Despite representing 70 million people in Latin America, access to comprehensive rehabilitation and participation in the community remains a challenge for persons with disability (PWDs) in the region. Through enactment of the Disability Law, Colombia has made improvements to recognize and address some of the barriers for PWDs, including access to comprehensive rehabilitation. However access remains limited with significant disconnect between perspectives of various stakeholders and the needs of the population. We examined the unique perceptions on access to comprehensive rehabilitation services and participation of PWDs. We also explored the perspective of caregivers of PWDs, rehabilitation professionals, and other stakeholders on the experiences of PWDs. Our goal was to identify gaps in the implementation of comprehensive rehabilitation programs, and barriers to access resources for comprehensive rehabilitation or services that would impact participation of PWDs.Qualitative study conducted in 2017. Data was collected from a purposive sample of adults with physical disability, aged 18–44 years, who had received rehabilitation services at a local partner organization and with different backgrounds and experiences with disability. Purposive sampling was also conducted with caregivers, rehabilitation professionals, and other stakeholders. Socio-demographic information was collected and semi-structured interviews were conducted by a research team member, recorded, transcribed and analyzed using a thematic analysis method to identify main themes related to our aim. CES University ethical review board approved this study.n = 14) were PWDs. Three main themes were identified among all the participants: the meaning of rehabilitation, challenges to access services, and participation. Challenges to access services had three sub-themes: barriers to personal mobility, perceptions and knowledge on disability, and navigating the system.Thirty-two participants were interviewed: eight were male, 42.1 ± 11.1 years old, and 44%  contains supplementary material, which is available to authorized users. Seventy million people in Latin America live with a disability, and higher rates are observed among the poorest and most vulnerable . The EcoThe International Classification of Functioning, Disability, and Health (ICF) defines disability as an evolving concept that results from the complex interaction between a person with an impairment and personal and contextual factors, with the potential of hindering their participation in society under equal conditions than others . ParticiIn recent decades, Colombia has had significant advances developing its constitutional and legal framework to protect the human rights of PWD. Specifically, the constitutional reform of 1991 along with the signature (2009) and ratification (2011) of the CRPD, resulting in the enacted Disability Law in 2013 and led to the development of the National Disability Public Policy Plan  18]. Ho. Ho18]. In Colombia, there is a lack of reliable and up-to-date data on disability , 20. BasIn alignment with the Colombian Disability Research Agenda  with ourA qualitative study design was undertaken to explore the experiences related to access and use of comprehensive rehabilitation services and their impact on participation of PWDs in Envigado, Colombia. This work was developed in partnership with a community organization ALFIME. ALFIME offers physical therapy, physical activity, psychology, legal advice, independent living program to PWD, and educational talks offered to users with disabilities and their families. The programs are financed with public funds from the city of Envigado and offered to users based on a sliding scale fee. Many users receive the services at no cost. In consequence, ALFIME is one of the main resources available for PWDs in the city regardless of socioeconomic status.This study was conducted in the city of Envigado. a city of 240 thousand inhabitants in the metropolitan area of Medellin, the second largest city in Colombia. Envigado has lower development than Medellin and Bogota  and 40,5PWDs and caregivers were recruited through referrals by providers affiliated with ALFIME and other local organizations. Rehabilitation professionals were invited to participate from the pool of professionals affiliated with ALFIME, representing various disciplines and perspectives. Other stakeholders were identified through local government agencies, civil organizations, academia, and community leaders.In-person semi-structured interviews were conducted in Spanish by one of the research team members [MLTH]. An interview guide, with open-ended non-directive questions, was developed for each group of participants and each explored the areas of independence and autonomy, perceptions on disability, perceptions and available resources in rehabilitation, and citizenship. Members of the research team  developed the guides with insights from experts in ALFIME [see Additional file We used thematic content analysis to analyse the data. Techniques used in the analysis included analytical summaries, open coding, identification of thematic codes, and codebook development. Each interview was coded by two members of the team; disagreements were discussed and resolved through involvement of a third team member. Initial analysis allowed identification of major challenges regarding access and use of rehabilitation services. Through subsequent analyses, themes were refined, focused on exploring domains such as the meaning of rehabilitation, challenges to accessing services, and community participation. We generated matrices to compare and contrast patterns across the perspectives of PWDs, caregivers, rehabilitation professionals, and other stakeholders. We assessed data saturation through an iterative analytical process that included conducting the interviews, reviewing field notes, reading and coding the data, and developing matrices. Even though our sample size was limited by financial restrictions, our saturation assessment alongside with data triangulation across participants made us confident that the key themes were saturated.The trustworthiness aspects of the study are based in the criteria proposed by Nowel et al. . First, A total of 35 people were invited to participate. Three men in the PWDs group were not eligible due to age or because they did not have the means to meet for the interview. We interviewed 8 participants per group of interest, for a total of 32 participants. Table Three main themes emerged from the interviews: the meaning of rehabilitation, challenges to access services, and community participation. Table Rehabilitation was conceptualized differently across participant groups. For PWDs, rehabilitation is focused on restoring function and includes physical therapy, mental health, practicing a sport, and accessing education. Rehabilitation was also viewed as necessary to overcome fear of leaving their house and needed to improve their lives. The value of rehabilitation is demonstrated through interaction with peers that have been successful with physical rehabilitation (they can do better). Similarly, for caregivers, rehabilitation related to regaining function and revolved around physical and psychological therapy, as well as the use of assistive technologies that improve the physical abilities of a PWD. Through rehabilitation comes acceptance of the disability. Most importantly, rehabilitation also meant less work for caregivers, as the PWD would be able to perform tasks without their help. Some caregivers also recognized the detrimental effect of over doing things for the PWD, which interfered in the rehabilitation process.For professionals, rehabilitation was a strategy to promote independence for both the PWD and their caregiver, which should be tailored to individual needs. Professionals viewed independence not only as the ability to perform activities without help, but also the capability of a PWD to make decisions about their own life. Moreover, rehabilitation is viewed as a multilevel and multi sectorial process where professionals, families, and communities work alongside to achieve progress. From their perspective rehabilitation should include not only physical therapy, but assistive technology, education, employment training, sports, arts and recreation, where caregivers and families are also engaged. Other stakeholders had a similar perspective as rehabilitation professionals, seeing rehabilitation in most cases as a multilevel / multi sectorial process that “frees” the person with disability and their family. However, some professionals and other stakeholders still perceive rehabilitation as a strategy to go back to where the person was before an injury. See Additional file Three sub-themes emerged in the challenges to access services: barriers to personal mobility, perceptions and knowledge on disability, and navigating the system. See Additional file All participants agreed on the challenges related to personal mobility. First, PWDs homes are inaccessible: they live in high floors without elevators, have small bathrooms or need to climb stairs to enter the home. All of these barriers significantly limit independence at home and the ability to leave the home. Second, lack of accessible and affordable public transportation posed a constant challenge to access available rehabilitation services and participate in the community. PWDs, rehabilitation professionals, and other stakeholders recognized that improvements have been made in the public transportation system: the Metro has stair platforms and there are buses with platforms for people with mobility impairments. However, they also acknowledged that the stair platforms are not always in working condition, not all buses have platforms, and the frequency in which buses with the platform circulate is unknown. In consequence, people often need to use taxis for transportation which is significantly more costly, and taxi drivers are not always willing to take a wheelchair in their car. Not having means to afford transportation was reported as one of the main causes for not accessing rehabilitation services and limiting participation in the community.Moreover, all participants reported that public places  were not accessible as many had stairs without ramps or alternative elevators, ramps were too steep, or bathrooms were too small. Several participants, across all groups, mentioned that sidewalks are inaccessible, forcing people to travel on the street increasing the risk of road accidents, plus the hilly condition of the city. The combination of the aforementioned challenges resulted in the PWDs needing an aid to be able to go outside of their home, increasing the cost of participation to account for the aids time and transportation.Some PWDs, rehabilitation professionals, and other stakeholders discussed the negative impact of not having access to an appropriate wheelchair or prosthetic device that meets user’s needs, or the training on how to appropriately use them.Perceptions and knowledge about disability emerged as a challenge to access resources by all the participants. PWDs