High-throughput evolutionary optimization of the induction medium towards recombinant protein production in BY-2 tobacco.

Bright yellow (BY-2) tobacco cells combined with the XVE chemically inducible system are one of the most promising plant-based platforms for recombinant protein production. This offers a range of benefits, including the separation of the cell growth and heterologous gene expression, lack of risk of infecting the end product with prions and human viruses or appropriate protein glycosylation and folding. However, low protein productivity remains a major obstacle that limits the extensive commercialization of bioproduction in plants. A number of molecular, cell culture and down processing approaches have been made to overcome this problem. Media development for the specific nutritional and hormonal requirements of transgenic plant cells is one of the most efficient cell-culture approaches. We optimized the induction medium towards recombinant protein production in BY-2 and demonstrated the usefulness of evolutionary medium optimization for high-yield protein production in liquid plant cultures. A reliable XVE/GFP model, parallel conducting experiments in a microscale on 96-well plates, and dedicated Gene Game evolutionary optimization software allowed for an effective search of 7611 possible solutions of 11-component media. Within the 4608 formulations tested, the Induct X medium was found with a significant 107.14% increase in protein expression in relation to the standard BY-2 medium.

Virus-Like Particles Produced Using the Brome Mosaic Virus Recombinant Capsid Protein Expressed in a Bacterial System.

Virus-like particles (VLPs), due to their nanoscale dimensions, presence of interior cavities, self-organization abilities and responsiveness to environmental changes, are of interest in the field of nanotechnology. Nevertheless, comprehensive knowledge of VLP self-assembly principles is incomplete. VLP formation is governed by two types of interactions: protein-cargo and protein-protein. These interactions can be modulated by the physicochemical properties of the surroundings. Here, we used brome mosaic virus (BMV) capsid protein produced in an E. coli expression system to study the impact of ionic strength, pH and encapsulated cargo on the assembly of VLPs and their features. We showed that empty VLP assembly strongly depends on pH whereas ionic strength of the buffer plays secondary but significant role. Comparison of VLPs containing tRNA and polystyrene sulfonic acid (PSS) revealed that the structured tRNA profoundly increases VLPs stability. We also designed and produced mutated BMV capsid proteins that formed VLPs showing altered diameters and stability compared to VLPs composed of unmodified proteins. We also observed that VLPs containing unstructured polyelectrolyte (PSS) adopt compact but not necessarily more stable structures. Thus, our methodology of VLP production allows for obtaining different VLP variants and their adjustment to the incorporated cargo.

Poly(C)-binding Protein 2 Regulates the p53 Expression via Interactions with the 5'-Terminal Region of p53 mRNA.

The p53 protein is one of the major transcriptional factors which guards cell homeostasis. Here, we showed that poly(C)-binding protein 2 (PCBP2) can bind directly to the 5' terminus of p53 mRNA by means of electrophoretic mobility shift assay. Binding sites of PCBP2 within this region of p53 mRNA were mapped using Pb2+-induced cleavage and SAXS methods. Strikingly, the downregulation of PCBP2 in HCT116 cells resulted in a lower level of p53 protein under normal and stress conditions. Quantitative analysis of p53 mRNA in PCBP2-downregulated cells revealed a lower level of p53 mRNA under normal conditions suggesting the involvement of PCBP2 in p53 mRNA stabilisation. However, no significant change in p53 mRNA level was observed upon PCBP2 depletion under genotoxic stress. Moreover, a higher level of p53 protein in the presence of rapamycin or doxorubicin and the combination of both antibiotics was noticed in PCBP2-overexpressed cells compared to control cells. These observations indicate the potential involvement of PCBP2 in cap-independent translation of p53 mRNA especially occurring under stress conditions. It has been postulated that the PCBP2 protein is engaged in the enhancement of p53 mRNA stability, probably via interacting with its 3' end. Our data show that under stress conditions PCBP2 also modulates p53 translation through binding to the 5' terminus of p53 mRNA. Thus PCBP2 emerges as a double-function factor in the p53 expression.

Development of consensus-based guidelines for managing communication of individuals with Rett syndrome.

