With global climate warming, recurring extreme heat and high temperatures irreversibly damage plants. Raspberries, known for their nutritional and medicinal value, are in high demand worldwide. Thus, it is important to study how high-temperature stress (HTS) affects raspberries. The physiological and biochemical responses and molecular genetic mechanisms of raspberry leaves to different HTS treatments were investigated: mild high temperature at 35°C (HT35), severe high temperature at 40°C (HT40), and the control at room temperature of 25°C (CK). The physiological results suggested that leaves in both the 35°C and 40°C treatments showed maximum relative conductivity at 4 d of stress, increasing by 28.54% and 43.36%, respectively, compared to CK. Throughout the stress period (0–4 d), malondialdehyde (MDA) and soluble protein contents of raspberry leaves increased under HT35 and HT40 treatments, while soluble sugar content first decreased and then increased. Catalase (CAT) activity increased, superoxide dismutase (SOD) activity first increased and then decreased, and peroxidase (POD) activity gradually decreased. Photosynthetic and fluorescence responses of raspberry leaves showed the most severe impairment after 4 d of stress. Transcriptomics results revealed significant alterations in 42 HSP family genes, two SOD-related differentially expressed genes (DEGs), 25 POD-related DEGs, three CAT-related DEGs, and 38 photosynthesis-related DEGs under HTS. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that these DEGs were mainly enriched in photosynthesis-antenna proteins, pentose and glucuronide interconversion, phenylpropane biosynthesis, and indole alkaloid biosynthesis. HTS induced excessive ROS accumulation in raspberry leaves, causing oxidative damage in plant cells and subsequently reducing photosynthesis in raspberry leaves. This reduction in photosynthesis, in turn, affects photosynthetic carbon fixation and starch and sucrose metabolism, which, combined with phenol propane biosynthesis, mitigates the HTS-induced damage. However, extreme heat stress (HS) weather is becoming more frequent, with high-temperature stress (HTS) disrupting intracellular environmental balance and severely inhibiting plant performance or even causing death . MoreoveRubus idaeus L.), a deciduous perennial shrub of the genus Rubus, family Rosaceae, is also known as pallet, mulberry, plucked berry, red berry, etc. They are eaten fresh, as well as processed into wine, juice, and jam  showed altered amino acid and sugar metabolism, antioxidant metabolite accumulation, phytohormone-related MAPK signaling pathways, and α-linolenic acid metabolism , with 25°C used as the control (CK). This was followed by studying how short-term moderate and severe HTS affects the physiological and molecular properties of red raspberry leaf cell membrane permeability, lipid peroxidation level, osmoregulation, antioxidant enzyme system, photosynthesis and chlorophyll fluorescence parameters, and transcriptome sequencing. Thus, the study results provide a more comprehensive insight into the raspberry HSR response, thus aiding in risk assessment and the development of essential measures to mitigate the adverse effects of heat on raspberry seedlings.22.1The HTS test was conducted on 22 August, 2020. Vigorously growing ‘Polka’ raspberry seedlings were selected under stable growing conditions, seedlings were ~11 cm high and 3 months old. They were then placed in a light incubator at 25°C with a 14-h photoperiod for 4 d. They were then subjected to HS at 35°C and 40°C, while 25°C served as a control for a 4-day treatment. Moisture was maintained during the stress treatment period. The experiment comprised three treatments with 10 plants per replicate and three replications, which totaled up to 90 plants. Each treatment was sampled at 0, 2, and 4 d post stress-treatment to determine their physiological indicators, and photosynthetic and chlorophyll fluorescence parameters. Transcriptome sequencing was performed on leaves at 0 and 4 d of stress treatment.2.2Ec1. Ec2 was the sample boiling water bath for two hours and then cooled to room temperature. Relative conductance=(Ec1/Ec2) × 100%  × 100% . Dried l) × 100% . Antioxi) × 100% . MDA con) × 100% .2.3nd–3rd leaf position, for photosynthetic parameter determination. The net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO2 concentration (Ci) were measured using a Li-6800 portable photo synthesizer (LI-COR of United States) equipped with a red and blue light source inside the leaf chamber. The light intensity was set at 800 μmol·m -2·s-1 and the flow rate was at 500 μmol·s-1. The average values were subsequently calculated.The photosynthetic performance of raspberry ‘Polka’ of each treatment was assessed around 10:00 am on 0 d, 2 d, and 4 d of stress. Three replicates of functional leaves from raspberry ‘Polka’ tissue culture seedlings were chosen from each treatment, specifically from the 2nd–3rd leaf position were subjected to dark treatment for 20 min. Using a Pocket PEA fast fluorometer, the initial fluorescence (Fo), maximum fluorescence (Fm), and variable fluorescence (Fv) of the leaves were measured. The experimental data were analyzed using the IBM SPSS Statistics 27.0.1 software.Chlorophyll fluorescence of the raspberry ‘Polka’ seedlings from each treatment was measured around 10:00 am on 0 d, 2 d, and 4 d of stress. Three functional leaves selected from the 22.4http://eggnogdb.embl.de), GO, KEGG (www.genome.jp/kegg), Pfam (http://pfam.xfam.org/), NCBI-nr, Swiss-Prot, and other databases were used to annotate the genome functions.Three replicate test materials were selected from the same batch of red raspberry ‘Polka’ seedlings used for evaluating the physiological and biochemical indicators and were sent to Hangzhou Lianchuanr Biotechnology Ltd. . Total RNA was extracted using the Tiangen RNA prep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit. Then cDNA library construction and transcriptome sequencing (RNA- seq) were performed. After obtaining raw data, sequencing junctions were removed using cutadapted and unqualified sequences were filtered out using fqtrim to obtain clean data. eggNOG . Quantitative reverse transcription PCR (qRT-PCR) was performed using the 2X SG Fast qPCR Master Mix  according to the manufacturer’s protocol. The specific primers used for qRT-PCR were designed using Primer Premier 5.0 Table S133.1The normal physiological metabolism of plants is disrupted under HTS, resulting in external changes in plant leaves, including delayed height and diameter growth, wilting, color fading, and leaves dropping . The pla3.2Ci is the main determinant of the photosynthetic rate. Chlorophyll a fluorescence determination can quickly, accurately, and effectively evaluate the plant photosynthetic response. With increasing HTS time, the Pn of red raspberry ‘Polka’ showed a significant downward trend  and 13,191 Unigene (27.21%) distributed within 200–300 nt and 300–700 nt  technology. The cDNA libraries of each treatment were sequenced, and the results showed high validity (94.9–97.8%), with Q20 > 98%, Q30 > 96%, and GC content > 46%, indicating good sequencing quality and could thus be used for subsequent analysis. The 0–700 nt Table S23.43.5The GO notes had three functional categories including “molecular function,” “cellular component,” and “biological processes” Figure 4In terms of biological processes, the combinations HT35_vs_CK and HT40_vs_CK demonstrated defense responses, responses to heat, flavonoid biosynthesis processes, responses to abscisic acid, and oxidized lipid biosynthesis processes. HT35_vs_CK specifically showed enrichment in response, oxidative stress response, and response to osmotic stress, while HT40_vs_CK displayed enrichment in photoinhibition, redox process, and photosynthesis  in plants, thereby enhancing antioxidant enzyme activity. The increase in antioxidant enzyme activity promotes the elimination of different types of HTS-generated ROS  ] . The incl (OH−)] . Additiol (OH−)] . Our tra3.9+ reduction enzyme were all reduced, specifically TRINITY_DN25263_c0_g6, TRINITY_DN23229_c0_g7, and TRINITY_DN25713_c0_g3. Additionally, cell pigmentation, B6F complex (CYTB6F), PSI, and PSII were injured. Among the genes encoding CYTB6F, one gene was downregulated, with nine and seven genes encoding PSI and PSII, respectively, also being downregulated.Photosynthesis is an essential process for plant growth, and chlorophyll a fluorescence shows temperature sensitivity. The result shows that the number of photosynthetic response genes differentially expressed between HT40_vs_HT35, HT40_vs_CK was higher than HT35_vs_CK Figure 8Haematococcus pluvialis subjected to abiotic stress, genes associated with photosynthetic antennae proteins such as LHCA1, LHCB2, LHCB3, and LHCB7 were partially upregulated  of PSI and PSII, also known as the optical protein composite, was damaged. At 35°C, there were six DEGs encoding LHCII in the PS II, with five downregulated and one upregulated. At 40°C, the differential expression of LHCI coding genes was observed in photosystem I, with all four genes being downregulated. In PS II, there were 11 DEGs encoding LHCII, with one gene upregulated and 10 genes downregulated. In egulated .3.10TRINITY_DN24223_c0_g3 and TRINITY_DN25717_c1_g9, with both being upregulated , which further reduces cinnamaldehyde through the actions of 4CL and CCR (3.11p<0.05) in the expression levels of all six genes after high-temperature treatment in red raspberry. The gene expression validation results were generally consistent with the gene expression trends determined by transcriptomic results, thus confirming the confidence and precision of this study’s RNA-Seq analysis.qRT-PCR detected the expression levels of six key genes under HTS. 44.1S. viminalis  (GAPA), phosphotrisaccharide isomerase (TPI), fructose bisphosphate aldolase class I (ALDO) enzyme and ribose 5-phosphate isomerase (rpiA) decreased, while the HT35 treatment showed no significant change  are important for regulating the internal plant cell pressure and maintaining the plant’s physiological stability. Osmoregulations are found to accumulate with increasing external abiotic stress, and starch can be hydrated to glucose in addition to its role in photosynthesis . Endogluvia the D-galacturonate, myo-inositol, and UDP-glucose pathways. The ugdase gene, which controls UDP-glucose synthesis, is downregulated in red raspberries under HTS  was reduced by 20–25% compared to the wild type, with salicylic acid levels also being reduced (Studies have shown that PAL and cinnamic acid coenzyme A ligase (4CL) play a key role in lignin biosynthesis. PAL is the main rate-limiting enzyme that converts L-phenylalanine into trans-cinnamic acid by eliminating ammonia, with its isomers playing a unique but redundant role in plant growth and stress response . Moreove reduced . Cinnamo reduced . Lowerin reduced  while do reduced . In this5Therefore, a working model is proposed to explain the impact of HS on raspberry leaves (The original contributions presented in the study are included in the article/JG and RY were responsible for writing the original manuscript. ZF conducted the research. JG and SC compiled the data. GQ, SG, PJ, and HL contributed to the resources. XZ and JG were responsible for funding acquisition and writing. XZ was responsible for funding acquisition and writing, reviewing, and editing. All authors contributed to the article and approved the submitted version.


