Conservation of shibire and RpII215 temperature-sensitive lethal mutations between Drosophila and Bactrocera tryoni.

The sterile insect technique can suppress and eliminate population outbreaks of the Australian horticultural pest, Bactrocera tryoni, the Queensland fruit fly. Sterile males mate with wild females that produce inviable embryos, causing population suppression or elimination. Current sterile insect releases are mixed sex, as the efficient removal of unrequired factory-reared females is not yet possible. In this paper, we assessed the known Drosophila melanogaster temperature-sensitive embryonic lethal alleles shibire (G268D, shits1) and RNA polymerase II 215 (R977C, RpII215ts) for potential use in developing B. tryoni genetic sexing strains (GSS) for the conditional removal of females. Complementation tests in D. melanogaster wild-type or temperature-sensitive genetic backgrounds were performed using the GAL4-UAS transgene expression system. A B. tryoni wild-type shibire isoform partially rescued Drosophila temperature lethality at 29°C by improving survivorship to pupation, while expressing B. tryoni shits1 failed to rescue the lethality, supporting a temperature-sensitive phenotype. Expression of the B. tryoni RpII215 wild-type protein rescued the lethality of D. melanogaster RpII215ts flies at 29°C. Overexpressing the B. tryoni RpII215ts allele in the D. melanogaster wild-type background unexpectedly produced a dominant lethal phenotype at 29°C. The B. tryoni shibire and RpII215 wild-type alleles were able to compensate, to varying degrees, for the function of the D. melanogaster temperature-sensitive proteins, supporting functional conservation across species. Shibire and RpII215 hold potential for developing insect strains that can selectively kill using elevated temperatures; however, alleles with milder effects than shits1 will need to be considered.

Drosophila melanogaster models of MPS IIIC (Hgsnat-deficiency) highlight the role of glia in disease presentation.

Sanfilippo syndrome (Mucopolysaccharidosis type III or MPS III) is a recessively inherited neurodegenerative lysosomal storage disorder. Mutations in genes encoding enzymes in the heparan sulphate degradation pathway lead to the accumulation of partially degraded heparan sulphate, resulting ultimately in the development of neurological deficits. Mutations in the gene encoding the membrane protein heparan-α-glucosaminide N-acetyltransferase (HGSNAT; EC2.3.1.78) cause MPS IIIC (OMIM#252930), typified by impaired cognition, sleep-wake cycle changes, hyperactivity and early death, often before adulthood. The precise disease mechanism that causes symptom emergence remains unknown, posing a significant challenge in the development of effective therapeutics. As HGSNAT is conserved in Drosophila melanogaster, we now describe the creation and characterisation of the first Drosophila models of MPS IIIC. Flies with either an endogenous insertion mutation or RNAi-mediated knockdown of hgsnat were confirmed to have a reduced level of HGSNAT transcripts and age-dependent accumulation of heparan sulphate leading to engorgement of the endo/lysosomal compartment. This resulted in abnormalities at the pre-synapse, defective climbing and reduced overall activity. Altered circadian rhythms (shift in peak morning activity) were seen in hgsnat neuronal knockdown lines. Further, when hgsnat was knocked down in specific glial subsets (wrapping, cortical, astrocytes or subperineural glia), impaired climbing or reduced activity was noted, implying that hgsnat function in these specific glial subtypes contributes significantly to this behaviour and targeting treatments to these cell groups may be necessary to ameliorate or prevent symptom onset. These novel models of MPS IIIC provide critical research tools for delineating the key cellular pathways causal in the onset of neurodegeneration in this presently untreatable disorder.

Leveraging AI in Postgraduate Medical Education for Rapid Skill Acquisition in Ultrasound-Guided Procedural Techniques.

