Staging laparoscopy(SL) is a recommended technique for the staging of Gastric Cancer(GC) and provides the indication for a radical surgery. Considering the medical practice in China, the standardized and regular usage of SL is yet to be spread. However, existing guidelines vary and make an ambiguity of indication for SL. Besides, the specific indication for Chinese patients remains a niche. This study aims to the essential, missing information of Chinese patients and tries to normalize the indication of LS in medical practice in China.The study is a prospective, multicenter cohort study being conducted in China with a total of 450 patients, all diagnosed with locally advanced gastric cancer  through Computed Tomography(CT) and/or Endoscopic Ultrasonography(EUS). Peritoneal lavage is regularly performed during the SL. Multivariate Cox regression model and receiver-operator characteristic(ROC) analysis will be used to analyze the significant risk factors of intra-abdominal metastasis.This confirmatory study will provide us with the specific positive rate of intraabdominal metastasis of GC in China, compared with empirical evidence of 20%. We expect this trial will contribute to our discovery of the specific risk factors of intra-abdominal metastasis of Chinese patients and to the stimulating and performing of minimally invasive surgical procedures.ClinicalTrials.gov: registration number NCT02172690. Gastric cancer is a major cause of cancer death worldwide with a 40 percentage of cases occurring in China . IndividMost of the Chinese patients are diagnosed with locally advanced stage, suffering the substantial intra-abdominal metastases with higher risks. Precise preoperative staging is particularly important for therapeutic strategies and prognostic evaluation.Precise staging is a foremost method that benefits the successful surgical intervention of cancer, contributed to the improvement of medical tools: despite the fact that preoperative imaging techniques have significantly improved the diagnostic accuracy of wall infiltration and lymph node involvement, the detection of peritoneal dissemination still needs to be developed; positive intra-peritoneal findings of free cancer cells has been proved to be related with poor survival yet could only be confirmed during surgical procedure , 3.SL has been suggested useful in detecting unrecognized peritoneal dissemination, and in collecting peritoneal lavage fluid for cytological examination with minimal invasion. However, indication for SL remains controversial. NCCN guideline recommends an indication for SL of T3 and/or N+; ESMO, IB-III; JGCA, none, merely mentioned SL only on the condition of neoadjuvant Chemoradiotherapy –6. No spThe aim of this study is to acquire a basic data on the positive rate of SL in Chinese patients and to identify the risk factors associated with intra-abdominal metastasis.This study evaluates the efficacy of SL with peritoneal cytology, and determine the indications if possible.Patients who meet the inclusion criteria will be enrolled consecutively, with their preliminary data collected using a web-based case report form. When the whole data-collecting is completed, factors associated with intra-abdominal metastasis will be analyzed by multivariate Cox regression model. Receiver-operator characteristic (ROC) will be used to find out risk factors of intra-abdominal metastasis. Figure Patients with pathologically confirmed gastric adenocarcinoma are recruited. If the patient meets all inclusion criteria, following data will be recorded: basic information including age, gender, height, body weight et al., clinical information including CT stage(enhanced CT is mandatory required), EUS stage, result of blood examinations such as tumor marker , blood cell count, albumin et al. If there been any differences between CT & EUS, it is required to record them separately.If the patient is confirmed at stage cT2-4bN0-3 M0, which means no sign of distant metastasis detected by CT/EUS, he/she undergoes SL. Following data will be recorded: intraoperative finding including the peritoneal dissemination, and the peritoneal lavage; video of surgery and pictures demonstrating the extent of peritoneal dissemination is required.Postoperative hospital stay and complications will be recorded, the follow-up time will be 30 days after surgery. Only those who undergoes DL alone will be included into safety analysis since the complications will be confused if the patient undergo radical surgery immediately after SL.The primary target of this study is to obtain the result of SL. Either peritoneal dissemination or a positive result of cytological examination will be recorded as a positive result. If the pathological or cytological examination describes “suspicious”, the result will be characterized as positive, consistent with our current practice.The secondary aim is to evaluate the safety of SL.Age ≥ 18;Histological confirmed diagnosis of primary gastric carcinoma (including esophagus-gastric junction).Karnofsky score ≥ 70;Abdominally enhanced spinal CT and endoscopic ultrasonography (EUS) showed a clinical stage of T2-4bN0-3bM0;Fit for radical gastrectomy or neo-adjuvant chemotherapy;Willing to comply with protocol, and provide informed consent forum.Unfit for laparoscopic operation or general anesthesia ;Underwent emergency surgery to relieve obstruction, bleeding or perforation.The exclusion criteria included:SL was performed under general anesthesia. The patient is placed in a supine position. A 10-mm disposable trocar (observing hole) is inserted into the sub-umbilicus, and a 30° telescope is used. Another 10-mm trocar and a 5-mm trocar (operating hole) are inserted into the right upper quadrant and left upper quadrant, respectively. Prior to any manipulation, 250 ml of warm normal saline is infused into subphrenic space, subhepatic space, omentum, bilateral paracolic sulci and the pouch of Douglas. The irrigation should be carefully operated to avoid the primary tumor. At least 100 ml of fluid is aspirated from the subphrenic space, subhepatic space and Douglas’ pouch. The fluid is immediately sent for centrifugation and cytological examination and a systematic inspection of the abdominal cavity is performed clockwise from the right quadrant. Any suspicious lesion will be biopsied and sent for frozen pathologic examination.This study is a single arm OPC (objective performance criteria) design where the primary evaluation index is the positive rate of SL. Based upon literature, expected event rate is 20%, and clinical acceptable rate is above 15%. Type I error as bilateral 5%, dropout rate as 5%, power as 80%, calculated sample size is 450. The recruitment process is estimated to be completed by December 2016.P values < .05 are considered statistically significant. All statistical analyses are carried out using the SPSS statistical software package (version 20).Clinical characteristics of the DL- and DL+ patients are compared using the Independent samples group t test and chi-square test. To determine the risk factors, all variables found to be significant by the univariate analysis are assessed by binary logistic regression analysis . The ROC curve and AUC analyses are used to determine sensitivity, specificity, and corresponding cut-off value of each factor. All This study is conducted to prospectively evaluate the positive rate of the result of SL based on samples of Chinese patient and try to discover risk factors of intra-abdominal metastasis. SL has been recommended by many guidelines yet different indications is suggested. The standardized operational procedure remains controversial for such practice along with possibly unknown bias. Druing the process of information collecting, data will be prospectively recorded using web-based case report forms.The results of the study will provide the first prospective multi-center database of SL in China, according to our knowledge, which will provide important figure of 40% of patients of the world.


Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. So far, no Western medicine treatment can completely inhibit or reverse the progress of SSc, while at the same time, our previous series of studies have shown that the treatment of SSc by the Wenyang Huazhuo Tongluo formula (WYHZTL), a Chinese herbal decoction, shows a delightful prospect. The aim of this study is to further investigate the mechanism of anti-fibrosis of WYHZTL formula in SSc mouse model.The Bleomycin-induced SSc mouse model was treated with saline (BLM), high-dosage of WYHZTL formula (WYHZTL-H), medium-dosage of WYHZTL formula (WYHZTL-M), low-dosage of WYHZTL formula (WYHZTL-L) and XAV-939, a small molecule inhibitor of Wnt/β-catenin signaling pathway, by the intragastric administration and intraperitoneal injection, respectively. The mRNA and protein levels of Wnt/β-catenin signaling pathway associated genes, fibrosis markers and histopathology were detected by reverse transcription-quantitative polymerase chain reaction, Western blotting and hematoxylin/eosin-staining. The levels of Wnt1, CTGF and DKK1 protein in serum were detected by enzyme-linked immunosorbent assay.Compared with BLM group, the WYHZTL formula and XAV-939 could significantly inhibit the thickness of the skin tissue of the SSc mouse model. The mRNA expression levels of GSK3β and DKK1 in the WYHZTL formula and XAV-939-treated group were significantly higher than those in the BLM group, while Wnt1, β-catenin, TCF4, cyclin D1, survivin, VEGF, CTGF, FN1, collagen I/III were decreased. Compared with BLM group, the protein expression levels of GSK3β and DKK1 in the WYHZTL formula and XAV-939-treated group were upregulated, while Wnt1, β-catenin, cyclin D1, survivin, CTGF, FN1, collagen I/III were downregulated. WYHZTL formula and XAV-939 could inhibit expression of Wnt1 and CTGF, but promoted DKK1 in serum. Furthermore, WYHZTL-H seemed more effective than WYHZTL-M and/or XAV-939 on regulating Wnt1, β-catenin, TCF4, GSK3β, DKK1, cyclin D1, survivin, VEGF, CTGF, FN1 and collagen I/III.This present study demonstrates that WYHZTL formula has anti-fibrosis effect in Bleomycin-induced SSc mouse model in a dosage-dependent manner, and the molecular mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway.The online version of this article (10.1186/s13020-018-0175-z) contains supplementary material, which is available to authorized users. Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. Fibrosis is a typical pathological feature of SSc, which is closely related to its prognosis and mortality , 2. So fStudies have shown that the occurrence, development and prognosis of SSc fibrosis depends on the “net effect” of the synthesis and degradation processes of extracellular matrix (ECM). Fibrosis occurs when collagen fibers are excessively synthesized and relatively less degraded , 6. Wnt Previous studies showed that Wnt/β-catenin signaling pathway was closely related to fibrosis in skin, lung, liver and kidney –15, whicThe current western medicine treatment of SSc is not satisfactory, so it is essential to explore another treatment option. In the traditional Chinese medicine treatment of SSc, Wenyangchubi formula and Yiqihuoxue formula were representative examples. Studies have shown that Wenyangchubi formula could reduce the expression of connective tissue growth factor (CTGF) and collagen I, improve the skin fibrosis of SSc mouse model . And YiqThe aim of this study was to further explore the molecular mechanism of anti-SSc fibrosis of WYHZTL formula, and investigate the effect of WYHZTL formula on Wnt/β-catenin signaling pathway in SSc mouse model.The Minimum Standards of Reporting Checklist contains details of the experimental design, and resources used in this study  C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. . By local injection of Bleomycin in 7-week-old female mice for 3 weeks, the skin fibrosis was induced. Briefly, Bleomycin dissolved in sterile saline to a concentration of 200 μg/ml, and subcutaneous injection of 100 μl of Bleomycin to the restricted area of the upper back each day . 100 μl Eight mice on a C57BL/6 background were randomly selected as normal groups. Then, the Bleomycin-induced SSc mouse models were randomly divided into five groups, and each group has eight mice. The five groups were named as Bleomycin group (BLM), WYHZTL formula low-dosage group (BLM + WHHZTL-L), WYHZTL formula medium-dosage group (BLM + WYHZTL-M), WYHZTL formula high-dosage group (BLM + WYHZTL-H), and XAV-939 group (BLM + XAV-939), respectively. The PBS group (PBS) received PBS administered daily by subcutaneous injection into defined areas of upper back of normal C57BL/6 mouse, while the normal group  didn’t receive any administration. Normal, PBS, and BLM group received saline (1 ml/day) administered daily by intragastric administration, while BLM + WYHZTL-L group, BLM + WYHZTL-M group, BLM + WYHZTL-H group received low-dosage of WYHZTL formula (11.75 g/kg/d), medium-dosage of WYHZTL formula (23.50 g/kg/day), high-dosage of WYHZTL formula (47.00 g/kg/day) administered daily by intragastric administration of Bleomycin-induced SSc mouse models, respectively. BLM + XAV-939 group received XAV-939 (10 mg/kg/day) administered daily by intraperitoneal injection of Bleomycin-induced SSc mouse models. All the treatments lasted for 4 weeks, then the mice of each group were anaesthetized and then euthanized, and skin tissues were harvested, stored at − 80 °C, to analyze expression levels of target genes and proteins.The measurement of skin thickness, skinfold (pinch) thickness and breaking strength of skin were performed as previously described protocol . BrieflySkin tissue samples were fixed with 4% paraformaldehyde, and embedded in paraffin, then cutted into 3 μm-thick sample sections for hematoxylin/eosin staining . For hemThe effects of the WYHZTL formula on Wnt/β-catenin signaling pathway was analyzed by Western blotting as previously described protocol . Briefly−ΔΔCt method [Total RNA of each skin tissue samples was extracted using TRIzol reagent . All handling procedures perform under a laminar flow hood and RNAse-free conditions. The extracted RNA was dissolved in 30 μl RNase-free water and quantified by Nanodrop 2000 ultraviolet spectrophotometer , then stored at − 80 °C until used. RNA (2 µg) was reverse transcribed into complementary DNA (cDNA) using M-MLV Reverse Transcriptase  according to the manufacturer’s instructions. RT-qPCR analysis was performed using ABI Power SYBR Green PCR Master Mix Kit on an ABI Step One Plus instrument . The reaction condition follows denaturation for 2 min at 95 °C, 40 cycles including 95 °C for 15 s, 60 °C for 20 s and 72 °C for 30 s. Each experiment was performed 3 times independently. PCR products were mixed with 6× loading buffer  and then electrophoresed in 1.5% agarose gels containing ethidium bromide. The images were captured using a 4600SF Gel Image System . The relative expression level of target gene was