A common polymorphism in the Intelectin-1 gene influences mucus plugging in severe asthma.

By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC+ mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma.

Utility of eosinophil peroxidase as a biomarker of eosinophilic inflammation in asthma.

BACKGROUND: The relative utility of eosinophil peroxidase (EPX) and blood and sputum eosinophil counts as disease biomarkers in asthma is uncertain. OBJECTIVE: We sought to determine the utility of EPX as a biomarker of systemic and airway eosinophilic inflammation in asthma. METHODS: EPX protein was measured by immunoassay in serum and sputum in 110 healthy controls to establish a normal reference range and in repeated samples of serum and sputum collected during 3 years of observation in 480 participants in the Severe Asthma Research Program 3. RESULTS: Over 3 years, EPX levels in patients with asthma were higher than normal in 27% to 31% of serum samples and 36% to 53% of sputum samples. Eosinophils and EPX correlated better in blood than in sputum (rs values of 0.74 and 0.43, respectively), and high sputum EPX levels occurred in 27% of participants with blood eosinophil counts less than 150 cells/µL and 42% of participants with blood eosinophil counts between 150 and 299 cells/µL. Patients with persistently high sputum EPX values for 3 years were characterized by severe airflow obstruction, frequent exacerbations, and high mucus plug scores. In 59 patients with asthma who started mepolizumab during observation, serum EPX levels normalized in 96% but sputum EPX normalized in only 49%. Lung function remained abnormal even when sputum EPX normalized. CONCLUSIONS: Serum EPX is a valid protein biomarker of systemic eosinophilic inflammation in asthma, and sputum EPX levels are a more sensitive biomarker of airway eosinophilic inflammation than sputum eosinophil counts. Eosinophil measures in blood frequently miss airway eosinophilic inflammation, and mepolizumab frequently fails to normalize airway eosinophilic inflammation even though it invariably normalizes systemic eosinophilic inflammation.

Managing the unexpected: considerations for new pharmacy leaders.

In an effort to expedite the publication of articles, AJHP is posting manuscripts online as soon as possible after acceptance. Accepted manuscripts have been peer-reviewed and copyedited, but are posted online before technical formatting and author proofing. These manuscripts are not the final version of record and will be replaced with the final article (formatted per AJHP style and proofed by the authors) at a later time.

Voting with their feet: Primary care provider choice and its implications for public sector primary care services in India.

Expanding networks of government primary health centers (PHCs) to bring health services closer to communities is a longstanding policy objective in LMICs. In pluralistic health systems, where public and private providers compete for patients, PHCs are often not the preferred source for care. This study analyzes the market for primary care services in the Indian state of Bihar to understand how choice of primary care provider is influenced by distance, cost and quality of care. This study is based on linked surveys of rural households, PHCs, and private primary care providers conducted in 2019 and 2020. Most rural residents lived in proximity to a primary care provider, though not a qualified one. Within a 5-km distance, 60% of villages had a PHC, 90% had an informal provider, 35% an Indian systems of medicine practitioner, and 10% a private MBBS doctor. Most patients sought care from informal providers irrespective of PHC distance; only 25% of patients living in the PHC's vicinity sought care there. Reducing distance to the PHC by 1 km marginally increased the likehood of the PHC being selected, and reduced the likelihood of private clinics being selected. Reducing patient's costs at PHCs increased the likelihood of the PHC being selected and reduced the likelihood of private clinics and private hospitals being selected. Improved clinical quality at PHCs had no effect on patient selection of PHCs, private clinics, or hospitals. Illness severity reduced the likelihood of PHCs or private clinics being selected, and increased the likelihood of private hospitals selected. Wealthier patients were marginally more likely to use PHCs, substantially more likely to use private hospitals, and less likely to use private clinics. Expanding PHC network coverage or improving their quality of care is not sufficient to make PHCs more relevant to local health needs. An orientation towards essential public health functions, as well as, a community-centered approach to the organization of primary health care system is necessary.

A Novel Air Trapping Segment Score Identifies Opposing Effects of Obesity and Eosinophilia on Air Trapping in Asthma.

