Background: Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Variants in the LRRK2 gene have been shown to be associated with PD. However, the clinical characteristics of LRRK2-related PD are heterogeneous. In our study, we performed a comprehensive pooled analysis of the association between specific LRRK2 variants and clinical features of PD.Methods: Articles from the Medline, Embase, and Cochrane databases were included in the meta-analysis. Strict inclusion criteria were applied, and detailed information was extracted from the final original articles included. Revman 5.3 software was used for publication biases and pooled and sensitivity analyses.Results: In all, 66 studies having the clinical manifestations of PD patients with G2019S, G2385R, R1628P, and R1441G were included for the final analysis. The prominent clinical features of LRRK2-G2019S-related PD patients were female sex, higher rates of early-onset PD (EOPD), and family history . PD patients with G2019S were more likely to have high scores of Schwab & England , low GDS scores, high UPSIT scores , and good response to L-dopa . Further, G2019S carriers had higher LEDD  and were more likely to develop motor complications, such as dyskinesia and motor fluctuations  than non-carriers. G2385R carriers were more likely to have family history  than non-G2385R carriers and lower H-Y and higher MMSE scores . G2385R carriers had higher LEDD and tended to develop motor complications, such as motor fluctuations  than non-carriers. Other clinical presentations did not feature G2019S or G2385R. We observed no distinct clinical features for R1628P or R1441G. Our subgroup analyses in different ethnic group for specific variant also presented with relevant clinical characteristics of PD patients.Conclusions: Clinical heterogeneity was observed among LRRK2-associated PD in different variants in total and in different ethnic groups, especially for G2019S and G2385R. Parkinson's disease (PD) is the second-most common neurodegenerative disease, with major clinical features comprising motor symptoms (MS) and non-motor symptoms (NMS). MS are characterized by four cardinal symptoms: bradykinesia, resting tremor, rigidity, and postural instability. NMS include olfactory dysfunction, constipation, depression, and sleep disturbance  is considered the most common genetic cause of PD  is still debatable. Some researchers believe that LRRK2-related PD has similar clinical onset features to IPD, such as resting tremor, good response to L-dopa, and a benign clinical course . To further explore the role of LRRK2 SNPs in PD clinical features, here, we conducted a complete analysis of clinical features in specific LRRK2 variants related to PD.Our previous comprehensive meta-analysis demonstrated the importance of *,” “PD,” “LRRK2,” and “PARK8.” The data were assessed online on February 10, 2018. Overlapping articles from different databases were excluded. Two researchers (Li Shu and Yuan Zhang) performed the search independently. In case of disagreements, a third researcher (Qiying Sun) was consulted to arrive at a consensus.Medline database in PubMed, Embase database in Ovid, and the Cochrane databases were electronically searched by the authors for publications in English. The key words used were “ParkinsonThe PICOS  principle was applied in the inclusion process.LRRK2 variants.Participants: all PD patients were diagnosed according to widely accepted criteria . If there were disputes in the process, a third author was asked to solve the problem , a fixed model (FM) was used for further analysis. However, if the heterogeneity was significant , a random model (RM) was applied. Publication biases were measured using funnel plots. Sensitivity analyses were performed by eliminating papers one at a time, and the changes in the total results were observed.Revman 5.3 software was used for all statistical analyses. Pooled odds ratio (ORs) or pooled mean difference (MD) and 95% CIs were calculated to estimate dichotomous data or continuous data about the importance of polymorphisms to the risk of phenotypes. Q statistic and ILRRK2 variants related to PD. Forty-six articles comprising 16,016 patients were included for analysis of the clinical features of LRRK2-G2019S-related PD. Seventeen articles involving 6,767 patients were included for analysis of clinical presentations of LRRK2-G2385R-related PD; out of these, 5 LRRK2-R1628P–related articles comprising 2,369 patients and 5 LRRK2-R1441G–related articles comprising 1,222 patients were included in the final meta-analysis. The characteristics of the included studies in each analysis are shown in Table A flowchart depicting the publication search process is shown in Figure In terms of the clinical features of specific variants, the present meta-analysis showed unique clinical manifestations in G2019S, G2385R, R1628P, and R1441G separately. In total, 40 specific clinical features or rating scales belonging to 13 classifications . With respect to NMS, G2019S carriers tended to have lower Geriatric Depression Scale (GDS) scores and higher University of Pennsylvania Smell Identification Test (UPSIT) scores than non-carriers . In terms of the response to treatment, the G2019S carriers showed good response to L-dopa  and higher Schwab and England Activity of Daily Living Scale scores . Further, the G2019S carriers received a statistically higher Levodopa equivalent daily dose (LEDD)  and were more likely to develop motor complications, such as dyskinesia and motor fluctuations than non-carriers . Other clinical presentations did not feature G2019S.Our data show that G2019S carriers were predominantly female and had higher rates of EOPD and family history . In European/West Asians, the G2019S carriers tended to have earlier age at onset, be female, have higher rates of EOPD, family history and higher LEDD than non-carriers . In Hispanics, family history characterized G2019S carriers . In mixed ethnic group, the G2019S carriers were more likely to be female, have family history . And the carriers tended to develop akinetic-rigid motor phenotype, dyskinesia, have lower GDS scores, better response to levodopa (l-dopa), higher LEDD and smoking rates . There were not enough data to analyze G2019S-related clinical features in East Asians in PD . With respect to the NMS, G2385R carriers had higher Mini Mental State Examination (MMSE) scores than non-carriers . In terms of MS, G2385R carriers had lower Hoehn and Yahr rating (H-Y) than non-carriers . In terms of treatment, G2385R carriers received statistically higher LEDD  and were more likely to develop motor fluctuations than non-carriers . In terms of the other clinical features of G2385R, no statistically significant differences were observed between carriers and non-carriers.In total, 20 specific clinical features or rating scales belonging to eight classifications . There were not enough data to analyze G2385R-related clinical features in other ethnic groups in PD . This previous analysis further suggested that specific demographic features, such as female sex, EOPD, or family history may be used to select a targeted population for LRRK2 screening when conducting or planning research.With respect to demographic features, G2019S carriers were more likely to be female and have higher rates of EOPD and family history, while G2385R carriers were more likely to have family history. The reason that LRRK2-G2019S is associated with abnormal olfactory function as a result of effects on Lewy body pathology in the rhinencephalon  gene have been shown to be involved in L-dopa-induced dyskinesia  and the phenotypes of PD. Therefore, more comprehensive data are needed to perfect this meta-analysis. Second, heterogeneities existed among original studies in our pooled analysis; with a greater availability of original articles, it is advisable to adjust the pooled results and aim for more robust evidence. Third, co-occurrence and interaction between the factors could not be analyzed in the present meta-analysis, which may have confounded the results.LRRK2-associated PD among different variants, especially for G2019S and G2385R, was found to occur. We observed no distinct clinical features for R1628P or R1441G. The prominent clinical features of LRRK2-G2019S-related PD patients were female sex and higher rates of EOPD and family history. Further, G2019S carriers were less likely to be depressive and have olfactory dysfunctions, had better response to L-dopa and better quality of life than non-carriers. Furthermore, carriers tended to be treated with higher dose of L-dopa and were more likely to develop motor complications, such as dyskinesia and motor fluctuations. With respect to the clinical symptoms of G2385R carriers, this group was more likely to have more family history, lower H-Y rating, and was less likely to develop cognitive dysfunctions than non-carriers. High-dose L-dopa treatment and related motor fluctuations were more likely to occur in PD patients carrying the G2385R variant. Other clinical presentations did not feature G2019S or G2385R. No distinct clinical features were found in R1628P or R1441G variants. Our subgroup analyses in different ethnic group also presented with relevant clinical characteristics of PD patients with G2019S and G2385R but not of R1628P or R1441G.Clinical heterogeneity in LS and YZ have contributed equally to this work and are co-first authors. LS, YZ, and QS chose the topic and designed the experiments; LS, YZ, and QS performed the analysis; LS, YZ, QS, and BT analyzed the data; LS, YZ, and QS wrote the manuscript; HP, QX, and JG: data management and figure modification.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective: Chronic periaortitis (CP) is a rare fibro-inflammatory disorder that incorporates idiopathic retroperitoneal fibrosis, inflammatory abdominal aortic aneurysms, and perianeurysmal retroperitoneal fibrosis. CP is included in the spectrum of IgG4-related disease. Since CP patients rarely undergo diagnostic biopsies, serum IgG4 levels are often used to classify CP as IgG4-related. However, the clinical and prognostic significance of serum IgG4 in CP is unknown.Methods: We measured serum IgG4 in active CP patients and compared the clinical characteristics, response to therapy and outcome of patients with high and normal levels. We also tested the diagnostic significance of IgG4 by comparing its levels in CP patients, healthy and disease controls .Results: We studied 113 consecutive patients with active CP. Twenty-four (21.2%) had high serum IgG4 (>135 mg/dL). The demographic, laboratory, and clinical characteristics of patients with high and normal IgG4 were similar, and so were the rates of ureteral obstruction and the disease characteristics on CT, MRI, and 18F-FDG-PET. Patients with high IgG4 only had a higher frequency of extra-retroperitoneal fibro-inflammatory lesions (p = 0.005). There were no significant differences in response to therapy and relapses between the two groups. Serum IgG4 levels did not discriminate CP from controls.Conclusions: Serum IgG4 levels are high in a minority of CP patients and do not identify specific clinical or prognostic subgroups; only a higher frequency of extra-retroperitoneal lesions is found in high-IgG4 patients. Serum IgG4 levels do not help in the differential diagnosis between CP and its mimics. Chronic periaortitis (CP) is a rare disease characterized by a peri-aortic and peri-iliac fibro-inflammatory