normalized to GAPDH and calculated with 2t method . The prig for 10 min, and obtain upper serum carefully to assay immediately. ELISA analysis was performed using enzyme-linked immunosorbent assay kit  on a SpectraMax iD3 microplate reader instrument . Reagents, and samples or standards were prepared, and experiments were performed according to the manufacturer’s instructions. Briefly, standard or sample were added to each well separately, and incubated at 37 °C for 2 h. Then standards or samples were removed without washing. Add Biotin-antibody to each well, and incubate at 37 °C for 1 h. Remove liquid of each well and wash. Add HRP-avidin to each well, and incubate at 37 °C for 1 h. Remove liquid of each well and wash. Add TMB Substrate to each well, and incubate at 37 °C for 20 min. Add Stop Solution to each well, and immediately detect the optical density of each well by a SpectraMax iD3 microplate reader instrument set to 450 nm. At last, the concentration of target protein was calculated according to standard curve. Each experiment was performed three times independently.One day after WYHZTL formula and XAV-939 administration, the animals were sacrificed. Collecting blood sample and standing to clot for 2 h at room temperature. Then centrifugation at 1000×P < 0.05 and there was no significant variance in homogeneity. All statistical analyses were performed with Graphpad Prism 6.0 Software. P < 0.05 was considered to indicate a statistically significant difference. All data and statistical analysis complied with the recommendations on experimental design and analysis in pathology.Results were represented as mean ± SD. Data were analyzed by one-way analysis of variance, as appropriate, followed by Bonferroni post hoc test if F achieved P < 0.05, P < 0.01, P < 0.01, P < 0.01, respectively)     staining. The results of HE staining showed that there was excessive accumulation of collagen and other extracellular matrix components in BLM group, which was attenuated with WYHZTL formula or XAV-939 treatment Fig. a. The skIn order to determine whether the therapeutic effect of WYHZTL formula on SSc mouse model was related to Wnt/β-catenin signaling pathway, we collected the skin tissues of mice in each group, and detected the components of Wnt/β-catenin signal pathway using RT-qPCR and Western blotting.P < 0.01), while BLM + WYHZTL-L, BLM + WYHZTL-M groups had no significant changes (P > 0.05)   , rather than BLM + XAV-939 group (P > 0.05) , while BLM + WYHZTL-L group had no significant changes (P > 0.05)  and BLM + XAV-939 (P < 0.05) group while the expression level of Wnt1 in BLM + WYHZTL-H were lower than BLM + WYHZTL-M (P < 0.01), other than BLM + XAV-939 group (P > 0.05)   Fig. d. And thly) Fig. d. The exly) Fig. e. And th05) Fig. e. Compar05) Fig. a, b. And05) Fig. a, b. Com01) Fig. c.Fig. 2TP > 0.05), and among BLM + WYHZTL-M, BLM + WYHZTL-H, and BLM + XAV-939 group, there were no significant difference (P > 0.05). However, in this study, we didn’t detect any significant difference among Normal group, PBS group and BLM group. And WYHZTL formula or XAV-939 also had no effect on the expression level of TCF4 protein.As shown in Fig. P < 0.001, P < 0.01, respectively), while BLM + WYHZTL-L and BLM + WYHZTL-M group didn’t have significant difference with BLM group (P > 0.05). And the expression level of DKK1 and GSK3β protein in BLM + WYHZTL-H were all higher than BLM + WYHZTL-M group , but not BLM + XAV-939 group (P > 0.05).As shown in Fig. In order to further study on regulating SSc fibrosis by WYHZTL formula via Wnt/β-catenin signaling pathway, we investigated the expression level of target genes of Wnt/β-catenin signaling pathway and fibrosis markers in the skin tissues of each mouse model by RT-qPCR and Western blotting.P < 0.05, P < 0.01, P < 0.01; all P < 0.01; all P < 0.01, respectively), while BLM + WYHZTL-L group didn’t significantly change (P > 0.05) , while BLM + WYHZTL-L and BLM + WYHZTL-M group didn’t significantly change (P > 0.05)   Fig. i. The mRly) Fig. h, k.P < 0.01, P < 0.01, P < 0.05, respectively), while the mRNA expression levels of FN1, collagen I in BLM + WYHZTL-H group were even lower than BLM + XAV-939 group    Fig. g, i–l. Bly) Fig. f, l.P < 0.01, P < 0.001, P < 0.001, P < 0.001; all P < 0.001; all P < 0.001; all P < 0.001, respectively), while CTGF in BLM + WYHZTL-M, BLM + WYHZTL-H and BLM + XAV-939 group was decreased . In addition, the protein expression levels of cyclin D1, CTGF, FN1 in BLM + WYHZTL-H group were even lower than BLM + WYHZTL-M group , while the protein expression levels of collagen I in BLM + WYHZTL-H group was lower than BLM + XAV-939 group (P < 0.01). Besides that, the protein expression levels of CTGF in BLM + XAV-939 group was even lower than BLM + WYHZTL-H group (P < 0.01).As shown in Fig. P < 0.001), but there was no significant difference among BLM, BLM + WYHZTL-L, BLM + WYHZTL-M, BLM + WYHZTL-H and BLM + XAV-939 group (P > 0.05).Furthermore, compared to Normal and PBS group, we found that VEGF was upregulated in BLM group , while DKK1 was upregulated , in which WYHZTL-H became more effective on promoting DKK1 (P < 0.001), while inhibiting Wnt1 and CTGF  than WYHZTL-M. Furthermore, compared with BLM + XAV-939 group, WYHZTL-H was more effective in downregulating Wnt1, and upregulating DKK1 .As shown in Fig. Wnt/β-catenin signaling pathway could induce fibrosis. Wnt1 and human skin fibroblasts were co-incubated to significantly stimulate the expression of α-SMA and type II collagen, which meant that Wnt1 could induce the transformation of resting fibroblasts to myofibroblasts, and stimulate collagen release , 37. PreFibrosis, atrophy and vascular occlusive vasculitis of connective tissue were basic pathological changes of SSc . FibrosiThe overexpression of target genes of Wnt/β-catenin signaling pathway were closely related to SSc fibrosis. The target genes include c-myc, cyclin D1, survivin, vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF), endothelin-1 (ET-1), and extracellular matrix components, such as fibronectin (FN) . TherefoIn addition, considering that Wnt1, DKK1 and CTGF are secreted proteins, we detected the protein expression levels of Wnt1, DKK1 and CTGF in serum of mice by ELISA. We found that WYHZTL formula and XAV-939 could significantly reduce the protein level of Wnt1 and CTGF, while increase the protein level of DKK1 in serum, which was consistent with the results in the study of skin tissue, suggesting that WYHZTL formula could indeed inhibit the development of fibrosis by regulating Wnt/β-catenin signal pathway.XAV-939 was widely used as an inhibitor of Wnt/β-catenin signaling pathway . Our preIn conclusion, our results validated the function of anti-fibrosis in SSc treatment for WYHZTL formula, and also proposed a new molecular mechanism of anti-fibrosis by inhibiting Wnt/β-catenin signaling pathway in Bleomycin-induced SSc mouse model Fig. . At presAdditional file 1. Minimum Standards of Reporting Checklist.


