Canine-assisted interventions (CAI) are becoming more popular in hospital settings, representing a crucial intersection between animals, veterinary medicine, and society. However, standardized policies and procedures to minimize risk and maximize benefit to vulnerable humans and protect therapy dog welfare are lacking, posing a challenge to safe practice. Few intervention programs are evaluated to document efficacy compounding the potential risk. This paper presents a rationale for CAI in hospitals and describes the evidence, issues, and challenges to establishing and maintaining safe and effective programs for humans and animals. Recommendations are made for best practices based on the existing scientific evidence and a model program in place in a major medical center for 19 years. Scientific and practical implications are considered. Hospitalized patients represent a vulnerable population as they face not only the challenges of their medical conditions and treatments, but also separation from familiar settings and social supports. Recognizing the distress associated with hospitalization, administrators, and clinicians seek novel, complementary, and cost-effective interventions to provide comfort and support to patients. Perhaps in part due to broad media claims of patient benefits, canine-assisted interventions (CAI) are becoming more popular in hospital settings. However, both a lack of consistent practice standards and evidence of program efficacy pose risks to patients, hospital employees, therapy dogs, and dog handlers. This paper describes the evidence, issues, and challenges for CAI programs to practice safely and effectively in hospital settings and provides recommendations for best practice based on scientific evidence and a model program.Humans have shared their lives with companion animals for thousands of years, yet it has only been over the past three to four decades that researchers have turned their attention to the possible health benefits of this relationship . The heaOther studies documenting reduced cardiovascular reactivity in pet owners followed. Married couples owning dogs or cats had lower baseline diastolic and systolic blood pressure than couples without pets and showed lower reactivity and faster recovery to mental and physical stressors . Lower cLower systolic and diastolic blood pressure were found in older adults with pre- to mild hypertension when their pet dogs were present during their normal daily lives . SimilarAfter a critical review of studies such as these, an American Heart Association scientific statement was published concluding that “pet ownership, particularly dog ownership, is probably associated with reduced cardiovascular risk” and “pet ownership, particularly dog ownership, may have some causal role in reducing cardiovascular disease risk” [Levine et al. , p. 2356A natural extension of this body of work is to examine whether humans in settings without dogs, such as hospitals, can benefit from safely interacting with unfamiliar dogs, as is the case when interacting with visiting therapy dogs. An exploratory study compared physiological benefits of dog owners interacting with their own therapy dog vs. an unfamiliar therapy dog in a clinic setting . This smAlthough studies have emerged supporting the benefits of CAI in hospital settings, little consistency exists in the clinical populations studied, outcomes, and methodologies emphasizing the need for more studies in this area. Inpatient psychiatry is one area with more consistent evidence of patient benefits. An early study of the effect of CAI on anxiety in hospitalized psychiatry patients yielded promising findings. Patients with mood, cognitive, psychotic, or other disorders had lower anxiety scores on the State-Trait Anxiety Inventory after participating in recreational therapy incorporating a CAI . AlthougOther studies reporting CAI benefits for hospitalized psychiatric patients found reductions in fear prior to electroconvulsive therapy in patients randomly assigned to a 15-min CAI vs. magazines , qualitaAlthough several studies have investigated the effects of CAI on pediatric patients, results have been mixed with two randomized controlled studies reporting no effect on biobehavioral stress , or selfOther hospitalized patient populations have been the focus of one or two studies of CAI indicating potentially positive effects, but further study is needed with these populations to confirm results. For example:Patients randomly assigned to CAI prior to cystoscopy had lower anxiety and stress levels than a treatment as usual control group Patients randomly assigned to physical therapy incorporating CAI following total joint arthroplasty reported less pain and higher satisfaction with their hospitalization than those assigned to standard physical therapy .Following total joint replacement surgery, patients from two hospitals were compared in a retrospective study of matched samples. Patients participating in CAI used less oral pain medication than the control group .Patients with moderate or greater anxiety in an emergency room had lower anxiety following CAI than those in a control group .Patients with heart failure selected to walk with a dog had a lower ambulation refusal rate (7.2 vs. 28%) and achieved greater ambulatory distance than a historical sample not participating in CAI .Cancer patients participating in CAI during chemotherapy had greater reductions in depression and increased arterial oxygen saturation compared with a control group .In addition to the benefits of CAI reported for patients, hospital healthcare professionals were also found to benefit from CAI in a pilot study documenting reduced physiological stress. Significant reductions in both serum and salivary cortisol were detected 45 min after 5 and 20 min of CAI . No cortThe promising evidence of CAI benefits for various hospital populations coupled with the low-cost of CAI programs utilizing community volunteer therapy dog teams support CAI as a feasible complementary intervention to traditional medicine. However, the vulnerability of hospital patients and unique environment of the hospital setting pose challenges to conducting CAI safely and effectively for patients, staff, dogs, and volunteers.Therapy dog visitation programs in hospitals present many potential issues and challenges stemming from multiple sources. Hospitalization is stressful for patients and their families  and evidHumans, including patients, staff, and visitors, may experience a variety of psychological and physiological stress reactions that are both acute and chronic in nature . Well-trHandlers are faced with a variety of potential challenges in hospital settings including routine practical issues such as what items to bring with them, where to park their car or exercise their dog, to more complex issues like how to safely avoid exposure to infectious diseases, and what topics of conversation are appropriate with each patient or visitor. In most, but not all cases, the dog handler is a person who is volunteering their time to bring their own dog to the hospital for visits. The handler has typically invested a sizeable amount of time and energy into training, testing, and registering their dog with an appropriate therapy dog organization . Research on human–dog attachment has shown that attachment styles tend to mirror those found in mother–child relationships  and simiCanine-assisted interventions (CAI) handlers often train their dogs to be in close proximity to, and interact with, strangers which appears to create behavioral adaptations during CAI, such that the dog may maintain greater eye contact with their handler as a way of maintaining contact during uncertainty . AttachmIf a handler notices that their dog is stressed, their role is to remove the animal from the situation, but they are frequently confronted with opposing pressures to extend the visit. For example, the handler may see that a person is enjoying interacting with the dog, or the person may be in pain  and taking great comfort from the dog, or the person may be alone and near the end of life and may simply want to touch the dog for a little longer. Handlers frequently struggle with what they may perceive as the morality of ending the visit under these more extreme circumstances. For this reason, it is important to prepare handlers for these challenging situations, give them tools to help them to deal with each individual situation appropriately, and provide ongoing support. Furthermore, there is some evidence to indicate that owners may not be well-prepared to recognize signs of stress in their dogs, and that they may benefit from educational efforts to improve their ability to recognize and interpret signs of stress in their own dog .Like hospital personnel, handlers are subject to the stresses inherent in a hospital environment. Exposure to patients who are suffering, facing terminal illness, and who die during hospitalization may contribute to handler anxiety and stress and secondary traumatization. Handlers who regularly visit a service may see a patient several times and 1 day finds the patient gone. Since they do not have access to confidential patient information, handlers are left wondering about the status of the patient. Unlike hospital personnel who can debrief with colleagues about patient conditions, including the death of a patient, and access employee assistance programs, handlers are left to process such situations alone unless support services are made available to them. Compassion fatigue is a risk for handlers that may lead to burnout, withdrawal from volunteering, or compromise their ability to safely provide CAI .Freedoms , the therapy dog registering agency , and local program administrators and evaluators.N = 17 studies) indicated that the use of aversive training methods jeopardizes both the physical and mental health of dogs . The recent COVID-19 pandemic has highlighted the fact that it is not possible to completely eliminate the risk of disease transmission  among humans and dogs, but monitoring the ongoing health of both species can reduce the risk and allow for early detection and treatment of potential health issues.Safe dog–human interactions require the understanding of dogs' signaling behaviors and there is a striking lack of knowledge of these behaviors in the general public . ShepherThere are many potential risks to dogs in a hospital setting, requiring the handler to be vigilant in monitoring their dog's safety. For example, something may have been dropped on the floor  of a patient room or spilled  in the hallway. Equipment can be both fragile and top heavy, so if it is inadvertently knocked over the dog may become injured. Equipment and people can block or crowd a pathway, creating obstacles that may be intimidating to the dog, requiring the handler to recognize and carefully navigate unexpected situations.Patients can also be a source of risk to the dog. Some patients may have difficulty with gross or