claimed the need to be acknowledged as individuals, without labels or classifications derived from their health condition. According to PWDs and caregivers, some rehabilitation professionals assume that people with the same health condition have the same functioning, ignoring what the PWD has to say about their needs and what they are able to do. Many caregivers referred that when their family member with a disability was discharged from hospitalization (after acquiring a disability) they did not receive enough training on how to face this new life. There is general mistrust in medical doctors, as initial diagnoses were given in a negative way and their outcomes in life have been more than what was initially predicted by doctors. Rehabilitation professionals, perceived similar experiences and described how the lack of training to work with PWDs results in poor communication, limiting the information professionals can share with PWDs and their families. From their perspective, it is common that PWDs do not receive information on what are the possibilities beyond the diagnosis. In addition, professionals perceived that current rehabilitation programs do not pay enough attention to sexuality and other ludic activities.Some participants perceived that other professionals beyond the health care sector, such as architects and engineers, lack knowledge on disability and accessibility, which limits the universal access design of roads, buildings, houses and other physical spaces. Moreover, rehabilitation professionals and other stakeholders stated that publicly sponsored programs such as education, sports, and assistive technology are managed by people without experience working with PWDs. Appropriate training on disability and accessibility was identified as a general need. Both rehabilitation professionals and other stakeholders recognized that professionals are not the only ones responsible for promoting participation as they claimed that many PWDs and their caregivers are not interested in learning, do not to put enough effort, and expect the government and society to provide for them. In their perspective this leads to limitations for participation and disconnect between expectations and what the programs can offer. For example: many participants with disabilities were waiting for personnel in an employment center (e.g. public program) to find them a job and were not actively looking for one. Other stakeholders recognized that PWDs need better training about their rights and how to advocate for them, as well as improved self-awareness to envision their possibilities.All acknowledged that the care pathway for PWDs is unclear for them and their families. Prescribed rehabilitation services may take several months to be approved by the health insurance and the number of office visits that are approved are often perceived as not enough, impacting the continuity of services. However, a contradicting view is explained by one PWD who criticized the lack of clear objectives in a rehabilitation process, suggesting users stay for years in the same rehabilitation program without a clear goal to strive for. Most PWDs and caregivers mentioned having to use legal resources because they were denied health and rehabilitation services, without receiving an explanation for the denial. Other stakeholders agreed that it is common for PWDs to legally appeal for services that are denied. In addition, caregivers shared that alternative services, such as equine therapy, are not covered by the health insurance and need to be paid out-of-pocket. One caregiver shared their concern with a bill that was being discussed at the moment of the interview that intended to cut out support for assistive technology by the healthcare system.Last, rehabilitation professionals manifested that disability is not a priority in the public policy agenda, which affects the continuity of the public programs that are in place, making them dependent on the governor in office. Along the same lines, there is lack of governmental funding for sports (including adaptive equipment), artistic, and recreational programs. Other stakeholders indicated that there is a lack of articulation between programs and resources.PWDs view participation as fundamental throughout the rehabilitation process. According to their experiences, education and employment are important because it engages them with social activities. Other stakeholders talked about the importance of employment to improve the quality of life of PWDs and their family environment, and the key role of education in improving participation. Nonetheless, some PWDs and other stakeholders mentioned that companies prefer not to hire people with disabilities due the legislation that protects PWDs from being fired due to their disability. See Additional file Several PWDs mentioned activities they enjoy in their communities, mostly related to leisure and recreational activities . Caregivers are supportive of these types of activities. PWDs, rehabilitation professionals, and other stakeholders also recognized the importance of sports to improve independence and create a sense of responsibility and commitment. Rehabilitation professionals and other stakeholders also stated that lack of interest and commitment from PWDs and their families limit their own community participation.PWDs believe they can be influential by raising awareness on disability and advocating to make their communities more accessible. Another important topic raised discussed by PWDs, professionals, and other stakeholders, is the need for PWDs to lead initiatives related to disability. One PWD was very critical in stating that most disability-related programs do not hire PWDs.Our findings provide evidence of the challenges that perpetuate the inequalities in access to rehabilitation services in a community of PWDs in Colombia. Despite de recent changes in the country’s legal framework, there is still significant disconnect between the needs of PWDs and the perspectives of other stakeholders. For instance, a national decree enacted in 2003, mandated accessibility for all new public buses starting in 2005 . Our resLack of consensus on the meaning of rehabilitation across PWDs and stakeholders, limits the efficacy of advocacy efforts to achieve equitable access to these services. It is also an indication that many still understand disability from the medical model: the individual with a disability has something wrong that needs to be fixed . PreviouOne potential strategy to foster a paradigm shift is through the explicit incorporation of disability in the curriculum of all levels of education, interventions can be developed to train professionals to work with PWDs, becoming facilitators instead of obstacles to participation , 39. HigEven though many participants defined rehabilitation from the perspective of the medical model of disability, when referring to the challenges to access services, almost all were placed outside of the individual  . SpecifiThe lack of a formal care pathway for PWDs and their families negatively contributes to their ability to timely access comprehensive rehabilitation services. The Colombian Disability Law, and later the National Disability Public Policy in 2013, mandated the Ministry of Health to provide a care pathway model for comprehensive rehabilitation, with clear directions on how different sectors should articulate and collaborate , 18. ThiParticipation in activities that one enjoys, has a positive impact on the quality of life . There iBy engaging stakeholders representing different sectors of the community, we were able to characterize the diversity in their perspectives on comprehensive rehabilitation, and identify actionable gaps, as our sample size allowed us to reach saturation among the main themes of interest. We acknowledge that our purposive sample may select PWDs, caregivers, rehabilitation professionals, and other stakeholders who are more engaged with services and the community. However, through our sampling strategy we ensured that our sample would represent diverse experiences by identifying participants who where heterogeneous in their physical disability, gender, socioeconomic status, education level, and role. Although the majority of our informants were women, this is consistent with gender differences in health and social services professions , 58 and Even though this study depicts the experience of the city of Envigado, the challenges could be transferable to other contexts with an evolving legal disability framework. One of our limitations is that the perspectives presented in this work only those of people with physical disabilities. To better inform inclusive strategies aimed at reducing inequalities in rehabilitation and health for all PWDs, the perspectives of people with other types of impairments must be taken into consideration. For instance, for people who are deaf communication may be one of the most significant barriers to accessing health services with equity , while fParticipation in the community is the ultimate aim of comprehensive rehabilitation. Significant gaps in the translation of the national disability framework such as outdated disability constructs, ongoing architectural and mobility challenges, lack of trained personnel, and difficulty to understand and navigate the system, limit access to comprehensive rehabilitation programs. By identifying actionable gaps, communities can become empowered and lead advocacy efforts to achieve equitable access to comprehensive rehabilitation for PWDs and their families.Additional file 1:Semi-structured interview guides in Spanish per type of participant. (DOCX 142 kb)Additional file 2:Testimonies depicting the meaning of rehabilitation by type of participant. This additional file includes a table with the testimonies by type of participant for their meaning of rehabilitation. (DOCX 124 kb)Additional file 3:Testimonies illustrating the challenges to access comprehensive rehabilitation services. This additional file includes a table with the testimonies by type of participant per sub-theme: barriers to personal mobility, perceptions and knowledge on disability, and navigating the system. (DOCX 124 kb)Additional file 4:Testimonies illustrating the community participation theme that emerged from participants experiences. This additional file includes a table with the testimonies by type of participant. (DOCX 122 kb)Additional file 5:manuscript in Spanish. (DOCX 110 kb)