Difficulties with communication have a profound impact on the lives of individuals with Rett syndrome and their caregivers. Globally, many families report difficulty accessing appropriate and timely information and services from professionals with expertise in augmentative and alternative communication (AAC) as it pertains to Rett syndrome. To address this need, international consensus-based guidelines for managing the communication of individuals with Rett syndrome were developed by combining available evidence and lived experience with expert opinion. A two-phase Delphi survey was built on statements and recommendations extracted from a review of over 300 pieces of literature combined with survey responses from communication professionals and caregivers. All statements that reached a pre-determined threshold of ≥70% agreement were incorporated into guidelines that consist of 268 statements and recommendations relating to (a) rights of the individual; (b) beliefs and attitudes of communication partners; (c) professional knowledge and team work; (d) strategies to optimize engagement; (e) assessment; and (f) intervention (targets and goals, techniques), including the use of AAC. To date, this project is the largest of its kind, with 650 participants from 43 countries contributing to development of consensus-based guidelines for Rett syndrome.

The many faces of Dicer: the complexity of the mechanisms regulating Dicer gene expression and enzyme activities.

There is increasing evidence indicating that the production of small regulatory RNAs is not the only process in which ribonuclease Dicer can participate. For example, it has been demonstrated that this enzyme is also involved in chromatin structure remodelling, inflammation and apoptotic DNA degradation. Moreover, it has become increasingly clear that cellular transcript and protein levels of Dicer must be strictly controlled because even small changes in their accumulation can initiate various pathological processes, including carcinogenesis. Accordingly, in recent years, a number of studies have been performed to identify the factors regulating Dicer gene expression and protein activity. As a result, a large amount of complex and often contradictory data has been generated. None of these data have been subjected to an exhaustive review or critical discussion. This review attempts to fill this gap by summarizing the current knowledge of factors that regulate Dicer gene transcription, primary transcript processing, mRNA translation and enzyme activity. Because of the high complexity of this topic, this review mainly concentrates on human Dicer. This review also focuses on an additional regulatory layer of Dicer activity involving the interactions of protein and RNA factors with Dicer substrates.

Six Medicago truncatula Dicer-like protein genes are expressed in plant cells and upregulated in nodules.

KEY MESSAGE: Here we report the existence of six putative Dicer-like genes in the Medicago truncatula genome. They are ubiquitously expressed throughout the plant and significantly induced in root nodules. Over the past decade, small noncoding RNAs (sncRNA) have emerged as widespread and important regulatory molecules influencing both the structure and expression of plant genomes. One of the key factors involved in sncRNA biogenesis in plants is a group of RNase III-type nucleases known as Dicer-like (DCL) proteins. Based on functional analysis of DCL proteins identified in Arabidopsis thaliana, four types of DCLs were distinguished (DCL1-4). DCL1 mainly produces 21 nt miRNAs. The products generated by DCL2, DCL3, and DCL4 belong to various classes of siRNAs that are 22, 24 and 21 nt in length, respectively. M. truncatula is a model legume plant closely related to many economically important cultivable species. By screening the recent M. truncatula genome assembly, we were able to identify three new DCL genes in addition to the MtDCL1-3 genes that had been earlier characterized. The newly found genes include MtDCL4 and two MtDCL2 homologs. We showed that all six M. truncatula DCL genes are expressed in plant cells. The first of the identified MtDCL2 paralogs encodes a truncated version of the DCL2 protein, while the second undergoes substantial and specific upregulation in the root nodules. Additionally, we identified an alternative splicing variant of MtDCL1 mRNA, similar to the one found in Arabidopsis. Our results indicate that DCL genes are differently activated during Medicago symbiosis with nitrogen fixing bacteria and upon pathogen infection. In addition, we hypothesize that the alternative splicing variant of MtDCL1 mRNA may be involved in tissue-specific regulation of the DCL1 level.

Aspects of speech-language abilities are influenced by MECP2 mutation type in girls with Rett syndrome.

This study investigates relationships between methyl-CpG-binding protein 2 gene (MECP2) mutation type and speech-language abilities in girls with Rett syndrome. Cross-sectional data on 766 girls, aged 15 years and under, with genetically confirmed Rett syndrome was obtained from the Australian Rett Syndrome Database (ARSD) (n = 244) and the International Rett Syndrome Phenotype Database (InterRett) (n = 522). Relationships between MECP2 mutation type and age of regression in speech-language abilities, and the level of speech-language abilities before and after this regression were investigated. The females had a median age of 4.95 years in the ARSD and 5.25 years in InterRett. The majority (89%, 685/766) acquired speech-language abilities in the form of babble or words at some point in time. Of those who acquired babble or words, 85% (581/685) experienced a regression in these abilities. Those with a p.Arg133Cys mutation were the most likely to use one or more words, prior to (RRR = 3.45; 95% CI 1.15-10.41) and after (RRR = 5.99; 95% CI 2.00-17.92), speech-language regression. Girls with Rett syndrome vary in their use of speech and language, and in their experience of speech-language regression and these variations are partly explained by genotype.