fine motor movements and may stumble onto the dog, or roughly grab at the dog. Other patients can become overly interactive with the dog and hug them tightly or attempt to pick them up or move them in ways the dog does not enjoy. Some patients will attempt to share their food or drinks with the dog. Children may pull their hair or tail, and I.V. bags and lines may startle the dog if they become entangled in them.The handler carries a heavy responsibility in monitoring their dog, their interactions with humans, and any potential risks in the environment. It is important for the CAI program and hospital to provide education and support to handlers in carrying out these responsibilities.Patient and healthcare provider safety is a top priority for all healthcare facilities, including hospitals. Therapy dog presence represents a risk to that safety in the form of potential zoonotic pathogen transmission, cross-transmission of human pathogens, injury, and symptom exacerbation.Insufficient research has been conducted investigating the role of dogs as a vector for zoonotic disease transmission in hospitals . We wereTwo recent surveys conducted in the United States examined CAI health and safety policies and practices. One study surveyed 45 hospitals, 45 senior care facilities, and 27 therapy dog organizations with results showing inconsistent policies to safeguard patient health and animal welfare . ParticuClostridioides difficile, methicillin-resistant Staphylococcus aureus, or vancomycin-resistant Enterococci.Feeding a raw meat diet remains a contentious subject among some therapy dog owners. A study of salmonella and other potential pathogen risk in therapy dogs in Canada fed a raw meat diet found dogs fed raw meat at some point during the yearlong study were more likely to test positive for salmonella and extended-spectrum cephalosporinase than dogs not fed raw meat . No diffIn the absence of studies providing more conclusive evidence on the role of canines in zoonotic pathogen transmission and cross-transmission of human pathogens, strict infection prevention and control procedures, including the ability to contact trace CAI interactions, must be established for CAI to maximize patient, healthcare provider, and CAI team safety. Two recent publications address this issue and provide guidelines for the safe practice of CAI in hospitals , 57.In addition to zoonotic disease transmission, therapy dogs represent a risk for injury to patients, visitors, and healthcare personnel as well as to other therapy dogs if providing CAI with other therapy dog teams. Although evaluated for health and temperament by therapy dog organizations to minimize such risk, dogs still have the potentially to trip, scratch, or bite someone in the environment or accidently interfere with equipment . Hospital policies and procedures must address such risks, provide direction for handling such incidents, consider liability issues, and develop procedures to minimize risks.Dogs in the environment also present a concern for patients, visitors, and staff with allergies to canine dander and fear of dogs. CAI programs must address these potential risks to maximize human safety.An additional potential issue for handlers, patients, and hospital personnel is the illness and/or death of a therapy dog. For the handler, serious illness or death of their therapy dog often represents a significant loss , 59. NotA less obvious issue for handlers and patients is the ability of therapy dog teams to meet the demand for CAI. Patients and staff requesting CAI may be disappointed if the visit does not materialize. This can be particularly difficult for pediatric patients who may be informed that a therapy dog will be coming or observe the CAI team visiting other patients and leaving the unit without seeing them. Staff training in understanding the limits of CAI resources and presenting the intervention as requested but not guaranteed can help minimize negative reactions when a requested visit cannot be fulfilled.For CAI to be successful, hospital staff must understand basic information about the approved CAI program. Staff must be informed of areas approved for CAI, criteria for determining patient appropriateness to participate, and procedures to request CAI for their patients. An understanding of therapy dog and handler requirements to deliver CAI is important to maximize appropriateness of requests. Ideally key hospital administrators and staff are involved in the development and ongoing evaluation of any CAI program in their setting.Best practice is defined by Merriam-Webster  as “a prDogs on Call is a therapy dog program established in the Center for Human–Animal Interaction at Virginia Commonwealth University (VCU) School of Medicine in 2001. The program model depicted in proposed as a standard suitable for widespread adoption  and selepublished efficacy studies providing evidence basetime-tested intervention operating in a major academic medical center since 2001established 19-year history of safety for humans and canines.Dogs on Call policies and procedures were developed in concert with internal representatives from diverse disciplines including epidemiology, veterinary, and human medicine, and healthcare administration as well as informed by relevant professional guidelines related to infection control, animal-assisted interventions, and animal welfare. Policies and procedures are reviewed and updated on a regular basis to reflect changes in hospital policies and procedures, new knowledge from relevant fields, and results of formal and informal evaluation efforts. The program is manualized to promote standardized interventions delivered throughout the medical center.Dogs on Call requirements include documentation of external therapy dog registration (Pet Partners or Alliance of Therapy Dogs), completion of VCU Medical Center volunteer services training, completion of Dogs on Call training, and adherence to manualized policies and procedures. The owner/handler and dog are evaluated and approved to participate together as a dyad in CAI programs. The unique bond and communication between them contribute to the safe practice of CAI and attention to canine welfare. Biannual meetings are held to provide program updates, reinforce program fidelity, provide continuing education, and solicit feedback. Teams are evaluated individually at least biennially to monitor program fidelity. Handlers participate in ongoing CAI related continuing education in such areas as canine behavior, patient safety, responding to difficult requests, and compassion fatigue. Program efficacy is documented through ongoing research and evaluation, disseminated through professional publications and internal and external education, and utilized in making program revisions. For detailed information on the Dogs on Call program see Barker et al. , and visAny novel program being considered by a hospital has a higher chance of being established, continued, and successful with administrative and employee involvement and support. Establishing such relationships and ongoing awareness of relevant research and practice are important foundations for any CAI program. For example, the involvement of medical, nursing, legal, volunteer services, veterinary, and other medical center administrators in developing Dogs on Call program policies and procedures were key to the program's credibility, acceptance, and ongoing support as a medical center program. One avenue for facilitating such involvement is establishing CAI executive and/or advisory committees involving key high-level administrators to build a sense of ownership and oversight by the hospital. Whether the CAI program is internal as is Dogs on Call, or external as with visiting community-based CAI programs, such committees serve to facilitate communication between the CAI program and hospital employees at all levels. Establishing liaison relationships with key hospital staff on services targeted for CAI promotes coordination and feedback regarding CAI activities. Liaisons can facilitate CAI scheduling, establish any needed service-specific orientation  for handlers, and provide CAI teams with unit-level assistance and support. Building relationships with frontline staff increases the likelihood of staff comfort with the CAI program and willingness to contact the program with any concerns, questions, or requests. These liaisons also enhance the CAI program's ability to monitor program implementation and resolve potential problems. Patient conditions in the hospital can change very quickly making real-time screening of patient appropriateness by front-line clinical staff critical to patient safety. Staff knowledge of the CAI provides an important framework for making informed decisions in screening patients and gauging the milieu for appropriateness for CAI on any given day.In addition to comprehensive knowledge of HAI and AAI, input from internal and external content experts representing other relevant disciplines is critical for developing policies and procedures to maximize therapy dog welfare and human safety in the hospital setting. At a minimum, representatives from human health , veterinary medicine, hospital risk management, hospital administration, and volunteer services should be involved. Their ongoing involvement provides a conduit for updating CAI policies and procedures based on evidence and regulatory changes from the diverse fields affecting a CAI program in a healthcare setting. For example, when COVID-19 cases began emerging in Virginia in March of 2020, Dog on Call operations were guided by the medical center's infection control department and hospital administration. Since Infection Control personnel and hospital leadership were knowledgeable of the Dogs on Call program, rapid infection control and visitation guidance was provided from an informed perspective of both Dogs on Call practices as well as COVID-19 risk to patients, CAI teams, and staff.CAI policies and procedures are strengthened by incorporating relevant published guidelines. The publication of the Society for Healthcare Epidemiology of America guidelines for animals in health care facilities provides recommendations for service and therapy animals informed by science and current practice . A 2019 It is important to note that at a minimum, most CAI programs require some type of handler-dog training and initial assessment to determine that minimum competencies are met. Therapy dog organizations providing such training and assessment vary in not only their initial requirements, but also requirements for renewal and their policies for members. Some renewals are payment-based with documentation of animal wellness while others require periodic re-evaluations of the dog/handler team. Such variability in requirements and policies highlight the need for hospitals to go beyond therapy dog organization requirements and develop comprehensive informed policies and procedures to maximize human and canine safety.Policies and