Research on incarcerated fathers tends to accentuate the harmful familialconsequences of parental incarceration and discuss how having children mightprompt incarcerated fathers to desist from crime. Less attention has focused onhow narratives of fatherhood shape the day-to-day dynamics of incarceration.Drawing on 93 qualitative interviews with incarcerated fathers in WesternCanada, we focus specifically on our participants’ parenting narratives. Suchnarratives are significant interventions in the world, allowing incarceratedfathers to frame their identities in particular ways while simultaneouslyshaping personal behaviour. Our research, 1. Identifies important fatherhoodnarratives provided by our participants, and 2. Details how such narrativesoperate in prison, allowing our participants to advance personal agendas thatare themselves related to the dynamics of incarceration. In doing so, we provideinsights into incarcerated fathers’ situations and advance criminologicalefforts to appreciate how different actors entangled in the criminal justicesystem conceive, manage, and narrate their situation. I hustle. I make money. That’s what I do. It’s not about dope or nothing,about girls or nothing. It’s just about I’ll go out and I’ll make $10,000 ina day. And I’ll give it to you because I’ll make $10,000 tomorrow. It don’tmean shit to me.Titus’s reputation gave him considerablestanding on the street and in prison, which he reflected in his biography. In fact,he intertwined his gang membership and identity so closely that we often paused toclarify whether the “brothers” Titus was describing were other gang members, or hisbiologically-related “brother-brothers,” as he would call them. Given the centralrole that Titus’s criminal activities played in his biography, the followingtransition in his narrative was a jarring surprise:I was sitting in the penitentiary and my son came. He came to visit me, andhe had this little [gang] tattoo, little eagle head. And he was like, “Dadjust like you, warrior.” And my whole mind just went blank, and I was like:“Fuck that.” I don’t want this for my babies. I don’t want this life for mykids. I’m doing enough time for everybody. They don’t deserve to have thislife. He grew up with a mom that was a prostitute. And she just hung herselfa month and a half ago. And they’ve got a dad that’s looking at another tenyears [in prison] that they’ve been waiting for thirteen years to getout.Until this point in our discussion, Titus hadexclusively framed himself as a tough and dangerous man on the streets, recountinghis drug dealing accomplishments and penchant for violence. However, when he begandiscussing his children, his story’s narrative arc dramatically shifted, and hebegan foregrounding a different set of characterizations focused on himself as aparent. In particular, he began weaving a far more complex narrative of failure,loss, and redemption, one where his paternal identity provided him with the strengthhe needed to survive incarceration.Titus was large, heavily tattooed, and physically intimidating. A powerful man in hisearly forties, he was charismatic and had a well-documented talent for fighting,which he had parlayed into a high-ranking position in a local street gang. Thesurrounding halogen-lit concrete walls gave credence to Titus’s tales of drug dealsand robbery: we spoke deep in the heart of one of Canada’s most volatilemaximum-security prisons. Most of our conversation focused on Titus’s criminal career:Children and family are a frequent topic of conversation for incarcerated people, andmany researchers have commented on the pain incarcerated persons experience fromfamily separation . The acaWe approach the dynamics of incarcerated fatherhood from a different angle. Drawingon 93 qualitative interviews with incarcerated fathers in Western Canada, we focuson how men recount parenting narratives to deal with the stressful and painfulexperience of incarceration . We consdo in the community is contrasted with theaccuracy of what they say, especially in the case of parentingfailures  self-identified as Indigenous, while 29 (or 31%) identified asCaucasian. Ten identified as Black, Asian, or “Middle Eastern,” while 19 did notspecify their ethnic background. These profiles are representative of the massiveover-incarceration of Indigenous men and women in Western Canada, where Indigenouspeople consistently account for between 40 and 75% of the prison population despitecomprising only 10% of the general populace .In line with the interviewing practices standard in narrative criminology, we allowedindividuals’ unique accounts to drive the discussion. We commenced each interview byinviting participants to “tell us whatever you would like us to know aboutyourself,” leaving it up to the participants what they wanted to share at theoutset. As the discussion progressed, we prompted into topics relating to ourparticipants’ upbringing, relationships, spouses, children and other supportnetworks. This resulted in an interactional situation that enabled us to collect andco-create a larger story-arc of how fatherhood identities shaped their incarcerationexperiences .After we completed the interviews, we assigned randomly-generated pseudonyms to eachparticipant and transcribed the interviews verbatim. Following transcription, theauthors and three research assistants reread each interview, identifying themes andnarratives emerging from the data . We usedOur participants articulated three fatherhood narratives relating to their prisonexperience. These were, 1. redemption, 2. illicit provider, and 3. departures fromthe prison code. Not all men identified in the same way with each narrative: someidentified with more than one, while others emphasized a single, dominant narrative.However, the favourable cultural meanings of fatherhood provided opportunities forparticipants to invoke their paternal status to advance different personal goals. These nI found myself again. Sometimes you have to be alone to find out who youreally are. I found myself again. I found out who I really was, why Iwas struggling. Why was I always blaming myself for this and that? And Itold myself, I never even learned how to not be in here. They just useme and throw me away. Right? I have to be a man for my son. That’s why Isay jail saved me.Men like Julian used their parentalstatus to reframe incarceration, narrating why “this time” was different, andpointing to their fatherhood as motivating their quests for personaltransformation. Dylan went even further:I think everyone should come to jail once … I lost my [four] kids, and itwas downhill from there right. But, I mean like jail, it’s almost likeit helps me to get back to [being] me … once you get over the first weekor two, it’s almost – for me I rebuild myself.Men likeDylan and Julian characterized prison as an important and redemptive moment intheir journeys as fathers: Dylan “rebuilt” himself in prison, while Julian“learned his lesson” about how to “be a man for his son.”Facing the daily challenges of imprisonment, fathers in our sample narrativelyreframed their family experiences, using their paternal status to reclaim thestressful and often-humiliating experience of incarceration . InkeepI learned six different recipes to cook methamphetamines. I’ve never doneit, but off the top of the head I can tell you recipes right now. Idon’t spread that information anymore, because I’m on a different path.I’m here now to make a positive impact. I have two seven-year-olddaughters. Even though my father is who he is, and I still can travelthat path, I won’t. I made that decision a long time ago, but I wasstill stuck in between. Now, coming here, I’ve realized I’ve built thetools to enable myself to build a whole new ego. I’ve created a newidentity to work with … I’ve made somethingnew.Narratives like Stan’s allowed men to draw a positivepicture of their lives, their futures, and their present. Incarcerated fathersused these stories to portray themselves as changed or changing men, who werestepping away from destructive street lives. Crucially, these narratives allowedmen to reclaim agency over their incarceration and prison experience, whilesimultaneously addressing the masculinity crises engendered by being forciblyremoved from their lives and families  even provide information to thepolice, to continue the transformation into becoming a better man for one’schildren. Masculinity proves flexible here: “being a man” in prison may beinterpreted in unexpected ways when masculine behavioural codes are narrated throughthe lens of fatherhood . What isThe narratives about fatherhood in prison ultimately offer insights into the hopesand dreams of a large group of incarcerated men. Irrespective of whether thesenarratives point to a viable future , they of


In recent decades, advances in methods in molecular biology and genetics have revolutionized multiple areas of the life and health sciences. However, there remains a global need for the development of more refined and effective methods across these fields of research. In this current Collection, we aim to showcase articles presenting novel molecular biology and genetics techniques developed by scientists from around the world. Since ancient times, humankind has recognized the influence of heredity, based on familial resemblance, selective breeding of livestock, and climate-adapted crops. Prior to Gregor Johann Mendel’s work in the nineteenth century, there was no clear scientific theory to explain heredity. Mendel’s work remained essentially theoretical until the discovery of DNA and confirmation of its role as the principal agent of heredity in organisms in the twentieth century . In addiIn recent decades, advances in methods in molecular biology and genetics have revolutionized multiple areas of life and health sciences . As a main vitro nucleotide synthesis technologies [The development of sequencing technologies and their iterative improvements have been instrumental in advancing the understanding of DNA and RNA, their identification, association with various proteins, their covalent modifications, the function of the genes they carry, and the function of the non-coding portion of DNA and RNA in normal and diseased cells, in pathogenic bacteria and viruses, and in plants , 9. By pnologies .Gene editing technologies, such as restriction endonuclease digestion, transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR-Cas) system, are an additional development in the field of molecular biology that has aided in the understanding of DNA and genes. There is optimism about the use of CRISPR-Cas9 technology in the treatment of a wide variety of diseases, such as cancer, blood-related diseases, hereditary blindness, cystic fibrosis, viral diseases, muscular dystrophy, and Huntington´s disease, due to its precision and its constant improvement, in comparison with other gene-editing technologies .There is a global need for the development of novel methods for molecular biology and genetics. Particularly, in the area of human health, there is a need for further approaches that facilitate point-of-care molecular analysis (particularly miniaturized and portable platforms), for infectious and non-transmissible diseases , the devIn addition, three key factors would also help push this field forward: additional research comparing the performance of different methods for molecular biology , the broAlthough older techniques, such as x-ray crystallography, gene cloning, PCR, and sequencing, have been instrumental in the study of various aspects of genetics, these techniques have several limitations that result in gaps, missing links, and incomplete understanding of the genome. Advances in these techniques are needed to fill in these missing pieces of the puzzle to better comprehend genetics and accelerate the discovery of the causes of various genetically linkeddiseases. From a technological standpoint, the accuracy of sequencing and coverage across the genome remain major issues, especially for GC-rich regions and long homopolymer stretches of DNA. Furthermore, the short read lengths generated by the majority of current platforms severely restrict our ability to accurately characterize large repeat regions, numerous indels, and structural variation, rendering large portions of the genome opaque or inaccurate. Fragmentation of the genome for sequencing continues to be a major source of disruption in the continuity of the correct genomic sequence , 15.in vivo delivery of the Cas system components, and immune responses. This requires a substantial amount of technological advancement or the creation of new, superior methods to combat severe diseases with minimal side effects [Recent advances in CRISPR technology provide hope for the medical treatment of cancer and other fatal diseases. Despite significant advances in this field, a number of technical obstacles remain, including off-target activity, insufficient indel or low homology-directed repair (HDR) efficiency,  effects , 16.As high-throughput, automated methods commonly produce very large amounts of data, deeper interaction between wet-lab and dry-lab researchers is required, to facilitate the design of efficient assays  and alloFurther collaborations between scientists from academia and industry (in addition to researchers from government agencies)  would heIn this current Collection, we are calling for articles showcasing novel methods from molecular biology and genetics, written by scientists from around the world. It is our goal to compile a set of articles that will help to address the challenges faced by the fields of molecular biology and genetics and broaden our understanding of genetic disorders and potential treatment strategies. We invite researchers working on such methods to consider submitting to our collection.