3D Puzzle at the Nanoscale-How do RNA Viruses Self-Assemble their Capsids into Perfectly Ordered Structures.

The phenomenon of RNA virus self-organization, first observed in the mid-20th century in tobacco mosaic virus, is the subject of extensive research. Efforts to comprehend this process intensify due to its potential for producing vaccines or antiviral compounds as well as nanocarriers and nanotemplates. However, direct observation of the self-assembly is hindered by its prevalence within infected host cells. One of the approaches involves in vitro and in silico research using model viruses featuring a ssRNA(+) genome enclosed within a capsid made up of a single type protein. While various pathways are proposed based on these studies, their relevance in vivo remains uncertain. On the other hand, the development of advanced microscopic methods provide insights into the events within living cells, where following viral infection, specialized compartments form to facilitate the creation of nascent virions. Intriguingly, a growing body of evidence indicates that the primary function of packaging signals in viral RNA is to effectively initiate the virion self-assembly. This is in contrast to earlier opinions suggesting a role in marking RNA for encapsidation. Another noteworthy observation is that many viruses undergo self-assembly within membraneless liquid organelles, which are specifically induced by viral proteins.

"I'm the Family Ringmaster and Juggler": Autistic Parents' Experiences of Parenting During the COVID-19 Pandemic.

Background: Little is known about autistic parenthood. The literature that exists suggests that autistic parents can find it difficult to manage the everyday demands of parenting and domestic life. While emerging research has also highlighted more positive parenting experiences, greater understanding of autistic parenthood is needed. Objective: This study sought to understand autistic parents' parenting experiences during the initial phase of the COVID-19 pandemic. Methods: Thirty-five Australian autistic parents (95% women) of autistic children (aged 4-25 years) took part in semi-structured interviews designed to elicit their experiences of life during lockdown. We used reflexive thematic analysis using an inductive (bottom-up) approach to identify patterned meanings within the data set. Results: Autistic parents repeatedly spoke of how the lockdown brought some initial relief from the intensity of their usual lives caring for their children. Nevertheless, most autistic parents felt that the "cumulative stress" of trying to juggle everything during lockdown proved very challenging, which eventually took its toll on parents' mental health. Parents were aware that they needed support but found it difficult to reach out to their usual social supports (including autistic friends) for help, and formal supports were virtually nonexistent. Consequently, they felt "very much forgotten." Nevertheless, they described how their connections with their children grew stronger over lockdown as they focused on nurturing their children's "mental health ahead of everything else." Conclusions: Our analysis shows how challenging conventional life can be for autistic parents. Parenting requires grappling with a distinctive set of demands, which are usually partially manageable through the informal supports many autistic parents draw upon. The relative absence of informal supports during the pandemic, however, left them reliant on more formal supports, which were not forthcoming. Research is urgently needed to identify the most effective formal supports for autistic parents, ideally in partnership with autistic parents themselves.

Why is this an important issue?: There is very little research about what autistic parents think about being a parent and how they manage in their everyday lives. What was the purpose of this study?: We, a group of autistic and non-autistic researchers, worked together to look at autistic people's experiences of life during the COVID-19 pandemic. In this study, led by an autistic parent, we focused on understanding what parenting was like for autistic parents during this time. What did we do?: We spoke to 35 autistic parents, mostly from Australia, about their experiences of life during the first COVID-19 lockdown. Almost all parents were women. They each spoke to us for about 1 hour. We discussed questions like, "What has been difficult for your child about being required to stay at home? And what has been positive about it? What about you? How have you found having to stay at home?". What were the results of the study?: Autistic parents told us that life before COVID-19 could "be really hard." Their days were spent focusing on their children and supporting them, but they felt life became a bit simpler during lockdown. They also did not have "to go out in the non-autistic world," which they found exhausting. But lockdown made things difficult, too. Autistic parents told us that they were stressed about trying to juggle everything at home, such as home learning, working from home, and cleaning the house. They also felt that they were the ones supporting everyone else. This meant they did not get the rest they needed. They were also worried about COVID-19. These worries and pressure often had a terrible effect on autistic parents' mental health. They wanted to reach out to their friends­especially autistic friends­for help but often could not. There were few other supports available to help them. Even when things were difficult, they told us that they nonetheless felt connected with their children during lockdown and that they were putting their children's mental health first, above everything else. What do these findings add to what was already known?: This study shows how hard everyday life can be for autistic parents. These parents usually rely on their friends for support. But they could not do that during lockdown. This meant they had to get help from more official supports and services. But these often just were not available. As a result, autistic parents felt "very much forgotten" as a group. What are potential weaknesses in the study?: Most of our participants were women, well educated, and White. We do not know if our autistic parents' experiences would be the same for other autistic parents, such as autistic fathers or those from other racial/ethnic backgrounds. How will these findings help autistic adults now or in the future?: These findings show how important it is to understand how we can support autistic parents and make their lives better during times of crisis and in more normal times. This needs to be done in partnership with autistic parents.

