High flavonoid accompanied with high starch accumulation triggered by nutrient starvation in bioenergy crop duckweed (Landoltia punctata)

Background: As the fastest growing plant, duckweed can thrive on anthropogenic wastewater. The purple-backed duckweed, Landoltia punctata, is rich in starch and flavonoids. However, the molecular biological basis of high flavonoid and low lignin content remains largely unknown, as does the best method to combine nutrients removed from sewage and the utilization value improvement of duckweed biomass. Results: A combined omics study was performed to investigate the biosynthesis of flavonoid and the metabolic flux changes in L. punctata grown in different culture medium. Phenylalanine metabolism related transcripts were identified and carefully analyzed. Expression quantification results showed that most of the flavonoid biosynthetic transcripts were relatively highly expressed, while most lignin-related transcripts were poorly expressed or failed to be detected by iTRAQ based proteomic analyses. This explains why duckweed has a much lower lignin percentage and higher flavonoid content than most other plants. Growing in distilled water, expression of most flavonoid-related transcripts were increased, while most were decreased in uniconazole treated L. punctata (1/6 x Hoagland + 800 mg center dot L-1 uniconazole). When L. punctata was cultivated in full nutrient medium (1/6 x Hoagland), more than half of these transcripts were increased, however others were suppressed. Metabolome results showed that a total of 20 flavonoid compounds were separated by HPLC in L. punctata grown in uniconazole and full nutrient medium. The quantities of all 20 compounds were decreased by uniconazole, while 11 were increased and 6 decreased when grown in full nutrient medium. Nutrient starvation resulted in an obvious purple accumulation on the underside of each frond. Conclusions: The high flavonoid and low lignin content of L. punctata appears to be predominantly caused by the flavonoid-directed metabolic flux. Nutrient starvation is the best option to obtain high starch and flavonoid accumulation simultaneously in a short time for biofuels fermentation and natural products isolation

Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin.

Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that myosin VI was recruited to SGs in a Ca(2+)-dependent manner. Interfering with myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These myosin VI SI-specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially via c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane

Heat inactivation of clinical COVID-19 samples on an industrial scale for low risk and efficient high-throughput qRT-PCR diagnostic testing.

We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 266 million confirmed cases, over 5.26 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as 'direct to PCR' assays that do not involve RNA extraction or chemical neutralisation methods

Mutations in the Neuronal Vesicular SNARE VAMP2 Affect Synaptic Membrane Fusion and Impair Human Neurodevelopment

VAMP2 encodes the vesicular SNARE protein VAMP2 (also called synaptobrevin-2). Together with its partners syntaxin-1A and synaptosomal-associated protein 25 (SNAP25), VAMP2 mediates fusion of synaptic vesicles to release neurotransmitters. VAMP2 is essential for vesicular exocytosis and activity-dependent neurotransmitter release. Here, we report five heterozygous de novo mutations in VAMP2 in unrelated individuals presenting with a neurodevelopmental disorder characterized by axial hypotonia (which had been present since birth), intellectual disability, and autistic features. In total, we identified two single-amino-acid deletions and three non-synonymous variants affecting conserved residues within the C terminus of the VAMP2 SNARE motif. Affected individuals carrying de novo non-synonymous variants involving the C-terminal region presented a more severe phenotype with additional neurological features, including central visual impairment, hyperkinetic movement disorder, and epilepsy or electroencephalography abnormalities. Reconstituted fusion involving a lipid-mixing assay indicated impairment in vesicle fusion as one of the possible associated disease mechanisms. The genetic synaptopathy caused by VAMP2 de novo mutations highlights the key roles of this gene in human brain development and function

Improving the efficiency and effectiveness of an industrial SARS-CoV-2 diagnostic facility.