Supplemental Digital Content is available in the text. NRP1(neuropilin-1) acts as a coreceptor for VEGF  with an essential role in angiogenesis. Recent findings suggest that posttranslational proteolytic cleavage of VEGF receptors may be an important mechanism for regulating angiogenesis, but the role of NRP1 proteolysis and the NRP1 species generated by cleavage in endothelial cells is not known. Here, we characterize NRP1 proteolytic cleavage in endothelial cells, determine the mechanism, and investigate the role of NRP1 cleavage in regulation of endothelial cell function.2+ ionophore, and reduced by pharmacological inhibition of metalloproteinases, by small interfering RNA-mediated knockdown of 2 members of ADAM  family, ADAMs 9 and 10, and by a specific ADAM10 inhibitor. Furthermore, VEGF upregulates expression of these NRP1 species in an ADAM9/10-dependent manner. Transduction of endothelial cells with adenoviral constructs expressing NRP1 C-terminal domain fragments inhibited VEGF-induced phosphorylation of VEGFR2 (VEGF receptor tyrosine kinase)/KDR (kinase domain insert receptor) and decreased VEGF-stimulated endothelial cell motility and angiogenesis in coculture and aortic ring sprouting assays.NRP1 species comprising the carboxy (C)-terminal and transmembrane NRP1 domains but lacking the ligand-binding A and B regions are constitutively expressed in endothelial cells. Generation of these C-terminal domain NRP1 proteins is upregulated by phorbol ester and CaThese findings identify novel NRP1 species in endothelial cells and demonstrate that regulation of NRP1 proteolysis via ADAMs 9 and 10 is a new regulatory pathway able to modulate VEGF angiogenic signaling. Production of an sNRP1 species was reported to be induced by the Ca2+ ionophore, ionomycin, in COS-7 and mouse embryo fibroblasts via ADAM 10-dependent pathway.15 Furthermore, NRP1 exodomain shedding in axons via ADAM10 and ADAM17 has been shown to play an important physiological role in mediating axonal desensitization to Sema3A essential for embryonic neuronal homing.16 However, the relevance of ADAM-dependent NRP1 cleavage in endothelial cells is unclear. Furthermore, neither the nature nor the function of NRP1 species generated by cleavage is known.Alternative splicing of mRNA encoding NRP1 gives rise to several sNRP1 (soluble NRP1) isoforms containing the a1/a2 and b1/b2 domains but lacking the MAM (c), transmembrane, and cytoplasmic domains.Herein, we report the expression in endothelial cells of novel NRP1 protein species containing the cytoplasmic domain, but lacking the regions of the extracellular domain essential for binding of VEGF and semaphorin ligands. We demonstrate that expression of NRP1 cytoplasmic domain species in endothelial cells is mediated through ADAM-dependent cleavage and is stimulated by VEGF. Overexpressing NRP1 species containing the cytoplasmic domain inhibited VEGF stimulation of receptor signaling, cell migration, and angiogenesis. These findings identify a novel mechanism in endothelial cells that may potentially regulate the VEGF/NRP1 signaling network, with implications for understanding the control of angiogenesis.The data that support the findings of this study are available from the corresponding author on reasonable request.Human umbilical vein endothelial cells (HUVECs) were purchased from TCS CellWorks  and cultured in endothelial basal medium  supplemented with gentamicin-ampicillin, epidermal growth factor, bovine brain extract , and 10% fetal bovine serum . HUVECs used in experiments were no more than passage 6. Human coronary artery smooth muscle cells were purchased from PromoCell .7 PMA, GI254023X, marimastat, chloroquine, and lactacystin were purchased from Sigma Aldrich ; Ionomycin was purchased from Merck Millipore .Antibodies to the NRP1 carboxy terminus , glyceraldehyde 3-phosphate dehydrogenase , ADAM9 (sc-23290), ADAM17 , and VEGFR2/KDR  were from Santa Cruz Inc . NRP1 extracellular domain antibody  was from R&D Systems . Phospho-VEGFR2  and Notch intracellular cleavage domain (number 4147) antibodies were from Cell Signaling Technology Inc . Antibody to ADAM10 was from Sigma Aldrich . Predesigned small interfering RNAs (siRNAs) targeted against ADAMs 9, 10, 17 (Table) or scrambled control were purchased from GE Healthcare  and used for transfection as previously described.All reagents used for the generation of adenovirus constructs were from Thermo Fisher Scientific. Adenoviruses (Ad) expressing human wild-type NRP1 (Ad.NRP1 WT), NRP1 lacking the intracellular domain (Ad.NRP1ΔC), NRP1 containing only the cytoplasmic and transmembrane domains (Ad.NRP1Cyt-TM), NRP1 containing the cytoplasmic, transmembrane, and juxtamembrane domains (Ad.NRP1Cyt-JM), and NRP1 containing the cytoplasmic, transmembrane, juxtamembrane, and MAM domains (Ad.NRP1Cyt-MAM) were generated using the Gateway system. Briefly, NRP1 open reading frames were subcloned into the pENTR/D-TOPO vector by PCR amplification with primers designed according to the manufacturer’s instructions (listed below) and using directional TOPO cloning. NRP1 adenoviral expression vectors (pAd/CMV/V5-DEST) were generated by recombination, and adenovirus was produced by transfection into host human embryonic kidney 293A cells. Viral particles were released from the human embryonic kidney 293A cells by 3 freeze-thaw cycles and purified using the Adenopure adenovirus purification kit , the virus titers were then determined using QuickTiterTM Adenovirus Titer quantification kit , and purified adenoviruses were stored at −80°C. HUVECs were infected with one of the Ads described above or Ad.LacZ at a multiplicity of infection of 100. The primers used for the generation of Ad.NRP1WT, Ad.NRP1ΔC, Ad.NRP1Cyt-TM, Ad.NRP1Cyt-JM, and Ad.NRP1Cyt-MAM were as follows:Ad.NRP1ΔC:Forward: 5′-GCTGTCTGTGGGGTCGTGCTGTAGTGTGCCTGTTGGCATAATGG-3′ and,Reverse: 5′-CCATTATGCCAACAGGCACACTACAGCACGACCCCACAGACAGC-3′Ad.NRP1WT: Forward: 5′-CACCATGGAGAGGGGGCTGCC-3′,Ad.NRP1Cyt-TM: Forward: 5′-CACCATGATCCTCATCACCATCATAGCC-3′,Ad.NRP1Cyt-JM: Forward: 5′-CACCATGGACATTAGTATTAATAACCACATTTCACAA-3′,Ad.NRP1Cyt-MAM: Forward: 5′-CACCATGACCATACAATCAGAGTTTCCAACA-3′The reverse primer for the WT, Cyt-TM, Cyt-JM, and Cyt-MAM constructs is: Reverse: 5′-TCATGCCTCCGAATAAGTACTCTGTG-3′.http://rsb.info.nih.gov/ij/).Cytoplasmic and nuclear extracts were obtained using a ProteoJet Cytoplasmic and Nuclear Protein extraction kit  according to the manufacturer’s instructions. For immunoblotting, cells and cell extracts were prepared by addition of a solution containing 50 mmol/L Tris-HCl (pH 7.5), 1% Triton X-100, 150 mmol/L NaCl, 5 mmol/L EDTA, complete protease inhibitor , and phosphatase inhibitors I and II (Sigma) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 4% to 12% Bis-Tris gels , followed by electrotransfer on to Invitrolon polyvinylidene difluoride membranes (Life Technologies). Membranes were blocked with 5% (wt/vol) nonfat dry milk and 0.1% (vol/vol) Tween 20 in phosphate-buffered saline for 1 hour at room temperature before being probed with the primary antibody by overnight incubation at 4°C, followed by incubation for 1 hour at room temperature with a horseradish peroxidase-linked secondary antibody (Santa Cruz) and detection using ECL plus reagents  in accordance with the manufacturer’s protocol. For immunoblotting of shed NRP1 ectodomain, supernatants from treated cells were pooled together and concentrated using Amicon centrifugal filtration devices with a 10 kDa cut off according to the manufacturer’s instructions, and samples were then prepared for immunoblotting as described above. All immunoblots were quantified by scanning films with a calibration strip and analyzed by densitometry using ImageJ . The lysates were immunoprecipitated with an antibody to VEGFR2  using the Dynabeads Protein G Kit (Life Technologies), as per the manufacturer’s instructions. The immunoprecipitated proteins were separated on a 4% to 12% SDS-PAGE gel, transferred to a PVDF membrane and probed with antibodies.HUVECs cultured in 15 dishes  were infected with Ad.NRP1. After 48 hours of infection, they were stimulated with Phorbol ester for 24 hours to stimulate the generation of cytoplasmic fragments. Protein lysates were prepared in IP buffer (PBS containing 1% NP-40) and immunoprecipitated with the C-19 antibody using Dynabeads G, as per the manufacturer’s instructions. The immunoprecipitated complex was resolved on a 4% to 12% SDS-PAGE gel, transferred as described above and the PVDF membrane was stained with Ponceau S (Sigma). All positively stained bands <25 kDa were sent for automated N-terminal sequencing by Edman degradation to Alta Biosciences, University of Birmingham.5 per well in serum-free endothelial basal medium were added to the top chamber and incubated at 37°C for 4 hours. Cells that had not migrated or had only adhered to the upper side of the membrane were removed before the membrane was fixed and stained with a Reastain Quik-Diff kit  using the manufacturer’s protocol and mounted on a glass slide. Cells that had migrated to the lower side of the membrane were counted at ×8 magnification.Transwell cell culture inserts  were inserted into a 24-well plate. Serum-free medium with or without the indicated growth factors or the vehicle was placed in the bottom chamber, and cells in suspension  on the day of harvest. Medium containing either no addition (control) or supplemented with 30 ng/mL VEGF, was replaced and fresh growth factors supplemented every 2 to 3 days. After 1 week, the aortic rings were fixed with 4% formalin for 30 minutes at room temperature. The fixed rings were permeabilized with PBS containing 0.25% Triton X-100 for 15 minutes (2×), blocked with casein solution for 30 minutes and incubated overnight with DyLight 594 Labeled GSL I-isolectin B4  and anti-NRP1 antibody  at 4°C. Negative controls were either incubated with PBS (for the isolectin B4-DyLight 594 antibody conjugate control) or rabbit IgG (for the Nrp1 antibody control) instead of primary antibody. Next morning, rings were washed 3× with PBS, and incubated for 1 hour in the dark with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Life Technologies), mounted on slides and imaged under a fluorescent microscope (Zeiss Axio Imager A1). The isolectin stained images were analyzed using Image J to evaluate the total area of outgrowth and number of branch points by an observer blinded to the treatments given.The murine aortic ring angiogenesis assay was set up as described previously.HUVECs were plated onto glass coverslips precoated with attachment factor (Life Technologies). The following day cells were infected with the adenoviral constructs and after an overnight incubation, the medium was replaced with fresh endothelial basal medium containing 10% fetal bovine serum. Forty-eight hours after infection, cells were rinsed 3× with PBS and fixed in 4% paraformaldehyde for 10 minutes followed by permeabilization with 0.1% Triton X-100 in PBS for 10 minutes. Cells were blocked in 1% BSA for 30 minutes at room temperature and incubated overnight at 4°C with C-19 antibody in PBS containing 1% BSA, or normal goat IgG for the negative control. After washing with PBS, cells were then incubated for 1 hour in the dark with Alexa Fluor 488-conjugated donkey anti-goat IgG (Life Technologies). Cells were then rinsed 3× with PBS then mounted using ProLong Gold antifade reagent with DAPI (Life Technologies). Images were acquired using a Leica TCS SP2 confocal microscope using a Planapo 63×/1.25 oil immersion objective and images were acquired in the horizontal (x-y) and in the vertical (x-z) planes by the LAS-AF software. Offline analysis was performed using ImageJ.7VEGFR2 phosphorylation was determined as described previously.Values have been presented as scatterplots with