Rationale: Density thresholds in computed tomography (CT) lung scans quantify air trapping (AT) at the whole-lung level but are not informative for AT in specific bronchopulmonary segments. Objectives: To apply a segment-based measure of AT in asthma to investigate the clinical determinants of AT in asthma. Methods: In each of 19 bronchopulmonary segments in CT lung scans from 199 patients with asthma, AT was categorized as present if lung attenuation was less than -856 Hounsfield units at expiration in ⩾15% of the lung area. The resulting AT segment score (0-19) was related to patient outcomes. Measurements and Main Results: AT varied at the lung segment level and tended to persist at the patient and lung segment levels over 3 years. Patients with widespread AT (⩾10 segments) had more severe asthma (P < 0.05). The mean (±SD) AT segment score in patients with a body mass index ⩾30 kg/m2 was lower than in patients with a body mass index <30 kg/m2 (3.5 ± 4.6 vs. 5.5 ± 6.3; P = 0.008), and the frequency of AT in lower lobe segments in obese patients was less than in upper and middle lobe segments (35% vs. 46%; P = 0.001). The AT segment score in patients with sputum eosinophils ⩾2% was higher than in patients without sputum eosinophilia (7.0 ± 6.1 vs. 3.3 ± 4.9; P < 0.0001). Lung segments with AT more frequently had airway mucus plugging than lung segments without AT (48% vs. 18%; P ⩽ 0.0001). Conclusions: In patients with asthma, air trapping is more severe in those with airway eosinophilia and mucus plugging, whereas those who are obese have less severe trapping because their lower lobe segments are spared.

Self-organizing behaviors of cardiovascular cells on synthetic nanofiber scaffolds.

In tissues and organs, the extracellular matrix (ECM) helps maintain inter- and intracellular architectures that sustain the structure-function relationships defining physiological homeostasis. Combining fiber scaffolds and cells to form engineered tissues is a means of replicating these relationships. Engineered tissues' fiber scaffolds are designed to mimic the topology and chemical composition of the ECM network. Here, we asked how cells found in the heart compare in their propensity to align their cytoskeleton and self-organize in response to topological cues in fibrous scaffolds. We studied cardiomyocytes, valvular interstitial cells, and vascular endothelial cells as they adapted their inter- and intracellular architectures to the extracellular space. We used focused rotary jet spinning to manufacture aligned fibrous scaffolds to mimic the length scale and three-dimensional (3D) nature of the native ECM in the muscular, valvular, and vascular tissues of the heart. The representative cardiovascular cell types were seeded onto fiber scaffolds and infiltrated the fibrous network. We measured different cell types' propensity for cytoskeletal alignment in response to fiber scaffolds with differing levels of anisotropy. The results indicated that valvular interstitial cells on moderately anisotropic substrates have a higher propensity for cytoskeletal alignment than cardiomyocytes and vascular endothelial cells. However, all cell types displayed similar levels of alignment on more extreme (isotropic and highly anisotropic) fiber scaffold organizations. These data suggest that in the hierarchy of signals that dictate the spatiotemporal organization of a tissue, geometric cues within the ECM and cellular networks may homogenize behaviors across cell populations and demographics.

What made primary health care resilient against COVID-19? A mixed-methods positive deviance study in Nigeria.

INTRODUCTION: The SARS-CoV-2 (COVID-19) pandemic overwhelmed some primary health care (PHC) systems, while others adapted and recovered. In Nigeria, large, within-state variations existed in the ability to maintain PHC service volumes. Identifying characteristics of high-performing local government areas (LGAs) can improve understanding of subnational health systems resilience. METHODS: Employing a sequential explanatory mixed-methods design, we quantitatively identified 'positive deviant' LGAs based on their speed of recovery of outpatient and antenatal care services to prepandemic levels using service volume data from Nigeria's health management information system and matched them to comparators with similar baseline characteristics and slower recoveries. 70 semistructured interviews were conducted with LGA officials, facility officers and community leaders in sampled LGAs to analyse comparisons based on Kruk's resilience framework. RESULTS: A total of 57 LGAs were identified as positive deviants out of 490 eligible LGAs that experienced a temporary decrease in PHC-level outpatient and antenatal care service volumes. Positive deviants had an average of 8.6% higher outpatient service volume than expected, and comparators had 27.1% lower outpatient volume than expected after the initial disruption to services. Informants in 12 positive deviants described health systems that were more integrated, aware and self-regulating than comparator LGAs. Positive deviants were more likely to employ demand-side adaptations, whereas comparators primarily focused on supply-side adaptations. Barriers included long-standing financing and PHC workforce gaps. CONCLUSION: Sufficient flexible financing, adequate PHC staffing and local leadership enabled health systems to recover service volumes during COVID-19. Resilient PHC requires simultaneous attention to bottom-up and top-down capabilities connected by strong leadership.