Ultrasound-guided techniques are increasingly prevalent and represent a gold standard of care. Skills such as needle visualisation, optimising the target image and directing the needle require deliberate practice. However, training opportunities remain limited by patient case load and safety considerations. Hence, there is a genuine and urgent need for trainees to attain accelerated skill acquisition in a time- and cost-efficient manner that minimises risk to patients. We propose a two-step solution: First, we have created an agar phantom model that simulates human tissue and structures like vessels and nerve bundles. Moreover, we have adopted deep learning techniques to provide trainees with live visualisation of target structures and automate assessment of their user speed and accuracy. Key structures like the needle tip, needle body, target blood vessels, and nerve bundles, are delineated in colour on the processed image, providing an opportunity for real-time guidance of needle positioning and target structure penetration. Quantitative feedback on user speed (time taken for target penetration), accuracy (penetration of correct target), and efficacy in needle positioning (percentage of frames where the full needle is visualised in a longitudinal plane) are also assessable using our model. Our program was able to demonstrate a sensitivity of 99.31%, specificity of 69.23%, accuracy of 91.33%, precision of 89.94%, recall of 99.31%, and F1 score of 0.94 in automated image labelling.

CRISPR/Cas9 Mutagenesis to Generate Novel Traits in Bactrocera tryoni for Sterile Insect Technique.

Sterile Insect Technique (SIT) is a biocontrol strategy that has been widely utilized to suppress or eradicate outbreak populations of insect pests such as tephritid fruit flies. As SIT is highly favored due to it being species-specific and environmentally friendly, there are constant efforts to improve the efficiency and efficacy of this method in particular at low pest densities; one of which is the use of genetically enhanced strains. Development of these desirable strains has been facilitated by the emergence of the CRISPR/Cas genome-editing technology that enables the rapid and precise genomic modification of non-model organisms. Here, we describe the manual microinjection of CRISPR/Cas9 reagents into tephritid pest Bactrocera tryoni (Queensland fruit fly) embryos to introduce ideal traits as well as the molecular methods used to detect successful mutagenesis.

Scar Assessment Tools: How Do They Compare?

Healing after dermal injury is a complex but imperfect process that results in a wide range of visible scars. The degree of disfigurement is not the sole determinant of a scar's effect on patient well-being, with a number of other factors being critical to outcome. These include cosmetic appearance, symptoms such as itch and pain, functional loss, psychological or social problems, and quality of life. An accurate assessment of these domains can help clinicians measure outcomes, develop, and evaluate treatment strategies. A PubMed literature search was performed up to 31st March 2020. Ten objective scar measurements, four Clinician-Reported Outcome Measures (CROMs), six Patient-Reported Outcome Measures (PROMs), and one combined measure were evaluated for their reliability, clinical relevance, responsiveness to clinical change, and feasibility. Many quantitative tools were limited in their clinical relevance and feasibility, whereas few qualitative CROMs and PROMs have undergone rigorous assessment. This review examines currently available assessment tools, focusing primarily on subjective scar measurements (CROMs, PROMs), and offers a perspective on future directions in the field.

Molecular Biology of the WWOX Gene That Spans Chromosomal Fragile Site FRA16D.

It is now more than 20 years since the FRA16D common chromosomal fragile site was characterised and the WWOX gene spanning this site was identified. In this time, much information has been discovered about its contribution to disease; however, the normal biological role of WWOX is not yet clear. Experiments leading to the identification of the WWOX gene are recounted, revealing enigmatic relationships between the fragile site, its gene and the encoded protein. We also highlight research mainly using the genetically tractable model organism Drosophila melanogaster that has shed light on the integral role of WWOX in metabolism. In addition to this role, there are some particularly outstanding questions that remain regarding WWOX, its gene and its chromosomal location. This review, therefore, also aims to highlight two unanswered questions. Firstly, what is the biological relationship between the WWOX gene and the FRA16D common chromosomal fragile site that is located within one of its very large introns? Secondly, what is the actual substrate and product of the WWOX enzyme activity? It is likely that understanding the normal role of WWOX and its relationship to chromosomal fragility are necessary in order to understand how the perturbation of these normal roles results in disease.

Lessons from Drosophila: Engineering Genetic Sexing Strains with Temperature-Sensitive Lethality for Sterile Insect Technique Applications.

