Teachers as first responders: classroom experiences and mental health training needs of Australian schoolteachers.

BACKGROUND: Schoolteachers are often the first to respond when a student presents with a mental health issue in the classroom. This places a burden on schools that impacts school staff, healthcare workers and teachers. More broadly, it places a responsibility on the education system to address students' mental health. This study examines Australian teachers' classroom experiences and the training areas identified by teachers as necessary to manage these issues. METHOD: Interviews were undertaken with 18 in-service teachers between 2020 and 2021 from Catholic, Independent and Public schools. Data were gathered via multiple interviews and analysed using thematic content analysis. RESULTS: The major mental health issues identified by teachers related to mental disorders, depression, anxiety, and a complex range of negative emotional states. Teachers requested training in child and adolescent mental health, counselling skills, early detection and intervention, and training skills to manage the complex relationship with parents and external health and community personnel. Teachers also reported the need to access mental health resources, support and training, which were differentially accessed along socioeconomic status and postcodes. CONCLUSION: The data show that teachers are often placed as first responders when a student has a mental health issue but feel inadequately trained to manage these issues in the classroom. We identified mental health issues presenting in Australian classrooms and documented critical features of mental-health training asked for by teachers in order to address those issues. Given the increasing demands on teachers to address the mental health of children and adolescents, we argue that an urgent review of mental health training for teachers is needed.

Two Oyster Species That Show Differential Susceptibility to Virus Infection Also Show Differential Proteomic Responses to Generic dsRNA.

Viral diseases are a significant cause of mortality and morbidity in oysters, resulting in significant economic losses. We investigated the proteomic responses of these two species of oysters to generic double-stranded RNAs (poly I:C and poly A:U). Analysis of proteomic data using isobaric tags for relative and absolute quantitaion (iTRAQ) indicated that there were significant differences in the proteomic responses of the two oyster species resulting from this treatment. Gene ontology analysis showed that several biological processes, cellular components, and molecular function were unique to the different data sets. For example, a number of proteins implicated in the TLR signaling pathway were associated with the Saccostrea glomerata data set but were absent in the Crassostra gigas data set. These results suggest that the differences in the proteomic responses to dsRNA may underpin the biological differences in viral susceptibility. Molecular targets previously shown to be expressed in C. gigas in response to OsHV1 infections were not present in our proteomic data sets, although they were present in the RNA extracted from the very same tissues. Taken together, our data indicate that there are substantial disparities between transcriptomic and proteomic responses to dsRNA challenge, and a comprehensive account of the oysters' biological responses to these treatments must take into account that disparity.

Dose-dependent effects of metals on gene expression in the sydney rock oyster, Saccostrea glomerata.

In the current study, we tested the effects of common environmental contaminants (the metals zinc and lead) on gene expression in Sydney rock oysters (Saccrostrea glomerata). Oysters were exposed to a range of metal concentrations under controlled laboratory conditions. The expression of 14 putative stress response genes was then measured using quantitative, real-time (q) PCR. The expression of all 14 genes was significantly affected (p < 0.05 vs. nonexposed controls) by at least one of the metals, and by at least one dose of metal. For 5 of the 14 target genes (actin, calmodulin, superoxide dismutase, topoisomerase I, and tubulin) the alteration of expression relative to controls was highest at intermediate (rather than high) doses of metals. Such responses may reflect adaptive (acclimation) reactions in gene expression at low to intermediate doses of contaminants, followed by a decline in expression resulting from exposure at higher doses. The data are discussed in terms of the intracellular pathways affected by metal contamination, and the relevance of such gene expression data to environmental biomonitoring.

Efficacy and Effectiveness of Universal School-Based Wellbeing Interventions in Australia: A Systematic Review.

