Most degenerative rotator cuff tears (RCTs) are associated with a limited range of motion (ROM) of the shoulder joint. Additionally, patients with diabetes mellitus (DM) show a higher frequency of limited ROM. Recently, advanced glycation end products (AGEs) of proteins have been observed to cause tissue fibrosis, primarily through abnormal collagen cross-linking and oxidative stress. In this study, we investigated the effect of AGEs on ROM limitation in the shoulder capsule and its relationship with DM in the patients with RCTs.Sixteen patients (eight in the DM and non-DM groups) who underwent arthroscopic surgery for RCT with limited shoulder ROM were included in this study. AGE-related pathologies in both groups were compared, and the relationship between AGE accumulation and shoulder joint ROM was evaluated. Shoulder capsule tissue was harvested and subjected to histological and in vitro evaluation.The DM group displayed high levels of AGEs and reactive oxygen species (ROS), and reduced cell viability. There was a significant positive correlation between ROS expression, apoptosis, and preoperative hemoglobin A1c. ROS expression, apoptosis, and ROM of the shoulder joint showed a negative correlation. The NADPH oxidase (NOX) expression and collagen III/I ratio were significantly higher in the DM group than in the non-DM group.The DM group showed significant AGEs deposition in the shoulder capsule. Additionally, there was a significant association between AGEs and ROM limitation. Collectively, the findings suggest that the oxidative stress induced by AGEs deposition, which leads to fibrosis and local inflammation, might contribute to the limited ROM of the shoulder joint in patients with RCTs accompanied by DM. Passive range of motion (ROM) limitation of the shoulder is common in patients with rotator cuff tears (RCTs), with more than 40% of patients with full-thickness tears showing mild or moderate limitation . The patAdvanced glycation end products (AGEs), the final glycation products of proteins, are formed by a non-enzymatic reaction between the ketone group of reducing sugars and the free amino group of proteins, resulting in progressive rearrangement, dehydration, and condensation . Owing tThe accumulation of AGEs is associated with the duration of hyperglycemia . Aging aThe Ethics Committee of our institute approved this study and informed consent was obtained from all the patients involved. Patients with limited ROM of the shoulder joint associated with degenerative RCT were included in the study . To conform to ethical standards, we did not collect shoulder capsule samples from patients with RCT who did not have limited ROM of the shoulder joint.Sixteen patients , who underwent surgical treatment for RCT with limited ROM of the shoulder joint at our hospital over a two-year period starting in January 2019, were chosen for the study. The passive ROM of the shoulder joint was measured under general anesthesia at the time of surgery. Patients with limited ROM and manipulation compared to the healthy side with normal findings on radiographs  were included in this study. To evaluate the association of AGEs with pain associated with RCTs and ROM limitation due to immobilization, patients with adhesive shoulder arthritis without RCTs were excluded from the study. For ethical reasons, patients with no ROM limitation were also excluded. Finally, eight cases with DM (DM group) and eight without DM (control group) were evaluated in this study. The DM group was defined as those with a preoperative HbA1c level of ≥6.5 . The sam3 and cultured. The minced tissue was cultured in a monolayer on a 100 mm-diameter culture dish in Dulbecco’s modified Eagle’s medium  mixed with 10% fetal bovine serum , 100 U/mL penicillin, and 100 μg/mL streptomycin. Cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2/95% air and passaged for 1–2 weeks. Cells in passages 2 and 3 were used in this study.Tissue from the shoulder capsule was obtained intraoperatively, and the tissue specimens were excised by the same surgeon (Y. M.). The shoulder capsule tissue was harvested using basket forceps from a site medial to the subscapularis tendon and adjacent to the joint labrum, as previously described . A portiAfter 24 h, formalin-fixed tissues were dehydrated and embedded in paraffin wax. Five micrometer sections were mounted on slides. Hematoxylin and eosin (H&E) staining Fig.  and immuCell viability was measured with the water-soluble tetrazolium salt (WST) assay using the Cell Counting Kit-8 , and 5000 cells were seeded in each of the 96 wells and cultured in DMEM for 24 h. After incubation, 10 μL of WST was added to each well and incubated at 37 °C for 4 h, after which soluble formazan was quantified in viable cells. Cell viability was evaluated by measuring the absorbance of the reduced formazan at 450 nm using the WST assay.4 cells/mL) were incubated with the oxidation-sensitive fluorescent probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) at a final concentration of 10 μM for 60 min in the dark at 37 °C. After incubation, the cells were washed with PBS and resuspended in trypsin. For quantification, the number of ROS-positive cells and diamidino-2-phenylindole (DAPI)-positive cells in the four fields of view (0.75 mm × 1.0 mm) of each slide were counted and the mean value was calculated.Fluorescence immunostaining was used to assess the effect of ROS on cells derived from the shoulder capsule. Based on previous reports , the ROSTwo days after incubation, immunofluorescence staining was performed to compare the apoptosis rates of the cells. The rate of apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining using the APO-DIRECT kit  according to the manufacturer’s protocol. The ratio of green-stained nuclear fragments to DAPI-stained cells was calculated for each of the four fields of view.5 cells/well and cultured in DMEM for 48 h. Total RNA was extracted using the RNeasy Mini Kit  according to the manufacturer’s protocol. Oligo (deoxythymidine)-primed first-strand cDNA was synthesized using the High Capacity cDNA Transcription Kit . Quantitative real-time PCR was performed in a 20 mL reaction mixture using the SYBR Green Master Mix reagent (Applied Biosystems) on an ABI Prism 7500 Sequence Detection System (Applied Biosystems). The PCR conditions were as follows: one cycle at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, and 40 cycles at 60 °C for 1 min.Cells were seeded in 12-well culture plates at a density of 1.0 × 10The messenger RNA (mRNA) levels of type I collagen COL1), type III collagen (COL3), RAGE, NOX1, NOX4, interleukin-6 (IL-6), and IL-1β were evaluated using the method described above. The primer sequences are listed in Table , type IIRelative gene expression levels were calculated using the DD-Ct method, with GAPDH as a reference. The expression of each gene was compared between the control and DM groups.p-value < 0.05 were considered significant. The data were analyzed using SPSS v23.0 .All data are expressed as the mean ± standard deviation. Cell viability and real-time PCR results were expressed as n-fold differences from the baseline control at the corresponding time point. Mann–Whitney’s U test was performed to compare the control group with the DM group. The relationship between the amount of AGEs and ROM was evaluated using Pearson’s correlation coefficient. Results with a p = 0.030 for AE, p = 0.002 for ER).The DM group consisted of four men and four women with a mean age of 64.0 years. The control group consisted of five men and three women with a mean age of 65.5 years. There was no significant difference in the average age between the two groups. In the DM group, the mean preoperative HbA1c was 7.3 ± 1.2 (6.5–9.7). The mean preoperative HbA1c level in the control group was 5.9 ± 0.2 (5.6–6.3). The mean preoperative passive ROM of the shoulder joint was 126.4 ± 36.3° of AE and 36.4 ± 6.9° of external rotation (ER) in the control group. In contrast, in the DM group, the mean ROM of AE was 90.0 ± 8.9° and that of the ER was 26.4 ± 11.2°. The ROM for both AE and ER was significantly greater in the control group than in the DM group  and the DM group (p = 0.008), respectively. The type III/I collagen ratio was significantly higher in the DM group (there was an increase by 1.00-fold and 2.57-fold in the control and DM groups, respectively. p = 0.01; Fig. Gene expression was compared between the control and DM groups. The gene expression of NOX1, NOX4, and RAGE, which are involved in the increase of ROS, was significantly higher in the DM group (In this study, we evaluated the effect of AGEs on the limited ROM of the shoulder with RCTs. AGEs were observed to accumulate in the shoulder capsule with limited ROM and was accompanied by increased oxidative stress and cell apoptosis. Increased oxidative stress and apoptosis were significantly correlated with HbA1c, an indicator of DM, and had a significant negative correlation with the ROM of the shoulder joint. Although previous reports have shown an association between DM and AGEs , this isAGEs are reported to cause aging in the skin, visceral organs, bone and cartilage, and lens, mainly because of the abnormal cross-linking of collagen . ExtraceRAGE and NOX and increased ROS production. The increase in ROS levels was accompanied by an increase in apoptosis, a decrease in cellular viability, and an increase in inflammatory cytokines. As a result, fibrosis and adhesion to surrounding tissues can occur in the joint capsule, leading to limited ROM.The binding of AGEs to RAGE activates NOX and increases ROS production, which is a source of oxidative stress . IncreasThe risks of AGE accumulation include aging and DM , 31. HwaThis study has several limitations. First, shoulder capsule tissues from patients without ROM limitation were not harvested owing to ethical reasons. Second, although a power analysis was performed, the sample size was not large. Further studies with more samples are required to confirm these results. Third, we did not compare the results according to the duration of DM. In addition, all patients in the DM group in this study had a preoperative HbA1c of 6.5 or higher, and the preventive effect of lowering the HbA1c to normal was not evaluated. HbA1c is considered an indicator of DM control, and its correlation with oxidative stress and apoptosis suggests that DM control could be important in preventing the ROM limitation. However, long-term prospective studies on the preventive effect of DM control are required. Finally, only DM has been examined as a contributing factor to limited ROM. The accumulation of AGEs may also be affected by age, and patient factors such as their daily schedules may also affect joint ROM. Further studies are required to confirm the results of this study.The DM group showed significant AGE deposition in the shoulder capsule. The findings of this study demonstrated a significant association between AGEs and ROM limitation. This study suggests that the oxidative stress induced by AGEs deposition, which leads to fibrosis and local inflammation, might contribute to the limited ROM of the shoulder joint in patients with RCTs accompanied by DM.