procedures should address CAI oversight as well. Identifying those responsible for the program establishes needed accountability and provides CAI handlers and hospital employees with individuals to contact with questions and concerns. In addition to monitoring therapy dog and handler ongoing compliance, oversight must include attention to canine welfare, and a system for monitoring where CAI is being conducted at any given time. This information will be needed in the event contact tracing is required. When a Dogs on Call handler thought his dog might have ringworm, he reported the incident to the hospital's Dogs on Call coordinator who immediately contacted Infection Control and contact tracing was initiated. Although the handler called later that day to confirm a veterinary consult ruled out ringworm (or any contagion), the contact system was able to be effectively initiated because CAI teams are monitored for time and location.Oversight responsibility also includes addressing canine or handler problems or complaints that arise. Addressing any non-compliance with policies and procedures must be handled immediately for the safety of humans and canines and to ensure program integrity. For canine misbehavior, policies should detail clear steps to remediate the behavior  or terminate program participation . Oversight also includes implementing methods to acknowledge the contributions of CAI teams and to address ongoing recruitment and retention.The most rigorous and well-informed CAI policies and procedures can only be effective if implemented as intended. Ongoing evaluation is an important component of any successful program to maintain program integrity and assess program worth and effectiveness . Dogs onHospital administrators are more inclined to support and continue programs that are shown to be effective. Providing evidence of efficacy involves evaluation. In addition to informing decision-makers, evaluations can provide valuable information for improving CAI processes, determining which patients benefit from CAI and in what ways, and identifying resource needs. For example, during the development of Dogs on Call, a needs assessment was electronically sent to all nursing coordinators in the hospital to determine interest in and concerns about a CAI program on their services. The results provided an indication of program demand, but just as importantly, identified areas for staff education on CAI to address their concerns. Ongoing studies of Dogs on Call have identified clinical populations benefiting from brief CAI  , 30 as wAlthough primary responsibility for canine welfare resides with the therapy dog owner, hospitals must also share some of that responsibility when CAI is approved on their premises. Hospital administrators are uniquely qualified to identify potential safety issues for canines in their facilities and develop strategies to minimize risk. For example, VCU Medical Center recognized the potential danger that dropped medication poses for therapy dogs. They proactively involved Dogs on Call representation on their medication safety subcommittee addressing this issue. Dogs on Call team orientation emphasizes continuous handler surveillance of the environment for potential risks for their dogs, including dropped medications, fluids, and food.In addition to addressing risks, CAI programs must emphasize canine wellness in their policies and procedures as well. CAI programs benefit from involving hospital administrators in addressing therapy dog welfare. Such collaboration facilitates identifying adequate parking to accommodate safely unloading dogs, designating adequate exercise, rest, and elimination areas, identifying on-site or local veterinary resources for emergent issues, and developing processes for CAI team check-in and check-out to enable contact tracing, not only for possible canine-to-human zoonotic transmission but human-to-canine transmission as well.In setting firm time limits for dogs in the hospital, the CAI program can address a canine welfare issue inconsistently addressed by therapy animal registration organizations . SettingIt is important to emphasize canine welfare in CAI orientation and continuing education, including understanding canine body language and recognizing when the dog does not enjoy the environment. Some dogs may not be comfortable in highly stimulating settings  but are very comfortable in more predictable surroundings . Handlers become very invested in participating in CAI and may not recognize changes in their dogs over time that may indicate they are fatigued or no longer enjoying the hospital setting. Periodic monitoring of the therapy dog team by CAI program staff can assist with identifying when a dog may need a break or retirement from CAI. Continuing education for handlers normalizing retirement can be helpful, but in the end the CAI program must be willing to administratively retire a therapy dog if it is in the best interest of the dog.This paper began by summarizing the evidence supporting benefits of CAI in hospital settings while noting the existence of conflicting evidence and the need for further research. Several studies have documented positive results of CAI, but there is little consensus in the clinical populations studied, outcomes and measurements selected, and methodological rigor. The exceptions to these disparate positive findings are accumulated studies showing CAI benefits for cardiac and psychiatric patients.To advance research on CAI in hospital settings, established CAI programs are needed. Yet the hospital setting presents unique challenges for the delivery of CAI. Sources of potential issues and challenges for humans and dogs involved in CAI in the hospital setting were presented based on the literature and the authors' extensive experience administering and conducting CAI. Best practice recommendations for CAI program processes and oversight were then provided with examples from a model best practice program operating in a major medical center.For people interested in establishing a CAI program in a hospital setting, gaining entry may seem like a daunting task. Identifying one or more key allies in the hospital can facilitate this process and provide valuable guidance on preparing a proposal that has a higher likelihood of being accepted by administrators. Hospital employees likely represent the surrounding community and their opinions can provide insight into views regarding pet ownership and the potentially favorable impact of a CAI program. However, it would be a mistake to assume that hospital employees are aware of hospital-based CAI and evidence supporting benefits. Our initial needs assessment at VCU Medical Center revealed many misconceptions and concerns about therapy dogs in the environment, including that dogs would be dirty, noisy, and disruptive in the environment and bring fleas, ticks, and other sources of illness to patients. An initial educational approach to introducing CAI may lay the groundwork for acceptance and support of the program.Ultimately approval, support, and endorsement of CAI at the highest administrative levels is desired, but the process of securing such widespread support takes time, planning, and patience. The antecedent of Dogs on Call was a small pilot project conducted by the first author on inpatient psychiatry. Suggesting a pilot project as an entrée to CAI has advantages for those approving the project. “Pilot” implies temporary and therefore can be discontinued if the process and outcomes are not considered valuable or beneficial. Including an evaluation component can provide documented outcomes to support the program. Outcomes can include informal feedback from patients and staff and/or simple assessments measuring mood or anxiety. More than any other single factor, results documenting positive patient outcomes contributed to the growth in credibility and widespread support and institutional integration of Dogs on Call at VCU Medical Center.Cost-effectiveness is a key consideration for hospitals considering any new program. A benefit of CAI is that therapy dogs reside with their owners in the community. Owners assume the costs of ownership including veterinary care, food, training, therapy dog registration, etc. They provide CAI as volunteers, incurring only a modest cost to the hospital for providing volunteer orientation and required training, vaccinations, and record-keeping. CAI programs need adequate funding to develop, implement, maintain, and evaluate the program. Identifying funding sources is challenging for CAI in any context with competing interests vying for a limited pool of financial resources. Successfully competing for funding against requests for healthcare and allied health personnel, equipment, patient needs, information technology, infrastructure and the myriad of other hospital needs is enhanced with evaluation data documenting the value of CAI in terms of benefits to patients, staff, and the organization.Some programs, including Dogs on Call, establish themselves as non-profits eligible for tax-deductible donations and seek support from community foundations, grants, and individuals. Again, evaluation is key to demonstrate program efficacy to potential sponsors. Some CAI programs charge a nominal annual fee for membership and require members to purchase branded merchandise  while others raise funds to provide membership and merchandise at no charge to members.As the popularity of a CAI program grows, so does the demand for services. Recruiting, training, and monitoring additional CAI teams can strain existing program resources. Experiencing such demands, Dogs on Call developed a Leads Program to assist with these efforts. Experienced Dogs on Call members are recruited, educated, and trained to assist with recruiting and onboarding of new members and monitoring for program fidelity. Meeting regularly with Leads members enhances their program involvement, visibility, and recognition as senior level volunteers. Their experienced insights and feedback strengthen and inform program policies and procedures.Canine-assisted interventions (CAI) have the potential to complement traditional medical treatments in contributing to the health of hospitalized individuals. The hospital setting presents unique challenges to humans, dogs, and dog handlers in providing CAI effectively and safely. Recommended best practices are presented based on the literature and a model best practice program to guide hospitals and CAI programs in implementing programs that maximize canine welfare and human safety. More evidence of CAI efficacy with hospitalized patients is needed. To advance existing research, studies must be undertaken utilizing rigorous methodologies to investigate CAI programs that meet best practice criteria.SB and NG conceived the idea for the paper together. SB provided the initial organization of the paper. All authors wrote the document in shared collaboration.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.


