Models of KPTN-related disorder implicate mTOR signalling in cognitive and overgrowth phenotypes.

KPTN-related disorder is an autosomal recessive disorder associated with germline variants in KPTN (previously known as kaptin), a component of the mTOR regulatory complex KICSTOR. To gain further insights into the pathogenesis of KPTN-related disorder, we analysed mouse knockout and human stem cell KPTN loss-of-function models. Kptn -/- mice display many of the key KPTN-related disorder phenotypes, including brain overgrowth, behavioural abnormalities, and cognitive deficits. By assessment of affected individuals, we have identified widespread cognitive deficits (n = 6) and postnatal onset of brain overgrowth (n = 19). By analysing head size data from their parents (n = 24), we have identified a previously unrecognized KPTN dosage-sensitivity, resulting in increased head circumference in heterozygous carriers of pathogenic KPTN variants. Molecular and structural analysis of Kptn-/- mice revealed pathological changes, including differences in brain size, shape and cell numbers primarily due to abnormal postnatal brain development. Both the mouse and differentiated induced pluripotent stem cell models of the disorder display transcriptional and biochemical evidence for altered mTOR pathway signalling, supporting the role of KPTN in regulating mTORC1. By treatment in our KPTN mouse model, we found that the increased mTOR signalling downstream of KPTN is rapamycin sensitive, highlighting possible therapeutic avenues with currently available mTOR inhibitors. These findings place KPTN-related disorder in the broader group of mTORC1-related disorders affecting brain structure, cognitive function and network integrity.

Breaking the Binary: How Clinicians Can Ensure Everyone Receives High Quality Reproductive Health Services.

The gender binary has historically defined the study and practice of reproductive and sexual healthcare. However, the male and female binary categorization of sex does not adequately define patients seeking reproductive and sexual health. Comprehensive sexual healthcare should consider diverse gender identity and non-heteronormative sexual practices, family planning, sexually transmitted infections, cancer prevention, and sexual function. Recent research suggests clinicians do not feel prepared to provide care for sexual and gender minority (SGM) patients. In this narrative review, we focus on reproductive and sexual health within the scope of obstetric and gynecologic (OB/GYN) clinical practice. We used traditional medical subject headings to summarize data from publications in peer-reviewed journals published in the last 10 years and identified barriers, facilitators and best practices for de-gendering reproductive healthcare. Following a roughly chronological care path across the lifespan, we categorized findings into the following topics: Early Care for SGM, Sexual Health, Family Planning, and Care Later in Life for SGM. We include recommendations for creating a welcoming environment, SGM inclusive training for clinicians and staff, and best practices for individualized counseling. We review suggested practices related to service delivery and clinical considerations in the provision of sexual and reproductive health care for gender and sexual minority patients.

A spatially resolved atlas of the human lung characterizes a gland-associated immune niche.

Single-cell transcriptomics has allowed unprecedented resolution of cell types/states in the human lung, but their spatial context is less well defined. To (re)define tissue architecture of lung and airways, we profiled five proximal-to-distal locations of healthy human lungs in depth using multi-omic single cell/nuclei and spatial transcriptomics (queryable at lungcellatlas.org ). Using computational data integration and analysis, we extend beyond the suspension cell paradigm and discover macro and micro-anatomical tissue compartments including previously unannotated cell types in the epithelial, vascular, stromal and nerve bundle micro-environments. We identify and implicate peribronchial fibroblasts in lung disease. Importantly, we discover and validate a survival niche for IgA plasma cells in the airway submucosal glands (SMG). We show that gland epithelial cells recruit B cells and IgA plasma cells, and promote longevity and antibody secretion locally through expression of CCL28, APRIL and IL-6. This new 'gland-associated immune niche' has implications for respiratory health.

The use of inertial measurement units to quantify forearm loading and symmetry during gymnastics vault training sessions.