Informal carers of patients with Chronic Obstructive Pulmonary Disease (COPD) have unmet support needs. Evidence relating to carers’ support needs in chronic conditions informed version 3 of the Carer Support Needs Assessment Tool (CSNAT) which forms part of an intervention to identify and address carer support needs. Aim of study: to establish the face and content validity of CSNAT v3 for use with COPD carers and explore their views on delivery of the CSNAT Intervention in practice.n = 2) attended one group to facilitate carer attendance, the impact of which is discussed. Most participating carers were female (n = 10); carers’ ages ranged 52–79 years.Focus groups conducted September-October 2019 in non-clinical settings recruited eleven COPD carers (two to six participants per group). COPD patients (CSNAT v3 was easy to understand and complete, and all 15 domains were considered relevant and appropriate, suggesting good face and content validity. The demeanour, relational skills, and knowledge of the CSNAT facilitator appeared more important to carers than being a certain practitioner type.COPD carers considered the CSNAT Intervention an acceptable way of identifying and responding to their needs. The intervention could potentially be delivered through a range of services. Carers are often key in supporting patients with chronic or progressive conditions such as Chronic Obstructive Pulmonary Disease (COPD): an estimated 77% of people with advanced COPD have carers. This supportive role can impact on carers’ health and wellbeing; many have support needs that go unidentified or unaddressed. A recent review found areas of unmet need related to insufficient information provision, poor support to manage emotional distress, social isolation and access to services. Carers are also patients with their own direct support needs. Policy increasingly emphasizes the importance of carers in sustaining health and social care services whilst recognising the need to provide them with person-centred support.47Informal carers are “lay people in a close supportive role who share in the illness experience of the patient and who undertake vital care work and emotion management.” The CSNAT Intervention consists of: (1) an evidence-based tool completed by the carer which is integrated into (2) ‘The CSNAT Approach’, a five-stage person-centred process of assessment and support that is practitioner facilitated but carer led. Following introduction (Stage 1) and completion of the tool (Stage 2), a needs-led conversation between carer and practitioner identifies and prioritises the carer’s unmet support needs using the carer’s self-completed tool (Stage 3). The carer and practitioner together then tailor responses to the prioritised needs (Stage 4); outcomes are reviewed at a later point (Stage 5) and the process repeated at an agreed time. Specific responses depend on the need identified, carer preference and available resources but may include active listening , education, signposting or onward referral.Health and social care practitioners are well-placed to enable identification and addressing of COPD carers’ support needs: the Carer Support Needs Assessment Tool (CSNAT) Intervention could facilitate this. Micklewright and Farquhar therefore conducted a systematic search and narrative review of COPD carer support needs and mapped these to the fourteen domains (questions) on CSNAT v2 to determine its comprehensiveness for COPD carers. The review concluded that, while CSNAT v2 encompassed most COPD carer support needs, the addition of a domain relating to relationship management would enhance comprehensiveness for COPD carers. In parallel, and independently, CSNAT’s developers completed work with carers of patients with Motor Neurone Disease (MND) and came to the same conclusion. As a result, CSNAT v3 includes an additional evidence-based fifteenth domain relating to relationship management.The CSNAT itself (the tool) was initially developed with carers of people predominantly with end-stage cancer: its relevance to COPD carers was relatively unknown. Further, confirmation of face and content validity would enhance practitioner confidence in the tool with COPD carers.This study therefore aimed to investigate the face and content validity of the 15-domain CSNAT v3 (including relationship management) with carers of patients with COPD and explore their views on mechanisms for delivery of the CSNAT Intervention with COPD carers in clinical practice. Being a carer-completed tool, establishing face and content validity is essential in order to recommend CSNAT for use with COPD carers in practice. Face validity could demonstrate acceptability to carers and, as the CSNAT was also designed to be holistic, demonstrating content validity would confirm breadth of coverage and suitability for purpose. Focus groups can achieve greater depth of understanding through their unique features enabling retrieval of data that may otherwise be missed: interactions between participants, within-session discussion of diverse viewpoints and experiences, and the greater ease participants can feel in an informal group setting. Ethical approval was obtained from University of East Anglia’s Faculty of Medicine and Health Sciences Research Ethics Committee (Reference: 201819–101).The study design drew on the pragmatist paradigm, utilising the method best suited to the research question: in this case, focus groups.Participants were recruited via Breathe Easy support groups currently, or previously, affiliated with the British Lung Foundation; the groups provide peer support and education for people living with respiratory conditions. East Anglian group leaders were invited to share study information with their members. Recruitment packs  were then provided to interested group leaders for distribution to members with COPD, or those who supported someone with COPD (carers). Patients were asked to pass on packs to their carer if this person did not attend the group. If returned reply slips indicated interest, the study researcher (KM) made contact to answer any questions and arrange the focus group for the locality. Following focus group completion, Breathe Easy groups were given a modest donation for facilitating recruitment. Refreshments were provided and travel costs reimbursed. Before the focus group discussion commenced, written informed consent was obtained and participants completed a brief demographics questionnaire. Groups were co-facilitated by both authors, lasted approximately 90 minutes, and were audio-recorded (with permission); fieldnotes documented non-verbal behaviour between participants.Four focus groups were held (September–October 2019). To enable participation, we used previously employed successful strategies including hiring attractive venues chosen with participant accessibility, comfort and convenience in mind: hotel meeting rooms (n = 3) and a community centre.A topic guide ensured key topic coverage. Participants were first given ground rules and reminded that the group’s purpose was data gathering for a research study rather than as a support group . The group was first asked what support they received as a carer and from whom. The CSNAT and CSNAT Approach (which comprise the CSNAT Intervention) were then explained to the group, before participants were given printed copies of CSNAT v3 (the tool). Participants were then asked to share initial thoughts on its layout and wording, followed by discussion of each of the 15 CSNAT domains  the most appropriate individuals to deliver the intervention, (2) appropriate settings for discussions around support needs identified by CSNAT, and (3) how these might be followed up, including when the CSNAT should be completed again.At focus group closure participants were thanked and advised to contact the research team  if they needed support resulting from participation.Audio-recordings were professionally transcribed. The study researcher (KM) checked and anonymised transcripts against the audio-recordings , whilst enhancing data familiarity. Interviewee transcript review (ITR) was not conducted in order to accurately preserve what was said; ITR can sometimes lead to alteration or removal of relevant data. Focus group discussions were largely structured around the 15 CSNAT v3 domains. The domains thus provided the initial framework which was then added to as analysis progressed, enabling identification of consistencies and divergences in thoughts and experiences across participants. Coding was completed in NVivo 12 Pro, then charted into a framework matrix in Excel by KM. Regular research team meetings involving iterative re-examination of transcripts ensured participants’ voices were retained whilst successfully distilling data consistently into appropriate codes to enable development of emerging themes.A narrative analytic approach was taken, utilising Framework Analysis.A Carer Advisory Group (CAG) consisting of bereaved and current COPD carers provided PPI (2 sessions). The CAG considered the acceptability and comprehensiveness of topic guide questions, ways to sensitively approach topics, resonance of the findings with their own experiences and the appropriateness of the researchers’ interpretations.n = 2) also attended one group to facilitate attendance of their carer: one was their carer’s husband while the other was their carer’s mother.In total, 62 recruitment packs were provided to group leaders, with a further four groups opting to send digital versions to members via email. The denominator is unknown therefore a response rate cannot be calculated. Thirteen carers responded: one was ineligible as the person they supported did not have COPD, while another declined participation stating that caring had not impacted on them significantly . Eleven COPD carers were successfully recruited and participated: most were female (n = 10), their ages ranged 52–79 years. COPD patients .Carers described support received or helpful services they had been signposted to. Helpful inputs included practitioners that took an interest in them, provided reablement care or equipment, or referred them to useful services (e.g. assistive technology or counselling sessions). However, this was not always the case: ‘…they’ve been extremely helpful in helping me to plan a way forward…’ . However, others reported having no support and several felt unsupported even if they went on to mention input that might be considered supportive: ‘I’ve been given lots of numbers and associations to go to but it’s very hard, it’s very slow, everything you either have to fight for or wait, so there is nothing there.’ Some carers were supported by their wider family. Carers also mentioned supportive organisations such as Breathe Easy and Carers Matter Norfolk : ‘And then he sits in the restaurant and somebody would come in and they’d say, “Ooh you look well”. And I’m thinking, “Do you know the effort to get us here today?”’ . Carers described life as less “spontaneous” because of COPD. Caring became more challenging over time as COPD (and comorbidities) progressed. Carers described the difficult balance between providing support and being considered overbearing: ‘But I do try to say nothing because they must think “Oh, here’s that busybody wife again”.’ . Most had few breaks from caring, compounded by patients being able to offer gradually less help with household tasks.COPD was challenging due to both its unpredictability and because patients often looked well, belying difficulties: ‘…my husband was under a super doctor at our surgery and he’s left…so we’ve got the job of, you know, getting to know a new doctor…we’ve got to sort of build up a relationship…’ . Factors negatively affecting patient care (and carers experiences) included: service and equipment access, lack of continuity in care and information, and poor interprofessional communication. Some carers felt healthcare professionals lacked time to talk to them: ‘I mean, you said this is for when a health professional talks to you about caring. When does anybody have-? Nobody’s ever done that.’ .Experiences of health and social care were mixed, with discussions largely negative. Carers spoke of staff that were knowledgeable, helpful and understanding of their situation, but when these staff moved on, or services suddenly ceased, adjustment was difficult: All carers agreed that CSNAT v3 was easy to understand, the instructions made sense and it was easy to complete. Summaries of their discussions of each of the 15 CSNAT domains are presented in None of the CSNAT v3 domains were considered redundant. While carers generally felt that, together, the domains were comprehensive, when asked whether there were additional concerns not covered by the tool two carers referred to travel issues: one mentioned travel insurance while another noted challenges in travelling with someone with COPD related to breathlessness, fatigue and managing medications, including oxygen equipment. Through the group discussion these carers agreed these concerns could be raised under other domains .‘The hospital’s too big and…is more specific to the illness and the medication and not general wellbeing…they’re there for the patient really rather than the person who’s with them.’ ; ‘You’d have to have somebody at the hospital that just helps with carers but then you’d have a queue out the door.’ . Some suggested that delivery would not necessarily need to be professionally led: ‘But what they would have to do is to be trained enough to understand this and be able to put it into action.’ Carers suggested a range of individuals could deliver the CSNAT Intervention including doctors, nurses, social workers, carer charity representatives and occupational health nurses. Suggested settings included GP surgeries, hospices, pulmonary rehabilitation sessions or during home visits. Hospitals were generally considered unsuitable: You know, if you’re going to do that, it’s not a five-minute job.’ . Good understanding of COPD and holistic appreciation of their situation was preferred. They also felt sessions should be one-on-one, with one carer stating a preference for someone not providing direct care for the patient: ‘I like to think there’s somebody I can go to – not that [Patient] can’t go to, but that [Patient] doesn’t go to.’ . Some carers wanted to discuss topics the patient avoided or found difficult, such as planning for the future and what to expect in terms of disease progression: ‘See, we [Carer and Patient] talk about the future but not as he is now, as he was, you know, it’s almost a sort of closed shop, it doesn’t really exist, and he can still do things.’ . The carers discussed talking to patients about future disease progression:‘And of all of them that’s the one that you don’t really want to have to talk about.’Jill: ‘No.’Phyllis: ‘No, we tend to talk about today or maybe tomorrow but I’m afraid we don’t talk about the other. But I would like to know.’ (FG4)Mabel: Carers stated that re-visiting their needs using CSNAT should happen at least six-monthly. They felt the time taken to action a response to any identified need would depend on the specific issue and acknowledged professionals’ time constraints. However, all carers wanted to be given a reasonable expectation of when things would be actioned or followed up:‘You don’t mind waiting a bit longer if you know from the outset that it’s-’Roger: ‘If it is going to happen, yes.’ (FG3)Amy: While carers identification of individuals and settings for CSNAT Intervention delivery were diverse, there was greater agreement on qualities carers would value pertaining to delivery such as continuity, rapport and not feeling rushed within CSNAT sessions: ‘ The findings also indicate content validity; no domains were superfluous and carers identified no missing support needs. Criterion validity was not assessed; previous validation work with CSNAT v1 suggests this is good. CSNAT is not a psychometric measure, hence investigation of construct validity and reliability were not appropriate.This paper primarily reports the face validity of CSNAT v3 with COPD carers. Confirmed face validity is important to reassure practitioners that the tool is acceptable to COPD carers.CSNAT v3 domains were interpreted in different ways by COPD carers, as evidenced by heterogenous responses to domains such as ‘Talking to your relative about his or her illness’. Responses included 1) speaking to the patient about COPD, 2) speaking to other relatives (e.g. adult children) about patient deterioration and 3) challenges encouraging patient exercise. CSNAT’s developers emphasise that carer-selected domains on the CSNAT are conversation openers and needs subsequently identified and discussed may not always obviously relate to selected domains. As such, domains initially selected are less important than the outcome of the carer-facilitator conversation.2224 The data also incidentally suggested relevance of this domain to carers of patients with cognitive impairments: at one group two carers discussed how patients’ comorbid dementia led to patient-carer relationship difficulties. The impact of dementia on relationships is well documented, with subsequent impact on carers’ physical and mental health.26CSNAT v3 included the additional relationship management domain. Although no emphasis was placed on this domain, nor mention made that it was a recent addition, reactions to it were strong with most indicating high relevance. The few carers who were initially less certain of its relevance went on to discuss numerous familial tensions (including patient-carer relationships), thereby endorsing the domain. Further support comes from literature on carers of people with long-term conditions suggesting that managing relationships is an important, yet challenging, aspect of caring in which carers are rarely supported.COPD carers’ views regarding CSNAT Intervention delivery are also presented. While considerable variation was found regarding carer preferences for where the intervention should be delivered and by whom, they were consistent in describing qualities relating to delivery that would facilitate sessions. This may reflect heterogeneity in services and practitioners carers interact with; they were more likely to suggest practitioners they had positive impressions of regardless of role. Some carers also suggested that CSNAT Intervention facilitators would not necessarily need to be professionals. Together, these findings suggest three implications: (1) that facilitators’ relational skills, demeanour, knowledge and personal qualities are more important than being a specific practitioner type, (2) this element could be incorporated into CSNAT training, and (3) embedding the CSNAT Intervention flexibly into services with varying structures may be possible. However one carer suggested that, due to the busy nature of acute hospitals, staff dedicated to addressing carer support might help: a concept previously mooted. Focusing on carers can help both patients and carers acknowledge the carer’s role, legitimising the idea that carers may have their own support needs.28 Carers may wish to discuss topics the patient would not e.g. our participants identified “the future” and end-of-life issues. Carers stated that while patients may be reluctant to engage with these topics, they remain important for carers; they can struggle to explore these in front of patients. These findings support recent work with carers of patients with Motor Neurone Disease (MND); carer privacy when completing the CSNAT was identified as a key consideration for facilitating CSNAT Intervention delivery with this group.Some carers preferred the idea of one-on-one CSNAT Intervention delivery sessions (as opposed to jointly with the patient). Practitioners “making space” for carers when discussing support needs has been identified in previous CSNAT studies.Interestingly, the two unplanned patient participants provided contrasting perspectives on this. We had initial concerns that patients’ presence in one focus group might compromise candid discussion between carers, but open discussion of difficulties within carer-patient relationships and how patient and carer support needs may differ suggested this was unfounded. Both patients were supportive of the CSNAT Intervention, however, even though they were appreciative of their carers, it appeared that prior discussion of carer needs had not taken place between these carers and patients. Inclusion of patients in the focus groups enabled observation of patient-carer dyad interactions and interactions with other carers. When one carer stated her (co-present) husband never went out without her, other carers within the group strongly encouraged him to engage with their local hospice day service to give the carer a break, prompting the patient to reconsider this. This suggests that, sometimes, patient presence could help highlight previously unaddressed patient-carer issues, prompting joint problem-solving to resolve them.This was one of several interesting interactions observed from the focus group format. Also noted was information trading between carers at every group e.g. useful organisations’ and services’ contact numbers. This suggests (1) that despite involvement in Breathe Easy and the extra information this may confer, carers still had unmet support needs and (2) the benefits of peer support. However no new findings were arising by the end of the final focus group, suggesting sufficient carers had been recruited to meet the study aim.This study has limitations. Recruitment difficulties have been previously documented regarding COPD carers, with issues relating to individuals not identifying with the ‘carer’ label and difficulties participating due to caring responsibilities (endorsing our enablement of patients’ attendance when requested).30 Future studies should explore the CSNAT Intervention’s utility with working carers, male carers and those from ethnic minorities. Recruitment was also limited to one region due to budgetary restraints, though participants were from localities across it. Using Breathe Easy support groups as the sole recruitment source, whilst pragmatic, may have biased the sample as group members are likely to be more informed on support. However, finding that many participants did not feel supported may suggest that carers within the general population could be even more likely to benefit from the CSNAT Intervention.Carer participants, whilst diverse in age, were all of white British ethnicity; all but one had retired or given up work. Almost all were also women: this is not uncommon in carer studies, and reflects lower numbers of male carers in the general population.32 As the topic guide required consideration of all 15 CSNAT domains, this was particularly advantageous. Interactions between group members further enriched the data and provided novel insights addressing the study’s aims. An added strength was the integration of PPI: the CAG endorsed the study findings, suggesting trustworthiness in terms of data collection and analytic inferences.Recruitment numbers and localities resulted in four small focus groups, however data collected were rich, with the smaller group sizes enabling sufficient time for all participants to contribute.Previous work by the authors suggests that COPD carers often have unmet support needs threatening both carer wellbeing and their ability to continue providing patient support. This study suggests that the CSNAT Intervention, using CSNAT v3, is acceptable and valid for identifying and addressing COPD carer support needs.