Plant cell walls are the main physical barrier encountered by pathogens colonizing plant tissues. Alteration of cell wall integrity (CWI) can activate specific defenses by impairing proteins involved in cell wall biosynthesis, degradation and remodeling, or cell wall damage due to biotic or abiotic stress. Polygalacturonase (PG) depolymerize pectin by hydrolysis, thereby altering pectin composition and structures and activating cell wall defense. Although many studies of CWI have been reported, the mechanism of how PGs regulate cell wall immune response is not well understood.ltn-212 showed obvious cell death and accumulation of H2O2 in leaf tips. The defense responses were activated in ltn-212 to resist bacterial blight pathogen of rice. Map based cloning revealed that a single base substitution (G-A) in the first intron caused incorrect splicing of OsPG1, resulting in a necrotic phenotype. OsPG1 is constitutively expressed in all organs, and the wild-type phenotype was restored in complementation individuals and knockout of wild-type lines resulted in necrosis as in ltn-212. Transmission electron microscopy showed that thicknesses of cell walls were significantly reduced and cell size and shape were significantly diminished in ltn-212.Necrosis appeared in leaf tips at the tillering stage, finally resulting in 3–5 cm of dark brown necrotic tissue. OsPG1 encodes a PG in response to the leaf tip necrosis phenotype of ltn-212. Loss-of-function mutation of ltn-212 destroyed CWI, resulting in spontaneous cell death and an auto-activated defense response including reactive oxygen species (ROS) burst and pathogenesis-related (PR) gene expression, as well as enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo). These findings promote our understanding of the CWI mediated defense response.These results demonstrate that The online version contains supplementary material available at 10.1186/s12284-021-00478-9. Oryza sativa L.) is not only the world’s most important food crop for more than half of the world’s population, but also a model species for genetic, cytological, and agricultural studies  mutant results in excessive accumulation of phosphate in shoots and thus causes plants to develop a leaf tip necrotic phenotype  supply  resulted in dwarfism and withered leaf tips, ultimately causing early leaf senescence  activity   and ADPG2 are essential for silique dehiscence; ADPG2 and QUARTET2 (QRT2) contribute to floral organ abscission; and all three genes contribute to anther dehiscence  and PGX2 function in cell expansion in seedling growth and rosette expansion in adult plants; overexpressing them resulted in enhanced hypocotyl elongation . Under natural summer field conditions, the leaf tip of ltn-212 had a small necrosis region and dark brown necrotic spots were formed at the tillering stage -derived mutant population in the JiaHe212 background, which we named ltn-212 significantly reduced plant height, 1000-grain weight, and grain length, compared to wild-type , but there were no changes in grain width or tiller number  and Evans blue staining. DAB staining results in the formation of reddish-brown formazan precipitates indicative of H2O2 accumulation. The red brown precipitate was observed on the ltn-212 leaf tip, whereas no precipitate was observed in WT  and superoxide dismutase (SOD) and peroxidase (POD) increased in ltn-212 plants, by 17.15 U/mgprot, 1.28 U/mgprot and 32.35 U/mgprot in ltn-212 leaf tips, respectively, which were approximately 2.1-, 1.6- and 1.5-fold higher than the levels of 8.08 U/mgprot, 0.82 U/mgprot and 21.19 U/mgprot in the WT, respectively , indicating that the leaf tip necrotic phenotype of ltn-212 was controlled by a single recessive nuclear gene. To map the gene responsible for the ltn-212 mutant phenotypes, an F2 mapping population was constructed by crossing ltn-212 with the indica variety ZH8015. Eight F2 plants with the ltn-212 phenotype were used for primary mapping, and the mutation was located on the short arm of chromosome 1 between markers 1–12 and RM493  had a single base substitution (G-A) in the first intron  of OsPG1 consists of 1512 nucleotides, and encodes a putative PG with a 504 amino acid protein. PG is one of the hydrolases of pectin and is involved in controlling the stability of cell wall structure. The typical PG genes in plants and fungi have four conserved domains: Domain I (SPNTDG), Domain II (GDDC), Domain III (CGPGHGISIGSLG), and Domain IV (RIK). Sequence alignment of PGs homologous among rice, Arabidopsis thaliana, Brassica rapa L. ssp. pekinensis, Ciboria shiraiana, Rhizoctonia solani, and Glycine max revealed that OsPG1 was highly conserved  reporter gene driven by the native promoter of OsPG1 to more precisely examine the spatial and temporal expression patterns of OsPG1. GUS staining showed activity of the OsPG1 promoter in all tissues examined . The results revealed that the cell wall thicknesses of bundle sheath fiber cells in ltn-212 were altered . Thus, we speculated that the necrotic leaf tips of ltn-212 were due to cell death and OsPG1 is a general suppressor of PCD in rice. The production of intracellular ROS is closely associated with cell death . The ltn-212 mutant was obtained from the mutant population generated by EMS treatment of JiaHe212. All plants used in this study were grown in paddy under natural conditions in Hangzhou, Zhejiang province and Lingshui, Hainan province, China. For disease characterizations, the plants were grown in a greenhouse with a photoperiod of 12h white light at 30 °C and 12 h dark at 25 °C.The wild-type used in this study was ‘JiaHe212’. The full length product was inserted to the pCAMBIA1300 vector using an In-Fusion Advantage Cloning kit . For Total RNA was extracted from rice leaves, roots, stems at different developmental stages using the RNAprep Pure kit for plants . Frist-strand cDNA was synthesized with a ReverTra Ace qPCR RT kit (Toyobo). The qRT-PCR assays were performed with Lightcycler 480 SYBR green  using a LightCycler 480 II real-time PCR instrument . The expression level of the target genes was normalized to that of the UBQ gene. The sequences of all primers used for qRT-PCR are given in Additional file OsPG1 was amplified and inserted to the pCAMBIA 1305 vector. The transgene rice tissues were stained with GUS staining solution . After incubating at 37 °C in the dark for 12 h, the chlorophyll was removed by boiling in 95% ethanol until removed completely.A 2.8-Kb native promoter region of Top leaves were collected at heading stage and immediately submerged in 10% SDS. After 10 min, the leaves were subsequently submerged in a 1 mg/mL solution of DAB or 0.5 mg/mL solution of Evans Blue incubated for 8 h in the dark at room temperature, respectively. Samples were then decolored in 95% boiling ethanol for 30 min and soaked for 48 h in 95% ethanol until all of the chlorophyll had been removed. The cleared leaves were then photographed.202 content of wild type and ltn-212 top leaves were measured from heading stage plants and using kits from Nanjing Jiancheng Bioengineering Research Institute.Malondialdehyde (MDA) content, Peroxidase (POD), Super Oxide Dismutase (SOD), Catalase (CAT) activity, Soluble Protein (SP) and H4 in phosphate buffer for 1-2 h and washed three times in the phosphate buffer  for 15 min at each step. For TEM, the samples were subsequently dehydrated through a graded series of ethanol and then embedded in acrylic resin. Ultrathin sections (70-90 nm) were double stained with uranyl acetate and lead citrate aqueous solutions and observed with a Hitachi H-7650 transmission electron microscope (JEOL) at 80 kV. The content assay of cell wall components was entrusted to Convinced-Test company .The materials were cut into small pieces and fixed in 2.5% glutaraldehyde and 0.1 M phosphate buffer at 4 °C overnight and washed three times in the phosphate buffer  for 15 min at each step; postfixed with 1% OsOOsPG1 and ospg1 were amplified from WT and ltn-212 using KOD FX, respectively. All the sequence alignments were used MegAlign and Gendoc software. The amino acid sequences of the OsPG1 and Homologous proteins were downloaded from NCBI BLAST server (http://www.ncbi.nlm.nih.gov/). Sequence alignment was performed with ClustalW . The Pathogen infection were performed as described previously . B. Leaf tip necrosis identification of WT and ltn-212 at tillering stage . Figure S2. Data statistics of agronomic traits. A-B. Comparison of tiller numbers and plant height of WT and ltn-212 (n = 10). C-D. Comparison of grain length and thousand grain weight of WT and ltn-212 (n = 10). E. Comparison of panicles and culm length of WT and ltn-212 (n = 10). ** indicates significance at P ≤ 0.01 and * indicates significance at P ≤ 0.05 (Student’s t test). Figure S3. Genome DNA and cDNA alignments between WT and ltn-212. Red star indicates start codon and stop codon, respectively. The red box indicates the incorrected splicing sequences. The red arrow indicates the mutant site. Figure S4. Amino acid sequence alignment of PG homologous. ADPG2: Arabidopsis thaliana, BCMF9: Brassicarapa L. ssp. Pekinensis, CsPG1: Ciboria shiraiana, RSPG1: Rhizoctonia solani, SDPG: Glycinemax. Figure S5. Phylogenetic analysis of OsPG1 with other homologues in rice. Figure S6. Cell wall structure of leaf bottom, middle, and tip Statistical analysis of the primary cell wall, secondary cell wall, and middle lamella thicknesses of bundle sheath fiber cells in leave bottom(A), middle(B), and tip(C) of WT and ltn-212. Data are means ± SD of 20 cells. ** indicates significance at P ≤ 0.01. Table S1. Primers used in this study.