Glycogen storage disease type I (GSD-I) is an autosomal recessive disorder of carbohydrate metabolism, resulting in limited production of glucose and excessive glycogen storage in the liver and kidneys. These patients are characterized by life-threatening hypoglycemia, metabolic derangements, hepatomegaly, chronic kidney disease, and failure to thrive. Liver transplantation (LT) has been performed for poor metabolic control and delayed growth. However, renal outcome was diverse in pediatric GSD patients after LT. The aim of this study was to investigate the long-term outcome of renal function in pediatric GSD-I patients after living donor LT (LDLT), and to identify modifiable variables that potentially permits LT to confer native renal preservation.The study included eight GSD-Ia and one GSD-Ib children with a median age of 9.0 (range 4.2–15.7) years at the time of LT. Using propensity score matching, 20 children with biliary atresia (BA) receiving LT were selected as the control group by matching for age, sex, pre-operative serum creatinine (SCr) and pediatric end-stage liver disease (PELD) score. Renal function was evaluated based on the SCr, estimated glomerular filtration rate (eGFR), microalbuminuria, and morphological changes in the kidneys. Comparability in long-term renal outcome in terms of anatomic and functional parameters will help to identify pre-LT factors of GSD-I that affect renal prognosis.2, P = 0.268; median urine microalbuminuria to creatinine ratio: 16.0 vs. 7.2 mg/g, P = 0.099, respectively) after LT. However, in the subgroups of the GSD cohort, patients starting cornstarch therapy at an older age (≥ 6-year-old) before transplantation demonstrated a worse renal outcome in terms of eGFR change over years (P < 0.001). In addition, the enlarged kidney in GSD-I returned to within normal range after LT.The clinical and biochemical characteristics of the GSD and BA groups were similar, including immunosuppressive regimens and duration of follow-up (median 15 years) after LT. Overall, renal function, including eGFR and microalbuminuria was comparable in the GSD-I and BA groups (median eGFR: 111 vs. 123 ml/min/1.73mPost-LT renal function was well-preserved in most GSD-I patients. Early initiation of cornstarch therapy before preschool age, followed by LT, achieved a good renal prognosis.The online version contains supplementary material available at 10.1186/s13023-021-02026-6. Glycogen storage diseases (GSD) are inborn errors of metabolism with abnormal storage or utilization of glycogen, caused by enzyme deficiency, affecting glycogen synthesis or breakdown, or from mutations in proteins regulating glycogen metabolism . GSD typDevelopment of renal dysfunction in GSD-I cases was first reported by Chen et al. in 1988 . GSD nepThe principle of treatment for GSD is to maintain normoglycemia by continuous or frequent nutrition therapy with glucose, meals, or cornstarch day and night, affecting the quality of life for patients and their parents , 8. NotaIn contrast to glucose therapy, LT provides a healthy liver graft that not only corrects the genetically acquired error of metabolism but also mitigates the risk of developing adenoma growth or liver cirrhosis , 14. AltKaohsiung Chang Gung Memorial Hospital, Taiwan, maintains a longitudinal database of primarily living donor liver transplantation (LDLT) recipients and records all demographic, pre-operative, peri-operative, pathological, and follow-up information. A total of 339 children underwent pediatric LDLT in our institution from June 1994 to December 2019. There were 11 GSD-I cases, however, two patients died before 2019 (one due to pancreatitis and the other one from chronic rejection), with nine surviving GSD-I patients undergoing regular surveillance at an out-patient basis. For all GSD recipients, liver and renal function, metabolic biochemistry, growth development, and gene expression of GSD mutation were studied. To minimize bias associated with complications related to the LDLT procedures and long-term immunosuppression which may affect renal function, we enrolled biliary atresia (BA) patients receiving LDLT as control. To adjust for bias due to variations in baseline characteristics, we applied propensity score -matching analysis in the biliary BA groups in our center for comparison with the GSD groups. Propensity scores were calculated by logistic regression, adjusting for the following preoperative covariates: age, sex, preoperative serum creatinine (Scr) level, and pediatric end-stage liver disease (PELD) score. A 1:2 match was performed using the nearest-neighbor matching method. Patients’ characteristics have been summarized in Table In this retrospective study, all living GSD-I patients (n = 9) who underwent LDLT due to poor response to medical treatment were included; patients who died before 2019 have been excluded. All GSD-I (n = 9) and selected BA (n = 20) recipients consented to participate in the study with no subsequent dropouts from loss of follow-up. The study has been approved by the Institutional Review Board (IRB no. 201800281B0C101) and enrolled patients were provided with written informed consent.2 lasting more than three months, irrespective of etiology.The estimated glomerular filtration rate (eGFR) was calculated using the updated Schwartz formula for children (1–18 years old) and shifted to Modification of Diet in Renal Disease (MDRD) formula upon reaching adulthood , 17. MicAcute kidney injury (AKI) in children was defined by KDIGO criteria and classified into three stages by the increase in Scr : stage 1Ultrasound examinations were used to determine the kidney length, measured as the maximum pole-to-pole distance along the longitudinal plane in centimeters (cm). Bilateral kidney length measured were expressed in z score as corresponding to the normal distribution within the same age group , 23. NepDemographic and operative variables included age, sex, and underlying etiology of liver disease, in addition to the coincident diagnosis of hepatic adenoma on histologic examination of the explanted liver. Preoperative variables measured include PELD score, body height (BH), body weight (BW), body mass index (BMI), serum albumin, aspartate transaminase (AST), total bilirubin (Bil), SCr, eGFR, fasting glucose, uric acid (UA), total cholesterol (Chol), and triglyceride (TG) levels. Perioperative variables include intraoperative blood loss and graft-to-recipient weight ratio (GRWR). Postoperative data included de novo hypertension (HTN), AKI, growth development, as well as liver and renal functions, being continuously monitored every three to six months in the out-patient-clinic.The primary outcome in this study was the dynamic long-term changes of renal function post-LT as evaluated by serum Cr, eGFR, presence of albuminuria and changes in sonographic kidney length. Secondary outcomes included the GSD genetic study of Taiwanese, correction of metabolic disturbance, and recipients’ age-adjusted growth parameters  after LDLT.The pre-operative assessment included psychological examination and radiological assessment of the hepatic vasculo-biliary anatomy of both the donor and recipient with liver computed tomography (CT) angiography, magnetic resonance imaging and echography. The decision to proceed to LDLT was made in weekly multidisciplinary meetings.All LDLT patients in the cohort received an initial standard triple immunosuppression regimen of cyclosporin A (CyA), prednisolone, and azathioprine. Target serum CyA levels were gradually decreased from 1000 to 100–150 ng/ml within the first month after LDLT. Prednisolone was weaned off over one to two years, and azathioprine was discontinued at one-year post-LDLT. When rejection occurred or pediatric patients transitioned into adulthood, CyA was switched to orally taken tacrolimus (FK) as an alternative calcineurin inhibitor (CNI). In patients with elevated SCr during follow-up, mammalian target of rapamycin (mTOR) inhibitor was given in place of CNIs to preserve renal function.G6PC and SLC37A4 gene of the patients’ DNA  followed by manufacturer's protocol. Primers were designed for the exon sequencing of U test. Pre-LT and post-LT data were compared using the Wilcoxon signed-rank test. The annual change in the eGFR (mean ± 2 SDs) were grouped and calculated by generalized estimating equation. The association between age of starting cornstarch and microalbuminuria was performed with Spearman’s test. A P value < 0.05 was considered statistically significant.Data was collected and analyzed using IBM SPSS version 20 statistical software . Qualitative variables in both GSD and BA groups were expressed as frequency of events and cumulative incidence (in percentage) and compared using the chi-squared test. Quantitative variables were expressed by their median with range and compared using the Mann–Whitney P = 0.001) and hypoalbuminemia  can be explained by the underlying pathophysiologic course of BA, wherein the main clinical outcome in patients include obstructive jaundice and subsequent biliary cirrhosis. Intraoperative blood loss and GRWR between the two groups were similar. The histopathologic finding of adenoma exclusive to the GSD-I group, more specifically, all included patients in the group who underwent LT beyond the age of 9 . However, 33% of GSD-I patients were switched to mTOR inhibitor because of elevated SCr  and cyclosporin related post-transplant lymphoproliferative disease . The rejection rate was comparable between the two groups , p.R83H, p.H119L and p.I341N in the G6PC gene were found in eight of the GSD-I patients (Table SLC37A4 gene in case no.27 (Table 3 pre-LT to 1996/mm3 post-LDLT.All exons and splicing sites of the P = 0.008), lactate (P = 0.012), AST (P = 0.011), TG (P = 0.008), Chol (P = 0.051), UA (P = 0.008), and growth parameters by percentile according to age group in terms of BH (P = 0.012), BW (P = 0.086), and BMI (P = 0.051) are visualized in Fig. The line graphs showing the pre-to post-LT linear changes of measured variables including fasting glucose . GSD-I patient No. 18 underwent renal transplantation 16 years after LT for progressive renal deterioration; conversely, patient No. 28 demonstrated improved