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.

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.

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.

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.

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.

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.

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.

Accuracy of MRCP Compared to ERCP in the Diagnosis of Bile Duct Disorders

AIM: To determine the accuracy of Magnetic Resonance Cholangiopancreatography compared to the gold standard Endoscopic Retrograde Cholangiopancreatography in the diagnosis of bile duct disorders at our institution. PATIENTS AND METHODS: 52 patients with suspected pancreatobiliary pathology were included in this prospective observational study. MRCP was performed in the 24-hour period prior to the ERCP. RESULTS: MRCP had sensitivity; specificity; positive and negative predictive values of 87; 80; 83.3and 84.2respectively for choledocholitiasis which correlates well with results obtained in other parts of the world. CONCLUSION: At our institution; MRCP has high diagnostic accuracy for bile duct calculi. Due to a small study population; results for other biliary pathology were inconclusive

Routes of pyruvate synthesis in phosphorus-deficient lupin roots and nodules.

Here, nodulated lupins (Lupinus angustifolius (cv Wonga)) were hydroponically grown at low phosphate (LP) or adequate phosphate (HP). Routes of pyruvate synthesis were assessed in phosphorus (P)-starved roots and nodules, because P-starvation can enhance metabolism of phosphoenolpyruvate (PEP) via the nonadenylate-requiring PEP carboxylase (PEPc) route. Since nodules and roots may not experience the same degree of P stress, it was postulated that decreases in metabolic inorganic phosphorus (Pi) of either organ, should favour more pyruvate being synthesized from PEPc-derived malate. Compared with HP roots, the LP roots had a 50% decline in Pi concentrations and 55% higher ADP : ATP ratios. However, LP nodules maintained constant Pi levels and unchanged ADP : ATP ratios, relative to HP nodules. The LP roots had greater PEP metabolism via PEPc and synthesized more pyruvate from PEPc-derived malate. In nodules, P supply did not influence PEPc activities or levels of malate-derived pyruvate. These results indicate that nodules were more efficient than roots in maintaining optimal metabolic Pi and adenylate levels during LP supply. This caused an increase in PEPc-derived pyruvate synthesis in LP roots, but not in LP nodules.

Pearl millet transformation system using the positive selectable marker gene phosphomannose isomerase.

Fertile transgenic pearl millet plants expressing a phosphomannose isomerase (PMI) transgene under control of the maize ubiquitin constitutive promoter were obtained using the transformation system described here. Proliferating immature zygotic embryos were used as target tissue for bombardment using a particle inflow gun. Different culture and selection strategies were assessed in order to obtain an optimised mannose selection protocol. Stable integration of the manA gene into the genome of pearl millet was confirmed by PCR and Southern blot analysis. Stable integration of the manA transgene into the genome of pearl millet was demonstrated in T1 and T2 progeny of two independent transformation events with no more than four to ten copies of the transgene. Similar to results obtained from previous studies with maize and wheat, the manA gene was shown to be a superior selectable marker gene for improving transformation efficiencies when compared to antibiotic or herbicide selectable marker genes.

Effect of biological contamination on dentine bond strength of adhesive resins.