The objective of this study is to analyze and summarize the characteristics of the clinical data of patients with systemic lupus erythematosus (SLE) complicated with liver failure, and to improve the cognition of the disease. The clinical data of patients with SLE complicated with liver failure hospitalized in Beijing Youan Hospital from January 2015 to December 2021 were collected retrospectively, including general information and laboratory examination data, and the clinical characteristics of the patients were summarized and analyzed. Twenty-one SLE patients with liver failure were analyzed. The diagnosis of liver involvement was earlier in 3 cases than that of SLE, and later in 2 cases. Eight patients were diagnosed with SLE and autoimmune hepatitis at the same time. The medical history is between 1 month and 30 years. This was the first case report of SLE complicated with liver failure. We found that: (1) among the 21 patients, organ cysts (liver and kidney cysts) were more common and the proportion of cholecystolithiasis and cholecystitis was higher than that in previous studies, but the proportion of renal function damage and joint involvement was lower. (2) The inflammatory reaction was more obvious in SLE patients with acute liver failure. The degree of liver function injury in SLE patients with autoimmune hepatitis was less than that in patients with other liver diseases. (3) The use of glucocorticoid in SLE patients with liver failure was worthy of further discussion. Systemiclupus erythematosus (SLE) is one of the typical manifestations of autoimmune diseases. The global morbidity is 20–150/100,000 people . PatientClinically, most SLE with liver dysfunction is not only caused by SLE itself, but also caused by drug toxicity or other liver diseases, such as fat infiltration and overlapping autoimmune liver disease, virus infection, and alcoholism –11. End-This study firstly explored the clinical characteristics of liver failure in SLE patients, to provide some evidence for clinical diagnosis and treatment.This study is a retrospective study in a single center. We used the information retrieval system to screen all SLE patients hospitalized in Beijing Youan Hospital from January 2015 to December 2021. The hospitalization record of each patient could be queried in the medical record system by the case number (ID). The diagnosis of SLE was referred to “the guidelines for the diagnosis and treatment of systemic lupus erythematosus” issued by the rheumatology branch of the Chinese Medical Association in 2010 . A totalRoutine laboratory examinations include blood routine test, urine routine test, coagulation indexes, biochemical indexes of liver injury, biochemical indexes of renal injury, blood lipid series, autoantibody series, and immunoassay of special proteins. Abdominal CT examination recorded the appearance and size of the liver and spleen, parenchymal echo, bilateral signs of the gallbladder wall, gallbladder sound shadow, sound enhancement, stones, etc.Liver failure is a serious liver damage caused by many factors, resulting in critical impairment or decompensation of synthesis, detoxification, metabolism, and biotransformation. It presents severe symptoms with jaundice, coagulation dysfunction, hepatorenal syndrome, hepatic encephalopathy, and ascites as the main clinical manifestations, resulting in high mortality. Generally, the major cause of liver failure in China is hepatitis virus.Acute liver failure (ALF) is characterized by acute onset, grade II or above hepatic encephalopathy within 2 weeks, as well as the following manifestations: (1) extreme fatigue, accompanied with obvious anorexia, abdominal distension, nausea, vomiting, and other serious gastrointestinal symptoms; (2) jaundice deepens progressively in a short time, TBIL ≥ 10 upper limit of normal (ULN) or daily rise ≥ 17.1 μmol/L; (3) bleeding tendency, prothrombin activity (PTA) ≤ 40%, or INR ≥ 1.5, and other reasons are excluded; (4) progressive reduction of liver volume.Subacute liver failure (SALF) is characterized by relatively acute onset as well as the following manifestations in 2–26 weeks: (1) extreme fatigue with obvious gastrointestinal symptoms; (2) jaundice deepens rapidly, TBIL ≥ 10 × ULN or daily rise ≥ 17.1 μmol/L; (3) with or without hepatic encephalopathy; (4) bleeding tendency, PTA ≤ 40%, or INR ≥ 1.5, and other reasons are excluded.Acute-on-chronic liver failure (ACLF) is characterized by acute jaundice, coagulation dysfunction, and other liver failure symptoms triggered by various factors on the basis of chronic liver disease. It could be combined with hepatic encephalopathy, ascites, electrolyte disorder, infection, hepatorenal syndrome, hepatopulmonary syndrome, and other complications, as well as extrahepatic organ failure. Usually, the patient shows rapid deepening of jaundice with TBIL ≥ 10 × ULN or daily rise ≥ 17.1 μmol/L, hemorrhage, PTA ≤ 40% or INR ≥ 1.5.Chronic liver failure (CLF) is characterized by slow progressive decline and decompensation of liver function on the basis of cirrhosis: (1) TBIL rises, usually < 10 × ULN; (2) albumin (ALB) decreased significantly; (3) platelet decreased significantly, PTA ≤ 40% or INR ≥ 1.5, and other reasons are excluded; (4) intractable ascites or portal hypertension; (5) hepatic encephalopathy.We obtain clinical data and quality indicators related to laboratory examination through the medical record system.t-test or Mann–Whitney u test (p < 0.05) was considered statistically significant.SPSS 26.0 software was used to analyze the data. The mean values of data conforming to a normal distribution are expressed as mean ± SD or median (range); classified variables were indicated as frequency or ratio. The mean values of data that did not conform to normal distribution were expressed as median ± quartile. Comparison of results between groups for continuous data using Student’s In this study, 21 patients of SLE with liver failure were included, including 4 male patients (19.05%); the average age was 42.75 ± 10.54 years and 17 female patients (80.95%); and the average age was 48.12 ± 3.41 years. Among the 21 SLE patients with liver failure included in this study, there were 3 patients of drug-induced liver disease, 3 patients of non-basic liver disease, 5 patients of chronic hepatitis, 8 patients of AIH, and 1 patient of alcoholic liver disease; the medical history is between 1 month and 30 years. There were 3 patients diagnosed with liver involvement earlier than SLE, 2 patients later than that, 8 patients diagnosed with SLE and AIH at the same time, 5 patients combined with renal injury, and 3 patients with abnormal thyroid function. Clinical examination showed that ascites could be found in 12 patients, hyperammonemia/hepatic encephalopathy in 10 patients, splenomegaly in 9 patients, pleural effusion in 9 patients, and hydropericardium in 2 patients. Additionally, compared with previous studies, we found more patients with organ cyst, such as liver cyst in 5 patients, renal cyst in 7 patients, gallbladder stone in 5 patients, and cholecystitis in 8 patients. In urine examination, the protein of cases 7, 11, 13, and 20 was positive, and the occult blood of cases 3, 11, 13, 19, and 20 was positive. Officially, the liver biopsy is considered the gold standard for diagnosis of various liver diseases. The pathological results of liver biopsy revealed that case 9 had an acute attack of chronic hepatitis B , case 11 had massive necrosis of liver tissue which was consistent with the diagnostic criteria of ALF, case 18 had multi-hepatic lobular necrosis accompanied with hepatocyte regeneration, and case 20 had moderate hepatitis (G2) and liver cirrhosis. The results are shown in Table The auto-antibody examination was detected in 19 patients, of which ANA positive in 18 patients (94.7%), ACA positive in 4 patients 21.1%), anti-parietal cell antibody (APCA) positive in 2 patients (10.5%), anti-proliferating cell nuclear antigen antibody (PCNA) positive in 1 patient (5.3%), and anti-mitochondrial antibody (AMA) positive in 2 patients (10.5%). The ANA spectrum was detected in 18 patients, of which nRNP positive in 3 patients (16.7%), ACA-B positive in 3 patients (16.7%), ARPA positive in 3 patients (16.7%), anti-Ro-52 antibody positive in 3 patients (16.7%), SSA in 2 patients (11.1%), SSB in 1 patient (5.6%), anti-SM antibody positive in two patients (11.1%), dsDNA positive in 6 patients (33.3%), Jo-1 positive in 1 patient (5.6%), ANuA positive in 4 patients (22.2%), and AHA positive in 3 patients (16.7%). Moreover, the complement and immunoglobulin (Ig) were detected in twenty patients, of which C3 was 0.54 (0.172–2.46) decreased in 18 patients (90%), C4 was 0.27 (0.03–2.82) decreased in 13 patients (65%), IgG was 20.96 (1.22–53.70) increased in 15 patients (75%), and IgM was 1.71 (0.22–6.88) increased in 5 patients (25%). Since the detection value of IgA in 1 patient was lower than the lower limit, the mean value of other 19 patients was 3.74 (< 0.233–9.72), of which 5 patients increased (25%). The results are shown in Table 1.1%, antp = 0.096). The hemoglobin (Hgb) of patients in the chronic liver failure group was 84.33 ± 12.34 g/L, which was mild anemia, lower than that of the other three groups and might be caused by cirrhosis. The platelet of patients in the ACLF group was fluctuated at 27 − 177 × 109/L, lower than that of the other three groups. The liver function of patients in the acute liver failure group was the most seriously damaged, and the elevation of transaminase , bilirubin , and ALP was most obvious compared with the other three groups, which might be related to the liver injury caused by acute inflammation. Meanwhile, the coagulation function of patients in the acute liver failure group was worse than that of the other three groups. And there was no significant difference in ALB among the four groups.As shown in Table As shown in Table Patients with SLE associated with liver dysfunction are more common clinically. Currently published articles mainly include research studies on SLE with liver dysfunction , 15, SLEAmong the 21 SLE patients with liver failure included in this study, there were 3 patients of drug-induced liver disease 14.29%), 3 patients of non-basic liver disease (14.29%), 3 patients of chronic hepatitis B (14.29%), 1 patient of chronic hepatitis C 4.76%), 8 patients of AIH (38.10%), and 1 patient of alcoholic liver disease (4.76%); the medical history is between 1 month and 30 years. In previous cohort studies, NAFLD and chronic hepatitis C were the most common reasons of liver dysfunction %, 8 pati, 6, the .29%, 3 pIt has been found in this study the level of GGT in patients with ALF (485.60 ± 920.08 U/L) > SALF (183.83 ± 125.09 U/L) > ACLF (96.43 ± 41.49 U/L) > CLF (19.33 ± 7.51 U/L). And the GGT level of patients with AIH (124.63 ± 133.20 U/L) was lower than that of patients with other liver diseases (251.31 ± 567.08 U/L). As is well known, GGT has a strong pro-inflammatory effect . Considep < 0.01) [p < 0.01) [p < 0.05). Previous articles reported that low level of C3 suggested worse prognosis [The complement was detected in 20 patients in this study. The mean value of C3 was 0.54 (0.172–2.46), of which 18 patients (90%) were decreased, and the mean value of C4 was 0.27 (0.03–2.82), of which 13 patients (65%) were decreased. It was consistent with the previous study that the incidence rates of simultaneous reduction of C3 and C4 levels in SLE patients (73.42%) were significantly higher than those in non-SLE patients ( < 0.01) . The inc < 0.01) . The dec < 0.01) . Meanwhi < 0.01) . Previourognosis , and therognosis . In thisGlucocorticoid is a powerful anti-inflammatory and immunosuppressive drug, which could effectively inhibit the immune system , and hasThis was the first series case reports on SLE patients with liver failure. We found that SLE patients complicated with organ cysts (liver and kidney cysts) were more common and the proportion of cholecystolithiasis and cholecystitis was higher than that in previous studies, but the proportion of renal function damage and joint involvement was lower. The inflammatory reaction was more obvious in SLE patients with acute liver failure. The degree of liver function injury in SLE patients with AIH was less than that in patients with other liver diseases. The use of glucocorticoid in SLE patients with liver failure was worthy of further discussion.To sum up, there were several limitations in our study. Due to the small sample capacity and only a few laboratory examination items, there were differences between the four groups, but no statistical significance. Moreover, the patients were not followed up for a long time in this retrospective study. In general, more research is urgently needed to explore the characteristics and mechanism of SLE patients with liver failure in order to improve the prognosis.