renal function after adjustments in the immunosuppression therapy , while significant deterioration in renal function in group C when compared with group B.We compared the dynamic change in mean eGFR among the GSD-I and BA recipients Fig. . The patP = 0.099; incidence of macroalbuminuria: 22% vs. 0%, P = 0.089). But there was a correlation between age of starting cornstarch and microalbuminuria , and a higher prevalence of macroalbuminuria  than group B.As shown in Table P = 0.028 and 0.075, respectively). For the other two GSD-I recipients, No. 215 and 235, no significant changes in kidney size were observed after LDLT. These changes have been graphically represented in Fig. Routine pre-LDLT renal ultrasonography for GSD-I cases was only protocolized in January 2004. Data of GSD-I patients with pre-LDLT ultrasonography (n = 6) was analyzed Table . RegressWhether correction of liver derangement prevents complications of GSD-I remains inconclusive. The liver transplant cases seem to be a good model to explore this possibility. However, such research is quite commonly- limited by the small study populations. In addition, the progression of late complications such as nephropathy may be best observed with long-term surveillance. While LT is theorized to help maintain a healthy metabolic environment, the necessary use of immunosuppressive agents has been a source of inquiry as to whether it compound the risk of developing the sequelae that LT claims to mitigate, that is renal dysfunction.2, P = 0.168; median ACR: 16.0 vs. 7.2 mg/g, P = 0.099). Patients with enlarged kidney in GSD-I patients may return to within normal range for age after LDLT. However, a subgroup analysis of the GSD-I cohort showed that patients who initiated cornstarch therapy at or beyond 6 years of age before LDLT had a propensity to develop poorer renal outcomes as measured in terms of albuminuria (P = 0.030) and eGFR changes (P < 0.001) over time.Here, we have provided a follow-up data of up to 22 years (median follow-up of 15 years), with multiple timepoints of assessment, in 9 GSD-I patients who underwent LDLT in our institution. Our results revealed that the overall renal function, particularly with eGFR and albuminuria as quantitative measures, were not statistically different between the two groups  medical records, as well as missing parameters from transferred medical records of patients from other hospitals. Second, the study was conducted at a single medical center, and postoperative prognosis may vary among different hospitals by virtue of differences in management. Third, the GFR was calculated using the updated Schwartz formula for children and the MDRD formula for adults, both of which may overestimate or underestimate the true GFR. However, we used the formula in both groups for comparability to minimize bias. Fourth, we may underestimate the incidence of AKI because only SCr values were used as the main criterion in the definition of AKI and urinary output was eliminated from the equation. Finally, the study recruited only 9 GSD-I and 20 BA patients. Therefore, to surpass the limitations, a larger, prospective, randomized controlled trial would be ideal.Our clinical data demonstrated that post-LT renal function was well preserved in most GSD-I patients. LT cannot reverse the preoperative renal dysfunction but may prevent or slow the progression of albuminuria and CKD. The timepoint of starting cornstarch therapy in GSD-I patients of pre-school age may be critical for long-term renal function. Early initiation of the treatment results in a good renal prognosis.Additional file 1: Supplementary Table 1. Primers for the exon sequencing of G6PC and SLC37A4 gene.


Zanthoxylum) is extensively used as spice and traditional medicine in eastern Asian countries. Recently, an emergent yellow-flower disease (YFD) break out in green Chinese prickly ash  at Chongqing municipality, and then leads to a sharp reduction in the yield of Qinghuajiao, and thus results in great economic losses for farmers. To address the molecular response for the emergent YFD of Qinghuajiao, we analyzed the transcriptome of 12 samples including the leaves and inflorescences of asymptomatic and symptomatic plants from three different towns at Chongqing by high-throughput RNA-Seq technique. A total of 126,550 genes and 229,643 transcripts were obtained, and 21,054 unigenes were expressed in all 12 samples. There were 56 and 164 different expressed genes (DEGs) for the AL_vs_SL (asymptomatic leaf vs symptomatic leaf) and AF_vs_SF (asymptomatic flower vs symptomatic flower) groups, respectively. The results of KEGG analysis showed that the “phenylpropanoid biosynthesis” pathway that related to plant–pathogen interaction were found in AL_vs_SL and AF_vs_SF groups, and the “Plant–pathogen interaction” found in AF_vs_SF group, implying that this Qinghuajiao YFD might cause by plant pathogen. Interestingly, we detected 33 common unigenes for the 2 groups, and almost these unigenes were up-regulated in the symptomatic plants. Moreover, most of which were homologs to virus RNA, the components of viruses, implying that this YFD was related to virus. Our results provided a primary molecular basis for the prevention and treatment of YFD of Qinghuajiao trees.Chinese prickly ash ( According to its peel color, Huajiao is divided into red Chinese prickly ash and green Chinese prickly ash . The fruits of Huajiao trees are largely used as a popular condiment in traditional cooking and medicine with a long history in China, which makes Chinese prickly ash an important economically fruit tree6. In particular, green Chinese prickly ash (Zanthoxylum schinifolium) is one of the most important commercial crops in Chongqing municipality. For instance, in 2018, approximately 530,000 acres of this variety were planted in Jiangjin district with a half billion dollars sale value. However, a yellow-flower disease broke out in Qinghuajiao at Chongqing in recent years, the infected trees showed phenotypes with pistil degeneration and stamen enlargement. Given that the normal green prickly ash was parthenogenesis, the infected plants could not bear fruit. On the other hand, with the growth of infected time, the leaves of diseased trees curled, and the root necrosis, which accelerated the death of Qinghuajiao trees, and then caused a serious impact on the production of Qinghuajiao, and thus resulted in huge economic losses.Chinese prickly ash  belongs to the Zanthoxylum genus of the Rutaceae family, and is a deciduous shrub native to East Asian countries7. Unlike a relatively stable genome, transcriptome alters with the change of biological conditions, stages of development, and external environmental factors. High-throughput transcriptome sequencing (high-through-put RNA-seq) is a powerful method to analyze the relationship between phenotype and genotype. It can accurately determine the gene expression level, differential splicing and specific expression of transcription products under specific conditions, so as to better understand the growth and development of cells and the underlying pathways and molecular mechanisms of disease progression9. The application of high-throughput RNA-Seq technology has promoted the study of plant–pathogen interactions with relatively complex genomes. Gray leaf spot (GLS), is one of the most impactful diseases in maize. Two maize cultivars ‘Yayu889’ (resistant) and ‘Zhenghong532’ (susceptible) were challenged with the GLS disease. The RNA-Seq results showed that there were 4666, 1733, and 1166 differentially expressed genes (DEGs) for ‘Yayu889’ cultivar, and 4713, 881, and 722 DEGs for ‘Zhenghong532’ cultivar after 8-, 10-, and 12-day infection, respectively, and which were mainly enriched in response to salicylic acid, protein phosphorylation, REDOX process, and carotenoid biosynthesis, and were especially active in the resistant cultivar10. Stripe rust is an important fungal foliar disease of wheat. Zhang et al. reported the transcriptome of wheat that infected by the powdery mildew E09 and stripe rust Cry31, and the results showed that Seven KEGG pathways were identified in response to the infection of powdery mildew and four pathways in response to the infection of stripe rust11. The RNA-Seq results of stripe rust infected wheat showed that the genes closely related to innate immunity of plants, such as biosynthesis and response genes related to plant stress hormones salicylic acid, jasmonic acid, ethylene and abscisic acid, were all significantly altered after strip rust infection. Meanwhile, genes encoding proteins with antimicrobial properties were found to be significantly enriched after the infection, including disease-related proteins, chitinases and cysteine-rich repeat proteins12.Recently, with the rapid development of high-throughput sequencing technology, the study of transcriptomics has achieved greatly progresses. Transcriptome refers to the complete set of transcripts of an organism, a specific tissue, and a cell at a specific developmental stage or under a specific physiological condition13, it allows us to define the global gene expression profiles for YFD infected Qinghuajiao trees. Here, we analyzed the transcriptome of 4 groups, 12 samples from three towns at Chongqing with severe disease by high-throughput RNA-Seq techniques. We found 56 and 164 different expressed genes (DEGs) for the leaf and flower samples of diseased trees. The results of KEGG pathway analysis showed that the DEGs for the diseased trees were involved in phenylpropanoid biosynthesis and plant–pathogen interaction pathways. In addition, we found 33 DEGs were shared by all samples from diseased trees, and most of these common genes were up-regulated in diseased trees and were highly homologous to the virus RNA, the components of viruses. These genes might be closely related to the YFD of Qinghuajiao. Our results provides an effective way for rapidly solving the outbreak diseases in agriculture.Given that RNA-Seq does not require a genome sequenceRecently, an emergent yellow-flower disease (YFD) was occurred in the green Chinese prickly ash (Qinghuajiao) trees in Chongqing, and showed a spread and dissemination tendency Figure . The heaTo