The purpose of this in vitro study was to determine the effect of saliva (S) and blood (B) contamination on the dentine bond strength of two single-component dentine bonding systems. The occlusal thirds of 120 recently extracted, human molars were removed with a low speed saw and subsequently embedded in Bencor rings by means of self-curing, acrylic resin. The occlusal surfaces were ground wet on 600-grit silicone carbide paper in a polishing machine to expose superficial dentine and to create a smear layer. The teeth were randomly divided into 12 groups (n = 10). All the dentine surfaces were etched with 34% phosphoric acid for 15 seconds rinsed with water, air-dried for 3 seconds, leaving the surfaces visibly moist. For the control groups (C) the etched dentine surfaces were treated with either, Scotchbond 1 (SB1, 3M) or Prime & Bond NT (PBNT, Dentsply) according to the manufacturer's instructions. In the contaminated groups, the saliva or blood was applied by means of a disposable brush, left undisturbed for 1 minute, and the excess then thinned by air spray. The dentine bonding systems were then applied, also according to manufacturer's instructions. Composite (Z250 and TPH) and Compomer (F2000 and Dyract AP (D-AP)) stubs were packed and cured incrementally to the corresponding pretreated dentine surfaces. All specimens were stored for 24 hours under water at 37 degrees C. The bonds were then stressed to failure with a Zwick testing machine, operating at a crosshead speed of 0.5 mm/min. Fractured samples were examined in a Scanning Electron Microscope. The data were statistically analysed (Student-t test). The mean SBS (MPa) were. SB1 with Z250: C = 19.1 +/- 4.4; S = 17.3 +/- 3.5; B = 2.6 +/- 0.9; SB1 with F2000: C = 11.8 +/- 3.3; S = 9.7 +/- 1.8; B = 4.7 +/- 1.6. PBNT with TPH: C = 9.2 +/- 3.2; S = 6.5 +/- 3.0; B = 4.3 +/- 1.5; PBNT with D-AP: C = 10.2 +/- 3.6; S = 9.3 +/- 2.9 and B = 7.3 +/- 2.5. There was no statistical significant difference in shear bond strengths between the control and the saliva-contaminated samples for both systems. There was, however, a significant difference in bond strengths between the control and the blood-contaminated samples. Blood contamination negatively influenced bond strength of bonding systems to dentine.

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.

Analysis of sucrose accumulation in the sugar cane culm on the basis of in vitro kinetic data.

Sucrose accumulation in developing sugar cane (Saccharum officinarum) is accompanied by a continuous synthesis and cleavage of sucrose in the storage tissues. Despite numerous studies, the factors affecting sucrose accumulation are still poorly understood, and no consistent pattern has emerged which pinpoints certain enzyme activities as important controlling steps. Here, we develop an approach based on pathway analysis and kinetic modelling to assess the biochemical control of sucrose accumulation and futile cycling in sugar cane. By using the concept of elementary flux modes, all possible routes of futile cycling of sucrose were enumerated in the metabolic system. The available kinetic data for the pathway enzymes were then collected and assembled in a kinetic model of sucrose accumulation in sugar cane culm tissue. Although no data were fitted, the model agreed well with independent experimental results: in no case was the difference between calculated and measured fluxes and concentrations greater than 2-fold. The model thus validated was then used to assess different enhancement strategies for increasing sucrose accumulation. First, the control coefficient of each enzyme in the system on futile cycling of sucrose was calculated. Secondly, the activities of those enzymes with the numerically largest control coefficients were varied over a 5-fold range to determine the effect on the degree of futile cycling, the conversion efficiency from hexoses into sucrose, and the net sucrose accumulation rate. In view of the modelling results, overexpression of the fructose or glucose transporter or the vacuolar sucrose import protein, as well as reduction of cytosolic neutral invertase levels, appear to be the most promising targets for genetic manipulation. This offers a more directed improvement strategy than cumbersome gene-by-gene manipulation. The kinetic model can be viewed and interrogated on the World Wide Web at http://jjj.biochem.sun.ac.za.

Population screening for isoniazid acetylator phenotype.

PURPOSE: To establish a useful method for acetylator phenotypification and therapeutic drug monitoring of patients receiving isoniazid. METHODS: Sixty patients with uncomplicated pulmonary tuberculosis were given a 5-mg/kg oral dose of isoniazid each. Plasma concentrations of isoniazid and its metabolite, acetyl-isoniazid, were determined by HPLC analyses at various post-dose times. From the isoniazid concentration and the concentration ratio of acetyl-isoniazid and isoniazid (metabolic ratio), phenotypification methods were assessed. RESULTS: The metabolic ratios at 3 h post-dose revealed a trimodal distribution; a fast, intermediate and slow acetylator phenotype group. The 2-h and 6-h data showed different bimodal combinations of these phenotype groups. The metabolic ratio phenotypification method could be simplified by using the HPLC data directly without converting it to absolute concentrations. CONCLUSIONS: A single-sample test based upon the plasma isoniazid concentration, combined with the metabolic ratio of acetyl-isoniazid and isoniazid, appears to be a reliable parameter for phenotype discrimination and for bioavailability testing.