Phenotypic and molecular characterisation of novel Vicia faba germplasm with tolerance to acetohydroxyacid synthase-inhibiting herbicides (AHAS) developed through mutagenesis techniques.

BACKGROUND: Faba bean (Vicia faba L.) is an important crop in Australian farming systems, however, weed control is a major constraint due to a lack of in-crop broadleaf herbicide options. To address this, we developed acetohydroxyacid synthase (AHAS) inhibitor herbicide tolerance in faba bean using mutagenesis techniques. Dose-response experiments, agronomic field evaluation and DNA sequencing of the AHAS gene were used to quantify and validate tolerance traits. RESULTS: Four M2 faba bean single-plant biotypes (IMI-1, IMI-2, IMI-3 and IMI-4) at a frequency of 3.63 × 10-6 were successfully recovered. Molecular characterisation of the AHAS gene identified two known target site mutations (resulting in protein substitutions Ala205Val and Ser653Asn) conferring tolerance. Phenotypic characterisation found that both mutations conferred high levels of tolerance to the imidazolinone herbicide imazapyr. However, although the Ala205Val substitution showed improved levels of cross-tolerance to a range of sulfonylurea chemistries, the Ser653Asn substitution did not. In the field, IMI-3 showed the highest level of agronomic tolerance across a range of imidazolinone herbicides. CONCLUSIONS: Mutagenesis techniques were successful in the development of tolerance to AHAS inhibitor herbicides in faba bean, and could facilitate the first safe in-crop broadleaf herbicide control option in Australian faba bean production. © 2019 Society of Chemical Industry.

The hepatic soluble guanylyl cyclase-cyclic guanosine monophosphate pathway mediates the protection of remote ischemic preconditioning on the microcirculation in liver ischemia-reperfusion injury.

BACKGROUND: Remote ischemic preconditioning (RIPC) protects against liver ischemia reperfusion (IR) injury. An essential circulating mediator of this protection is nitric oxide (NO) induced by lower limb RIPC. One of the mechanisms through which NO generally acts is the soluble guanylyl cyclase-cyclic GMP (sGC-cGMP) pathway. The present study aimed to assess the role of hepatic sGC-cGMP in lower limb RIPC-induced protection against liver IR injury. METHODS: Mice were allocated to 4 groups: 1.Sham; 2.IR: 40 min of lobar hepatic ischemia and 2 hr reperfusion; 3.RIPC+IR: 6 cycles of 4x4 min IR of the lower limb followed by IR group procedure; (4) 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ)+RIPC+IR: ODQ (sGC inhibitor) was administered followed by RIPC+IR group procedure. Hepatic microcirculatory blood flow (MBF) was measured throughout the experiment. Plasma transaminases, hepatic histopathological and transmission electron microscopy studies were performed at the end of the experiment. Hepatic cGMP levels were measured in groups 1-3 in addition to an RIPC alone group. RESULTS: Compared to liver IR alone, RIPC+IR increased hepatic MBF during liver reperfusion (P<0.05), and reduced plasma transaminases (P<0.05) and ultrastructural markers of injury. In contrast compared to RIPC+IR, ODQ+RIPC+IR decreased hepatic MBF (P<0.05) and ultrastructural markers of injury. However, plasma transaminases were not significantly different in the ODQ+RIPC+IR compared to the RIPC+IR group. Hepatic cGMP levels were significantly elevated in the RIPC compared to sham group. CONCLUSIONS: The hepatic sGC-cGMP pathway is required for mediating the protective effects of lower limb RIPC on hepatic MBF in liver IR injury.

Role of insulin-like growth factor binding protein-4 in prevention of colon cancer.

BACKGROUND: Insulin-like growth factors (IGFs) are important for the proliferation of cancer cells. One of their binding proteins, known as insulin-like growth factor binding protein -4 (IGFBP-4) is well known for its inhibitory action on IGFs in vitro. We assessed the effect of IGFBP-4 in prevention of development of colon cancer in vivo. METHODS: Nude mice were subcutaneously inoculated with HT-29 colon cancer cells and they were also simultaneously injected either gene construct containing mammalian expression vector pcDNA3 with or without IGFBP-4 gene or phosphate buffered saline. The effect was assessed 4 weeks later by evaluating the tumours for mitosis, necrosis, apoptosis, and expressions of IGFBP-4, Bcl-2 and Bax proteins. RESULTS: The results showed that the IGFBP-4 gene therapy did not prevent the tumour establishment but it increased the tumour apoptosis which was associated with an increase in Bcl-2 and Bax expressions. The IGFBP-4 protein was low in tumours which received IGFBP-4 gene construct which may be due to a feed back mechanism of IGFBP-4 upon its own cells. CONCLUSION: IGFBP-4 gene therapy in the form localised gene transfer did not prevent colon cancer initiation and establishment but it resulted in increased apoptosis and Bax protein expression and a decrease in tumour cellular mitosis.