Using a retrospective cohort design with patients in the Department of Veterans Affairs healthcare system, we use doubly robust regression and matching to estimate the association between baseline use of α1-AR antagonists and likelihood of death due to COVID-19 during hospitalization. Having an active prescription for any α1-AR antagonist  at the time of admission had a significant negative association with in-hospital mortality  and death within 28 days of admission . In a subset of patients on doxazosin specifically, an inhibitor of all three alpha-1 adrenergic receptors, we observed a relative risk reduction for death of 74%  compared to matched controls not on any α1-AR antagonist at the time of admission. These findings suggest that use of α1-AR antagonists may reduce mortality in COVID-19, supporting the need for randomized, placebo-controlled clinical trials in patients with early symptomatic infection.Effective therapies for coronavirus disease 2019 (COVID-19) are urgently needed, and pre-clinical data suggest alpha-1 adrenergic receptor antagonists (α The viral replication phase in Coronavirus disease 2019 (COVID-19) can be followed by a hyperinflammatory host immune response, hereafter referred to as COVID-19-associated hyperinflammation, which can lead to acute respiratory distress syndrome (ARDS), multiorgan dysfunction, and death despite maximal supportive care . While dde novo and signal in an autocrine/paracrine self-regulatory fashion  (1-AR) antagonist that inhibits all three receptor subtypes  can prevent cytokine storm and death by blocking deleterious catecholamine signaling and immune responses  compared to non-users  are monoamine hormones that signal through adrenergic receptors (ARs) expressed on tissues including cells of the immune system –10. Cell fashion , 11. Bey, MIP-2) , 10–12. on-users . Similarammation . We thernfection , but dat1-AR antagonists with in-hospital mortality in patients with COVID-19. Here, we analyzed a large cohort of patients hospitalized at Veterans Health Administration (VA) hospitals, in whom α1-AR antagonists are commonly used to treat unrelated diseases such as benign prostatic hyperplasia (BPH), post-traumatic stress disorder (PTSD), or arterial hypertension  criteria .1-AR antagonists at the time of admission for the index hospitalization. Active prescriptions of α1-AR antagonists  were identified and defined by the patient having medication on hand on the day of the index admission, regardless of dosage. Secondary analyses examined the effect of tamsulosin  and doxazosin  individually. Finally, with in-hospital therapies evolving during the pandemic, we repeated the analysis by week and VA hospital to ensure results were not driven by any particular time or location.The primary outcomes were death during the index hospitalization and death within 28 days of admission. The primary exposure variable was the use of αWe obtained data on patient demographics, vital signs, and prescription drugs from the VA's corporate data warehouse (CDW). Patient comorbidities were captured based on the International Classification of Diseases, Version 10 codes from VA care in the year prior to index admission. Other physiologic variables, including oxygen saturation and temperature, were defined at time of inpatient admission.1-AR antagonist prescriptions to all other patients with COVID-19 using Fisher's exact-test. We then estimated propensity scores and trimmed the sample to ensure overlap in the propensity score distributions of the exposed and unexposed groups. On this reduced sample, the adjusted analysis used inverse propensity-weighted logistic regression adjusting for patient age at admission , calendar week, location of hospitalization, and comorbidities diagnosed any time in the two years prior to the index inpatient stay. This approach is “doubly robust” in that it uses the observed confounders in both the calculation of the propensity score and the odds ratios. Comorbidities included in the matching procedure were diabetes mellitus, arterial hypertension, heart failure, ischemic heart disease, acute myocardial infarction, chronic obstructive pulmonary disease (COPD), end-stage renal disease (ESRD), and PTSD. We also included an indicator variable for oxygen saturation under 94 percent on the day of admission.Analyses followed the methodology of a companion paper examining patients with acute respiratory distress and pneumonia . UnadjusAll of the control variables reflect information on patients prior to admission with COVID-19. As noted above, we controlled for secular changes in COVID-19 care using calendar week, starting with February 20, 2020. We chose not to examine endpoints during the hospital stay, such as use of a ventilator or admission to the ICU, given this is based on physician coding or data structures that we cannot assure were handled uniformly, especially during surges. We also chose not to control for processes of care during the stay given this could introduce bias in the analysis.p-values. We also present relative risk reductions (RRR) for the matched cohorts, and the pre- and post-matching balance of covariates is shown in We then conducted a 5:1 matched analysis using the same covariates as the adjusted model . This ap1-AR antagonist at time of admission. Of those taking α1-AR antagonists, 73% of patients were on tamsulosin , 12% on terazosin (n = 679), 10% on prazosin (n = 581), 4% on doxazosin (n = 215), 3% on alfuzosin (n = 186), and <1% were on silodosin (n = 5)  . One hun1-AR antagonist, compared to non-users, had an 18% relative risk reduction for death during their hospitalization , in-hospital mortality was 6% overall and 5.5% in our sample. Patients with confirmed COVID-19 taking any αn = 25,130). The bottom panel expands the denominator to also include patients with suspected COVID-19 . The dark green odds ratios in 1-AR antagonists, while the lighter green represent doxazosin. Results were similar for the suspected COVID-19 sample. Patients taking any α1-AR antagonists, compared to non-users, had an 20% relative risk reduction for death (p ≤ 0.001) in this cohort , than for a pooled population of patients treated with any α1-AR antagonist in whom tamsulosin was the most common drug (72%). This was similarly true for patients treated exclusively with tamsulosin, a “uroselective” α1-AR antagonist on α1A- and α1D-ARs without clinically relevant inhibition of α1B-ARs expressed by immune cells and the peripheral vasculature  may be required to effectively interrupt autocrine and paracrine catecholamine signaling in monocytes and other immune cells that enhance inflammatory injury  may therefore be consistent with a redundancy in catecholamine signaling pathways which are globally inhibited by doxazosin, whereas tamsulosin allows for continued signaling through the α1B-AR. These findings have practical implications for the selection of α1-AR antagonists for the prevention of inflammatory injury and suggest that the immunomodulatory benefits may not be uncoupled from inhibition of α1B-ARs expressed on the peripheral vasculature.Immune cells can induce expression of all three α α1B-ARs , and cat α1B-ARs . As suchy injury , 20. Indsyndrome . The mar1-AR blockade in the prevention of inflammatory and autoimmune injury. In a model of encephalitis, early α1-AR inhibition reversed neutrophil infiltration in lungs and prevented hemorrhagic pulmonary edema  may reduce in-hospital and 28-day mortality in patients with COVID-19 and highlight the need for randomized placebo-controlled clinical trials to examine the efficacy of α1-AR antagonists for improving survival and preventing adverse outcomes from COVID-19. Importantly, α1-AR antagonists are inexpensive, administered orally, do not require refrigeration, and have a well-established safety profile. Thus, if trials confirm these results, α1-AR antagonists could be widely deployed to reduce mortality from inflammatory injury. Importantly, α1-AR antagonists are immunomodulatory, but not immunosuppressive drugs. Long-term use of doxazosin does not appear to be associated with the development of opportunistic infection in human studies  which confer an increased risk of opportunistic infection.Our results suggest that inhibition of catecholamine signaling with doxazosin  at the time of admission. Among different α1-AR antagonists, doxazosin was associated with a 74% relative risk reduction for death, while tamsulosin had a more modest 18% relative risk reduction for death. A clinical trial testing the efficacy and safety of α1-AR antagonists such as doxazosin to prevent hyperinflammation and reduce mortality in COVID-19 would appear warranted.In summary, patients hospitalized with COVID-19 had lower odds of in-hospital and 28-day death if they had an active prescription for any αThe original contributions generated in the study are included in the article/The studies involving human participants were reviewed and approved by Stanford IRB. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.JV, BV, CB, and MK conceived of the idea. LR, LG, AK, MP, and RX conducted statistical analyses and results presentation. AK, MP, RX, ZS, and SA developed the methodology. LR, MK, JV, and TW wrote the manuscript with input from all authors. MK and TW are co-senior authors. All authors reviewed the final manuscript.In 2017, The Johns Hopkins University (JHU) filed a patent application on the use of various drugs to prevent cytokine release syndromes, on which BV and KK are listed as inventors. JHU will not assert patent rights from this filing for treatment related to COVID-19. MK received personal fees from Bristol-Myers Squibb and Celltrion, unrelated to the manuscript. BV and KK are founders of and hold equity in Thrive Earlier Detection. KK is a consultant to and is on the Board of Directors of Thrive Earlier Detection. BV and KK are founders of, hold equity in, and serve as consultants to Personal Genome Diagnostics. KK and BV are consultants to Sysmex, Eisai, and CAGE Pharma and hold equity in CAGE Pharma. BV is also a consultant to Nexus. KK and BV are consultants to and hold equity in NeoPhore. CB is a consultant to Depuy-Synthes and Bionaut Pharmaceuticals. CB, BV, and KK are also inventors on technologies unrelated or indirectly related to the work described in this article. Licenses to these technologies are or will be associated with equity or royalty payments to the inventors, as well as to JHU. The terms of all these arrangements are being managed by JHU in accordance with its conflict of interest policies. SA is an advisor and holds an equity stake in two private companies, Prealize  and Dr. Consulta (Brazil). Prealize is a healthcare analytics company, and Dr. Consulta operates a chain of low-cost medical clinics in Brazil. The remaining 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.