A major obstacle of sterile insect technique (SIT) programs is the availability of robust sex-separation systems for conditional removal of females. Sterilized male-only releases improve SIT efficiency and cost-effectiveness for agricultural pests, whereas it is critical to remove female disease-vector pests prior to release as they maintain the capacity to transmit disease. Some of the most successful Genetic Sexing Strains (GSS) reared and released for SIT control were developed for Mediterranean fruit fly (Medfly), Ceratitis capitata, and carry a temperature sensitive lethal (tsl) mutation that eliminates female but not male embryos when heat treated. The Medfly tsl mutation was generated by random mutagenesis and the genetic mechanism causing this valuable heat sensitive phenotype remains unknown. Conditional temperature sensitive lethal mutations have also been developed using random mutagenesis in the insect model, Drosophila melanogaster, and were used for some of the founding genetic research published in the fields of neuro- and developmental biology. Here we review mutations in select D. melanogaster genes shibire, Notch, RNA polymerase II 215kDa, pale, transformer-2, Dsor1 and CK2α that cause temperature sensitive phenotypes. Precise introduction of orthologous point mutations in pest insect species with CRISPR/Cas9 genome editing technology holds potential to establish GSSs with embryonic lethality to improve and advance SIT pest control.

White pupae phenotype of tephritids is caused by parallel mutations of a MFS transporter.

Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp- mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of wp- mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.

Precise single base substitution in the shibire gene by CRISPR/Cas9-mediated homology directed repair in Bactrocera tryoni.

BACKGROUND: Pest eradication using the Sterile Insect Technique (SIT) involves high-density releases of sterilized males that mate with wild females and ultimately suppress the population. Sterilized females are not required for SIT and their removal or separation from males prior to release remains challenging. In order to develop genetic sexing strains (GSS), conditional traits such as temperature sensitive lethality are required. RESULTS: Here we introduce a known Drosophila melanogaster temperature sensitive embryonic lethal mutation into Bactrocera tryoni, a serious horticultural pest in Australia. A non-synonymous point mutation in the D. melanogaster gene shibire causes embryonic lethality at 29 °C and we successfully used CRISPR/Cas9 technology to recreate the orthologous shibire temperature sensitive-1 (shits1) mutation in B. tryoni. Genotypic analyses over three generations revealed that a high fitness cost was associated with the shits1 mutant allele and shits1 homozygotes were not viable at 21 °C, which is a more severe phenotype than that documented in D. melanogaster. CONCLUSIONS: We have demonstrated the first successful use of CRISPR/Cas9 to introduce precise single base substitutions in an endogenous gene via homology-directed repair in an agricultural pest insect and this technology can be used to trial other conditional mutations for the ultimate aim of generating genetic sexing strains for SIT.

Health-Related Quality of Life in Pediatric Patients with Leukemia in Singapore: A Cross-Sectional Pilot Study.

There has been a paradigm shift in health service delivery to a more holistic approach, which considers Quality of Life (QoL) and overall functioning. Health-Related Quality of Life (HRQoL) is a multidimensional construct that encompasses physical functioning as well as psychosocial aspects of emotional and social functioning. This study explored factors related to HRQoL in Asian pediatric patients with leukemia in Singapore. The available variables included: age, treatment duration, household income, gender, ethnicity, religion, diagnosis, and phase of treatment. It is hypothesized that the relationships will be significant. In the current study, there were 60 patients (60% males) with leukemia; their ages ranged from 1 to 21 years (Mean = 8.03, Standard Deviation = 4.55). The hypothesis was partially supported. Age had a significant positive relationship with physical functioning, r(60) = 0.28, p < 0.05, physical health, r(60) = 0.28, p < 0.05, and the total HRQoL score, r(60) = 0.29, p < 0.05. Treatment duration had a positive relationship with school functioning, r(60) = 0.28, p < 0.05. All other correlations were statistically non-significant. The effects of the available psychosocial variables of gender, ethnicity, and religion were examined on scores from the Pediatric Quality of Life Inventory (PedsQL). Ethnicity had a significant effect on social functioning, U = 292.00, p < 0.05, r = 0.3 (medium effect size). Specifically, Chinese (Median = 85.00, n = 33) had significantly higher scores on social functioning than others (Median = 70.00, n = 27). The remaining comparisons were statistically non-significant. The current findings added to QoL research, and provided an impetus for more research in the area of HRQoL for children with leukemia in Singapore.