tissue that frequently entraps retroperitoneal structures such as the ureters, causing ureteral obstruction and renal failure. CP includes non-aneurysmal [idiopathic retroperitoneal fibrosis (IRF)] and aneurysmal forms  –3. It maCP can also arise in the context of a systemic fibro-inflammatory condition known as IgG4-related disease (IgG4-RD) , whose cPrevious studies have demonstrated that, based on histological findings, up to 50% of CP cases can be classified as IgG4-related –16. No sIn this study, we evaluated the IgG4 status  of a large cohort of CP patients at the time of active disease, and compared the clinical presentation, laboratory and imaging findings of IgG4-related and—unrelated cases based on serum levels. Next, we investigated whether serum IgG4 predict response to immunosuppressive therapy and risk of disease relapse. Also, we explored the role of serum IgG4 in the differential diagnosis of CP.In this retrospective observational study we reviewed the clinical records of all patients with CP referred to or diagnosed at our center from January 2010 through June 2017 and investigated the diagnostic, clinical and prognostic significance of serum IgG4. Inclusion criteria were: (i) diagnosis of CP ; (ii) avIn order to test the significance of serum IgG4 in the differential diagnosis between CP and other conditions, we measured IgG4 in age- and sex-matched healthy controls and in patients affected by aortitis (in the context of Takayasu or giant cell arteritis), Erdheim-Chester disease (ECD), retroperitoneal malignancies , and granulomatosis with polyangiitis ; all of these conditions were included because they can cause aortic or periaortic retroperitoneal inflammation and fibrosis , 20, 21.We retrieved data about patient demographics, clinical manifestations, and routine laboratory tests  performed at the time of active disease (first presentation or relapse). Serum IgG4 were considered high when >135 mg/dL. CT and MRI scans were independently reviewed by two experienced radiologists who were unaware of the patient IgG4 status. The following features were considered when CT and MRI scans were reviewed: localization of the soft-tissue mass, presence of hydronephrosis, aneurysmal aortic dilation, and maximal thickness of the retroperitoneal peri-aortic and/or peri-iliac tissue.We also analyzed, where available, data regarding treatment and outcome after therapy, including relapses during the post-treatment follow-up. For follow-up imaging studies, measurements  were performed using the same imaging technique (either CT or MRI) at the same peri-aortic or peri-iliac level chosen for the scans performed at baseline.Remission was defined as disappearance of disease-related symptoms , normalization or reduction to <30% of the baseline ESR and CRP levels, and hydronephrosis resolution without need of ureteral stents or nephrostomies; all of the above three criteria had to be met. Relapses were defined as an increase in disease activity represented by recurrent or new symptoms, hydronephrosis, or mass enlargement on CT or MRI , or by any combination of these.18F-fluorodeoxyglucose (FDG)–positron emission tomography (PET) performed at baseline and at the end of therapy, in order to identify any differences between the groups of patients based on the IgG4 status. 18F-FDG uptake was graded as follows: 0 = no uptake; 1 = lower than liver uptake; 2 = similar to liver uptake; and 3 = higher than liver uptake. The metabolic responses on FDG-PET were assessed according to PERCIST criteria as: complete (complete resolution of FDG uptake), partial (decline of at least 30% in FDG uptake), no response/stable metabolic disease , or progressive metabolic disease (increase of at least 30% in target lesion activity or appearance of new areas of uptake), as previously described . In each case, the antigen-antibody reactions were performed in immediately subsequent histological sections in order to explore highly comparable pictures. This immunohistochemical staining was carried out following the manufacturers' protocols; the negative control procedures omitted the primary antibody. The number of IgG4+ and IgG+ plasma cells was assessed in 10 microscopic fields for each of the tested slides using a light microscope  at a magnification of 400x (10x eyepiece and 40x lens). The IgG4+/IgG+ plasma cell ratio was calculated dividing the total number of IgG4+ plasma cells by the total number of IgG+ plasma cells, and multiplying this value by 100. The presence of an IgG4+/IgG+ plasma cell ratio >40% was considered indicative of IgG4-related CP .n (%). Differences between continuous variables were analyzed using the Mann Whitney test. Categorical variables were compared with Fisher's exact test. Correlations were analyzed using Spearman's rank correlation coefficient. All reported P-values are two-sided. P < 0.05 were considered statistically significant. The statistical analysis was performed using GraphPad Prism 5.Continuous variables are presented as median and interquartile range (IQR), while categorical variables as p = 0.01) and in CP patients than in patients with aortitis (p = 0.02) (p = 0.21) or GPA (p = 0.42). Receiver operating characteristic (ROC) analysis of the sensitivity and specificity of IgG4 in discriminating CP vs. healthy controls or aortitis patients showed poor reliability of this marker (One hundred and thirteen CP patients were included in the study. Twenty-four patients (21.2%) had high serum IgG4 (>135 mg/dL) and their median serum IgG4 level was 216 mg/dL (IQR 153–263.5). In addition to CP patients, 51 healthy controls, 41 patients with retroperitoneal malignancies or ECD, 22 patients with aortitis (secondary to Takayasu or giant cell arteritis), and 18 with GPA were tested for serum IgG4; all patients were tested at the time of diagnosis, before the start of treatment. Serum IgG4 levels were significantly higher in CP patients than in healthy controls ( = 0.02) . No stats marker .p = 0.09). The two groups did not differ in terms of comorbidities (cardiovascular or autoimmune diseases) or presenting signs or symptoms (p = 0.19). The main laboratory parameters were also similar between the two groups. Baseline serum IgG4 levels did not correlate with ESR, CRP, serum creatinine, hemoglobin, and leukocyte count (data not shown).The main demographic and clinical characteristics of the patients at disease onset are reported in symptoms ; the ratp = 0.005)  compared to patients with normal serum IgG4  . None of18F-FDG uptake grade between patients with normal and high IgG4 (p = 0.09).Sixty patients (53%) had undergone FDG-PET at baseline, before the start of treatment; of them, 47 (78%) had normal serum IgG4 levels and 13 (22%) had high levels. No differences were found in the igh IgG4 . In addiData about treatment and post-treatment follow-up were available for 104 patients, of whom 84 (80.8%) had normal serum IgG4 and 20 (19.2%) had high IgG4 at baseline . Sixty-op = 1.00). In addition, the median percent change in the mass thickness on follow-up CT or MRI (month 9 of treatment vs. baseline) did not differ between these two groups of patients (p = 0.63).Ninety patients (86.5%) achieved remission following treatment. The percentage of patients who achieved remission was similar between the groups with normal and high IgG4 levels   . The prep = 0.08). Only two patients (8.7%) had a tissue IgG4+/IgG+ plasma cell ratio >40%. One of them had high serum IgG4 levels and also extra-retroperitoneal localization of IgG4-RD (thyroiditis). The other one had normal serum IgG4 levels and no extra-retroperitoneal disease localizations.Retroperitoneal tissue samples were available from 23 patients, of whom 18 had normal serum IgG4 levels and five high levels. Nineteen biopsies (82.6%) exhibited an active inflammatory disease, with dense lymphoplasmacytic infiltrates admixed with fibrosis, while in four (17.4%) the inflammatory infiltrate was almost absent and fibrosis dominated. The median number of IgG+ plasma cells and of IgG4+ plasma cells infiltrating the tissue did not significantly differ between patients with normal or high serum IgG4, although it tended to be higher in the latter group , probably because of the small number of biopsies analyzed.No statistically significant correlations were found between serum IgG4 levels and plasma cell tissue infiltration (CD138+ cells) or between serum IgG4 levels and the IgG4+/IgG+ plasma cell ratio in the tissue  is quite similar. When we compared the clinical and laboratory characteristics of the two subgroups at presentation, we found analogous frequencies of disease-related symptoms, hydronephrosis, and acute kidney failure. Likewise, the levels of acute-phase reactants such as ESR and CRP were comparable, and so was the frequency of positive anti-nuclear antibodies and associated autoimmune diseases. In keeping with previous studies , 26, we The only phenotypic difference we observed with respect to the IgG4 status lies in the more frequent occurrence of extra-retroperitoneal fibro-inflammatory lesions in the high IgG4subgroup, thus suggesting that patients with a more pronounced IgG4 response are more likely to have a systemic disease than those with normal levels, as previously demonstrated , 14–16.We also evaluated response to treatment and relapses in the two subgroups. Most of our patients had received conventional therapies based on glucocorticoids, alone or in combination with immunosuppressants, with no significant differences in the distribution of these treatment regimens between the IgG4-related and—unrelated groups. Response to therapy was assessed as percentage of patients achieving remission   as well We classified patients with CP into IgG4-related and—unrelated based on serum IgG4 levels rather than on tissue evidence of IgG4-plasma cell infiltration. This is remarkably different from most of the previous studies, and it must be acknowledged that up to 40–50% of patients with biopsy-proven IgG4-RD have normal serum IgG4 –34. The We observed that only 21% of CP patients had high serum IgG4, which is lower than the percentage reported by other authors  , 25, 26;Despite these limitations of our study, and also considering its retrospective nature, we believe that the data we provide are quite solid, and that the large size of our cohort, the long follow-up, the accurate assessment of clinical presentation and outcomes are important strengths. It appears therefore that IgG4 cannot help differentiate subsets of patients with different disease severity or prognosis, thus new biomarkers are warranted. Certainly, based on our findings, treatment or follow-up strategies should not be tailored on the IgG4 status of CP patients.In conclusion, the clinical phenotype, inflammatory activity, response to treatment, and relapse probability of CP patients with high or normal serum IgG4 levels are comparable. Patients with high IgG4 levels are more likely to have multisystemic involvement, and should therefore be investigated more carefully for the occurrence of extra-retroperitoneal lesions.The datasets generated for this study are available on request to the corresponding author.FM: study design, data collection and analysis, manuscript drafting; RR, EA, CM, MU, PF, GB, and AP: data collection; MN and DC: data collection ; FP: statistical analysis; AAP: radiological evaluation- data collection; GE: study design, data collection; NP: data collection, manuscript drafting; AV: study design, data collection and analysis, manuscript writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Community-acquired pneumonia (CAP) remains a major cause of death worldwide. Mechanisms underlying the detrimental outcome despite adequate antibiotic therapy and comorbidity management are still not fully understood.To model timely versus delayed antibiotic therapy in patients, mice with pneumococcal pneumonia received ampicillin twice a day starting early (24 h) or late (48 h) after infection. Clinical readouts and local and systemic inflammatory mediators after early and late antibiotic intervention were examined.Early antibiotic intervention rescued mice, limited clinical symptoms and restored fitness, whereas delayed therapy resulted in high mortality rates. Recruitment of innate immune cells remained unaffected by antibiotic therapy. However, both early and late antibiotic intervention dampened local levels of inflammatory mediators in the alveolar spaces. Early treatment protected from barrier breakdown, and reduced levels of vascular endothelial growth factor (VEGF) and perivascular and alveolar edema formation. In contrast, at 48 h post infection, increased pulmonary leakage was apparent and not reversed by late antibiotic treatment. Concurrently, levels of VEGF remained high and no beneficial effect on edema formation was evident despite therapy. Moreover, early but not late treatment protected mice from a vast systemic inflammatory response.Our data show that only early antibiotic therapy, administered prior to breakdown of the alveolar–capillary barrier and systemic inflammation, led to restored fitness and rescued mice from fatal streptococcal pneumonia. The findings highlight the importance of identifying CAP patients prior to lung barrier failure and systemic inflammation and of handling CAP as a medical emergency.The online version of this article (10.1186/s13054-018-2224-5) contains supplementary material, which is available to authorized users. Streptococcus pneumoniae being the most prevalent causative pathogen [Community-acquired pneumonia (CAP) is a significant cause of morbidity and mortality worldwide, with pathogen , 2. Sincpathogen . Severe pathogen –6. Antibpathogen , 8. To ipathogen –14. HoweDifferences in survival of CAP patients receiving antibiotic therapy result from a wide range of contributors, which can be pathogen, drug or host related. The host’s immune response may aggravate detrimental pulmonary barrier failure and lung edema development –19. PartA detailed methodology and materials are presented in Additional file Female C57BL/6 N mice  were housed under specific-pathogen-free conditions. All animal studies have been ethically reviewed and approved by the State Office for Health and Social Services, Berlin, Germany.6 colony-forming units (CFUs) of S. pneumoniae serotype 3 in 20 μl PBS. Control mice received sham infection (PBS). Starting at 24 h or 48 h p.i., ampicillin (0.4 mg/mouse) or 0.9% NaCl (solvent control) was injected intraperitoneally every 12 h  suspensions were used to determine the bacterial burden. Supernatant (BAL fluid (BALF)) was frozen at − 80 °C. BAL cells were used for leukocyte differentiation.2 prior to CFU counting.Serial dilutions of EDTA-blood and BAL suspensions were plated onto Columbia agar with 5% sheep blood (BD) and incubated for 24 h at 37 °C under 5% COMice were sacrificed prior to histological analysis. Lungs were immersion-fixed in 4% buffered formalin, embedded in paraffin, cut into 2-μm sections and stained with hematoxylin and eosin (H&E) as described previously , 28. LunAST and urea levels were measured using Cobas-8000-C701 (Roche) by LABOKLIN .BAL cells were preincubated with blocking antibody and stained with anti-CD11c, anti-CD11b, anti-F4/80, anti-Ly6G, anti-Ly6C and anti-MHCII antibodies. Cells were measured and analyzed with FACS Canto II (BD) and FACSDiva software. Cell numbers were calculated using CountBright Absolute Counting Beads (ThermoFisher Scientific).Mouse serum albumin (MSA) levels in BALF and serum were measured by ELISA (BETHYL). The alveolar–capillary barrier permeability index was calculated as the ratio of BALF/serum MSA.Mouse cytokine/chemokine levels were measured in BALF and serum with the ProcartaPlex Custom Mix & Match according to the manufacturer’s instructions (AffymetrixBioscience). BALF levels of CXCL2 and CXCL5 were measured by ELISA (RD).Details for construction of the network are described in Additional file S. pneumoniae-infected mice at the start of therapy. Grouped analyses were performed using two-way ANOVA/Sidak’s multiple comparison test. p < 0.05 was considered significant.GraphPad Prism 6 software was used for statistical analyses. Survival curves were analyzed using the Kaplan–Meier method and log-rank (Mantel–Cox) test. The Kruskal–Wallis test/Dunn’s multiple comparisons test and one-way ANOVA/Dunnett’s multiple comparisons test were used for comparison to S. pneumoniae-infected mice were treated with ampicillin starting at different times p.i. Mice receiving ampicillin therapy starting 24 h p.i. showed a significantly higher survival rate (97.1%) than animals with late (48 h p.i.) therapy (20.4%)  Fig. . Clinica4%) Fig. , weight 4%) Fig. , decreas4%) Fig. . Hence,  rate 97.% than anNotably, antibiotic therapy failure was not responsible for different outcomes in survival. At 24 h and 48 h p.i., before initiation of an antibiotic regimen, all mice analyzed at these time points had developed bacteremia. Within 12 h both regimens reduced the mean bacterial load in the blood by over 99% and in the alveolar spaces by over 85%  to 90 times (CCL3) lower than prior late antibiotic therapy at 48 h p.i. Early antibiotic intervention prevented dramatic increases of G-CSF, CCL2 and CXCL1 in serum, with significant differences from the respective solvent-treated control groups. Early therapy did not lead to a significant change in CCL3 levels. After late antibiotic therapy, systemic CXCL1, G-CSF and CCL2 concentrations decreased, but no significant differences from the respective control groups were observed . This indicates that recruiting mechanisms like chemokine gradients were less affected by antibiotic treatment. However, pronounced antibiotic-dependent reduction in inflammatory mediators suggests that, contrary to cellular numbers, the activation state and effector functions of alveolar immune cells were dampened by antibiotic-governed bacterial eradication.An intact blood–air barrier is crucial for gas exchange, and fatal barrier breakdown during streptococcal pneumonia, leading to acute respiratory distress syndrome (ARDS), is well described (reviewed in –37). AccS. pneumoniae in pulmonary immune cells like macrophages and epithelial cells [The evident local and systemic inflammation might as well have occurred due to release of proinflammatory cytosolic components upon cell death processes. Necroptosis, a form of programmed necrosis, can be initiated by al cells , 39. In al cells , stable S. pneumoniae-infected rats that early and late antibiotic regimens similarly eliminated bacterial burdens whereas only early therapy helped to prevent lung damage. However, the authors started early therapy just 1 h p.i., an important difference to our protocol whereby infected mice already developed pneumonia prior to antibiotic treatment. We therefore conclude that our model represents the patients’ situation in a more compatible fashion.In severe pneumonia, only early antibiotic therapy, administered prior to barrier breakdown, prevented systemic inflammation, development of pleuritis, steatitis and elevated AST levels, which was followed up by restoration of fitness and rescue of mice from fatal outcome. Gracia et al.  have shoNonetheless, our mouse model remains a species-different approach to a highly complex human disease. It does not include therapeutic measures conducted in the ICU such as additional intake of fluids, (high flow) oxygen or macrolide therapy. Furthermore, laboratory mice which did not experience pathogen contact before generally show a modified immune response as humans do, for example, since they are missing mucosal memory cells . NeverthAs a consequence, we stress the importance of defining CAP as an emergency and to identify and treat CAP patients at risk prior to lung barrier failure and systemic inflammation , 14, 46.Our mouse model of antibiotic-treated CAP will aid in investigating how barrier-stabilizing interventions can prevent fatal disease progression in mice. Conclusively, proof-of-concept analyses indicating to which extent this mouse model adequately mirrors a significant range of patients must follow.By combining a mouse model of severe pneumococcal pneumonia with antibiotic treatment, we identified host factors of high relevance for successful therapy outcome. We were able to show that antibiotic treatment effectively reduces local pulmonary inflammation independent of treatment start and without beneficial effect on survival rates. However, despite being unable to reverse lung barrier dysfunction, lung edema and systemic inflammation once established, timely antibiotic intervention effectively prevented their development, significantly increasing survival. Handling CAP as an emergency along with new lung barrier stabilizing therapies might thus aid in finally lowering mortality rates.Additional file 1:Table S1. Total numbers of mice analyzed per group per analysis time point. Table S2. Total numbers of mice analyzed per group per histopathological analysis time point. Table S3. Murine pneumonia scoring system. Figure S1. Infection, antibiotic regimen and analysis time points (PDF 272 kb)Supplementary Materials and methods. Additional file 2:Figure S2. Body temperature and histopathological analysis. Figure S3. Early versus late antibiotic regimen at 60 h and 72 h p.i. Figure S4. Innate immune cell gating strategy. Figure S5. Innate immune cell analysis in BAL and blood. Figure S6. Chemokine and cytokine levels in BAL fluid. Figure S7. Histopathological analysis of lung inflammation. Figure S8. Cytokine–cell network. Figure S9. Cytokine, chemokine and urea levels in serum. Figure S10. Histopathological analysis of edema development (PDF 1638 kb)