In the sub-analysis, the score improved significantly for the group with families (p = 0.050). Improvement was observed in the group living alone, but the difference was not significant (p = 0.428). The shopping rehabilitation intervention improved the Kihon Checklist score. Continuous observations and research are necessary to measure the long-term effects of shopping rehabilitation and the mechanisms that foster their maintenance and effects.In an aged society, the deterioration of physical and cognitive functions is prevalent. To motivate the rehabilitation of older persons, an initiative known as “shopping rehabilitation” incorporates shopping as an element of a nudge. The purpose of this study was to clarify motor function changes and cognitive functions of participants during shopping rehabilitation, through a semi-experimental study. We measured changes in the Kihon Checklist score before and after rehabilitation interventions. A paired t-test was used to analyze changes in the overall score of the basic checklist before and after the rehabilitation intervention. In December 2020, 59 participants answered the Kihon Checklist after their shopping rehabilitation intervention. During the 6-month intervention period, the number of participants with a checklist score of 8 or higher was significantly reduced after the intervention ( In aging societies, the deterioration of physical and cognitive functions of older persons has become a paramount concern, which is related to older people’s motivation to engage in rehabilitation. Several systematic reviews of older people aged 65+ years) report that regular exercise improves physical and cognitive functions ,2,3. Pro+ years rAs a method to overcome this issue, the concept of a nudge for human behavioral changes is becoming popular. A nudge is a strategy that changes human behavior based on behavioral science, with concepts such as “softly boosting” and “pushing lightly with elbows” . Nudge-bIn addition, a meta-analysis summarized the effects on physical activity of the game application “Pokémon GO”, which is a nudge-based intervention that uses gamification. The app allows users to capture characters by walking and using the GPS function of their smartphone. The motivation to capture a character is used as a nudge. The game increases walking time, distance, and the number of steps per day among its players . These eShopping can be used as a nudge for rehabilitation, which could drive rehabilitation among older people. This is known as “shopping rehabilitation”, which allows older people to enjoy shopping and naturally engage in rehabilitation . In thisIn rural areas, stores may be relatively far from places of residence, which can limit older people’s ability to go shopping ,12. ResiShopping rehabilitation that includes transportation systems is expected to relieve people who cannot use transportation easily and revitalize the local economy. Shopping rehabilitation for older people and the mechanisms underlying its effects have not been clarified. Therefore, we hypothesized that shopping rehabilitation would improve the cognitive and motor functions of older persons. Clarification of such effects could drive rehabilitation in rural communities with collaboration among rural shops and transportation systems, which could additionally improve rural economies. Therefore, this study clarified the effects of shopping rehabilitation on the motor and cognitive functions of older persons.This study used a semi-experimental method with older people who participated in a shopping rehabilitation program in Unnan City, Japan, from June to December 2020.Unnan City is one of the most rural cities in Japan. It is located to the southeast of the Shimane Prefecture. In 2020, the total population of Unnan was 37,638 , with 39% of residents aged over 65 years; this number is expected to reach 50% by 2025 .Marcherises, where this study was conducted, is the largest commercial facility in Unnan City, and many older people visit the area daily for shopping. At Hikari Salon Unnan, an initiative known as “shopping rehabilitation” is being carried out in its commercial facility.The participants were recruited in Unnan City. Information regarding the shopping rehabilitation was provided via brochures and local newspapers to eligible people. Those eligible for shopping rehabilitation had one or more Kihon Checklist scores available; the Japanese government constructed the checklist to detect people that required possible care . The eliRegarding the shopping rehabilitation, 95% of the participants used shuttle cars to travel from their homes to commercial facilities. After clerks performed health checks, such as assessments of blood pressure, pulse rate, and temperature in the reception area, nine to 12 participants moved around the facility while following the instructions of two occupational therapists (OT). For this, a uniquely crafted shopping cart was used. This cart is manufactured by Hikari project Co., Ltd which is located at Unnan City, Shimane, Japan. and designed to promote effective rehabilitative movements during shopping. The Japanese government registered this cart as safe. Participants move their bodies up and down to pick up the product they want to purchase under the direction of the OT. In addition, the OT monitored participants’ postures during movements and guided them to make their exercises effective. Rehabilitation using the special shopping cart was performed for 30 min, followed by 60 min of gymnastics conducted in the Hikari Salon on the second floor which can accommodate more than 20 participants, followed by a second 30 min shopping session. Therefore, one complete session lasted approximately 120 min in total. The shopping rehabilitation was held four times a month on average. For 10 min, OTs instructed the participants regarding home exercises . A logicChanges in Kihon Checklist scores before and after shopping rehabilitation, which were shown to correlate with prognosis, were measured . The KihValues before and after participation in shopping rehabilitation were used as the independent variable.We used a questionnaire to collect the background data of participants. The contents were age, gender , medical history, family structure (living with family or living by oneself), dependency status , level of dementia , housing environment (detached house or apartment), and history of smoking and drinking. Based on the participant’s medical history, a Charlson Comorbidity Index (CCI) score was calculated for each participant to assess the severity of their medical conditions . This inp-value < 0.05. All statistical analyses were performed using EZR , a graphical user interface for R  [The necessary sample size was calculated as 34 participants, given 80% statistical power and 5% type 1 error to detect a difference of 0.1 points between pre-and post-intervention groups, with standard deviation of 0.2 in parted t-test. The participants were categorized into two groups—those with families and those living independently—to investigate the differences in demographic data related to isolation. Student’s t-test was used to assess parametric data, and the Mann–Whitney U test was performed on non-parametric data. Numerical variables were dichotomized, namely care dependency (≥1 and 0) and the dementia scale (≥1 and 0). Differences were assessed in the pre-and post-intervention proportions of total Kihon Checklist scores ≥ 8 . Cases wAustria) .The hospital was assured of the anonymity and confidentiality of patients’ information. Details of this study were posted on the hospital website without disclosing any details concerning the patients. To address any questions regarding this study, the contact information of the hospital representative was also listed on the website. The purpose of this study was explained to all participants, and informed consent was obtained by the clerks of shopping rehabilitation. The Clinical Ethics Committee of our institution approved this study .In June 2020, 72 older people participated in shopping rehabilitation and answered the Kihon Checklist. Finally, 59 participants answered the Kihon Checklist in December 2020 after the intervention. Consent was obtained from all participants during this period. The mean age of participants in this study was 86.32 years (standard deviation = 4.67), with 93.2% being female. There was no significant difference in the background data between groups living independently and with families .p = 0.050). In the sub-analysis, the score improved significantly for the group with families (p = 0.050). Improvement was observed in the group living alone, but the difference was not significant (p = 0.428; During the 6-month intervention period, the number of participants with a checklist score of 8 or higher was significantly reduced after the intervention (This study shows that incorporating the element of shopping into rehabilitation can improve the Kihon Checklist score. In addition, as the proportion of persons with a total score ≥ 8 decreases, shopping rehabilitation can improve the independence period and survival rate. The element of shopping can enhance the participation of older people who do not go shopping by themselves, improve their daily activities, and reduce the risk of frailty, leading to a better quality of life.The improvement of the Kihon Checklist score shows the effectiveness of shopping rehabilitation, depending on living situations. Shopping rehabilitation contains physical and cognitive training factors during shopping, which can contribute to improving the score. Furthermore, participants with families improved their scores significantly. This improvement might correlate with their living conditions. Older people often receive help from their families, so they may lose the opportunity to go shopping by themselves ,21. In rIn contrast, in this study, a higher proportion of participants living alone had a checklist score of 8 or higher and did not improve in physical or cognitive function through the shopping rehabilitation. Since such persons lack help in their daily life, they might not collect their utilities and groceries themselves. Furthermore, isolation can promote health issues such as frailty. This can limit an individual’s ability to travel and communicate with others, which might lead to further deterioration in their physical and cognitive functions . This reThe possibility of improving the independence period and survival rate through shopping rehabilitation, as shown in this research, can be critical to sustaining aged societies. Rehabilitation itself can be effective for supporting people with disabilities in medical facilities. Most rehabilitation is performed in medical facilities, but the need for physical and cognitive rehabilitation can be high in communities, even in rural areas ,29,30. SIn this research, most participants were female, which is likely related to their previous routine in a rural context. In rural regions, the woman’s role is often housework, including purchasing groceries . HoweverIn different contexts, older people often lose their routines. As with shopping rehabilitation, regaining these routines as a nudge could revitalize older people’s activities, which could help them sustain their roles in families, leading to the preservation of their physical and cognitive functions. The investigation of lost routines among older people in communities should be studied to find new ways to revitalize such persons.Furthermore, during the COVID-19 pandemic, rural older people could not visit various destinations because of the risk and fear of infection. COVID-19 reduced the number of activities people could undertake outside of their houses, which could cause older people’s frailty to worsen ,35. VariThis study had several limitations. First, as it was performed in a single shopping mall located in a Japanese rural area, the study’s setting may not be applicable to older people in all developing and developed countries. However, the results are applicable to rural areas with a lack of social resources, aged societies, and isolated older people. Future studies should investigate these constructs in other rural settings, such as remote islands or developing countries. Another limitation pertains to the sampling method. Possible confounding factors were considered in this study; however, the randomization of the sampling process could further address potential confounds. Therefore, future studies should implement randomization to overcome this limitation.The shopping rehabilitation intervention improved Kihon Checklist scores. Continuous observation and research are needed to measure the long-term effects of shopping rehabilitation on participants, regarding not only improvements in physical and cognitive abilities but also loneliness, the mechanisms that foster health maintenance, and other potential impacts.