We inferred the intercellular communication network in each experimental AAA model and human AAA and predicted commonly altered signaling pathways, paying particular attention to thrombospondin (THBS) signaling between different cell populations. Together, our analysis inferred intercellular signaling in AAA based on single-cell transcriptomics. This work provides important insight into cell-cell communications in AAA and has laid the groundwork for future experimental investigations that can elucidate the cell signaling pathways driving AAA.Cell-cell communication coordinates cellular differentiation, tissue homeostasis, and immune responses in states of health and disease. In abdominal aortic aneurysm (AAA), a relatively common and potentially life-threatening vascular disease, intercellular communications between multiple cell types are not fully understood. In this study, we analyzed published single-cell RNA sequencing (scRNA-seq) datasets generated from the murine CaCl Abdominal aortic aneurysm (AAA), defined as a focal dilation of the abdominal aorta beyond 50% of its normal diameter, is a common and potentially lethal aortic disease . DecadesSingle-cell RNA sequencing (scRNA-seq) is a powerful research tool that has been recently employed by multiple groups to investigate transcriptomic profiles of human and experimental aortic aneurysm tissue at single-cell resolution , 8–11. T2-, elastase-, or Angiotensin II (Ang II) induced mouse experimental AAA as well as human AAA scRNA-seq datasets were downloaded from the NCBI GEO data repository . Data preprocessing, normalization, scaling, and cell clustering were performed with Seurat package (version 4.0.3) in R (version 4.1.1) environment  interaction strength is the communication probabilities of the incoming (or outgoing) signaling to (or from) a cell population. The overall information flow for a given signaling pathway is the sum of communication probability among all pairs of cell groups in the inferred network .2 model by applying CellChat to the scRNA-seq dataset published by our lab (GSE164678). In that study, we perivascularly treated the infrarenal abdominal aortas of C57BL/6J mice with 0.5 M CaCl2 (AAA group) or NaCl (sham group). Aortas were collected 4 days after AAA induction , two smooth muscle cell (SMC), and three macrophage (Maph) populations, as well as several other cell types such as endothelial cell (EC), neutrophil (Neutro), dendritic cell (DC), T and natural killer cell (T/NK), and B cell  . In thiser Mki67 . SMC-1 e7 , THBS signaling was prominent in the elastase treated group however absent in the control. All macrophage populations also showed elevated incoming COLLAGEN signaling and increased outgoing SPP1, MIF, and GALECTIN signaling. Outgoing COLLAGEN signaling was also upregulated, but only in Maph-2 and Maph-3. Similarly, COLLAGEN signaling was also the most increased incoming signaling pathway in all SMC populations and the most enhanced outgoing signaling pathway in SMC-1 and SMC-2 . All fibApoe−/− mice were infused with 1,000 ng/kg/min Ang II via osmotic pumps for 28 days. No control group was included in this data set. Our analysis identified nine cell populations, including two SMC, two fibroblast, two EC populations, and macrophage, T/NK, and B cell populations  (7 (3). Inulations . Specifiulations , was theulations . As showDavis et al. conducted scRNA-seq on infrarenal abdominal aortas of patients undergoing open aortic aneurysm repair (AAA group) or open aortobifemoral bypass (control group) (GSE166676) . We idenAs demonstrated in THBS1—in two murine AAA models, we examined THBS signaling in a greater detail. Consistent with our previous reports (Since we have previously reported the importance of thrombospondin-1 (TSP1)—the matricellular protein encoded by  reports , 18, THB reports , 9–11. H2 model, SMC-2 and Fib-1 were the major cell types sending out THBS signaling. Upon AAA induction, THBS signaling sent from SMC-2 and Fib-1 populations was diminished, while signaling generated by Maph-2 was elevated. Specifically, THBS signaling sent from Maph-2 to SMC-1 or SMC-2 was most abundant in AAA , we predicted that SMCs were actively sending and receiving signals in both sham and AAA groups. CaCl2 treatment increased signal sending from Maph-2 (pro-inflammatory macrophages) to SMC-1 (contractile SMCs). Among all the signaling pathways altered by AAA, SPP1 signaling was the most up-regulated outgoing signal in Maph-2, and the most elevated incoming signal in SMC-1. Similarly, in an early-stage of the elastase model (Day 7), macrophages also sent out more SPP1 signals compared to control, and increased incoming SPP1 signaling was detected in SMCs. Spp1 encodes osteopontin, which participates in vascular calcification and is associated with the synthetic SMC phenotype  to other cell types, especially SMCs. This finding is consistent with our previous publication in which we showed that macrophages are the major source of TSP1 in murine CaCl2, Ang II models, and human AAA tissues  were the main source of THBS signaling in the sham group of CaCl tissues . By anal2 model, the Thbs1-Sdc4 pathway was also the most increased pathway sent from pro-inflammatory macrophages (Maph-1) to SMCs . Of note, Thbs1-Sdc4 signaling was increased the most at Day 7, and slightly decreased at Day 14. Expression of each THBS signaling gene also showed that, in Maph-1, Thbs1 was robustly increased at Day 7 and slightly decreased at Day 14 compared to control. Thbs1-Sdc4 and Thbs1-Cd47 were the major signaling pathways sent from SMCs (SMC-3) to macrophages. In contrast to observations in macrophages, these two signaling pathways were most elevated at Day 14, consistent with the up-regulated expression of Thbs1 in SMC-3 at Day 14. These results suggested that THBS signaling from pro-inflammatory macrophages to SMCs was activated at early stage, and from SMCs to macrophages at a later stage.In the elastase model, THBS signaling was relatively quiescent in the control group, but was induced by elastase stimulation. Similar to the CaClThis study has several limitations. First, our analysis infers cell-cell communication based on gene expression of ligands and their receptors and cofactors, while cell signaling ultimately occurs at the protein level. In the setting of post-transcriptional and post-translational modifications, as well as multi-subunit protein complex assembly, gene expression may not always accurately reflect protein level. Second, the proximity of cells, ligands, cofactors, and receptors to one another is critically important to cell signaling. Many ligands activate signaling cascades either by diffusing through the extracellular environment from a sender cell to a nearby receiver cell, or through gap-junctions between directly adjacent cells. Unfortunately, this spatial information is not captured in scRNA-seq data . In addi2 model, elastase model, and Ang II model, as well as in human AAA. Our analysis also predicted commonly altered signaling pathways in AAA, paying particular attention to THBS signaling between different cell populations. Our data provide a guide for future experimental investigations to elucidate the cell-cell communications driving AAA.In conclusion, we inferred intercellular communication networks in the murine CaClThe 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/HY, TZ, and BL designed research studies. HY analyzed data. TZ, ED, and BL wrote the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the National Institute of Health  and the American Heart Association (17POST33680095 and 20CDA35350009 to TZ).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.