Diverse sequences related to HcRNAV were successfully amplified from marine sediments. Amplicons contained conserved and variable regions; the latter were predicted to be located on the outer surface of the capsid. Our approach provides insights into the diversity of viruses that are difficult to isolate in the environment and will enhance rapidly growing metagenome sequence repositories.Heterocapsa circularisquama RNA virus (HcRNAV) is the only dinoflagellate-infecting RNA virus that has been isolated to date. We herein investigated the diversity of the major capsid protein gene of HcRNAV and related viruses using degenerate PCR and  Sediments were then subjected to total RNA extraction and reverse-transcribed. Amplicons obtained from degenerate PCR were sequenced on an Illumina MiSeq platform (300-bp paired-end reads). Sequencing reads were processed and amplicon sequence variants (ASVs) were generated and identified using Divisive Amplicon Denoising Algorithm 2   were useAlvernaviridae) and Alvernaviridae-like clades. However, the attribution of ASV_065 remains unknown  or HcRNAV-related (Alvernaviridae-like) viruses; however, we were unable to conclude whether the amplified sequences were derived from the narnavirus and/or sobemovirus (The results of the BLASTn search were as follows: 27 ASVs showed homology (>92%) to HcRNAV, 1 to another virus, and 11 to cellular organisms , whereas 89 showed no significant search hits . Nucleot unknown A. The Be unknown A were noemovirus A.Alvernaviridae (HcRNAV-derived reads) accounted for more than 50% of the total reads, whereas at a depth of 0–9 cm, its abundance was minimal (<10%) and Alvernaviridae-like reads were dominant  Degenerate PCR Targeting the Major Capsid Protein Gene of HcRNAV and Related Viruses. https://doi.org/10.1264/jsme2.ME21075Supplementary Material


Advanced genomic techniques have now been incorporated into diagnostic practice in neuro-oncology in the literature. However, these assays are expensive and time-consuming and demand bioinformatics expertise for data interpretation. In contrast, single-gene tests can be run much more cheaply, with a short turnaround time, and are available in general pathology laboratories. The objective of this study was to establish a molecular grading scheme for adult gliomas using combinations of commonly available single-gene tests. We retrospectively evaluated molecular diagnostic data of 1,275 cases of adult diffuse gliomas from three institutions where we were testing for IDH1/2 mutation, TERTp mutation, 1p19q codeletion, EGFR amplification, 10q deletion, BRAF V600E, and H3 mutations liberally in our regular diagnostic workup. We found that a molecular grading scheme of Group 1 , Group 2 , Group 3 , Group 4 , Group 5 , and Group 6  could significantly stratify this large cohort of gliomas for risk. A total of 1,028 (80.6%) cases were thus classifiable with sufficient molecular data. There were 270 cases of molecular Group 1, 59 cases of molecular Group 2, 248 cases of molecular Group 3, 27 cases of molecular Group 4, 117 cases of molecular Group 5, and 307 cases of molecular Group 6. The molecular groups were independent prognosticators by multivariate analyses and in specific instances, superseded conventional histological grades. We were also able to validate the usefulness of the Groups with a cohort retrieved from The Cancer Genome Atlas (TCGA) where similar molecular tests were liberally available. We conclude that a single-gene molecular stratification system, useful for fine prognostication, is feasible and can be adopted by a general pathology laboratory. There has been a vast increase in our knowledge of the molecular pathology of central nervous system (CNS) tumors, and a new WHO classification will soon be published . The WHONew England Journal of Medicine, in addition to using IDH and 1p19q, introduced also TERT mutation to stratify low-grade gliomas (Isocitrate dehydrogenase (IDH) genotype has been shown to be a major classifier and prognosticator for gliomas , 11 and  gliomas . BRAF is gliomas –18. Phil gliomas –24.We hypothesized that combinations of genes IDH1/2, 1p19q, TERTp, EGFR, BRAF, TERTp, 10q, and H3 status can stratify gliomas for risk and may even be superior to conventional histological grading for prognostication. We recognize that this gene list is not exhaustive as regards the vast amount of literature concerning the molecular pathology of gliomas. However, since there is a limit as to how many single-gene tests an in-house facility can possibly carry out with a reasonable turnover time, we hypothesize that such a list would adequately molecularly grade a glioma and that it could be superior to conventional histological grading in some instances.A total of 1,275 adult diffuse gliomas diagnosed as astrocytic or oligodendroglial from patients aged 18 years or above were unselectively and retrospectively retrieved from the pathology archives of three institutions: Prince of Wales Hospital, Chinese University of Hong Kong, Huashan Hospital, Fudan University, Shanghai and First Affiliated Hospital of Zhengzhou University, Zhengzhou. Pediatric gliomas were excluded due to the vastly different molecular pathology between adult and pediatric gliomas , 26. SomDemographics and survival data were obtained from the hospital information systems of the three hospitals. The protocol for treatment for adult gliomas of Grades II–IV in all three hospitals was maximal safe resection in the first instance. Some but not all patients received adjuvant radio-chemotherapy due to individual clinical situations Table 1,in situ hybridization (FISH) for CDKN2A homozygous deletion was also evaluated in some cases as previously described  containing the genomic sequences of the 7p11.2 and a centromere probe  was used for EGFR amplification detection. Vysis LSI PTEN/CEP 10 FISH Probe Kit was applied for detection of 10q loss. Vysis 1p36/1q25 and 19q13/19p13 FISH Probe Kit (Vysis) was used to examine 1p19q codeletion. Vysis LSI CDKN2A SpectrumOrange/CEP 9 SpectrumGreen Probes (Vysis) was employed to study CDKN2A. At least 100 non-overlapping signals were counted and analyzed in each case. EGFR amplifications were considered when >5% recorded cells displayed many tight clusters or a ratio of the target (red) to reference (green) signal >2 , 36. DelA cohort with a similar gene set from TCGA was also retrieved , 14. GraStatistical analysis was conducted on IBM SPSS software v22. A chi-square test was used to identify the correlation between molecular grade and clinical parameters. Survival curves were evaluated by the Kaplan–Meier (KM) method, and the log-rank test was done to compare survival distribution between groups. All statistical tests performed were two-sided, with p-values less than 0.05 as the threshold for significance.https://www.surgery.cuhk.edu.hk/btc/hsbc/.A summary of the clinical characteristics of this study cohort is shown in We stratified the adult gliomas into 6 molecular grades based on IDH mutations, 1p19q codeletion, TERTp mutation, BRAF mutation, EGFR amplification, 10q loss, and H3 mutations. The molecular grading scheme was as follows:Molecular Group 1: IDH mutant, 1p19q codeletedMolecular Group 2: IDH mutant, 1p19q non-codeleted, TERT mutantMolecular Group 3: IDH mutant, 1p19q non-codeleted, TERT wild typeMolecular Group 4: IDH wild type, BRAF mutantMolecular Group 5: IDH wild type, BRAF wild type, with no evidence showing TERTp mutation, EGFR amplification, 10q loss, and H3 mutationMolecular Group 6: IDH wild type, positive for TERTp mutation, EGFR amplification, 10q loss, or H3 mutationsThe numbers of different tests carried out on this cohort are shown in Overall, there were 270 cases of molecular Group 1, 59 cases of molecular grade 2, 248 molecular grade 3, 27 cases of molecular grade 4, 117 cases of molecular Group 5, and 307 cases of molecular grade 6 Table 1.As shown in We also tried to compare the power of prognostication of the molecular groups against histological grades in different scenarios. In three scenarios, the molecular groups were better than histological grades in prognostication. We next retrieved an unselected adult glioma cohort with relevant clinical and molecular data from TCGA . The proThe diagnostic protocols for brain tumors have been vastly changed in the advanced centers due to the advent of next-generation sequencing, for both DNA and RNA and also genome-wide methylation profiling. The former can evaluate gene mutations or gene fusion on a panel of a large number of genes, while the latter can map all brain tumors according to their methylation profiling into 91 methylation classes . Copy nuMost general pathology laboratories in the world are not in a position to carry out next-generation sequencing or genome-wide methylation profiling routinely on all their neurosurgical specimens, as these tests remain expensive and demanding on expertise for interpretation, e.g., t-distributed stochastic neighbor embedding (t-SNE) plots. Most national types of health finance systems are unable to support such extensive genomic tests regularly. However, most pathology departments in the more advanced societies will have the capacity for single-gene studies and molecular tests like Sanger sequencing, for example, for BRAF V600E, IDH1 R132, and IDH2 R172H. Similarly, FISH is also widely available in pathology departments, especially in the context of Her-2 testing for breast cancers. We therefore believe that there is a need to develop a relatively simple molecular grading algorithm for the gliomas based on single-gene testing based on current literature on the molecular pathology of gliomas , 40–42. Mutations of the promoter region of TERT have been suggested to be a key terminal event in the evolution of glioblastomas, but it has an intriguing property of being mutated also in oligodendrogliomas, which are mostly low-grade gliomas . The reaOther than lower-grade gliomas, there have also been a few studies in the literature devoted to molecular-grade special groups of gliomas by single-gene studies , 33, butThe biomarkers used in this study were known in the literature on their own. Group 1, IDH mutant, 1p19q codeleted is the regular oligodendroglioma as currently defined by WHO Classification, and Group 6 constitutes the bulk of the regular IDH wild-type glioblastomas as defined by cIMPACT-NOW update 3 . Group 5For glioblastomas, cIMPACT and the new WHO 2021) Classification recommended the use of EGFR amplification, TERT, and chromosome 7+/10− as criteria for diagnosis of molecular glioblastoma in IDH wild-type adult gliomas, which do not show diagnostic histological features like necrosis and microvascular proliferation  Classifi. For IDHThe molecular grading scheme we propose can easily be made available in general pathology departments. Adoption of this system provides a fine prognostication for gliomas and in, some instances, is superior to the histological grading, as shown in We also recognize the limitations of our study, as we used retrospective analyses of existing data, and it would be impossible for us to re-do a new panel of single-gene markers for 1,275 cases according to the recommendation of the new WHO Classification. Ours is not a proposal to replace the new WHO Classification (The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The studies involving human participants were reviewed and approved by The Joint Chinese University of Hong Kong–New Territories East Cluster Clinical Research Ethics Committee; the Ethics Committees of Huashan Hospital, Shanghai; and The First Affiliated Hospital of Zhengzhou University, Zhengzhou. The patients/participants provided their written informed consent to participate in this study.The authors confirm contribution to the paper, as follows: study conception and design: AC, W-SP, HL, and H-KN. Data acquisition: Z-FS, KL, W-WW, HC, NC, DC, X-ZL, Z-YZ, and YM. Analysis and interpretation of data: AC, Z-FS, KL, NC, and H-KN. Drafting of the manuscript: AC and H-KN. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This study was supported by the Health and Medical Research Fund (HMRF), the Food and Health Bureau of Hong Kong (07180736); the National Natural Science Foundation of China ; and Shanghai Municipal Science and Technology Major Project (2018SHZDZX01).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Electrospun hybrid nanofibers, based on functional agents immobilized in polymeric matrix, possess a unique combination of collective properties. These are beneficial for a wide range of applications, which include theranostics, filtration, catalysis, and tissue engineering, among others. The combination of functional agents in a nanofiber matrix offer accessibility to multifunctional nanocompartments with significantly improved mechanical, electrical, and chemical properties, along with better biocompatibility and biodegradability. This review summarizes recent work performed for the fabrication, characterization, and optimization of different hybrid nanofibers containing varieties of functional agents, such as laser ablated inorganic nanoparticles (NPs), which include, for instance, gold nanoparticles (Au NPs) and titanium nitride nanoparticles (TiNPs), perovskites, drugs, growth factors, and smart, inorganic polymers. Biocompatible and biodegradable polymers such as chitosan, cellulose, and polycaprolactone are very promising macromolecules as a nanofiber matrix for immobilizing such functional agents. The assimilation of such polymeric matrices with functional agents that possess wide varieties of characteristics require a modified approach towards electrospinning techniques such as coelectrospinning and template spinning. Additional focus within this review is devoted to the state of the art for the implementations of these approaches as viable options for the achievement of multifunctional hybrid nanofibers. Finally, recent advances and challenges, in particular, mass fabrication and prospects of hybrid nanofibers for tissue engineering and biomedical applications have been summarized. Electrospinning has emerged as a principal technique for the fabrication of 3D nanofibers. This technique has seen enormous growth in the last decade, leading to a generation of comprehensive research, demonstrating effective implementation of electrospinning  to fabriThe fabrication of nanofibers using electrospinning requires the fulfilling of certain conditions. Significantly, it requires the processability of the polymers, including their stability in commonly used solvents, molecular weight etc. ,24. ThesThe characteristics of nanocompartments obtained simply from polymer blends are still restrictive, and there is a lot to be desired when it comes to their mechanical, electrical, or optical properties. The improvement of these properties through the immobilization of various functional agents could provide a multifunctional characteristic to nanofibers. These functional agents come in many forms and include inorganic/organic nanoparticles (NPs), drug molecules, perovskites, biomolecules such as proteins, dendrimers, carbohydrates, lipids, growth factors, and hormones, among others ,43,44,452 accepts electrons and facilitates their transfer across the membrane . Subseqnofibers . However3TMO4 for skin cancer treatment [Felipe et al. had also electrospun chitosan using PEO as co-spinning agent. The nanofibers were functionalized with carboxymethyl-hexanoyl chitosan/dodecyl sulphate nanoparticles loaded with pyrazoline Hreatment . PolymerSimilarly, inorganic polymers, which are an interesting class of materials containing highly electronegative domains such iron, phosphorous, and boron in their backbone, can be spun into nanofibers by co-electrospinning . The preUsing the co-spinning method, long, cylindrical, homogeneous nanofibers without beads were fabricated from blends on smart, inorganic polymers Fe A, Fe B, and PEO/PS. Nanofibers exhibited an average diameter ranging from 623 nm to 478 nm for Fe A/PS and Fe B/PS blends, respectively. The average diameter of nanofibers, prepared from the solution blended with PEO, had an average diameter of 525 nm and 491 nm for Fe A/PEO and Fe B/PEO blends, respectively . ThermalThese studies demonstrate the effectivity of co-electrospinning approach to obtain nanofibers from highly desirable polymers such as chitosan, which inherently have low electrospinnability. Using the co-electrospinning approach has resulted in the fabrication of environmentally sustainable nanofibers with good biocompatibility for applications such as wound dressing materials with hydrophilicity, water retention, and antimicrobial capabilities. Additionally, it has established nanofibers as a platform for modulated drug-delivery systems, localized cancer treatment, photothermal therapy, catalysis, and sensors, among others. Nanofibers functionalized with inorganic nanoparticles provide a combination of unique properties with a very large surface area to volume ratio. The immobilization of nanoparticles on nanofibers can be achieved by either solubilizing the nanoparticles directly in an electrospinning solution ,141, red2, cellulose nanocrystals, and hydroxyapatite at 1% (w/v) with respect to the polymer [2 and Al2O3. These hybrid nanofibers had higher thermal stability and demonstrated improved cycling performance. Moreover, the presence of inorganics enhanced the mechanical properties of fibers as a scaffold and their wettability, leading to efficient performance. While designing biomaterials for biomedical applications, there are many structural, physical, chemical, and biological challenges to be overcome. The engineering and growth of tissues require a complex environment difficult to emulate ,147,148. polymer . The res polymer . This ap polymer . Jaritph polymer . AdditioRecently, an effective immobilization of inorganic (titanium nitride) nanoparticles in a PCL matrix was demonstrated . Here, lw/v) PCL in dichloromethane/acetone (3:2) (v/v) provided bead-free and smooth nanofibers (400 nm to around 1 micron). The presence of TiN NPs in the nanofibers’ matrix led to a slight decrease in both the degradation initiation temperature and the temperature at which the maximum mass loss takes place. Similar behavior was observed in DSC where pristine PCL nanofibers had a slightly lower temperature corresponding to the melting temperature of nanofibers (Tm). TiN NPs seemed to act as heating spots in the nanofibers. However, no drastic changes were observed with a changing concentration of TiN NPs themselves in a nanofiber matrix [After the optimization of electrospinning parameters, functionalized nanofibers were obtained, as shown in , containr matrix . Finallyr matrix . In the The presented studies have shown the immobilization of inorganic nanoparticles in nanofibers to fabricate hybrid materials provided a bouquet of unique properties. These characteristics could potentially be used in biomedical applications such as wound dressing, stent coating, cosmetics, nerve guide conduits, drug delivery, etc., with improved antibacterial and mechanical properties. Further, such hybrid structures could be useful for providing a plasmonic effect, photothermal therapy, and improved stability and electrochemical behavior of separators used in batteries. Tissue-engineering approaches appear highly promising for the regeneration of injured tissues . In dentRecently, gene therapy using nanofibers as a drug-delivery system was reported by Qi et al. Hybrid PLGA/gelatin nanofibers loaded with microRNA-181a/b, induced osteogenic differentiation of adipose-tissue derived MSCs in vitro . The funA recent experimental study showed that a hydrolytically modified poly(L-lactide-co-caprolactone) (PLCL) electrospun scaffold revealed suitable parameters for human dental pulp stem cells (hDPSCs) growth and differentiation towards osteoblasts . It was Advances in tissue engineering need to focus on combinations of dental stem cells with nanofibrous scaffolds, which will induce the appropriate microenvironment and enhance the regenerative potential of dental stem cells.Manufacturing of medical devices is a very promising application of composite nanofibers. Their main functions are drug delivery, used to manufacture a controlled drug-release system, bone regeneration, wound healing, and for antibacterial and antitumor applications. Ideal drug-delivery systems (DDS) should release drugs through a specified time frame ,184,185,3C2) activity. Composite nanofibers were produced from a mixture of poly(L-lactide), other polyhydroxyalkanoate, and MXene nanoparticles. The nanocomposite enhanced cells’ differentiation to osteoblasts. Zhong et al.  preparedHaidar et al.  used chi4 nanoparticles for efficient glucose sensing applications. They obtained carbon nanofibers (CNF) using a two-step process, initially electrospinning polyacrylonitrile (PAN) and then carbonizing scaffold at a high temperature under nitrogen. The immobilization of nanoparticles was performed by in situ growth of NiMoO4 on a CNF surface. The resulting nanofibers showed good sensing response in a wide range of glucose concentrations (0.0003–4.5 mM) at a very low limit of detection (LOD) (50 nM) and high sensitivity (301.77 µA mM−1 cm−2). Nanofibers were also analyzed to characterize their real-life response where their recovery rate was between 97.5–103.2%, similar to commercial glucometers [Nowadays, there is a huge focus on reducing the footprint of the biosensors used for health monitoring purposes. The idea is to develop flexible sensors, which conform to movements of the user with minimal invasiveness. Here, electrospinning has emerged as an ideal platform for the development of such sensors, as they provide a large surface area to volume ratio for sensing activity and the possibility of the inclusion of