Callus induction, regeneration and transformation of sugarcane (Saccharum officinarum L.) with chitinase gene using particle bombardment

This study was carried out to optimize the conditions for introducing a chitinase gene into the sugarcane cv. Phil 66-07 calli by particle bombardment. Young leaves were cultured on the modified Murashige and Skoog (MS) medium supplemented with varied concentrations of 2,4- dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid (NAA), yeast extract and coconut water (CW). The maximum percentage of callus induction was obtained from the MS medium supplemented with 3 mg/l 2,4-D and 15% (v/v) coconut water. Multiple shoots were achieved by transferring sugarcane calli to the MS medium amended with 1 mg/l benzyl aminopurine (BA) and 0.5 mg/l indole-3-butyric acid (IBA). Additional experiments were also performed to determine the effect of antibiotics on regeneration of sugarcane. It was found that growth of sugarcane calli and plantlets were completely inhibited by hygromycin concentrations of 25 and 50 mg/l, respectively. The genetic transformation was achieved via particle bombardment with an optimal helium pressure of 900 psi and the stopping screen set at 9 cm. Sugarcane was transformed with either GUS or a chitinase gene and a gene for hygromycin selection. GUS transformed calli were produced to optimize the particle bombardment protocol. Using the optimized protocol, the chitinase gene was transformed into sugarcane and polymerase chain reaction (PCR) was used to verify the integration of a chitinase gene, 35S promoter and nitric oxide synthase (NOS) terminator in transgenic sugarcane.Key words: Sugarcane, genetic transformation, particle bombardment, chitinase gene

Repeated-batch ethanol fermentation from sweet sorghum juice by free cells of Saccharomyces cerevisiae NP 01

Low-cost inoculum preparation (IP) media for ethanol production were developed. It was found that sweet sorghum juice (SSJ) containing 100 g l-1 of total sugar without nutrient supplement could be used as the low-cost IP medium instead of the typical IP medium or yeast extract malt extract (YM) medium. Ethanol production from the SSJ (total soluble solids of 24 °Bx) by the inoculum of Saccharomyces cerevisiae NP 01 in batch and repeated-batch fermentations was then investigated. The fermentations were carried out under static condition in 500 ml air-locked Erlenmeyer flasks at 30°C and the initial yeast cell concentration was 1×108 cells ml-1. In the batch fermentation, the concentration (P), productivity (Qp) and yield (Yp/s) of ethanol were 110.09 ± 0.81 g l-1, 2.29 ± 0.01 g l-1 h-1 and 0.51 ± 0.02, respectively. In the repeated-batch fermentation, the yeasts could be used at least eight successive batches without a marked decrease in ethanol production. The repeated-batch fermentation with fill and drain volume at 75% of the working volume gave higher ethanol production efficiencies than those at 50% of the working volume in terms of total ethanol production rate (g h-1). The average P, Qp and Yp/s of the eight successive batches at 75% fill and drain volume in a 2 L bioreactor at the agitation rate of 100 rev min-1 were 93.30 ± 9.44 g l-1, 1.21 ± 0.43 g l-1 h-1 and 0.48 ± 0.03, respectively.Key words: Repeated-batch, ethanol fermentation, low-cost nutrient, sweet sorghum juice, Saccharomyces cerevisiae

Types of gene effects governing the inheritance of oleic and linoleic acids in peanut (Arachis hypogaea L.)

Oleic and linoleic acids are major fatty acids in peanut determining the quality and shelf-life of peanut products. A better understanding on the inheritance of these characters is an important for high-oleic breeding programs. The objective of this research was to determine the gene actions for oleic acid, linoleic acid, the ratio of oleic to linoleic acids (O/L ratio) and percentage oil (% oil) in peanut. Georgia-02C, SunOleic 97R (high-oleic genotypes) and KKU 1 (low-oleic genotypes) were used as parents to generate P1, P2, F2, F3, BC11S and BC12S. The entries were planted in a randomized complete block design with four replications in the rainy season (2008) and the dry season (2008/2009). Gas liquid chromatography (GLC) was used to analyze fatty acid compositions. The data were used in generation means analysis to understand gene effects. The differences in season, generation and generation X season interactions were significant for oleic acid in the crosses Georgia-02C X KKU 1 and SunOleic 97R X KKU 1. Additive, dominance and epistasis gene effects were significant for oleic acid, linoleic acid, O/L ratio and % oil. Initial selection can be carried out in early segregating population, and final selection in late generations.Keywords: Breeding, gene actions, generation mean analysis, groundnut, oil qualit

The use of dried spent yeast as a low-cost nitrogen supplement in ethanol fermentation from sweet sorghum juice under very high gravity conditions