The upper limbs are important in gymnastics vaulting, as the success of the flight phase is dependent on a quick and forceful push-off from the vault. This places the upper limbs under stress, which has been associated with pain and injury. This study aimed to quantify forearm segmental loading and symmetry when performing foundation to advanced-level vaulting skills during training. Twelve advanced-level artistic gymnasts (female, n = 6; male, n = 6) wore bilaterally forearm-mounted inertial measurement units while completing their vaulting sessions. The peak resultant acceleration for the leading and non-leading forearms during contact were calculated. Female gymnasts performed variations of Yurchenko vaults, while male gymnasts performed Handspring and Tsukahara vaults. Descriptive statistics (median and inter-quartile range), symmetry index scores, and total session impact load (measure of cumulative loading) were calculated between the lead and non-lead forearms. High asymmetrical loading was identified for some Yurchenko, Handspring, and Tsukahara vaults, with large variations identified between gymnasts. Some gymnasts experienced greater loading at their lead forearm, while others experienced greater loading at their non-lead forearm. Results indicate that limb loading patterns in advanced gymnastics are highly individualised, indicating that individual analysis is needed to identify gymnasts (or limbs) at an increased risk for overuse injury.

Free Achilles tendon strain during selected rehabilitation, locomotor, jumping, and landing tasks.

A better understanding of the strains experienced by the Achilles tendon during commonly prescribed exercises and locomotor tasks is needed to improve efficacy of Achilles tendon training and rehabilitation programs. The aim of this study was to estimate in vivo free Achilles tendon strain during selected rehabilitation, locomotor, jumping, and landing tasks. Sixteen trained runners with no symptoms of Achilles tendinopathy participated in this study. Personalized free Achilles tendon moment arm and force-strain curve were obtained from imaging data and used in conjunction with motion capture and surface electromyography to estimate free Achilles tendon strain using electromyogram-informed neuromusculoskeletal modeling. There was a strong correspondence between Achilles tendon force estimates from the present study and experimental data reported in the literature (R2 > 0.85). The average tendon strain was highest for maximal hop landing (8.8 ± 1.6%), lowest for walking at 1.4 m/s (3.1 ± 0.8%), and increased with locomotor speed during running (run 3.0 m/s: 6.5 ± 1.6%; run 5.0 m/s: 7.9 ± 1.7%) and during heel rise exercise with added mass (BW: 5.8 ± 1.3%; 1.2 BW: 6.9 ± 1.7%). The peak tendon strain was highest during running (5 m/s: 13.7 ± 2.5%) and lowest during walking (1.4 m/s: 7 ± 1.8%). Overall findings provide a preliminary evidence base for exercise selection to maximize anabolic tendon remodeling during training and rehabilitation of the Achilles tendon.NEW & NOTEWORTHY Our work combines medical imaging and electromyogram-informed neuromusculoskeletal modeling data to estimate free Achilles tendon strain during selected rehabilitation, locomotor, jumping, and landing tasks in trained middle-distance runners. These data may potentially be used to inform Achilles tendon training and rehabilitation to maximize anabolic tendon remodeling.

Differentiation of human induced pluripotent stem cells into cortical neural stem cells.

