpose of this study was to evaluate toothpaste currently available to the South African public regarding total soluble and soluble ionic fluoride. A variation of the method used by Grobler et al. (1983) and Du Preez et al. (1993) in previous studies conducted in South Africa was used in preparing the samples for fluoride analysis. Measurements of fluoride concentrations were done using an ionselective electrode. For all toothpaste studied (n=62), large variations occurred for the soluble ionic fluoride (mean = 318.44 microg F/g toothpaste; median = 139.69; standard deviation = 398.77). Correspondingly a mean total soluble fluoride of 848.84 microg F/g toothpaste was found (standard deviation = 189.35; median = 857.38). From this study it can be concluded that fluoride concentrations in toothpaste available to the South African public are in line with those found in the two previous South African studies. In the absence of artificially fluoridated water, toothpaste remains the most cost-effective strategy in the prevention of dental caries in South Africa.

Caries prediction factors in children with primary dentition.

Previous studies have indicated an association between the dmft and the lactobacilli counts in small children. This study evaluated and compared a number of salivary factors that could have an effect on caries progression in two groups of children with primary dentition (group 1 = 3-6 years; group II = 9 years). The average dmft score was higher for group II. The dmft score of group I consisted mainly of a large dt component, while in group II a large ft component was found. Lactobacilli were present in 44.83% of group I and in 77.27% of group II. Significant positive correlations were found for group I between the dt component of the dmft and lactobacilli count (P < 0.05, r = 0.48) as well as the total dmft and lactobacilli count (P < 0.05, r = 0.45). Significant positive correlations were found for group II between the dmft and lactobacilli count (P < 0.05, r = 0.39) and the plaque index and lactobacilli count (P < 0.05, r = 0.31). Significant correlations between the dmft and the prevalence of lactobacilli in the oral cavity were also indicated (group I: P < 0.05, r = 0.45; group II: P < 0.05, r = 0.36). Significant correlations confirmed the association of lactobacilli with the caries process and indicated the reliability of lactobacilli counts to determine caries activity. Correlations between the dmft and the prevalence of lactobacilli in the oral cavity indicated the possibility of an excellent but simple test for the prediction of caries susceptibility in children.