individual data points. Data were tested for normality using the Shapiro-Wilk test and equality of variance using the Levene test. Where necessary data were log transformed before being analyzed using either 1-way or 2-way ANOVA as appropriate with the Bonferroni correction for multiple pairwise comparisons.online-only Data Supplement and results not shown). To examine the specificity of the NRP1 immunoreactivity of these species, NRP1 knockdown was performed using NRP1-targeted siRNA. As shown in Figure Western blots of lysates of HUVECs with an antibody specific for the cytoplasmic domain of NRP1 detected the major full-length NRP1 protein of 130 kDa, but additionally recognized several smaller species with molecular weights of ≈10, 15, 25, and 30 kDa, primarily in the cytoplasmic compartment with less expression detected in nuclear extracts . Treatment of HUVECs with chloroquine, which blocks endosomal acidification and membrane trafficking of VEGFR2 in endothelial cells,17 also resulted in a marked increase in the level of C-terminal NRP1 species . These findings indicate that NRP1 C-terminal domain species undergo endocytosis and degradation via both proteasomal and lysosomal pathways, but are not themselves the products of these degradative pathways.To test the possibility that expression of small C-terminal NRP1 fragments could result from proteasomal or lysosomal degradative pathways, we examined whether inhibitors of endocytotic trafficking and lysosomal or proteasomal degradation had any effect on expression of NRP1 cytoplasmic domain species.online-only Data Supplement). In contrast, treatment of HUVECs with a broad-specificity inhibitor of metalloproteinases, marimastat, significantly reduced expression of the 10 and 15 kDa NRP1 fragments  and increase intracellular Ca2+.19 Because generation of NRP1 C-terminal domain species might be a consequence of NRP1 extracellular domain shedding, we therefore examined the effects of PMA and the ionophore ionomycin on expression of these protein species. As shown in Figure It was next investigated whether expression of C-terminal NRP1 fragments could result from posttranslational proteolytic cleavage mediated by a membrane-bound or extracellular proteinase, such as a matrix metalloproteinase or γ-secretase. Treatment with the γ-secretase inhibitor, DAPT, had no significant effect on expression of C-terminal NRP1 species, but markedly inhibited constitutive expression of the Notch cytoplasmic domain, which is produced specifically via γ-secretase cleavage . Knockdown of ADAM15 also caused no significant reduction in expression of NRP1 C-terminal species (data not shown). To further validate the conclusion that NRP1 C-terminal domain fragments were generated via an ADAM-dependent pathway, we also examined the effect of the specific ADAM10 inhibitor, GI254023X.22 Treatment of endothelial cells with GI254023X markedly reduced endogenous generation of 10 and 15 kDa NRP1 C-terminal bands . VEGF-induced NRP1 ectodomain shedding was also inhibited by knockdown of ADAM10 and by ADAM10 inhibition using GI254023X, indicating that ADAM10 cleavage of NRP1 resulted in generation of a sNRP1 containing most of the extracellular domain comprising the VEGF and Sema3 ligand-binding regions, and NRP1 species containing the cytoplasmic, and possibly other domains including the transmembrane and MAM domains. We also examined the possibility that generation of NRP1 C-terminal domain fragments could be regulated by other cytokines. Our studies showed that treatment of endothelial cells with TNF (tumor necrosis factor)-α for 24 hours also significantly increased expression of the major 10 kDa NRP1 C-terminal fragment .Our findings supported the conclusion that generation of NRP1 C-terminal domain fragments was the consequence of a constitutive proteolytic process mediated via ADAMs 9 and 10. To investigate whether formation of these fragments was regulated by physiological stimuli, we tested the effects of VEGF on generation of NRP1 C-terminal domain species. As shown in Figure DISI (Asp Iso Ser Iso) juxtamembrane to the MAM domain, and TIQSE (Thr Iso Gln Ser Glu) near the N-terminal end of the MAM domain . NRP1 is strongly implicated in mediating VEGF stimulation of endothelial cell migration.8Therefore, initially we determined effects of NRP1 cytoplasmic domain-containing constructs in assays of VEGF-stimulated endothelial cell chemotactic migration. As shown in Figure 23 The effects of overexpressing NRP1 C-terminal domain species were next examined in an aortic ring assay of sprouting angiogenesis. VEGF induced new angiogenic sprouts in aortic rings transduced with either Ad.NRP1WT or Ad.LacZ, as determined by endothelial-specific staining and measurement of both branch points and total endothelial network area . In contrast, VEGF-induced angiogenesis was markedly reduced in aortic rings expressing either Ad.NRP1CytTM, Ad.NRP1CytJM, or Ad.NRP1CytMAM as compared with control aortic rings expressing Ad.NRP1WT or Ad.LacZ. We further examined the effect of NRP1 C-terminal domain constructs on angiogenesis in the coculture model of angiogenesis. Similar to results obtained in the aortic ring assay, overexpression of either Ad.NRP1CytTM, Ad.NRP1CytJM, or Ad.NRP1CytMAM, reduced endothelial network formation in the coculture angiogenesis assay in comparison to Ad.NRP1WT overexpression (data not shown).To investigate whether NRP1 C-terminal domain species generated by ADAM-dependent cleavage could exert functional effects in HUVECs independently of the NRP ligand-binding domain, we generated adenoviral (Ad) constructs encoding either NRP1WT (Ad.NRP1), or different NRP1 deletion mutants, and compared their effects on VEGF angiogenic signaling in endothelial cells. The constructs tested comprised either the cytoplasmic and transmembrane domains , the cytoplasmic, transmembrane, and juxtamembrane regions , or the cytoplasmic, transmembrane, juxtamembrane, and MAM domains . Furthermore, VEGF treatment caused no change in the degree of VEGFR2 co-immunopreciptation with small C-terminal domain NRP1 fragments . These findings indicate that the NRP1 C-terminal domain alone, independent of the NRP1 ligand-binding domain associates with VEGFR2 only weakly and that any association is not regulated by VEGF.The major angiogenic signaling receptor for VEGF in endothelial cells is VEGFR2/KDR. We next examined whether the inhibitory effects of NRP1 cytoplasmic domain constructs on VEGF-induced angiogenesis could be explained in part by reduced VEGFR2/KDR activation in response to VEGF. As shown in Figure Here, we report the identification of novel NRP1 protein species containing the cytoplasmic domain and the transmembrane domain, but lacking the regions of the extracellular domain essential for binding of VEGF and semaphorin ligands. We show that these species are endogenously expressed in human endothelial, vascular smooth muscle, and tumor cells. Importantly, we demonstrate that expression of these species occurs via a VEGF-regulated and ADAMs-mediated pathway resulting in NRP1 ectodomain cleavage and generating novel NRP1 species containing the C-terminal domain. We further identify proteolytic cleavage of NRP1 by ADAM 9 and 10 as the major mechanism mediating generation of these NRP1 fragments. These conclusions are supported by the following evidence: (1) siRNAs targeted to either ADAMs 9 and 10 significantly reduced endogenous and VEGF-induced expression of NRP1 C-terminal 10 and 15 kDa domain species, whereas ADAM17 knockdown had no effect; (2) generation of NRP1 C-terminal 10 and 15 kDa domain species was inhibited by the broad-specificity metalloproteinase inhibitor, marimastat, and by the specific pharmacological ADAM10 inhibitor, GI254023X; (3) levels of NRP1 C-terminal 10 and 15 kDa domain species were increased by PMA and ionomycin, known activators of ADAM-like proteolytic activity, whereas perturbation of alternative mechanisms, including degradative pathways and γ-secretase, did not inhibit expression of these fragments; and (5) the N-terminal sequences of NRP1 C-terminal fragments generated by cleavage were similar to known consensus sites of ADAMs cleavage.25 and there are few unambiguously identified sites of ADAM proteolysis. However, the protein sequencing of the 2 major C-terminal domain NRP1 fragments detected in endothelial cells, taken together with the fact that ADAM9/10 knockdown and ADAM10 inhibition strongly inhibited generation of these NRP1 fragments in endothelial cells indicates that NRP1 undergoes ADAM9/10-mediated cleavage at or near these sites  program grant RG/06/003  and BHF Project grant PG/12/65/29840 (to I. Zachary and C. Pellet-Many), and by funding from the European Union’s Seventh Program for research, technological development, and demonstration under grant agreement no. 278313, BIOMAGSCAR (V. Mehta and L. Fields).None.AdadenovirusesADAMa disintegrin and metalloproteinaseHUVEChuman umbilical vein endothelial cellsNRP1neuropilin-1siRNAsmall interfering RNAsNRP1soluble NRP1TNFtumor necrosis factorVEGFvascular endothelial growth factorVEGFR2VEGF receptor tyrosine kinase


Halobacteria, representing two functional groups: aerobic polysaccharide utilizers and sulfur-respiring anaerobes. The other four strains represent halophilic methanogens from the class Methanomicrobia and a recently discovered class Methanonatronarchaeia. A wide range of polar lipids were detected across the 13 strains including dialkyl glycerol diethers (archaeols), membrane-spanning glycerol tetraethers and diether-based cardiolipins. The archaeols contained a range of core lipid structures, including combinations of C20 and C25 isoprenoidal alkyl chains, unsaturations, and hydroxy moieties. Several diether lipids were novel, including: (a) a phosphatidylglycerolhexose (PG-Gly) headgroup, (b) a N,N,N-trimethyl aminopentanetetrol (APT)-like lipid with a methoxy group in place of a hydroxy group on the pentanetetrol, (c) a series of polar lipids with a headgroup with elemental composition of either C12H25NO13S or C12H25NO16S2, and (d) novel cardiolipins containing a putative phosphatidylglycerolphosphate glycerophosphate (PGPGP) polar moiety. We found that the lipid distribution of the 13 strains could be generally separated into two groups, the methanogens (group) and the Halobacteria (class) based on the presence of specific core lipids. Within the methanogens, adaption to a high or more moderate salt concentration resulted in different ratios of glycerol dialkyl glycerol tetraethers (GDGTs) to archaeol. The methanogen Methanosalsum natronophilum AME2T had the most complex diether lipid composition of any of the 13 strains, including hydroxy archaeol and macrocyclic archaeol which we surmise is an order-specific membrane adaption. The zwitterionic headgroups APT and APT-Me were detected only in the Methanomicrobiales member Methanocalculus alkaliphilus AMF2T which also contained the highest level of unsaturated lipids. Only alkaliphilic members of the Natrialbales order contained PGPGP cardiolipins and the PG-Gly headgroup. The four analyzed neutrophilic members of the