molecules promoting selectivity, response time, visual cues (colorimeter), and sensitivity along with flexibility/stretchability . For inscometers . Anothercometers .Ozoemena et al. presented a sensor for the detection of dopamine using nanofibers made from onion, such as carbon (OLC) blended with PAN, to enhance electrospinnability. The resulting nanofibers were carbonized to obtain pristine OLC nanofibers. Here, OLC nanofibers displayed the best surface area and pore size during morphology analysis. OLC nanofibers were effective in dopamine sensing with 1.42 µM LOD and 0.31 µA µM sensitivity . These e−1. However, there are several innovative principles for improving yield: (i.) multineedle electrospinning utilizes a high number of needle-based emitters arranged into arrays; (ii.) needleless electrospinning utilizes special emitters enabling the self-arrangement of multiple fiber jets on their surface (Nanofiber systems are revolutionizing and advancing in every possible domain. Nevertheless, there are several reservations when it comes to real life applications of nanofibers. Several challenges are holding back the potential of nanofibers, including yield, cost effectiveness, reproducibility, and inconsistent quality, especially at the commercial scale. Fortunately, many researchers are working in conjunction with industries to tackle some of these challenges. For instance, by applying new approaches for large-scale fabrication and providing novel nanofibers systems, the upscalability of nanofiber production has improved. Emerging production techniques of nanofibers offer a promising potential for obtaining consistently significant yields. The standard needle-based spinning techniques enable the production of only ~0.2 g h surface  [206,207Advances in the fabrication of hybrid nanofibers open new avenues for the next generation of multifunctional nanocompartments via utilization of novel combinations of functional nanomoieties. In this review, the fabrication, characterization, and optimization of nanofibers containing unique modalities, such as naturally occurring biocompatible and biodegradable polymers, are shown to present their own challenges. Combining vanities of polymers along with functional agents to form multifunctional nanocompartments demand new strategies. Herein, multiple such hybrid nanofibers have been presented, where the combination of a polymeric matrix and functional agents are utilized to generate multifunctional nanocompartments with unique collective properties. Co-electrospinning and template spinning approaches are merging polymers which possess excellent electrospinnability, higher processability, and immobilization with functional agents, such as laser ablated NPs, providing exceptional surface properties. The review presents these strategies which allow for the fabrication of stable, functional hybrid nanofibers. The generated nanofibers possess unique collective properties and facilitate a platform for a wide range of applications, such as biosensors, drug delivery, theranostics, tissue engineering. For instance, the immobilization of TiNPs within PCL nanofibers provide a potential platform for cancer cell theranostics and tissue engineering. These applications demand the implementation of novel combinations of hybrid nanocompartments, leading to further development and optimization of electrospun, hybrid nanofiber techniques. Such hybrid nanofiber systems comprising functional agents such as drugs, growth hormones, nutrients, nanoparticles, perovskites, etc., can be employed for tissue engineering and biomedical applications, among others, even though the fabrication of these hybrid nanofibers at the industrial scale is still at a very basic juncture. With extensive research, the potential of hybrid, multifunctional nanocompartments will be realized sooner than predicted.


Our study not only developed QSRR model for the prediction of the retention time of hydroxamic acid in HPLC but also proved the feasibility of using molecular interaction-based features as molecular descriptors.As a potent zinc chelator, hydroxamic acid has been applied in the design of inhibitors of zinc metalloenzyme, such as histone deacetylases (HDACs). A series of hydroxamic acids with HDAC inhibitory activities were subjected to the QSRR (Quantitative Structure–Retention Relationships) study. Experimental data in combination with calculated molecular descriptors were used for the development of the QSRR model. Specially, we employed PCA  to accomplish dimension reduction of descriptors and utilized the principal components of compounds  to execute GA -BP (error backpropagation) algorithm. We performed double cross-validation approach for obtaining a more convincing model. Moreover, we introduced molecular interaction-based features (molecular docking scores) as a new type of molecular descriptor to represent the interactions between analytes and the mobile phase. Our results indicated that the incorporation of molecular interaction-based features significantly improved the accuracy of the QSRR model, (R Moreove (HDAC))  (Ho et a (HDAC)) . HoweverChromatography is a method by which we can obtain a lot of precise, comparable, and repeatable retention data for structure-diverse compounds. At the same experimental conditions, we can get the almost same results on analytes’ retention data. In a chromatographic system, the molecular structure of a compound determines its properties, and further, affect relative affinity for the mobile and stationary phases and, therefore, its retention characteristics . The quaEach analyte has its retention behavior in a chromatographic system. To great extent, the analyte’s retention behavior depends on the stationary phase of the chromatographic column and its structure. Some studies have reported the models to characterize the conformation of ODS (Octadecylsilyl) material  and the Multiple variables can provide abundant information for the research technically. But in many cases, there may be correlations between variables, which increases the complexity of problem analysis. PCA  is one of the most widely used data dimension reduction algorithms. It can eliminate noise and some unimportant information and change the variables into a few independent integrated variables which can stand for the most kinds of information existing in each original variable . A certaIf not specified, all chemicals were of analytical grade. The methanol was purchased from Fisher Chemical (HPLC Grade), while the formic acid was obtained from Kermel. The ultra-pure water was obtained from Heal Force SPW ultra-pure water system. All the twenty-seven analytes, including the marketed drugs or the compounds reported by our group’s former work , were thAll the compounds were tested on Agilent 1,100 system , equipped with a quaternary pump, manual injector (20 μL sample loop), and VWD detector. The column used in this study was Phenomenex Luna 5u C18 (150*4.6 mm 5micron). The mobile phase consisted of 45% aqueous phase (contains 0.1% formic acid) and 55% methanol (contains 0.1% formic acid). The flow rate was 1 ml/min and the UV detection was performed at 254 nm. The retention time of each analyte was shown in The steps of descriptor generation were as follows. First, we sketched molecular structures with ChemDraw 14.0. Then the molecules were minimized using Tripos Field  in SybylThe Molecular Descriptors function of Schrödinger software was applied to descriptors calculation. The molecular descriptors, Schrödinger software can calculate, contain three main categories: Topological Descriptors, QikProp Properties, and Semiempirical Properties. The Handbook of Molecular Descriptors  details The ODS model was manually established using Sybyl-X 2.0. All the analytes were docked against the ODS model using Surflex-Dock module . The repBecause the descriptors were too much to get a good QSRR model rapidly, we performed the PCA on two matrixes, one consists of the descriptors without molecular interaction-based features and the other is made up of the descriptors with molecular interaction-based features, in Matlab  to achieThe matrixes we obtained through PCA were independent variables and we added the retention time of each compound as the dependent variable to form two new matrixes. The retention time of each compound was added into the matrixes consisted of principal components to form two new matrixes. Each matrix was separated into two groups randomly, of which one was the training group consisted of twenty samples and the other one was the testing group consisted of seven samples. Each line was a sample and every column except the last one was the independent variable and the last column was the dependent variable.α = adjustment constant between 1 and 10, and N = the number of training samples.GA-BP was performed on these two matrixes respectively. The matrix was divided into training group (20 compounds) which was used in the inner loop for the model training and selection, and test group (7 compounds) utilized to validate the precision of model externally. In the inner loop, we utilized 5-fold cross validation which means that we separate the training group into five portions and take four portions for training and one for validation for five times. Then we compared the mean absolute error (MAE) value of these five model in the internal validation and selected the one whose MAE value is lowest to process external prediction . Levenbe2, the root-mean-square error prediction (RMSEP) and MAE were measured for an independent set of analytes that were completely separate from the training set used in creating a QSRR model. R2 and RMSEP were defined as the function 2)   and 3) (ction 2)  respectii  values of a compound for all components in a model plus 1.28 times corresponding standard deviation  is lower than 3, then there is 90% probability that the Si values of that compound are lower than 3. Thus, when Snew value of a compound is lower than 3, then the compound can be considered to be not an X-outlier (if in the training group) or within the AD (if in the test group).  