Fusarium oxysporum, F. ipomoeae, F. solani, Penicillium citrinum, P. rotoruae, Aspergillus wentii, Mucor variicolumellatus (Mu. circinelloides species complex), and Macrophomina phaseolina. F. oxysporum was the predominant pathogen as this is the most common pathogen of sweet potato storage roots causing the surface rot disease, and M. phaseolina caused the most severe disease among the pathogens. Dual culture antagonistic assays were evaluated using Trichoderma harzianum strains CMML20–26 and CMML20–27. The results revealed that the two strains showed strong antifungal activity in different ranges against all tested pathogens. This study provides an understanding of diverse postharvest diseases in sweet potatoes and suggests potential biocontrol agents to manage the diseases. In addition, this is the first report of sweet potato storage root rot diseases caused by A. wentii, and P. rotoruae worldwide.Sweet potato is the 11th most important food crop in the world and an excellent source of nutrition. Postharvest diseases were monitored in sweet potato storage roots collected from the local markets in Korea during 2021. Several diseases including Fusarium surface and root rot, charcoal rot, dry rot, and soft rot were observed in the postharvest sweet potatoes. A total of 68 fungal isolates were obtained from the diseased samples, and the isolates were grouped into 8 different fungal colony types. Based on multilocus phylogeny and morphological analysis of 17 representative isolates, the isolates were identified as  Ipomoea batatas (L.) Lam.; Convolvulaceae) is regarded as one of the most important food crops in the world and is an alternative source of bioenergy, with an annual production area of 8.0 million hectares and a total global production of 106,569 million tons [The sweet potato , dry rot (A. niger and Diaporthe batatas), Fusarium surface rot (F. oxysporum), Fusarium root and end rot (F. solani), foot rot (Plenodomus destruens), soft rot (Rhizopus stolonifer and Rhizopus oryzae), blue mold (Penicillium spp.), java black rot (Botryodiplodia theobromae), circular spot (Sclerotium rolfsii), charcoal rot (M. phaseolina), and storage rot (Mucor sp.) [Fusarium spp. and M. phaseolina can survive in crop residue and soil from one season to another and enter the storage roots through wounding, causing diseases postharvest [F. solani, M. phaseolina, B. theobromae, and D. batatas [There are several biotic and abiotic factors limiting the production and commercialization of sweet potatoes. Among them, fungal diseases are the most prominent before and after harvest; fungi reduce the quality of storage roots ,12. The cor sp.) ,16,17,18tharvest . Other h batatas ,19,20.Cultural practices are generally applied to overcome or minimize the problems associated with the storage of sweet potatoes. In addition, chemicals can also be applied to remove pathogens from storage roots ,22. HoweT. harzianum strains.In the present study, sweet potato samples were collected from the local markets of three locations in Korea, and different symptoms of postharvest diseases were observed. Therefore, this study aimed (i) to investigate the postharvest fungal diseases of sweet potato in Korea and characterize the causal fungal isolates by molecular phylogenetic and morphological analyses and pathogenicity assay, and (ii) to test in vitro biocontrol activity against the fungal pathogens by two −1 of streptomycin, rifampicin, and kanamycin  to stop bacterial growth. After incubation at 25 °C for 3–10 days, individual hyphal tips of the developing fungal colonies were placed onto PDA and further incubated for 5–10 days for culture purity. Finally, representative isolates were selected, assigned an identification number (CMML21–1 to CMML21–17), and preserved in the Molecular Microbiology Lab, Dept. of Integrative Food, Bioscience and Biotechnology, Chonnam National University, Gwangju, Republic of Korea  supplemented with 50 μg mLof Korea . The funEF1–α), calmodulin (CAL), RNA polymerase II second largest subunit (RPB2), small subunit , and β-tubulin (BT) genes were amplified using the primer pairs ITS1–ITS4, EF1 728F–EF1 986R, CAL1–CAL2, RPB2 5F–RPB2 7cR, NS1–NS4, LROR–LR5, and B27a–B27b, respectively [Fusarium spp. and Macrophomina spp., primers of ITS and EF1-α regions were commonly used. Primers of ITS, SSU, and LSU regions were chosen for the identification of Mucor spp. To identify species level of Aspergillus and Penicillium, primer sets from ITS, BT, CAL, and RPB2 were used. The polymerase chain reaction (PCR) was carried out using a SimpliAmp™ PCR system by Applied biosystems®  in a 25 μL reaction volume containing 0.25 μL of Takara Ex Taq® DNA polymerase  (5U μL−1), 2.5 μL of 10× Ex Taq buffer, 2 μL of dNTP mixture (2.5 mM each), 1 μL of each primer (10 pmoles μL−1), 1 μL template DNA solution (100 ng μL−1), and sterilized distilled water up to 25 μL. The PCR amplification conditions were initial denaturation at 98 °C for 30 s, followed by 35 cycles of denaturation at 98 °C for 15 s, and annealing at 50 °C for ITS, SSU and LSU, 55 °C for EF1–α and CAL, 49 °C for RPB2, 52 °C for BT, with 30 s, and a final extension at 72 °C for 2 min. PCR products were sequenced in both directions by a commercial sequencing service provider .To confirm the identity of the fungi, total genomic DNA was extracted directly from the mycelia grown on PDA using the CTAB DNA-extraction method . Differeectively ,26. PCR http://www.ncbi.nlm.nih.gov) to obtain sequence similarity. Closely related sequences were obtained from GenBank , malt extract agar (MEA), Czapek yeast extract agar (CYA), and yeast extract sucrose agar (YES) at 25 °C in the dark for 7 days. To microscopically observe the conidia, mycelia of the isolates were scratched off after 7 days and incubated under NUV (near–ultraviolet) light in 12h–12h light–dark conditions for 3–5 days . The sizPenicillium and Aspergillus isolates were inoculated following the methods described by Paul et al. [5 spores mL−1 before inoculation. Identical amounts of sterilized distilled water served as the control. The storage roots were then kept in moistened clean boxes and incubated at 25 °C. The pathogenicity test was conducted at three different times with three replications each time. After one week of incubation, the artificially inoculated storage roots were observed for lesion development daily; after 3 weeks, pathogenicity was confirmed, and disease severity was measured. Diseased storage roots were used for the reisolation of the fungi on PDA media. After reisolating, the fungal morphology was observed to confirm Koch’s postulates.Pathogenicity assay was performed on a susceptible sweet potato variety, namely Beniharuka, with the isolates CMML21–2, CMML21–4, CMML21–5, CMML21–7, CMML21–8, CMML21–12, CMML21–13, CMML21–16, and CMML21–17. Each isolate was selected from each species group, and two of the isolates (CMML21–4 and CMML21–7) had not previously been identified in sweet potatoes anywhere in the world. Beniharuka is known to be susceptible to common sweet potato diseases. To perform the pathogenicity test, the fungal isolates were cultured on PDA for 7–10 days, and PDA plugs were used to inoculate sweet potato storage roots. The sweet potato storage roots were surface sterilized by dipping in 1% NaOCl for 10 min and washed with sterilized distilled water three times. The storage roots were then allowed to dry in a laminar airflow chamber. A 5 mm hyphal disc of each isolate was placed in a wound made in each root using a 5 mm cork borer. Blank PDA discs were used as control treatments . AdditioTrichoderma harzianum strains (CMML20–26 and CMML20–27) collected from the Molecular Microbiology Laboratory, Dept. of Integrative Food Bioscience and Biotechnology, Chonnam National University, Gwangju, Republic of Korea. Nine representative pathogenic species were selected for screening: F. oxysporum CMML21–2, A. wentii CMML21–4, P. expansum CMML21–5, P. rotoruae CMML21–7, F. ipomoeae CMML21–8, Mu. variicolumellatus CMML21–12, F. oxysporum CMML21–13, M. phaseolina CMML21–16, and F. solani CMML21–17. Five to seven days old antagonistic T. harzianum strains and pathogenic fungi were cultured on opposite edges of the PDA and incubated at 25 °C for 5–7 days. Each experiment was repeated three times. Antifungal activity was assessed by the size (diameter in mm) of the inhibition zones by following the formula explained by Ji et al. [A1 = radial growth of pathogenic mycelia without the T. harzianum strain (CMML20–26 or CMML20–27), A2 = radial growth of pathogenic mycelia with the T. harzianum strain.Pathogens causing postharvest diseases of sweet potatoes were screened for antifungal activity with two antagonistic i et al. .InhibitSymptoms of different diseases were observed on the collected storage roots; surface rot disease was most common, followed by several molds. Other diseases found on the storage roots included dry rot, charcoal rot, and end rot. Surface rot symptom was characterized by its circular, somewhat sunken enlarged spots on the surface of the storage roots. Charcoal rot disease was restricted to part of sweet potato so that one end desiccated, and the other end appeared intact. The symptoms of charcoal rot and end rot were very similar, and two different pathogens were obtained from these symptoms. In blue mold symptom, the bluish-green sporulation was abundantly visible on the surface of the storage roots. Dry rot symptom was also found on the surface of the storage roots and became dried A–F. SweeFusarium spp. were the most predominate fungi (67.6%) recovered in this experiment, followed by Macrophomina sp. (14.7%), Penicillium spp. (10.3%), Aspergillus sp. (5.9%), and Mucor sp. (1.5%)  .F. oxysporum. Two sequences (ITS and EF1–α) of these isolates showed 99–100% sequence similarity with the reference strains (SPL 15020 and SPL 16048), which had previously been reported to be F. oxysporum in sweet potato in Korea [F. oxysporum strains  were grouped together, with a high