Halobacteria were characterized by the presence of sulfur-containing headgroups and glycolipids. The presence of cardiolipins with one or more i-C25 alkyl chains, generally termed extended archaeol (EXT-AR), in one of the Methanonatronarchaeia strains was unexpected as only one other order of methanogenic archaea has been reported to produce EXT-AR. We examined this further by looking into the genomic potential of various archaea to produce EXT-AR.We analyzed the polar membrane lipids of 13 strains of halophilic euryarchaea from hypersaline lakes. Nine belong to the class  Halobacteria, commonly referred to as haloarchaea, have attracted attention both as an object of fundamental research in extremophiles and as producers of extremophilic enzymes with biotechnological potential , extended archaeol  and tetraethers with two glycerol moieties connected by two C40 isoprenoid chains [glycerol dialkyl glycerol tetraethers (GDGT)]. Membrane-spanning GDGTs can contain up to eight cyclopentane moieties . Membrane-spanning GDGTs have not been detected in members of the class Halobacteria. Archaea also produce bisphosphatidylglycerol diethers, analogous to the diacyl cardiolipins found in Bacteria and Eukarya  volcanii, there was a correlation between the amount of PGP-Me and the concentration of Mg2+ in the media during culturing experiments. Kellermann et al. (+ and K+) . However, Kellermann et al.  and high (5 M Na+) ends of the salt concentration range. While lipid analysis is an essential tool for understanding archaeal membrane composition, culturing conditions are known to affect this composition  thermophilum AMET1T and “Candidatus (Ca.) Methanohalarchaeum (Mh.) thermophilum” HMET1) are extremely halophilic methyl-reducers forming a novel class Methanonatronarchaeia  natronophilum AME2T and Methanocalculus (Mc.) alkaliphilus AMF2T) are haloalkaliphiles from soda lakes , while Ms. natronophilum AME2T is an extremely salt-tolerant alkaliphile . Five haloarchaeal isolates are aerobic polysaccharide utilizers: cellulolytic “Halococcoides (Hc.) cellulosivorans” HArcel1T, Natronobiforma (Nb.) cellulositropha AArcel5 and uncharacterised strain AArcel7 and two natronarchaeal chitinolytic strains “Natrarchaeobius (Na.) chitinivorans” AArcht4T and “Na. haloalkaliphilus” AArcht-S1T are members of the orders Halobacteriales and Natrialbales in the class Halobacteria. The last four strains belong to a group of anaerobic sulfur-respiring haloarchaea. Two halophilic strains comprise two novel genera in the family Halobacteriaceae: Halanaeroarchaeum (Hn.) sulfurireducens HSR2T, which grows exclusively by acetate-dependent sulfur respiration  formicicum HSR6T, which is capable of utilizing either formate or hydrogen as their electron donor, and elemental sulfur, thiosulfate or dimethylsulfoxide as electron acceptors  sulfurireducens”  and a new genus and species “Halalkaliarchaeum (Ha.) desulfuricum”  philic euryarchaea  isolatedMc. alkaliphilus AMF2T was grown at 0.6 M total Na+ and 30°C with formate as electron donor and acetate as carbon source and Ms. natronophilum AME2T at 2 M total Na+ with methanol as a sole substrate, both at pH 9.5, as described previously  and yeast extract as carbon source (100 mg L−1) at 50°C and pH either 9.5 (2 M Na+ as carbonates and 2 M NaCl) or 7.0 (4 M NaCl), respectively  or 50 mM formate (Hd. formicicum HSR6T) as the electron donor and 200 mg L−1 of yeast extract as carbon source at 37°C as described by Sorokin et al. (T)  or 10 mM N-acetylglucosamine (strains AArcht4T and AArcht-SlT) at pH 9.5 and 9.1, respectively, as described previously  and to obtain the remaining core lipids the freeze-dried biomass was hydrolyzed with HCl/MeOH (1.5 N) by refluxing for 3 h. The hydrolysate was adjusted with aqueous KOH to pH 5, extracted three times with dichloromethane, and dried over Na−1. This remained isocratic for 5 min followed by a linear gradient to 1.8% propanol at 45 min. The injection volume was 10 μL. An external standard of AR:GDGT-0  run in sequence before and after the samples was used to quantify AR and GDGT-0. GDGT-1 was assumed to have the same response as GDGT-0 and was in this way semi-quantified. Hereafter, the concentrations of AR and GDGTs are expressed as percent of their total combined concentration.The hydrolyzed biomass extracts were redissolved in hexane:isopropanol  and filtered through a 0.45 μm PTFE filter and analyzed using HPLC-atmospheric pressure chemical ionization/mass spectrometry (HPLC-APCI/MS) on an Agilent 1100/Hewlett Packard 1100 MSD instrument equipped with automatic injector and HP-Chemstation software according to Hopmans et al.  with the2 and trimethylsilylated using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and pyridine . Gas chromatography (GC) was performed using a Hewlett Packard 6890 gas chromatograph equipped with an on-column injector and a flame ionization detector (FID). A fused silica capillary column  with helium as a carrier gas was used. The samples were injected at 70°C. The GC oven temperature was subsequently raised to 130°C at a rate of 20°C min−1, and to 320°C at 4°C min−1. The temperature was then held constant for 15 min. Additionally, all hydrolyzed fractions were analyzed by GC–MS for compound identification. The structural designation of lipids was evaluated by the comparison of their retention times and mass spectral fragmentation patterns with standards and with published spectra  and He as the carrier gas at a constant flow rate of 2 mL min−1. The oven temperature program initiated at 70°C, increased first at a rate of 20°C min−1 to 130°C, and next at a rate of 4°C min−1 to the final temperature of 320°C, which was held for 10 min.Hydrolyzed biomass extracts were methylated with diazomethane in diethyl ether which was removed under a stream of NGC peak area response (FID) of the mono and diethers was recorded. One of the diethers, AR, had previously been quantified . The two separate hydrolysis-derived ether lipid data sets (GC-MS derived and HPLC-MS derived), were both normalized to AR and combined to give an estimate of the total ether distribution  at a concentration of 2 mg mL−1 and aliquots were filtered through 0.45 μm regenerated cellulose syringe filters .Lipids were extracted from freeze-dried biomass using a modified Bligh-Dyer procedure  and B = 2-propanol/water/formic acid/ 14.8 M NH3aq . The lipid extract was analyzed by an MS routine where a positive ion scan  was followed by a data dependent MS2 experiment where the base peak of the mass spectrum was fragmented  25, isolation width 5.0, activation Q 0.175). This was followed by a data-dependent MS3 experiment where the base peak of the MS2 spectrum was fragmented under identical fragmentation conditions. This process was repeated on the second to fourth most abundant ions of the initial mass spectrum. Identification of the IPLs was carried out by comparison of their masses and their diagnostic fragmentations in MS2 with those described in the literature  according to Sturt et al.  with mod+, [M+NH4]+, and [M+Na]+ ion (where present) for each individual IPL species. The peak areas for the individual species were summed for data handling in groups based on headgroups and/or core lipids.Intact polar lipids (IPLs) were quantified in terms of their MS peak area response. As different IPLs show different response behavior, the relative abundance of peak area does not necessarily reflect the actual relative abundance of the different IPLs. However, this method allows for comparison between the strains analyzed in this study, especially as they all contained at least one, but generally three, lipid groups in common. The peak areas were determined from extracted ion chromatograms of the combined [M+H]2) pressure, 35 arbitrary units (AU); auxiliary gas (N2) pressure, 10 AU; spray voltage, 4.0 kV; probe heater temperature, 275°C; S-lens 70 V. Target lipids were analyzed with a mass range of m/z 350–2,000 , followed by data-dependent tandem MS2 , in which the 10 most abundant masses in the mass spectrum were fragmented successively . The Q Exactive was calibrated within a mass accuracy range of 1 ppm using the Thermo Scientific Pierce LTQ Velos ESI Positive Ion Calibration Solution. During analysis dynamic exclusion was used to temporarily exclude masses (for 6 s) in order to allow selection of less abundant ions for MS2.Additional analysis of selected extracts was carried out using an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) method  1100 series HPLC with a thermostatted autoinjector, column oven, and a Foxy Jr. fraction collector . Aliquots of filtered Bligh-Dyer extract (200 μL) were injected onto a LiChrospher DIOL column , all fractions were eluted using the identical gradient and mobile phase composition described above for the HPLC-ITMS analysis, but at a flow rate of 3 mL min−1. The column effluent was collected in 3 mL fractions. Fractions from semi-preparative HPLC were screened for the presence of the target compound with flow injection analysis using HPLC-ITMS as described above, and the fractions containing the target compound were pooled. For further purification the combined fractions were re-injected and column effluent was collected in 15 s fractions.Specific IPLs were isolated from Bligh-Dyer extracts of N,N,N,-trimethyl aminopentanetetrol (APT)-AR and an unknown lipid (referred to below as IIa) were dissolved in 500 μL 99.9% CDCl3 at concentrations of 3.0 and 0.8 mg ml−1, and NMR experiments were performed at 298 K on a Bruker 900-MHz Avance III spectrometer equipped with 5 mm TCI cryoprobe, running under TOPSPIN 3.2. One-dimensional (1D) 1H and 1H-decoupled 1D 13C and Distortionless Enhancement by Polarization Transfer (DEPT)-135 experiments were recorded with spectral widths/offsets of 20 ppm/4.7 ppm, 200 ppm/100 ppm, and 200 ppm/100 ppm and with 16,384 (16 k), 4,096 (4 k), and 4,096 (4 k) complex points, respectively. 