of the developed model. The reliability of any QSRR model relies on the confident predictions of these new compounds based on the AD of the model, and therein lies the importance of the AD study. Therefore, we harnessed an approach which has been reported to test the compounds we used whether were suitable. If the means of the S group). .After the dimension reduction by the PCA, some components were obtained. The principal components were ranked according to the contribution rate and we selected the first few components whose cumulative contribution rate can reach 95% to form new matrixes. The matrix generated from the matrix without molecular interaction-based features was named matrix 1, the other one called matrix 2. The contribution rates of these principal components of two matrixes are shown in According to the results of PCA performed on different matrixes, when the molecular interaction-based features are added as new descriptors, one more component was gained and the contribution rates of principal components are different. Since the PCA can reduce the correlation of variables and change the closely related variables into as few new variables as possible, these new variables are not related in pairs. It suggests the molecular interaction-based features, to a certain extent, are independent of other descriptors.With the selection range of the number of hidden neurons existing, we used each number to test on each matrix 20 frequencies respectively to find the optimum number of hidden neurons. The Regression R values measure the correlation between outputs and targets, meaning predicted retention time and observed retention time here. An R-value of 1 means a close relationship and 0 means a random relationship. We took whether the Regression R-value reaches 0.95 as a simple judgment criterion of the training model and counted the number of frequencies that R-value reaches 0.95 when the different number of hidden neurons was chosen to run. The results are shown in According to the results above, the frequencies that R-value reaches 0.95 of the All Set have been improved apparently when we added the molecular interaction-based features as molecular descriptors, which suggested they are beneficial for the accuracy of QSRR models. Meanwhile, the frequencies that R values of four sets reach 0.95 at the same time are improved as well. Through the running with different numbers of hidden neurons on different matrixes, we decided to choose 11 hidden neurons to perform the GA-BP algorithm. Because the R values of Training Set, Validation Set, Test Set, and All Set , which were assigned randomly by the Levenberg-Marquart optimization algorithm, can reach 0.95 at the same time when we took eleven hidden neurons to perform the algorithm on the training group of matrix 1 and 2 .We took 11 hidden neurons and a double cross-validation approach to run GA-BP on the training groups of matrixes 1 and 2, and due to the random generation of weights initialization threshold, we take the same validation set to modelling 10 times respectively, then calculated the average of each training group . Compari2 values of both test groups are greater than 0.6, suggesting the models are not rejected irrespective of the absolute error , absolute (ABS) of average error (AE) with mean absolute error (MAE), mean positive error (MPE) with mean negative error (MNE). The data was shown in 2 (ith vs. (i-1)th residuals) > 0.5 for residuals sorted on Ypred; 5) R2 > 0.5 [57], even if our models are not satisfy, in the future work, we need more analytes to improve, due to not satisfying the standard of number (at least 10).Recording to the reported criterions: 1) NPE/NNE >5 or NNE/NPE >5; 2) ABS(MPE/MNE) > 2 or ABS(MNE/MPE) > 2; 3) MAE - ABS(AE) < 0.5×MAE; 4) RTo investigate the relationship between molecular interaction-based features and retention time of compounds, we used molecular docking scores alone to run the PLS algorism in Matlab. Fifteen analytes were used as a training group to generate the coefficients of each type of docking score and the constant term. The simulation training and predicting of the test group were following . We founvs 2.38 min), to investigate the relevance. The docking results are shown in We took the molecular docking results of two analytes . As a new type of molecular descriptors, molecular interaction-based features (docking scores) could contribute to the dimension reduction, selection of hidden neurons, model selection and prediction of retention time. Our QSRR model could be used to predict the retention time of hydroxamic acids.


DIPAB nanoparticles were grown in situ during the nanofiber formation. X-Ray diffraction (XRD) patterns and Fourier transform infrared spectroscopy (FTIR) proved that electrospinning of DIPAB doped PVDF solutions led to the formation of a highly electro-active β-phase in the nanofibers. Electrospinning in the presence of DIPAB inside PVDF led to very well aligned nanofibers with preferred (001) orientation that further enhanced the effective dipole moments in the nanofiber structures. The dielectric properties of the composite nanofibers were significantly enhanced due to the improved orientation, ionic and interfacial polarisation upon the applied electrospinning process, ionic nature of DIPAB and the interface between the PVDF nanofibers and equally dispersed DIPAB nanoparticles inside them, respectively. The relative dielectric constant of the PVDF nanofibers was improved from 8.5 to 102.5 when nanofibers were doped with 5% of DIPAB. Incorporating DIPAB in PVDF nanofibers has been shown to be an effective way to improve the structural and dielectric properties of PVDF.Diisopropylammonium bromide (DIPAB) doped poly(vinylidene difluoride) (PVDF) nanofibers  with improved and tunable dielectric properties were synthesised  via electrospinning.Diisopropylammonium bromide (DIPAB) doped poly(vinylidene difluoride) (PVDF) nanofibers  with improved and tunable dielectric properties were synthesised  In recent years, many researchers have started to study organic and molecular ferroelectric materials that became interesting because of their ability to be processed in solutions,7 as well as their stability and ease of use.8Ferroelectric materials are used in many electronic applications and have been studied for over a century for their multifunctional electro-active properties.9,10 It is a semicrystalline polymer that is flexible, lightweight, inexpensive and has a high electrical breakdown strength.11 PVDF belongs to the ‘order–disorder’ ferroelectric class in which the phase transition can take place from randomly oriented dipoles (paraelectric phase) to ordered dipoles (ferroelectric phase). PVDF has a crystallinity of around 50% and can be found in five different polymorphs. The monoclinic α-phase of PVDF is the most commonly occurring polymorph with TGTĞ {Trans–Gauche(+)–Trans–Gauche(−)} chain conformation. The β-phase shows piezoelectricity with an all-trans (TTTT) conformation forming an orthorhombic structure with all the fluorine atoms located on the same side in the polymer crystal structure.9 The other polymorphs include γ , δ and ε, which are polar and anti-polar equivalents to α-phase and γ-phase, respectively.Poly(vinylidene difluoride) (PVDF) has been a material of interest because it shows distinctive piezoelectric, pyroelectric and ferroelectric properties.Tc, higher than its melting temperature, Tm.12 These properties can be used in a wide range of applications including sensors,13 actuators,14 capacitors15 and non-volatile ferroelectric memories.16 The most common α-phase can also be converted easily into β-phase by electrical poling and mechanical drawing.17–19 Despite PVDF's potential as an alternative to the inorganic ferroelectric crystals, it suffers from low dielectric constant (∼8.5 for bulk PVDF) resulting in a low electrical energy density that can be stored within the material.20 This can be overcome by incorporating materials with high dielectric permittivity such as BaTiO3, BaxSrx1−TiO3, PbO3.21,22 Although these materials have advantages, they are still heavy metals with a high processing temperature.23PVDF has a long range of ordered dipole interactions, a strong ferroelectric phase and a Curie temperature, i.e. 1-F and 1-P.24 The 1-F (ferroelectric phase) belongs to the monoclinic crystal system, while the 1-P (paraelectric phase) belongs to the rhombohedral crystal system at room temperature. The 1-P phase can be easily converted into 1-F phase by heating DIPAB to 428 K. A spontaneous polarisation of 23 μC cm−2 (comparable to that of BaTiO3), a high Tc of 426 K, along with a room temperature dielectric constant of 85 and small loss tangent of 0.44% at the frequency of 400 Hz was reported for pure DIPAB crystals.23,25–27 Hence, DIPAB exhibits a good ferroelectric and piezoelectric response along with well-defined ferroelectric domains and can act as a molecular alternative to perovskite ferroelectrics.Diisopropylammonium bromide (DIPAB) is a molecular, organic and ferroelectric crystal having a room temperature polar point group and a high melting temperature. It exists in two different polymorphs, 9,28 These publications have demonstrated the nanocomposites' potential portraying an enhanced dielectric strength and a good chemical stability in harsh environments. Electrospinning of the PVDF at low temperatures along with fast solvent evaporation result to β-phase formation and it also influences the crystallinity of the nanofibers. Baji et al. showed that electrospinning is said to enhance ferroelectric and piezoelectric phase of PVDF because of uniaxial stretching taking place at high electric potentials.29 The diameter, surface tension and mechanical strength of the fibers are impacted by the electrospinning parameters and type of solvent used. So far, DIPAB–PVDF composites as nanofibers have not been reported.Very few PVDF–DIPAB nanocomposites have been reported in the literature and they were all in the form thin films.in situ nanocrystals with high surface area. The high β-phase PVDF nanofibers with varying loading content of DIPAB have been synthesised and studies with respect to the dielectric properties of the formed structures have been investigated. We found out that there was an improvement in the relative dielectric constant of the DIPAB doped PVDF nanofibers by more than 12 times in comparison to the regular bulk PVDF and this value is higher than the reported value of DIPAB–PVDF films reported earlier in the literature.9,28We studied the properties of uniaxially aligned DIPAB doped PVDF nanofibers formed using rotating drum electrospinning. DIPAB was used along with the PVDF due to its high dielectric constant value and affinity to form N,N-dimethylformamide (DMF), diisopropylamine and hydrobromic acid (HBr) were purchased from Sigma Aldrich.All chemicals that were used in the reaction were of analytical grade. The poly(vinylidene difluoride) (HSV 900) in powder form was purchased from MTI chemicals, USA. Diisopropylammonium bromide (DIPAB) crystals were obtained by the slow evaporation of the equimolar solution of diisopropylamine and hydrobromic acid in methanol. The obtained DIPAB crystals were collected and finely ground using mortar and pestle, and stored under inert gas atmosphere.A 9.8 wt% homogenous solution of PVDF powder in DMF was obtained by heating the mixture at 105 °C for 1.5 hours. Different amounts of DIPAB were then added to the solution. The mixtures were left to stir at room temperature overnight to ensure complete dissolution of DIPAB. The overnight stirring also leads to the removal of air bubbles in the solution.9 The sample with highest DIPAB loading (24.2 wt%) was considered based on the improved orientation in doping samples since the above this concentration, the polymer solution became too viscous to electrospin well and it was difficult to maintain the fibers orientation. Each sample was loaded in a plastic syringe fitted with a metallic needle tip (18G). The syringe containing the solution was then loaded on the top of an automatic syringe pump (Harvard Apparatus) and was connected to the high voltage power source (Gamma High Voltage Research) as shown in DIPAB doped PVDF solution (10 ml) with a doping ratio 0%, 5%, 10% and 24% by wt were prepared. The following doping ratios were considered based on the previous studies performed on DIPAB doped PVDF thin film samples where the highest dielectric constant was observed in samples below 10 wt% DIPAB doping.