Efficient and effective methods for converting human induced pluripotent stem cells into differentiated derivatives are critical for performing robust, large-scale studies of development and disease modelling, and for providing a source of cells for regenerative medicine. Here, we describe a 14-day neural differentiation protocol which allows for the scalable, simultaneous differentiation of multiple iPSC lines into cortical neural stem cells We currently employ this protocol to differentiate and compare sets of engineered iPSC lines carrying loss of function alleles in developmental disorder associated genes, alongside isogenic wildtype controls. Using RNA sequencing (RNA-Seq), we can examine the changes in gene expression brought about by each disease gene knockout, to determine its impact on neural development and explore mechanisms of disease. The 10-day Neural Induction period uses the well established dual-SMAD inhibition approach combined with Wnt/ß-Catenin inhibition to selectively induce formation of cortical NSCs. This is followed by a 4-day Neural Maintenance period facilitating NSC expansion and rosette formation, and NSC cryopreservation. We also describe methods for thawing and passaging the cryopreserved NSCs, which are useful in confirming their viability for further culture. Routine implementation of immunocytochemistry Quality Control confirms the presence of PAX6-positive and/or FOXG1-positive NSCs and the absence of OCT4-positive iPSCs after differentiation. RNA-Seq, flow cytometry, immunocytochemistry (ICC) and RT-qPCR provide additional confirmation of robust presence of NSC markers in the differentiated cells. The broader utility and application of our protocol is demonstrated by the successful differentiation of wildtype iPSC lines from five additional independent donors. This paper thereby describes an efficient method for the production of large numbers of high purity cortical NSCs, which are widely applicable for downstream research into developmental mechanisms, further differentiation into postmitotic cortical neurons, or other applications such as large-scale drug screening experiments.

Passive drag in Para swimmers with physical impairments: Implications for evidence-based classification in Para swimming.

The inherent hydrodynamic resistance force, or passive drag, of a swimmer directly influences how they move through the water. For swimmers with physical impairments, the strength of association between passive drag and swimming performance is unknown. Knowledge on this factor could improve the World Para Swimming classification process. This study established the relationship between passive drag and 100 m freestyle race performance in Para swimmers with physical impairments. Using a cross-sectional study design, an electrical-mechanical towing device was used to measure passive drag force in 132 international-level Para swimmers. There was a strong, negative correlation between normalized passive drag force and 100 m freestyle race speed in the combined participant cohort (ρ = -0.77, p < 0.001). Type of physical impairment was found to affect the relationship between passive drag and 100 m freestyle race speed when included in linear regression (R2  = 0.65, χ2  = 11.5, p = 0.025). These findings contribute to the body of evidence that passive drag can provide an objective assessment of activity limitation in Para swimmers with physical impairments. The effect of physical impairment type on the relationship between passive drag and swimming performance should be accounted for in Para swimming classification.

Family building desires among transgender and gender expansive adolescents: A longitudinal family-level analysis.

Introduction: Professional societies state that Transgender and gender expansive (TGE) adolescents and their families should be counseled about future family building options prior to initiating gender affirming therapy. While emerging data show that TGE adolescents have diverse desires regarding future family building, little is known regarding how these preferences are developed in a larger ecological context. Aim: The current study used Ecological Systems Theory as a framework to describe the family building attitudes of TGE adolescents, their caregivers, and their siblings. Methods: Participants were recruited from community-based venues in the New England region of the U.S. to participate in the TTFN Project, a longitudinal community-based mixed methods study. The sample for the current study included 84 family members from 30 families (30 TGE adolescents, 11 siblings, 44 caregivers). All participants completed a semi-structured qualitative interview about family building attitudes and desires for TGE and cisgender adolescents at two waves across 6-8 months. Interview transcripts were analyzed using a combination of immersion/crystallization, thematic analysis, and template organizing style approaches. The Transgender Youth Fertility Attitudes Questionnaire (TYFAQ) was employed to quantitatively describe the family building attitudes of TGE adolescents and their families. Results: Eight themes corresponding to the levels of the ecological systems model - individual-level (perceived reproductive potential, reproductive identity), family-level (communication about family building, familial experiences and expectations), community-level (community support and role models; community expectations and norms), and societal/institutional-level (medicalization of family building, external sociopolitical factors) - were developed from the interviews. Results from the TYFAQ indicated that compared to cisgender adolescents, TGE adolescents were less likely to value having biological children and more likely to consider adoption in comparison to their cisgender siblings. Discussion: Findings emphasize the importance of using Ecological Systems Theory to understand the family building attitudes and desires of TGE adolescents and their families.

The Free Achilles Tendon Is Shorter, Stiffer, Has Larger Cross-Sectional Area and Longer T2* Relaxation Time in Trained Middle-Distance Runners Compared to Healthy Controls.