Negotiation of collective and individual candidacy for long Covid healthcare in the early phases of the Covid-19 pandemic: Validated, diverted and rejected candidacy.

This analysis of people's accounts of establishing their need and experiences of healthcare for long Covid (LC) symptoms draws on interview data from five countries (UK, US, Netherlands, Canada, Australia) during the first ∼18 months of the Covid-19 pandemic when LC was an emerging, sometimes contested, condition with scant scientific or lay knowledge to guide patients and professionals in their sense-making of often bewildering constellations of symptoms. We extend the construct of candidacy to explore positive and (more often) negative experiences that patients reported in their quest to understand their symptoms and seek appropriate care. Candidacy usually considers how individuals negotiate healthcare access. We argue a crucial step preceding individual claims to candidacy is recognition of their condition through generation of collective candidacy. "Vanguard patients" collectively identified, named and fought for recognition of long Covid in the context of limited scientific knowledge and no established treatment pathways. This process was technologically accelerated via social media use. Patients commonly experienced "rejected" candidacy (feeling disbelieved, discounted/uncounted and abandoned, and that their suffering was invisible to the medical gaze and society). Patients who felt their candidacy was "validated" had more positive experiences; they appreciated being believed and recognition of their changed lives/bodies and uncertain futures. More positive healthcare encounters were described as a process of "co-experting" through which patient and healthcare professional collaborated in a joint quest towards a pathway to recovery. The findings underpin the importance of believing and learning from patient experience, particularly vanguard patients with new and emerging illnesses.

Co-design of an NHS primary care health check for autistic adults.

LAY ABSTRACT: Autistic people are on average more likely to experience poor health than people who are not autistic. Health checks have been shown to improve access to effective healthcare. This study investigated people's views about a primary care health check for autistic adults. We held discussion groups and interviewed autistic adults, adults with intellectual disabilities, supporters and health professionals. People wanted the health check to look at a person's physical and mental health, and how they were doing socially. They thought people should be able to share information about their needs and the reasonable adjustments they would like before the health check. They wanted healthcare services to change the way they communicate with autistic people, such as being able to book appointments online rather than by telephone. They wanted a choice in how the health check was completed, with video call or email offered as well as face-to-face appointments. People thought further training of primary care staff on autism was needed, to increase awareness of the diversity of experiences of autistic people and ways in which difficulties, such as pain, may present differently to non-autistic people. Clinicians raised questions about whether mental health and social care services could meet the additional needs that might be identified through the health check. We used this information to design an NHS primary care health check for autistic people in collaboration with autistic people, supporters and health professionals.

A trial of the AASPIRE healthcare toolkit with Australian adults on the autism spectrum.

BACKGROUND: Autistic adults experience barriers to accessing health care, such as service provider communication not meeting their needs, healthcare facilities causing sensory discomfort and feeling fear or anxiety regarding their healthcare visit. The Academic Autism Spectrum Partnership in Research and Education (AASPIRE) developed and trialled an online healthcare toolkit to reduce such barriers and improve healthcare interactions between autistic adults and their primary care providers in the United States. This preliminary study aimed to explore experiences of autistic adults using the AASPIRE Healthcare Toolkit in Australia. METHODS: Semi-structured interviews were conducted with six autistic adults about their experiences and perceptions of utilising the toolkit in an Australian healthcare setting. RESULTS: Participants identified that the toolkit facilitated their interactions with health professionals by providing structure to appointments, supplementing new knowledge and increasing individual confidence. They also offered suggestions to tailor the toolkit for use in Australia. CONCLUSIONS: Future research should seek to explore the experiences of autistic adults using a version of the toolkit adapted for Australian use, as well as exploring the views of health professionals utilising it.