In the original publication , there wSection 3, Section 3.1, Paragraph 1, from fifth to seventh sentences should be: “There were 22 female (55%) and 18 male (45%) patients negative for perineural invasion (Pn0 group) with a median age of 64.7 years (range 35–84). The group of patients positive for perineural invasion (Pn1 group) consisted of 242 female (45.6%) and 289 male (54.4%) individuals with a median age of 65.6 years (range 37–83). Tumor location in over 70% of both groups was the head of the pancreas , followed by the pancreatic tail  and pancreatic body .”p = 0.05). Pre-operative tumor markers  that well reflect the overall tumor burden, including micro-metastases, did not reveal strong correlations.”Section 3, Section 3.2, Paragraph 2, the last two sentence should be: “There was a significant difference between the BMI of the Pn0 and Pn1 group (p = 0.04).”Section 3, Section 3.3, the first sentence should be: “Studying long-term survivors  in our study cohorts, we discriminated a significantly increased number of LTS in the Pn0 group compared to Pn1 patients (p = 0.007). The majority of Pn1 tumors showed lymph node metastasis , while lymph node infiltration occurred only in 40% of Pn0 tumors . This also significantly correlated with lymphatic invasion; 44.1% of the Pn1 tumors were positive for lymphatic invasion .”Section 3, Section 3.2, Paragraph 1, from third to fifth sentences should be: “In our cohort, 30% of Pn0 patients presented with early stage pT1 tumors , while only 10.5% of Pn1 patients presented with pT1 tumors . However, vascular invasion and resection margin did not significantly correlate with either Pn0 or Pn1 tumors.”Section 3, Section 3.2, Paragraph 1, the last two sentence should be: ”There also appeared to be a detectable difference in the grading of Pn1 tumors compared to Pn0 tumors with a shift toward less-differentiated tumors in the Pn1 group :“Patients with incomplete medical history documentation, R-status, PNI status and tumor stage were excluded. Patients with in-hospital mortality  or who were lost to follow-up during that time were excluded for analyses of time to event outcomes .”In addition, we detected an error as we included patients starting from March 2008, not January. We request to modify the sentence as follows :“Patients undergoing curative intended surgical resection for PDAC at the Department of Surgery, Campus Charité Mitte|Campus Virchow, Charité—Universitätsmedizin, Berlin, Germany, between March 2008 and December 2019 were included.”For a validation of our results, we have used additional statistical software, now also included in the Section 2, Section 2.3, the first sentence:“For statistical analysis, the statistical software R , SPSS  and Prism  were used.”Section 4, Paragraph 5, the third sentence should be: “During the entire study period, 11.2% of the patients received neoadjuvant treatment.”A correction has been made to Funding section:Funding: Matthäus Felsenstein, Karl-Herbert Hillebrandt and Brigitta Globke are participants in the BIH-Charité Clinician Scientist Program, funded by the Charité–Universitätsmedizin Berlin and the Berlin Institute of Health.We also modified the Supplementary Table S1 for a better overview of adjuvant chemotherapy regimen across all patients, which was missing in the previously published version. There were no modifications needed in the table content/numbers.The authors state that the scientific conclusions are unaffected. This correction was approved by the Academic Editor. The original publication has also been updated.


The drug therapy for non-small cell lung cancer (NSCLC) have always been issues of poisonous side effect, acquired drug resistance and narrow applicable population. In this study, we built a novel network analysis method , which was based on the difference analysis, Spearman correlation network analysis, biological function analysis and Bayesian causality network analysis to discover new therapeutic target of NSCLC in the sequencing data of BEAS-2B and 7 NSCLC cell lines. Our results showed that, as a proteasome subunit coding gene in the central of cell cycle network, PSMD2 was associated with prognosis and was an independent prognostic factor for NSCLC patients. Knockout of PSMD2 inhibited the proliferation of NSCLC cells by inducing cell cycle arrest, and exhibited marked increase of cell cycle blocking protein p21, p27 and decrease of cell cycle driven protein CDK4, CDK6, CCND1 and CCNE1. IPA and molecular docking suggested bortezomib has stronger affinity to PSMD2 compared with reported targets PSMB1 and PSMB5. In vitro and In vivo experiments demonstrated the inhibitory effect of bortezomib in NSCLC with different driven mutations or with tyrosine kinase inhibitors resistance. Taken together, bortezomib could target PSMD2, PSMB1 and PSMB5 to inhibit the proteasome degradation of cell cycle check points, to block cell proliferation of NSCLC, which was potential optional drug for NSCLC patients. With the development of clinical diagnosis and treatment, the 5-year postoperative survival rate of early NSCLC patients has been significantly improved . UnfortuIndividualized precise treatment based on tumor pathology, cell biology and molecular biology has become the main development direction for clinical NSCLC treatment –14. The Network science is regarded as a sharp weapon to analyze complex systems. The reason is that by abstracting system components and relationships into nodes and edges, complex systems can be transformed into mathematical network models. With the widespread application of omics detection technology in tumor research, it is easier to obtain the omics data of tumor DNA, RNA, protein, metabolites and other molecules. On the basis of considering the structural variation or differential expression, introducing correlation or causality network can better fit the internal characteristics of the complex system of tumor, and it has become a new path for the exploration of therapeutic targets –22.In fact, network analysis has been widely used in disease research. In the enrichment analysis of tumor pathway, we usually rely on the correlation relationship between genes to annotate the function of target gene groups . At the We tried to integrate the existing methods of difference analysis, correlation analysis, functional enrichment and causality analysis to construct a new network analysis method of tumor transcriptome data, so as to discover the effective therapeutic targets. In this study, we built a new “difference-correlation-enrichment-causality-node” network construction and node capture technology process of tumor transcriptome data. Among them, “difference” refers to differential expression genes or differential network nodes, “correlation” refers to Pearson or Spearman correlation coefficient analysis, “enrichment” refers to functional enrichment analysis and “causality” refers to Bayesian probability analysis. This network analysis method is different from the previous network analysis methods. Based on the double test of correlation and causality, the molecular relationship described has higher authenticity and reliability. Relies on layer-by-layer dimensionality reduction and uses module segmentation and parallel operation, this method overcomes the problems of limited input data scale and slow operation speed of Bayesian probability calculation to a certain extent. Further, taking prognosis information as dependent variables and nodes as independent variables, we have accurately screened potential candidate genes by Cox regression analysis.In this study, we combined existing difference analysis, correlation analysis, functional enrichment and causality analysis to construct a transcriptome network analysis method for discovering new therapeutic target of NSCLC Fig. . PrognosFirst, the sequencing gene sets of seven NSCLC cell lines and one normal bronchial epithelial cell line were intersected to obtain an expression matrix consisting of 9639 common genes and 23 cell samples Fig. . Then, bP < 0.05, Fig. Based on univariate Cox regression analysis, we got 6 candidate genes  were associated with poor prognosis of NSCLC patients in TCGA database Fig. . STRING P < 0.001, Fig. P = 0.001, 0.014, Table n = 95) in the NSCLC tissue microarray   Fig. . It coulups Fig. . Meanwhi25 and IC50 of bortezomib in the 7 types of NSCLC cells. We found that there was no apparent activation or inhibition of phosphorylated proteins in all 7 types of NSCLC cells, and only a few cells were inhibited in the same signaling pathways. For example, with increasing dosage of bortezomib, in A549 and H358 cells, the expression of p-STAT3 was decreased; in A549, H358 and PC9OR cells, the expression of p-NF-κB was decreased; in H1299, H358 and H3122 cells, the expression of p-p38 MAPK was decreased; and in A549, H358, PC9 and PC9OR cells, the expression of p-ERK1/2 was decreased  and treated with bortezomib. Figure Combined with these results, we believe that by targeting proteasome coding genes of cell cycle check points, bortezomib could block the proteasome degradation of p21 and p27, hinder the formation of cyclin/CDK complex, arrest the cell cycle in G0/G1 phase, and then inhibit the proliferation of NSCLC cells  [RNA sequencing was performed on NSCLC cell lines, including H3122, H3122LR (Lorlatinib-resistant), PC9 and PC9OR (Osimertinib-resistant). Meanwhile, we downloaded the sequence data of A459, H1299, H358 and BEAS-2B  from the GSE72794, GSE77209 and GSE172222 (gov/geo) . The comA total of 180-spot NSCLC tissue microarray composed of 98 NSCLC and 82 adjacent cancer samples were obtained from Shanghai Outdo Biotech CO., Ltd. This study was approved by the Ethics Committee of Shanghai Outdo Biotech CO., Ltd, with the ethics approval number YBM-05-02 and the informed consent was obtained from all subjects. All patients underwent surgery between January 2008 and July 2013 and were followed up for 3–8.5 years. All patients had no special medical history and did not receive preoperative chemotherapy and radiotherapy. The no special medical history refers to (within 5 years): no other history of malignant tumors; no history of pulmonary fibrosis, interstitial pneumonia, pneumoconiosis, radiation pneumonia, drug-related pneumonia, severe damage to lung function, and other diseases; no history of infectious diseases; no history of immune deficiency; no history of organ transplantation. During subsequent data analysis, 3 NSCLC samples with incomplete clinical information were removed, and 95 NSCLC samples remained.For the expression matrix mentioned above, the “removeBatchEffect” function of the “limma” package  was usedP < 0.05, regardless of the logFC (fold change) (logFC = 0).The corrected matrix contains 5421 genes and 23 cell samples. According to NSCLC and normal cell groupings, difference analysis was performed by the “limma” package. In order to retain all differential genes, only screened by We used spearman correlation analysis  to calcuThen, we screened the functional clusters on the correlation network by the “MCODE” plugin of Cytoscape (3.7.2) , 62. AccWe used the “bnlearn” R package  to perfoThrough univariate and multivariate Cox regression analysis, we identified PSMD2 was the prognosis-related gene in NSCLC. The “survival” and “survminer” R packages were used for survival analysis. The Kaplan-Meier method was used to estimate the survival curve, and the log-rank test was used to analyze the difference in survival time.http://software.broadinstitute.org/gsea/index.jsp) [Based on the protein–protein interaction (PPI) analysis, we identified 269 PSMD2-related proteins. These proteins were subjected to GO terms enrichment analysis using “clusterProfiler” R package . Lung addex.jsp)  was perf50 of bortezomib, while the control group was incubated with corresponding amounts of DMSO at 37 °C in a 5% CO2 incubator for 1 h. (2) Discard the supernatant, digest the cells with trypsin into a 15 ml tube, centrifuge 300 × g for 3 min; Discard the supernatant and resuspend the cell precipitate with pre cooled 5 ml PBS solution, centrifuge 300 × g for 3 min; Discard the supernatant