Dried spent yeast (DSY) was used as a low-cost nitrogen supplement for ethanol fermentation from sweet sorghum juice under very high gravity (VHG) conditions by Saccharomyces cerevisiae NP 01. The fermentation was carried out at 30\ubaC in a 5-litre bioreactor. The results showed that DSY promoted ethanol production efficiencies. The ethanol concentration (P), productivity (Qp) and yield (Yp/s) of the sterile juice (total sugar of 280 g l-1) supplemented with 8 g l-1 of DSY were not different from those supplemented with yeast extract and/or peptone at the same amount. The initial yeast cell concentration of 5 x 107 cells ml-1 was found to be optimal for scale-up ethanol production. In addition, an increase in sugar concentration in inoculum preparation medium (from 10 to 100 g l-1) improved the ability of the inoculum to produce ethanol under the VHG conditions. When S. cerevisiae NP 01 grown in the juice containing 100 g l-1 of total sugar was used as the inoculum for ethanol fermentation, the P, Qp and Yp/s obtained were 108.98 \ub1 1.16 g l-1, 2.27 \ub1 0.06 g l-1 h-1 and 0.47 \ub1 0.01 g g-1, respectively. Similar results were also observed when the ethanol fermentation was scaled up to a 50-litre bioreactor under the same conditions. The cost of the sweet sorghum for ethanol production was US$ 0.63 per litre of ethanol. These results clearly indicate the high potential of using sweet sorghum juice supplemented with DSY under VHG fermentation for ethanol production in industrial applications

Ethanol production from sweet sorghum juice under very high gravity conditions: Batch, repeated-batch and scale up fermentation

Batch ethanol fermentations from sweet sorghum juice by Saccharomyces cerevisiae NP 01 were carried out in a 500 ml air-locked Erlenmeyer flask under very high gravity (VHG) and static conditions. The maximum ethanol production efficiency was obtained when 9 g l-1 of yeast extract was supplemented to the juice. The ethanol concentration (P), productivity (Qp) and yield (Yp/s) were 120.24 \ub1 1.35 g l-1, 3.01 \ub1 0.08 g l-1 h-1 and 0.49 \ub1 0.01, respectively. Scale up ethanol fermentation in a 5-litre bioreactor at an agitation rate of 100 rev min-1 revealed that P, Qp and Yp/s were 139.51 \ub1 0.11 g l-1, 3.49 \ub1 0.00 g l-1 h-1 and 0.49 \ub1 0.01, respectively, whereas lower P (119.53 \ub1 0.20 g l-1) and Qp (2.13 \ub1 0.01 g l-1 h-1) were obtained in a 50-litre bioreactor. In the repeated-batch fermentation in the 5-litre bioreactor with fill and drain volume of 50% of the working volume, lower P and Qp were observed in the subsequent batches. P in batch 2 to 8 ranged from 103.37 \ub1 0.28 to 109.53 \ub1 1.06 g l-1

Theoretical Engineering of the Gut Micro biome for the Purpose of Creating Superior Soldiers

The purpose of this review is to highlight research raising the possibility of exploiting the host-microbiome gut axis for military purposes. Through optimizing the gut-microbiome environment it is possible to enhance nutritional access to indigestible material, provide local and systemic analgesia, enhance psychological robustness to battlefield stress, produce endogenous steroids, reduce muscle fatigue, and promote peripheral wound healing. However, this approach is still in its early stages and thus has not been explored to its full potential. The challenges that are currently preventing the practical use of gut bacteria include the following: inconsistency of clinical outcomes, transient effects requiring continuous supplementation, the type of regimen selected, the initiation and cessation of regimen, and the broader clinical studies needed to validate this research. This review is intended to shed light on the numerous and varied positive impacts such an approach could have for the military if further developed

Veterinary Considerations for the Theoretical Resurrection of Extinct Species

The de-extinction of the dinosaur is a dubious possibility but its consideration brings forth some issues that are at least worthy of scientific discussion. In this review, we discuss two distinct issues that have implications for a de-extinct species such as a dinosaur: the ability, or lack thereof, to safely sedate a rare and potentially fractious animal capable of harming the veterinary staff tasked with its care; and, disease risks associated with a species that has been extinct for millions of years. To identify potential sedatives, comparative pharmacology will be needed to uncover the links between receptor pharmacology and the desired clinical outcomes of activating established alpha-2 adrenergic, opioid, and benzodiazepine receptors. Specific to disease control, it will be necessary to understand the unique susceptibility of the new species to current diseases as well as predicting their reservoir capacity for potential human and veterinary pandemic diseases. While the topics presented herein are not exhaustive, this review highlights some of the foremost research that should be conducted in order to serve the unique veterinary needs of a de-extinct species using the dinosaur as a paradigm. Addressing these issues should be considered if an intact dinosaur genome becomes available, regardless of the feasibility of dinosaur resurrection