In this paper, the properties of concrete containing zeolite and tuff as partial replacements of cement and sand were studied. The compressive strength, water absorption, chloride ion diffusion and resistance to acid environments of concretes made with zeolite at proportions of 10% and 15% of binder and tuff at ratios of 5%, 10% and 15% of fine aggregate were investigated. The results showed that the compressive strength of samples with zeolite and tuff increased considerably. In general, the concrete strength increased with increasing tuff content, and the strength was further improved when cement was replaced by zeolite. According to the water absorption results, specimens with zeolite showed the lowest water absorption values. With the incorporation of tuff and zeolite, the chloride resistance of specimens was enhanced significantly. In terms of the water absorption and chloride diffusion results, the most favorable replacement of cement and sand was 10% zeolite and 15% tuff, respectively. However, the resistance to acid attack reduced due to the absorbing characteristic and calcareous nature of the tuff. However, cement production is estimated to produce about 5% to 7% of total greenhouse gas emission each year. This has a significant impact on the environment and construction industries . The hig2 and Al2O3 contents), environmental advantages , and economic advantages (high reserves and low cost of production) [In many countries, such as Russia, Germany, Slovenia, Spain, China, Turkey and Iran, Zeolites have been used successfully in concretes providing both technical and environmental advantages over conventional Portland cement concrete. Some of the advantages that make zeolite an acceptable SCM in construction include: good physical properties , strong chemical properties (high reactive SiOduction) .The impact of zeolite on concrete properties has been investigated in previous research. Chan et al.  reportedOn the other hand, over the last few decades the increase in demand for sand for the rapid increase in construction activities  has placed immense pressure on this valuable resource. To overcome this problem, it is crucial to use substitute materials such as crushed sand, dune sand, limestone fines, natural pozzolana, blast-furnace slag and different wastes. However, with these palliative materials, the market expectations are still far from being satisfied. Consequently, research on other replacements would be required.Tuff is usually lighter than aggregates such as sand, gravels and ground rocks ,15. TherConcrete is a susceptible material to acid attack . Concret(i)Places where industrial waste exists. Industrial waste may often contain a large amount of sulphuric acid. The sulphuric acid in industrial waste may cause severe corrosion in concrete pipes in the short or long term period.(ii)Groundwater normally contains different kinds of sulfates, and free sulfuric acid may also be one of the products.(iii)Acid rain, which is formed by oxidation of sulfur dioxide, is a product of the combustion of coal and petroleum or other industrial processes. Acid rain decreases the pH of the concrete significantly and results in deterioration of concrete.Portland cement concretes usually do not have a high resistance to acid. Acids attack concrete by dissolving both hydrated products and unhydrated cement as well as calcareous aggregates. Concrete deterioration increases as the pH of the concrete decreases due to the acidic environment. Sulphuric acid can exist in many different places, such as:In the literature there is no research on examining the effect of both tuff as fine aggregate and zeolite as SCM together on durability of concrete or on its resistance against different hazardous acids. This research aimed to study the influence of tuff and zeolite on strength development, water absorption, chloride penetration and acid resistance properties of concrete samples.Portland cement Type I complying with the requirement of ASTM C150 was used. Tuff and zeolite were obtained from the same volcanic areas of the northern region of Iran. Chemical compositions and physical properties of the cement, zeolite and tuff are given in 3 was used to achieve the flowability of the mixtures. Sulphuric acid with 98% concentration and hydrochloric acid with 37% concentration were used to make an acidic solution of 5% concentration.Natural river sand with water absorption of 1.2% and a nominal maximum particle size of 4.75 was used as fine aggregate. Tuff with similar particle size distribution was used for partial replacement of fine aggregate. The fineness modulus of sand and tuff lay between 2.7 and 2.8, which indicated that they were medium-coarse. Limestone gravel with water absorption of 0.69% and a nominal maximum size of 12.5 mm was used as coarse aggregate. In this experiment, a total of 10 different concrete mixtures were made. Tuff was added at the ratios of 5%, 10% and 15% (by weight) as partial replacement of sand and zeolite was used at 10% and 15% (by weight) as replacement of cement. The water-to-binder ratio was 0.4 and kept constant for all mixtures. The abbreviations for the labeling of the mixtures used in this study are adopted in such a way that the number after T shows the percentage of tuff and the number after Z shows the percentage of zeolite. The mixture proportions of the specimens are presented in The mixing procedure used by Khayat et al.  was utilCompressive strength was tested in accordance with IS 516  at 28, 9The water absorption of hardened concrete was determined in accordance with the ASTM C642-13 standard . Water aC = chloride concentration, measured at depth x and exposure time t, mass %, sC = projected chloride concentration at the interface between the exposure liquid and test specimen that was determined by the regression analysis, mass %, iC = initial chloride-ion concentration of the cementitious mixture prior to submersion in the exposure solution, mass %, x = depth below the exposed surface (to the middle of a layer), m, aD = chloride diffusion coefficient, m2/s, t = the exposure time, s, and erf = the error function.Chloride diffusion testing was conducted on specimens after 90 days of curing according to ASTM 1556-11a Standard . In thisSulphuric acid attack and hydrochloric acid attack tests were conducted on hardened concrete samples at an age of 56 days. The concentration of the acid solution was kept constant at 5% by regularly adjusting the pH of the solution. Samples were immersed in sulphuric or hydrochloric acid solution for 180 days at a temperature of 23 ± 2 °C, thereafter, the samples were taken out and assessed visually. At the same time, compressive strength of specimens was measured. After leaving specimens in the oven at 100 °C for 24 h, the samples were weighted to measure the mass loss.2-pozzolan blend, the Ca(OH)2 hydrolysed, which resulted in a high pH value. Then the silicate or aluminosilicate network of the pozzolanic material was broken down due to the OH− attack, and the depolymerized species entered the solution as follows:2+ ions and the above products , crystalline calcium silicate and aluminate hydrate were created that were similar to those present in the hydration of cement. Such synthesized products tended to harden, and consequently, they promoted the increase in mechanical strength of the cementitious material. Besides, the crucial environmental and economic merits of zeolite should not be neglected, since the production of cement needs a lot of energy input which results in high CO2 release in the atmosphere.By mixing water and a Ca(OH)The high surface area of tuff accelerated the hydration rate and resulted in higher compressive strength.Compressive strength values of the concrete samples at 28, 90 and 236 days of age are presented in 2 during the hydration of calcium silicate which was more than the amount of Ca(OH)2 consumed in the pozzolanic reaction.Perraki et al.  reportedWater absorption results of the concrete samples are presented in On the contrary, a noticeable change in water absorption was not observed in concrete samples with tuff as sand replacement. For the specimen with 15% tuff, a slight reduction in water absorption was noted. As a consequence, zeolite was considered more effective than tuff to enhance concrete’s resistance to permeability. The most remarkable reduction occurred in mixture T15Z15, with a 35% water absorption reduction at the age of 90 days in comparison with the control sample. The results confirmed the beneficial effect of tuff and zeolite on the durability of concrete. The relatively high level of water absorption of samples incorporating tuff was related to the percentage of clay, the void area inside the tuff, the glassy matrix which connected the particles and the voids between these particles, and the compact nature of the tuff .The reduction in water absorption was attributed to the pozzolanic reaction of zeolite developed in the concrete . The ratChloride