Incorporating superabsorbent polymer (SAP), which has the abilities of absorption and desorption in concrete can achieve the effect of internal curing. The influences of the volume, particle size and ways of entrained water of SAP on the workability, compressive strength, shrinkage, carbonation resistance and chloride penetration resistance of concrete were analyzed through the macroscopic and microscopic test. The results show that pre-absorbed SAP can increase the slump of the mixture, but SAP without water absorption and pre-absorbed SAP with the deduction of internal curing water from mixing water can reduce the slump. The improvement effects of SAP on compressive strength of concrete increase gradually with the increase of age. Especially from 28 days, the compressive strength of concrete increases obviously. At later age, the compressive strengths of SAP concrete under natural curing environment exceed the strength of reference concrete under natural curing environment and nearly reach the strengths of reference concrete under standard curing environment. SAP effectively reduces the shrinkage of concrete, improves the concrete’s abilities of carbonation resistance and chloride penetration resistance. The microscopic test results show that SAP can effectively improve the micro structure and make the pore structure refined. When SAP is added into concrete, the gel pores and small capillary pores are increased, the size of big capillary pores and air pores are reduced. Concrete is a kind of porous, multiphase at all scales, and heterogeneous complex system . The degThe internal curing method has been studied for some years which may be an effective way to ensure the hydration process in concrete. Water is introduced into the concrete in advance by absorbent material like superabsorbent polymer (SAP), so that some of the water acts as internal curing water from the inside out, in order to achieve the effect of internal curing in the hydration process of the cementitious material. When the relative humidity inside the cement matrix gradually drops, the absorbent material can slowly release water to supplement the water consumption of cement hydration. The hydration process continued to generate hydration products to fill the pores of cement paste, reduce the micro cracks in cement matrix, and relieve the autogenous shrinkage and drying shrinkage. Then the strength and durability of the concrete can be improved ,8,9.SAP is a common water absorbent material with excellent water absorption and desorption capacity. In recent years, relevant research work on SAP concrete has been carried out internationally, and the influence of SAP on the construction performance, strength and durability of concrete has been studied ,11. It hThe incorporation of SAP increases the porosity of concrete which may affect the durability of concrete. However, SAP with the absorption and desorption properties can change the water distribution in concrete. So that the process of cement hydration is promoted, the microstructure of cement matrix is improved, and the adverse effects of voids released by SAP can be offset. Hasholt and Jensen  concludeIn this paper, the influence of the volume, particle size and ways of entrained water of SAP on the workability, compressive strength, shrinkage, carbonation resistance and chloride penetration resistance of concrete have been experimentally studied. Based on the microscopic test results of mineral compositions, pore structures and morphology of hydration products, the mechanism of SAP on the performance of concrete has been studied.An ordinary Portland cement  was used. The physical and chemical compositions of the cement are presented in According to the specification for mix proportion design of ordinary concrete (JGJ55-2011) , the speMixing program of materials was prepared as follows: (a) the raw materials which is taking as dry state should be weighted accurately. SAP was divided into non-absorbed and pre-absorbed of 10 times the weight of SAP, respectively; (b) The fine aggregate, coarse aggregate, cement and fly ash were poured into the wet mixing bucket and mixed 30 s to ensure the uniformity; (c) Water reducing agent and concrete mixing water were added to the mixture and mixed for 100 s; (d) Non-absorbed SAP or pre-absorbed SAP were added to the mixture and mixed for 60 s to ensure that SAPs were distributed homogenously in the mixture.All specimens were casted and kept indoors for 24 h, and then demoulded and numbered. The natural curing and standard curing methods were used for reference concrete specimens, which represent PC-Z and PC-B respectively. Only natural curing method was used for internal curing concrete specimens, which represent S. The natural curing concrete specimens were placed in the outdoor environment. The standard curing concrete specimens were stored in the standard curing room where the temperature was (20 ± 2) °C and relative humidity was above 95%.The workability test was carried out according to the standard for test method of performance on ordinary fresh concrete (GB/T50080-2016) , and theThe compressive strength test was carried out according to the standard for test method of mechanical properties on ordinary concrete (GB/T50081-2002) . A totalThe shrinkage test was carried out by non-contact method according to the standard for test method of long-term performance and durability of ordinary concrete (GB/T50082-2009) . A total2 could be achieved. The specimens were positioned in an accelerated carbonation chamber which was set at (20 ± 2) °C, (70 ± 5)% of relative humidity and (20 ± 3)% of CO2. The carbonation depths were measured at 3, 7, 14 and 28 days using a phenolphthalein solution on the exposed surfaces of split specimens.The carbonation resistance test was carried out according to the standard for test method of long-term performance and durability of ordinary concrete (GB/T50082-2009) . A total2 solution. Subsequently, the specimens were placed inside rubber cell. The top surface of the specimens was filled with a 0.3 mol/L NaOH anodic solution and the bottom surface of the specimens was filled with a 10% NaCl cathodic solution. Rapid chloride transport (RCM) tests were then performed. After the test, the specimen was taken out to flash the surface, and then cut along the direction of the axis parallel to the specimen. The fracture surface was sprayed with silver nitrate (0.1 mol/L AgNO3) solution. Finally the depth of chloride penetration after 15 min was measured and the chloride migration coefficient was computed.The chloride penetration resistance test was carried out by rapid chloride ions migration coefficient method (RCM) according to the standard for test method of long-term performance and durability of ordinary concrete (GB/T50082-2009) . A totalThe mercury intrusion porosimetry (MIP) test, X-ray diffraction (XRD) test and scanning electron microscope (SEM) test were carried out to research the microstructural characteristics of concrete. After being cured 56 days, the micro-specimens were taken from the center of samples and placed into anhydrous alcohol to stop cement hydration. The micro-specimens were placed for 24 h in an oven at 60 °C. Then the microscopic experiments were carried out. Mercury intrusion porosimetry test was carried out by using AutoPore IV 9500 automatic mercury intrusion apparatus  and the measured pore diameters was 0.003–1000 µm. For X-ray diffraction test, the micro-specimens were ground into powder in an agate bowl until the particle size was below 0.08 mm. After vacuum drying, the X ray diffraction test was carried out. The tests were performed over a Braggs angle (2θ) range of 5–60°. The fracture surfaces of the micro-specimens were sputtered a layer of gold. The micro-specimens were observed by scanning electron microscope at different degrees of magnification.The influence curves of different ways of entrained water and volume on concrete slump for two particle sizes of SAP is shown in As can be seen from With the increase of ages, the differences of compressive strengths between SAP concrete and the reference concrete decrease gradually which is shown in As can be seen from In the later age, the compressive strength of the internal curing concrete is greatly affected by the particle size of SAP. The strength of SAP-b concrete is generally higher than that of SAP-a concrete, which shows that SAP-b is more suitable than SAP-a in playing the role of internal curing. This is because the change of the particle size of SAP has very much to do with the water absorption and desorption properties , and theCompared with the reference concrete, the shrinkage value is slightly decreased when non-absorbed SAP or pre-absorbed SAP with the deduction of internal curing water from mixing water is added. However, the total shrinkage of pre-absorbed SAP is reduced remarkably. It indicates that pre-absorbed SAP can better improve the shrinkage of concrete. This is because with the change of ways of entrained internal curing water, the storage form of SAP changed. Especially the pre-absorbed SAP can effectively store additional water and improve the effectiveness of internal maintenance. In the process of concrete hardening, SAP release water to the capillary pore of cement matrix in order to extend the period of saturation as well as alleviate the shrinkage of concrete. The added internal curing water and the absorption of free water from the mixture have different effects on the internal humidity of the concrete, which results in different degrees of shrinkage relieving.− and enhances the resistance to chloride ion penetration.At the same time, the chloride ion diffusion coefficient is greatly affected by the ways of entrained water of SAP. And the Chloride ion diffusion coefficients of S-10K, S-0 and S-10 decrease by 27%, 17% and 15% respectively in different ways of entrained water. This shows that the diffusion coefficients of chloride ion decrease with the decrease of the effective water binder ratio. In addition, the SAP without water absorption and the pre-absorbed SAP with the deduction of internal curing water from mixing water can reduce the total pore volume and porosity of concrete, so that the compactness of concrete is improved. Other scholars ,13,37 fo2 and ettringite in cement-based materials is carried out by the XRD-Rietveld full spectrum fitting method. Compared with the PC-Z reference concrete, the content of Ca(OH)2 and ettringite in the internal curing concrete is generally reduced by more than 40% by adding proper amount of SAP. It shows that the incorporation of SAP provides fly ash and Ca(OH)2 with water that needed for second hydration reaction. This effectively promotes the formation of hydration products and makes the Ca(OH)2 of the interface area of aggregate and mortar reduced greatly. A large amount of stable hydrated calcium silicate is generated, which is filled in the micro pores of the cement mortar, so that the Ca(OH)2 is surrounded by the impermeable C-S-H gel, and the quantity and the size of the Ca(OH)2 crystal are reduced. It also reduces the formation of expansive salts, which is called ettringite. The reduction of the number and size of Ca(OH)2 crystals and ettringite enhances the strength of cement paste, improves the microstructure of matrix, the strength and compactness of concrete. The SEM image test shows that it is difficult to find Ca(OH)2 crystal in the cement matrix mixed with SAP, which further confirms the fact that the content of Ca(OH)2 decreases in XRD test.The quantitative analysis of XRD test on the component of SAP concrete at 56 days age is shown in 2 crystal is more. The surface hydration reaction of fly ash is low and there is less attachments on the surface. The surface of concrete is not dense and the structure is relatively loose. Compared with the PC-Z reference concrete, the volume of 0.2% SAP-b concrete with different ways of entrained water is shown in 2 and ettringite enrichment are found. There is a dense product C-S-H gel of the second hydration. And the fly ash is wrapped with a large number of dense gels. The internal curing effect of SAP contributes to the second hydration reaction of fly ash as well as improving the compactness of concrete microstructure. This is because the SAP absorbs additional internal curing water before the addition to mixture or absorbs free water after the addition to mixture. And then SAP gradually releases water, which needed for the second hydration reactions within the concrete. This makes the active SiO2 and Al2O3 of fly ash well involved in the hydration reaction, and consumes a large amount of Ca(OH)2. It