Identification of Y-chromosome scaffolds of the Queensland fruit fly reveals a duplicated gyf gene paralogue common to many Bactrocera pest species.

Bactrocera tryoni (Queensland fruit fly) are polyphagous horticultural pests of eastern Australia. Heterogametic males contain a sex-determining Y-chromosome thought to be gene poor and repetitive. Here, we report 39 Y-chromosome scaffolds (~700 kb) from B. tryoni identified using genotype-by-sequencing data and whole-genome resequencing. Male diagnostic PCR assays validated eight Y-scaffolds, and one (Btry4096) contained a novel gene with five exons that encode a predicted 575 amino acid protein. The Y-gene, referred to as typo-gyf, is a truncated Y-chromosome paralogue of X-chromosome gene gyf (1773 aa). The Y-chromosome contained ~41 copies of typo-gyf, and expression occurred in male flies and embryos. Analysis of 13 tephritid transcriptomes confirmed typo-gyf expression in six additional Bactrocera species, including Bactrocera latifrons, Bactrocera dorsalis and Bactrocera zonata. Molecular dating estimated typo-gyf evolved within the past 8.02 million years (95% highest posterior density 10.56-5.52 million years), after the split with Bactrocera oleae. Phylogenetic analysis also highlighted complex evolutionary histories among several Bactrocera species, as discordant nuclear (116 genes) and mitochondrial (13 genes) topologies were observed. B. tryoni Y-sequences may provide useful sites for future transgene insertions, and typo-gyf could act as a Y-chromosome diagnostic marker for many Bactrocera species, although its function is unknown.

Neuronal-specific impairment of heparan sulfate degradation in Drosophila reveals pathogenic mechanisms for Mucopolysaccharidosis type IIIA.

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder resulting from the deficit of the N-sulfoglucosamine sulfohydrolase (SGSH) enzyme that leads to accumulation of partially-degraded heparan sulfate. MPS IIIA is characterized by severe neurological symptoms, clinically presenting as Sanfilippo syndrome, for which no effective therapy is available. The lysosomal SGSH enzyme is conserved in Drosophila and we have identified increased levels of heparan sulfate in flies with ubiquitous knockdown of SGSH/CG14291. Using neuronal specific knockdown of SGSH/CG14291 we have also observed a higher abundance of Lysotracker-positive puncta as well as increased expression of GFP tagged Ref(2)P supporting disruption to lysosomal function. We have also observed a progressive defect in climbing ability, a hallmark of neurological dysfunction. Genetic screens indicate proteins and pathways that can functionally modify the climbing phenotype, including autophagy-related proteins (Atg1 and Atg18), superoxide dismutase enzymes (Sod1 and Sod2) and heat shock protein (HSPA1). In addition, reducing heparan sulfate biosynthesis by knocking down sulfateless or slalom expression significantly worsens the phenotype; an important observation given that substrate inhibition is being evaluated clinically as a treatment for MPS IIIA. Identifying the cellular pathways that can modify MPS IIIA neuropathology is an essential step in the development of novel therapeutic approaches to prevent and/or ameliorate symptoms in children with Sanfilippo syndrome.

Expressing a moth abcc2 gene in transgenic Drosophila causes susceptibility to Bt Cry1Ac without requiring a cadherin-like protein receptor.