ions can ingress into concrete in different ways including diffusion, capillary suction, migration or a combination of these mechanisms. Diffusion is the movement of dissolved ions due to a concentration gradient in a saturated porous material. This causes a net flow from regions of higher concentration to regions of lower concentration of the diffusing substance . The chl−12 m2/s, which was reduced by up to 55% when 15% tuff and 10% zeolite were used. Thus, it is identified that the resistance of the specimens to chloride diffusion was significantly enhanced by the presence of tuff and zeolite.As illustrated in 2 and Al2O3 in concrete can convert Ca(OH)2 into C-S-H gel and aluminates and eventually improve the micro-structure of concrete.In this experiment, specimens were made, cured and examined in identical conditions so that the rate of ingress of chloride into the concrete was highly related to the pore structure of the concrete, which was affected by different factors including the ingredients, particle packing, and the degree of reaction of the cementitious materials. This was also influenced by the inclusion of supplementary cementing materials which serve to subdivide the pore structure . The addIt can be seen that the chloride resistance results are in good agreement with the water absorption results, and it is apparent that the high resistance to water and chloride penetration may be related to the reduced porosity of concrete containing zeolite and stuff. It is believed that the addition of 15% zeolite and 15% tuff could reduce the porosity substantially. However, the effect of tuff on permeability was found to be higher when compared with the effect of zeolite. When these two materials were used together, a dense structure with improved resistance against chloride diffusion was observed. Previous work has also observed similar effect of zeolite on reducing the chloride diffusion. Ahmadi et al.  reported2SO4) solution. The results indicated that tuff did not have a positive impact on sulphuric acid resistance of concrete samples. All the specimens were found to have increased damage when tuff was used in the concrete. The deterioration of samples was mainly due to the decomposition of hydration products, in which the reaction of acid with lime increased the amount of highly soluble calcium by-products. A similar reduction in chemical resistance was also observed by other researchers [earchers .Another reason that made specimens with tuff more vulnerable to acid attack was that sand without pozzolanic function was replaced by a material with pozzolanic properties (tuff with high silica content). In this case, the specimens were made with more paste volume which was less resistant to the acid attack. However, the addition of zeolite seemed to alleviate the undesirable impacts of the addition of tuff in concrete specimens, since it contained 2.24% calcium in comparison with cement that contained 63.75% calcium. Consequently, addition of zeolite as a cement replacement is highly recommended in acidic environments, whereas the incorporation of tuff alone as a sand replacement in acidic environments does not appear to be beneficial.Sulphuric acid reacted with the calcium hydroxide and converted it to calcium sulphateThe calcium sulphate from the previous reaction reacted with calcium aluminate to form ettringite that could cause cracking and expansion.The calcium silicate hydrate reacted with sulphuric acid and the product was the silica gel.The mass loss in the control sample was 3.3% and the mass reduction of T5, T10 and T15 were 54%, 88% and 106% higher than the control sample. The mass loss in the concrete can be explained by the following reactions of sulphuric acid in concrete:2. The amount of Ca(OH)2 was not sufficient for pozzolanic reaction, and the concrete samples with tuff lost some parts of the C-S-H gel. The reduction in compressive strength was due to the following reasons:2 (at the beginning) and, later, C-S-H phases, dissolved, which formed new phases, such as calcium sulphates.Depth of reaction (DR): it is the depth which contains the weakened depth of the concrete specimen but is still attached to the concrete. In this weakened layer, components such as alkalis, Ca(OH)Depth of erosion (DE): it is the depth in which the paste can be totally extracted manually or by using a wire brush. The depth of erosion consists of the layer of the specimen from which the hardened cement paste is totally extracted from concrete. The DE was quantified after 236 days of immersion by measuring the dimensions of the samples. The results of average DE are presented in To study the effect of sulphuric acid on strength of concrete samples, the compressive strength of concrete samples was measured after 236 days of immersion. The compressive strength test results are given in On the other hand, the presence of zeolite as a cement replacement could rather compensate for the effects of tuff in concrete. The presence of zeolite could significantly reduce the calcium-containing compounds in the cement paste which led to less calcium hydroxide.The mass loss values of the specimens due to hydrochloric acid attack are given in The mass loss percentage in the control sample was 2.28% and the specimens with 5%, 10% and 15% tuff experienced 56%, 91% and 102% higher mass loss in comparison with control sample. Slightly less sensitive to HCl aggression were the mixes of T5, T5Z10, T5Z15 and T10Z10, which seemed qualified to be more resistant to acidic aggression. It is clear that the specimens immersed in hydrochloric acid showed similar degradation in terms of the mass reduction percentage. The chemical reaction of hydrochloride acid with lime is:2) is a highly soluble and harmful salt that can easily leach out of the concrete and therefore results in deterioration of concrete. Cherrak et al. [Calcium chloride (CaClk et al.  reportedConcrete containing 15% tuff and 10% zeolite showed the highest compressive strength.Replacement of cement by zeolite substantially decreased the water absorption. However, in concrete samples with tuff as sand replacement, considerable absorption was not observed.The chloride diffusion coefficient of the control specimen diminished by up to 55% in T15Z10 sample. Thus, it is identified that the chloride resistance of the specimens was significantly enhanced by the use of tuff and zeolite.The compressive strength reduction of concrete samples in sulphuric acid after 236 days of immersion varied between 9.2% and 15.7% in tuff-included samples and also 13.07% and 21.4% in tuff- and zeolite-included samples, however the strength reduction of plain concrete sample was 5.01%.The highest mass loss of 4.8% was recorded for specimen 15T15Z after 180 days of exposure to hydrochloric acid.Based on the results, the following conclusions are drawn:


MET alterations in the plasma of patients with diverse malignancies.We analyzed clinical associations of Digital sequencing of circulating tumor DNA (ctDNA) (54–70 genes) was performed in 438 patients; 263 patients also had tissue sequencing (182–315 genes). The most represented tumor types were gastrointestinal (28.1%), brain (24.9%), and lung (23.2%). Most patients (71.2%) had recurrent/metastatic disease.MET alterations were observed in 31 patients (7.1%) and correlated with bone metastasis (P = 0.007), with TP53 (P = 0.001) and PTEN (P = 0.003) abnormalities, and with an increased number of alterations  . Patients with MET alterations demonstrated a significantly shorter median time to metastasis/recurrence  and a poorer survival . Of the 31 patients with MET alterations, 18 also had tissue testing; only two also had tissue MET alterations (11.1%); MET alterations were detected at a lower frequency in tissue (1.14%) compared to ctDNA (7.1%), with P = 0.0002.MET alterations by liquid biopsy is feasible. MET ctDNA alterations were associated with a poorer prognosis, higher numbers of genomic abnormalities, and bone metastases. The correlation with bone metastases may explain the higher frequency of MET alterations in blood ctDNA than in tissue (since bones are rarely biopsied) and the previous observations of bone-predominant responses to MET inhibitors. The high number of co-altered genes suggests that MET inhibitors may need to be combined with other agents to induce/optimize responses.In conclusion, the detection of The online version of this article (10.1186/s13045-018-0610-8) contains supplementary material, which is available to authorized users. MET, also called c-MET or hepatocyte growth factor (HGF) receptor, is a receptor tyrosine kinase discovered as an oncogene in the 1980s . IndepenMET mutations are found in diverse human cancer .http://www.guardanthealth.com/guardant360/), a Clinical Laboratory Improvement Amendment (CLIA)-certified and College of American Pathologists (CAP)-accredited clinical laboratory. The analytical and clinical validation of Guardant360 was conducted in accordance