Tendon geometry and tissue properties are important determinants of tendon function and injury risk and are altered in response to ageing, disease, and physical activity levels. The purpose of this study was to compare free Achilles tendon geometry and mechanical properties between trained elite/sub-elite middle-distance runners and a healthy control group. Magnetic resonance imaging (MRI) was used to measure free Achilles tendon volume, length, average cross-sectional area (CSA), regional CSA, moment arm, and T2* relaxation time at rest, while freehand three-dimensional ultrasound (3DUS) was used to quantify free Achilles tendon mechanical stiffness, Young's modulus, and length normalised mechanical stiffness. The free Achilles tendon in trained runners was significantly shorter and the average and regional CSA (distal end) were significantly larger compared to the control group. Mechanical stiffness of the free Achilles tendon was also significantly higher in trained runners compared to controls, which was explained by the group differences in tendon CSA and length. T2* relaxation time was significantly longer in trained middle-distance runners when compared to healthy controls. There was no relationship between T2* relaxation time and Young's modulus. The longer T2* relaxation time in trained runners may be indicative of accumulated damage, disorganised collagen, and increased water content in the free Achilles tendon. A short free Achilles tendon with large CSA and higher mechanical stiffness may enable trained runners to rapidly transfer high muscle forces and possibly reduce the risk of tendon damage from mechanical fatigue.

Biomechanics of starting, sprinting and submaximal running in athletes with brain impairment: A systematic review.

OBJECTIVES: Para athletes with brain impairment are affected by hypertonia, ataxia and athetosis, which adversely affect starting, sprinting and submaximal running. The aim was to identify and synthesise evidence from studies that have compared the biomechanics of runners with brain impairments (RBI) and non-disabled runners (NDR). DESIGN: Systematic review. METHODS: Five journal databases were systematically searched from inception to March 2020. Included studies compared the biomechanics of RBI (aged>14 years) and NDR performing either block-starts, sprinting, or submaximal running. RESULTS: Eight studies were included, analysing a total of 100 RBI (78M:22F; 18-38 years) diagnosed with either cerebral palsy (n=44) or traumatic brain injury (n=56). Studies analysed block-starts (n=3), overground sprinting (n=3) and submaximal running (n=2), and submaximal treadmill running (n=1). Horizontal velocity during starts, sprinting and self-selected submaximal speeds were lower in RBI. During sprinting and submaximal running, compared with NDR, RBI had shorter stride length, step length, and flight time, increased ground-contact time, increased cadence, and reduced ankle and hip range of motion. In submaximal running, RBI had decreased ankle-power generation at toe-off. CONCLUSIONS: There is limited research and small sample sizes in this area. However, preliminary evidence suggests that RBI had lower sprint speeds and biomechanical characteristics typical of submaximal running speeds in NDR, including increased ground-contact times and reduced stride length, step length, and flight times. Meaningful interpretation of biomechanical findings in RBI is impeded by impairment variability (type, severity and distribution), and methods which permit valid, reliable impairment stratification in larger samples are required.

Effects of digital filtering on peak acceleration and force measurements for artistic gymnastics skills.

Low-pass filters are ideal when filtering human movements, however the effectiveness of such filters relies on the correct selection of the cut-off frequency. The aim of this study was to determine the most appropriate filter cut-off for acceleration- and force-time data when measuring peak resultant acceleration (PRA) and ground reaction force (PRGRF) during gymnastics landings. Sixteen gymnasts executed backward handsprings and backward somersault landings onto a matted force plate while wearing four inertial measurement units (IMUs). Acceleration- and force-time data were filtered using a fourth-order Butterworth filter at different cut-off frequencies ranging from raw through to 250 Hz. Residual analysis plots were produced, and the PRGRF and PRA for all IMUs were calculated for each participant and skill at all cut-off frequencies. Descriptive statistics, model II linear regressions and Bland-Altman plots were conducted. Results indicated that a minimum 85 Hz cut-off is optimal. High cut-off frequencies (>80 Hz) showed good linear relationships and had minimal mean bias compared with raw values, indicating that either filtered (above ~85 Hz) or raw signals can be used. It is suggested that for applied sports settings no filtering is needed, however a minimum cut-off of 85 Hz should be implemented for research purposes.