New aspects of DNA recognition by group II WRKY transcription factor revealed by structural and functional study of AtWRKY18 DNA binding domain.

WRKY transcription factors (TFs) constitute one of the largest families of plant TFs. Based on the organization of domains and motifs, WRKY TFs are divided into three Groups (I-III). The WRKY subgroup IIa includes three representatives in A. thaliana, AtWRKY18, AtWRKY40, and AtWRKY60, that participate in biotic and abiotic stress responses. Here we present crystal structures of the DNA binding domain (DBD) of AtWRKY18 alone and in the complex with a DNA duplex containing the WRKY-recognition sequence, W-box. Subgroup IIa WRKY TFs are known to form homo and heterodimers. Our data suggest that the dimerization interface of the full-length AtWRKY18 involves contacts between the DBD subunits. DNA binding experiments and structural analysis point out novel aspects of DNA recognition by WRKY TFs. In particular, AtWRKY18-DBD preferentially binds an overlapping tandem of W-boxes accompanied by a quasi-W-box motif. The binding of DNA deforms the B-type double helix, which suggests that the DNA fragment must be prone to form a specific structure. This can explain why despite the short W-box consensus, WRKY TFs can precisely control gene expression. Finally, this first experimental structure of a Group II WRKY TF allowed us to compare Group I-III representatives.

Cryo-EM reconstructions of BMV-derived virus-like particles reveal assembly defects in the icosahedral lattice structure.

The increasing interest in virus-like particles (VLPs) has been reflected by the growing number of studies on their assembly and application. However, the formation of complete VLPs is a complex phenomenon, making it difficult to rationally design VLPs with desired features de novo. In this paper, we describe VLPs assembled in vitro from the recombinant capsid protein of brome mosaic virus (BMV). The analysis of VLPs was performed by Cryo-EM reconstructions and allowed us to visualize a few classes of VLPs, giving insight into the VLP self-assembly process. Apart from the mature icosahedral VLP practically identical with native virions, we describe putative VLP intermediates displaying non-icosahedral arrangements of capsomers, proposed to occur before the final disorder-order transition stage of icosahedral VLP assembly. Some of the described VLP classes show a lack of protein shell continuity, possibly resulting from too strong interaction with the cargo (in this case tRNA) with the capsid protein. We believe that our results are a useful prerequisite for the rational design of VLPs in the future and lead the way to the effective production of modified VLPs.

Exploring the Experience of Seeking an Autism Diagnosis as an Adult.

Background: Emerging research suggests that seeking an autism diagnosis as an adult is usually difficult and time-consuming but brings relief once a diagnosis is made. This study explored the experience of the pathway to an autism diagnosis during adulthood for adults living in Australia. Methods: We conducted a qualitative phenomenological study and interviewed 13 adults who identified as autistic about their pathway to autism diagnosis in their mode of choice. Spoken interviews were transcribed verbatim, and transcripts were analyzed by using a thematic approach. Results: Data analysis resulted in 6 themes and 20 meaning units that described the experiences of adults seeking an autism diagnosis in Australia. These themes involved two interwoven journeys that spanned before, during, and after the diagnostic process. The personal journey involved feeling different, considering autism, and living as autistic, whereas the clinical journey involved missed opportunities, varied diagnostic experiences, and absent supports. Conclusions: Given the potential benefits for adults obtaining a formal autism diagnosis and accessing post-diagnostic supports, it is important that health professionals and governments collaborate to reduce access barriers and ensure adequate services are available. The findings from this study informed the development of Australia's national guideline for autism diagnosis.