with evidentiary standards established by the Standards for Reporting of Diagnostic Accuracy (STARD), REporting of tumor MARKer Studies (REMARK), Evaluation of Genomic Applications in Practice and Prevention (EGAPP), and the recent Next-generation Sequencing: Standardization of Clinical Testing (Nex-StoCT) biomarker guidelines [Digital sequencing of ctDNA (DNA) in all patients was performed by Guardant Health, Inc. . For 272 patients, a 68-gene version of the original panel  was used, and for 22 patients, the most recent 70-gene panel version (further expanded to amplifications in 18 genes and fusions in 6 genes) was applied  (N = 182 to 315 gene panels).In addition, 263 patients (~ 60%) of the 438 patients with ctDNA test also had CLIA/CAP-accredited next-generation sequencing (NGS) performed on tumor tissue  or last follow-up date (patients who had not recurred/developed metastases at last follow-up were censored on that date). Overall survival (OS) was defined as the time from diagnosis to death or last follow-up date for patients who were alive . Estimations for the time to first metastasis/recurrence and OS were done using Kaplan-Meier analyses and were compared among subgroups by the log-rank test for univariable analysis or Cox regression models for multivariable analysis. All statistical analyses were performed by author MS with SPSS version 24.0.N = 228) of the population. The majority of patients were Caucasian . The most represented tumor types were gastrointestinal , brain , lung , and breast  cancers. The majority of the patients had recurrent or metastatic disease at the time of blood draw used for testing  . Women comprised 52.1% . Sixteen patients had a MET amplification only; 13 had a somatic mutation only; and two had both an amplification and a somatic MET mutation  associated with lung cancers (11.8%) and the presence of metastasis/recurrence at the time of blood draw and negatively associated with brain tumors 1.8%  . The latter conclusions remained unchanged if the patients with brain tumors, lymphoma/leukemia, thymoma, and NF were excluded, with P values of < 0.05 for association between MET alterations and lymph node or bone metastases and increased number of metastases. cancers 1.8% and P ≤ 0.05) in univariate analysis), the only variables that remained statistically associated with MET alterations were aberrations in TP53 (P = 0.001) and PTEN (P = 0.003) genes, as well as an increased incidence of bone metastasis (P = 0.007)   Table . The uniTP53, PIK3CA, BRAF, KRAS, or MYC genes as well as the presence of metastasis in the lymph node or adrenal tissue . A log rank test demonstrated a poorer survival (time from diagnosis until death or last follow-up date) for patients bearing MET alterations . In the multivariable analysis, only lung cancer as well as the presence of ARID1A, KRAS, ALK, and MYC alterations and liver metastasis remained significant predictors of a poorer survival  had died at the time of our analysis  versus 10.4 months (95%CI 6.9–14.0) (P = 0.003) , along with the presence of liver (P = 0.022) or lymph node metastases (P < 0.001). Inversely, breast cancer correlated with a longer time to metastasis/recurrence (P < 0.001) . In these two patients, the time interval between the ctDNA and tissue biopsies was 1.8 and 15.3 months, and both tissues used for the testing were from the primary tumor.Of the 31 patients with MET amplification was detected in the tissue and not in ctDNA . The tissue test was performed on a pancreatic tumor that was surgically removed, and the ctDNA test was done more than 1 year later (recent scans showed appearance of new pulmonary and liver nodules 2 months prior the ctDNA testing).In only one patient, a P = 0.0002. Further, of eight patients who harbored MET alterations in ctDNA, and had ctDNA and tissue testing within 2 months of each other, only one patient showed a similar MET alteration in the tissue. Six of the seven patients with only ctDNA positive for MET alterations had bone metastases.Overall, MET alterations were detected at a significantly lower frequency in tissue  compared to ctDNA , MET alteration detection through blood-derived ctDNA. Liquid biopsy is a non-invasive method to find genomic aberrations and is increasingly utilized in the clinical setting as reflected by the non-small cell lung cancer National Comprehensive Cancer Network (NCCN) guidelines [MET ctDNA alterations were detected in 7.1% of patients with solid tumors. This detection rate is higher compared to previous tissue studies [MET tissue amplification determined by fluorescent in situ hybridization (FISH) demonstrated that 2.6% of 1115 solid tumor specimens were positive [MET tissue alterations [MET alterations between our study and the other studies could be due to following reasons: (i) our technology detected both single nucleotide substitutions and amplifications, while the previous investigation by Jardim et al. at MD Anderson Cancer Center [MET alterations than the ctDNA NGS: 3 of 263 patients (1.1%)  were positive for MET alterations in tissue versus 31 of 438 ctDNA-tested patients (7%) being positive for MET alterations (P = 0.0002). Further, of 18 patients positive for MET ctDNA alterations who also had tissue NGS, only 2 (11.1%) were also positive for a MET alteration by tissue NGS. The biologic underpinnings of discordance between ctDNA and tissue NGS results have been previously documented and include spatial (intra-tumor and inter-tumor) and temporal heterogeneity in genomic anomalies in cancers along with the fact that ctDNA is comprised of DNA that has leaked into the circulation from diverse metastatic sites while tissue NGS reflects only the tissue specimen analyzed [This is the largest study interrogating the feasibility and utility of idelines , 19. Our studies , 20, 21.analyzed .MET alterations in ctDNA. Of interest in this regard, our study showed that bone metastases were independently correlated with MET alterations , albeit without prostate surface antigen (PSA) response [MET alterations preferentially localize to bone and may therefore explain bone responses after MET inhibitor therapy. Tissue biopsies are rarely performed on bones . Of interest in this regard, MET is prominently expressed (as determined by immunohistochemistry) at the site of bone metastases in renal cell cancer [MET alterations, which strongly and independently correlated with bone metastases in our study, reflect shed MET alteration-bearing DNA from bone lesions  with ctDNA analyses also had tissue sequencing and the median time between the two tests in the patients with MET-altered ctDNA was about 6 months. Second, our patients did not have bone biopsies done, which would be of interest to determine if MET alterations are found in such biopsies, since ctDNA MET alterations correlate independently with the presence of bone metastases. Third, though our study with 438 patients is the largest to date, the rarity of MET alterations suggests that investigations of even greater numbers of patients may be worthwhile in order to best understand the biology and correlations of MET alterations.Our study has several limitations. First, only 60% of our patients — identifies potential tumor-related genomic alterations within 54 cancer-related genes including amplifications in ERBB2, EGFR, and MET. Only non-synonymous alterations were analyzed. Table S2. 68-gene panel (N = 272 patients), comprising amplifications in 16 genes as well as some fusions and indels. Only non-synonymous alterations were analyzed. Table S3. 70-gene panel (N = 22 patients). Only non-synonymous alterations were analyzed. Table S4. Comparison of clinical characteristics in 438 patients with or without MET alterations . (DOCX 20 kb)