The World Health Organisation defines health in terms of wellbeing, and wellbeing has become both a construct and a measure of impact in early intervention and prevention programs in schools. In Australia, schools report on their wellbeing initiatives and there is a plethora of government-funded wellbeing programs already in place in schools. However, education systems and stakeholders worldwide are facing significant challenges with mixed evaluation results of program impact and intervention effect. To better support students, schools, school-based healthcare workers, and community, it is important to know about the effectiveness of school-based programs; yet in the last decade, there has been no national appraisal of these programs in Australia. This systematic review aims to report on the effectiveness of Australian school-based wellbeing programs through a search of 13 databases. Out of 2888 articles, 29 met inclusion criteria. The results found that seventeen interventions comprising 80% of the total number of participants reported no statistically significant intervention effect on wellbeing outcomes. We argue that supporting wellbeing through robust program intervention is important as wellbeing presents both an indication of later onset of more serious mental health issues, and an opportunity for early intervention to break the trajectory leading to full disorder.

Ultrastructural localization of highly variable 185/333 immune response proteins in the coelomocytes of the sea urchin, Heliocidaris erythrogramma.

The 185/333 proteins of sea urchins represent a family of highly variable immune response molecules with unknown functions. In this study, we show that 185/333 proteins are expressed by three cell types: amoebocytes, colourless spherule cells and gut-associated amoebocytes. A sub-population of amoebocytes express 185/333 proteins on the membranes of vesicles emanating from the trans-Golgi and which later fuse with the plasma membranes of the cells. The previously uncharacterized gut-associated amoebocytes also show a high level of 185/333 protein expression on their internal vesicles and plasma membranes. Colourless spherule cells contain 185/333 proteins within large spherules (specialized intracellular vesicles). In the presence of bacteria and yeast, the ultrastucture of colourless spherule cells changes and 185/333 proteins disappear. In contrast, 185/333 proteins were not found in the phagosomes of coelomocytes. The 185/333-positive gut amoebocytes were often associated with anuclear bodies, which appeared to incorporate material of microbial origin that was surrounded by 185/333 proteins. The association between 185/333 proteins on gut amoebocytes and anuclear bodies suggests that these proteins may be involved in the phagocytosis of microbes in the gut epithelium.

Purine Nucleoside Phosphorylase mediated molecular chemotherapy and conventional chemotherapy: a tangible union against chemoresistant cancer.

BACKGROUND: Late stage Ovarian Cancer is essentially incurable primarily due to late diagnosis and its inherent heterogeneity. Single agent treatments are inadequate and generally lead to severe side effects at therapeutic doses. It is crucial to develop clinically relevant novel combination regimens involving synergistic modalities that target a wider repertoire of cells and lead to lowered individual doses. Stemming from this premise, this is the first report of two- and three-way synergies between Adenovirus-mediated Purine Nucleoside Phosphorylase based gene directed enzyme prodrug therapy (PNP-GDEPT), docetaxel and/or carboplatin in multidrug-resistant ovarian cancer cells. METHODS: The effects of PNP-GDEPT on different cellular processes were determined using Shotgun Proteomics analyses. The in vitro cell growth inhibition in differentially treated drug resistant human ovarian cancer cell lines was established using a cell-viability assay. The extent of synergy, additivity, or antagonism between treatments was evaluated using CalcuSyn statistical analyses. The involvement of apoptosis and implicated proteins in effects of different treatments was established using flow cytometry based detection of M30 (an early marker of apoptosis), cell cycle analyses and finally western blot based analyses. RESULTS: Efficacy of the trimodal treatment was significantly greater than that achieved with bimodal- or individual treatments with potential for 10-50 fold dose reduction compared to that required for individual treatments. Of note was the marked enhancement in apoptosis that specifically accompanied the combinations that included PNP-GDEPT and accordingly correlated with a shift in the expression of anti- and pro-apoptotic proteins. PNP-GDEPT mediated enhancement of apoptosis was reinforced by cell cycle analyses. Proteomic analyses of PNP-GDEPT treated cells indicated a dowregulation of proteins involved in oncogenesis or cancer drug resistance in treated cells with accompanying upregulation of apoptotic- and tumour- suppressor proteins. CONCLUSION: Inclusion of PNP-GDEPT in regular chemotherapy regimens can lead to significant enhancement of the cancer cell susceptibility to the combined treatment. Overall, these data will underpin the development of regimens that can benefit patients with late stage ovarian cancer leading to significantly improved efficacy and increased quality of life.