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories

Role of Munc18-1 in regulating neuroexocytosis

Neuroexocytosis requires the formation of a protein complex called the SNARE complex, formed when the v-SNARE VAMP2 located on vesicles containing the neurotransmitter, bind to the t-SNAREs syntaxin-1 and SNAP25 on the target plasma membrane. Munc18-1 contributes to exocytosis not only through its interaction with syntaxin-1 during its transport to the cell surface, but also by promoting SNARE complex formation. The focus of this thesis is to characterise these two functions at the molecular level by investigating how Munc18-1 interaction with syntaxin-1 is affected in early infantile epileptic encephalopathy (EIEE), and to elucidate the trafficking pathway underpinning Munc18-1-mediated syntaxin-1 transport to the cell surface in neurosecretory cells. Munc18-1 binding to the syntaxin-1 N-terminus has been proposed to promote SNARE complex formation leading to fusion, suggesting that Munc18-1 might play a role in priming of exocytosis. In Chapter 2, the significance of the Munc18-1:Syntaxin-1 N-terminus interaction in exocytosis and syntaxin-1 transport to the cell surface was investigated. Munc18-1 recombinant protein harbouring specific mutations in the hydrophobic pocket (F115E and E132A) designed to selectively abrogate the N-terminus interaction slightly reduced the binding affinity of Munc18-1 to syntaxin-1 but fully blocked binding to the SNARE complex. Overexpression of the Munc18-1- F115E/E132A double mutant in Munc18-1 knock down PC12 cells (KD43) not only rescued the transport of syntaxin-1 to the plasma membrane but also stimulated secretion of hGH and NPY. Total internal reflection fluorescence microscopy analysis show that the secretory vesicles had a reduced rate of vesicle fusion at the onset of stimulation. The Munc18-1 hydrophobic pocket is therefore essential for SNARE complex binding but removal of the Munc18-1:Syntaxin-1 Nterminus binding produced a limited effect on Ca2+-dependent exocytosis in PC12 cells. This suggests that alternative mechanisms are in place to support Munc18-1-mediated SNARE complex formation and priming. Munc18-1 binds syntaxin-1 in a closed conformation, where the N-terminal Habc domain of syntaxin-1 folds back onto its C-terminal H3 domain (syntaxin-1 SNARE motif) with high affinity. This binding prevents syntaxin-1 from participating in SNARE complex formation, and thus inhibits exocytosis. In Chapter 3, we dissected the contribution of the closed conformation binding and the N-terminus using Munc18-1/-2 double knockdown PC12 (DKD PC12) cells. In these cells, endogenous syntaxin-1 is mislocalised and regulated secretion inhibited. The expressions levels of syntaxin-1, -2 and -3 were also greatly reduced in the DKD PC12 cells. The Munc18-1- K46E/E59K mutant, specifically designed to inhibit syntaxin-1 closed conformation binding, was unable to restore syntaxin-1 expression levels, syntaxin-1 transport to cells surface and regulated viii secretion in these PC12 cells. Munc18-1-F115E/E132A mutant, previously shown to have impaired binding to syntaxin-1 N-terminus, was able to rescue syntaxin-1 transport to the cell surface and exocytosis in DKD PC12 cells. These results suggest that Munc18-1/-2 regulate syntaxin expression levels, its transport to the cell surface and exocytosis in PC12 cells. The binding of Munc18-1 to the syntaxin-1 closed conformation is therefore necessary for Munc18-1 stimulatory action, whereas the binding to syntaxin-1 N-terminus plays a limited role in neurosecretion. The roles of Munc18-1 in neuroexocytosis was further dissected in Chapter 4 by analysing the importance of Munc18-1 domain 1 in mediating binding to syntaxin-1 closed conformation and its contribution in chaperoning syntaxin-1 during its transport to the cell surface. The binding affinity of Munc18-1 domain 1 mutants (D34N, M38V, K46E, E59K, K63E, E66A) was tested and their effect on syntaxin-1 transport to the plasma membrane and secretion in DKD PC12 cells investigated. We found that Munc18-1 domain 1 mutant’s ability to rescue syntaxin-1 transport to the plasma membrane and exocytosis in DKD PC12 cells was correlated to the mutant’s binding affinity to syntaxin-1. These results demonstrated that Munc18-1 domain 1 is critical for syntaxin-1 closed conformation binding and that this binary interaction underpins Munc18-1 chaperoning activity. The significance of Munc18-1:Syntaxin-1 interaction during syntaxin-1 transport/traffic to the cell surface was characterised in Chapter 5. Using Munc18-1-C180Y, a structurally unstable mutant linked to early infantile epileptic encephalopathy (EIEE), we investigated the trafficking pathway underlying syntaxin-1 transport to the plasma membrane in DKD PC12 cells. Overexpression of the Munc18-1-C180Y-EmGFP in DKD PC12 resulted in the formation of fluorescent aggregates and failure to restore syntaxin-1 plasma membrane localisation. Regulated exocytosis was also affected due to the lack of the t-SNARE synatxin-1 on the plasma membrane. Importantly, Munc18-1-C180Y binding affinity to syntaxin-1 was similar to that of wild type Munc18-1. Our results demonstrate that structural instability of Munc18-1-C180Y interferes with the Munc18-1-mediated syntaxin-1 transport to the plasma membrane, which may be an underlying factor in EIEE. Using immunoisolation we purified Munc18-1 trafficking cargoes and identified Rab33b as a critical player in this trafficking pathway. This thesis elucidates the molecular basis of Munc18-1-mediated syntaxin-1 transport/traffic to the plasma membrane, a necessary step for functional regulated exocytosis, and highlights a novel role for Rab33b in promoting Munc18-1-dependent syntaxin-1 traffic. Abolishing Munc18- 1:Syntaxin-1 binding impairs Munc18-1-mediated syntaxin-1 trafficking and contributes to the establishment of diseases such as EIEE