From the field of play to the laboratory: Recreating the demands of competition with augmented reality simulated sport.

Biomechanical analysis has typically been confined to a laboratory setting. While attempts have been made to take laboratory testing into the field, this study was designed to assess whether augmented reality (AR) could be used to bring the field into the laboratory. This study aimed to measure knee load in volleyball players through a jump task incorporating AR while maintaining the perception-action couplings by replicating the visual features of a volleyball court. Twelve male volleyball athletes completed four tasks: drop landing, hop jump, spike jump, and spike jump while wearing AR smart glasses. Biomechanical variables included patellar tendon force, knee moment and kinematics of the ankle, knee, hip, pelvis and thorax. The drop landing showed differences in patellar tendon force and knee moment when compared to the other conditions. The hop jump did not present differences in kinetics when compared to the spike conditions, instead of displaying the greatest kinematic differences. As a measure of patellar tendon loading the AR condition showed a close approximation to the spike jump, with no differences present when comparing landing forces and mechanics. Thus, AR may be used in a clinical assessment to better replicate information from the competitive environment.

Comparing inertial measurement units and marker-based biomechanical models during dynamic rotation of the torso.

Inertial measurement units (IMUs) enable human movements to be captured in the field and are being used increasingly in high performance sport. One key metric that can be derived from IMUs are relative angles of body segments which are important for monitoring form in many sports. The purpose of this study was to (a) examine the validity of relative angles derived from IMUs placed on the torso and pelvis; and (b) determine optimal positioning for torso mounted sensors such that the IMU relative angles match closely with gold standard torso-pelvis and thorax-pelvis relative angle data derived from an optoelectronic camera system. Seventeen adult participants undertook a variety of motion tasks. Four IMUs were positioned on the torso and one was positioned on the pelvis between the posterior superior iliac spines. Reflective markers were positioned around each IMU and over torso and pelvis landmarks. Results showed that the IMUs are valid with the root mean square errors expressed as a percentage of the angle range (RMSE%) ranging between 1% and 7%. Comparison between the IMU relative angles and the torso-pelvis and thorax-pelvis relative angles showed there were moderate to large differences with RMSE% values ranging between 4% and 57%. IMUs are highly accurate at measuring orientation data; however, further work is needed to optimise positioning and modelling approaches so IMU relative angles align more closely with relative angles derived using traditional motion capture methods.

Magnetic Resonance Imaging and Freehand 3-D Ultrasound Provide Similar Estimates of Free Achilles Tendon Shape and 3-D Geometry.

The purpose of this study was to assess the similarity of free Achilles tendon shape and 3-D geometry between magnetic resonance imaging (MRI) and freehand 3-D ultrasound (3-DUS) imaging methods. Fourteen elite/sub-elite middle-distance runners participated in the study. MRI and 3-DUS scans of the Achilles tendon were acquired on two separate imaging sessions, and all 3-D reconstructions were performed using identical methods. Shape similarity of free Achilles tendon reconstructed from MRI and 3-DUS data was assessed using Jaccard index, Hausdorff distance and root mean square error (RMSE). The Jaccard index, Hausdorff distance and RMSE values were 0.76 ± 0.05, 2.70 ± 0.70 and 0.61 ± 0.10 mm, respectively. The level of agreement between MRI and 3-DUS for free Achilles tendon volume, length and average cross-sectional area (CSA) was assessed using Bland-Altman analysis. Compared to MRI, freehand 3-DUS overestimated volume, length and average CSA by 30.6 ± 15.8 mm3 (1.1% ± 0.6%), 0.3 ± 0.7 mm (0.6% ± 1.9%) and 0.3 ± 1.42 mm2 (0.4% ± 2.0%), respectively. The upper and lower limits of agreement between MRI and 3-DUS for volume, length and average CSA were -0.4 to 61.7 mm3 (-0.2% to 2.3%), -1.0 to 1.5 mm (-3.2% to 4.5%) and -2.5 to 3.1 mm2 (-3.5% to 4.3%), respectively. There were no significant differences between imaging methods in CSA along the length of the tendon. In conclusion, MRI and freehand 3-DUS may be considered equivalent methods for estimating shape and 3-D geometry of the free Achilles tendon. These findings, together with the practical benefits of being able to assess 3-D Achilles tendon shape and geometry in a laboratory environment and under isometric loading, make 3-DUS an attractive alternative to MRI for assessing 3-D free Achilles tendon macro-structure in future studies.