12- by 12-ppm 2D correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), and nuclear Overhauser effect spectroscopy (NOESY) experiments were performed with 512 by 256 complex points (by 200 for the NOESY) and an offset frequency of 4.7 ppm. Mixing times were 60 and 120 ms for TOCSY and 250 ms for NOESY experiments. -heteronuclear single-quantum correlation spectroscopy (HSQC) and -heteronuclear multiple-bond correlation spectroscopy (HMBC) experiments were recorded with spectral widths/offsets of 12/4.7 ppm for protons, 200/100 ppm for 13C HSQC (512 x 64 complex points) and 150/75 ppm for the HMBC (1 k × 80 complex points). All spectra were calibrated with respect to internal residually protonated CHCl3 at 7.24 ppm (1H) and 77.0 ppm (13C).https://www.arb-silva.de/). A phylogenetic tree of 16S rRNA gene sequences was inferred with the Neighbor-Joining method  using two iteration steps using the annotated geranylfarnesyl disphosphate synthase of Aeropyrum (A.) pernix + at m/z 1171.4 and 1241.4  from the ion at m/z 994.8116. A further loss of C6H10NO5 from the m/z 832.7605 ion produced the most abundant product ion at m/z 653.6800 representing the AR core lipid). The fragment ion m/z 373.3675 was assigned as a phytanyl moiety attached to a glycerol moiety (C23H49O3). The elemental composition of this compound, taken in combination with its MS2 fragmentation allowed us to propose a putative structure of IIIa as having a headgroup with elemental composition C12H25NO13S and an AR core  and the corresponding C25 isoprenoid alkyl chain attached to a glycerol moiety . Based on accurate mass determination of fragments and losses the IIIb compound was identified as having a C12H25NO13S headgroup with an EXT-AR core.Four unknown IPLs with two closely related headgroups were detected in four strains  distributed across all three archaeal metabolic groups, namely in methanogenic ‘d 1241.4 . Structud 1241.4 . The MS2spectrum  of IIIa  AR core . The secspectrum : loss ofIIIc and IIId produced similar MS2 spectra to IIIa and IIIb  could not be established. Biomass was not available in great enough quantity to carry out the compound isolation that would be required for NMR analysis.and IIIb , but IIIer an AR  or EXT-Aer an AR  core. ThIVa-c) were found in three polysaccharide utilizing alkaliphilic haloarchaeal strains, “Na. chitinivorans” AArcht4T, Nb. cellulositropha AArcel5T and AArcel7. They exhibited [M+H]+ masses  identical to those of a series of unknown cardiolipins from the cell membrane of two species of alkaliphilic haloarchaea from the alkaliphilic genus Natronococcus that were previously reported by Angelini et al. (2 spectra did not support the presence of sugar groups. One of our three novel tetraether cardiolipins (IVa) exhibited a [M+H]+ at m/z 1676.4. Its MS2 spectrum  and at m/z 1023.7 . The most abundant ion at m/z 869.7 is also present in the MS2 spectrum of AR-PGP-AR  polar group (IVb) exhibited a [M+H]+ at m/z at 1746.5, 70 Da larger than IVa. This difference in mass suggests the presence of an EXT-AR core instead of an AR core. Indeed, the product ions in the MS2 spectrum arising from fragmentation of this ion mirrored the losses seen for IVa cardiolipin with addition of losses associated with an EXT-AR chain , m/z 1023.7, and 1093.8  and four ions at m/z 807.8, 869.7, 877.8, and 939.7, which represent the AR and EXT-AR containing halves of the parent ion, fragmented on either side of the central phosphate group  was 140 Da larger than the first, at m/z 1816.6, in agreement with the presence of two EXT-ARs instead of a single AR-group. The product ions in its MS2 spectrum are interpreted as follows: m/z 1536.2: loss of a phytanyl chain of 280 Da, m/z 1093.8: loss of EXT-AR of 723 Da, two ions at m/z 877.8 and 939.7, representing the two halves of the parent ion when fragmented at either side of the central phosphate group philic euryarchaea, all methanogens . These i6H10NO5 sugar with either one  or two  sulfate groups.A wide range of intact polar dialkyl glycerol diethers, membrane-spanning GDGTs and cardiolipins were identified in 13 strains of halophilic archaea in this study cf. . Many of+ and K+) is the most important membrane adaption for haloarchaea , was the presence of membrane-spanning GDGT lipids  contained more GDGT than AR  while the two members of the class Methanomicrobia  contained more AR than GDGTs . This difference may be an adaption to external salt concentration: while the two Methanonatronarchaeia are extremely halophilic methanogens (both cultured at 4 M Na+), the Methanomicrobia are haloalkaliphiles, in that Ms. natronophilum AME2T is an extremely salt-tolerant alkaliphile , while Mc. alkaliphilus AMF2T is a moderately salt-tolerant alkaliphile . The presence of GDGTs in the membrane of methanogens may be associated with the reduction of wasteful ion cycling under chronic energy stress conditions  and Methanosarcinales (Ms. natronophilum AME2T), but it was absent in any members of the Halobacteria. This polar headgroup has been described previously in a wide range of methanogenic archaea  acidocaldarius  barkeri  burtonii -OH-AR may be characteristic of this order rather than being related to e.g., cold adaption.The polar headgroup PS was present in a et al. . While PCa. Mh. thermophilum” HMET1 from the class Methanonatronarchaeia , but not before in any methanogenic members of the Euryarchaeota. Our results indicate that synthesis of BPGs is not restricted to the Halobacteriales. Indeed, Yoshinaga et al.  by upregulating cardiolipin levels, in order to counterbalance the absence of “cardiolipin-surrogates” that form at higher Mg2+ concentrations, between the Mg2+ cations and two anionic PG lipids , many of the analyzed Halobacteria strains showed a high proportion of EXT-AR in agreement with the other cultured genera from the class  the EXT-AR zipper configuration within the bilayer is not compatible with membrane-shielding PG-cation interactions . Therefore, alkaliphilic haloarchaea with high levels of EXT-AR would need to upregulate the amount of PGP-Me, to provide an alternative mode of Mg2+ buffering at the membrane surface. Indeed, in agreement with this idea, PGP-Me was the dominant headgroup . Kellermann et al. , Ms. natronophilum AME2T had the most complex diether lipid composition of any of the thirteen strains: based on GC-MS analysis  and the MAR and OH-AR core lipid adaptions of this strain may be order- or species-specific adaptions to high salt and high pH conditions.While the two analysis  it contaanalysis , S5. TheMethanomicrobiales member Mc. alkaliphilus AMF2T. While  N-methyl APT-Me are all novel compounds, trimethyl APT has been described in a range of methanogenic euryarchaea, including the members of the order Methanomicrobiales, but, in agreement with our findings, not in the order Methanosarcinales . This predominantly neutral membrane surface may have evolved to adapt to the conditions encountered by Mc. alkaliphilus AMF2T of moderate salt  and high pH (9.5–10). It is of note that Szekely et al. , APT and APT-Me were detected only in the single y et al.  found thMethanomicrobia class, Ms. natronophilum AME2T, also contained a high proportion of Uns-ARs . However, a higher salt concentration does not explain the high level of AR unsaturation in the two Methanomicrobia as both Ms. natronophilum AME2T and Mc. alkaliphilus AMF2T were cultured at the lowest salt concentrations of all 13 strains . This suggests that AR unsaturation in response to salt concentrations is a class specific adaption, manifested at lower salt concentrations by these Methanomicrobia than the Methanonatronarchaeia and Halobacteria, which are all more extreme halophiles.It should be noted that the other member of the ARs 16%, , whereasARs 16%,  studied ARs 16%,  observedNatrialbales included three PGPGP cardiolipins, one with two AR cores, one with an AR and an EXT-AR core and the final one with two EXT-AR cores  but in no other orders. The PG-Gly headgroup with a diacyl core has been described in halophilic gammaproteobacteria of the genus Halomonas , which was also seen in methanogenic “Ca. Mh. thermophilum” HMET1. The presence of these sulfur containing lipids may be a distinctive characteristic associated with these Halobacteriales members or may relate to the neutrophilic nature of these extreme halophiles. Indeed, while nine strains out of 13 examined were alkaliphilic, the remaining four neutrophilic strains  and not in any of the alkaliphilic strains  did not contain any EXT-AR, whereas all analyzed Halobacteria did . Bulky amino acids such as phenylalanine (F) block the length of the isoprenoids at C20, while smaller amino acids such as serine (S) allow for elongation to C25 and beyond  involved in the elongation of isoprenoids  FARM motif. Indeed, replacement of bulky F at the fifth position with a smaller amino acid residue such as serine (S) has been described as the most effective way to elongate beyond a C20 chain  that distinguishes the enzymes' ability to form extended isoprenoids beyond the C20 length. Indeed, in the case of A. pernix it is the presence of the relatively small and compact A at the eighth position which potentially determines the C25 chain length of the product  as pointed out in Many of the examined archaeal genomes encode multiple putative polyprenyl synthases, generally separated in function by the nature of the specific FARM amino acids described above . A majorMethanonatronarchaeia presently known in culture, “Ca. Mh. thermophilum” HMET1 and Mn. thermophilum AMET1T and a single-cell genome sequence from Nereus Deep in the Red Sea. All three only contained one putative polyprenyl synthase with two Is, and the relatively bulky F at the 11th, 8th, and 5th positions, a motif associated with short chain polyprenyl synthase. However, these putative polyprenyl synthases cluster far apart from all the other short chain polyprenyl synthases and cluster closer to the long chain polyprenyl synthases philic representatives of Euryarchaeota, novel diether-based lipids were detected including a PG-Gly headgroup combined with a diether core, an APT-like lipid with an additional methoxy group in place of a hydroxy group on the pentanetetrol and finally headgroups with elemental composition of either CHalobacteria (class) based on the presence of specific core lipids. Within the methanogen group adaption to high or more moderate salt concentration resulted in the extremely halophilic class Methanonatronarchaeia containing more GDGTs than AR, while the less halophilic members of the class Methanomicrobia contained more AR than GDGTs. The methanogen Ms. natronophilum AME2T had the most complex diether lipid composition of any of the 13 strains, including OH-AR and MAR which we surmise is an order-specific membrane adaption. The zwitterionic lipids APT and APT-Me headgroups were detected only in the Methanomicrobiales member Mc. alkaliphilus AMF2T, which also contained the highest proportion of unsaturated lipids. Many of the Halobacteria strains contained a high proportion of the EXT-AR core and PGP-Me headgroup in agreement with the other cultured genera from the class. Within the Halobacteria only alkaliphilic members of the Natrialbales order contained PGPGP cardiolipins and the PG-Gly headgroup. The four neutrophilic strains belonging to Haloferacales and Halobacteriales orders were characterized by the presence of sulfur-containing headgroups and glycolipids.We compared the distribution of membrane lipids across the 13 strains to examine what factors determined lipid composition, including the phylogenetic relationships between the strains, the three different metabolic processes they carryout and adaptions associated with the pH. In general we found that the lipid distribution of the 13 strains could be generally separated into two, the methanogens (group) and the All datasets generated for this study are included in the manuscript and/or the supplementary files.NB carried out data analysis and wrote manuscript. DS isolated and cultured archaea. EH oversaw analytical methods and carried out data analysis. MK and WR carried out extractions and data analysis. LV carried out homology search and phylogenetic analyses. HW carried out NMR analysis and JS conceived and supervised the project.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.