elucidate the molecular response for this YFD of Qinghuajiao, we collected the leaves and inflorescences of symptomatic and asymptomatic Qinghuajiao trees of the same variety from the experimental field of Fruit Research Institute of Chongqing Academy of Agricultural Sciences, in Diaojia Town, Wutan Town and Mixin Town at March 21, 2019 , we assembled all clean data from scratch, and obtained 126,550 unigenes and 229,643 transcripts. The smallest and largest length were both 201 bp and 14,553 bp for the assembled unigenes and transcripts, and the N50 length was 1095 bp for unigenes, 1524 for transcripts  software, we obtained 56 and 164 differentially expressed genes  value < 0.001) for AF_vs_SF and AL_vs_SL groups, respectively. There were 147 up-regulated and 17 down-regulated DEGs for AF_vs_SF group, and 35 up and 21 down DEGs for AL_vs_SL group, respectively , SF , AL , and SL . In order to avoid the influence of environmental factors, the samples collected from three different towns were used as replicated samples. For instance, DAF, WAF, and MAF were used as replicates for AF samples. By using DESeq2 . For AL_vs_SL group, 22 DEGs were related to molecular function, in which “catalytic activity” (12) and “binding” (9) were the mainly GO terms; 25 DEGs touched upon biological process, and “metabolic process” (9) and “cellular process” (8) occupied a high percentage; 27 DEGs referred to cellular component, and “membrane part” (8) and “cell part” (6) made a major contribution to this category  and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis by using the free online tools of Majorbio Cloud Platform  by using the assembled sequences. Among the 33 genes, 9 genes are homologous to ZPNe1 RNA1 (Zhuye pepper nepovirus isolate ZPNe1 segment RNA1), 10 genes are homologous to ZPNe1 RNA2, 5 genes are homologous to CYMVLS RNA (Chicory yellow mottle virus large satellite RNA), which are all components of viruses; and 8 genes are homologous to other genes in plants , a badnavirus, leads to vein clearing symptoms in green Chinese prickly ash18. These reports are all from the plant pathogen view to elaborate disease of Chinese prickly ash. In this study, we detected the transcriptome alteration of Qinghuajiao leaves and inflorescences that encountered the YFD disease, and obtained a total of 220 DEGs, which were involved in the molecular response of Qinghuajiao to this YFD. To our knowledge, this is the first transcriptome report in response to Qinghuajiao YFD.Several pathogens including fungi and phytoplasmas caused diseases have been reported in Chinese prickly ash. Infection of 19. Based on the KEGG pathway and GO enrichment of transcriptome data, Guo et al. showed a complex regulatory network for PM resistance in pumpkin that may also involve hormone signal transduction pathways20. Chen et al. reported that genes associated with plant hormone signal transduction, detoxification, phenylpropanoid biosynthesis, photosynthesis and chlorophyll metabolism were significantly affected by CMV infection in yellow passion fruit21. Transcriptome results showed that the moderate resistance of Pinus pinaster to Fusarium circinatum may be explained by the expression profiles pertaining to early recognition of the pathogen, the induction of pathogenesis-related proteins and the activation of complex phytohormone signaling pathways22. Some KEGG annotations of Qinghuajiao encountered with YFD were also belong to these previously reported to be response to plant–pathogen interaction by transcriptomic analysis. For instance, the “Plant–pathogen interaction” and “plant hormone signal transduction” pathways for AF_vs_SF group, and the “phenylpropanoid biosynthesis” for both groups. These results are important first step for insights on the molecular mechanism for the interaction between Qinghuajiao and YFD, and suggest that the phenylpropanoid biosynthesis and plant hormone signal transduction pathways were involved in the responses of Qinghuajiao to YFD.The transcriptome profiling of cucumber leaves infected with powdery mildew (PM) revealed that the complex regulatory network for PM resistance involves plant hormone signal transduction, phenylpropanoid biosynthesis, plant–pathogen interaction and the MAPK signalling pathway23. The grapevines that infected by an Enamovirus member, GEV-1 (Grapevine enamovirus-1) exhibit redden or yellowing and downward rolling leaves24. Tomato black ring virus (TBRV), a typical member of Nepovirus genus within the Secoviridae family, infects a wide range of economically important plants worldwide, including tomato, strawberry, potato, celery, and artichoke25. Gaafar et al. reported that a caraway yellows virus (CawYV) infection results in systemic yellowing in Caraway26. Grapevine fan leaf virus (GFLV), a picorna-like plant virus, is severely in responsible for a widespread disease in vineyards worldwide27. The blast results of the up-regulated 30 common genes showed that most of these genes has highly homolog with virus RNA, implying that the YFD of Qinghuajiao might be related to virus. Further study will be conducted at the isolation of the virus that resulted in YFD of Qinghuajiao, the molecular character and the transmission routes of these viruses, so as to get the methods to suppress the transmission of these viruses, and thus to inhibit the further spread of YFD. In addition, although all most virus-infected plant diseases cause economic losses, Beaver-Kanuya and Harper had reported that four viruses infecting the pollen of Prunus species might have implications for biosecurity28. While the symptomatic Qinghuajiao trees have intumescent stamens, which could be used to improve male sterility in other crops.Several virus diseases have been reported in plants. A subgroup B nepovirus, Potato virus B (PVB) infected the potatoes in central Peru, and leading to calico-symptom leavesTaken together, through high-throughput RNA-Seq analysis of 12 samples from three points, we revealed the molecular response of YFD of Qinghuajiao. We found a total 220 DEGs for the symptomatic trees. The Gene Ontology (GO) analysis showed that the major related GO terms of the DEGs were “catalytic activity” and “binding” to molecular function category, “metabolic process” (9) and “cellular process” to biological process category, and “membrane part” and “cell part” to cellular component category, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) results showed that the DEGs were divided into 10 categories for AL_vs_SL group and were referred to 24 categories for AF_vs_SF group. Particularly, the “phenylpropanoid biosynthesis” pathway that related to plant–pathogen interaction were found in both groups, and the “Plant–pathogen interaction” founded in AF_vs_SF group, implying that this Qinghuajiao YFD might cause by plant pathogen. Interestingly, we found 33 shared DEGs for the AL_vs_SL and AF_vs_SF groups, and most of them were all up-regulated in the samples from diseased trees. Among which, 24 genes were homologs to virus RNA, implying that this YFD of Qinghuajiao was related to virus. Although the mechanisms underlying the response of Qinghuajiao to YFD still require further research, the knowledge obtained from this study will serve as a useful genetic resource to facilitate further investigations of YFD in Qinghuajiao, and provide many possible directions for prevention and treatment of YFD of Qinghuajiao. Additionally, our study also provides a quick and preliminary approach to identify the causes of serious diseases occurring in plants with complex genomes under natural conditions.Zanthoxylum schinifolium, Qinghuajiao) was grown in Chongqing at natural conditions. The in inflorescences and leaves of asymptomatic and symptomatic Qinghuajiao trees of same variety (Qinghuajiao) were collected from the experimental field of Fruit Research Institute of Chongqing Academy of Agricultural Sciences, at Diaojia town , Mixin town , and Wutan town  in Chongqing as biological replicates to avoid environmental influences. The plants for photograph and samples collection were 5-year old, all leaves are fully developed antepenultimate leaves, and the flowers are shoot apical flowers. The experimental research on the plants described in this study comply with institutional, national and international guidelines.The green Chinese prickly ash  magnetic beads. The sample libraries were qualified and quantified by Agilent 2100 Bioanaylzer and Nanodrop2000. The library products were sequenced via Illumina Novaseq 6000. The raw data were filtered with the FASTQ_Quality_Filter tool from the FASTX-toolkit . And then using Trinity software  to assemble all sample clean data from scratch13, and using TransRate 30 and BUSCO 31 to Optimize and evaluate assembly. All assembled transcripts were compared with six major databases . The transcriptome was quantified by RSEM 32, and the amount of gene expression was expressed in the words TPM (Transcripts Per Million reads)33. The different expression gene (DEG) was analyzed by DESeq2  as described by Love et al34. The significance threshold for the differential expression was FDR  < 0.05 and a |log2 fold change | > 1. All DEGs were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment using KOBAS software (http://www.kegg.jp/kegg/kegg1.html)35. The corrected P-value is tested by Fisher's exact test, when the corrected P-value is < 0.05, significant enrichment of KEGG pathway is considered. The heat map was built by D3  with modification by Shanghai Majorbio Bio-pharm Technology Co., Ltd.The transcriptome data was analyzed on the free online tools of Majorbio Cloud Platform . First-strand cDNA was synthesized from 1 µg total RNA using a reverse transcription kit with genomic DNA remover . Gene-specific primers are designed and listed in Table The plant materials used in this study were collected from the experimental field of Fruit Research Institute of Chongqing Academy of Agricultural Sciences, and field permission was not necessary to collect the plant samples for this study. The authors declared that experimental research on the plants described in this study comply with institutional, national and international guidelines.Supplementary Information.