Bt toxins ingested by insect pests can bind to midgut receptors and cause death, although several steps in this process remain unclear. Multiple Bt toxin receptors have been identified in Lepidoptera, including a cadherin-like protein (CaLP), which is central to several models explaining Bt toxins' mode of action. Mutations in the Plutella xylostella ATP-dependent binding cassette transporter C2 (Px-abcc2), rather than CaLP, are genetically linked with Bt Cry1Ac resistance. Here we expressed Px-abcc2 in Drosophila and performed larval bioassays to determine whether this protein acts as an effective Bt receptor. Cry1Ac had no effect on larvae expressing Px-abcc2 in salivary glands, yet larvae expressing Px-abcc2 in the midgut were highly susceptible to both Cry1Ac protoxin and trypsin activated toxin. Furthermore, the CaLP orthologue has been lost from the Drosophila genome, making this a useful system for investigating the role of CaLP peptides from Manduca sexta (CR12-MPED), which are known to act as Bt synergists in larval feeding assays. Drosophila larvae expressing Px-ABCC2 in the midgut were fed LD50 concentrations of Cry1Ac toxin or protoxin, plus purified CR12-MPED cloned from M. sexta or P. xylostella. The M. sexta CR12-MPED protein acted synergistically with Cry1Ac protoxin and activated toxin significantly more effectively than the P. xylostella peptide. This work demonstrates ABCC2 is the major functional Cry1Ac receptor for P. xylostella and the importance of CaLP proteins in Bt mode of action may vary between different lepidopteran species.

Tumor suppressor WWOX moderates the mitochondrial respiratory complex.

Fragile site FRA16D exhibits DNA instability in cancer, resulting in diminished levels of protein from the WWOX gene that spans it. WWOX suppresses tumor growth by an undefined mechanism. WWOX participates in pathways involving aerobic metabolism and reactive oxygen species. WWOX comprises two WW domains as well as a short-chain dehydrogenase/reductase enzyme. Herein is described an in vivo genetic analysis in Drosophila melanogaster to identify functional interactions between WWOX and metabolic pathways. Altered WWOX levels modulate variable cellular outgrowths caused by genetic deficiencies of components of the mitochondrial respiratory complexes. This modulation requires the enzyme active site of WWOX, and the defective respiratory complex-induced cellular outgrowths are mediated by reactive oxygen species, dependent upon the Akt pathway and sensitive to levels of autophagy and hypoxia-inducible factor. WWOX is known to contribute to homeostasis by regulating the balance between oxidative phosphorylation and glycolysis. Reduction of WWOX levels results in diminished ability to respond to metabolic perturbation of normal cell growth. Thus, the ability of WWOX to facilitate escape from mitochondrial damage-induced glycolysis (Warburg effect) is, therefore, a plausible mechanism for its tumor suppressor activity.

Tumor Suppressor WWOX Contributes to the Elimination of Tumorigenic Cells in Drosophila melanogaster.

WWOX is a >1 Mb gene spanning FRA16D Common Chromosomal Fragile Site, a region of DNA instability in cancer. Consequently, altered WWOX levels have been observed in a wide variety of cancers. In vitro studies have identified a large number and variety of potential roles for WWOX. Although its normal role in vivo and functional contribution to cancer have not been fully defined, WWOX does have an integral role in metabolism and can suppress tumor growth. Using Drosophila melanogaster as an in vivo model system, we find that WWOX is a modulator of TNFα/Egr-mediated cell death. We found that altered levels of WWOX can modify phenotypes generated by low level ectopic expression of TNFα/Egr and this corresponds to altered levels of Caspase 3 activity. These results demonstrate an in vivo role for WWOX in promoting cell death. This form of cell death is accompanied by an increase in levels of reactive oxygen species, the regulation of which we have previously shown can also be modified by altered WWOX activity. We now hypothesise that, through regulation of reactive oxygen species, WWOX constitutes a link between alterations in cellular metabolism observed in cancer cells and their ability to evade normal cell death pathways. We have further shown that WWOX activity is required for the efficient removal of tumorigenic cells from a developing epithelial tissue. Together these results provide a molecular basis for the tumor suppressor functions of WWOX and the better prognosis observed in cancer patients with higher levels of WWOX activity. Understanding the conserved cellular pathways to which WWOX contributes provides novel possibilities for the development of therapeutic approaches to restore WWOX function in cancer.

Composite scaffold of poly(vinyl alcohol) and interfacial polyelectrolyte complexation fibers for controlled biomolecule delivery.