Vaccination Utilization and Subnational Inequities during the COVID-19 Pandemic: An Interrupted Time-Series Analysis of Administrative Data across 12 Low- and Middle-Income Countries.

BACKGROUND: During and after the SARS-CoV-2 (COVID-19) pandemic, many countries experienced declines in immunization that have not fully recovered to pre-pandemic levels. This study uses routine health facility immunization data to estimate variability between and within countries in post-pandemic immunization service recovery for BCG, DPT1, and DPT3. METHODS: After adjusting for data reporting completeness and outliers, interrupted time series regression was used to estimate the expected immunization service volume for each subnational unit, using an interruption point of March 2020. We assessed and compared the percent deviation of observed immunizations from the expected service volume for March 2020 between and within countries. RESULTS: Six countries experienced significant service volume declines for at least one vaccine as of October 2022. The shortfall in BCG service volume was ~6% (95% CI -1.2%, -9.8%) in Guinea and ~19% (95% CI -16%, 22%) in Liberia. Significant cumulative shortfalls in DPT1 service volume are observed in Afghanistan (-4%, 95% CI -1%, -7%), Ghana (-3%, 95% CI -1%, -5%), Haiti (-7%, 95% CI -1%, -12%), and Kenya (-3%, 95% CI -1%, -4%). Afghanistan has the highest percentage of subnational units reporting a shortfall of 5% or higher in DPT1 service volume (85% in 2021 Q1 and 79% in 2020 Q4), followed by Bangladesh (2020 Q1, 83%), Haiti (80% in 2020 Q2), and Ghana (2022 Q2, 75%). All subnational units in Bangladesh experienced a 5% or higher shortfall in DPT3 service volume in the second quarter of 2020. In Haiti, 80% of the subnational units experienced a 5% or higher reduction in DPT3 service volume in the second quarter of 2020 and the third quarter of 2022. CONCLUSIONS: At least one region in every country has a significantly lower-than-expected post-pandemic cumulative volume for at least one of the three vaccines. Subnational monitoring of immunization service volumes using disaggregated routine health facility information data should be conducted routinely to target the limited vaccination resources to subnational units with the highest inequities.

Vaccine hesitancy among healthcare workers in low- and middle-income countries during the COVID-19 pandemic: Results from facility surveys across six countries.

BACKGROUND: Vaccine hesitancy remains a critical barrier in mitigating the effects of the ongoing COVID-19 pandemic. The willingness of health care workers (HCWs) to be vaccinated, and, in turn, recommend the COVID-19 vaccine for their patient population is an important strategy. This study aims to understand the uptake of COVID-19 vaccines and the reasoning for vaccine hesitancy among facility-based health care workers (HCWs) in LMICs. METHODS: We conducted nationally representative phone-based rapid-cycle surveys across facilities in six LMICs to better understand COVID-19 vaccine hesitancy. We gathered data on vaccine uptake among facility managers, their perceptions of vaccine uptake and hesitancy among the HCWs operating in their facilities, and their perception of vaccine hesitancy among the patient population served by the facility. RESULTS: 1,148 unique public health facilities participated in the study, with vaccines being almost universally offered to facility-based respondents across five out of six countries. Among facility respondents who have been offered the vaccine, more than 9 in 10 survey respondents had already been vaccinated at the time of data collection. Vaccine uptake among other HCWs at the facility was similarly high. Over 90% of facilities in Bangladesh, Liberia, Malawi, and Nigeria reported that all or most staff had already received the COVID-19 vaccine when the survey was conducted. Concerns about side effects predominantly drive vaccine hesitancy in both HCWs and the patient population. CONCLUSION: Our findings indicate that the opportunity to get vaccinated in participating public facilities is almost universal. We find vaccine hesitancy among facility-based HCWs, as reported by respondents, to be very low. This suggests that a potentially effective effort to increase vaccine uptake equitably would be to channel promotional activities through health facilities and HCWs.However, reasons for hesitancy, even if limited, are far from uniform across countries, highlighting the need for audience-specific messaging.