Many recent studies highlight the fundamental importance of viruses. Besides their important role as human and animal pathogens, their beneficial, commensal or harmful functions are poorly understood. By developing and applying tailored bioinformatical tools in important virological models, the Marie Skłodowska-Curie Initiative International Training Network VIROINF will provide a better understanding of viruses and the interaction with their hosts. This will open the door to validate methods of improving viral growth, morphogenesis and development, as well as to control strategies against unwanted microorganisms. The key feature of VIROINF is its interdisciplinary nature, which brings together virologists and bioinformaticians to achieve common goals. The Innovative Training Network (ITN) is one of the Marie Skłodowska-Curie Actions of the European Commission, which aims to train a new generation of creative, entrepreneurial and innovative early-stage researchers, able to face current and future challenges and to convert knowledge and ideas into products and services for economic and social benefit. Within the ITN VIROINF, we aim to understand virus-host interactions, by bridging virology and bioinformatics being an emerging field, see The main themes of VIROINF are: (i) Modelling virus-host interactions, including virus identification, host prediction, virus-host interactions, virus regulation, and virus products; and (ii) Modelling virus evolution in hosts, including microevolution (virus quasispecies) and macroevolution  in Jena in 2017, a broad range of applied bioinformaticians and virologists met to guide developments in virus bioinformatics. The interactions between the members resulted in the identification of a broad range of opportunities for collaboration between the research groups. In order to create a framework for collaboration between these research groups we propose the implementation of the VIROINF ITN  and requThis part of the ITN aims at developing fundamental bioinformatical tools for modelling virus-host interactions and understanding these interactions mechanistically. In order to describe virus-host interactions, interacting viruses need to be identified and hosts need to be determined.en masse, but different research groups use different protocols to analyse these samples and prepare them for sequencing. All the current approaches result in relative distributions, which lack absolute quantitation. Sample preparation, methods and standardisation are prerequisites for meaningful and comparative work. On the bioinformatical side, the most frequently used similarity-based virus identification approaches are largely limited by available databases, which are incomplete and often poorly annotated. VIROINF aims to set up a multiple layer database for phages and their hosts, which with the help of the European Virus Bioinformatics Center will be performed in the first months of VIROINF.Despite large ongoing efforts to identify viruses in humans, animals and the environment, there are still multiple challenges remaining in the identification of novel viruses. These challenges concern both the wet-lab as well as the bioinformatical side. On the wet-lab side, divergent viruses can now be identified from an infected sample VIROINF aims to quantify viruses by the following four approaches: (i) spiking in known amounts of selected viruses into a sample as internal controls; (ii) counting virus particles using flow cytometric approaches in collaboration with Corina Brussaard from the Netherlands Institute for Sea Research; (iii) test Minion sequencing technology on unamplified extracted viral nucleic acids, and (iv) NGS-based quantification developed in collaboration with non-academic partner Baseclear. The novel wet-lab approaches will be combined with newly developed advanced bioinformatical tools to identify viruses from metagenomics datasets (ESR-UMG), using sequence dependent and independent approaches. Motif searches, k-mer frequencies, and genome organisation are de novo approaches that will complement homology-based methods, together allowing the detection of known and unknown viruses, yielding a comprehensive detection pipeline of all types of viruses ranging from well-studied human pathogens to completely novel bacteriophages. A range of available tools for taxonomic classification will be combined into a rapid pipeline that will be used from many beneficiaries: ESR-FU, ESR-HMGU, ESR-JMU, ESR-ETH, ESR-RKI, ESR-UZH and ESR-UL. These tools will be made available in the public domain by VIROINF. Together, these developed wet-lab and bioinformatical approaches will be used by ESR-UL to provide an in depth in silico and in vivo characterization of the bacteriophage population infecting the most important members of the honeybee gut microbiota, as well as studying their role in honey bee health and development.ESR-UL—Development of more quantitative viral metagenomics approaches to investigate the role of bacteriophages in the honey bee gut and their effects on health and development. Very few is known about the role of the gut virus population (the virome) in health and disease of honey bees. Recently the NetoVIR protocol was developed to purify viral particles from biological samples, before deep sequencing using Illumina NGS technology [chnology . Using tThe third objective of this project is to provide an in depth in silico and in vivo characterization of the bacteriophage population infecting the most important members of the honeybee gut microbiota, as well as studying their role in honey bee health and development. Both the bacteriome (16 s) and the virome (shotgun) inhabiting the digestive tract of these honey bees will be characterized. Furthermore, bacteria will be isolated and characterized from the crop, midgut and hindgut of the collected honeybees, and finally these bacterial isolates will be used to isolate bacteriophages followed by their genome sequencing and functional gene annotation. Depending on the available time, we may also start with inoculation experiments of life honey bees see .ESR-UMG—A machine learning pipeline to identify and characterise novel viruses in metagenome data. The wide use of deep sequencing technology in biology and medicine has led to huge publicly available sequencing data bases, offering novel insights into the genetic and genomic makeup of life. Similarly, metagenome sequencing projects offer a deep view into microbiomes. Only a minuscule fraction of the virome has so far been identified; however, many hitherto unknown viral sequences lie hidden in already available data. The objective is to develop efficient computational pipelines to search for novel viruses in genome or metagenome sequencing data, as well as in public DNA and RNA sequencing databases such as NCBI Genbank/SRA. Template-based search strategies will be developed to search for unknown viruses that are related to a known virus family which is used as search template [template . We willHosts of viruses infecting higher eukaryotes have been well described in the past, whereas the specific hosts of bacteria-infecting viruses are usually unknown. The aims of VIROINF are to investigate how viruses of Bacteria or Archaea (bacteriophages) are functionally linked to each other and to their unicellular hosts. This is crucial for understanding how phages impact the microbiome as they act as key regulators of bacterial population size , and canStaphylococcus aureus. Phages are important mediators of bacterial virulence and hence impact human health. Understanding how bacterial gene content is influenced by this lateral gene transfer mechanism is important for understanding how viruses impact both the specific bacteria and the human host [VIROINF will, for the first time, address this topic using a combined experimental and computational approach: ESR-HMGU develops methods to experimentally link phages to their hosts as described by Li Deng , as wellman host . The tooESR-HMGU—Bacteriophage–host relationships prediction. This project seeks to understand how (bacterio)phages are functionally linked to their Bacteria or Archaea hosts both using experimental and bioinformatics approach. Phages identified using cultivation independent viral metagenomic sequencing approach lack sequence similarity to any of previously sequenced ones and can hardly be assigned to their hosts. Different methods exist to predict hosts for phage sequence, e.g., single or multiple features , or using these features in machine learning models. Therefore, we aim to integrate features of our unique experimental identified, host-linked viral metagenomic data in a deep learning model to improve the prediction performance. Particular attention will be given to phages of anaerobic bacteria which are underrepresented comparing to those infecting aerobic bacteria [bacteria . ESR-HMGbacteria ,10,11. IESR-UG—Computational prediction of virus-host interactions in the microbiome. Virus-host interactions are highly specific as viruses (including bacteriophages/phages) have co-evolved with the systems they infect and depend on for replication. As a consequence, phylogenetic relatedness and shared genomic properties of host species are a strong predictors of host infectivity. However, our knowledge of host relationships remains sparse and is unknown for many viruses and phages, particularly for metagenomically assembled data. In this project, existing resources and genomic signals will be leveraged to infer missing/unobserved and probable hosts, e.g., where infection has not been observed due to host immunity. Machine learning models will be trained on this data to predict virus-host/ phage-bacteria interactions at the species and strain levels  . A new aESR-JMU—Conservation of regulatory elements and effector mechanisms in lytic and latent cytomegalovirus infection. The human cytomegalovirus (HCMV) is a herpesvirus prevalent worldwide responsible for life-threatening infections in individuals