Highly variable immune-response proteins (185/333) from the sea urchin, Strongylocentrotus purpuratus: proteomic analysis identifies diversity within and between individuals.

185/333 genes and transcripts from the purple sea urchin, Strongylocentrotus purpuratus, predict high levels of amino acid diversity within the encoded proteins. Based on their expression patterns, 185/333 proteins appear to be involved in immune responses. In the present study, one- and two-dimensional Western blots show that 185/333 proteins exhibit high levels of molecular diversity within and between individual sea urchins. The molecular masses of 185/333-positive bands or spots range from 30 to 250 kDa with a broad array of isoelectric points. The observed molecular masses are higher than those predicted from mRNAs, suggesting that 185/333 proteins form strong associations with other molecules or with each other. Some sea urchins expressed >200 distinct 185/333 proteins, and each animal had a unique suite of the proteins that differed from all other individuals. When sea urchins were challenged in vivo with pathogen-associated molecular patterns (PAMPs; bacterial LPS and peptidoglycan), the expression of 185/333 proteins increased. More importantly, different suites of 185/333 proteins were expressed in response to different PAMPs. This suggests that the expression of 185/333 proteins can be tailored toward different PAMPs in a form of pathogen-specific immune response.

Comparative proteomic analysis of a sea urchin (Heliocidaris erythrogramma) antibacterial response revealed the involvement of apextrin and calreticulin.

Echinoderms evolved early in the deuterostome lineage, and as such constitute model organisms for comparative physiology and immunology. The sea urchin genome sequence (Strongylocentrotus purpuratus) revealed a complex repertoire of genes with similarities to the immune response genes of other species. To complement these genomic data, we investigated the responses of sea urchins to the injection of bacteria using a comparative proteomics approach on a closely related species. In the sea urchin, Heliocidaris erythrogramma, the relative abundance of many proteins was altered in response to the injection of both bacteria and saline, suggesting their involvement in wounding responses, while others were differentially altered in response to bacteria only. The identities of 15 proteins that differed in relative abundance were determined by mass spectrometry. These proteins revealed a significant modification in energy metabolism in coelomocytes towards the consumption of glutamate and the production of NADPH after injection, as well as an increased concentration of cell signalling molecules, such as heterotrimeric guanine nucleotide-binding protein. The injection of bacteria specifically increased the abundance of apextrin and calreticulin, suggesting that these two proteins are involved in the sequestration or inactivation of bacteria.

Immunosuppressive effects of environmental stressors on immunological function in Pinctada imbricata.

This study assessed the effects of mechanical agitation, hypo-saline conditions, and exposure to the air on the Akoya pearl oyster, Pinctada imbricata, focusing specifically on the immunological activity of haemocytes. Both phagocytosis and phenoloxidase activity decreased significantly when oysters were exposed to all three stressors. Transient decreases were also evident in total haemocyte counts after mechanical stress and exposure to air, while significant increases in total haemocyte counts were evident after exposure to low salinity. Acid phosphatase activity increased significantly when oysters were exposed to air. The frequency of granulocytes in the haemolymph increased significantly when oysters were stressed by hypo-saline conditions, whilst the relative frequency of granulocytes did not differ significantly after mechanical agitation or exposure to air. The total protein content of haemolymph increased significantly when oysters were stressed by mechanical agitation and low salinity. These results suggest that fluctuations in environmental conditions affect circulating haemocytes and their cytochemistry, and that the different immunological parameters tested were influenced uniquely according to the type of stressor.

Echinoderm immunity.