and resuspend the cell precipitate with 1.5 ml pre cooled PBS containing 1% protease inhibitor, transfer to a new 2 ml EP tube. (3) Take 100 μl of cell suspension from each treatment group and control group into a 200 μl PCR tube, ensuring that every 100 μl contains 3 × 106 cells. Set the temperature range between 50 °C and 70 °C using a PCR instrument, with 14 types of temperature wells. Heat the sample for 3 min, then place the PCR tube at room temperature for 3 min, and then transfer it to liquid nitrogen for quick freezing. (4) Repeat freeze-thaw twice on the cell suspension using liquid nitrogen and a 25 °C constant temperature metal bath. After each thaw, a vortex is required, and finally the cell lysate is placed on ice. (5) Transfer the cell mixture from all PCR tubes to a new 1.5 ml EP tube, centrifuge 17,000 × g at 4 °C for 20 min, and collect 90 μl of supernatant into the new 1.5 ml EP tube on ice. (6) BCA protein assay kit  detects protein concentration, and then adds 5× loading buffer to heat in a 100 °C metal bath for 10 min for subsequent experiments.Experimental procedures for thermal shift assay have been reported . (1) Prehttp://vina.scripps.edu/manual.html) [The SDF file of bortezomib structure was downloaded from the PubChem database . The PDBal.html) . Bortezoal.html) . The PLIal.html) . PyMol wal.html) .NSCLC cell lines  and normal lung bronchial epithelial cells BEAS-2B were purchased from Shanghai Academy of Science . H358, A549, H1299, HCC827 and H3122 cells were maintained in RPMI-1640 medium . BEAS-2B and PC9 were maintained in DMEM medium . All media were supplemented with 1% penicillin and streptomycin  and 10% fetal bovine serum . The dose of bortezomib (PS-341)  in NSCLC cells was mentioned below.Briefly, knockout of PSMD2 was performed using sgPSMD2  and lentiCRISPRv2 plasmids (Zhang Lab) compared to a negative control of sgNC encoding a nonspecific 20nt guide RNA. The cells were co-transfected with lentiCRISPRv2 plasmids with CRISPR/Cas9 sgRNA. Two days after the transfection, cells were selected by puromycin for 1 week to obtain stable cell lines.Cells in logarithmic growth phase were taken and seeded into 96-well plates at 3000 cells per well. Five parallel control wells were set for each group of cells, and 4, 24, 48, 72 and 96 h were set as detection time points. After adding CCK-8 , the 96-well plate was placed in a constant temperature incubator for 1 h. Finally, use the microplate spectrophotometer  to detect the OD value at a wavelength of 450 nm, and calculate the relative cell growth rate at each time point.NSCLC cells were lysed in RIPA buffer  with protease and PMSF inhibitor cocktails. Protein concentration was determined using BCA protein assay kit . An equal amount of proteins (30 µg) for each sample were loaded on 10% SDS-polyacrylamide gels and then transferred to PVDF membranes . Membranes were subsequently blocked with TBST containing 5% skim milk at room temperature for 1 h and incubated with diluted primary antibody at 4 °C overnight. The next day, incubated the secondary antibody and exposed the bands with ECL luminescent solution . Primary antibodies used were as follows: Rabbit anti-PSMD2 , Rabbit anti-CDK1 , Rabbit anti-p21 , Rabbit anti-p27 , Rabbit anti-p16 , Rabbit anti-p57 , Rabbit anti-CDK2, Rabbit anti-CDK4 , Rabbit anti-CDK6 , Rabbit anti-Ki67 , Rabbit anti-p-IκBα , Rabbit anti-IκBα, Rabbit anti-p-NF-κB p65, Rabbit anti-NF-κB p65 , Rabbit anti-p-ERK1/2 , Rabbit anti-ERK1/2 , Rabbit anti-STAT3 , Rabbit anti-p-STAT3 , Rabbit anti-p44/42 MAPK , Rabbit anti-p-p44/42 MAPK , Rabbit anti-AKT , Mouse anti-p-AKT , Rabbit anti-cyclin D1 , Rabbit anti-cyclin A2 , Rabbit anti-cyclin B1 , Rabbit anti-cyclin E1 , Rabbit anti-PSMB1 , Rabbit anti-PSMB5 , Mouse anti-GAPDH  and Mouse anti-β-actin .The NSCLC cells were seeded in 6-well plates (500 cells per well) treated with bortezomib in the next day with culture medium for 2 weeks. The cells were washed twice with PBS and fixed with 4% paraformaldehyde for 30 min followed by 0.1% crystal violet staining for 15 min. Afterwards, the cells were re-washed twice with PBS and imaged by a digital camera.Cells were harvested when NSCLC cells reached 90% confluence. After fixation with 70% ethanol at 4 °C overnight, cells were re-washed twice, and then stained with propidium iodide (PI)/RNase Staining Solution  for 30 min in the dark. The proportion of cell cycle phases was detected by flow cytometry .For apoptosis assay, cells were treated with Annexin V-FITC Apoptosis Detection Kit  and determined by cell sorting. Approximately 20,000 cells were collected per experiment and assays were repeated three times independently.Formalin-fixed and paraffin-embedded sections were baked at 65 °C for 4 h, then deparaffinized, hydrated, and subjected to antigen retrieval. Sections were incubated with Rabbit anti-PSMD2 , Rabbit anti-p21 , Rabbit anti-p27 , Rabbit anti-CDK4  and Rabbit anti-CDK6  overnight at 4 °C. Subsequently, these tissue sections were incubated with horseradish peroxidase-conjugated secondary antibody  for 5 min at 37 °C. Then, sections were stained with DAB+ substrate chromogen solution  for 1 min at room temperature, followed by counterstaining with hematoxylin. Two researchers scored the staining results in a blinded fashion independently.The protein expression was evaluated by both staining intensity and percentage of staining positive cells based on a semi-quantitative scoring system . Stainin6 NSCLC cells. Tumor size was measured every 3 days (tumor size = a × b2/2). When the tumor size had reached 80–120 mm3, the mice were randomly divided into two groups (no blinding). Mice were injected subcutaneously twice a week (3 weeks) with 0.5 mg/kg bortezomib . Then, all mice were sacrificed on day 45, xenograft tumors were dissected and its size were measured. Finally, tumor tissues were fixed with 4% formalin, and the corresponding analysis was performed by immunohistochemistry.Female nude mice were purchased from GemPharmatech Co., Ltd  and housed in facilities approved by Animal Care and Use Committee of West China Hospital, Sichuan University. Nude mice were housed in an SPF environment for 1 week, and then each mouse was subcutaneously injected with 100 μl of 5 × 10t-test or Mann–Whitney U test. The Wilcoxon test was used to compare the expression of PSMD2 in normal lung and tumor tissues. The chi-square test was used to analyze the correlation between the IHC score of PSMD2 and the clinical features of NSCLC patients. The effects of bortezomib on colony formation, cell cycle and apoptosis in NSCLC cells were analyzed using one-way ANOVA. All data with normal distribution and homogeneity of variance were expressed as mean ± standard deviation. All results were considered statistically significant at P < 0.05 .Statistical analysis of all experimental data was performed using SPSS 22.0  and graphical presentation of experimental results was performed using Graphpad Prism 8.0 . Semi-quantitative results of all CCK-8 assays and western blot were analyzed using Supplementary Figure S1-S9Table S1Table S2Table S3Table S4Table S5Table S6Table S7


Dual localization or dual targeting refers to the phenomenon by which identical, or almost identical, proteins are localized to two (or more) separate compartments of the cell. From previous work in the field, we had estimated that a third of the mitochondrial proteome is dual-targeted to extra-mitochondrial locations and suggested that this abundant dual targeting presents an evolutionary advantage. Here, we set out to study how many additional proteins whose main activity is outside mitochondria are also localized, albeit at low levels, to mitochondria (eclipsed). To do this, we employed two complementary approaches utilizing the α-complementation assay in yeast to uncover the extent of such an eclipsed distribution: one systematic and unbiased and the other based on mitochondrial targeting signal (MTS) predictions. Using these approaches, we suggest 280 new eclipsed distributed protein candidates. Interestingly, these proteins are enriched for distinctive properties compared to their exclusively mitochondrial-targeted counterparts. We focus on one unexpected eclipsed protein family of the Triose-phosphate DeHydrogenases (TDH) and prove that, indeed, their eclipsed distribution in mitochondria is important for mitochondrial activity. Our work provides a paradigm of deliberate eclipsed mitochondrial localization, targeting and function, and should expand our understanding of mitochondrial function in health and disease. It has become evident in recent years that targeting proteins to multiple locations is more abundant than was initially assumed ,4,5,6,7.Since the precise subcellular localization of a protein is critical for its function, one of the challenges facing post-genomic biology is exploring protein subcellular localization to characterize location-specific functions. Indeed, there is a growing number of studies designed to address this challenge by developing global screens of protein localization ,24,25,26Saccharomyces cerevisiae (from here on termed simply yeast) employed an α-complementation technology that uses a split β-galactosidase system to determine protein subcellular localization   mass speTo complement our above efforts and create a list of eclipsed protein candidates, we initiated a hypothesis-based approach in which we bioinformatically examined for traits associated with mitochondrial proteins as a means to directly identify eclipsed candidates. We considered those proteins that were predicted to have a high-scoring amino terminal MTS by the MitoProt II algorithm ,33,38. OWe chose the ~200 highest-scoring proteins  enzymes. TDH enzymes constitute a protein family of three unlinked, highly homologous genes, TDH1, TDH2, and TDH3  methionine and further incubated for 30 min at 30 °C. When required, carbonyl cyanide chlorophenylhydrazone (CCCP) was added to a final concentration of 20 μM at the start of labeling. Labeling was stopped by the addition of 10 mM sodium azide. The labeled cells were collected by centrifugation and resuspended in TE buffer  containing 1 mM phenylmethylsulfonylfluoride (PMSF), broken with glass beads, and centrifuged to obtain the supernatant fraction. Supernatants were denatured by boiling in 1% SDS, immunoprecipitated with the indicated anti-serum and protein A–Sepharose  or magnetic Dynabeads protein A . Samples were then analyzed by SDS-PAGE, followed by visualization with the BAS2000 image analyzing system  and autoradiography.Yeast cultures were grown in 5 mL SD–glucose or SD–galactose at 30 °C or as indicated to OD600 nm = 1.5. Mitochondria were isolated as described previously [Induced yeast  cultures were grown to ODeviously . SpheropS. cerevisiae cells were harvested using lysis buffer containing the following: 10 mM Tris pH 8, 1 mM EDTA, and 100 mM PMSF. Protein concentrations were determined using the Bradford method [d method . SamplesS. cerevisiae cultures grown in galactose medium for translation induction were harvested and resuspended in TE buffer (pH 8.0) containing 1 mM PMSF, broken with glass beads, and centrifuged to obtain the supernatant fraction. Glyceraldehyde-3-phosphate dehydrogenase activity was assayed in 1.0-mL reactions containing 0.1 M potassium phosphate (pH 7.4), 1.0 mM NAD+, 10 mM EDTA, 0.1 mM dithiothreitol, and 4.0 mM glyceraldehyde 3-phosphate [hosphate . NADH fo600 nm = 0.1 in ethanol-acetate medium, and 180 µL were seeded in Seahorse XF96 microplate coated in poly-L-lysine (22 µL of 0.1 mg/mL per well). The loaded plate was centrifuged at 500× g for 3 min at room temperature, following incubation for 30 min at 30 °C. Basal Oxygen Consumption Rate (OCR) was measured following an injection of 20 µM of FCCP and 2.5 µM of antimycin A according to the manufacturers’ manual Seahorse XF Cell Mito Stress Test (Agilent). OCR was normalized to cells at OD600 nm = 0.1, and basal respiration, maximal respiration, and spare respiration capacity were calculated according to Agilent protocol.Mitochondrial function was assessed using the XF96 extracellular flux analyzer  