Why is this an important issue?: The experience of being diagnosed as autistic as an adult is not well understood, particularly in Australia. Research from other places, such as New Zealand and the United Kingdom, suggests that receiving an autism diagnosis in adulthood is difficult and time-consuming, but brings relief. We do not know whether this is the same for adults in Australia. What was the purpose of this study?: This study aimed at exploring the experience of seeking an autism diagnosis during adulthood in Australia. What did the researchers do?: We conducted interviews with 13 adults who identified as autistic about their pathway to an autism diagnosis. Three sets of interview questions were used, depending on whether they had already obtained an autism diagnosis, were going through the assessment process, or were self-diagnosed. Adults completed the interview in their mode of choice. Spoken interviews were transcribed word-for-word, and the transcripts were analyzed to identify common themes. What were the results of the study?: We identified six themes that described the experiences of adults seeking an autism diagnosis in Australia. These themes involved two related journeys that spanned before, during, and after the diagnostic process. The personal journey involved feeling different, considering autism, and living as autistic. The clinical journey involved missed opportunities, varied diagnostic experiences, and absent supports. Before starting the diagnostic process in adulthood, participants described always feeling different and many missed opportunities to receive an autism diagnosis in their younger years.During the diagnostic process, participants described beginning to consider whether they were autistic and the varied pathways they underwent to confirm this. After the diagnostic process, participants described their experiences of living as autistic and a lack of post-diagnostic supports tailored to their needs. What do these findings add to what was already known?: Our findings were similar to previous research findings from other countries, including the complex journey to diagnosis, relief and understanding on identifying as autistic, and lack of post-diagnostic services. However, to our knowledge, this is the first qualitative study to explore the experiences of adults seeking an autism diagnosis in Australia. Further, we included participants who did not have a formal diagnosis of autism. This group of people is often excluded from autism research, and their experiences of seeking an autism diagnosis are largely unknown. What are potential weaknesses in the study?: The weaknesses of our study included recruiting a relatively small sample of mostly Caucasian females, and we did not consult with our study participants or other autistic adults to see whether our final themes aligned well with their experience. However, no new findings emerged in later interviews and our findings were similar to international literature. Future research should recruit more diverse groups of autistic adults and involve greater levels of autistic input. How will these findings help autistic adults now or in the future?: Our findings informed the development of the "National Guideline for the Assessment and Diagnosis of Autism Spectrum Disorder in Australia," a first step toward improving autism diagnosis in Australia.

COVID-19, social isolation and the mental health of autistic people and their families: A qualitative study.

LAY ABSTRACT: In this study, we show that autistic people and their families have found it very difficult to deal with the lockdowns during the COVID-19 pandemic. Autistic and non-autistic researchers spoke to 144 people, including 44 autistic adults, 84 parents of autistic children and 16 autistic young people (12-18 years old). We asked them about their everyday lives and mental health during lockdown. People told us that they enjoyed having fewer obligations and demands compared to pre-COVID-19 life. They felt that life was quieter and calmer. But people also told us again and again how much they missed meeting people in real life, especially their friends, and their therapists and support workers. People told us that their mental health suffered because they did not have contact with their friends and services. Importantly, many people (including researchers) think that autistic people do not want friends or to be around people. But our results show that is not true. Many autistic people do want friends and to be around other people. Some people's mental health has been damaged by not being able to see people during COVID-19. Autistic people need support in many areas of life so they can keep socialising and seeing their friends even through difficult times, like pandemics.

Views about primary care health checks for autistic adults: UK survey findings.

BACKGROUND: Compared with the general population, autistic adults experience higher rates of physical and mental health conditions, premature morbidity and mortality, and barriers to health care. A health check for autistic people may improve their health outcomes. AIM: To establish the views of autistic people towards a primary care health check for autistic people. DESIGN & SETTING: Cross-sectional questionnaire study in England and Wales. METHOD: A questionnaire was sent to autistic adults with physical health conditions in England and Wales. A total of 458 people (441 autistic adults and 17 proxy responders) completed the questionnaire. RESULTS: Most responders (73.4%, n = 336) thought a health check is needed for all autistic people. Around half of the participants thought a health check should be offered from childhood and the health check appointment should last between 15 and 30 minutes. Autistic people were positive about providing primary care staff with contextual information regarding their health and the reasonable adjustments they would like before their health check appointment. Training about autism and the health check was considered important, alongside adequate time for discussions in the health check appointment (all by over 70% of responders). The clinician's autism knowledge, seeing a familiar clinician, environmental adaptations, appropriate information, and accessible appointments were considered particularly important in making a health check accessible. CONCLUSION: Autistic people and relatives were supportive of a primary care health check for autistic people. Information gathered was used to support the design of a primary care health check for autistic adults.

Strong interactions between Salp15 homologues from the tick I. ricinus and distinct types of the outer surface OspC protein from Borrelia.