with an impaired or immature immune system. During the first 1–2 h after virus entry into a cell, a decision is made whether this results in lytic, latent or abortive infection. Previous single cell RNA-seq (scRNA-seq) approaches did neither provide the necessary temporal resolution nor sensitivity to analyse this crucial phase of virus-host interaction. To overcome these limitations, we recently developed single-cell SLAM-seq ESR-RUMC—Impact of host immune pathways on virus evolution Evolution of viruses is strongly affected by antagonistic co-evolution of virus and host. Host immune pathways select for viruses that evade, avoid, or suppress the immune response, which in turn may drive counter-adaptations in host immune genes. For example, the antiviral RNA interference (RNAi) mechanism is actively suppressed by many insect viruses [Drosophila melanogaster and other invertebrates [Drosophila C virus, widespread pathogens of Drosophila that suppress RNAi at different stages [ viruses , likely tebrates ,21. Whett stages ,23.ESR-WZ—Elucidating the role of RNA modification during cytomegalovirus infection. N6-methyladenosine (m6A) plays critical roles in RNA metabolism and function. In addition to the internal m6A, N6,2′-O-dimethyladenosine (m6Am) is present at the transcription start nucleotide of capped mRNAs. The methyltransferase that adds this modification, PCIF1, was recently identified but the functional importance of this modification still remained enigmatic.Human cytomegalovirus (HCMV) is an important human pathogen that replicates in the nucleus exploiting cellular machineries. We will investigate if HCMV utilises the m6Am modification by conducting m6A-IP and high throughput sequencing using primary fibroblasts and investigate its functional importance especially to viral titers in the absence of PCIF1. Our objectives are: (a) We will examine m6Am modification landscape during viral infection  by conducting m6A-IP on HCMV infected primary fibroblasts and analysing the enrichment of the first base compared to input control. (b) We will dissect the effect of this modification on viral propagation by generating PCIF1 KO cells. (c) We will analyse how m6Am modification affects viral RNA lifecycle using RNA-seq , 4sU-seq  and Ribo-seq (RNA translation) applied on infected PCIF1 KO and control cells.Viruses circulate predominantly in their natural reservoir host but frequently also infect other species. However, to establish a new lineage the virus has to adapt to the new environment by acquiring advantageous new mutations. These mutations also influence the secondary structure of the viral DNA and RNA, in turn affecting the replication process. ESR-RKI, will study the fundamental correlation between virus evolution and RNA secondary structures. Being more specific, in case of segmented genomes ) packaging is influenced by the secondary structure of the virus genomic RNA segments. In cells co-infected with different viruses, new variants with mixed genomes can emerge that have the potential to overcome the natural host species barrier and to cause a devastating pandemic. Mixing of viral segments, also designated genetic reassortment, is possible due to a very efficient incorporation of different genome segments into one virus particle. ESR-CNRS will systematically generate reassortant viruses between two human IAVs and select those that replicate most efficiently for further functional studies. Additionally, ESR-CNRS will use chemical probing to compare the genome structure in parental and those reassortant viruses. ESR-FSU will study genetic reassortment events of IAV and especially the role of RNA-RNA interaction that are supposed to coordinate the interaction of the different genomes which are finally incorporated into viral particles. The focus of ESR-FSU is to develop a computational tool that can predict the complex interplay of the RNA-RNA interaction required for genome packing between quite diverse IAV based on experimental data.ESR-FSU—Deciphering the RNA genome packaging code of influenza A viruses. Currently, bioinformatical tools are not specifically designed for viruses. However, viruses bring unique features, which require specific bioinformatical tools to trace virus-host interaction. For example the number of sequences in a quasispecies is massively high due to their high mutation rate, but only a few interact again with the host cells. Some viruses, such as IAV, are segmented RNA viruses, which urgently require tools with specific features: RNA viruses should include standardised secondary structure predictions, leading to RNA-RNA interaction prediction necessary for the packaging of segmented RNA viruses, as preliminary depicted in [icted in ,25. TherESR-CNRS—Mechanism of coordinated incorporation of segmented virus genomes into viral particles. Genetic reassortment between divergent IAVs in the host is a highly biased process in which only a small fraction of all possible genotypes are efficiently produced. This restriction is due to the limited compatibility between packaging signals of divergent IAVs and the limited conservation of RNA-RNA interactions required for genome packaging, but might also be influenced by the host as environment. Thus, analyses of genetic reassortment not only shed light on how viruses with pandemic potential are produced, but also provides crucial information about the coordinated incorporation of the segmented IAV genome. Therefore, we will (a) generate reassortants between two human IAVs that will contain seven segments from one parental virus and one segment from the second parental virus. (b) Compare the secondary structure of the genomic RNA segments in the two parental and up to eight selected reassortant viruses grown in human cells using chemical probing. This analysis will be performed with ESR FU1 and highlights differences in RNA-RNA interactions between parental and reassortant viruses  metagenomics samples. We aim for large-scale identification of phage sequences, functional annotation and in vitro validation of phages as treatment against harmful bacteria.ESR-UZH—Generation of experimental datasets for phage-bacterial interactions in complex communities and mutational spectra analysis following exposure to selective pressure to support artificial intelligence applications for R&D of bacteriophage products. Bacteriophages are the most abundant organisms on Earth, making it difficult to select which ones have potential for therapeutic or other commercial applications. Current methods rely on non-iterative in vitro elaboration for product development, requiring >6 months per phage product. In the current project, we aim to generate input data from experiments to build deep learning algorithms that will guide and support phage product development by (a) revealing which phages compete best under which specific conditions and combinations, (b) identifying mutations important for phage activity and survival under environmental conditions , and (c) demonstrating the population effects of phage infection in complex community structures. In this project, we will use a top-down approach to characterise the resulting community composition and mutational spectra from in vitro assays and/or clinical samples to measure: (i) competition between multiple phages; (ii) phage-interaction in complex communities to simulate microbiome and polymicrobial infection settings; and (iii) exposition of phage to multiple environmental conditions using genomic re-sequencing and/or cycling temperature capillary electrophoresis (CTCE) to identify regions of mutations associated with specific stimuli.ESR-UV—Computational methods for the analysis of metagenomic datasets to extract viral sequences within the context of commercial data-mining/R&D support for bacteriophage products. Bacteriophages are increasingly being developed for the treatment of bacterial infections, and their role as modulators of the microbiome is of a growing area of extension of phage use. The sheer number and diversity of phage particles makes it impossible to isolate and test each of them individually. This project aims to identify and harness phage sequences for commercial use by developing a method for large-scale identification of phage sequences, functional annotation, and in vitro validation of findings in a bottom-up approach. We have four specific aims: (i) Identification of phage sequences within >50 metagenomic datasets . Reconstruction of viral genomes sequences from metagenomic data ; Reconstruction of cellular genome sequences (MAGs) from the same/related metagenomic datasets, in order to extract potential phage-host pairs from co-occurrence patterns; Prediction of prophage sequences in MAG in order to facilitate host prediction of phage sequences (in collaboration with ESR-UMG). (ii) Extraction and testing of sequences to validate WP 1 findings ie., hypothesis testing by either isolating the phages themselves or having their genome synthesised (in collaboration with ESR-UZH). (iii) Sub-analysis of geographical and temporal variation in metagenomic datasets that could justify product composition or modification analysis of geographic and temporal variation of phage sequences according to the local epidemiology  is examined in the viral population, also known as ‘virus quasispecies’. In the area of macroevolution, the long term evolution (millions of years) is examined to determine the relation of virus families.Viruses display high genetic diversity both within and among viral species, as well as within and among infected hosts. The composition of mixed samples can be assessed by metagenomics approaches , such asVIROINF will search for patterns which can emerge when host affiliation is projected onto viral taxonomy. Branch permutation techniques will be used to statistically determine at which level the viruses and hosts co-evolve. These patterns will reveal the evolution of virus-host associations for different classes of viruses. The