as previously described , with miGlass-bottom, 384-well microscopy plates  coated with Concanavalin A  were used for imaging. Cells in the mid-logarithmic phase were adhered to the plates by incubating at RT for 15 min. Upon adherence to the plate, media was replaced with media containing 50 nM MitoTracker , and cells were incubated at RT for 10 min, washed once, and imaged using automated inverted fluorescence microscope system  harboring a spinning disk high-resolution module . Images of cells in the 384-well plates were using a 60× oil lens (NA 1.42) and with a Hamamatsu ORCA-Flash 4.0 camera. Mitottracker was excited by a 561 laser, and the signal was detected through a 617/73 nm emission filter. For each well, 9 positions were imaged, and over 6000 yeast cells were segmented for mitochondria, according to the mitotracker signal, and analyzed using ScanR software .https://scholarbank.nus.edu.sg/handle/10635/236754, accessed on 2 May 2023.An amount of 100 μL of no more than 110 μg proteins of each fraction sample  with 10 μL of 1 M TEAB and 2 μL of 5% SDS was reduced with 5 μL of 100 mM TCEP and capitalized with 5 μL of 200 mM MMTS. An amount of 20 mL of 150 mM TEAB with 0.05% SDS was used to dialyze each capitalized sample overnight using Tube-O-DIALYZER™, Micro 1K MWCO. An amount of 2–3 μg trypsin was used to trypsinize each dialyzed sample. iTRAQ labeling of trypsinized samples was conducted using a SCIEX kit. The 8 differently labeled samples were then pooled together. The pooled mixture was then subjected to cation exchange chromatography followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which was conducted by Protein and Proteomics Centre (PPC), NUS Biological Sciences. Data processing information can be found at p-value was calculated for unpaired and non-parametric tests using the Mann-Whitney test (to compare ranks) or the Kolmogorov-Smirnov (to compare cumulative distribution). GO enrichment analysis in p < 0.05.All statistical analyses in


In vivo experiments with new-born and adult mice induced body tremors, hind limb paralysis, weakness, lethargy, and mortality. Taken together, the data suggest the detection of a potentially disease-causing orbivirus in cattle in Kenya. Its impact on livestock, as well as its potential economic damage, needs to be addressed in future studies using targeted surveillance and diagnostics.Arboviruses are among emerging pathogens of public and veterinary health significance. However, in most of sub-Saharan Africa, their role in the aetiologies of diseases in farm animals is poorly described due to paucity of active surveillance and appropriate diagnosis. Here, we report the discovery of a previously unknown orbivirus in cattle collected in the Kenyan Rift Valley in 2020 and 2021. We isolated the virus in cell culture from the serum of a clinically sick cow aged 2 to 3 years, presenting signs of lethargy. High-throughput sequencing revealed an orbivirus genome architecture with 10 double-stranded RNA segments and a total size of 18,731 bp. The VP1 (Pol) and VP3 (T2) nucleotide sequences of the detected virus, tentatively named Kaptombes virus (KPTV), shared maximum similarities of 77.5% and 80.7% to the mosquito-borne Sathuvachari virus (SVIV) found in some Asian countries, respectively. Screening of 2,039 sera from cattle, goats, and sheep by specific RT-PCR identified KPTV in three additional samples originating from different herds collected in 2020 and 2021. Neutralizing antibodies against KPTV were found in 6% of sera from ruminants (12/200) collected in the region. IMPORTANCE The genus Orbivirus contains several viruses that cause large outbreaks in wild and domestic animals. However, there is little knowledge on the contribution of orbiviruses to diseases in livestock in Africa. Here, we report the identification of a novel presumably disease-causing orbivirus in cattle, Kenya. The virus, designated Kaptombes virus (KPTV), was initially isolated from a clinically sick cow aged 2 to 3 years, presenting signs of lethargy. The virus was subsequently detected in three additional cows sampled in neighboring locations in the subsequent year. Neutralizing antibodies against KPTV were found in 10% of cattle sera. Infection of new-born and adult mice with KPTV caused severe symptoms and lead to death. Together, these findings indicate the presence of a previously unknown orbivirus in ruminants in Kenya. These data are of relevance as cattle represents an important livestock species in farming industry and often is the main source of livelihoods in rural areas of Africa. Orbivirus  consists of 22 classified virus species (–Orbiviruses are arthropod-borne viruses (arboviruses) that are commonly transmitted by mosquitoes, sandflies, biting midges, and ticks –4 to a w–5– species , 2. Abou species , 5, 12.  species . The VP2 species –18. In sspecies –. The intspecies –, 19. Orbspecies –, 18.The genus contains several viruses that can cause severe disease in wild and domestic animals, such as Bluetongue virus (BTV), African horse sickness virus (AHSV), and Epizootic hemorrhagic disease virus (EHDV). BTV mainly causes disease with high mortality in sheep but can also infect cattle, goats, buffalo, antelope, deer, elk, and camels –22. AHSV–Orbiviruses are distributed worldwide and their emergence and distribution depend on the abundance of competent vectors and environmental conditions that favor virus transmission between vectors and hosts –25. In K–26–In this study, we report the discovery and molecular and phenotypic characterization of a novel orbivirus designated Kaptombes virus (KPTV) from a diseased cow with evidence of circulation in indigenous cattle and local breeds of small ruminants in Kenya.Of the 1,500 serum samples from cattle, goat, and sheep used for blind virus isolation, one serum sample obtained from a 2- to 3-year-old cow with symptoms of emaciation and lethargy located in Kaptombes village in Kapkuikui, Baringo County , inducedBrahminy myna) in India in 1963, and later found in healthy cattle from Japan in 2005 (Infectious cell culture supernatant was applied to high-throughput sequencing (HTS) revealing the genome of an orbivirus with 10 segments of double-stranded (ds) RNA . The tot in 2005   in their VP3 protein. The closest genetic relative of KPTV was SVIV with nucleotide (nt) and amino acid (aa) identities of 77.46% and 89.5%, as well as 80.71% and 94.44% in their VP1 (RdRp) and VP3 (T2) proteins, respectively, suggesting that both viruses may belong to the same virus species. The orbivirus outer-core VP7 (T13) is more conserved than the outer-capsid proteins, VP2 (OC1) and VP5 (OC2), and is considered virus-species specific and therefore targeted for serological assays , 15, 30.An SDS-PAGE analysis confirmed the presence of 10 dsRNA segments of different sizes in KPTV and in the BTV BLUVAX vaccine strain, which was used as a control . Genome 10 copies/mL at 2- and 3-dpi in mosquito- and rodent-derived cell lines. In contrast, peak genome copies were about 10-fold lower and reached at 4 dpi in cell lines derived from cattle and nonhuman primates. These findings, together with the phylogenetic grouping within the mosquito-borne orbiviruses, suggest a mosquito and vertebrate transmission cycle for KPTV, similar to those of other mosquito-borne orbiviruses like SVIV, Umatilla virus and Skunk River Virus among others , primate Vero E6), and rodent (BHK 21) cells, as well as the mosquito cell lines C6/36 and Aag2. All cell lines were broadly susceptible for KPTV infection with peak genome copy numbers in cells derived from mosquitoes and hamster . KPTV re, and rodg others , 15, 31.6, 1 × 106, 5 × 105, 2.5 × 105, 1.25 × 105, 6.25 × 104, 3.125 × 104, 1.563 × 104, 7.8 × 103, and 3.9 × 103 PFU/mL) or mock-infected via intracerebral inoculation in groups of 10. Intracerebral inoculation of KPTV into 3- to 4-day-old Swiss Albino suckling mice resulted in severe clinical disease and induced tremors, hind-limb paralysis, and prostration. None of the clinically diseased mice recovered and lethal outcome was observed 1 to 7 days after onset of symptoms , respectively. All 10 mice of both study groups infected with the highest virus concentrations succumbed to the infection by day 8 and 9. Onset of symptom presentation and lethal outcome after onset of symptoms was observed up to 6 and up to 3 days later, respectively, in mice infected with lower KPTV doses  compared to mice infected with higher virus doses . All infected mice exhibited high infectious virus concentrations in the brain of 2.9 × 106 PFU/mL (mean virus titer).New-born Swiss Albino suckling mice were infected with a range of 10 doses of KPTV  collected at different locations in 2020 and 2021 . Phylogen = 100, sheep n = 50, and goats n = 50) from the same geographical area where KPTV originated and screened them by plaque reduction neutralization test (PRNT) for neutralizing reactivity against KPTV. In total, 12 samples  neutralized the virus with titers ranging from 1:20 to 1:320  (P = 0.035). All antibody positive samples were negative for KPTV RNA by qPCR and none of the PCR positive samples contained KPTV neutralizing antibodies.To further assess the dispersal of KPTV, we selected randomly 200 livestock serum samples (cattle  [1/50]) . The preIn vivo infection experiments with new-born mice induced body tremors, hind limb paralysis, weakness, lethargy, and mortality as from 4 to 5 dpi. Taken together, these findings provide evidence of a novel virus infecting ruminants in Baringo county, Kenya.We report the detection and isolation of a novel mosquito-borne orbivirus designated Kaptombes virus from cattle in Kenya. KPTV was initially isolated from a bovine with clinical symptoms aged 2 to 3 years. The virus was subsequently also detected in two cows sampled in the same year in other animal herds from the nearby villages and in another cow within the same subcounty, roughly 70 km away sampled 1 year later. Neutralizing antibodies against KPTV were detected in cattle (10%) and to a small extend in small ruminants (1%). An aliquot of the serum sample was sent to the Central Veterinary Laboratory (CVL) Kabete, Kenya for routine diagnostics where no positive results were obtained for tested diseases, including Rift Valley fever . At this time, we cannot confirm the orbivirus infection to be the cause nor dismiss its contribution to the clinical state and symptoms of the animal. The other animals from which the virus was later detected in serum samples showed no symptoms of disease.P = 0.035). Orbivirus specificity to a certain vertebrate host is common and has been observed among some host species, notably, horses which are known to be more susceptible to AHSV or sheep that are more susceptible to BTV. However, these AHSV and BTV can also infect many other host species  screened. However, neutralizing antibodies were also detected in sheep and goats, although with a significant lower prevalence than in cattle. Our data support the previous detection of its closest relative, SVIV in cattle, signifying a probable higher susceptibility of cattle to the virus or a preferred host range to large ruminants . An over species –22. ThisBrahminy myna) in India, and later isolated from sera of healthy cattle from Japan in 2005  at the termini of all segments which vary depending on the virus. Mosquito-borne orbiviruses have an overall NCRs of 5.03% to 5.695% , 15. Theo 45.89% , 35. KPTSVIV has so far only been reported in three Asian countries  , 13, 32.36–We report the identification of a novel presumably disease-causing orbivirus in cattle. These findings expand our knowledge of circulating orbiviruses in livestock. Cattle represents one of the most important livestock species in farming industry and often is the main source of livelihoods in rural areas of Africa . Thus, iicipe.Serum samples were collected from randomly selected cattle, sheep, and goats between 2020 and 2022, twice a year in May/June and September/October after the short and long rains in the semiarid areas of Marigat, Baringo County  and Nguruman, Kajiado County  in Kenya. Serum was collected and transported in