Design of a framework for the deployment of collaborative independent rare disease-centric registries: Gaucher disease registry model.

Orphan drug clinical trials often are adversely affected by a lack of high quality treatment efficacy data that can be reliably compared across large patient cohorts derived from multiple governmental and country jurisdictions. It is critical that these patient data be captured with limited corporate involvement. For some time, there have been calls to develop collaborative, non-proprietary, patient-centric registries for post-market surveillance of aspects related to orphan drug efficacy. There is an urgent need for the development and sustainable deployment of these 'independent' registries that can capture comprehensive clinical, genetic and therapeutic information on patients with rare diseases. We therefore extended an open-source registry platform, the Rare Disease Registry Framework (RDRF) to establish an Independent Rare Disease Registry (IRDR). We engaged with an established rare disease community for Gaucher disease to determine system requirements, methods of data capture, consent, and reporting. A non-proprietary IRDR model is presented that can serve as autonomous data repository, but more importantly ensures that the relevant data can be made available to appropriate stakeholders in a secure, timely and efficient manner to improve clinical decision-making and the lives of those with a rare disease.

A web-based, patient driven registry for Angelman syndrome: the global Angelman syndrome registry.

Angelman syndrome (AS) is a rare neurodevelopmental disorder that is characterised by severe global developmental delays, ataxia, loss of speech, epilepsy, sleep disorders, and a happy disposition. There is currently no cure for AS, though several pharmaceutical companies are anticipating drug trials for new therapies to treat AS. The Foundation for Angelman Therapeutics (FAST) Australia therefore identified a need for a global AS patient registry to identify patients for recruitment for clinical trials.The Global AS Registry was deployed in September 2016 utilising the Rare Disease Registry Framework, an open-source tool that enables the efficient creation and management of patient registries. The Global AS Registry is web-based and allows parents and guardians worldwide to register, provide informed consent, and enter data on individuals with AS. 286 patients have registered in the first 8 months since deployment.We demonstrate the successful deployment of the first patient-driven global registry for AS. The data generated from the Global AS Registry will be crucial in identifying patients suitable for clinical trials and in informing research that will identify treatments for AS, and ultimately improve the lives of individuals and their families living with AS.

Gene-enriched draft genome of the cattle tick Rhipicephalus microplus: assembly by the hybrid Pacific Biosciences/Illumina approach enabled analysis of the highly repetitive genome.

The genome of the cattle tick Rhipicephalus microplus, an ectoparasite with global distribution, is estimated to be 7.1Gbp in length and consists of approximately 70% repetitive DNA. We report the draft assembly of a tick genome that utilized a hybrid sequencing and assembly approach to capture the repetitive fractions of the genome. Our hybrid approach produced an assembly consisting of 2.0Gbp represented in 195,170 scaffolds with a N50 of 60,284bp. The Rmi v2.0 assembly is 51.46% repetitive with a large fraction of unclassified repeats, short interspersed elements, long interspersed elements and long terminal repeats. We identified 38,827 putative R. microplus gene loci, of which 24,758 were protein coding genes (≥100 amino acids). OrthoMCL comparative analysis against 11 selected species including insects and vertebrates identified 10,835 and 3,423 protein coding gene loci that are unique to R. microplus or common to both R. microplus and Ixodes scapularis ticks, respectively. We identified 191 microRNA loci, of which 168 have similarity to known miRNAs and 23 represent novel miRNA families. We identified the genomic loci of several highly divergent R. microplus esterases with sequence similarity to acetylcholinesterase. Additionally we report the finding of a novel cytochrome P450 CYP41 homolog that shows similar protein folding structures to known CYP41 proteins known to be involved in acaricide resistance.