Identifying optimal dosing of antibiotics has proven challenging—some antibiotics are most effective when they are administered periodically at high doses, while others work best when minimizing concentration fluctuations. Mechanistic explanations for why antibiotics differ in their optimal dosing are lacking, limiting our ability to predict optimal therapy and leading to long and costly experiments. We use mathematical models that describe both bacterial growth and intracellular antibiotic-target binding to investigate the effects of fluctuating antibiotic concentrations on individual bacterial cells and bacterial populations. We show that physicochemical parameters, e.g. the rate of drug transmembrane diffusion and the antibiotic-target complex half-life are sufficient to explain which treatment strategy is most effective. If the drug-target complex dissociates rapidly, the antibiotic must be kept constantly at a concentration that prevents bacterial replication. If antibiotics cross bacterial cell envelopes slowly to reach their target, there is a delay in the onset of action that may be reduced by increasing initial antibiotic concentration. Finally, slow drug-target dissociation and slow diffusion out of cells act to prolong antibiotic effects, thereby allowing for less frequent dosing. Our model can be used as a tool in the rational design of treatment for bacterial infections. It is easily adaptable to other biological systems, e.g. HIV, malaria and cancer, where the effects of physiological fluctuations of drug concentration are also poorly understood. In this era of rising concerns about antibiotic resistance, the rational design of optimal antibiotic treatment regimens remains an important unrealized goal. At this time, the characteristics of antibiotic treatment regimens  are determined largely based on costly in vivo experiments. The sheer number of possible dosing strategies that must be tested contributes to the delay and cost of the development of new drugs and may limit the feasibility of finding optimal regimen characteristics. Here, we demonstrate how modeling the chemical kinetics of drug-target binding can identify the best time-concentration profile of antibiotics. Using both analytical approaches and numerical simulations, we find that the physicochemical characteristics of drug-target binding are sufficient to explain the pharmacodynamics of commonly used antibiotics such as ampicillin, isoniazid and tetracycline. In practical terms, our models can be used as a tool in the rational design of treatment for bacterial infections. Because of the generality of drug-target binding kinetics, these approaches may also be adapted to other diseases where the effects of physiological fluctuations of drug concentration are also poorly understood, such as HIV, malaria and cancer. The rise of antibiotic resistance underlines the need for employing existing antibiotics prudently. Although antibiotic dosing regimens have been investigated for more than half a century , we do nCmax) or iii) the time during which the concentration exceeds a specific threshold  would be optimized by employing different dosing strategies, for example by using large intermittent doses to increase Cmax or by employing extended release formulations to increase TC>MIC  is given by Surprisingly, we find that the system approaches equilibrium more quickly when ions see . Initial quickly .fc, i.e. the time of onset of the antibiotic action (tonset), and can be compared to the blue and green lines in The delay until an antibiotic is effective depends on many physiological and biochemical factors. Since this model focuses on the reaction kinetics alone (ignoring diffusion barriers and concentration gradients), Model 1 provides a lower bound for the expected delay until onset of antibiotic action. Even here, for reasonable parameter settings, we find that even this delay can extend for several hours. One potential approach for speeding antibiotic-target binding and reducing delay to onset of action is to increase antibiotic exposure through higher dosing. Lower thresholds and slower turnover are associated with delays until antibiotic action; these effects can be overcome by increasing the drug concentration . The ligfc may have additional benefits if they extend bacterial suppression beyond the time that the antibiotic concentration exceeds the MIC.Bacterial growth often remains suppressed after the antibiotic concentration drops below the MIC (i.e. the post-antibiotic effect). This effect occurs because drug-target complex dissociation is not instantaneous. Therefore, high drug concentrations that saturate the target beyond the threshold required for antibiotic action tbound. When the threshold required for antibiotic action fc is very high, the antibiotic stops working very rapidly and the length of the post-antibiotic effect is brief and relatively insensitive to the half-life of the drug-target complex. Conversely, when there is both a low threshold and a slow turnover time of drug-target binding, the post-antibiotic period may last for several hours.We use our model to identify the conditions in which high antibiotic concentrations are expected to prolong antibiotic action. For simplicity, for these simulations we assume that at the time of antibiotic withdrawal, 99.9% of the target is bound and that the antibiotic concentration both inside and outside of the bacterial cell immediately drops to zero. Under these assumptions, Eq  can be sNext, we investigate the dynamics of drug target binding under different dosing regimens. We use ampicillin as an example, because a large body of literature describes the time-dependent action of beta-lactam antibiotics, both in experimental models as well as in patients –30. In afc.Beta-lactams acetylate penicillin-binding proteins , and thereby inhibit cell wall synthesis. The acetylation of PBPs consumes beta-lactams, and therefore the drug-target reaction is not reversible. However, PBPs are constantly de-acetylated and the effects of the antibiotic are therefore reversible. The kinetics of PBP acetylation and de-acetylation as well as target occupancy at MIC have been determined experimentally . In singTC>MIC, AUC or Cmax are the best predictors of antibiotic efficacy, we model three simplified dosing strategies: i) an idealized bolus injection where the drug concentration immediately reaches its peak and then declines exponentially, ii) a hypothetical pharmacokinetic curve where the antibiotic concentration is maintained just above the MIC (1.01x MIC) for the same length of time >MIC as in i) and then falls instantaneously to 0, and iii) a curve of similar shape to ii) that retains the same area under the curve as i)  see . In ii),kr[AT] for the differential equation describing the intercellular antibiotic [A]i. For all three dosing strategies, antibiotic action starts immediately after the drug concentration rises above MIC and stops as soon as the drug concentration falls below MIC, i.e. increasing the AUC alone without increasing TC>MIC does not change antibiotic action substantially  and the half-life of the drug-target complex (tbound). A long exposure to ampicillin at MIC is expected to result in a target saturation that just reaches the critical threshold fc = 95.4% , [T], [AT] rather than the absolute number of molecules. We assume that the total concentration of target/cell [T0] is constant. In this case, the concentration of free target can be described as [T] = [T0] − [AT]. Assuming that cells are treatment-naïve, i.e. there are no bound target molecules at the beginning, the kinetics of antibiotic-target reaction can then be described by a single differential equation, which can be simplified if we assume the intracellular antibiotic concentration [A] is constant:The dynamics of this system are governed by the concentrations fc, is reached. Thus, the MIC is the antibiotic concentration at which the equilibrium fraction of bound antibiotic is exactly fc: i.e.At a certain point, the fraction of bound target reaches a critical threshold at which the net growth of the bacterial population is zero. In this framework, the MIC is characterized as the minimal antibiotic concentration at which this critical percentage of bound target, xMIC) and thereby replacing [A] with Expressing all antibiotic concentrations as fold-MIC .Ae, the number of extracellular antibiotic molecules, Ai, the number of intracellular antibiotic molecules, T, the number of free target molecules, and AT, the number of drug-target complexes. For bolus injections, the model is described by the following set of equations:Model 2 includes the following compartments: c and after a specified time (tend) is assumed to fall instantaneously to 0 (i.e. intravenous dosing), the extracellular antibiotic concentration is given by:To model an alternative drug administration approach in which the antibiotic concentration is maintained at a constant level xMIC) using Eq , target binding occurs after drug activation to the adduct INH•NAD  which depends on NAD content and oxygen saturation. Here, we focus on INH binding to the enoyl reductase InhA, which is then present in its inactive form InhAi. Assuming NAD and target molecule concentration as well as oxygen saturation remain constant, the number of molecules in each compartment is described by the following set of equations:Here, we use the same equations and parameters as in Figure 7 in , extendeThis set of equations is based on  and we ar is proportional to ff = 1- fc and that there is no growth when the fraction of free ribosomes falls below this critical threshold:Finally, Model 3 expands on Model 2 by allowing the reproduction of target molecules that would occur as a result of bacterial replication and also allows for unspecific binding.  