associations will be validated by analysing both, known and predicted, host-associations independently. Moreover, virus-host associations will be used to validate the co-evolutionary signal.ESR-ETH—Development of new computational methods for characterisation and analysis of intra-host viral populations. Viruses exist in their hosts as populations of genetically heterogeneous particles, often referred to as viral quasispecies. Intra-host genomic diversity can result in phenotypic heterogeneity, and it has been linked to viral pathogenesis and virulence. NGS can be used to assess the genomic heterogeneity of virus populations in a cost-efficient manner, but this approach is challenging due to sequencing errors and short read length. In this project, we will develop improved tools for the reconstruction of viral genomic diversity from both short-read and long-read NGS data, and we will leverage the power of the inferred population structure to inform models of virus micro-evolution, to detect selection, and to correlate population features to viral and host phenotypes.ESR-RKI—Genotype-Phenotype mapping and inference of epistatic interactions driving adaptation in viral hosts. Viruses hijack the host cellular machinery for replication. This hijacking is driven by the interaction of viral proteins and non-coding RNAs with host-cellular components. Viral genomic sites instrumental to these interactions are thus conserved through evolution, yet bioinformatics analysis of evolutionary conservation cannot tell apart and quantify the functional relevance of genomic sites under selection. We have previously shown using the Mutational Interference Mapping Experiment (MIME) that in vitro [in vitro  and in vin vitro  evolutioin vitro  to identDrosophila virus) to predict phenotypic contributions to virus growth, mortality and host transcriptional responses.On the technical side, (a) we will learn maximum entropy models  from nucleotide abundances in functionally selected and de-selected virus quasispecies to identify single- and interacting sites. (b) As an alternative approach and a means to validate a), we investigate the application of deep learning approaches to identify higher-order epistasis in collaboration with secondment Bernhard Renard (HPI). (c) We then derive algebraic expressions from kinetic modelling of the investigated selection process to interpret the derived coupling terms mechanistically . This allows to characterise the complex (and possibly highly constraining) fitness landscape on which adaptation takes place. In the development phase our computational methods will be benchmarked by simulating the selection experiments and the corresponding NGS data sets, as well as with existing NGS datasets and phenotypic endpoints from previous work . Subsequently, we will apply the tools to NGS data generated by in vitro evolution experiments of ESR-CNRS/secondment R. Smyth (HIRI) to investigate domains in the influenza genomic RNA, which are responsible for viral packaging/re-assortment, in collaboration with AllGenetics (AG). Moreover, we will apply the methods to in vivo evolution experiments conducted by ESR-FU (deformed wing virus), as well as ESR-RUMC , but thESR-FU—Evolution of Deformed wing virus (DWV) in bees.Varroa destructor, which can vector the virus. In this project, we will carry out experiments with the aim of understanding the genomic basis of virus virulence (see This project seeks to understand the evolution and impact of virus virulence. The honeybee virus Deformed wing virus (DWV) is an emerging pathogen that is responsible for widespread declines of honeybees in the Northern hemisphere. The impact of the virus has been exacerbated by the arrival of an ectoparasitic mite: ence see . TogetheLayer 1: project specific training (bioinformatics and virology) and scientific and transferable skill courses at the host institution; Layer 2: training through project-specific ESR-ESR partnerships , and through secondments; Layer 3: training through network-wide events. Yellow stars indicate additional training for the ESRs ).We hope to be a nucleus that not only conducts fundamental research, but also reaches out into both research communities—virologists and bioinformaticians—to foster further collaborations. We are open to discussions about integrating your appreciated research in any way possible, from joint talks or conferences, to pair-wise collaborations, to association in our consortium. Please do not hesitate to contact us (


Later, a sucrose/acid gallic solution was concentrated through block freeze concentration (BFC) at three cycles. Thus, each solution was a mixture with the respective combination of alginate/cornstarch. The final solution was added drop-wise on a CaCl2 solution, allowing the formation of calcium alginate-cornstarch hydrogel beads filled with sucrose/acid gallic solution or cryoconcentrated solution. The results showed that alginate at 2% (w/w) and cornstarch at 2% (w/w) had the best efficiency to encapsulate any solution, with values close to 63.3%, 90.2%, 97.7%, and 75.1%, and particle sizes of approximately 3.09, 2.82, 2.73, and 2.64 mm, for initial solution, cycle 1, cycle 2, and cycle 3, respectively. Moreover, all the samples presented spherical shape. Therefore, the appropriate content of alginate and cornstarch allows for increasing the amount of model cryoconcentrated solution inside of the hydrogel beads. Furthermore, the physicochemical and morphological characteristics of hydrogel beads can be focused for future food and/or pharmaceutical applications, utilizing juice or extract concentrated by BFC as the solution encapsulated.The objective of this study was to evaluate the optimal conditions to encapsulate cryoconcentrate solutions via ionic gelation technique. Hydrogel beads were prepared using alginate (1%, 2% and 3% ( Recently, many studies have shown the potential of block freeze concentration (BFC) to improve different physicochemical properties and bioactive compounds from fruit juices, such as total soluble solid content , color , polypheDespite all the benefits and interesting properties in the cryoconcentrated juices, there are limitations on their properties in terms of stability, bioactivity, and bioavailability, due to factors such as processing , environmental features , storage time , and eve2+), allowing the construction of a polymeric microparticle, also called hydrogel beads [Hence, in the last few decades, the encapsulation by ionic gelation has emerged as an effective technology to contain liquid samples in biopolymer matrices due to the low temperatures involved in the encapsulation process, and thus, this method allows minimal modification of the quality properties in the encapsulated solution . Specifiel beads . Wherebyel beads , betaxanel beads , and ellel beads , anthocyel beads ,20, and el beads . HoweverAdditionally, the use of an experimental design (DOE) allows one or more variables (factors) to be deliberately manipulated to determine the effects on a variable of interest  . SpecifiThus, the objective of this work was to investigate the effects of encapsulation of cryoconcentrated solution  obtained by BFC through the ionic gelation method to form stable calcium alginate hydrogel beads filled with cryoconcentrated solution. Additionally, total soluble solid content, gallic acid content, particle size, shape, efficiency of encapsulation, loading capacity, water activity, moisture content, and bulk density were determined. Hence, the innovation of this study is the first step on encapsulation of cryoconcentrated model solution for future formation of calcium alginate hydrogel beads filled with cryoconcentrated samples obtained from different liquid foods, such as fruit juices or plant extracts. The results provided information for the best alternative in terms of alginate and cornstarch concentration to encapsulate cryoconcentrate solutions.2), sodium carbonate (Na2CO3), Folin–Ciocalteu reagent, and gallic acid (GA) were supplied from Merck . Distilled water was used throughout. All of the chemical products have high purity grade (99.9%) which were used in this study. Commercial corn starch was purchased from a local market .Sodium alginate, sucrose, ethanol, calcium chloride . Therefore, it is very important to control the freezing rate, since, when the freezing rate was higher than 8 μm/s, the freezing occurred too rapidly to expect a considerable separation of the concentrated solution from the ice crystals, i.e., the ice occluded solutes during the freezing process . Thereby2 powder was dissolved into distilled water with constant agitation using a magnetic stirrer, preparing a concentration of 0.5 M. Thus, the sodium alginate/cornstarch solution was mixed with a sucrose/GA solution  under mild agitation (250 rpm) for 30 min. Once homogenized, each solution was added drop-wise on the CaCl2 solution (in constant mild agitation with a magnetic stirrer) through a fine stainless-steel needle , with a height between the tip and the surface of the CaCl2 solution close to 15 cm. Then, the beads were maintained in the gelling bath (CaCl2) for 30 min, and later, the beads were filtered and subsequently washed with distilled water -.The encapsulation of initial sucrose/GA solutions and cryoconcentrated samples was prepared by the ionic gelation method as described by Fathordoobady et al. , with moThe TSSC in the initial sucrose/GA solution and cryoconcentrated samples was determined using a handheld refractometer  and the results were expressed as °Brix. Distilled water was used as the calibration substance.2CO3 solution was added to the mixture. Thus, the mixture was allowed to stand in the dark at room temperature for 90 min (incubation), and later, the absorbance at 760 nm was recorded, using gallic acid as the standard. The samples were measured using a UV–vis spectrophotometer . The results were expressed as mg GA equivalents (GAE) per liter of sample (mg GAE/L).The GA content in the initial