bootstrap value (99%). In addition, isolates CMML21–8 and CMML21–9 were identified as F. ipomoeae, supported by high sequence similarity with the reference F. ipomoeae strains, and they formed a monophyletic group comprising the two isolates and F. ipomoeae strains (CQ1099 and LC12163), with a bootstrap value of 97%. A BLASTN search of ITS and EF1–α sequences from isolate CMML21–17 indicated that the isolate had high sequence similarity (99–100%) with F. solani reference strains, and the isolate was grouped with three F. solani strains with a high bootstrap value (%)  .Aspergillus species, three genes, including ITS, BT, and CAL, of isolates were sequenced. BLASTN search analysis and maximum likelihood phylogenetic analysis using three gene sequences (ITS+BT+CAL) revealed that the two isolates matched well with the reference strains (CBS 118.34 and CBS104.07) of A. wentii and formed a single clade in the phylogenetic tree with a high bootstrap value (100%) (For the identification of two isolates (CMML21–3 and CMML21–4) of e (100%) .Penicillium species causing mold disease in sweet potatoes were recovered. Isolates CMML21–5 and CMML21–6 were identified as P. citrinum based on a BLASTN search and molecular phylogenetic analyses using multigene sequences . The two isolates and three reference P. citrinum strains  were grouped with a high bootstrap value (100%). In addition, isolate CMML21–7 was identified as P. rotoruae, supported by the high sequence similarity with the P. rotoruae strain CBS 14.534 and forming a monophyletic group comprising the isolate and P. rotoruae strain, with a bootstrap value of 99% . Molecular phylogeny and the maximum likelihood tree constructed from the ITS, SSU, and LSU sequences showed that the isolate matched well with the reference strain of Mu. variicolumellatus (CBS 236.35) and was closely related to Mu. circinelloides f. lusitanicus (CBS 108.17 and CBS 276.49)  (The isolate CMML21–12 obtained from soft rot-related disease in sweet potato was identified as  276.49) . The caund CPMM) .Fusarium species to characterize morphologically were CMML21–2, CMML21–8, and CMML21–17. The size of the macro and microconidia of the isolate CMML21–2 ranged from 14.5–33.0 × 2.7–4.4 µm to 5.6–10.2 × 2.5–3.8 µm  at 25 °C in darkness to facilitate colony morphology, texture, color, and the sizes and shapes of conidia and conidiophores. Representative isolates from 5–3.8 µm A. The maF. ipomoeae [F. solani [Morphology study of the isolate CMML21–8 revealed that the colony on PDA after 7 days at 25 °C was white, fluffy, and grew moderately quickly; cotton-like aerial mycelia were common. Conidia formation was hard; conidia were slightly curved and tapered at the apex. Conidial size varied and ranged between 17.5–49.0 × 3.1–5.6 to 5.1–8.0 × 3.1–4.7 µm, and 3–5 septations was common C. Microcipomoeae ,33,34,35. solani .P. citrinum. CMML21–5 was cultured on MEA, CYA, and YES at 25 °C for 7 days. The reverse colors on CYA and YES were brownish-yellow and yellow or orange yellow, respectively. The size of the colonies varied with the media; the diameters on MEA, CYA, and YES were 34–40, 25–28, and 23–26 mm, respectively. Conidia were globose to sub-globose with smooth walls . Conidiogenesis generally was not observed but was abundant at 30 °C. Stipes were smooth-walled and rarely roughened; conidia subglobose to slightly ovoid and smooth-walled.Note: All four sequences matched well with the fungus p < 0.05). The isolate CMML21–16, which was confirmed as M. phaseolina, showed the highest disease severity by producing charcoal rot disease on sweet potato storage roots. The average length and depth of lesions were 112.95 and 42.80 mm, respectively  and F. solani (CMML21–17). The newly isolated pathogen A. wentii showed the lesion length and depth of 15.32 and 7.80 mm, respectively. In addition, the average lesion length and depth caused by the pathogen P. rotoruae were 15.55 and 7.23 mm, respectively, and the species caused higher lesion length and depth than P. citrinum (The least infected sweet potato was caused by the pathogen citrinum .T. harzianum strains (CMML20–26 and CMML20–27) were tested against eight different pathogenic species isolated from sweet potato storage roots. Dual culture activity results revealed that the two T. harzianum strains showed strong antifungal activity against all tested pathogens. Two Trichoderma strains showed strong antagonistic activity against F. oxysporum isolates (CMML21–2 and CMML21–13), and the rate of pathogen inhibition was 72.88–81.14%. The newly recovered pathogen in sweet potato A. wentii (CMML21–4) was inhibited 73.93–77.04% by the two antagonists. Lower inhibition was observed against P. citrinum (CMML21–5) (P. rotoruae (CMML21–7), was inhibited by CMML20–26 and CMML20–27 by 69.40 and 58.13%, respectively. The inhibition of F. ipomoeae (CMML21–8) by two T. harziaznum strains was 56.99–57.06%. The lowest inhibition rate (42.66 to 50.21%) was observed against Mu. variicolumellatus (CMML21–12). The pathogen inhibition rate of M. phaseolina (CMML21–15) and F. solani (CMML21–17) was 75.01–78.22% and 64.94–68.83%, respectively, by these antagonistic fungal strains (Two antagonistic MML21–5) . Another strains .Fusarium spp., F. solani, Sclerotium rolfsii, Rhizopus and Mucor spp., Penicillium spp., Diaporthe batatas, and M. phaseolina, respectively [Sweet potato is the eleventh most important food crop globally  and is vectively ,15,16,17F. oxysporum, F. ipomoeae, P. citrinum, and M. phaseolina were frequently isolated. The most frequent pathogen was F. oxysporum, which caused surface rot disease. The disease is more damaging than any other storage disease of sweet potato worldwide [F. solani, M. phaseolina, Lasiodiplodia theobromae, and D. batatas [F. solani was isolated from the end rot disease, and the M. phaseolina isolates were isolated from charcoal rot diseases. Charcoal rot of sweet potato is widespread in the tropics [Mucor sp. was isolated from rotten sweet potatoes. Multigene molecular phylogeny identified the pathogen as M. varricolumellatus, which Wagner named in 2020 [Mu. circinelloides species complex group. Mu. circinelloides (Syn. M. racemosus) was previously isolated as a pathogen from sweet potato [In the present study, surface rot, blue mold, and charcoal rot diseases were commonly observed, and orldwide . In addi batatas . F. sola tropics ; decay u in 2020 . Detailet potato .M. phaseolina isolate. Macrophomina spp. are the most severe and frequent species among fungal isolates recovered from sweet potato stems and storage roots with rot symptoms in Brazil [M. phaseolina deserves to be highlighted as it is a widely distributed plant pathogen that can produce microsclerotia that survive in the soil for a long time [The pathogenicity test is essential to observe the environmental condition producing the disease and whether the pathogen isolated was virulent or not and to confirm Koch’s postulates. All the pathogens tested for pathogenicity in the present study showed disease symptoms in a different lesion length. The most severe disease was observed on sweet potato storage roots inoculated by the n Brazil . M. phasong time ,44. The ong time ,15,20. Pong time . TherefoA. wentii and P. rotoruae, were isolated in the storage roots of sweet potato. A. wentii caused black mold or dry rot disease, and P. rotoruae caused blue mold disease. The pathogen A. niger causes black mold rot in storage roots and has been reported from Bangladesh, China, India, Nigeria, and the USA [Penicillium spp. including P. oxalicum and P. citrinum [P. rotoruae was discovered and described as a new species in New Zealand [Two new postharvest disease pathogens,  the USA ,47. Moldcitrinum ,47,48,49 Zealand , and theFusarium spp., M. phaseolina, and many other postharvest pathogens enter in such a way [Aspergillus spp. and Penicillium spp., which produce mycotoxins that can be lethal to humans [Trichoderma have been used for postharvest biocontrol agents in crops such as papayas, strawberries, tomatoes, apples, pears, and bananas. Three Trichoderma species, T. asperellum, T. viride, and T. harzianum, showed strong antagonistic activities against different fungal pathogens [Trichoderma spp. use various mechanisms against pathogens, including production of antifungal compounds, competition for nutrients, parasitism or inhibition of pathogens, antibiosis, and production of lytic enzymes [T. harzianum strains CMML20–26 and CMML20–27 for dual culture antagonistic activity and observed that these two biocontrol agents could reduce the pathogenic fungal growth up to 85%. A similar antagonistic agent T. asperellum was isolated as a pathogen from the sweet potato storage roots, which inhibited the growth of pathogens such as F. oxysporum, F. proliferatum, L. theobromae, and Rhizopus nigricans [Trichoderma spp. as well as other biocontrol agents.Postharvest diseases are caused by several factors, such as low pH, moisture content, nutritional composition, improper handling, and management of storage roots during harvest, transport, and marketing. Improper handling causes wounds in storage roots and allows pathogens to enter, thus infecting the storage roots. ch a way . Infesteo humans . Therefoathogens . The bio enzymes ,52. In tigricans . Therefo