−1 to study the presence of β-phase of PVDF. Scanning electron microscopy (SEM) was performed on a Jeol SEM-6500 microscope to study the morphologies of obtained nanofibers. The samples were coated with Platinum to prevent charging. The XRD, FTIR and SEM were performed on the sheets of aligned PVDF nanofibers with different DIPAB ratios. The dielectric measurements were performed on the same batch of the samples. High resolution transmission electron microscopy (HR-TEM) was further performed using a Jeol TEM-2100F instrument to confirm the uniform doping of DIPAB and observe the formation of DIPAB nanocrystals inside the fibers. The composite nanofibers were spun directly on carbon coated lacey copper grids. The uniform doping of DIPAB was also proven by performing an Energy dispersive X-ray spectroscopic (EDXS) analysis on the sample using the same equipment for SEM imaging. The dielectric properties of the nanofibers were measured using parallel RLC circuit with Agilent 4294A impedance analyser and Agilent 16451B dielectric test fixture at 500 mV for all the measurements. The impedances of nanofibers were measured in the range of 40 Hz to 1 MHz. Smoothening was performed on the collected dielectric data to reduce the noise. The observed noise might have been caused by the improper contact of the electrodes with some nanofibers during the measurements.X-Ray diffraction (XRD) was performed on a Panalytical X-ray diffractometer using Cu-Kα at an operating voltage of 45 kV and current of 40 mA at room temperature to study the crystal structure of the nanocomposite fibers. Fourier transform infrared spectra (FTIR) was recorded over a range of 650 to 1000 cmθ peaks in the XRD at 12.5°, 17.0°, 22.4°, 25.2° and 27.6°. These peaks correspond to the monoclinic system of DIPAB crystals.−1 (CF2 stretching), 598 cm−1 (CF2 waging) and 840 cm−1, (CF2 asymmetric stretching and CH2 rocking) represent the electroactive β-phase. The electrospinning of nanofibers result to higher β-phase content when it is performed at room temperature (below 80 °C) along with uniaxial stretching at high electric fields. The intense vibration occurring at 840 cm−1 confirms the presence of a very high concentration of the β-phase. The β-phase was calculated for all the nanofibers sample using the equation:where Aα and Aβ are the corresponding wavenumber values at 762 cm−1 and 840 cm−1, and the Kα and Kβ are the absorption coefficients for the α and β-phases with a value of 6.1 × 104 cm2 mol−1 and 7.7 × 104 cm2 mol−1, respectively.30 The high β-phase of ∼93.8 wt% was observed in all the DIPAB doped PVDF samples.The crystalline structure and β-phase of PVDF nanofibers with different concentrations of DIPAB was investigated by Fourier transform infrared (FTIR) spectroscopy. The incorporation and dispersion of DIPAB crystals inside the PVDF nanofibers was confirmed using Scanning Electron Microscopy (SEM). in situ was in the range of ∼80–250 nm.High resolution Transmission Electron Microscopy (TEM) was further performed to confirm the uniform doping of DIPAB in the PVDF nanofibers. The frequency dependent measurements were performed parallel as well as perpendicular to the nanofibers' orientation. This was done to prove the enhanced performance of nanofibers in the preferred orientation. The measurements parallel to the nanofibers required the formation of a cylinder of standing nanofibers with a height of less than 1 cm—C), loss tangent (tan δ), admittance magnitude (Y) and phase (θ) of the samples were measured to calculate the relative dielectric constant (εr) and the AC conductivity (σAC) using the following equations:d and A represent the thickness and area of the samples, respectively, and ε0 is the constant vacuum permittivity with a value of 8.854 × 10−12 F m−1.The capacitance . The interfacial polarisation works smoothly at lower frequencies as the charges have enough time to move. At higher frequencies, the mismatch between frequency and charge carrier movements results in a decrease in dielectric constant.38 The dielectric constant was also affected positively when DIPAB was added to PVDF because of the highly ionic nature of the DIPAB crystals that contributed to the dielectric constant by ionic polarisation.The frequency dependent properties of DIPAB doped PVDF nanofibers with varying DIPAB content ratio at room temperature in the range of 40 Hz to 1 MHz is shown in The two-way relative dielectric constant measurements  were undertaken to prove the alignment of dipoles along the direction of the nanofibers. During the electrospinning process, the PVDF nanofibers were spun under a very high applied voltage (ranging from 10 kV to 17.5 kV). This resulted in the poling in the PVDF structure where the alignment of the dipoles is along the direction of spinning of nanofibers that cause an increase in the effective dipole moment in these structures. This phenomenon is known as orientation polarisation. It can be seen from e.g. DIPAB 24%) to be lower than DIPAB 5% sample. This can be attributed to the formation of in larger DIPAB particles at higher doping. The increased DIPAB particle size results to decreased surface to volume ratio and hence decreased surface area between the two materials. The decreased surface area resulted to a lower interfacial polarisation as it directly dependent on the interface area. The dielectric constant should increase with the increasing DIPAB concentration since DIPAB has relatively higher εr but the decreasing trend of εr proved that the ionic polarisation was dominated by the interfacial polarisation. This proves domination of interfacial polarisation at low frequencies. The ionic polarisation exceeds the interfacial polarisation at high DIPAB concentration. This can be confirmed by the dielectric measurements performed perpendicular to the alignment of the nanofibers. The dielectric constant was found to be highest in the DIPAB 24% sample. The dielectric measurements performed parallel to the nanofibers proved that interfacial polarisation play a huge role in enhancing the dielectric properties. The ionic and interfacial polarisation can usually be attributed to the distinguishable dielectric loss factor peaks. However, the complex network of DIPAB–PVDF in this work cannot be represented by a simple model and the specific dielectric loss factor peaks associated with different polarisations could not be plotted. The individual fibers and the conjunction between the fibers corresponded to multiple peaks where individual peaks were indistinguishable from each other. Overall, the dielectric constant is most effected by orientation of dipoles followed by the interfacial polarisation as a result of charge trapping followed by the ionic polarisation.The maximum dielectric constant recorded was ∼102 in 5 wt% DIPAB doped PVDF (DIPAB 5%) nanofiber samples when the measurements were performed parallel to the nanofibers. To the best of our knowledge, this was by far the highest value ever recorded for this composite material. The dielectric value when measured perpendicular to the nanofibers was much lower (∼19 at 40 Hz) for the DIPAB 5% sample. This proves that most of the dipoles present in the nanofibers were contributing to the dielectric behavior of nanofibers in the direction along the length of the nanofibers showing the effect of orientation polarisation. The values are lower in all the samples when measured perpendicular to nanofibers and they followed the trend like other nanofibers with increasing frequency. It is expected that dielectric constant should increase with the increase in the doping of DIPAB in the PVDF nanofibers owing to the high spontaneous ionic polarisation of DIPAB. The dielectric constant of DIPAB 5 wt% sample was observed to be the highest at low frequencies as a result of the formation of DIPAB nanoparticles that are equally dispersed in the nanofibers. vs. frequency values are shown in 28 The DIPAB 5% sample had a loss tangent of 0.3 at 40 Hz parallel to the nanofibers. The highest loss tangent of 0.55 was shown by the DIPAB 10% sample at low frequency. The loss tangent for all the samples measured perpendicular to the nanofibers' orientation was very low (<0.1). The loss tangent tends to increase at higher frequency, which is caused by conductance. The AC conductivity increased with increasing frequency as shown in −9 S cm−1 at 40 Hz. The DIPAB 5% sample had an AC conductivity of 0.43 × 10−9 S cm−1 at 40 Hz. The bump in the dielectric constant, loss tangent and AC conductivity data for all the samples was observed in the range of 100 Hz to 500 Hz when the measurements were performed parallel to the nanofibers. These features are constant in all the sample which proves that it is not the noise. It could be the result of complex pathways formation in the composite structure. The limit of detectability of the impedance analyser, for example, the AC conductivity measurements where the value is as low as 2.2 × 10−10 S cm−1 for DIPAB 5% sample makes it difficult to conclude the real reason for the above feature. εr decreased gradually with an increasing amount of DIPAB doping. The values of εr, when measured perpendicular to the nanofibers, were very low for the same fibers because of a very low number of effective dipoles. This also proves that the orientation polarisation and presence of DIPAB play a significant role in the enhancement of the dielectric properties of the material.The loss tangent For the first time DIPAB, which is a highly ferroelectric compound was successfully dispersed in PVDF nanofibers. The electrospinning and addition of DIPAB helped to obtain very high β-phase, improved alignment and crystallinity in the fibers in comparison to the thin-film samples. The dielectric properties were significantly improved and by far the highest relative dielectric constant was observed in this composite material. Incorporating DIPAB in PVDF is an effective way to produce anisotropic and highly dielectric nanofibers that may be ideal candidates for applications such as electric-energy-storage capacitors and actuators.There are no conflicts to declare.RA-009-C9RA06470B-s001