cold chain (dry ice) to the Martin Lüscher Emerging Infectious Diseases Laboratory at l-glutamine (200 mM), 2% Gibco antibiotic-antimycotic (100×). The cells were rinsed with Gibco PBS, pH 7.4, and 50 μL serum was added followed by incubation at 37°C, 5% CO2  for 1 h, rocking after every 15 min to allow virus adsorption. After incubation, Gibco DMEM maintenance medium (MM) with 5% Gibco FBS, 2% Gibco l-glutamine (200 mM), and 2% Gibco Antibiotic-Antimycotic (100×) was added. Cells were incubated at 37°C, 5% CO2  and observed daily for signs of cytopathic effects (CPE). The CPE positive sample was passaged in 25-cm2 cell culture flasks  and frozen at –80°C before harvesting by thawing and centrifuging at 3,000 rpm for 10 min. The infectious supernatant was stored at –80°C until further use.Vero CCL-81 (ATCC CCL-81) cells were seeded in 24-well tissue culture plates  to 80% confluence in Gibco Dulbecco’s modified Eagle’s medium (DMEM) containing 10% Gibco fetal bovine serum (FBS), 2% Gibco Clarified infectious cell culture supernatant was filtered using 0.22-μm filters  to remove possible cellular residues. RNA was extracted using QIAamp viral RNA minikit  following the manufacturer’s protocol. RNA was quantified using a Nanodrop 2000 spectrophotometer  and Qubit RNA 2.0 fluorometer using the Qubit RNA HS assay kit . Libraries for sequencing were prepared using TruSeq stranded mRNA kit , following the manufacturer’s protocol with the modification to exclude the poly(A)-containing mRNA purification steps. Reverse transcription was done using Superscript III reverse transcriptase  and random hexanucleotide primers . This was followed by second-strand synthesis using DNA polymerase I and RNase H, provided with the library preparation kit. Purification was performed using AMPure XP beads  after which the purified double-strand cDNA fragments were end repaired by adding a single A’ nucleotides to the 3′ end of the blunt fragments. Ligation of the adapters was performed, and the products purified and enriched by PCR to create the final library. Libraries were normalized, pooled, and sequenced using the Illumina platform.Aedes albopictus C6/36, Aedes aegytpi Aag-2) and vertebrates  were infected with KPTV in duplicates at a multiplicity of infection (MOI) of 0.1. Aliquots of infectious cell culture supernatants from vertebrate and insect cells were harvested every 24 h for a period of 5 days. Amount of viral genome copy numbers were quantified by real-time RT-PCR using plasmid-based quantification standards  of it were intracerebrally inoculated through the cranial wall of the skull into 3- to 4-day-old Swiss Albino suckling mice. All 10 pups of one litter in a cage were infected with the same virus dose. The doses used in the experimental infection were quantified by plaque assays in Vero CCL-81 cells and the corresponding viral titers were as follows: 2 × 106, 1 × 106, 5 × 105, 2.5 × 105, 1.25 × 105, 6.25 × 104, 3.125 × 104, 1.563 × 104, 7.8 × 103, 3.9 × 103 PFU/mL. Noninfectious MEM was included as negative control. All mice were observed 2 times/day for up to 14 days for signs of infection and any severely moribund mice separated and preserved at –80°C immediately after death. Brain samples were homogenized in 1 mL of cell culture media and inoculated in Vero CCL-81 cells. Infectious viral particles were quantified via plague assay.Swiss Albino mice obtained from the KEMRI animal unit were used. Eleven female lactating mice with a litter size of 10 were selected for the study and the neonates observed for 2 to 3 days before the experiment in a Biosafety Level 2 (BSL 2) laboratory. Mice were fed 6 PFU/mL diluted in phosphate-buffered saline . Control mice were injected with equal amount of PBS. Infected mice were observed daily for 20 days for specific symptoms presentation (SSP)  on the lower abdomen through the abdominal wall and intracerebrally (IC) with 500 μL of the virus inoculum at a concentration of 1.2 × 10on (SSP) , 44.(i) RNA extraction. For dsRNA segment analyses, 50 μL of prewarmed 1M sodium acetate (NaAc) containing 1% sodium dodecyl sulfate (SDS), pH 5.0 was added to 450 μL of infectious cell culture supernatant from either KPTV or the BTV BLUVAX vaccine from KEVEVAPI (Kenya Veterinary Vaccine Production Institute) isolate, vortexed for 10 s and incubated for 15 min at 37°C. Next, 500 microliters phenol-chloroform (1:1) was added to the tube, vortexed for 1 min then incubated for 15 min at 56°C in a water bath. After incubation, the sample was vortexed for 1 min then centrifuged for 5 min at 12,000 rpm. The upper aqueous phase containing the dsRNA was pipetted into a clean Eppendorf tube and 40 μL (~1/10 volume) 3M NaAc added and the tube filled with ice-cold (–20°C) absolute ethanol, mixed gently by turning the tube over and over 4 to 6 times then incubated at –20°C overnight. The tubes were centrifuged at 4°C for 20 min at 12,000 rpm to pellet the dsRNA, the supernatant was poured off and tubes were air dried. The pellet was resuspended in 30 μL PAGE sample dye containing 10 mg bromophenol blue, 5 mL of Spacer gel buffer, and 1 mL of glycerol before loading on a PAGE gel.(ii) PAGE analysis. A total of 10%1.5 mm Resolving Gel was prepared using 15.8 mL distilled water, 10 mL of 30% acrylamide stock, 3.75 mL resolving buffer (pH 8.9) containing 36.3 g Tris Base and 48 mL 1N HCL, 15 μL Tetramethylethylenediamine (TEMED), and 450 μL of 10% ammonium persulphate. The gel was immediately poured between the thick gel spacers up to the gel mark after cleaning with 96% ethanol then overlaid with 1 mL layer of water before polymerization. The resolving gel was allowed to polymerise for 2 h, after which the water was poured off and 1.5 mm 3% spacer gel prepared by 6.8 mL distilled water, 1.6 mL of 30% acrylamide stock, 1.25 mL spacer buffer (pH 6.7) containing 5.98 g Tris Base and 50 mL distilled water, 5 μL TEMED, and 150 μL of 10% ammonium persulphate was poured on top after positioning the combs and allowed to polymerize for 45 min. After polymerization, the combs were removed, and the glass plate assembled onto the electrophoresis apparatus with electrophoresis buffer prepared by 200 mL 5% Tris-glycerine buffer with 800 mL of distilled water. The dsRNA of KPTV and BTV BLUVAX vaccine extracted as earlier described and diluted in 30 μL PAGE-dye was loaded to the wells and run for 18 h at 100V.3) staining solution then washed twice in distilled water for 2 min each wash. To remove the black precipitate, approximately 50 mL of developing solution  was used, agitated for 30 s, poured off then 200 mL added, and incubated for 5 min, drained off before adding stopping solution (10 mL acetic acid and 200 mL dH2O) to prevent further color development for 10 min and rinsing the gel in distilled water for 10 min. Distilled water was added to the gels and left until ready to dry.After electrophoresis, the gel was washed and fixed with 200 mL fixing solution 1, prepared with 80 mL ethanol, 110 mL distilled water, and 10 mL acetic acid then incubated for 30 min on an orbital shaker. The fixing solution 1 was drained off and replaced with 200 mL of fixing solution 2 containing 20 mL ethanol, 180 mL distilled water, and 1 mL acetic acid and incubated for 30 min on the orbital shaker. The fixing solution 2 was drained off and the gel stained for 30 min using silver nitrate (AgNOAGCGAGGTGGATAGTGAAGA-3′ and R 5′-CTCCGCCCTAACATCCAATAAA-3′) and real-time RT-PCR primers  were designed based on Segment 2 using the IDT PrimerQuest Tool.KPTV specific primers  and positive samples purified using ExoSAP-IT PCR Product Clean-up Reagent (Applied Biosystems) according to the manufacturer’s instructions, then sequenced in both directions. The sequencing services were outsourced to Macrogen, Europe B.V.A total of 248 livestock serum pools made up of 83/248 (33.5%) cattle, 85/248 (34.3%) goats, and 80/248 (32.2%) sheep from Baringo county were screened for presence of KPTV RNA. Each pool consisted of five to seven individual samples based on species and location. Viral RNA was extracted from 140 μL pooled serum and the CPE positive sample (KPTV) using the QIAamp Viral RNA minikit  according to the manufacturer’s protocol. cDNA was synthesized using SuperScript III Reverse Transcriptase according to the manufacturer’s instructions. The samples were tested for presence of KPTV genome copies using a 25 μL PCR containing 15.65 μL PCR water, 2.50 μL 10 × PCR Buffer, 1.25 μL Mg (50 mM), 0.50 μL 10 mM dNTPs, 1.5 μL of 10 μM forward and reverse primers, 0.10 μL Platinum-T target probe, 2 μL Mg (50 mM), 2.5 μL 10 × Buffer, 0.5 μL dNTPS (10 mM), 0.2 μL Invitrogen Platinum Taq, and 2 μL cDNA template.The 25 μL qRT-PCR total reaction volume included 16.05 μL of water, 0.75 μL forward and reverse primer each, 0.25 μL FAMn = 100, sheep n = 50, and goats n = 50) were aliquoted in volumes of 30 μL, heat inactivated at 56°C for 30 min and then tested for neutralizing antibodies to Kaptombes virus in 2-fold serum dilutions from 1:20 to 1:320 using PRNT90 as previously described  Seroprevalence,  The seroprevalence, in vivo, and in vitro studies data were entered into Microsoft Excel v. 2016, cleaned then imported to R version 4.2.0 for analysis. Comparison of KPTV seroprevalence between the different livestock species was done using Fisher exact test. The 95% confidence intervals (CIs) were estimated using the Agresti-Coull method. All tests were performed at a 5% significance level.(ii) Sanger sequence and phylogenetic analysis. Sequences were analyzed using Geneious Prime and queried against the NCBI GenBank database using the Basic Local Alignment Search Tool (BLAST) ( (BLAST) . Multipl (BLAST)  and maxi (BLAST) , using t (BLAST) . In tota(iii) NGS data analysis. Raw sequence reads were initially subjected to cleaning using Trim Galore v0.6.5 to remove adapters and Prinseq Lite v0.20.4 to remove low-quality reads using the following parameters: minimum length, 50 bp; maximum length, 301 bp; and minimum mean Q score, 30. Further, filtering of the reads was performed by using ribo Picker v0.4.3, to remove rRNA sequences by comparing them to the SILVA rRNA database, release 138.1. Paired-end reads were merged using PEAR 0.9.8, and preliminary analysis was performed using the MG-RAST server to identify reads taxonomically. De novo assembly of cleaned reads was done using trinity program with default parameters. The cleaned reads were mapped back to the assembled contigs and filtered to retain only contigs in which at least 90% of nucleotides had a 5-time coverage. Contigs were compared to the NCBI database. Sequences were further analyzed in Geneious Prime software (https://www.geneious.com).The study was approved by the Kenya Medical Research Institute’s Scientific and Ethics Review Unit (under SERU No.3312) after gaining approval by the animal care and use committee. Additional approval for the study was accorded by the University of Pretoria, Faculty of Health Science’s Research Ethics Committee (Ethics Reference No. 568/2020).OQ122118 to OQ122127, as well as fragments of S2 of KPTV B97, KPTV B215, KPTV B128, and KPTV B364 under the accession numbers OQ122128 to OQ122131, respectively.The sequences of this study can be found under the GenBank accession numbers KPTV B215 genome