can contribute to the formation of dense and uniform gel, improve the compactness of concrete as well as improve the durability and mechanical properties of SAP concrete. Compared to S-10 and S-0, fly ash microspheres in S-10K are wrapped with a large amount of C-S-H gel, which is the production of second hydration reaction. It explains that fly ash can play a better role in the pozzolanic effect, and further explains the reasons for the increase of compressive strength and durability.With the incorporation of SAP, the slump of concrete is influenced by SAP volume and different entrained ways of internal curing water. The pre-absorbed SAP increase the slump of mixture. And with the increase of SAP volume, the slump increases gradually. But the SAP without water absorption and the pre-absorbed SAP with the deduction of internal curing water from mixing water can reduce the slump of mixture. And with the increase of SAP volume, the decreasing range increased.At early age, the concrete mixed with SAP does not have effect on internal maintenance. Then the internal curing effect of SAP gradually begins to work. At later age, the internal curing effect of SAP is more obvious, and the compressive strength of the internal curing concrete with SAP increases continuously. In addition, SAP with the particle size of 150–180 μm has an optimum particle size range to improve the compressive strength of concrete.The SAP can alleviate the total shrinkage of concrete. When the non-absorbed SAP or pre-absorbed SAP with the deduction of internal curing water from mixing water are added, the total shrinkage value of SAP concrete decreases slightly. However, the effect of reducing total shrinkage of concrete by adding pre-absorbed SAP is remarkable.SAP can significantly improve the abilities of carbonation resistance and chloride penetration resistance of concrete. And different ways of entrained water have great impact on the abilities of carbonation resistance and anti-chloride erosion of concrete. When the pre-absorbed SAP with the deduction of internal curing water from mixing water is added, the abilities of carbonation resistance and anti-chloride erosion of concrete are relatively better.When SAP is added into concrete, the gel pores and small capillary pores increase, the size of big capillary pores and air pores are reduced. The pre-absorbed SAP can increase the porosity and total pore volume of concrete. But the SAP without water absorption and the pre-absorbed SAP with the deduction of internal curing water from mixing water can reduce the porosity and total pore volume of concrete.The influence of volume, particle size, and ways of entrained water of SAP on the workability, compressive strength, shrinkage, carbonation resistance and chloride penetration resistance of concrete has been studied through macroscopic and microscopic test. Based on mercury intrusion porosimetry (MIP) test, X-ray diffraction (XRD) test and scanning electron microscope (SEM) test results, the pore structure, morphology of hydration products and mineral compositions of concrete have been further analyzed, and the mechanism of SAP on the performance of concrete has been studied. The main conclusions are as follows:


Overuse or inappropriate use of antibiotics in agriculture has been implicated in the development of antibiotic resistant bacteria, a significant and growing public health threat. In a previous study, we found that Missouri goats had a higher percentage of antibiotic residues at slaughter than predicted based on the national average, so we undertook this study to understand contributing factors. As farmers are typically the ones administering antibiotics to their animals, we set out to investigate Missouri goat farmers’ knowledge and attitudes regarding antibiotics, veterinarians, and antibiotic resistance using qualitative research interview methods. Our aims were to determine circumstances leading to farmers’ administration of antibiotics, farmers’ decision process resulting in the use of antibiotics, the role of veterinarians, and farmers’ perceptions about antibiotic resistance. The following themes emerged: how farmers detect illnesses in individual goats, herd health management, where farmers obtain antibiotics, and farmers’ thoughts about antibiotic resistance. Our findings highlighted the need for more emphasis on goat health management during veterinary education and the need for improved working relationships between veterinarians and farmers to promote appropriate antibiotic use and prevent the emergence of antibiotic resistant bacteria.Use of low dose, prophylactic antibiotics contributes to the emergence of antibiotic resistant bacteria. In one study, goat meat in Missouri was found to have a higher percentage of antibiotic residues at slaughter than the national average, so we attempted to identify factors related to goat production that may contribute to this issue. Using the knowledge, attitude, and behavior (KAB) model, we interviewed 11 Missouri goat farmers about factors affecting antibiotic use. Most of the farmers did not have specific protocols for managing illnesses and only relied on veterinarians for major health issues. Many felt veterinarians lacked knowledge about goat medicine so instead relied on other farmers’ or their own experiences for treatment modalities. While most agreed that antibiotic resistance was a concern, only 4 of the 11 indicated that they only used antibiotics when prescribed by the veterinarian. Veterinarians should be relied on and valued for their medical expertise, but they are not always being utilized in this manner. Therefore, veterinary education should emphasize goat health management to a greater extent than it currently does, and soft skills to build collaborative relationships with farmers should be taught to promote preventative health measures and more judicious use of antibiotics. Animal production practices have been cited as major contributors to the emergence of antibiotic resistant bacteria, contributing to over two million illnesses and 23,000 deaths annually ,2. This The beef, swine, and poultry industries have had increased scrutiny and oversight of antibiotic use over the years, but the goat industry has not received similar attention despite its increasing market share. According to the 2013 Red Book, which includes the results of the 2011 meat inspections performed by the United States Department of Agriculture’s (USDA) Food Safety Inspection Service (FSIS), goat meat was reported as having the highest percentage of drug residues compared with other types of meat . These lVeterinarians play an important role in preventing the overuse of antibiotics in goats. The Netherlands saw a 56% reduction in antimicrobial use for farm animals from 2007 to 2012 when it made efforts to support veterinary and farmer relationships, including setting up a task force comprised of veterinarians, farmers, and other stakeholders . HoweverThe knowledge, attitude and behavior (KAB) model suggests that behavior  follows knowledge and attitudes . For exaIn the present study, we investigated farmers’ knowledge and attitudes about antibiotics, veterinarians, and antibiotic resistance with the goal of understanding their possible role in the burden of antibiotic-positive goat carcasses in Missouri. Our specific aims were to determine why farmers administered antibiotics to their herd, their decision process leading to the use of antibiotics, the role of their veterinarians, and farmers’ perceptions about antibiotic resistance.This study was approved by the Institutional Review Board at Saint Louis University (protocol #27522). Goat farmers who consented to participate were interviewed using a focus group-tested questionnaire and interview guide, developed to capture (1) age, gender (2) flock size, (3) whether antibiotics were used and, subsequently, (4) reason using antibiotics, (5) role of veterinarian in the decision to use or not to use antibiotics and (6) their thoughts on antibiotic resistance.http://www.agrilicious.org/ was used to find farmers’ contact information in Missouri since it has a database of farmers who can be identified by state, species, and products sold  up to the feed dish. The ones that don’t run up to the feed dish. The last ones in the line up to the barn are the ones that get checked [for illnesses].”(F5)“We check the animals daily and if we have an animal that is not acting the way it should we do not carry. We check it over and narrow down the cause of the problem and treat accordingly.”(F8)The first emerging theme found among the farmers was how they identify illnesses in goats. Most farmers did not have a specific protocol for determining the health of individual goats and any need for antibiotics. However, they felt confident in identifying illnesses. Several mentioned that they knew there was a problem with their goats when they would notice a change in their behavior, especially around meal time.“We do a weekly check, like a 6-point binary check on the animals. We try to do it every week, sometimes it’s every 2 weeks, but we check basically the eyes for anemia and/or discharge. We check, well I sort of start at the other end, the tail, for scours. And then we check the coat condition, body condition, check the eyes, check the nose, and then check the jaw for any edema, so…it sounds lengthy but it’s a quick pass over on each animal.”(F4)The farmers stated that after they notice something awry with the goats, they further investigate to determine what the main problem is. One farmer mentioned that when the goats are acting unusual, he checks their ears, head, tail, temperature, fecal sample, and entire body. Another farmer did mention that he performs a 6-point binary check on the goats on a regular basis:“Not much, I mean truthfully… When I call he knows it must be something horrible, a blood transfusion or something…I don’t call him every time I think something needs an antibiotic, no.”(F5)Another emerging theme found among the farmers was herd health management. The first subtheme identified was the role of the veterinarian in farm management and prescribing antibiotics. All the farmers stated that they had access to a veterinarian who could come to the farm and provide necessary treatment for their herds. However, it was noted by several farmers that they contact a veterinarian because of severe problems and not for minor infections. When asked to describe the role of the veterinarian on the farm, one farmer replied:“Um…it generally costs nearly as much as a goat’s worth to call in a veterinarian out here, so unless I’ve got a problem with the herd overall, then it would be a zero.”(F3)Cost was a subsequent subtheme that emerged from the interviews. Several farmers noted that service costs were a barrier for getting the veterinarian to the farm.One farmer did mention, however, that cost would never prevent getting appropriate care for the goat. This farmer mentioned that she is a member of an animal welfare approved program, so understands the importance of the veterinarian being involved in the treatment of the animals.