Controlled delivery of hydrophilic proteins is an important therapeutic strategy. However, widely used methods for protein delivery suffer from low incorporation efficiency and loss of bioactivity. The versatile interfacial polyelectrolyte complexation (IPC) fibers have the capacity for precise spatiotemporal release and protection of protein, growth factor, and cell bioactivity. Yet its weak mechanical properties limit its application and translation into a viable clinical solution. To overcome this limitation, IPC fibers can be incorporated into polymeric scaffolds such as the biocompatible poly(vinyl alcohol) hydrogel (PVA). Therefore, we explored the use of a composite scaffold of PVA and IPC fibers for controlled biomolecule release. We first observed that the permeability of biomolecules through PVA films were dependent on molecular weight. Next, IPC fibers were incorporated in between layers of PVA to produce PVA-IPC composite scaffolds with different IPC fiber orientation. The composite scaffold demonstrated excellent mechanical properties and efficient biomolecule incorporation. The rate of biomolecule release from PVA-IPC composite grafts exhibited dependence on molecular weight, with lysozyme showing near-linear release for 1 month. Angiogenic factors were also incorporated into the PVA-IPC grafts, as a potential biomedical application of the composite graft. While vascular endothelial growth factor only showed a maximum cumulative release of 3%, the smaller PEGylated-QK peptide showed maximum release of 33%. Notably, the released angiogenic biomolecules induced endothelial cell activity thus indicating retention of bioactivity. We also observed lack of significant macrophage response against PVA-IPC grafts in a rabbit model. Showing permeability, mechanical strength, precise temporal growth factor release, and bioinertness, PVA-IPC fibers composite scaffolds are excellent scaffolds for controlled biomolecule delivery in soft tissue engineering.

WWOX, the chromosomal fragile site FRA16D spanning gene: its role in metabolism and contribution to cancer.

The WWOX gene spans the common chromosomal fragile site FRA16D that is located within a massive (780 kb) intron. The WWOX gene is very long, at 1.1 Mb, which may contribute to the very low abundance of the full-length 1.4 kb mRNA. Alternative splicing also accounts for a variety of aberrant transcripts, most of which are devoid of C-terminal sequences required for WWOX to act as an oxidoreductase. The mouse WWOX gene also spans a chromosomal fragile site implying some sort of functional relationship that confers a selective advantage. The encoded protein domains of WWOX are conserved through evolution (between humans and Drosophila melanogaster) and include WW domains, an NAD -binding site, short-chain dehydrogenase/reductase enzyme and nuclear compartmentalization signals. This homology has enabled functional analyses in D. melanogaster that demonstrate roles for WWOX in reactive oxygen species regulation and metabolism. Indeed the human WWOX gene is also responsive to altered metabolism. Cancer cells typically exhibit altered metabolism (Warburg effect). Many cancers exhibit FRA16D DNA instability that results in aberrant WWOX expression and is associated with poor prognosis for these cancers. It is therefore thought that aberrant WWOX expression contributes to the altered metabolism in cancer. In addition, others have found that a specific (low-expression) allele of WWOX genotype contributes to cancer predisposition.

Common chromosomal fragile site FRA16D tumor suppressor WWOX gene expression and metabolic reprograming in cells.

The WWOX gene spans the FRA16D common chromosomal fragile site and is able to suppress tumor growth. FRA16D is a frequent site of DNA instability in cancer resulting in reduced levels of WWOX expression. Altered levels of WWOX have been shown to affect metabolism. Whereas metabolic reprograming of cells from oxidative phosphorylation to aerobic glycolysis is a major hallmark of tumors, the relationship between common chromosomal fragile site genes and altered metabolism has been unclear. Here we report that altering metabolism from glycolysis to oxidative phosphorylation causes stable increase in steady-state levels of transcripts of the WWOX gene. Consistent with this, exposure to hypoxic conditions, in which cells rely on glycolysis, causes a downregulation of WWOX mRNA. The function of WWOX is therefore intimately integrated with metabolism, as WWOX not only contributes to the metabolic state of cells, its transcript levels are also linked to intracellular metabolic state.