This cross-sectional, register-based study aimed to explore patterns of planned rehabilitation at discharge from stroke units in Sweden in 2011 and 2017 and identify explanatory variables for planned rehabilitation. Multivariable binary logistic regression was used to identify variables that could explain planned rehabilitation. There were 19,158 patients in 2011 and 16,508 patients in 2017 with stroke, included in the study. In 2011, 57% of patients were planned for some form of rehabilitation at discharge from stroke unit, which increased to 72% in 2017 (p < 0.001). Patients with impaired consciousness at admission had increased odds for planned rehabilitation , . Admission to a community hospital   or to a specialized non-university hospital ,  was associated with decreased odds of receiving planned rehabilitation compared to admission to a university hospital. As a conclusion severe stroke was associated with increased odds for planned rehabilitation and patients discharged from non-university hospitals had consistently decreased odds for planned rehabilitation. Multidisciplinary team-based rehabilitation as well as early, supported discharge with home-based rehabilitation are effective interventions for reducing the odds of death and functional dependency after stroke3. Access to neurorehabilitation has been identified as a critical problem in acute stroke care in Europe, including Sweden3. In Sweden, health care including rehabilitation services, is publicly funded through taxes, and rehabilitation should be offered in proportion to individual needs. However, decisions regarding healthcare vary depending on region.The global estimated disability adjusted life years (DALYs) due to stroke was more than 116 million in 20164. Disability or functional dependency prior to stroke, severe behavioral problems, and living far from a rehabilitation center were identified as unfavorable predictors of receiving inpatient rehabilitation4. The European Stroke Organization and Stroke Alliance for Europe stroke care targets for 2030 include national plans that encompass the entire chain of care, access to early rehabilitation for at least 90% of stroke survivors, and review of each patient’s rehabilitation needs at 3 months, 6 months, and annually thereafter5. The World Health Organization’s “Rehabilitation 2030” initiative stated that sufficient rehabilitation services are needed for an increasing population living with disability and requested that rehabilitation should be available to people regardless of their age6. There is a need to analyze the extent of reaching the targets for stroke rehabilitation in a country with publicly funded healthcare and to identify possible differences in factors associated with planned rehabilitation during two separate periods.Patients with normal cognition after stroke, ability to follow instructions, and presence of a spouse or relative have been shown to be more likely to receive inpatient rehabilitationThis study aimed to explore patterns of planned rehabilitation at discharge from acute stroke care in Sweden in 2011 and 2017 and identify eligible explanatory variables for planned rehabilitation.7. Patients 18 years or older who were diagnosed with stroke according to the International Classification of Disease-10 criteria  were included in the study. Patients who died during hospitalization or missing data on both of the following: planned rehabilitation after discharge from acute stroke care and discharge destination were excluded , and treatment received during hospitalizationThe study complied with the Declaration of Helsinki and was approved by the Gothenburg Regional Ethical Review Board, DNR 2019-02694. Quality registers are exempt to the general rule of patient consent according to the Personal Data Act (Swedish law No. SFS 1998:204), since the information is of public interest, but there is opt out possibility.Planned rehabilitation was based on registrations in the register when discharged from the stroke unit. The variable has several options. Patients were categorized as having planned rehabilitation if they were discharged to nursing homes, geriatric or rehabilitation clinics, inpatient rehabilitation, outpatient rehabilitation, day rehabilitation, or to early supported discharge. The level of rehabilitation service varies widely. At the nursing homes, occasional rehabilitation service is provided. In geriatric or rehabilitation clinics as well as inpatient rehabilitation, rehabilitation generally is provided by several professions in a team. In outpatients care, the rehabilitation is delivered usually by a single profession. Day rehabilitation on the other hand, usually is team based and delivered several times per week. In early supported discharge, the patient continues the rehabilitation with a multidisciplinary team , in the patient’s home environment. Patients were categorized as no planned rehabilitation if the following was noted: “rehabilitation need but no access,” “patient responsible for exercise,” “no rehabilitation need,” or “no rehabilitation planned.”The variable Stroke type was categorized as ischemic stroke (IS) or hemorrhage. Patients diagnosed with I64, unspecified cerebrovascular disease were included in the IS group. At arrival, a clinical assessment as well as a CT scan is part of the routine, and an acute bleeding would most likely be detected during the examination. Thus, it is reasonable to assume these patients had an IS.Hospitals were categorized according to the Riksstroke registry as university hospitals, specialized non-university hospitals, or community hospitals8.Level of consciousness on admission was assessed using the Reaction Level Scale (RLS-85) and was used to indicate stroke severity9. The RLS-85 categorizes levels of consciousness as follows: alert (RLS 1), drowsy (RLS 2–3), and unconscious (RLS 4–8)9.Region included the 21 regions in Sweden . The tax funded health care in Sweden is delivered in 21 independent regions, with own economic conditions. The level of rehabilitation facilities vary between regions but there are national recommendations. This variable was used to describe the patterns of planned rehabilitation.Reperfusion therapy was defined as thrombolysis, thrombectomy, or both.Dependency in basic activities of daily living (ADL) before stroke was defined as requiring another person’s help during the following tasks: walking indoors or outdoors, toilet activities, and/or dressing. Sociodemographic variables were also analyzed.2 test for dichotomized variables and the Mann–Whitney U test for continuous variables. Data regarding planned rehabilitation or discharge destination were converted to dichotomized variables (planned rehabilitation at discharge from acute stroke care and no planned rehabilitation). Proportions for the 21 regions in total, was computed to evaluate the national mean. The national mean was compared with the proportion for each region respectively.Data from 2011 and 2017 as well as for IS and hemorrhage were analyzed separately. Differences between included and excluded patients as well as between groups were calculated using Pearson’s χ11, and were as follows: consciousness on admission, age, pre-stroke ADL dependency, previous stroke, sex, living alone, and hospital type. Reperfusion therapy was included as an independent variable in the IS models. Patients with missing information on any of the independent variables were excluded to enable comparison between models with identical sample sizes. Maximum likelihood estimation was performed with backward removal using the Akaike Information Criterion12. The final model was selected based on the lowest Akaike Information Criterion value12 and compared with the model containing all the variables. The Hosmer and Lemeshow test (p > 0.05 indicates good fit) and receiver operating characteristic curves13 were computed to check the goodness of fit. Nagelkerke R2 was used to show the models’ explanatory value. Odds ratios (OR), 95% confidence intervals, and p-values from binary logistic regression were presented as the results.Binary logistic regression analyses were performed to identify eligible explanatory variables for planned rehabilitation at discharge from acute stroke care (coded as 1), which was the dependent variable. The independent variables were selected based on previous studieshttp://www.graphpad.com) was used for figures. Binary logistic regressions were performed in R version 4.0.2 . The packages Modern Applied statistics with S14, and Lasso and Elastic-Net Regularized Generalized Linear Models (Glmnet)15 were used. The licenses for SPSS and GraphPad prism were provided by the University of Gothenburg. R is a free software environment that can be downloaded from https://cran.r-project.org/bin/windows/base/.The level of significance was set at α = 5%. SPSS for Windows, version 25.0  was used for descriptive statistics. GraphPad prism  . Patients had lower odds of receiving planned rehabilitation if they had pre-stroke ADL dependency  Fig. .Figure 3Patients receiving reperfusion therapy had higher odds of receiving planned rehabilitation than those not receiving it . Patients with pre-stroke ADL dependency had lower odds of receiving planned rehabilitation  than those without it. Compared to patients admitted to university hospitals, patients admitted to specialized non-university hospitals and community hospitals had lower odds of receiving planned rehabilitation . Compared to patients admitted to university hospitals, patients who were admitted to specialized non-university hospitals or community hospitals had lower odds of receiving planned rehabilitation  and pre-stroke ADL dependency . Admission to a specialized non-university hospital  or community hospital  were associated with lower odds of receiving planned rehabilitation compared to admission to a university hospital Fig. .The results of this register-based study showed that regardless of stroke type, the proportion of patients receiving planned rehabilitation at discharge from a stroke unit increased from 2011 to 2017. Regional variation was observed in both years. Explanatory variables for planned rehabilitation were different depending on year and stroke type. Patients with impaired consciousness on arrival at the hospital had increased odds for planned rehabilitation regardless of year and stroke type. Admission to non-university hospitals was consistently associated with decreased odds of receiving planned rehabilitation.3. According to Swedish national stroke care guidelines, ESD should be offered to patients with mild to moderate stroke16. This knowledge has grown over the years, and has in the revisions of national guidelines given a higher priority each time. In this