“Uh, yeah…the way I would phrase that is I trust her to do anything structural, or you know, that she would have to do to get in there and save the animals life, but I don’t think that they have the information on goat care. I really like our veterinarian but I don’t really think she believes that dewormer resistance is a problem, so…”(F4)“Int: Do you trust your veterinarian’s recommendations?F5: Certainly.Int: So you use antibiotics…[when prescribed by a veterinarian]?F5: I combine it, but I combine it with my experience as well, because I have more experience, you know, but he [the veterinarian] has sometimes some really good ideas or alternative thoughts, you know, which, well part of our relationship is a good give and take.”(F5)Trust of veterinarians was another subtheme that came up. Overall the farmers noted that they trust their veterinarian. Most farmers felt that they could trust their veterinarian’s recommendations with regards to antibiotic use, although most veterinarians are not always contacted before administration of antibiotics.“That would probably just be myself. I use one of the vets a little bit. There’s not a lot of veterinarians that have a lot of knowledge about goats in our area, so it’s really hard to have a vet by your side to work with you on your farm and prescribe medications accordingly as I see fit for my goats. So, I think mostly I do most of the intervention for my farm… I’m not saying that I know more than a veterinarian because that’s not true at all, but I think I’m able to better care for them in my point of view than what they can help me with as of right now.”(F10)Another subtheme that emerged during interviews was farmers’ perception of how knowledgeable veterinarians were about goat herds compared to farmers. While farmers felt that they could trust veterinarians in regards to antibiotic recommendations, the majority did not feel that veterinarians had as much knowledge regarding goats as farmers do. Therefore, farmers felt that they could rely on their own knowledge and experience regarding treatment of their goats rather than reach out to the veterinarian. Regarding overall herd health, one farmer said that she relies on herself more than on the veterinarian:In addition, some farmers relied on other farmers and social media, such as Facebook groups where veterinarians respond to general questions, for their antibiotic recommendations rather than seeking out traditional veterinary care.“Well because we’re a participant of animal welfare approved program, which is now a greener world. We have to log every time we use antibiotics, in a book, with the date and the lot number. We have to double the amount of time for withdrawal period should we decide to slaughter that animal. So we just, you know, it’s all in the book.’’(F9)“Int: Can you describe some disadvantages [of using antibiotics]?F7: We only use them for sick animals. Yeah, disadvantages is withdrawal time. Obviously we have to destroy milk for a period.” (F7)When it comes to specific knowledge of drug withdrawal times, which is the minimum time allowed between the last dose of a medication being administered and slaughter or milking, 3 of the farmers indicated that they felt comfortable in their knowledge.“You know, I’m not going to say a situation couldn’t come up [where I would use antibiotics not prescribed by a veterinarian], but I can’t think of any other reason, other than a large wound, I can’t think of any other reasons I would be using them [antibiotics].”(F3)While farmers do not feel that their veterinarian is always knowledgeable about goats, 4 of the 10 farmers indicated they only use drugs prescribed by the veterinarian. The other farmers were not concerned about using drugs not prescribed by a veterinarian.“The only two I really use is Pen-G and Oxytetracycline and I keep those on hand in the refrigerator, when you need them you need them. Pen G is a good antibiotic. It’s good for gram negative, gram positive, and it works on 99% of everything. If I don’t get a good response with the Pen G then I’m calling my vet.” (F6)Several indicated that they have certain antibiotics on hand to use on a case by case basis.“No, I don’t really have any concerns. I’ve used them [antibiotics] in the past, and I feel that my knowledge about them [antibiotics] is pretty good.”(F11)When probed further about their use of antibiotics without a prescription, some farmers indicated they chose the particular antibiotics based on their own experiences. A couple even indicated no concerns when using antibiotics in this manner given their personal expertise in their herd management:“Well I mean if it has to be a veterinarian to give me the medication, like Nuflor and stuff like that, I’ve gotten bottles of that to use on my farm, so I guess it would be prescribed. They prescribe that to my farm, for me to use on my farm, I can’t just get that at a feed store or anything like that. It’s a little more stronger antibiotic, a little more aggressive than Penicillin, especially when there’s a goat with mastitis or a serious respiratory, you really need to act fast on those illnesses that you don’t have a lot of time with.” (F11)Speed of the illness progressing was mentioned by a couple of the farmers as a reason for administering the antibiotics before getting a veterinarian consult.All of the farmers indicated they only medicated the ill goat or goats.“F6: I order some through the mail and feed store and the vet.Int: Do you need a prescription for the ordering online or feed store?F6: No, I don’t get prescription antibiotics, I just get them at the feed store, because you can, so I just go to the vet and get any medicines that I can’t pick up on my own.”(F6)While many farmers interviewed relied on veterinarians for the specific drugs used on their goats, they also felt confident in getting antibiotics at a feed store or online. Only 4 of the 8 farmers indicated that they only use drugs prescribed by veterinarians.“I think they [farmers] do. Yeah, the last line, we’re farmers and the ones who actually administer the antibiotics, I do think that they do play a huge role in it.”(F7)The final emerging theme found among the farmers was their attitudes regarding antibiotic resistance. When asked about the disadvantages of antibiotic use, one farmer indicated that resistance was his biggest fear. All of them feel that antibiotic resistance is a problem and that farmers may play a role in the emergence of resistant bacteria.“Now there are some big-ticket operations and I’m sure, you know, that’s a whole different dynamic. That’s, they couldn’t, probably couldn’t function, couldn’t keep up their management practice without it [antibiotic administration].”(F5)Many indicated that they felt that the larger farms may have a harder time managing their goats’ health without using antibiotics.Since antibiotic resistant bacteria are becoming an increasing concern with livestock, it is prudent to use antibiotics only when necessary ,24,25. TIdentification of illnesses in individual animals was the first theme that emerged. Only one farmer had a specific protocol for determining the health and well-being of his animals. All the same, farmers felt that they had intimate knowledge of their goats’ behaviors and the ability to identify abnormalities that may indicate an illness: lack of appetite (as evidenced by being last to the food dish), limping, and any other visible problems. Farmers indicated that they felt comfortable acting independently on this knowledge. While all of the farmers regarded their goats as healthy and felt that they had a good understanding of herd health, protocols to assess health may help in preventing infections and thus reduce the use of antibiotics . A formaWhile all farmers had access to a veterinarian, they tended to rely on the veterinarian only for major health issues. This is consistent with the USDA National Animal Health Monitoring System (NAHMS) Goat 2009 study that found only a third of farming operations had consulted a veterinarian in the last year . Cost waThe biggest concern that emerged from the interviews, though, was the lack of knowledge or perceived knowledge of the veterinarians about goat medicine. This may seem a reasonable concern, as in the United States there is limited time spent in veterinary school focusing on small ruminants . AnotherDue to perceived lack of knowledge of their veterinarian, many of the farmers indicated that they relied either on their own experiences, Facebook groups , or other farmers for the care of their goats as well as their information regarding antibiotic use. In particular, many of the farmers stated that they felt they had the knowledge needed to identify the illnesses, understood how they needed to treat problems, and felt confident in their usage of antibiotics based on the instructions on the bottle and prior experiences. This is consistent with research from Kaler and Green , where tAn emerging theme coming from the interviews was where the farmers were able to get antibiotics. Even though the United States Food and Drug Administration (FDA) has repeatedly recommended that veterinarians be involved in all antibiotic prescription and administration protocols, most antimicrobial drugs can be easily purchased over the counter . Only foMost of the farmers interviewed felt that antibiotic resistance was a problem to which farmers contributed. However, the interviewees also felt that they did not use antibiotics very much compared to larger farms (over 200 goats). The farmers felt that their ability to care for their herd more closely and their low use of antibiotics prevented the emergence of antibiotic resistance on their own farms. To ensure antibiotic usage is low, the aforementioned formalized herd health checklist could help.This study helped ascertain some of the reasons for use of antibiotics by goat farmers. The KAB model pointed to two areas to address: knowledge of antibiotic use  for farmers as well as veterinarians  and attitude towards veterinarians. Overall, they felt they had good knowledge of the health of their goats. They also felt confident in their knowledge in the antibiotics used on their farms. While the farmers interviewed said that they trusted veterinarian recommendations, they did not feel it always necessary to contact the veterinarian when a problem arose, indicating that their behavior is not congruent with their attitude. There is a consensus among veterinarians that the information provided about small ruminant husbandry and medicine in veterinary schools is limited . TherefoWhile this study discovered valuable findings, there were several limitations. The sample size was small and did not include large-scale producers. The farmers interviewed indicated that large-scale operations were most likely to misuse antibiotics, as has been shown for other species ,36, so tOverall, the interview process was helpful in determining farmers’ feelings towards antibiotic use, about veterinarians, and the role of goat farming in the emergence of antibiotic resistant bacteria. Veterinarians should be the experts for farmers to call on to prevent and treat illnesses. Therefore, education about goat farming and tools for relationship building with farmers should be covered in veterinary school and continuing education courses. A productive client–veterinary relationship can boost preventative care and educate the farmers on best practices to reduce harmful use of antibiotics for minor problems. In addition, antibiotics should not be available for purchase over the counter, as per current recommendations by the FDA. In the future, we hope to get the veterinarians’ perspective on their role in the combat against the emergence of resistant organism by food animal production facilities.