Cerebral small-vessel disease (SVD) is prevalent in type 1 diabetes and has been associated with the haptoglobin variant allele Hp1. Contrarily, the Hp2-allele has been linked to cardiovascular disease and the role of haptoglobin-genotype in asymptomatic SVD is unknown. We, therefore, aimed to evaluate the alleles’ association with SVD.1c 8.1 ± 3.2% [65 ± 12 mmol/mol]). Examinations included genotyping  by polymerase chain reaction, clinical investigation, and magnetic resonance brain images assessed for SVD manifestations .This cross-sectional study included 179 neurologically asymptomatic adults with type 1 diabetes . Haptoglobin genotype frequencies by presence versus absence of SVD were 16.1%; 46.8%; 37.1% versus 15.4%; 41.9%; 42.7% (p = 0.758). Minor allele frequencies were 39.5% versus 36.3% (p = 0.553). Hp1 homozygotes and Hp2 carriers displayed equal proportions of SVD  and SVD manifestations . Hp1-1 was not associated with SVD  when adjusting for age, blood pressure, and diabetic retinopathy.SVD prevalence was 34.6%. Haptoglobin genotype frequencies were 15.6% (Hp1-1), 43.6% (Hp1-2), and 40.8% (Hp2-2). Only diastolic blood pressure differed between the genotypes Hp1-1, Hp1-2, and Hp2-2 (81 [74–83], 75 [70–80], and 75 [72–81] mmHg, Although the SVD prevalence was high, we detected no significant association between SVD and haptoglobin-genotype.The online version contains supplementary material available at 10.1007/s00592-023-02059-2. Cerebrovascular disease is among the most common causes of disability worldwide . In typeSVD affects the microvasculature of the brain and is visible on magnetic resonance images (MRI) by its manifestations, which include white matter hyperintensities, cerebral microbleeds, lacunar infarctions, and cortical superficial siderosis . AlthougHp is a plasma protein that binds to free haemoglobin, thereby affecting its oxidative activity. The protein is affected by genetic variation. Two variant alleles, Hp1 and Hp2 give rise to three genotypes Hp1-1, Hp2-1, and Hp2-2, of which Hp2-2 possesses the weakest antioxidative properties  and has On the other hand, also the less-frequently observed genotype Hp1-1 has been linked to cerebrovascular disease in the general population . In typeAlthough not known, the impact of a distinct genotype could vary for different cardiovascular outcomes, such that Hp2-2 relates to atherosclerosis and large-vessel disease, whereas Hp1-1 to SVD. It is yet unknown whether the Hp-genotype is associated with the prevalence of SVD, or if a particular manifestation occurs more often in certain genotypes. We, therefore, aimed to elucidate whether the Hp-genotype is associated with SVD, or any of its manifestations, in our cohort of 179 neurologically asymptomatic individuals with type 1 diabetes.Within the nationwide FinnDiane Study, we initiated a sub-study at the Helsinki University Hospital (HUS) study centre in 2010. This sub-study aims to identify early risk factors for cerebrovascular disease in type 1 diabetes. The FinnDiane study  and the Individuals entering the HUS study centre were consecutively recruited for a study visit that included a brain MRI if they met the inclusion criteria: age 18–50 years and type 1 diabetes onset before 40 years of age. Exclusion criteria were kidney failure , previous clinical signs of cerebrovascular disease, and contraindications for MRI. A total of 191 participants were enrolled and the Hp-genotype was successfully determined for 179 (93.7%) participants, all eligible for this study. With this sample size and significance level (α = 0.05) we were able to detect an effect of OR = 1.91 with 0.80 power.All participants entered a study visit. Their medical history was evaluated thoroughly, and data were verified from the participant’s medical records. Anthropometrics, office blood pressure, and current medication were recorded. Body mass index was calculated by dividing body mass by the square of the body height. Participants furthermore filled in questionnaires on lifestyle, such as smoking. Diabetic retinopathy was verified and categorized based on fundus images with “any retinopathy” indicating the presence of at least retinal microaneurysms, and proliferative diabetic retinopathy indicating neovascularization or previous treatment with retinal photocoagulation.1c), haemoglobin, creatinine, total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides were determined from blood samples. The glomerular filtration rate was estimated using the Chronic Kidney Disease Epidemiology Study (CDK-EPI)-formula [Glycated haemoglobin . Images were assessed for markers of SVD by an experienced neuroradiologist (J.M.). As markers, we considered white matter hyperintensities , cerebral microbleeds, lacunar infarctions, and superficial siderosis, based on standardised criteria . The neuContinuous parametric variables were analysed using one-way ANOVA, with central tendency presented as mean ± standard deviation. For analysis of non-parametric variables, the Kruskal–Wallis test was used with results presented as median (interquartile range). Categorical variables were compared using the chi-squared test, and Fisher’s exact test when observed frequencies were < 5.p value < 0.05 was considered statistically significant.To analyse the relationship between SVD and genotype Hp1-1, multiple logistic regression analysis was used. SVD was analysed as the dependent variable, with Hp-genotype and clinical confounders as independent variables. Clinical confounders were chosen based on their significance in univariable analysis. The analyses of SVD (any manifestation), and all its manifestations, were performed in separate models. Results are presented as odds ratios (OR) with 95% confidence interval (CI). A Power analyses were performed post hoc to evaluate the smallest detectable effect size in the cohort. The smallest detectable OR for SVD as a binary outcome (present vs absent) was calculated based on minor allele frequency, the proportion of SVD cases, and the number of participants using the R genpwr package version 1.0.4 . A signi1c was 8.1 ± 3.5% (65 ± 12 mmol/mol). None of the participants had peripheral arterial disease. A total of 62 (34.6%) participants had SVD. Cerebral microbleeds were observed in 40 (22.3%), white matter hyperintensities of Fazekas ≥ 1 in 31 (17.3%), and lacunar infarctions in four (2.2%). None of the participants had cortical superficial siderosis. White matter hyperintensities and cerebral microbleeds were observed simultaneously in nine participants. All four participants with lacunar infarctions had some additional manifestation: two had white matter hyperintensities and the other two had cerebral microbleeds.The study participants had a mean age of 38.8 ± 7.3 years, 95 (53.1%) were women, participants had a mean diabetes duration of 23.3 ± 10.1 years, and their mean HbAp = 0.647) and similar to frequencies previously observed in the Finnish population [A total of 28 15.6%) participants had the Hp1-1, 78 (43.6%) the Hp 2-1, and 73 (40.8%) the Hp2-2 genotype. The distribution was within the Hardy–Weinberg equilibrium (pulation . The min5.6% partp = 0.758). Likewise, minor allele frequencies by presence versus absence of SVD did not differ between the groups (p = 0.553). Also, when analysing the additive effect of the Hp1 alleles by simple logistic regression, we did not detect a significant association between SVD and the number of Hp1 alleles .Figure p = 0.988, and white matter hyperintensities 40.3% versus 36.8%, p = 0.605. As we had only four observations of lacunar infarctions, we could not perform statistical testing for those.We tested for differences in the prevalence of separate SVD markers across genotypes, by using the chi-squared test. Results are presented in Table p = 0.027), homozygotes for Hp1 did not significantly differ from the rest of the participants regarding clinical characteristics (data not shown).In further analysis, homozygotes for Hp1 (N = 28) were compared to Hp2 carriers . Except for diastolic blood pressure  for this number of participants, indicating that the study is well-powered to detect more prominent associations. Of note is, that although we would have been able to detect larger effects (OR > 1.91), the sample size required to detect more subtle effects (OR < 1.5) with a probability of 0.8 is 4,295 participants (α = 0.05). Regardless, we consider the standardised brain MRI and the detailed assessment of the images to be a substantial strength of our study. Another strength of our study is the unique and well-characterized study population.Our results suggest that there is no prominent association between asymptomatic SVD and genetic variation in Hp. Although our current study has a unique cohort with both MRI and Hp-genotype data of individuals with type 1 diabetes, and we have previously presented similar observations for stroke, i.e. overt disease, our results highlight a call for more comprehensive studies on the topic of SVD in type 1 diabetes.Supplementary file1 (PDF 105 kb)Below is the link to the electronic supplementary material.


Pseudomonas aeruginosa, we screened possible synergistic effects of AGXX on several antibiotic classes. We found that the combination of AGXX and aminoglycosides tested at sublethal concentrations led to a rapid exponential decrease in bacterial survival and restored the sensitivity of a kanamycin-resistant strain. ROS production contributes significantly to the bactericidal effects of AGXX/aminoglycoside treatments, which is dependent on oxygen availability and can be reduced by the addition of ROS scavengers. Additionally, P. aeruginosa strains deficient in ROS detoxifying/repair genes were more susceptible to AGXX/aminoglycoside treatment. We further demonstrate that this synergistic interaction was associated with a significant increase in outer and inner membrane permeability, resulting in increased antibiotic influx. Our study also revealed that AGXX/aminoglycoside-mediated killing requires an active proton motive force across the bacterial membrane. Overall, our findings provide an understanding of cellular targets that could be inhibited to increase the activity of conventional antimicrobials.The rapid dissemination of antibiotic resistance combined with the decline in the discovery of novel antibiotics represents a major challenge for infectious disease control that can only be mitigated by investments in novel treatment strategies. Alternative antimicrobials, including silver, have regained interest due to their diverse mechanisms of inhibiting microbial growth. One such example is AGXX, a broad-spectrum antimicrobial that produces highly cytotoxic reactive oxygen species (ROS) to inflict extensive macromolecular damage. Due to the connections identified between ROS production and antibiotic lethality, we hypothesized that AGXX could potentially increase the activity of conventional antibiotics. Using the gram-negative pathogen P. aeruginosa. The combination of AGXX and aminoglycosides not only reduces bacterial survival rapidly but also significantly re-sensitizes aminoglycoside-resistant P. aeruginosa strains. In combination with gentamicin, AGXX induces increased endogenous oxidative stress, membrane damage, and iron-sulfur cluster disruption. These findings emphasize AGXX’s potential as a route of antibiotic adjuvant development and shed light on potential targets to enhance aminoglycoside activity.The emergence of drug-resistant bacteria coupled with the decline in antibiotic development highlights the need for novel alternatives. Thus, new strategies aimed at repurposing conventional antibiotics have gained significant interest. The necessity of these interventions is evident especially in gram-negative pathogens as they are particularly difficult to treat due to their outer membrane. This study highlights the effectiveness of the antimicrobial AGXX in potentiating aminoglycoside activities against  Pseudomonas aeruginosa, a common cause of acute  and chronic infections   . P. aeruibiotics .2−) through a series of redox reactions where the oxidized Ag component is reduced by organic matter and donates electrons to the valent Ru, which subsequently generates O2− and other ROS  of conventional antibiotics required for treatment or possibly overriding and/or delaying antibiotic resistance development . Silver derivates have received increased attention in medical applications, for example, as antimicrobial surface coatings on catheters that protect from biofilm-forming bacteria and reduce the risk of nosocomial infections (P. aeruginosa infections in burn wound patients (3), and silverdene, we conducted survival studies of PA14 in the presence and absence of 25 µg/mL of each compound. Survival of PA14 was highly compromised when cells were exposed to AGXX394 (2DCFDA). H2DCFDA has been extensively demonstrated to detect various ROS, including peroxides as well as peroxyl and •OH radicals   . We founP. aeruginosa. Specifically, we were interested in determining whether combining AGXX with members of different antibiotic classes, such as inhibitors of DNA replication (fluoroquinolones), cell wall biosynthesis (β-lactams), folate biosynthesis, membrane integrity (polymyxins), and translation (aminoglycosides), shows increased bactericidal activity against P. aeruginosa. Here, we used AGXX720C, a formulation with significantly lower antimicrobial activity that allowed us to control dosage of AGXX more reliably . We determined the MICs of AGXX720C and 10 members of five different antibiotic classes in PA14, which were cultivated under aerobic conditions in Mueller-Hinton broth (MHB)  . Using tth (MHB) ; Fig. S3th (MHB) Fig. S3. 