Fibre-infused gel scaffolds guide cardiomyocyte alignment in 3D-printed ventricles.

Hydrogels are attractive materials for tissue engineering, but efforts to date have shown limited ability to produce the microstructural features necessary to promote cellular self-organization into hierarchical three-dimensional (3D) organ models. Here we develop a hydrogel ink containing prefabricated gelatin fibres to print 3D organ-level scaffolds that recapitulate the intra- and intercellular organization of the heart. The addition of prefabricated gelatin fibres to hydrogels enables the tailoring of the ink rheology, allowing for a controlled sol-gel transition to achieve precise printing of free-standing 3D structures without additional supporting materials. Shear-induced alignment of fibres during ink extrusion provides microscale geometric cues that promote the self-organization of cultured human cardiomyocytes into anisotropic muscular tissues in vitro. The resulting 3D-printed ventricle in vitro model exhibited biomimetic anisotropic electrophysiological and contractile properties.

Adverse birth outcomes among women with 'low-risk' pregnancies in India: findings from the Fifth National Family Health Survey, 2019-21.

Background: Despite substantial progress in improving maternal and newborn health, India continues to experience high rates of newborn mortality and stillbirths. One reason may be that many births happen in health facilities that lack advanced services-such as Caesarean section, blood transfusion, or newborn intensive care. Stratification based on pregnancy risk factors is used to guide 'high-risk' women to advanced facilities. To assess the utility of risk stratification for guiding the choice of facility, we estimated the frequency of adverse newborn outcomes among women classified as 'low risk' in India. Methods: We used the 2019-21 Fifth National Family Health Survey (NFHS-5)-India's Demographic and Health Survey-which includes modules administered to women aged 15-49 years. In addition to pregnancy history and outcomes, the survey collected a range of risk factors, including biomarkers. We used national obstetric risk guidelines to classify women as 'high risk' versus 'low risk' and assessed the frequency of stillbirths, newborn deaths, and unplanned Caesarean sections for the respondent's last pregnancy lasting 7 or more months in the past five years. We calculated the proportion of deliveries occurring at non-hospital facilities in all the Indian states. Findings: Using data from nearly 176,699 recent pregnancies, we found that 46.6% of India's newborn deaths and 56.3% of stillbirths were among women who were 'low risk' according to national guidelines. Women classified as 'low risk' had a Caesarean section rate of 8.4% (95% CI 8.1-8.7%), marginally lower than the national average of 10.0% (95% CI 9.8-10.3%). In India as a whole, 32.0% (95% CI 31.5-32.5%) of deliveries occurred in facilities that were likely to lack advanced services. There was substantial variation across the country, with less than 5% non-hospital public facility deliveries in Punjab, Kerala, and Delhi compared to more than 40% in Odisha, Madhya Pradesh, and Rajasthan. Newborn mortality tended to be lower in states with highest hospital delivery rates. Interpretation: Individual risk stratification based on factors identified in pregnancy fails to accurately predict which women will have delivery complications and experience stillbirth and newborn death in India. Thus a determination of 'low risk' should not be used to guide women to health facilities lacking key life saving services, including Caesarean section, blood transfusion, and advanced newborn resuscitation and care. Funding: Bill and Melinda Gates Foundation and the World Bank. The findings, interpretations and conclusions expressed in the paper are entirely those of the authors, and do not represent the views of the Gates Foundation or of the World Bank, its Executive Directors, or the countries they represent.

Availability and use of personal protective equipment in low- and middle-income countries during the COVID-19 pandemic.