Determination of sulfonamides in selected Malaysian swine wastewater by high-performance liquid chromatography

An analytical HPLC method for the simultaneous determination of eight sulfonamides in swine wastewater was developed. The samples were collected from three states in Malaysia. Sample clean up was carried out by employing solid-phase extraction using a 60 mg Oasis HLB (Waters) cartridge with 3 ml reservoir. The HPLC column used was Supelcosil C18 (250 mm × 4.6 mm I.D.) and elution was carried out using gradient mode. The mobile phases used were acetonitrile and 0.5% acetic acid in purified water. Antibiotics were detected using UV absorbance at 272 nm. Recoveries obtained for sulphanilamide ranged from 31.9 ± 5.1% to 36.2 ± 1.0%, while recoveries for other sulfa drugs studied were from 91.9 ± 5.0% to 106.0 ± 1.1%. The limit of quantitation (LOQ) for sulfamerazine, sulfamethazine and sulfamethoxypyridazine was 7.5 ng/L, while the LOQ for the other studied antibiotics was 5.0 ng/L. The method was used to analyse sulfonamides in wastewater collected from selected Malaysian swine facilities. Copyright © 2006 Elsevier B.V

Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin.

Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that myosin VI was recruited to SGs in a Ca(2+)-dependent manner. Interfering with myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These myosin VI SI-specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially via c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane

Mutations in the neuronal vesicular SNARE VAMP2 affect synaptic membrane fusion and impair human neurodevelopment

VAMP2 encodes the vesicular SNARE protein VAMP2 (also called synaptobrevin-2). Together with its partners syntaxin-1A and synaptosomal-associated protein 25 (SNAP25), VAMP2 mediates fusion of synaptic vesicles to release neurotransmitters. VAMP2 is essential for vesicular exocytosis and activity-dependent neurotransmitter release. Here, we report five heterozygous de novo mutations in VAMP2 in unrelated individuals presenting with a neurodevelopmental disorder characterized by axial hypotonia (which had been present since birth), intellectual disability, and autistic features. In total, we identified two single-amino-acid deletions and three non-synonymous variants affecting conserved residues within the C terminus of the VAMP2 SNARE motif. Affected individuals carrying de novo non-synonymous variants involving the C-terminal region presented a more severe phenotype with additional neurological features, including central visual impairment, hyperkinetic movement disorder, and epilepsy or electroencephalography abnormalities. Reconstituted fusion involving a lipid-mixing assay indicated impairment in vesicle fusion as one of the possible associated disease mechanisms. The genetic synaptopathy caused by VAMP2 de novo mutations highlights the key roles of this gene in human brain development and function