A survey for immune genes in the genome for the purple sea urchin has shown that the immune system is complex and sophisticated. By inference, immune responses of all echinoderms maybe similar. The immune system is mediated by several types of coelomocytes that are also useful as sensors of environmental stresses. There are a number of large gene families in the purple sea urchin genome that function in immunity and of which at least one appears to employ novel approaches for sequence diversification. Echinoderms have a simpler complement system, a large set of lectin genes and a number of antimicrobial peptides. Profiling the immune genes expressed by coelomocytes and the proteins in the coelomic fluid provide detailed information about immune functions in the sea urchin. The importance of echinoderms in maintaining marine ecosystem stability and the disastrous effects of their removal due to disease will require future collaborations between ecologists and immunologists working towards understanding and preserving marine habitats.

The marsupial CD8 gene locus: molecular cloning and expression analysis of the alpha and beta sequences in the gray short-tailed opossum (Monodelphis domestica) and the tammar wallaby (Macropus eugenii).

In eutherian mammals, CD8 is a key receptor of cytotoxic T cells and plays a pivotal role in the recognition and elimination of infected host cells by cell-mediated cytotoxicity. Here, we report the molecular cloning and expression analysis of CD8alpha and CD8beta cDNAs in two marsupial species, the gray short-tailed opossum and the tammar wallaby. The opossum and tammar CD8 sequences share a high degree of amino acid identity of 63% (CD8alpha) and 57% (CD8beta) to each other as well as 36-45% (CD8alpha) and 38-41% (CD8beta) with their eutherian counterparts. In addition, many of the signature features of eutherian CD8alpha and CD8beta are preserved in both marsupials including the two invariant cysteines that form the intra-chain disulphide bond in the extracellular IgSfV domain and the two hinge region cysteines involved in dimerisation between the two subunits. The p56(lck) binding motif in the cytoplasmic tail of the CD8alpha subunit is also conserved. Interestingly, the opossum CD8alpha and the tammar CD8beta sequences have a truncated cytoplasmic tail. RT-PCR analysis of CD8alpha and CD8beta transcripts in the tissues of the adult opossum and tammar showed broad tissue expression with a high level of expression observed in the lymphoid tissues of both marsupials. Furthermore, RT-PCR analysis of CD8alpha and CD8beta transcripts in the immune tissues of tammar young over the first 120 days of pouch life revealed a pattern of expression analogous to the maturation of the lymphoid tissues. This is the first report confirming the presence of CD8 in the tissues of a marsupial and will provide the tools to further analyse T cell subsets in this unique group of mammals.

Effects of noradrenaline on immunological activity in Sydney rock oysters.

This study investigates the effects of noradrenaline injection on a range of immunological activities in Sydney rock oysters (Saccostrea glomerata). Noradrenaline caused a decrease in most of the immunological parameters tested. Phenoloxidase activities in both whole hemolymph and serum decreased significantly within the first 60 min of noradrenaline injection, as did the total frequency of hemocytes in hemolymph, differential hemocyte frequencies, the frequency of phenoloxidase positive cells and phagocytic activity. All of these parameters started to return to normal levels within 120 min. In contrast, the total protein content of hemolymph, which also decreased after noradrenaline injection continued to decline throughout the experimental period. In vitro studies found that superoxide and peroxide production by hemocytes increased in the presence of noradrenaline, but acid phosphatase activity decreased significantly. Additional experiments showed that noradrenaline secretion was stimulated by altered salinity, altered temperature and physical agitation. This suggests that stressors commonly associated with oyster farming may result in noradrenaline-based stress responses that suppress immunological activity.

In vitro effects of noradrenaline on Sydney rock oyster (Saccostrea glomerata) hemocytes.

Our prior work has shown that the catecholamine hormone, noradrenaline, mediates environmental stress responses in Sydney rock oysters, resulting in impaired immunological function. In the current study, we tested the cellular basis of this stress response. Hemocytes were exposed to noradrenaline in vitro before cell morphology and viability were analyzed. Noradrenaline was shown to induce apoptotic markers, including the loss of mitochondrial membrane potential, DNA fragmentation and plasma membrane blebbing. F-actin appeared to play an important role in the changes observed in hemocytes, being concentrated mostly in the plasma membrane blebs of noradrenaline-treated hemocytes. This may explain why hemocyte adhesion and pseudopodia formation were inhibited by noradrenaline. Cellular dysfunction induced by norarenaline mainly affected the hyalinocyte sub-population of hemocytes, whilst the other major cell type, granulocytes, remained unaffected. Given that hyalinocytes are important immunological effectors, the results of this study help to explain why immunosuppression accompanies noradrenaline-mediated stress responses in oysters.