solution, cryoconcentration samples, and beads was quantified by the Folin–Ciocalteu assay , with slcTBC is the GA concentration released from the beads, and TBC0 is the respective initial GA concentration added during the encapsulation process .The beads were deposited in a mortar and crushed with a pestle . TherebyW0 is the weight of the beads before the drying process, and fW is the weight of the beads after the drying process.The beads were dried in an oven at 120 °C for 2 h . Thereby® software [The particle size of the beads was measured by digital image analysis (1600 × 1200 pixels) using ImageJsoftware , where fmaxd is the largest diameter passing through the bead centroid and mind is the smallest diameter perpendicular to maxd.The shape was quantified to identify the roundness of the beads using the sphericity factor (SF)  through w of the samples was determined using a dew-point hygrometer  at ambient temperature.The aThe moisture content was carried out according to the method defined by Kaderides and Goula . The samρB was obtained according to the method described by Abdullah and Geldart [ρB was calculated by dividing the mass of the beads by the volume occupied in the probe [The  Geldart . Specifihe probe .2, considering 2 factors at 3 levels (for each factor) was used to study the optimum combination of alginate and cornstarch solutions in the encapsulation of cryoconcentrated solution through the ionic gelation technique. The levels of independent variables used for the experiment were 1%, 2%, and 3% (w/w) in alginate solution and 0.5%, 1%, and 2% (w/w) in cornstarch solution. A total of nine experiments were performed separately for getting the experimental response for the dependent variables, such as particle size, shape, EE%, aw, moisture content, and bulk density.The experiments were conducted according to the experimental design given in p ≤ 0.05) and graphical analysis of the data (response surface methodology (RSM) and contour plots).All the experiments were conducted in triplicate, and the results were reported as mean ± standard deviation. Results were subject to analysis of variance (ANOVA) and least significant difference test (LSD) using Statgraphics Centurion XVI Software  to obtain the statistical analysis of the data of the experimental design (w/w) and cornstarch (0.5% w/w) presented the lowest EE% values, with 12.5%, 37.9%, 59.4%, and 19.3%, for initial solution, cycle 1, cycle 2, and cycle 3, respectively, but an increase in the concentration of cornstarch ) allowed a significate increase in the EE%, since the values were close to 21.3% and 25.8% for initial solution, 60.8% and 64.4% for cycle 1, 64.6% and 69.0% for cycle 2, and 22.1% and 32.2% for cycle 3, with cornstarch at 1% and 2% (w/w), respectively. Thus, alginate at 1% (w/w) with any concentration of cornstarch and encapsulated solution presented lower EE% values than the other alginate/cornstarch combinations. Thereby, a gradual increase in the concentrations of alginate and cornstarch indicated a significant increase in EE%, since alginate at 2% and 3% (w/w) presented EE% values ranged from 47.7% to 63.3%, and 39.0% to 57.2%, for initial solution, 75.9% to 90.2%, and 52.1% to 70.2%, for cycle 1, 82.9% to 97.7%, and 64.9% to 83.5%, for cycle 2, and 50.7% to 75.1%, and 40.0% to 53.6%, for cycle 3, from 0.5% to 2.0% (w/w) of cornstarch, respectively. Hence, the encapsulation process  with cryoconcentrated solution from cycle 2 presented the maximum EE% values in comparison to the initial solution, cycle 1, and cycle 3.Firstly, for all the cases, the minimum concentration of alginate  and cornstarch at 2% (w/w), since the EE% values were close to 63.3% for initial solution, 90.2% for cycle 1, 97.7% for cycle 2, and 75.1% for cycle 3, respectively. Moreover, alginate at 3% (w/w) with any concentration of cornstarch and solution encapsulated had EE% values lower than those of alginate at 2% (w/w) with the respective concentration of cornstarch and solution encapsulated.Then, from the EE% values, the best combination to encapsulate any solution  in comparison to the other combinations was alginate at 2% (w/w) with cornstarch at 2% (w/w) was chosen as the optimal combination to encapsulate the initial solution and each cycle of the BFC process, since these values present the maximum EE% values.Therefore, by considering all the results from this experimental design, the alginate at 2% (w/w) allows for better interlacing between the molecules than other concentrations, forming thicker connections, and thus, this phenomenon strengthens the hydrogel bead matrix, and in turn, reduces the size of the empty spaces (pores), decreasing the output of liquid encapsulated to the outside [w/w) produces hydrogel beads with weak connections, making it more prone to liquid leakage during the encapsulation process, and also, a concentration of cornstarch lower than 2% (w/w) is not enough to fill the pores generated by the cross-linking of the molecules that form the microsphere. In the same way, cryoconcentrate from the third cycle, alginate higher than 2% (w/w), and cornstarch higher than 2% (w/w) affects the properties of the mixture, since the mixture provokes a solution with high viscosity, causing difficult manipulation (extrusion) of the material encapsulated [The high EE% values for the selected combinations can be explicated due to the fact that the concentration of alginate at 2%  and cornstarch at 2% (w/w)) were higher than in the encapsulation through calcium-alginate (3% (w/w)) capsules containing cornstarch at 2% (w/w) of yerba mate (Ilex paraguariensis) extract [The EE% values  and cornstarch at 2% (w/w).Specifically, statistically significant differences were detected when compared to the hydrogel beads samples . Thereby, from initial solution to cycle 3, the LC% values had descending behavior, with 86.0%, 81.1%, 74.5%, and 68.3% for initial solution, cycle 1, cycle 2, and cycle 3, respectively. This performance displays a marked inverse relationship in relation to the efficiency (until cycle 2), since LC% have a very close connection to the water-soluble characteristic of the solution used . Therefore, the sucrose/GA solution encapsulated affects the water-soluble characteristic due to the progressive increase in the viscosity , helpingThe LC% results were lower than in previous studies in our laboratory, since Petzold et al.  achievedw/w) and cornstarch at 2% (w/w) in terms of particle size, SF, aw, moisture content, and ρB.w/w) and cornstarch at 2% (w/w), and thus, the alginate/cornstarch/solution mixture plays a primary role in the size of the pores, and it can be extrapolated on the particle size of the hydrogel beads.Firstly, the size of the hydrogel beads decreased significantly from 3.09  mm to 2.64 mm (cycle 3). This progressive decrease can be related to the interaction of the solution with the alginate/cornstarch mixture, since the initial solution, despite its possible high solubility with the alginate/cornstarch mixture , does not have a substantial influence in the molecular bond interactions to close interstitial empty spaces of the gel matrix . Therebyw/w) and cornstarch at 2% (w/w) presented a spherical shape. A similar behavior was reported by Rohman et al. [Rhodopseudomonas palustris KTSSR54 using alginate/cornstarch beads through the ionic gelation technique, since the authors indicated that a cornstarch concentration between 3% (w/v) to 4% (w/v), with alginate at 2% (w/v), allows for good sphericity (SF < 0.05) in the alginate beads.For SF, there are no significant differences between samples, since the SF values were close to 0.02, indicating that the hydrogel beads filled with any solution can be considered spherical, since Chan et al.  suggesten et al. , who encw and moisture content, there are significant differences between the samples, with a continuous decrease from the initial solution until cycle 3, with values from 0.98 to 0.92, and from 86.1% to 73.6%, respectively. This progressive decrease is related to the loss of water by syneresis due to the addition of the cryoconcentrated solution since a solution with a high TSSC produces a significant diffusion of water molecules in the hydrogel beads’ formation [w and moisture content of the samples.For aormation . Hence, ormation , since tρB, there is a significant increase in the values as the cycles increased, from 1.04  to 1.16 g/mL (cycle 3). This behavior can be related to the addition of solution encapsulated in the alginate/cornstarch mixture, since the cryoconcentrate from cycle 3 has a greater mass than the previous cycles  due to the high TSSC value . Thus, a solution treated by BFC has a considerable increase in its density as the cycles increase, affecting the density of the alginate/cornstarch/solution mixture [ρB, since the authors used different concentrations of cornstarch (0–600 g/L) to reduce the porosity of alginate hydrogel beads, and in turn, the results showed that the ρB of the capsules increased progressively as the cornstarch concentration increased.For  mixture . In the  mixture  showed aw/w) and cornstarch at 2% (w/w) allowed for better efficiency of encapsulation of sucrose/acid gallic concentrated by block freeze concentration assisted by centrifugation than other alginate/cornstarch mixtures, where cornstarch filled the empty space (pores) of the alginate beads. This mixture produced a more suitable matrix , and in addition, it increased the amount of model cryoconcentrated solution inside of the hydrogel beads, and in turn, it permitted final beads with spherical shape. Therefore, the results obtained in this study could be used as a first step to develop good sustained-release devices and the possible uses on gastrointestinal conditions or storage situations. Moreover, the hydrogel beads will be filled with juice or extract concentrated by BFC for future food, engineering, and/or pharmaceutical applications.The optimization process by RSM indicated that alginate at 2% (