P < 0.05), while adding UA decreased the RANKL-induced levels (P < 0.05) in RAW264.7 cells. Total RNA isolated from RANKL- or RANKL + UA-treated cells was sequenced, and the obtained transcriptome dataset revealed 2,399 differentially expressed genes. They were enriched in multiple pathways involved in rheumatoid arthritis, ERK1 and ERK2 cascade, regulation of inflammatory response, ECM-receptor interactions, and TNF signaling. Scanning electron microscopy showed that RANKL promoted bone resorption pits in bone biopsy specimens, whereas UA inhibited their formation. When bone morphogenic protein 2 (BMP2) was shRNA-silenced, the bone resorption pits were restored. Moreover, while RANKL significantly enhanced the levels of p-ERK2/ERK2, p-p38/p38, p-Akt1/Akt1, p-ERK1/ERK1, and osteoclast-related proteins (P < 0.05), UA reduced them. BMP2 silencing also reversed the UA inhibitory effect. Thus, UA represses the RANKL-induced osteoclast differentiation of RAW264.7 cells by regulating Akt1, p38, and ERK1/2 signaling, and BMP2 likely reverses the UA inhibitory effect via these pathways. We propose BMP2 as a potential drug target for treating bone metabolic diseases, such as osteoporosis.Urolithin A (UA) is an intestinal microbial metabolite derived from ellagitannins and a promising agent for treating osteoarthritis. However, its effects on osteoporosis are unclear. This study explored the effects of urolithin A (UA) on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclasts and its underlying molecular mechanisms. RANKL treatment significantly increased tartrate-resistant acid phosphatase (TRACP) or osteoclast marker levels (  Bone remodeling involves the dynamic coupling of osteoblast bone formation and osteoclast bone resorption. Under nUrolithin A (UA) is an intestinal microbial metabolite of ellagic acid and ellagitannins. It has high bioavailability and multiple health benefits, including anti-inflammatory and anti-proliferative effects against cancer, antioxidant activity, and improved lipid metabolism ,9. A firOsteoclasts are key participants in bone resorption and unique multinuclear cells originating from the mononuclear/macrophage lineage of the bone marrow. TherefoRAW264.7 is a mature pre-osteoclast cell line that can differentiate into osteoclasts after induction by specific growth factors, such as RANKL. RANKL, 2 at 37°C.RAW264.7 cells, a kind of the mouse macrophage cell line, were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences . The cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 100 U/mL penicillin–100 μg/mL streptomycin (Thermo Fisher Scientific). The cells were maintained in a humidified incubator containing 5% CO4 cells/well and cultured. The adhered cells were treated with 0, 0.5, 5, 25, 50, or 100 μM UA  and cultured at 37°C for 12, 24, 48, 72, and 96 h. Upon culturing, 10 μL of the CCK-8 reagent was added to each well, and the cells were incubated for another 2 h. The optical density (OD) was measured at 450 nm using a microplate reader (Thermo Fisher Scientific).A Cell Counting Kit-8  was used to assess the optimal concentration of UA for treating RAW264.7 cells before subjecting them to RANKL-stimulated differentiation. The cells were seeded into 96-well plates at a density of 1 × 10RAW264.7 cells were divided into three treatment groups: control, RANKL, and RANKL + UA groups. Cells in the RANKL and RANKL + UA groups were treated with 100 ng/mL RANKL and 100 https://ccb.jhu.edu/software/hisat2/index.shtml) was used for mapping, and gene expression levels were determined using the featureCounts software. The DESeq2 algorithm was used to identify differentially expressed genes (DEGs) based on the fold change (FC) cutoff of > 1.5 and false discovery rate (FDR) < 0.05. The DEGs were subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analyses. The GO terms and KEGG pathways were considered significantly enriched if P < 0.05.The RNA samples isolated from the RANKL- and RANKL + UA-treated cells were sequenced at Yanzai Biotechnology Co, Ltd  on an Illumina  sequencing platform. Clean data were obtained by removing low-quality sequences and adapter contamination. The HISAT2 software (BMP2 short hairpin RNA (shRNA) was synthesized by Yanzai Biotechnology Co, Ltd. The pGreenPuro vector was used to construct the BMP2 shRNA vector, as previously described[BMP2 shRNA vector suspension and cultured for 48 h. After 48 h, the complete medium containing 2 μg/mL puromycin was added to select for stable, transfected shBMP2-RAW264.7 cells[BMP2 primer sequences are shown in BMP2 expression.Using the sequence reported by Fujimoto et al. . The RNA quality and concentration were assessed using the 260/280 OD ratio. The isolated RNA with a high 260/280 ratio was reverse-transcribed into cDNA using PrimeScript RT Master Mix (Takara) and used as the template for RT-qPCR. The PCR assays were performed using SYBR Premix Ex Taq II  in an ABI 7500 detection system . The qPCR cycling conditions were as follows: 50°C, 2 min; 95°C, 2 min; 40 cycles at 95°C, 15s; 40 cycles at 60°C, 60s. All primer sequences are listed in GAPDH) was used as a housekeeping gene, and the relative mRNA expression levels were calculated using the 2−ΔΔCt method[Total RNA was extracted from RAW264.7 or shCt method.BMP2-RAW264.7 cells were seeded into the wells containing the biopsies. The biopsies with shNC-RAW264.7 or shBMP2-RAW264.7 cells were treated with 100 ng/mL RANKL or 100 ng/mL RANKL and 25 μM UA. Subsequently, the treated biopsy specimens were harvested and fixed with 2.5% glutaraldehyde. The images of the bone biopsies were obtained using a scanning electron microscope  and quantified using the NIH ImageJ software .Bone biopsies  were washed with medium and placed in 96-well plates. shNC-RAW264.7 cells (negative control) or shBMP2-RAW264.7 cells subjected to different treatments using radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology). Protein concentrations were determined using a bicinchoninic acid assay kit (Beyotime Biotechnology). The protein samples (20 μg) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel, transferred to polyvinylidene fluoride membranes, and blocked with 5% skim milk at 37°C for 2 h. After washing with PBST  three times, the membranes were incubated with anti-Akt1 , anti-phosphorylated (p)-Akt1 , anti-P38 , anti-p-P38 , anti-ERK , anti-phospho-ERK , anti-RANK , anti-cathepsin K , anti-integrin β3 , or anti-GAPDH  antibodies at 4°C overnight. After washing with PBST three times, the membranes were incubated with the secondary antibody  at 37°C for 2 h. After washing the membranes with PBST five times, they were subject to an enhanced chemiluminescence assay (Beyotime Biotechnology). The protein bands were detected in a chemiluminescence imaging apparatus .Total protein was isolated from RAW264.7 or sht-test was used to compare two groups, and a one-way analysis of variance to compare more than two groups. P < 0.05 was considered statistically significant.Data are reported as the mean ± standard deviation. GraphPad Prism 5  was used for statistical analyses. A Student’s P > 0.05, P > 0.05). The viability dropped only on 50 and 100 μM UA  but lower than the viability of untreated cells after 25, 50, and 100 μM UA . When RANKL-induced cells were UA-treated, their TRACP levels dropped to those similar in the control cells . By contrast, it was markedly decreased in UA-treated RANKL-induced cells (P < 0.05) and similar to that of control cells  and four repressed  DEGs were quantified by RT-qPCR to validate the sequencing data. The expression levels of BMP2, Cebpa, and Mapk1ip1 were significantly increased in RANKL + UA-treated cells compared with those in RANKL-treated cells  versus the control cells. Moreover, UA further increased BMP2 expression in the RANKL-treated cells (Four activated (ed cells . TherefoBMP2-specific shRNA was used to silence BMP2 in RAW264.7 cells to delineate BMP2 effects on osteoclast formation. The relative expression of BMP2 was considerably lower in the shNC-RAW264.7 cells (negative control) than in the shBMP2-RAW264.7 cells , clearly increasing in the RANKL + UA-treated shBMP2 group, relative to that in the RANKL + UA-treated shNC group , but after BMP2 knockdown, their levels rose again compared with those in the RANKL + UA-treated shNC group , and UA significantly decreased their RANKL-induced expression ).Protein expression levels of Akt1/P38/ERK pathway-related proteins and osteoclastic proteins  were assessed by Western blotting in RANKL or RANKL + UA-treated cells. Representative Western blot images are summarized in TRAP, NFATC1 and c-Fos are osteoclast-specific gene markers[Osteoporosis is a systemic bone disease that increases the susceptibility to fracture and may be caused by excessive bone resorption due to increased osteoclast activity ,26. UA ie markers. Our stuBMP2, Cebpa, and Mapk1ip1. BMP2 is a potent growth factor that regulates osteoblast and osteoclast formation. Durbano et al.