Design of the Familial Hypercholesterolaemia Australasia Network Registry: Creating Opportunities for Greater International Collaboration.

Familial Hypercholesterolemia (FH) is the most common and serious monogenic disorder of lipoprotein metabolism that leads to premature coronary heart disease. There are over 65,000 people estimated to have FH in Australia, but many remain undiagnosed. Patients with FH are often under-treated, but with early detection, cascade family testing and adequate treatment, patient outcomes can improve. Patient registries are key tools for providing new information on FH and enhancing care worldwide. The development and design of the FH Australasia Network Registry is a crucial component in the comprehensive model of care for FH, which aims to provide a standardized, high-quality and cost-effective system of care that is likely to have the highest impact on patient outcomes. Informed by stakeholder engagement, the FH Australasia Network Registry was collaboratively developed by government, patient and clinical networks and research groups. The open-source, web-based Rare Disease Registry Framework was the architecture chosen for this registry owing to its open-source standards, modular design, interoperability, scalability and security features; all these are key components required to meet the ever changing clinical demands across regions. This paper provides a high level blueprint for other countries and jurisdictions to help inform and map out the critical features of an FH registry to meet their particular health system needs.

An internet-based bioinformatics toolkit for plant biosecurity diagnosis and surveillance of viruses and viroids.

BACKGROUND: Detection and preventing entry of exotic viruses and viroids at the border is critical for protecting plant industries trade worldwide. Existing post entry quarantine screening protocols rely on time-consuming biological indicators and/or molecular assays that require knowledge of infecting viral pathogens. Plants have developed the ability to recognise and respond to viral infections through Dicer-like enzymes that cleave viral sequences into specific small RNA products. Many studies reported the use of a broad range of small RNAs encompassing the product sizes of several Dicer enzymes involved in distinct biological pathways. Here we optimise the assembly of viral sequences by using specific small RNA subsets. RESULTS: We sequenced the small RNA fractions of 21 plants held at quarantine glasshouse facilities in Australia and New Zealand. Benchmarking of several de novo assembler tools yielded SPAdes using a kmer of 19 to produce the best assembly outcomes. We also found that de novo assembly using 21-25 nt small RNAs can result in chimeric assemblies of viral sequences and plant host sequences. Such non-specific assemblies can be resolved by using 21-22 nt or 24 nt small RNAs subsets. Among the 21 selected samples, we identified contigs with sequence similarity to 18 viruses and 3 viroids in 13 samples. Most of the viruses were assembled using only 21-22 nt long virus-derived siRNAs (viRNAs), except for one Citrus endogenous pararetrovirus that was more efficiently assembled using 24 nt long viRNAs. All three viroids found in this study were fully assembled using either 21-22 nt or 24 nt viRNAs. Optimised analysis workflows were customised within the Yabi web-based analytical environment. We present a fully automated viral surveillance and diagnosis web-based bioinformatics toolkit that provides a flexible, user-friendly, robust and scalable interface for the discovery and diagnosis of viral pathogens. CONCLUSIONS: We have implemented an automated viral surveillance and diagnosis (VSD) bioinformatics toolkit that produces improved viruses and viroid sequence assemblies. The VSD toolkit provides several optimised and reusable workflows applicable to distinct viral pathogens. We envisage that this resource will facilitate the surveillance and diagnosis viral pathogens in plants, insects and invertebrates.