study, the proportion of patients who received ESD increased from 2011 to 2017 in line with the guidelines. This can be taken as an indicator for successful implementation of the guidelines in clinical praxis. The content and intensity of other types of rehabilitation interventions, remains unknown.The proportion of patients receiving planned rehabilitation at discharge from a stroke unit increased significantly from 2011 to 2017. Within this time period, two revisions of National stroke guidelines have been published. This may lead to increased awareness of the patient’s rehabilitation needs. The median length of stroke unit stay was 2 days shorter in 2017 than in 2011, and it is possible that earlier discharge corresponds with more frequent referral for rehabilitation. Development of rehabilitation facilities such as ESD, could also contribute to increased detection of rehabilitation needs. ESD has been shown to reduce the odds of death or dependencys had consistently lower odds of receiving planned rehabilitation compared to those treated at university hospitals. There are several possible explanations for this. First, non-university hospitals are often located in smaller towns with a larger geographical coverage than university hospitals; thus, it is possible that patients lived far from rehabilitation centers. Long distances between patients’ homes and rehabilitation centers have previously been associated with decreased probability of referral to rehabilitation4. Second, there may have been fewer rehabilitation facilities available in these areas. Third, university hospitals are also education centers, and may be staffed by professionals that smaller hospitals are unable to employ.Patients treated at specialized non-university hospitals or community hospital18. ADL dependency prior to stroke and older age have been shown to be unfavorably associated with rehabilitation access19. The results from this study indicate an increased awareness of rehabilitation needs for older adults and individuals with dependency in basic ADL. This may be due to the ongoing quality assurance in Swedish healthcare that aim to supply individualized rehabilitation to support healthiness in different circumstances of life.Patients with IS with pre-stroke ADL dependency and those who were older were less likely to receive planned rehabilitation in 2011. In contrast, higher age and ADL dependency were favorably associated with planned rehabilitation in patients with IS in 2017. Post-stroke referral patterns in Canada showed that younger and more functionally impaired patients were referred for inpatient and outpatient rehabilitation more often than older or functionally independent patientsRegional variation was observed in both 2011 and 2017 and are unlikely to be completely explained by differences in patient characteristics. Inequalities in access to rehabilitation based on geographical location, is unaccepted given that equal conditions to achieve health is stated in the Swedish law. Local differences in referral protocols could have contributed to the observed variation. In addition, there is no standardized tool to assess rehabilitation need after stroke in general. Taken altogether, there is a need to implement a strategy that ensure that the actual rehabilitation is planned and delivered to the patient according to the rehabilitation needs.9, was used to evaluate stroke severity. The RLS-85 is a part of the National Institutes of Health Stroke Scale, and consciousness level is considered as a reliable predictor of post-stroke survival9. Socioeconomic factors such as income, education level, and country of birth were not available from the Riksstroke registry, and these factors could possibly affect planned rehabilitation. Lastly, the present study is based on cohorts from 2011 and 2017, and if continuous data had been available between the years, perhaps a better understanding on patterns of planned rehabilitation could have been obtained.The current study was based on a large dataset from the Riksstroke registry. Tax funded health care gives the opportunity for each person to receive care. The present study had a few limitations. Register-based data commonly includes missing data, due to administrative issues, causing patient dropout. Furthermore, the types of rehabilitation eligible in the registry changed between 2011 and 2017 and data regarding rehabilitation and discharge destination was dichotomized, causing reduced generalizability to clinical practice. In the register, data regarding the patients’ National Institutes of Health Stroke Scale scores were missing. As a proxy, the consciousness on admission, assessed using the RLS-85Regardless of stroke type, the proportion of patients with planned rehabilitation from stroke units increased from 2011 to 2017. There were pronounced regional variations regarding patients with planned rehabilitation both years. Severe stroke was associated with increased odds for planned rehabilitation. Patients discharged from non-university hospitals had consistently decreased odds for planned rehabilitation. The increased proportion with planned rehabilitation in 2017 could indicate improved awareness for rehabilitation needs. The regional variations and the impact of hospital type though disclose a need for future development in line with the European Stroke Organization’s targets for 2030 and the World Health Organization’s “Rehabilitation 2030: a call for action”.Supplementary Tables.


Ideal Plant Architecture 1 (IPA1) encodes SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 14 (SPL14) with a pleiotropic effect on regulating rice development and biotic stress responses. To investigate the role of IPA1 in early seedling development, we developed a pair of IPA1/ipal-NILs and found that seed germination and early seedling growth were retarded in the ipa1-NIL. Analysis of the soluble sugar content, activity of amylase, and expression of the α-amylase genes revealed that the starch metabolism was weakened in the ipa1-NIL germinating seeds. Additionally, the content of bioactive gibberellin (GA) was significantly lower than that in the IPA1-NIL seeds at 48 h of imbibition. Meanwhile, the expression of GA synthesis-related gene OsGA20ox1 was downregulated, whereas the expression of GA inactivation-related genes was upregulated in ipa1-NIL seeds. In addition, the expression of OsWRKY51 and OsWRKY71 was significantly upregulated in ipa1-NIL seeds. Using transient dual-luciferase and yeast one-hybrid assays, IPA1 was found to directly activate the expression of OsWRKY51 and OsWRKY71, which would interfere with the binding affinity of GA-induced transcription factor OsGAMYB to inhibit the expression of α-amylase genes. In summary, our results suggest that IPA1 negatively regulates seed germination and early seedling growth by interfering with starch metabolism via the GA and WRKY pathways. Seed germination is a complex and diverse process that plays an important role in ensuring the continuity of life. Generally, germination starts with water uptake and the reactivation of diverse metabolism processes in the quiescent seed, followed by emergence of the radical and coleoptile . In riceα-amylase, β-amylase, debranching enzyme, and α-glucosidase [α-amylase is the most abundant hydrolase in rice. It catalyzes the hydrolysis of α-1,4-glucosidic bonds of starch at random sites to generate α-glucose and α-maltose [α-amylase is synthesized in the aleurone layer and scutellum to break down starch granules. Generally, the expression of rice α-amylase gene αAmys  is positively and negatively regulated by gibberellin and sugar, respectively [OsGAMYB , which directly binds to the GA response element (GARE) and activates the expression of αAmys [αAmys is strongly induced by sugar starvation and repressed by various sugars produced during mobilization of endosperm stores [Starch is the major reserve in rice seed that provides a major carbon source for generating energy and metabolites. During seed germination and early seedling growth, starch is degraded by the concerted action of various hydrolases, including cosidase . Among t-maltose ,6. Afterectively ,8. Afterof αAmys ,10. Meanm stores ,11,12. Tgermination-defactive1 (gd1) mutant, the expressions of GA biosynthesis-associated genes OsGA20ox1, OsGA20ox2, and OsGA3ox2 were suppressed, while the expression of the GA inactivation gene OsGA2ox3 was dramatically upregulated, resulting in decreased endogenous GA4 content and the inhibition of seed germination [OsGA20ox2 loss-of-function mutant, which resulted in a delayed seed germination [The GA contents in germinating seeds are determined by GA biosynthesis and metabolism. In brief, GA biosynthesis is catalyzed by the combined actions of ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurene acid oxidase (KAO), GA 20-oxidase (GA20ox), and GA 3-oxidase (GA3ox), whereas it is inactivated by GA 2-oxidase (GA2ox) ,14. The mination . Meanwhimination .Arabidopsis and rice, changes in the expression level of miR156 affect seed dormancy [Ideal Plant Architecture 1 (IPA1), which encodes a transcription factor OsSPL14, a member of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family in rice, is an important regulator of plant development [ipa1 mutant plants, one nucleotide substitution located in the miR156 recognition site perturbs the miR156-regulated degradation of IPA1 mRNA, leading to an ideal plant architecture with reduced tiller number, increased lodging resistance and panicle branches and, thus, an increase in yield [IPA1 was also identified as a vital regulator to participate in biotic stress response [IPA1/ipa1-NILs developed in our laboratory, we found that IPA1 negatively regulates the processes of seed germination and early seedling growth. To elucidate the mechanism, we examined the amylase activity, GA content, and expression of the related genes in the NILs. Meanwhile, the relationship between IPA1 and two transcriptional repressors (WRKY51 and WRKY71) was