[Cebpa is a key regulator of osteoclast lineage stereotyping, and its deficiency in osteoclasts can prevent bone loss[Mapk1ip1 expression is increased in osteosarcoma cells[MMP3, MMP9, and RUNX2. MMP3 and MMP9 are upregulated in chondrocytes in osteoarthritis[RUNX2 is critical for osteoblast differentiation and chondrocyte maturation and usually upregulated in chondrocytes in osteoarthritis[BMP2, may be involved in suppressing RANKL-induced osteoclast differentiation by UA.Total RNA was isolated from cells subject to RANKL treatment or the combined treatment with UA and sequenced to understand how UA inhibits RANKL-driven osteoclast differentiation. The transcriptome dataset revealed 2,399 DEGs, of which 587 were induced and 1,812 repressed. Among the notable activated genes were no et al. indicatebone loss. Furtheroma cells but nevearthritis. TranscrFunctional annotation analyses showed that the identified DEGs were associated with rheumatoid arthritis, the regulation of the ERK1/2 cascade, ECM-receptor interactions, the regulation of inflammatory response, and the TNF signaling pathway. The ERK1/2 cascade, for example, regulates various processes, such as cell proliferation, differentiation, adhesion, migration, and cell cycle progression. ActivatMSC-AS1 promotes osteogenic differentiation by binding miR-140-5p to upregulate BMP2, relieving osteoporosis[BMP2 induces osteogenic differentiation in various cells. In mesenchymal stem cells, it causes chondrogenic differentiation and endochondral ossification. It is also essential for bone engineering. Long noeoporosis. In addieoporosis. These f2 promotes osteoclast apoptosis through the ERK1/2 and Akt signaling pathways. We conclude that UA represses the RANKL-induced osteoclast differentiation of RAW264.7 cells by regulating Akt1, p38, and ERK1/2 signaling pathways, and that BMP2 may reverse the UA inhibitory effect via these pathways.Akt1 is an overactive proto-oncogene in most cancers. It is aRANKL/RANK signaling activates a central regulator of osteoclast generation NFATc1, inducing osteoclast gene expression. CathepsBMP2, and the signaling pathways require further investigation. The potential therapeutic effects of UA on osteolytic diseases require further confirmation by culturing osteoblasts with conditioned medium from osteoclast cultures and in vivo experiments. Our results uncover the role of UA in regulating osteoclast activity and provide insights into the underlying mechanisms. Moreover, our findings propose BMP2 as a potential drug target for the therapy of bone metabolic diseases, such as osteoporosis, based on its roles in osteoclast differentiation.We identified 2,399 differentially expressed genes in UA-promoted inhibition of osteoclast differentiation. UA may suppress the RANKL-induced osteoclast differentiation by regulating Akt1, p38, and ERK1/2 signaling pathways and repressing osteoclast-related proteins. Additionally, BMP2 was shown to be a novel UA target in inhibiting osteoclast differentiation. However, the regulatory roles of the identified genes, except


Cell adhesion to extracellular matrix proteins mediates resistance to radio- and chemotherapy by activating integrin signaling. In addition, mutual and cooperative interactions between integrin and growth factor receptor signaling contribute to the cellular radiation response. Here, we investigate to which extend the crosstalk between β1 integrins and growth factor receptor signaling determines the cellular radiation response of fibroblasts by assessing clonogenic survival and cell cycling. By utilizing growth factor signaling competent and either β1 integrin wildtype GD25β1A fibroblasts or β1 integrin mutant, signaling incompetent GD25β1B fibroblasts, we show basal clonogenic survival to depend on growth factor receptor but not integrin signaling. Our data further suggest the cooperation between β1 integrins and growth factor receptors to be critical for enhancing the radiation-induced G2/M cell cycle block leading to improved clonogenic radiation survival. By pharmacological inhibition of EGFR and PI3K, we additionally show that the essential contribution of EGFR signaling to radiogenic G2/M cell cycle arrest depends on the co-activation of the β1 integrin signaling axis, but occurs independent of PI3K. Taken together, elucidation of the signaling circuitry underlying the EGFR/β1 integrin crosstalk may support the development of advanced molecular targeted therapies for radiation oncology.The online version contains supplementary material available at 10.1007/s11626-022-00656-z. The extracellular matrix (ECM) is an important element of the tumor microenvironment that drives cancer therapy resistance  and 8 beta (β) subunits making up 24 integrin receptor pairs with specificity for different ECM ligands  as described recently  kindly provided the GD25β1A cells. S. Johansson  generously provided the GD25β1B cells. GD25β1A and GD25β1B were generated from GD25 fibroblasts derived from β1 integrin null stem cells by stably transfecting cDNAs encoding the murine integrin subunit β1A and β1B, respectively  24 h prior to X-ray irradiation as previously described  or fibronectin  of 240 kV X-rays with a dose rate of approximately 1 Gy/min at 13 mA filtered with 3 mm Beryllium . The absorbed dose was measured using a Duplex dosimeter .Inhibition of the EGFR was achieved by BIBX1382BS . For inhibition of PI3K, the compound LY294002  was used. Where indicated, cells were serum-starved or grown in complete medium for 16 h in the presence or absence of BIBX1382BS and LY294002 inhibitors prior to irradiation.et al. All cell cultures were grown for 72 h on PS or FN as indicated before cell cycle assessment as previously described , and complete protease inhibitor cocktail . Total protein amount in lysates was quantified by BCA assay (Thermo Fisher Scientific). After SDS–PAGE and transfer of proteins onto nitrocellulose membrane , probing of specific proteins was accomplished using indicated primary antibodies and horseradish peroxidase (HRP)–conjugated secondary antibodies. ECL Prime western blotting detection reagent  was used for the detection of proteins using the Fusion FX imaging device .Cells were lysed with modified RIPA buffer consisting of 50 mM Tris–HCl (pH 7.4), 1% Nonidet-P40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM NaVOAntibodies were purchased as follows: β-actin , β1 integrin , EGFR , αV integrin , α5 integrin , α8 integrin , α9 integrin , Caspase-3 , HRP-conjugated sheep anti-mouse secondary antibodies , and HRP-conjugated donkey anti-rabbit secondary antibodies .t-test. P values less than 0.05 are considered statistically significant.Data are represented as mean ± SD of two to three independent experiments as indicated. Statistical analysis of the data was performed with Microsoft Excel® using an unpaired To investigate the crosstalk between β1 integrin and growth factor receptor signaling, we employed GD25 β1 integrin knockout fibroblasts, expressing either a fully functional β1A integrin subunit (GD25β1A) or a signaling incompetent β1B integrin splice variant (GD25β1B)  competent β1 integrin signaling and growth factor presence seem to be key for clonogenicity upon X-ray irradiation and a pronounced radiogenic G2/M arrest, (ii) the EGFR dependency of the radiation dependent G2/M arrest relies on functional β1 integrin signaling, and (iii) the joint regulation of the G2/M arrest by the EGFR and β1 integrin occurs independent of PI3K.et al. et al. The formation of a G2/M phase block after ionizing radiation represents a natural reaction of cells to irradiation Below is the link to the electronic supplementary material.