Pre-operative immune cell numbers and ratios are associated with peri-operative adverse outcomes in transfused patients

Background and objectivesTransfusion-related immune modulation (TRIM) and associated adverse outcomes during major surgery are increasingly important to patients and health services internationally. A panel of pre-operative blood tests is an essential part of the pre-operative anaesthetic assessment. This panel of blood tests commonly considers numbers of immune cells (i.e., lymphocytes, monocytes, and neutrophils and cell ratios) that may be used as biomarkers to evaluate and potentially predict post-operative adverse outcomes.DesignThis retrospective data collection from eight hospital databases, within the Royal Brisbane and Women's Hospital, considered only patients who received blood transfusion during surgery (2016–2018) (n = 2,121). The association between pre-operative immune cell numbers and ratios and adverse outcomes were assessed. Adverse outcomes were coded using the International Classification of Diseases-10 (ICD-10) coding which specifically considered transfusion-related immune modulation. Results were adjusted for confounding factors.ResultsAfter adjustment, decreased pre-operative lymphocyte numbers and increased neutrophil/lymphocyte ratio (NLR) were associated with increased odds of developing infection; decreased NLR with decreased odds of developing adverse renal outcomes; and decreased lymphocyte numbers with decreased odds of developing adverse cardiovascular outcomes. Monocyte numbers, neutrophil numbers, and the lymphocyte/monocyte ratio (LMR) were not associated with increased adverse outcomes after adjustment.ConclusionPre-operative lymphocyte numbers and NLR are associated with adverse outcomes during peri-operative transfusion. Future assessment of peri-operative immune modulation should include the assessment of immune cell function and numbers

Characterizing the in vivo functions of Cyfip1 in the development of cardiovascular, hematopoietic, and nervous systems by precise targeted genome editing in zebrafish

Actin cytoskeleton is the most abundant and crucial protein in most eukaryotic cells, involving a broad range of essential cellular processes. The major regulator for actin dynamics is the WAVE regulatory complex (WRC). The activation and function of the WRC is governed through its own subunit called Cyfip. In vertebrates, there are two Cyfip isoforms, Cyfip1 and Cyfip2, which have been shown to have different expression patterns and distinct functions in various biological systems. However, there is a limited understanding of the in vivo functions of Cyfip proteins in animals, especially in vertebrates. With the development of CRISPR/Cas9-based gene editing technologies and zebrafish as a vertebrate model organism, we have established cyfip1 and cyfip2 knockout mutant in zebrafish using a newly developed and efficient CRISPR/Cas9-based short homology targeted integration strategy named GeneWeld. Together with a novel gene inactivation method called pPRISM-Stop vector, we explored the in vivo functions of each Cyfip isoform in various biological systems, including cardiovascular, hematopoietic, retinotectal, and spinal motor nervous systems. With the high efficiency of the GeneWeld method for precise targeted integration of pPRISM-Stop cassette into each cyfip locus, we were able to recover cyfip1 and cyfip2 germline transmitting adults with on-target integration with frequencies at 13% for both cyfip loci (3/24 for cyfip1 and 2/16 for cyfip2). Despite an unexpected integration of the vector backbone into cyfip2 locus uncovered later, we were able to successfully establish stable lines of true cyfip1 knockout mutant to investigate the phenotypes from its homozygous deletion. Intriguingly, we discovered that cyfip1 abolishment during early stage of development led to mismigration or stalled development of endothelial cells and stenotic vessels accompanied by disrupted blood circulation, substantial reduction of the HSPCs in various hematopoietic tissues, as well as aberrant axon branching and abnormal axon terminals. Taken together, our study demonstrated efficient targeted integration at zebrafish cyfip1 locus using CRISPR/Cas9 short homology targeted GeneWeld strategy and pPRISM-Stop-mediated gene inactivation method to establish, for the first time, stable cyfip1 knockout mutant zebrafish lines to analyze cyfip1 knockout phenotypes and characterize the in vivo functions of cyfip1 in cardiovascular, hematopoietic, retinotectal and spinal motor nervous systems. Although additional samples and further analysis is necessary to make final conclusions, the pronounced morphological and microscopic phenotypes discovered in this study suggested the promising essential in vivo functions of cyfip1 in the development of cardiovascular, hematopoietic, retinotectal, and spinal motor nervous systems, which worth investigating more profoundly to fully characterize the in vivo functions and identify molecular mechanisms of cyfip1, and the WRC-mediated actin remodeling, in these physiological systems