Precision plant genome engineering holds much promise for targeted improvement of crop traits via unprecedented single-base level control over the genetic material. Strigolactones (SLs) are a key determinant of plant architecture, known for their role in inhibiting shoot branching (tillering).Oryza sativa) for targeted disruption of CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), which controls a key step in SL biosynthesis. The ccd7 mutants exhibited a striking increase in tillering, combined with a reduced height, which could be rescued by application of the synthetic SL analog GR24. Striga germination assays and liquid chromatography–mass spectrometry analysis showed that root exudates of ccd7 mutants were also SL deficient.We used CRISPR/Cas9 in rice  contains supplementary material, which is available to authorized users. Technologies that facilitate efficient, robust, and precise engineering of the plant genome can be used for targeted improvement of crop traits, and will pave the way for increasing plant yield and improving food security . Plant aGenome engineering requires molecular scissors capable of making precise double strand breaks (DSBs) in the genome , 49. SucStriga perceive SLs as a germination signal ensuring the presence of a nearby host ..carotenoase DWARF in rice recursor .Fig. 1OsThe first step in SL signal transduction is the binding of SL to the receptor, DWARF14 (D14) in rice, an α/β-hydrolase that hydrolyzes SL ligands and forms a covalent bond with one of the two hydrolysis products (D-Ring) , 24, 55.OsCCD7 point mutation (C-T) in the Nanjing 6 background, htd1 (high-tillering and dwarf 1), causes a dwarf phenotype and production of a high number of tillers [OsCCD7. In this work, we report the engineering of SL biosynthesis by CRISPR/Cas9-mediated mutation of OsCCD7. Our data provide a proof-of-principle for translational application of knowledge on SL biosynthesis for improvement of plant architecture.Targeted mutation of key enzymes in SL biosynthesis or perception can be used to engineer plant architecture in ways that improve yield. Moreover, in contrast to traditional, time-consuming approaches involving breeding natural alleles into elite varieties, genome editing provides a targeted, precise and rapid method to improve key plant traits in agronomically relevant, locally adapted varieties. OsCCD7 catalyzes a key step in SL biosynthesis, and an  tillers , 59. ThiOryza sativa L. ssp. japonica cv Nipponbare was used for all experiments. The pRGEB32 vector was used for callus transformations [OsUbiquitin promotor, and the gRNA was expressed as a polycistronic tRNA-gRNA under the OsU3 promoter. The OsCCD7 (LOC_Os04g46470) was used as a target locus. Two gRNAs were designed and transformed independently. gRNA-1 was designed to target the genomic sequence 289 to 308 bp (5′- ACCTACTACCTCGCCGGGCCGGG-3′); the underlined GGG represents the PAM sequence. gRNA-2 was designed to target the genomic sequence 2416 to 2435 bp (5′- AAGAACCTCACTTTTCCAATGGG-3′); the underlined GGG represents the PAM sequence. The potential off-targets were predicted using Cas-OFFinder [d3–1 and d17–1 mutants, in Shiokari background , were used for comparison.rmations . The expAgrobacterium-mediated rice transformation was performed as described previously [eviously . Transge− 2 s− 1) for 7 days. One-week-old uniform seedlings were selected to grow in a microfuge tube, fixed on top of 50-ml tubes  filled with half-strength modified Hoagland nutrient solution [d3, and d17 were measured at final harvest.Rice seeds were surface-sterilized with 2.5% sodium hypochlorite for 10 min. The seeds were then washed thoroughly with sterile MilliQ water and imbibed in water for 2 days at 30 °C in the dark. The pre-germinated seeds were shifted to 90-mm Petri dishes on a filter paper moistened with 5 ml ½-strength Murashige and Skoog medium . The seasolution . After 1Striga bioassays. For this purpose, SPE C18 columns (Grace Pure) was pre-conditioned by solvation (6 ml of methanol) and equilibration (6 ml of water). After adding the internal standard D6–5-Deoxystrigol (0.672 ng per 50 ml root exudate), root exudates were run through the preconditioned SPE C18 column. After washing with 6 ml of water, SLs were eluted with 5 ml of acetone. The SL fraction (acetone-water solution) was concentrated to SL aqueous solution (∼1 ml), followed by extraction with 1 ml of ethyl acetate. Then 750 μl of SL-enriched organic phase was transferred to a 1.5-ml tube and evaporated to dryness. The sample was re-dissolved in 100 μl of acetonitrile:water  and filtered through a 0.22-μm filter for LC-MS/MS analysis. SLs were analyzed using HPLC-Q-Trap-MS/MS with MRM mode. Chromatographic separation was achieved on an Acquity UPLC BEH C18 column . Mobile phases consisted of water:acetonitrile  and acetonitrile, both containing 0.1% formic acid.One week old rice seedlings were established as described above and grown hydroponically in a growth cabinet with half-strength modified Hoagland nutrient solution under normal phosphorus (Pi) supply for 1 week. Then, rice seedlings were kept in a Pi-deficient nutrient solution for another week. On the day of root exudate collection, rice seedlings were first refreshed with Pi-deficient Hoagland nutrient solution for 6 h, and root exudates were then collected from each tube. SLs were then extracted from root exudates for LC/MS-MS analysis and Striga hermonthica seed germination bioassay was conducted as described previously . The Striga seeds were preconditioned for 10 days at 30 °C under moist conditions. The pre-conditioned Striga seeds were supplied with 50 μl acetone-free SL extract collected from the root exudates for each mutant line as described above. Wild type and the d17 mutant were included as positive and negative controls, respectively. After SL application, Striga seeds were incubated at 30 °C in the dark for 2 days. Germinated (seeds with radicle) and non-germinated seeds were counted under a binocular microscope to calculate germination rate (%).The OsCCD7 to generate variants for translational research and to enhance our understanding of the diverse functions of this protein. The OsCCD7 gene (LOC_Os04g46470) has 7 exons encoding a protein of 609 amino acids  including deletions of 1–27 bp. Similarly, for sgRNA-2, all of the plants were bi-allelic with indels. Some plants, however, exhibited only monoallelic mutations. These data show the high efficiency of targeted mutagenesis for CCD7 by gRNAs targeting the 1st and 7th exons. We used the nomenclature d17-like (dl) for mutants produced by sgRNA-1. Two mono-allelic mutants, dl1 and dl2 with one- and two-bp deletions, respectively, were used for further studies . Seven bi-allelic mutants hl1–hl7 were used for further studies  plants and this decreased to 1 tiller per plant with GR24 treatment . The LC-MS/MS data indicated that all the mutants showed defective and very poor synthesis of strigolactone, which cannot be detected and quantified by LC-MS/MS , tomato (Solanum lycopersicum), and maize (Zea mays) [Use of the CRISPR/Cas9 system to precisely manipulate the plant genome in a user-defined manner opens myriad possibilities for translational applications in agriculture , 42, 49.ea mays) , 47. Howea mays) , 47. MorCCD7 to produce rice mutants with reduced SL biosynthesis. For this purpose, we designed two sgRNAs that target two exons in CCD7 with the aim of generating knockout phenotypes. The deletion of one or two nucleotides at the sgRNA-1 target site in the first exon produced the alleles dl1 and dl2, respectively, and resulted in truncated CCD7 proteins. Targeting the terminal region, the last 7th exon by sgRNA-2 produced seven different alleles labelled hl1 to hl7. The C-terminus of CCD7 protein is an important region to target as any substitution in this region might disrupt the function of the protein [dl and hl gave rise to a significant reduction in plant height and an increase in tiller number  and hl1 the lowest (61 tillers per plant). It can be assumed that these difference are a result of regeneration from tissue culture. In the next segregating generation, some of mutants were lacking the T-DNA


Synthetic lethality exploits the phenomenon that a mutation in a cancer gene is often associated with new vulnerability which can be uniquely targeted therapeutically, leading to a significant increase in favorable outcome. DNA damage and survival pathways are among the most commonly mutated networks in human cancers. Recent data suggest that synthetic lethal interactions between a tumor defect and a DNA repair pathway can be used to preferentially kill tumor cells. We recently published a method, DiscoverSL, using multi-omic cancer data, that can predict synthetic lethal interactions of potential clinical relevance. Here, we apply the generality of our models in a comprehensive web tool called Synthetic Lethality Bio Discovery Portal (SL-BioDP) and extend the cancer types to 18 cancer genome atlas cohorts. SL-BioDP enables a data-driven computational approach to predict synthetic lethal interactions from hallmark cancer pathways by mining cancer’s genomic and chemical interactions. Our tool provides queries and visualizations for exploring potentially targetable synthetic lethal interactions, shows Kaplan–Meier plots of clinical relevance, and provides in silico validation using short hairpin RNA (shRNA) and drug efficacy data. Our method would thus shed light on mechanisms of synthetic lethal interactions and lead to the discovery of novel anticancer drugs. HER2, BCR-ABL, EGFR, and BRAF [Drug treatment of cancer depends on the notion that mutations that give rise to the development of cancer also bring about a weakness that can be exploited therapeutically. Large-scale cancer genome sequencing efforts have catalogued mutations in various cancer types that can be explored as tumor-specific vulnerabilities . These gand BRAF . From a One promising way to tackle this challenge is based on the concept of synthetic lethality (SL). SL describes the relationship between two genes whereby inactivation of either gene alone results in a viable phenotype, while their combined inactivation is lethal. SL has long been considered a foundation for the development of selective anticancer therapies ,5,6,7,8,Given the importance of SL, considerable work has been devoted to finding such interactions in cancer—both experimentally ,5,6,7,8 BRCA1, BRCA2, TP53, PTEN, ATM, ATR, KRAS, HRAS, BRAF, EGFR, MET, and PIK3CA. Many of the predicted SL interactions of these 12 genes across 18 cancers were also present in the genetic dependency CRISPR screen  “GENES” tab: using a primary gene of interest (the model currently includes 623 cancer genes commonly mutated in cancers), (2) “CANCER” tab: tumor tissue of origin (currently includes 18 cancer histology types), and (3) “DRUG” tab: drugs of interest . Additionally, potential synergistic drug combinations for different cancers can be browsed from the “INFERRED DRUG SYNERGY” tab. An example of search and browsing functionalities in SL-BioDP is illustrated in p-value for expression correlation, mutual exclusivity of mutations, conditional gene essentiality from RNAi screens, and a change in the SL interactor amplification profile in the presence of a primary gene mutation. For validation purposes, to show conditional gene essentiality, we provided an in silico validation approach using shRNA screening data from cancer cell lines [p < 0.05) where downregulation of the synthetic lethal interactor gene shows better disease-free survival in the presence of the mutation in the primary gene are ideal cases of clinically relevant synthetic lethality. Together with the amplification status of the SL interactor gene, we can hypothesize if a favorable clinical outcome can be seen by selective targeting of the interactor gene (Gene2). In addition, we provide reported SL interactions and supporting references of our findings whenever it is feasible.