BACKGROUND: Availability and appropriate use of personal protective equipment (PPE) is of particular importance in Low and Middle-Income countries (LMICs) where disease outbreaks other than COVID-19 are frequent and health workers are scarce. This study assesses the availability of necessary PPE items during the COVID-19 pandemic at health facilities in seven LMICs. METHODS: Data were collected using a rapid-cycle survey among 1554 health facilities in seven LMICs via phone-based surveys between August 2020 and December 2021. We gathered data on the availability of World Health Organization (WHO)-recommended PPE items and the use of items when examining patients suspected to be infected with COVID-19. We further investigated the implementation of service adaptation measures in a severe shortage of PPE. RESULTS: There were major deficiencies in PPE availability at health facilities. Almost 3 out of 10 health facilities reported a stock-out of medical masks on the survey day. Forty-six percent of facilities did not have respirator masks, and 16% did not have any gloves. We show that only 43% of health facilities had sufficient PPE to comply with WHO guidelines. Even when all items were available, healthcare workers treating COVID-19 suspected patients were reported to wear all the recommended equipment in only 61% of health facilities. We did not find a statistically significant difference in implementing service adaptation measures between facilities experiencing a severe shortage or not. CONCLUSION: After more than a year into the COVID-19 pandemic, the overall availability of PPE remained low in our sample of low and middle-income countries. Although essential, the availability of PPE did not guarantee the proper use of the equipment. The lack of PPE availability and improper use of available PPE enable preventable COVID-19 transmission in health facilities, leading to greater morbidity and mortality and risking the continuity of service delivery by healthcare workers.

RUNX1 mutations mitigate quiescence to promote transformation of hematopoietic progenitors in Fanconi anemia.

Many inherited bone marrow failure syndromes (IBMFSs) present a high risk of transformation to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). During transformation of IBMFSs, hematopoietic stem and progenitor cells (HSPCs) with poor fitness gain ectopic, dysregulated self-renewal secondary to somatic mutations via undefined mechanisms. Here, in the context of the prototypical IBMFS Fanconi anemia (FA), we performed multiplexed gene editing of mutational hotspots in MDS-associated genes in human induced pluripotent stem cells (iPSCs) followed by hematopoietic differentiation. We observed aberrant self-renewal and impaired differentiation of HSPCs with enrichment of RUNX1 insertions and deletions (indels), generating a model of IBMFS-associated MDS. We observed that compared to the failure state, FA MDS cells show mutant RUNX1-mediated blunting of the G1/S cell cycle checkpoint that is normally activated in FA in response to DNA damage. RUNX1 indels also lead to activation of innate immune signaling, which stabilizes the homologous recombination (HR) effector BRCA1, and this pathway can be targeted to abrogate viability and restore sensitivity to genotoxins in FA MDS. Together, these studies develop a paradigm for modeling clonal evolution in IBMFSs, provide basic understanding of the pathogenesis of MDS, and uncover a therapeutic target in FA-associated MDS.

Resilience of front-line facilities during COVID-19: evidence from cross-sectional rapid surveys in eight low- and middle-income countries.

Responsive primary health-care facilities are the foundation of resilient health systems, yet little is known about facility-level processes that contribute to the continuity of essential services during a crisis. This paper describes the aspects of primary health-care facility resilience to coronavirus disease 2019 (COVID-19) in eight countries. Rapid-cycle phone surveys were conducted with health facility managers in Bangladesh, Burkina Faso, Chad, Guatemala, Guinea, Liberia, Malawi and Nigeria between August 2020 and December 2021. Responses were mapped to a validated health facility resilience framework and coded as binary variables for whether a facility demonstrated capacity in eight areas: removing barriers to accessing services, infection control, workforce, surge capacity, financing, critical infrastructure, risk communications, and medical supplies and equipment. These self-reported capacities were summarized nationally and validated with the ministries of health. The analysis of service volume data determined the outcome: maintenance of essential health services. Of primary health-care facilities, 1,453 were surveyed. Facilities maintained between 84% and 97% of the expected outpatient services, except for Bangladesh, where 69% of the expected outpatient consultations were conducted between March 2020 and December 2021. For Burkina Faso, Chad, Guatemala, Guinea and Nigeria, critical infrastructure was the largest constraint in resilience capabilities (47%, 14%, 51%, 9% and 29% of facilities demonstrated capacity, respectively). Medical supplies and equipment were the largest constraints for Liberia and Malawi (15% and 48% of facilities demonstrating capacity, respectively). In Bangladesh, the largest constraint was workforce and staffing, where 44% of facilities experienced moderate to severe challenges with human resources during the pandemic. The largest constraints in facility resilience during COVID-19 were related to health systems building blocks. These challenges likely existed before the pandemic, suggesting the need for strategic investments and reforms in core capacities of comprehensive primary health-care systems to improve resilience to future shocks.