Ticks belonging to the genus Ixodes are parasites feeding on vertebrate blood and vectors for many pathogenic microbes, including Borrelia burgdorferi sensu lato spirochetes, the causative agent of Lyme borreliosis. The tick saliva contains a mixture of bioactive molecules showing a wide range of properties for efficient engorgement. One of the most extensively studied components of tick saliva is a 15-kDa salivary gland protein (Salp15) from Ixodes scapularis. This multifunctional protein suppresses the immune response of hosts through pleiotropic action on a few crucial defense pathways. Salp15 and its homologue from I. ricinus Iric1 have been also shown to bind to Borrelia burgdorferi sensu stricto outer surface protein C (OspC) permitting the spirochetes to evade antibody-mediated killing in the human host. Further studies revealed that Salp15 and Iric1 protected B. burgdorferi s. s. and B. garinii expressing OspC against the complement system. OspC is the most variable protein on the outer surface of Borrelia, which in addition to Salp15 can also bind other ligands, such as plasminogen, fibrinogen, fibronectin or complement factor 4. So far several OspC variants produced by B. burgdorferi s. l. spirochetes were shown to be capable of binding Salp15 or its homologue, but the protection against borreliacidal antibodies has only been proven in the case of B. burgdorferi s. s. The question of Salp15 contribution to Borrelia survival during the infection has been comprehensively studied during the last decades. In contrast, the organization of the OspC-Salp15 complex has been poorly explored. This report describes the binding between three Salp15 homologues from the tick Ixodes ricinus (Iric1, Iric2 and Iric3) and OspC from four B. burgdorferi sensu lato strains in terms of the binding parameters, analyzed with two independent biophysical methods - Microscale thermophoresis (MST) and Biolayer interferometry (BLI). The results of both experiments show a binding constant at the nanomolar level, which indicates very strong interactions. While the Iric1-OspC binding has been reported before, we show in this study that also Iric2 and Iric3 are capable of OspC binding with high affinity. This observation suggests that these two Salp15 homologues might be used by B. burgdorferi s. l. in a way analogous to Iric1. A comparison of the results from the two methods let us propose that N-terminal immobilization of OspC significantly increases the affinity between the two proteins. Finally, our results indicate that the Iric binding site is located in close proximity of the OspC epitopes recognized by human antibodies, which may have important biological and medical implications.

3D Domain Swapping Dimerization of the Receiver Domain of Cytokinin Receptor CRE1 From Arabidopsis thaliana and Medicago truncatula.

Cytokinins are phytohormones regulating many biological processes that are vital to plants. CYTOKININ RESPONSE1 (CRE1), the main cytokinin receptor, has a modular architecture composed of a cytokinin-binding CHASE (Cyclases/Histidine kinases Associated Sensory Extracellular) domain, followed by a transmembrane fragment, an intracellular histidine kinase (HK) domain, and a receiver domain (REC). Perception of cytokinin signaling involves (i) a hormone molecule binding to the CHASE domain, (ii) CRE1 autophosphorylation at a conserved His residue in the HK domain, followed by a phosphorelay to (iii) a conserved Asp residue in the REC domain, (iv) a histidine-containing phosphotransfer protein (HPt), and (v) a response regulator (RR). This work focuses on the crystal structures of the REC domain of CRE1 from the model plant Arabidopsis thaliana and from the model legume Medicago truncatula. Both REC domains form tight 3D-domain-swapped dimers. Dimerization of the REC domain agrees with the quaternary assembly of the entire CRE1 but is incompatible with a model of its complex with HPt, suggesting that a considerable conformational change should occur to enable the signal transduction. Indeed, phosphorylation of the REC domain can change the HPt-binding properties of CRE1, as shown by functional studies.

Recognising, supporting and understanding Autistic adults in general practice settings.

BACKGROUND: The health and wellbeing of Autistic people are well below those of their non-Autistic counterparts. With studies showing a reduced life expectancy and poor physical and mental health, it is important that healthcare professionals strive towards improving the lives of Autistic people by recognising those who are undiagnosed and/or misdiagnosed and providing meaningful support. OBJECTIVE: The aim of this article is to empower general practitioners (GPs) to feel comfortable in recognising potentially undiagnosed Autistic adults. The authors aim to further the understanding of the Autistic experience, including frequently co-occurring physical and mental health conditions, and to give GPs some tools to make the experience of accessing healthcare less problematic for Autistic people. DISCUSSION: The healthcare experiences of Autistic adults will be greatly improved with a knowledgeable, understanding and accepting GP as the cornerstone of their care.