Molecular characterisation and expression of CD4 in two distantly related marsupials: the gray short-tailed opossum (Monodelphis domestica) and tammar wallaby (Macropus eugenii).

The gene and corresponding cDNA for CD4 in the gray short-tailed opossum, Monodelphis domestica, and the cDNA sequence for CD4 in the tammar wallaby, Macropus eugenii, have been characterised. The opossum CD4 homolog reveals conserved synteny, preserved genomic organisation and analogous structural arrangement to human and mouse CD4. Opossum and tammar CD4 exhibit typical eutherian CD4 features including the highly conserved p56(lck) binding motif in the cytoplasmic region and the invariant cysteine residues in extracellular domains 1 and 4. Interestingly, the marsupial CD4 sequences substitute a tryptophan for the first cysteine in domain 2 negating the formation of a disulphide bond as seen in other eutherian CD4 sequences except human and mouse. Overall the marsupial CD4 sequences share amino acid identity of 59% to each other and 37-41% with eutherian mammals. However, in contrast to eutherian homologs, the marsupial CD4 sequences were found to be truncated at the terminal end of the cytoplasmic tail. This is the first report confirming the presence of CD4 in a marsupial and describing its key features.

Characterization of phenoloxidase activity in Sydney rock oysters (Saccostrea glomerata).

Phenoloxidase (PO) activity was studied in Sydney rock oysters (Saccostrea glomerata). As in other molluscs, PO was found to exist as a pro-enzyme (proPO) in hemocytes. ProPO could be activated to PO by exogenous proteases (trypsin and chymotrypsin), exposure of hemocytes to pathogen-associated molecular patterns (PAMPs) and by the detergents, Triton X-100 and sodium dodecyl sulphate (SDS). Inhibition studies confirmed the proPO activating system of Sydney rock oysters is a proteinase cascade in which Ca2+ dependent serine proteinases proteolytically convert proPO into active PO. Activated PO was found to be a tyrosinase-like enzyme that is responsible for both monophenolase and diphenolase activity. The bifunctional PO had higher affinity for the monophenol, hydroquinine monomethyl ether (4HA) (Km=4.45+/-1.46 mM) than for the diphenol, l-DOPA (Km=10.27+/-1.33 mM). Maximum enzyme activity was evident at 37 degrees C, pH 8 and at salinities of between 30 and 37 ppt. Melanogenesis catalysed by the active enzyme is a composite of eumelanin and the product of a sclerotin pathway combining DOPA decarboxylase with PO activity.

Double stranded RNA is processed differently in two oyster species.

Ostreid herpes virus causes serious disease in the Pacific oyster (Crassostrea gigas), but not in the Sydney Rock Oyster (Saccostrea glomerata). To investigate differences in disease progression, we injected oysters with double stranded RNA (dsRNA). dsRNA is known to mimic viral infection, and can evoke immune responses when Toll-like receptors detect the dsRNA, leading to the production of type 1 interferon and inflammation cytokines. The uptake and processing of dsRNA was tracked in gill and mantle tissue of Crassostrea gigas and Saccostrea glomerata after injection of fluorochrome labelled poly (I:C) dsRNA. The two species showed significant differences in tissue uptake and clearance, and differences in immune responses confirmed by real time PCR. These results showed that S. glomerata was more efficient in processing dsRNA than C. gigas, and that the gill tissue is an important site of dsRNA processing and response.

A second form of collagenous lectin from the tunicate, Styela plicata.