investigated, respectively. Our work suggests that the elevated levels of IPA1 expression retards seed germination and early seedling growth through GA and WRKY pathways. These findings provide new insight into understanding the function of IPA1 in seedling development.In dormancy ,18. As aelopment ,20,21. Iin yield ,20. In aresponse ,23. HereIPA1 gene in rice seedling development, we developed a pair of IPA1/ipa1-NILs and compared the expression pattern of IPA1 in germinating seeds. We found that IPA1 expression was significantly higher in the ipa1-NIL seeds after 24 h imbibition compared with the IPA1-NIL seeds  [indica cultivar Yuetai B (IPA1/IPA1) to develop the Yuetai B/SNJ F1 plants. Then, the F1 plants were backcrossed with Yuetai B to develop the BC1F1 plants. Subsequently, one of the BC1F1 plants was self-crossed for six generations to develop Yuetai B/SNJ BC1F7 plants. Using marker-assisted selection, a BC1F7 plant with the genotype IPA1/ipa1 on the IPA1 locus was obtained. In the self-crossed progeny of this BC1F7 plant, the plants with the genotype IPA1/IPA1 were identified as IPA1-NIL, whereas the plants with the genotype ipa1/ipa1 were identified as ipa1-NIL.To generate near-isogenic lines (NILs) of ng (SNJ)  was cros3, or 30 μM GA3. Three replicates (more than 50 seeds/replicate) were analyzed for each treatment. The seed germination rate was calculated at different time points after imbibition. A seed was considered to have germinated when the radicle or shoot length was longer than 1 mm.Sterilized NIL seeds were incubated at 30 °C in the dark with water, 10 μM GAα-amylase activity, 1 mL crude enzyme extract was incubated in the water at 70 °C for 15 min to inhibit the activity of β-amylase. The additional 1 mL enzyme extract was not heated and used to measure the total amylase activity. Then, each enzyme extract was mixed with 1 mL of 1% (w/v) soluble starch dissolved in citric acid buffer (pH = 5.6). After heating in a water bath at 40 °C for 5 min, 2 mL 3,5-dinitrosalicylic acid reagent was added, and the mixture was boiled for 5 min. The absorbance of the mixture was measured at wavelengths of 540 nm. Three replicates were analyzed for each treatment.The amylase activity was quantitatively determined by a modified version of the 3,5-dinitrosalicylic acid method, as described in previous studies ,38. Briev/v) ethanol. The mixture was heated at 80 °C for 30 min, cooled, centrifuged at 3000 rpm (664× g) for 10 min, and diluted to a volume of 2 mL with 80% (v/v) ethanol. The reaction mix contained 200 μL extract and 1 mL reagent was heated at 95 °C for 15 min. The absorbance of samples was finally recorded at 620 nm. The soluble content was calculated using glucose as the standard. Three replicates were analyzed for each treatment.The soluble sugar content was quantified by the anthrone method  with minv/v) methanol at 4 °C. After centrifugation at 12,000× g for 15 min at 4 °C, the supernatant was dried under a stream of N2. Then, the residues were dissolved in 30% methanol and centrifuged. The supernatant was collected for LC-MS analysis.The endogenous GA contents in seeds were determined as described in . In brieTM real-time PCR Detection System . The primers used for qPCR are listed in OsActin gene expression was used as an internal control to normalize the expression of target genes.After 12, 24, 36, and 48 h incubation, total RNA from the germinating seeds was extracted with TRIzol reagent . First-strand cDNA was synthesized from 2 μg RNA in a total of 20 μL reaction system with an ABScript III RT Master Mix with gDNA Remover  according to the manufacturer’s instructions. Synthesized cDNAs were used for qPCR with 2× Universal SYBR Green Fast qPCR Mix  on a CFX384OsWRKY51 and OsWRKY71 was amplified from the genomic DNA  under control of the CaMV 35S promoter was used as an internal control. The cDNA of IPA1 was amplified (primer pairs listed in Ubi:IPA1 effector construct. The combined reporter and effector plasmids were co-transformed into rice protoplasts as described in [The dual-luciferase assays were performed according to a previously described method . The proribed in . An emptSBP-AD construct. Various truncated fragments of the promoter regions of OsWRKY51 and OsWRKY71 were amplified and ligated into the pABAi vector to generate the bait vectors. After linearization by the BstBI enzyme, the bait vector co-transformed with the prey vector into the yeast strain Y1HGold. The transformants were grown on SD/−Ura/−Leu plates at 30 °C for 3 days, then grown on SD/−Ura/−Leu/AbAi plates. Yeast strains containing the empty pGADT7 in combination with the bait vector were used as the negative control.The coding region of IPA1-SBP was amplified by PCR and cloned into a pGADT7 vector to produce an IPA1


By continuously activating β1-AR, β1-AA can induce cytotoxicity, leading to cardiomyocyte apoptosis and heart dysfunction. However, the mechanism underlying the persistent activation of β1-AR by β1-AA is not fully understood. Receptor endocytosis has a critical role in terminating signals over time. β2-adrenoceptor (β2-AR) is involved in the regulation of β1-AR signaling. This research aimed to clarify the mechanism of the β1-AA-induced sustained activation of β1-AR and explore the role of the β2-AR/Gi-signaling pathway in this process. The beating frequency of neonatal rat cardiomyocytes, cyclic adenosine monophosphate content, and intracellular Ca2+ levels were examined to detect the activation of β1-AA. Total internal reflection fluorescence microscopy was used to detect the endocytosis of β1-AR. ICI118551 was used to assess β2-AR/Gi function in β1-AR sustained activation induced by β1-AA in vitro and in vivo. Monoclonal β1-AA derived from a mouse hybridoma could continuously activate β1-AR. β1-AA-restricted β1-AR endocytosis, which was reversed by overexpressing the endocytosis scaffold protein β-arrestin1/2, resulting in the cessation of β1-AR signaling. β2-AR could promote β1-AR endocytosis, as demonstrated by overexpressing/interfering with β2-AR in HL-1 cells, whereas β1-AA inhibited the binding of β2-AR to β1-AR, as determined by surface plasmon resonance. ICI118551 biasedly activated the β2-AR/Gi/G protein-coupled receptor kinase 2 (GRK2) pathway, leading to the arrest of limited endocytosis and continuous activation of β1-AR by β1-AA in vitro. In vivo, ICI118551 treatment attenuated myocardial fiber rupture and left ventricular dysfunction in β1-AA-positive mice. This study showed that β1-AA continuously activated β1-AR by inhibiting receptor endocytosis. Biased activation of the β2-AR/Gi/GRK2 signaling pathway could promote β1-AR endocytosis restricted by β1-AA, terminate signal transduction, and alleviate heart damage.Heart failure is the terminal stage of many cardiac diseases, in which β The myocardial cell line HL-1 was cultured in confocal dishes. A TIRF inverted microscope  equipped with an EMCCD camera and an oil immersion objective lens  was utilized. The depth of field, 120-130 nm, was selected to observe β1-AR fluorescence upon the plasma membrane but not the cytoplasm. Changes in cell surface fluorescence were recorded at 0, 5, 10, and 30 minutes (min) after cell administration. Recordings were made every 30 seconds (s) using MetaMorph software version 7.8.8.0. Image fluorescence intensity was analyzed using ImageJ 1.51J8 [1-AR-GFP and β-arrestin-RFP, we recorded images at 5 min after drug treatment and utilized ImageJ 1.53c for colocalization analysis. Before analysis, all images were adjusted with ImageJ 1.53c to the same brightness and color. Pearson’s R value (above threshold) was used as an indicator of the colocalization of β1-AR-GFP and β-arrestin-RFP [Total internal reflection fluorescence microscopy (TIRF) was utilized to determine βstin-RFP .2+ fluorescent probe Fluo-4  for 90 min before the experiment. At the time of the experiment, the cell culture medium was changed to FluoroBrite DMEM . Intracellular Ca2+ levels were measured using a turntable confocal microscope  [HL-1 cells were cultured at a density of ~30% in confocal dishes. The cells were incubated with the CaoX, USA) .Please refer to supplementary table 1-AA  [1-AA injection . In β2-AR inverse agonist treatment group, 32.7 μg/kg ICI118551 was injected intraperitoneally every week for 2 months, accompanied by β1-AA injection every two weeks. Left ventricular function was determined in all mice by echocardiography, and morphological changes in the heart were observed by HE staining.A mouse monoclonal β, China)  was mixe1-AR endocytosis in the different groups were analyzed by 2-way ANOVA followed by a Bonferroni post hoc test. One-way ANOVA followed by Dunnett’s test was used to compare multiple groups, and the t-test was used to compare the mean between two groups. P < 0.05 was considered statistically significant.In this study, GraphPad Prism 8.0.2 software was used for statistical analysis and statistical graph production. The experimental results are expressed as the mean ± SEM. The differences in dynamic changes in NRCM-beating frequency, cAMP concentration, and βThe detail of other material and methods was described in the supplementary material.1-AA and negative IgG used in the current study originated from mouse-derived hybridoma cells and sera from healthy Balb/c mice, respectively. We first compared the effect of mouse-derived β1-AA with that from HF patients. As shown in Fig. 1-AA (+)-IgG  elicited persistent activation of β1-AR, evidenced by the increasing NRCM-beating frequency for 1 hour, an effect partially inhibited by the β1-AR blocker metoprolol  and completely neutralized by the peptide corresponding to the extracellular second loop of β1-AR   for 30 min to block the α1-adrenergic receptor (α1-AR), NE (10−5 M) increased the NRCM-beating frequency for a much shorter time, ~5 min  also continuously increased NRCM-beating frequency through β1-AR, whereas negative IgG had no effect . This effect could be blocked by the β1-AR-ECII peptide. In contrast, after NE stimulation, the intracellular Ca2+ increased rapidly and then decreased rapidly, maintaining for only 5 s  Fig. . After pmin Fig. . The mouect Fig. . Moreoveels Fig.  and S8. 