(1) Search using “GENE” tab: search can be initiated by using either the official gene symbol or Entrez gene id of the primary gene. SL-BioDP presents cancer-specific synthetic lethality information in multiple levels. First, the mutation and expression profiles of the primary genes in each cancer type are presented. Then, the predicted synthetic lethal partners of the chosen primary genes in selected cancer types are presented. The predicted synthetic lethal pairs in SL-BioDP are ranked by the synthetic lethal score calculated by the DiscoverSL model. Additional parameters for assessment of synthetic lethality for each predicted SL pair are shown as: a ll lines . Assessm(2) Search using “CANCER” tab: users can select from a drop-down list containing 18 tumor histology types. By selecting a particular histology, users are shown the top genes which are differentially expressed between the tumor vs. adjacent normal tissues from that histology. Then by selecting one or multiple gene(s) of interest, users can see their predicted SL partners, view enriched pathways, and search for drugs targeting the SL partners (as described above).(3) Search using “DRUG” tab: users can search by the name of a drug of their interest and choose from any of the two databases, “DrugBank” or “DGIdb”, to see their targets. From the resulting drug-target page, the users can select one or multiple target gene(s) to search the primary genes for which the chosen target genes are SL partners. Or in other words, the primary genes which when mutated can be potentially treated with the drugs of interest, by targeting the SL partner genes.p < 0.05) in cancer cells carrying the mutation in Gene1 (compared to cells not carrying the mutation in Gene1) is calculated from GDSC (Genomics of drug sensitivity in cancer) portal drug screening data (as explained above and in the methods section). In our database portal SL-BioDP, the potential synergy between cancer drugs in different cancer types, inferred from the predicted synthetic lethal interaction between their targets, can be explored in the tab “Inferred Drug Synergy”. The reasoning behind potential drug synergy is explained in (4) Browsing the “INFERRED DRUG SYNERGY” tab: users can browse for the potential synergistic drug combinations in the 18 cancer types. The inferred drug synergy table for each cancer type shows the potential synergistic drugs based on the predicted SL interactions of a primary mutated gene. The tables also include published references (if any) for the inferred synergistic drug combinations. An interesting inference coming from the gene mutation-based drug sensitivity study is the potential synergy between drugs targeting the primary gene (mutated) and the SL interactor gene. The potential synergy between two drugs targeting the primary gene and the SL interactor gene is inferred from this hypothesis: if the loss of function (presence of the mutation) of the primary gene (Gene1) makes cancer cell lines sensitive to certain drugs targeting the SL interactor gene(s), then it is possible that drugs that target the primary gene(s) will make cancer cells sensitive to the drugs that target the SL interactor gene(s). Information for drugs targeting the primary gene (Gene1) and the SL interactor gene (Gene2) are collected from the two databases, DrugBank and DGIdb. Gene1 and Gene2 SL interaction predictions are taken from SL-BioDP. Drug2 (targeting Gene2) sensitivity (BRAF in cancer lung adenocarcinoma (LUAD), a form of non-small-cell lung cancer , compared to solid tissue normal samples in TCGA tumor sample data. By searching for the predicted mutation-based synthetic lethal targets of BRAF in LUAD (either by selecting the “LUAD” histology from the “GENE” search results page or by selecting “BRAF” from the “CANCER” search results page), we get predicted synthetic lethal interactors. BRCA1 is a predicted SL partner of BRAF in LUAD , which was also reported in published SL screens. Alternately, when a search for a drug target is performed using the “DRUG” tab in SL-BioDP, e.g., for the drug temozolomide, we first get its targets from one of the databases, DrugBank or DGIdb (chosen by the user). Then, the user can select one or multiple target genes of the drug to search for the primary (mutated) genes across the 18 tumor histology types in SL-BioDP. From this page, one or more histology type(s) can be selected to search for gene alteration patterns  in the chosen histology  which can be potentially treated by the drug of interest  using a synthetic lethal approach, that is by targeting the synthetic lethal partner . Survival analysis (Kaplan–Meier plot) shows better disease-free survival in patients with underexpression of BRCA1 compared to overexpression, in LUAD samples with BRAF mutations. Pathway enrichment for the SL interactors of the primary gene (BRAF) can be seen. Next, drug targets for selected SL interactors can be seen by searching either of the two databases: DGIdb or DrugBank. A search for drugs for the SL pair BRAF–BRCA1 from DGIdb shows drugs targetable for either the SL gene, BRCA1 or the primary gene, BRAF.The utility of SL-BioDP is shown with a case study on the primary gene g cancer . BRAF mu in LUAD . Two theresponse . SearchiBRCA1/BRCA2 mutations is a widely documented example where synthetic lethality is used for effective treatment of cases with driver alterations [BRCA1 and BRCA2 mutations that can be treated by PARP inhibitors are identified from this study. Also, there are reports on the use of PARP inhibitors for treating tumors with mutations in the gene ATM [MAPK1, MAPK8, MAP2K4, and MAP4K3), that can be potentially targeted by the PARP inhibitor, olaparib. When we performed pathway enrichment analysis on all the primary genes for which we detected sensitivity to the drugs  inhibitor drugs for treating breast cancers or ovarian cancers with erations ,22. PARPgene ATM . Our stuBRCA1 gene, and in general the homologous recombination repair pathway. In addition, the analysis offers to look into new potentially effective gene mutation-based drug therapies. We extended our analysis to infer potential synergy between drugs targeting the predicted SL interactors. We reasoned that if the presence of a mutation (or loss of function) in a primary gene makes cancer cells sensitive to certain drugs targeting an SL interactor, then it is highly likely that the drugs targeting the primary gene will also make the cancer cells sensitive to those drugs targeting the SL interactor. These inferred drug synergies can be searched in SL-BioDP for each of the 18 tumor histologies included in SL-BioDP. In support of our predictions of synergistic drugs in cancers, we collected literature references and reports of ongoing clinical trials, which is included in Synthetic lethality has immense potential in cancer therapeutics. Where the driver genes or oncogenes cannot be targeted, synthetic lethal interactors can potentially serve as drug targets in the presence of mutations in the driver genes, as the mutant tumor cells are dependent upon the synthetic lethal interactors for their survival. Recent advances in RNAi and CRISPR technologies have enabled large-scale synthetic lethality screens for individual genes to be performed in human cell lines. The genetic dependency map (DepMap) project is dedicated towards finding genetic dependencies from the published shRNA/CRISPR or drug screening data in cancer cell lines. While identifying genetic dependencies or essentiality in the context of specific genetic alterations is important for screening potential targets for cancer therapy, identification of clinically relevant synthetic lethal pairs in cancers has been a challenge. A recent work tried to address this limitation with an in silico analysis of clinical relevance of the SL interactions identified from in vitro SL screens and reported that only a fraction of these SL interactions hold up to be clinically significant when tested on TCGA tumor data . Also, tThe primary source of tumor genomic and clinical data of 18 tumor types is the cancer genome atlas (TCGA) project . AdditioFor validation purposes, we collected shRNA screening data in cancer cell lines from the Achilles project version 2.4.3 . GenomicDrug-protein interaction data was collected from the databases, DrugBank and DGIdb ,19. For Synthetic lethal partners for cancer genes in all 18 cancer types in SL-BioDP were calculated using the recently published DiscoverSL algorithm . Discovep-value for changes in the amplification status of Gene2 was calculated; (3) to assess the clinical outcome of underexpression vs. overexpression of the predicted SL gene (Gene2) in cases with mutations in the primary gene (Gene1), a Kaplan–Meier survival analysis was performed on disease-free survival in TCGA clinical data; and (4) to assess the potential drug sensitivity, a p-value was calculated using LNIC50 values  between primary gene mutated vs. non-mutated cells from the Genomics of Drug Sensitivity in Cancer (GDSC) project data [We used multiple methods to validate the significance of SL pair interactions: (1) to assess the effect of silencing the SL gene (gene2) in cancer cell lines where the primary gene (gene1) was mutated, significance of difference was calculated by t-test using shRNA screening data from Achilles 2.4.3 project ,28; (2) ect data .The SL-BioDP web application was built in PHP with MySQL as the primary data repository. It was constructed as a multi-tier architecture, which provides greater flexibility in development and maintenance. A runtime view of the architecture is shown in https://SL-BioDP.nci.nih.gov and includes a detailed documentation tutorial.The resource is freely available at In conclusion, the SL-BioDP web portal is an easily accessible, open source platform to compare multiple cancers and predict potentially targetable SL interactions and drug combinations for future experimental validations. The query options provide flexibility in user-defined searches with intuitive visualization graphs and tables. For the predicted SL interactions, we provided in silico shRNA validation and a clinically relevent Kaplan–Meier analysis from patient data. However, cancers are heterogeneous and more complicated. It is very likely that cross talk between multiple genes and mutual impacts on each other is possible. In the current state, it is beyond our predictions to provide mechanistic insight into cross talk interactions. In the future versions of SL-BioDP, we plan to include updated information from new genetic dependency screens and a new set of tumor genomic and clinical data for better interpretation of our model.