Changes in clinical markers observed from pharmacist-managed cardiovascular risk reduction clinics in federally qualified health centers: A retrospective cohort study.

BACKGROUND: Reductions in hemoglobin A1c (HbA1C) have been associated with improved cardiovascular outcomes and savings in medical expenditures. One public health approach has involved pharmacists within primary care settings. The objective was to assess change in HbA1C from baseline after 3-5 months of follow up in pharmacist-managed cardiovascular risk reduction (CVRR) clinics. METHODS: This retrospective cohort chart review occurred in eight pharmacist-managed CVRR federally qualified health clinics (FQHC) in Indiana, United States. Data were collected from patients seen by a CVRR pharmacist within the timeframe of January 1, 2015 through February 28, 2020. Data collected include: demographic characteristics and clinical markers between baseline and follow-up. HbA1C from baseline after 3 to 5 months was assessed with pared t-tests analysis. Other clinical variables were assessed and additional analysis were performed at 6-8 months. Additional results are reported between 9 months and 36 months of follow up. RESULTS: The primary outcome evaluation included 445 patients. Over 36 months of evaluation, 3,803 encounters were described. Compared to baseline, HbA1C was reduced by 1.6% (95%CI -1.8, -1.4, p<0.01) after 3-5 months of CVRR care. Reductions in HbA1C persisted at 6-8 months with a reduction of 1.8% ([95%CI -2.0, -1.5] p<0.01). The follow-up losses were 29.5% at 3-5 months and 93.2% at 33-36 months. CONCLUSIONS: Our study augments the existing literature by demonstrating the health improvement of pharmacist-managed CVRR clinics. The great proportion of loss to follow-up is a limitation of this study to be considered. Additional studies exploring the expansion of similar models may amplify the public health impact of pharmacist-managed CVRR services in primary care sites.

Development of transgenic composite Stylosanthes plants to study root growth regulated by a ß-expansin gene, SgEXPB1, under phosphorus deficiency.

KEY MESSAGE: A highly efficient transformation procedure to generate transgenic Stylosanthes roots was established. SgEXPB1 is involved in Stylosanthes root growth under phosphorus deficiency. Stylo (Stylosanthes spp.) is an important forage legume widely applied in agricultural systems in the tropics. Due to the recalcitrance of stylo genetic transformation, functional characterization of candidate genes involved in stylo root growth is limited. This study established an efficient procedure for Agrobacterium rhizogenes-mediated transformation for generating transgenic composite plants of S. guianensis cultivar 'Reyan No. 5'. Results showed that composite stylo plants with transgenic hairy roots were efficiently generated by A. rhizogenes strains K599 and Arqual, infecting the residual hypocotyl at 1.0 cm of length below the cotyledon leaves of 9-d-old seedlings, leading to a high transformation efficiency of > 95% based on histochemical ß-glucuronidase (GUS) staining. Notably, 100% of GUS staining-positive hairy roots can be achieved per composite stylo plant. Subsequently, SgEXPB1, a ß-expansin gene up-regulated by phosphorus (P) deficiency in stylo roots, was successfully overexpressed in hairy roots. Analysis of hairy roots showed that root growth and P concentration in the transgenic composite plants were increased by SgEXPB1 overexpression under low-P treatment. Taken together, a highly efficient A. rhizogenes-mediated transformation procedure for generating composite stylo plants was established to study the function of SgEXPB1, revealing that this gene is involved in stylo root growth during P deficiency.