1-AR activation, we transiently transfected the β1-AR-GFP plasmid into an HL-1 cell line  endocytosis can terminate the downstream signal over time. To investigate the potential role of endocytosis in long-term βine Fig. . TIRF wamin Fig. , respectups Fig. .1-AR activation duration, we assessed the changes in β1-AR-mediated signaling and the effect after decreasing or increasing β1-AR endocytosis. As shown in Fig. 1-AR with Phe (10−5 M), NE (10−5 M) induced a short-term increase in NRCMs beating and intracellular cyclic adenosine monophosphate (cAMP) levels. In contrast, after preincubation with the GPCR endocytosis inhibitor Dynasore (10−4 M) for 30 min, NE increased the beating frequency of NRCMs for 60 min and the cAMP level for 30 min, similar to that caused by β1-AA , β1-AR-GFP fluorescence decreased by 42% in the β-arr1 OE group  of purified β1-AR protein were added. Surface plasmon resonance (SPR) detection showed that purified β1-AR and β2-AR proteins could bind directly in a dose-dependent manner; the KD value was 2.147 μM  for 5 min, 10 min, and 30 min, the fluorescence intensities of β1-AR-GFP on the cell membrane were reduced by approximately 1%, 5%, and 11%, respectively, in the siRNA β2-AR group, which was markedly lower than that of the siRNA control group . This indicated that the lack of β2-AR restricted β1-AR endocytosis  administration decreased the fluorescence intensities of β1-AR-GFP by 14%, 24%, and 27% at 5 min, 10 min and 30 min after β2-AR overexpression, respectively. These results indicated the promoting effect of the increased β2-AR expression on β1-AA-induced β1-AR endocytosis  also acted as a biased agonist of the β2-AR/Gi-signaling pathway  with or without ICI118551 (10−5 M). As shown in Fig. 1-AA-induced increased beating frequency and the intracellular cAMP content of NRCMs were significantly relieved by ICI118551 supplementation, but ICI118551 alone had no effects on these parameters  and ICI118551 (10−5 M) costimulation and ICI118551 single stimulation increased GRK2 phosphorylation  stimulation showed no significant effect  that measured the colocalization of β1-AR with β-arrestin1 and β-arrestin2 were 0.160 ± 0.024 and 0.330 ± 0.083 as the mean ± SEM, respectively, whereas in the ICI118551 plus β1-AA stimulation group, the Pearson coefficient was increased to 0.687 ± 0.110 (β-arrestin1) and 0.753 ± 0.068 (β-arrestin2) as the mean ± SEM  and protein kinase A (PKA) are two predominant kinases that promote βion Fig. , whereasect Fig. . Howeverion Fig. . These rSEM Fig. . In addired Fig. . These r2-AR/Gi-signaling pathway protected β1-AA-induced cardiac injury in vivo, wild-type Balb/c male mice (6–8 weeks old) without β1-AA were divided into four groups: saline , β1-AA , and therapy groups β1-AA combined with ICI118551 and ICI118551 alone   and left ventricular end systolic diameter (LVESD) increased by 15% and 42%, respectively. However, LVEF, LVESD, or LVEDD in the saline control group and the ICI118551 treatment group showed no significant change compared with preadministration measures  Fig. . Eight woup Fig. . M-mode res Fig. . Hematox1 Figure . These r1-AA promotes the occurrence and development of HF by continuously activating β1-AR. However, the specific mechanism is not fully understood. Receptor endocytosis is one of the important mechanisms for terminating signal transduction. The main study findings were that β1-AA continuously activated β1-AR by restricting β1-AR endocytosis and that biased activation of the β2-AR/Gi pathway could activate GRK2, promote β1-AR endocytosis restricted by β1-AA in vitro and alleviate the morphologic and functional damage in the mouse heart in vivo Fig. 7.Previous studies have shown that β1-AA is produced against β1-AR-ECII [1-AA have been detected in a variety of cardiovascular diseases, especially in HF [1-AA and HF has gradually attracted researchers’ attention. β1-AA elicits various pathological effects, such as triggering cardiomyocyte apoptosis [1-AR is a common mechanism underlying these effects. Elucidating the related mechanism is conducive to drug development and clinical treatment. As an agonistic ligand control, NE was selected. It is an endogenous α1/β1-AR selective agonist belonging to the catecholamine category.β-AR-ECII . In 1987-AR-ECII  found thly in HF , 18, 19.poptosis , reducinpoptosis , to caus1-AR is a complicated process. The agonist ligand binds to β1-AR and induces its structural change, which in turn exposes the Gs protein-binding sites. The Gs protein undergoes the GTP cycle to convert to GTP-Gs and binds to β1-AR. Then, GTP-Gs activates AC and mediates signal transduction through cAMP-PKA [1-AR needs to be desensitized and endocytosed over time. GPCR desensitization is divided into transient desensitization and long-term desensitization. Transient desensitization proceeds through the interaction of β-arrestin and GPCRs over a short period of time (minutes). Long-term desensitization proceeds through GPCR degradation in the lysosome over a long period of time (hours or days), during which the mRNA level of GPCR is reduced [1-AR, and then, β-arrestin1/2 in the cell recognizes and binds to phosphorylated β1-AR, blocking the binding between Gs protein and β1-AR and promoting the endocytosis of β1-AR [1-AA endocytosis restricted by β1-AA in HL-1 cells.As a GPCR, the activation of βcAMP-PKA , 22, 23. reduced . In thisof β1-AR , 25. β-aof β1-AR . In the 1-AR activation and endocytosis, NRCMs and HL-1 cells were used. NRCMs are rhythmic and their contraction profile just meets the needs of beating frequency experiments [2+ determination. Thus, a mouse myocardial HL-1 cell line, with good adhesion, was chosen as well. It has been studied that HL-1 cells can be used as a tool cell for β1-AR and β2-AR signaling pathways [1-AR, β2-AR, β-arrestin1, and β-arrestin2 were expressed in HL-1 cells (Fig. 1-AR, whereas HL-1 cells do not, because NE has a higher affinity for α1-AR than β1-AR in cardiomyocytes, whereas HL-1 cells do not express α1-AR (Fig. To detect the βeriments . Howeverpathways –30. Herells Fig. . Further-AR Fig. .2+ upon NE stimulation was transient. However, a long time of elevated cytosol Ca2+ was observed under the β1-AA condition. The sustained high concentration of Ca2+ in the cytosol of cardiomyocyte was a fascinating phenomenon, which we cannot fully explain yet. We speculated that it may be owing to the change of activity of Ca2+ channels or calcium ATPase in sarcoplasmic reticulum when stimulated by β1-AA.In HL-1 cells, the change in cytosol Ca2-AR is also important in cardiomyocytes because it can bind to either Gs or Gi protein [2-AR inhibits AC activation to block β1-AR signaling and exerts cardioprotective effects [2-AR can target the C-terminus of β1-AR in a phosphodiesterase 4-dependent manner to limit signal diffusion to avoid cytotoxicity after β1-AR activation [1-β2-AR heterodimer strengthens the Gs signaling pathway in normal adult mouse cardiomyocytes and restricts β2-AR endocytosis [1-AR and β2-AR in different situations. Here, we verified that β2-AR could promote β1-AR endocytosis upon stimulation with either β1-AA or NE.β protein , 31. By tivation . Howeverocytosis , 34. The2-AR [2-AR/Gi [2-AR/Gi pathway by reducing the contraction of myocytes in failing hearts [2-AR/Gi pathway, ICI118551 could increase GRK2 activity, enhance the recruitment of β-arrestin1/2 to β1-AR, and improve β1-AA-induced β1-AR endocytosis. GRK2 is the predominant GRK and the primary regulator of β1-AR and β2-AR desensitization in the heart. GRK2 can phosphorylate activated β-adrenergic receptors, which are recognized by β-arrestin and initiate receptor endocytosis [1-AA upregulated β1-AR phosphorylation, whereas ICI118551 supplementation reduced it (Fig. 1-AR phosphorylation was decreased in the ICI118551 treatment group, the exposed phosphorylation sites were prone to coupling with β-arrestin. Of course, more work is needed to test this hypothesis.ICI118551 is used as a specific antagonist of β2-AR . However[2-AR/Gi , 36. ICIg hearts  and reduocytosis . Contrar it Fig. . As repo1-AA and HF is known, how to effectively block β1-AA remains difficult. β1-AR blockers are among the most commonly used medications in treating HF and the most convenient method because of their oral administration. However, β1-AR blockers cannot sufficiently block the continuous activation of β1-AR induced by β1-AA [1-AA and β1-AR blockers. Metoprolol recognizes the β1-AR ligand-binding pocket [1-AA binds to the second extracellular loop of β1-AR [2-AR agonist fenoterol and β1-AR blocker metoprolol can improve the heart function of rats with HF. In addition, preferentially activating β2-AR/Gi can antagonize the apoptosis-promoting effect of β1-AR on cardiomyocytes through the PI3K-Akt pathway [2-AR/Gi could exert a cardioprotective effect by promoting β1-AR endocytosis and terminating its signal in a timely manner. Therefore, biased activation of the β2-AR/Gi-signaling pathway may be a new strategy for the treatment of HF patients with β1-AA. However, the effectiveness of this program in clinical treatment needs to be further confirmed.Although the close relationship between βby β1-AA . This prg pocket , 41, 42,of β1-AR . Animal  pathway . Here, wSupplemental materialIntracellular Ca2+ fluorescence intensity of HL-1 cells increased temporarily under NE stimulation.Intracellular Ca2+ fluorescence intensity of HL-1 cells increased temporarily under NE stimulation.The fluorescence intensity of Ca2+ in HL-1 cells continuously increased after being treated by β1-AA.Table S3Table S4Table S5Table S6Table S7Table S8Table S9Table S10Table S11Table S12Table S13Table S14Table S15Table S16Table S17Table S18Table S19Table S20Table S21Table S22Table S23Table S24Table S25Table S26Table S27Table S28Table S29Table S30Table S31Table S32Table S33Table S34Table S35Table S36