This review also sums the crosstalk between these compounds. Besides, those signaling molecules can regulate the expressions of some genes during AR development, including cell division genes, auxin-related genes, and adventitious rooting-related genes. We conclude that these small-molecule compounds enhance adventitious rooting by regulating antioxidant, water balance, and photosynthetic systems as well as affecting transportation and distribution of auxin, and these compounds further conduct positive effects on horticultural plants under environmental stresses. Hence, the effect of these molecules in plant AR formation and development is definitely a hot issue to explore in the horticultural study now and in the future.Adventitious root (AR) is a kind of later root, which derives from stems and leaf petioles of plants. Many different kinds of small signaling molecules can transmit information between cells of multicellular organisms. It has been found that small molecules can be involved in many growth and development processes of plants, including stomatal movement, flowering, fruit ripening and developing, and AR formation. Therefore, this review focuses on discussing the functions and mechanisms of small signaling molecules in the adventitious rooting process. These compounds, such as nitric oxide (NO), hydrogen gas (H The root system of a plant is made up of the primary, lateral, and adventitious roots. The primary root originates during embryogenesis and will later elongate after germination. The AR is initiated and developed post-embryonically from differentiated cells; however, the later root is derived from pericycle cells, which is also defined anatomically as meristems ,2. AR is2S) were regarded as toxic molecule compounds in animals. Gradually, small signaling molecules have proven to be resistant to various abiotic stresses in plants, such as chilling, osmotic stress, drought stress, flooding, salt stress, heavy metal stress , and UV-A irradiation [2, CH4, ethylene (ETH), and H2O2 have been indicated to induce AR formation and development in plants [Many different kinds of molecules transmit information between the cells of multicellular organisms, including small-molecule compounds. These molecules are produced by signaling cells and subsequently bind to receptors in target cells, acting as ligands and chemical signals to coordinate responses and a variety of biological processes both in animals and plants ,9. Over adiation ,7,10. NOadiation ,12. Besiadiation ,13,14,15n plants ,17,18,19AR development is a complicated process and influenced by many abiotic factors such as mineral nutrition , light, temperature, and various biotic factors including ectomycorrhizas and agrobacterium rhizogenes ,20. In a2+, of plant signal transduction in the growth and development of plants, such as seed germination and dormancy, stomatal movement, and photosynthesis [NO plays a crucial role as a second messenger molecule, after Caynthesis . NO alsoynthesis . Tagetes erecta L. ‘Marvel’) [Cucumis sativus L.) [In plants, NO is synthesized through two synthetic pathways: enzymatic and nonenzymatic. The enzymatic pathway includes nitrate reductase (NR) and NO synthase (NOS)-like enzymes. Previous studies of NO showed that NR played crucial roles in the process of physiological activities in NO production . It was Marvel’)  and AR divus L.) , suggestivus L.) ,25. Addiivus L.) . Ample eivus L.) ,27. ThesDendranthema morifolium ‘Beiguozhicun’), and the explants of cucumber and marigold [It has been widely reported that NO may regulate the developmental process of adventitious rooting. Application of NO significantly enhanced AR length and number in the cuttings of ground-cover chrysanthemum  [NO can also regulate AR development through adjusting enzyme activities, such as peroxidase (POD), polyphenol oxidase (PPO), and indoleacetic acid oxidase (IAAO). Furthermore, NO accumulation significantly increased the activity of pro-oxidants including triphosphopyridine nucleotide (NADPH oxidase) and antioxidants such as superoxide dismutase (SOD), catalase (CAT), POD, ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) enzymes in AR development in mountain ginseng (. Meyer) . NO dono. Meyer) . Exogeno. Meyer) ,28. Thesw, SOD, CAT, and APX activities and chlorophyll content in cucumber explants [Oryza sativa L. cv. Komal) seedlings under arsenate (AsV) stress by increasing APX content [NO also regulates AR development under abiotic stresses. Exogenous NO enhanced the root number and root fresh weight in marigold explants under drought stress . And NO explants . Moreove content . Therefo2) is a colorless and tasteless molecule, and hydrogen is one of the most abundant elements in the universe, constituting nearly 75% of the mass of the universe [2 has come to the forefront in therapeutic medical gas research. Recently, the attention on the role of H2 has changed from animals to plants. It has been found that H2 could regulate the growth and development of plants, including seed germination, seedling growth, stomatal closure. and root elongation [2 enhances the tolerance of plants to abiotic stress conditions such as salt stress, osmotic stress, drought, cadmium toxicity, aluminum stress, mercury toxicity, and UV-A irradiation [Hydrogen gas  significantly induced adventitious rooting and enhanced NO content in a time-dependent manner, which reached a maximum at 24 h during the treatment. Also, the application of H2 triggered transition from G1 to S phase in plant cell cycle to enter a new cell cycle in a synchronous manner [Many studies have shown that Hs manner . And HRWs manner . Further2 exerted a positive effect on AR development under stress conditions. Fifty percent HRW significantly induced AR length and number and enhanced the RWC during rooting in cucumber explants under drought stress [2 dramatically increased leaf chlorophyll content, Fv/Fm, ɸPSII, and qP as well as the activities of SOD, POD, CAT, and APX enzymes during drought [2O2), superoxide radical (O2−), thiobarbituric acid reactive substances (TBARS), ascorbic acid (AsA), and reduced glutathione (GSH), as well as relative electrical conductivity (REC), lipoxygenase (LOX) activity, AsA/docosahexaenoic acid (DHA) ratio, and GSH/oxidized glutathione (GSSG) ratio, while increasing DHA and GSSG content. HRW + Cd treatment also significantly increased in the activity and related gene expressions of APX, DHAR, monodehydroascorbate reductase (MDHAR), and GR. Additionally, HRW + Cd treatment increased the contents of osmotic adjustment substances, as well as the activities of POD and PPO, while significantly decreasing IAAO activity [2 induces adventitious rooting under Cd stress by decreasing the oxidative damage, increasing osmotic adjustment substance content, and regulating rooting-related enzyme activity.Also, Ht stress . Also, e drought . Additio drought . Compareactivity . These r2S) is a naturally occurring, colorless, highly soluble, flammable, and toxic gas [2S has been found to be involved in abiotic stresses of plants such as salinity, drought, extreme temperatures, and heavy metals [2S regulated many aspects of plant development, like seed germination and adventitious root induction [2S, 10 μM sodium hydrosulfide (NaHS) significantly induced AR primordia in cucumber explants [Salix matsudana var. tortuosa Vilm) and soybean (Glycine max L.) seedlings [Hydrogen sulfide  in dose- and time-dependent manners [A number of studies showed that CO could trigger AR development. CO treatment induced adventitious rooting in the hypocotyl of mung bean , and ultraviolet radiation [4 on various plant developmental processes. Eighty percent methane-rich water (MRW) dramatically triggered the increase of AR length and number in cucumber [4-induced adventitious rooting in cucumber explants required γ-glutamyl cysteine synthetase (γ-ECS) [4 can be regarded as a crucial inducer during AR process.CH animals . CH4 alsstresses . It was adiation ,46. Howecucumber . CH4-ind (γ-ECS) ,47. ThesArabidopsis  [Arabidopsis mutant (max2 and max4) plants in a dose-dependent manner [ETH is a gaseous plant hormone and involved in many growth and development processes in plants, such as seed germination and dormancy, cell division and expansion, leaf senescence, and fruit ripening ,49. Zimmhaliana) . Howevert manner . The appt manner . 2O2 is viewed mainly as a type of ROS and a signaling messenger. A number of studies reveal that H2O2 is involved in many biological processes of plants, such as stomatal closure [H closure , floweri closure , leaf se closure , cellula closure , lateral closure ,59. 2O2 production, which showed that 3-O-C10-HL is likely to participate in auxin-prompted AR formation by H2O2-dependent cGMP signaling. Li et al. (2007) [2O2 could significantly increase the number of AR, while treatment with 10–50 μM H2O2 significantly increased the fresh weight of AR in cucumber. Moreover, in marigold, the treatment of 200 μM H2O2 significantly induced root length and root number, but its scavenger CAT alleviated its positive effects [2O2 and the synthetic phytohormone indole-3-butyric acid (IBA) may act synergistically to regulate adventitious rooting dependent on the auxin pathway in marigold explants [2O2, suggesting that H2O2 may be the downstream signal molecule in the auxin signaling cascade [2O2 promoted the AR length and number in marigold, and treatment with indole-3-butyric acid (IBA) enhanced the endogenous level of H2O2 in explants [2O2. In ground-cover chrysanthemum, 200 μM H2O2 was the most suitable dosage to promote AR development, at which this dosage increased the activities of PPO, IAAO, and WSC, as well as total nitrogen content, and decreased the total polyphenol content [2O2 could enhance AR development via increasing some enzyme activities, and carbohydrate and nitrogen contents, and inhibiting polyphenol production. According to Bai et al. (2012) , in mung. (2007)  found th effects . It has explants . Exogeno cascade . Also, 2explants , which s content . These r2O2 treatment significantly promoted root length and number in marigold, attenuated the destruction of the mesophyll cell ultrastructure, and increased leaf chlorophyll content, chlorophyll fluorescence parameters , and hypocotyl soluble carbohydrate and protein content, while decreasing starch content of marigold [2O2 on the ultra-microstructure of mesophyll cells improves the photosynthetic performance of blades and lightens the adverse impacts of drought on the build-up of carbohydrates and nitrogen in explants, boosting adventitious rooting. An overview of the effects and mechanisms in plant AR-related studies of small-molecule compounds under no stress and stress conditions are shown in Under drought stress, 600 μM Hmarigold . It implNO and CO are two toxic gases, but they function as second messenger molecules in decreasing vascular tone