This study characterised a 90 kDa lectin from an invertebrate chordate, the tunicate Styela plicata. One- and two-dimensional electrophoresis showed that the apparent molecular weight of this protein is maintained under both reducing and non-reducing conditions, suggesting that its native form is a monomer. The 90 kDa lectin was localised within a single type of hemocyte (morula cells), but was secreted from those cells when tunicates were challenged with the inflammatory elicitor, zymosan. Functional studies showed that the 90 kDa protein binds to galactose-based sugars in a divalent cation-dependent manner. Amino acid composition analysis and N-terminal amino acid sequencing indicated that the 90 kDa lectin is related to a previously characterised, collagenous lectin from S. plicata, splic43. However, peptide mass fingerprinting identified numerous differences between the two proteins. This suggests that the 90 kDa molecule represents a novel protein that is involved in host defence.

Macroarray analysis of coelomocyte gene expression in response to LPS in the sea urchin. Identification of unexpected immune diversity in an invertebrate.

The purple sea urchin, Strongylocentrotus purpuratus, is a member of the phylum Echinodermata, which is basal to the phylum Chordata within the deuterostome lineage of the animal kingdom. This relationship makes the analysis of the sea urchin immune system relevant to understanding the evolution of the deuterostome immune system leading to the Vertebrata. Subtractive suppression hybridization was employed to generate cDNA probes for screening high-density arrayed, conventional cDNA libraries to identify genes that were upregulated in coelomocytes responding to lipopolysaccharide. Results from 1,247 expressed sequence tags (ESTs) were used to infer that coelomocytes upregulated genes involved in RNA splicing, protein processing and targeting, secretion, endosomal activities, cell signaling, and alterations to the cytoskeletal architecture including interactions with the extracellular matrix. Of particular note was a set of transcripts represented by 60% of the ESTs analyzed, which encoded a previously uncharacterized family of closely related proteins, provisionally designated as 185/333. These transcripts exhibited a significant level of variation in their nucleotide sequence and evidence of putative alternative splicing that could yield up to 15 translatable elements. On the basis of the striking increase in gene expression in response to lipopolysaccharide and the unexpected level of diversity of the 185/333 messages, we propose that this set of transcripts encodes a family of putative immune response proteins that may represent a major component of an immunological response to bacterial challenge.

Meta-analysis of studies using suppression subtractive hybridization and microarrays to investigate the effects of environmental stress on gene transcription in oysters.

Many microarray and suppression subtractive hybridization (SSH) studies have analyzed the effects of environmental stress on gene transcription in marine species. However, there have been no unifying analyses of these data to identify common stress response pathways. To address this shortfall, we conducted a meta-analysis of 14 studies that investigated the effects of different environmental stressors on gene expression in oysters. The stressors tested included chemical contamination, hypoxia and infection, as well as extremes of temperature, pH and turbidity. We found that the expression of over 400 genes in a range of oyster species changed significantly after exposure to environmental stress. A repeating pattern was evident in these transcriptional responses, regardless of the type of stress applied. Many of the genes that responded to environmental stress encoded proteins involved in translation and protein processing (including molecular chaperones), the mitochondrial electron transport chain, anti-oxidant activity and the cytoskeleton. In light of these findings, we put forward a consensus model of sub-cellular stress responses in oysters.

Exocytosis of a complement component C3-like protein by tunicate hemocytes.

This study investigates the exocytic responses of invertebrate hemocytes to pathogen-associated antigens. It demonstrates that a homologue of complement component C3, a key defensive protein of the innate immune system, is expressed by phagocytic hemocytes (non-refractile vacuolated cells) of the tunicate, Styela plicata. C3-like molecules are localized in sub-cellular vesicles and are rapidly exocytosed after stimulation with bacterial, fungal or algal cell surface molecules. Signal transduction analysis indicated that the induced secretion of C3-like molecules is mediated by a G-protein dependent signaling pathway, which modulates tubulin microtubules. All of this evidence indicates that hemocytes can contribute to host defense responses by rapidly exocytosing C3-like proteins at sites of infection.