Understanding subnational variation in age-specific fertility rates (ASFRs) and total fertility rates (TFRs), and geographical clustering of high fertility and its determinants in low-income and middle-income countries, is increasingly needed for geographical targeting and prioritising of policy. We aimed to identify variation in fertility rates, to describe patterns of key selected fertility determinants in areas of high fertility.I statistic and did a spatial cluster analysis using the Getis-Ord Gi* local statistic to examine the geographical clustering of fertility and key selected fertility determinants. Descriptive analysis was used to investigate the distribution of ASFRs and of selected determinants in each cluster.We did a subnational analysis of ASFRs and TFRs from the most recent publicly available and nationally representative cross-sectional Demographic and Health Surveys and Multiple Indicator Cluster Surveys collected between 2010 and 2016 for 70 low-income, lower-middle-income, and upper-middle-income countries, across 932 administrative units. We assessed the degree of global spatial autocorrelation by using Moran's TFR varied from below replacement (2·1 children per women) in 36 of the 932 subnational regions , to rates of 8 and higher in 14 subnational regions, located in sub-Saharan Africa and Afghanistan. Areas with high-fertility clusters were mostly associated with areas of low prevalence of women with secondary or higher education, low use of contraception, and high unmet needs for family planning, although exceptions existed.Substantial within-country variation in the distribution of fertility rates highlights the need for tailored programmes and strategies in high-fertility cluster areas to increase the use of contraception and access to secondary education, and to reduce unmet need for family planning.Wellcome Trust, the UK Foreign, Commonwealth and Development Office, and the Bill & Melinda Gates Foundation. An awareness of fertility rate is crucial for describing the demographic profile of a country, opportunities for development, change in population size, and for assessing challenges to women's reproductive health.6One aim of the UN Sustainable Development Goals (SDGs) is to address issues concerning the reproductive health and wellbeing of women of all ages.11Evidence before this studyEstimates of age-specific fertility rates (ASFRs) and total fertility rates (TFRs) are extensively used in all forms of demographic, development, and health-related analyses. Since the 1990s, national estimates of ASFRs and TFRs have been produced by the UN Population Division, among others, but an increased need has been recognised for estimates at the subnational level to target policy and geographical prioritisation. ASFRs and TFRs at the subnational level can be estimated through nationally representative household surveys and can serve a wide range of analyses, highlighting in-country heterogeneities. Although subnational fertility estimates have been used in previous studies, these have not looked at geographical clustering of TFRs and at patterns of fertility determinants and background characteristics within those clusters across a wide area in low-income, lower-middle-income, and upper-middle-income countries. We searched PubMed, Google Scholar, and Scopus for articles in English related to subnational data assembly, mapping, and analysis of ASFRs in low-income and middle-income countries between Jan 1, 2006, and Dec 1, 2019. We used the search terms ‘subnational mapping’, ‘total fertility rates’, “age-specific fertility rates”, ‘fertility determinants’ and combinations of each, as well as regional and country-specific terms to capture studies covering low-income and middle-income countries.Added value of this studyThis study provides a comprehensive descriptive analysis of the geographical heterogeneities in fertility rates within countries, by assembling subnational ASFRs and TFRs from nationally representative household surveys for a total of 932 administrative units in 70 low-income, lower-middle-income, and upper-middle-income countries. This study was the first to assemble such datasets at this scale, and to investigate geographical clustering of fertility and key determinants. Such heterogeneities are masked when examining only national-level data. The study also highlights areas of clustering of high and low fertility which cross country borders, as well as patterns of characteristics and behaviours in each cluster. The analyses explore how fertility clusters relate to fertility determinants, such as the use of contraception, women's secondary education or higher, and unmet needs for family planning. These findings highlight contiguous subnational geographical areas with similar characteristics, indicating that both subnational and national strategies are needed to reduce inequalities in access to contraception and education.Implications of all the available evidenceThis study can help inform the allocation of resources for family planning and women's education. The relationship between high-fertility clusters and areas of low use of contraception, unmet needs for family planning, and low rates of women's education emphasise the need for targeting and maximising impact in the context of resource scarcity. Resources should be targeted to areas where levels of unmet need for family planning are high, and where access to contraception and women's access to education are low. Interventions that respond to these subnational variations will enable progress towards the achievement of the UN Sustainable Development Goals.Global fertility rates have decreased considerably since the 1970s,In this study, firstly we aimed to identify statistically significant pockets of relative high fertility  and relative low fertility , by using local spatial autocorrelation statistics for subnational fertility rates across 70 low-income, lower-middle-income, and upper-middle-income countries as defined by the World BankWe did a subnational analysis of cross-sectional surveys collected between 2010 and 2016 to analyse fertility rates across 70 countries, including 932 administrative units in Africa, Asia, and Latin America. Fertility data from each administrative unit were obtained from the most recent publicly available and nationally representative Demographic and Health Surveys (DHS) and UNICEF Multiple Indicator Cluster Surveys (MICS). DHS and MICS are typically designed using a stratified two-stage probability sampling design, and provide nationally representative surveys for a number of low-income and middle-income countries. When appropriate data were available from both surveys, we used the most recent source. Data were gathered across 70 low-income, lower-middle-income and upper-middle-income countries. Only a subsample of low-income, lower-middle-income, and upper-middle-income countries is included in this work, as the DHS and MICS do not collect data for all countries . EthicalEstimates of ASFRs and TFRs were used to generate subnational ASFRs and TFRs using methods described by the DHS Guide to Statistics.We did spatial cluster analyses for the TFR indicator to test the presence or absence of significant spatial clustering across subnational areas in the dataset and to characterise those clusters in terms of the relationships among neighbouring subnational areas. Here, cluster refers to agglomerations of adjacent subnational areas, which can show, for example, areas of high concentrations of the observed characteristic surrounded by neighbouring areas with high rates (high–high); or, areas of low concentration surrounded by neighbours with low concentrations (low–low).I statistic . The global Moran's I statistic indicates whether spatial clustering of a characteristic exists across the area, compared with a null hypothesis of a spatially random distribution. In the context of this study, the spatial units used are administrative units and global Moran's I values closer to 0 indicate little or no spatial clustering, whereas positive values indicate spatial clustering where neighbouring units tend to have similar values, and negative values indicate that neighbouring units tend to have different values. We considered each of the 932 subnational regions as spatial units in the context of this study, and the values we assessed under spatial cluster analysis were TFR and key selected fertility determinants.We first assessed the degree of global spatial autocorrelation, or the degree to which one spatial unit shares similar or different characteristics with its neighbouring spatial unit, by using the Moran's We used the Gi* statisticI) and the local Getis-Ord Gi* hot spot analysis were done using GeoDa (version 1.12.1.161).I for the three regions), applied randomisation (using n=99 999 permutations), and specified a pseudo p value for clusters of less than 0·01 as the cutoff value. In this study, we did a separate spatial cluster analysis for each of the three regions to capture specific characteristics of each region; therefore, high-fertility clusters and low-fertility clusters are relative to each region. Although clusters are not comparable across regions , between-cluster patterns of ASFR can be compared across the regions. Within each cluster, we plotted the distribution of ASFRs and explored the variation of ASFR patterns between high-fertility clusters and low-fertility clusters. We also extracted from the surveys key selected determinants of fertility data , including the percentage of women with unmet needs for family planning (defined as the percentage of women who do not want to become pregnant but are not using contraception), the percentage of women using any contraceptive method, the percentage of women using modern contraception ,Both the spatial autocorrelation analysis  and created maps using Esri ArcGIS (version 10.7).The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.We analysed 70 nationally representative household surveys from the DHS and MICS for the period 2010–16. TFRs were heterogeneous across the three regions . HighestI , and inconsistencies in survey data might affect comparability, especially in areas where fertility rates are changing rapidly. Only a subsample of low-income and middle-income countries have been used in this study, due to the low availability of data from the DHS and the MICS surveys. This limitation could have influenced the clustering; high-fertility and low-fertility clusters are defined as relative to all areas included in the analysis. Furthermore, it was necessary to perform the cluster analysis by region to maintain comparability within region and to be able to identify cross-country hot spots and cold spots, but this approach meant that high-fertility clusters and low-fertility clusters have different magnitudes in each region. Including more data as they become available could affect the cluster analysis and will be a focus of ongoing work. Some limitations exist in the survey data that were used to construct the indicators. Data quality issues such as recall bias, misreporting, and omissions might arise when using fertility data based on birth history data, although a study done in 69 countries using 182 DHS surveys has shown that DHS fertility estimates are of good or acceptable quality in the majority of surveys.WorldPop project,Future work will aim to broaden the scope of the analyses by expanding the work to global scales, examining multitemporal trends, and integrating other sources of data, including high resolution birth maps and pregnancies for Africa, Latin America, and the Caribbean developed by the The input data used to produce this work are freely available after approval of registration and with a signed data access agreement on the websites of the data providers . The outputs produced in this paper can be made available upon request to the corresponding author.We declare no competing interests.