and inhibiting platelet aggregation by simultaneously inducing cGMP content in animals . The bioIn plants, NO and CO are able to act as two typical downstream signals in auxin-induced adventitious rooting. In mung bean seedlings, addition of the CO donor hematin induced AR formation in dose- and time-dependent manners, similarly with the phenotype induced by NO donor SNP. Furthermore, the NO-specific scavenger cPTIO suppressed the entire NO induced by CO, and the root numbers of the mung bean hypocotyls were decreased by NOS inhibitor L-NAME and hematin treatments when compared to that under hematin treatment alone. However, the inhibitor of HO-1 zinc protoporphyrin IX (ZnPPIX) could not affect the SNP action, suggesting that the CO-induced adventitious rooting process is mediated by the NO/NOS pathway, and NO may act downstream of CO during AR formation in mung bean hypocotyls .heme oxygenase1 (CsHO1) gene was cloned and proved to exhibit HO activity, which was the inducible isoform of heme oxygenase, and it participated in the heme degradation pathway while concomitantly releasing CO. The nitric oxide donor SNP, as well as other AR inducers, could increase the level of the CsHO1 transcript and the corresponding protein, which further suggested the crosstalk between endogenous HO-1/CO and NO during adventitious rooting and their signal transduction pathway [In cucumber, the  pathway . Subsequent research found that application of CO and hemin to cucumber explants induced the production of NO and the NOS-like enzyme inhibitors, and the NO scavenger blocked NO content and AR formation stimulated by CO, but not the NR inhibitors. Simultaneously, researchers found that NO might act as a downstream regulator of CO during adventitious rooting in a cGMP-dependent manner .2 in AR development in cucumber and marigold. The NO scavenger cPTIO, NOS inhibitor NG-nitro-L-Arg methyl ester hydrochloride (l-NAME) or NR inhibitor sodium azide (NaN3), and tungstate could partly reduce the effect of H2-triggered AR in cucumber, which indicated that NO acts as the downstream signaling molecule, and that NOS and NR might be responsible for NO generation during H2-induced AR formation [2 induced AR development through regulating the cell cycle and the activity of root organogenesis-related enzymes POD, IAAO, and PPO [It was reported that NO had a close relation with Hormation . Moreove and PPO .3, suggesting that the positive role of ETH on AR formation was partially NO-dependent [The marigold and cucumber explants treated with ETH and NO together exhibited a better phenotype of AR formation than those treated with ETH or NO alone. These effects of ETH were restrained by cPTIO, L-NAME and NaNependent . It was ependent ,27. In mependent . Togethe4 in cucumber (C. sativus ‘Lufeng’) explants triggered generation of NO and was followed by adventitious rooting, which was similar to the effects of the NO donor SNP and NONOate, suggesting the involvement of NO in CH4-elicited root organogenesis. Further studies illustrated that CH4 induced endogenous NO production through NOS-like enzymes and diamine oxidases (DAOs), and it triggered AR development via controlling expressions of NO-targeted genes [The applied CHed genes . Ipomoea batatas L.), application of the H2S donor NaHS induced ARs in a dose-dependent manner. Treatment of NaHS increased endogenous H2S, IAA, and NO contents, and H2S mediated AR, which was blocked by NPA and cPTIO, This indicated that IAA and NO participate in H2S-induced adventitious rooting, and this regulation mechanism could also be applicable to root organogenesis in willow (S. matsudana var. tortuosa Vilm) and soybean (G. max L.) stem cuttings [In sweet potato , researchers further discovered that CO may be involved in H2-triggered adventitious root formation through adjusting RWC, photosynthesis, and metabolic constituent content and alleviating oxidative damage under drought stress conditions [In alfalfa seedlings, Hstresses . In cucustresses . In cucunditions .2S donor NaHS induced adventitious rooting, and the expression of CsHO-1 as well, and ZnPPIX significantly suppressed the effects induced by NaHS. Furthermore, NaHS regulated similar target genes of HO-1/CO in AR formation, but the H2S scavenger hypotaurine (HT) could not influence HO-1/CO AR formation, which suggested that HO-1/CO acts downstream of H2S during AR formation [In cucumber, the Hormation .2+ pathway in the MRW-induced AR process was certified. Together, CH4 could stimulate adventitious rooting through partially adjusting HO-1/CO and Ca2+ pathways [In cucumber, MRW significantly induced AR formation in IAA-depleted explants, and the inducible affect was still effective in soybean and mung bean explants. Meanwhile, auxin signaling-related genes and cell cycle regulatory genes exerted by MRW during AR formation were blocked by ZnPPIX and further reversed by CO, and the involvement of the Capathways .2S and CH4 mediated adventitious rooting in cucumber explants. Further study demonstrated that CH4 induced endogenous generation of H2S during adventitious rooting, and the H2S scavenger HT restrained the development of adventitious root primordia and gene expressions as well as S-sulfhydration level elicited by CH4, indicating the important role of endogenous H2S in participating in CH4-induced cucumber adventitious root development [Previous results showed that Helopment .2 increased adventitious root mass, ginsenoside contents, and polysaccharides, while CO2 and C2H4 were found to operate opposite impacts as O2 on secondary metabolism in plant cell cultures [During the adventitious root bioreactor culture of ginseng, the enrichment of Ocultures . 2, NO, CO, ETH, and other small-molecule compounds work together by regulating antioxidant, water balance, and photosynthetic systems, and they affect the transportation and distribution of auxin as well as expedite plant adventitious rooting. The mechanisms of the cross-talk between NO and other small signal molecules during plant adventitious rooting are summarized in In summary, Hcyclins  and two CDK (CDKA and CDKB) genes are found to analyze the effect of cell division on AR formation in plants. In cucumber, NO and H2 treatment up-regulated the expression of the cell-cycle regulatory genes CycA, CycB, CDKA, and CDKB, which participate in mediating cell division in internode growth and root meristem induction to boost the process of AR development. This result suggests that both NO and H2 take part in regulating the cell cycle progress to the mitotic phase [CsCDPK1 and CsCDPK5 genes in cucumber explants [4 could also up-regulate cell cycle regulatory genes in cucumber, including CsCDC6, CsCDPK1, CsCDPK5, and CsDNAJ-1 [It is well known that cell cycle regulation genes are often related to the regulation of cell division in plant internode growth. Two ic phase . Also, Nexplants . The appCsDNAJ-1 ,47.CsDNAJ-1 and CsCDPK1/5 genes. However, when NO and ETH were applied alone, the relative expressions of CsDNAJ-1, CsCDPK1, and CsCDPK5 genes were significantly higher than that of any other treatments. The CsDNAJ-1 and CsCDPK1/5 target genes were up-regulated by the application of H2 and HO-1/CO during AR development in cucumber [2S donor NaHS triggered up-regulation of target genes responsible for HO-1/CO-induced cucumber adventitious root formation, including CsDNAJ-1 and CsCDPK1/5 [CsDNAJ-1 and CsCDPK1/5 genes were the possible target genes in the MRW-induced HO-1/CO-mediated cucumber adventitious development [CsHO1 can inhibit antioxidant processes in some abiotic stresses. CsHO1 was found to be involved in cucumber adventitious rooting via NO, H2S, and CO treatments [CsmiR160 and CsmiR167 were increased or decreased, respectively, by the application of MRW, indicating that CH4 may play a positive effect on AR development in cucumber [2, CO, H2S, and CH4 are able to induce high expressions of the CsDNAJ-1, CsCDPK1/5, and CsHO1 genes to mediate adventitious rooting.Furthermore, molecular evidence illustrates that the corresponding genes of DNAJ-like proteins and calcium-dependent protein kinases (CDPKs) are related to the initiation and development of adventitious roots. Xu et al. 2017)  found thcucumber . The H2SsCDPK1/5 . CsDNAJ-elopment . Also, t  found teatments . In addicucumber . TherefoARF6 and ARF8, which were targeted by miR167, had important effects on the regulation of AR development by adding ETH in Arabidopsis; however, ARF17, a target of miR160, was a negative regulator [CsAUX22B-like and CsAUX22D-like were target genes to HRW-induced AR formation [CsAux22D-like and CsAux22B-like in cucumber explants [4 could induce two auxin-signaling genes, such as CsAux22D-like and CsAux22B-like, which are responsible for AR formation in cucumber [Besides, some auxin-response genes are associated with AR formation. Previous studies showed that the auxin response factors ormation . Recentlexplants . Also, Ccucumber . Table 32S, were previously considered as toxic. They recently were assumed to be the gaseous substances to induce AR development. This review is based on a number of studies to show that small-molecule compounds such as NO, H2, H2S, CO, CH4, and ETH can be necessary to trigger adventitious rooting. However, the mechanisms of these compounds in adventitious rooting should still be illustrated and consummated. Many results have reported that some small molecules exert interactions with each other. For instance, H2 is involved in CO-induced AR development, but the concrete pathway is still unclear. Therefore, the combined effects of these compounds can mediate the induction of AR development. Besides, some target genes responsible for adventitious rooting, including CsDNAJ-1 and CsCDPK1/5, could also be regulated by these small molecules.In recent years, there have been increasing studies about the effects on the small signaling molecules of AR formation and development. Some molecules, like CO and HAbove all, these small-molecule compounds are involved in AR development through modifying antioxidant defenses, water balance, photosynthesis, and auxin transportation. In addition, the small compound molecules are widely and easily applied in various plant species and regulate many processes in plants, and they are more efficient and cheaper than some chemicals. Therefore, it is very meaningful to study the influence of small molecules on AR development and formation. However, the mechanism of the promotion of AR development by these small molecules has not been clear. Thus, further research is required to investigate their mechanisms. Finally, the question of whether there any other signaling compounds that can be used to promote AR formation and development should be considered. In the future, related work should be done to improve our knowledge in AR promotion and the future application of signaling molecules to horticultural products.