3’s synergizing effects on aminoglycosides were reduced in minimal media . Although we do not probe the reasons behind the effects of casamino acids in minimal media, it is evident that AGXX synergizes with aminoglycosides in both rich and minimal media.It has been reported that AgNOal media . We therP. aeruginosa strains. Like many other P. aeruginosa strains, PA14 is intrinsically resistant to many different antibiotics, including the aminoglycoside kanamycin. Under the conditions tested, our PA14 strain showed a MIC of 240 µg/mL for kanamycin. We then exposed PA14 to 75 µg/mL AGXX720C, 50 µg/mL kanamycin, or a combination of 75 µg/mL AGXX720C and 50 µg/mL kanamycin and determined their survival over the time course of 3 h as described earlier. As early as 60 min posttreatment, the combination of AGXX720C and kanamycin reduced colony survival by ~1.5 log, which increased to about 4 log  difference in survival after 3 h -detoxifying enzyme catalase resulted in a significant decline in H2DCFDA fluorescence  dye as well as hydroxyphenyl fluorescein (HPF) to examine intracellular concentrations of H2O2 and •OH levels, respectively  and 100-fold (ibpA) increased when PA14 was treated with the combination of AGXX720C and gentamicin, indicating significant macromolecular damage  fairly well and showed about 2.5 log higher survival relative to aerobically grown cells exposed to the same treatment. However, when we increased the Gm concentration to 1 µg/mL (0.5× MIC) in the presence of AGXX, PA14 survival also declined albeit at a lower rate than aerobically growing cells under the same conditions , katA , katB , and dps (encodes an iron acquisition protein in response to oxidative stress) to sublethal concentrations of AGXX720C (25 µg/mL), tobramycin (0.25 µg/mL), or the combination thereof for 3 h and compared their colony survival to that of the corresponding wild-type strain PA14 . Overall, our data suggest a potential role of free iron for the ROS-mediated toxicity of aminoglycoside exposure when in synergy with AGXX.Fe/S cluster are essential cofactors in various metabolic enzymes, including members of the electron transport chain and the tricarboxylic acid (TCA) cycle . Fe/S clbination , we wondbination . The los2 stress , 48. To 2 stress . HoweverN-phenyl-1-naphthylamine (NPN), which has diminished fluorescence in the presence of an intact outer membrane but significantly increases when bound to exposed phospholipid groups in a disrupted lipopolysaccharide monolayer , a compound that efficiently inhibits the PMF, we determined whether the synergy between AGXX and aminoglycosides is dependent on the membrane potential. Not surprisingly, PA14 killing by a lethal dose of gentamicin (1 µg/mL) could be reduced by almost 2 logs when the cells were preincubated with CCCP . Likewise, pretreatment with CCCP restored PA14 survival to a similar degree in the presence of 0.25 µg/mL Gm and 50 µg/mL AGXX720C for 3 h  in exponentially growing PA14 in the presence or absence of AGXX720C. Treatment of PA14 with a combination of AGXX720C and TR-Gm resulted in a significant increase in TR-Gm uptake similar to treatments with the membrane-targeting antibiotic polymyxin B , 54. As  for 3 h . SurprisP. aeruginosa. We show that AGXX serves as an efficient tool to potentiate the activity of aminoglycoside antibiotics by reducing bacterial survival up to 50,000-fold. Aside from a 1-log increase in killing found when the Ag/Ru compound was combined with the membrane-targeting antibiotic polymyxin B, AGXX did not influence the activity of antibiotics targeting DNA replication, cell wall synthesis, or folate metabolism  , given that catalase cannot readily cross the bacterial cell envelope. The combination of AGXX and aminoglycosides concertedly increased endogenous ROS levels, including H2O2 and •OH. Potentially facilitated by released silver ions, the increased ROS level may disrupt iron-sulfur clusters in metabolic enzymes such as aconitase, resulting in the release of free iron, which ultimately triggers •OH formation in a Fenton reaction  , 16. Whireaction . IncreasP. aeruginosa. AGXX itself has been proposed to exert its antimicrobial action through the generation of ROS rather than the release of silver ions. AGXX-mediated ROS production likely starts with the generation of O2−, which subsequently can be dismutated by cellular superoxide dismutases into hydrogen peroxide . We further found that disrupting membrane potential using an ionophore increased bacterial survival under combined AGXX and gentamicin stress . The procedure was originally described in Guridi et al.  and lateP. aeruginosa PA14 strains (N-morpholino) propanesulfonic acid minimal media containing 0.2% glucose, 1.32 mM K2HPO4, and 10 µM thiamine (MOPSg) and incubated at 37°C at 150 rpm. Growth under anaerobic conditions was performed as described previously  screw cap culture tubes filled with minimal room. Overnight PA14 cultures were diluted to an OD600 = 0.002 in MHB containing 1% KNO3 and grown until OD600 ~0.2 after which the treatments were applied for 4 h.Unless stated otherwise, overnight  strains  were groeviously . In brie600 of 0.002 and distributed into 96-well plates that contained increasing concentrations of ciprofloxacin, nalidixic acid, carbenicillin, imipenem, trimethoprim, polymyxin B, gentamicin, amikacin, tobramycin, streptomycin, and kanamycin, respectively. Plates were subsequently incubated at 37°C for 16 h at 300 rpm. MIC assays were performed in duplicates. The MIC is defined as the lowest antibiotic concentration that inhibited growth.MIC assays were performed in 96-well plates in a total volume of 200 µL per well. Overnight PA14 cultures were diluted into MHB to an OD600 = 0.1 in a six-well sterile cell culture plate. Sublethal concentrations of AGXX720C and antibiotics of interest were added individually and in combination as indicated and plates incubated at 37°C for 3 h at 150 rpm under aerobic conditions. For thiourea, CCCP and BIP pretreatments, overnight PA14 was grown in MOPSg to exponential phase. Thiourea, CCCP or BIP was then added 60 min prior to AGXX/aminoglycoside treatments whenever indicated. At 1 h intervals, OD600 of cultures were recorded, sample volumes were normalized to the lowest optical density measured, serially diluted in PBS (pH 7.4) and plated on LB agar to quantify CFUs after 16 h incubation. Percentage of survival was calculated as the ratio of surviving colonies from treated to untreated samples.Overnight PA14 cultures were diluted ~25-fold into MHB by normalizing cultures to an OD2DCFDA) (Thermofisher Scientific). Exponentially growing PA14 cultures were left untreated or treated with 0.25 µg/mL Gm, 50 µg/mL AGXX720C or a combination of both for 1 h. Samples were collected and normalized to an OD600 = 1.0. Cells were washed twice, resuspended in prewarmed PBS containing 10 µM H2DCFDA, and incubated in the dark at 37°C for 30 min before samples were washed twice again in PBS and DCF fluorescence measured at excitation/emission wavelengths of 485/535 nm (Tecan 200 plate reader). To quench cellular ROS, cells were pretreated with 50 mM thiourea prior to the stress treatments.Intracellular ROS levels were quantified using the redox-sensitive dye 2′,7′-dichlorodihydrofluorescein diacetate  in the PE-CF594 channel. At least 10,000 events were recorded, and figures generated using FCSalyzer .600nm of 1.0 after washing twice with PBS (pH 7.4). Cells were then stained with 10 µM HPF and incubated in the dark for 30 min at 37°C. Cells were washed twice and resuspended in prewarmed PBS. Samples were analyzed using the flow cytometer (BD FACS Melody) in the FITC channel. At least 10,000 events were recorded, and figures generated using FCSalyzer (The fluorescent probe HPF (Invitrogen) was used to determine the amount of cellular hydroxyl radicals (•OH) produced . ExponenCSalyzer .600 = 0.1 and grown to mid-log phase (OD600nm = 0.3). Cultures were left untreated or treated with 0.25 µg/mL Gm, 50 µg/mL AGXX720C, or the combination thereof for 60 min. Transcription was stopped by the addition of an equal volume of ice-cold methanol. Total RNA was extracted from three biological replicates using a commercially available RNA extraction kit (Macherey & Nagel). Remaining DNA was removed using the TURBO DNA-free kit (Thermo Scientific). mRNA was reverse transcribed into cDNA using the PrimeScript cDNA synthesis kit (Takara). The following primer pairs were used for gene amplification: ibpA, TTCCGTCATTCCGTAGG/AGGTCTTCTTCCTGG; sulA,ACTGTTCCAGGAAGCGTTCT/AGCGAAAGTTCGCTAAAGGC; rrsD,TATCAGATGAGCCTAGGTCGGATTA/TTTACAATCCGAAGACCTTCTTCAC.Overnight PA14 cultures were diluted into MOPSg media to an ODrrsD gene and relative fold changes in gene expression were calculated using the 2-ΔΔCT method . Transcript levels of the indicated genes were normalized against transcript levels of the 16S rRNA-encoding T method .Exponentially growing PA14 were treated with sublethal concentrations of AGXX720C (100 µg/mL), Gm (0.6 µg/mL), or the combination thereof. Aconitase activity was measured from cell lysates using the Aconitase Assay Kit (Abcam) according to the manufacturer’s instructions. One unit of aconitase is defined as the amount of enzyme that isomerizes 1 µmol of citrate to isocitrate per min at pH 7.4 and 25°C.600 = 0.5 in HEPES-sodium buffer (pH 7.2). 10 µM NPN was added, and samples incubated in the dark for 15 min. NPN fluorescence was measured at excitation/emission wavelengths of 350/420 nm. Increased NPN uptake (indicating outer membrane damage) was calculated using the following equation (The NPN uptake assay was used to detect outer membrane damage as described in reference equation :600 = 0.5. Propidium iodide (Thermo Fisher Scientific) was added to a final concentration of 500 nM and samples incubated in the dark for 30 min. Fluorescence intensities were measured at excitation/emission wavelengths of 535/617 nm. Samples treated with polymyxin B at a sublethal concentration (2 µg/mL) were included as a positive control.Inner membrane integrity was determined by the cellular uptake of propidium iodide following antimicrobial treatments. Exponentially growing PA14 cells were either left untreated or treated with 0.25 µg/mL Gm, 50 µg/mL AGXX720C, or a combination thereof. Cells were harvested after 1 h, washed twice, and resuspended in PBS (pH 7.4) at an OD600 = 0.2. Samples were stained with SYTO9 (6 µM) and PI (30 µM) and incubated in the dark for 15 min at room temperature. Cells were then transferred onto a glass slide and covered with 1% agarose prior to visualization using a Leica SP8 confocal system equipped with a DMi8 CS inverted microscope. Samples treated with polymyxin B at a sublethal concentration (2 µg/mL) were included as a positive control.Exponentially growing PA14 cells were either left untreated or treated with 0.25 µg/mL Gm, 50 µg/mL AGXX720C, or a combination thereof. Cells were harvested after 1 h, washed twice, and resuspended in PBS (pH 7.4) at an ODN,N-dimethylformamide at 4°C (2CO3 (pH 8.5) to a final concentration of 10 mg/mL at 4°C. 20 µL Texas Red was slowly added dropwise to 700 µL Gm to allow for the conjugation reaction to obtain the Texas Red-labeled gentamicin (TR-Gm) (Texas Red-succinimidyl ester (Invitrogen) was dissolved to a final concentration of 20 mg/mL in high-quality anhydrous e at 4°C . Gm was  (TR-Gm) . The TR- (TR-Gm) .All statistical analyses were performed in GraphPad Prism version 8.0.