This model describes antibiotics that only suppress bacterial growth but do not increase bacterial killing (i.e. bacteriostatic agents). For bacteriostatic translation inhibitors such as tetracycline, the bacterial replication rate depends linearly on the fraction of free ribosomes . We therB  that can reproduce until they reach a maximal carrying capacity K, the extracellular and intracellular number of antibiotics Ae and Ai, and the intracellular concentration of drug-target complexes AT and unspecifically bound antibiotic AU. The rates kf and kr describe specific binding and dissociation, the rates ku,f and ku,r describe the rates for unspecific binding and dissociation. Data indicate that the total number of ribosomes increases linearly with cell volume; this means that the intracellular concentration within a single cell between the time of its “birth” and the split into two daughter cells remains relatively constant [T = BT0 − AT with T0 describing the fixed number of total target molecules per cell. The growth of bacteria exposed to sub-MIC concentrations of a translation inhibitor can then be described by the following set of differential equations :Here, we track bacterial cells constant . We can Again, this equation is based on , in addiS1 Fig(A) replication rate h-1; (B) elongation rate in pixel/min. Experimental mean in 5 min intervals (thick black line), experimental mean for entire pre-, peri- and post-exposure period (blue line), experimental minimum and maximum in 5 min intervals , experimental minimum and maximum for entire pre-, peri- and post-exposure period (blue shaded area). 20 bacteria were observed and exposed to 6 mg/L ampicillin (0.8x MIC) for 16 h. For details see [Cells were grown in a flow chamber supplied with medium without antibiotics for 4 h , exposed to antibiotic for 16h , followed by growth in medium that did not contain antibiotics for 4h ). ails see , 71.(TIF)Click here for additional data file.S2 Figfc. The dotted vertical lines indicate beginning and end of antibiotic action. The time the antibiotic is active, TC>MIC and AUC are given in the figure title. Graphs in the first column depict bolus injections with an initial antibiotic concentration of 50MIC and a half-life of 1/2h, the half-life in the second column is 1h and the half-life in the third column is 2h. All graphs show drug-target binding expected based on physicochemical characteristics of ampicillin drug-target binding from the literature ((A) Includes no diffusion barrier, (B) includes a diffusion barrier with p = 10−4, and (C) an diffusion barrier with p = 10−5.The x-axes show the time after initiation of antibiotic therapy in hours, the y-axes the current antibiotic concentration in fold MIC  and the % bound target . The green line shows the antibiotic concentration outside and inside the cell (assuming that there is a negligible diffusion barrier), the violet line shows the amount of bound target (refers to y-axis on the right). The grey area indicates that either the antibiotic concentration is below MIC or the fraction of bound target is below the inhibitory threshold terature . (A) Inc(PDF)Click here for additional data file.S3 Figfc. The dotted vertical lines indicate beginning and end of antibiotic action. Graphs in the first column depict bolus injections with an initial antibiotic concentration of 50MIC and a half-life of 1h. The second column shows a hypothetical dosing regimen with a constant concentration just above the MIC (1.01 MIC) that has the same TC>MIC as in the first column. The third column shows a hypothetical dosing regimen with a constant concentration just above the MIC (1.01 MIC) that has the equivalent area under the curve (AUC) as in the first column. Note the different timescale in the third column. All graphs show drug-target binding expected based on physicochemical characteristics of ampicillin drug-target binding from the literature ((A)tbound = 19h 15 min (kr = 10−5). (B)fc = 10%.The x-axes show the time after initiation of antibiotic therapy in hours, the y-axes the current antibiotic concentration in fold MIC  and the % bound target . The green line shows the antibiotic concentration outside and inside the cell (assuming that there is a negligible diffusion barrier), the violet line shows the amount of bound target (refers to y-axis on the right). The grey area indicates that either the antibiotic concentration is below MIC or the fraction of bound target is below the inhibitory threshold terature  with the(TIF)Click here for additional data file.S4 FigTC>MIC, AUC and Cmax)were kept, only Cmax is significantly correlated with tonset. The statistical programming software R was used, output of the function summary(step(lm)).We used the Akaike Information Criterion as implemented in the function step in R to identify the model that best describes the data. This function would drop all explanatory variables that do not improve model quality. While all explanatory variables ((PNG)Click here for additional data file.S1 Moviefc. The dotted vertical lines indicate beginning and end of antibiotic action.These graphs show the expected dynamics of antibiotic-target reaction according to model 2 with all parameters adapted to ampicillin see . The lef(MOV)Click here for additional data file.S2 MovieThe setup is the same is in movie 1, but this movie corresponds to (MOV)Click here for additional data file.S3 Moviep = 10−4 /s and a threshold fc of 10%.The setup is the same as in movie 1, but we assume a diffusion barrier of (MOV)Click here for additional data file.S4 Moviep = 10−4 /s and a threshold fc of 10%.The setup is the same as in movie 1, but we assume a diffusion barrier of (MOV)Click here for additional data file.S1 TableCmax and short half-lives, the critical threshold could not be reached, i.e. the dosing strategy did not have an antibacterial effect. Values are given in hours.Note that for combinations of low (DOC)Click here for additional data file.


We report a case of membranous nephropathy associated with type 1 autoimmune pancreatitis. A 58-year-old man presented with anorexia. Work-up revealed a mass in the pancreatic head, which was subsequently resected. Pathological examination showed diffuse infiltration of immunoglobulin (Ig) G4-positive plasma cells, which was compatible with the diagnosis of type 1 autoimmune pancreatitis. Serum IgG4 was elevated. He developed nephrotic syndrome around the time of the surgery. Kidney biopsy confirmed the diagnosis of membranous nephropathy. Immunofluorescent staining showed predominant glomerular IgG4 deposit among IgG subclasses. Tubulointerstitial nephritis, which is usually a dominant feature of renal involvement in IgG4-related disease, was not observed. The patient was treated with prednisolone and several immunosuppressants. During the course, the degree of proteinuria was associated with the serum IgG4 level. Serum antibody against phospholipase A2 receptor was negative. These findings together with IgG4-dominant glomerular deposit suggest that IgG4 may play a unique role in the pathogenesis of secondary membranous nephropathy caused by IgG4-related diseases. Immunoglobulin (Ig) G4-related disease (IgG4-RD) is characterized by increased serum IgG4 levels, caused by the infiltration of IgG4-positive cells into various organs –3. RenalWe describe a unique case of MN associated with IgG4-RD. The patient presented with IgG4-dominant deposit in the glomerular capillary wall and no associated TIN. The degree of proteinuria and serum IgG4 levels were associated and serum anti-PLA2R antibody was negative.The patient is a 58-year-old Japanese man with no previous medical history. In September 2010, he presented with anorexia. He was not on any medications. Serum transaminase and alkaline phosphatase levels were elevated. Urinalysis, renal function, serum total protein, and albumin levels were as follows: urinalysis: 3+ protein and 3+ occult blood, blood urea nitrogen 17 mg/dL (6.0 mmol/L), serum creatinine 0.67 mg/dL (51.1 μmol/L), total protein 6.2 g/dL (62 g/L), albumin 3.1 g/dL (31 g/L). Enhanced computed tomography of the abdomen showed localized tumor on the pancreatic head with no kidney abnormalities. Magnetic resonance cholangiopancreatography showed tumor on the pancreatic head without pancreatic duct dilation and stenosis. He underwent subtotal stomach-preserving pancreatoduodenectomy for localized tumor on the pancreatic head. Histological examination of the pancreas showed diffuse infiltration of plasma cells and lymphocytes without evidence of malignancy  and 24-h urinary protein excretion was 15.7 g. He also developed portal vein thrombosis.In December, he was transferred to our hospital for the evaluation of nephrotic syndrome. On admission, he was taking candesartan, furosemide, spironolactone, warfarin, aspirin, famotidine, and voglibose. On examination, he was normotensive and had bilateral lower extremity edema. Urinalysis showed 4+ proteinuria and 2+ occult blood. Other laboratory data were as follows (reference range in parentheses): blood urea nitrogen: 13 mg/dL (4.6 mmol/L), serum creatinine: 0.7 mg/dL (53.4 μmol/L), albumin: 1.8 g/dL (18 g/L), total cholesterol: 140 mg/dL, C-reactive protein: 0.01 mg/dL (9.52 nmol/L), hepatitis B surface antigen: negative, hepatitis C antibody: negative, antinuclear antibody: negative, C3: 85.3 (65–135) mg/dL, C4: 22.0 (13–35) mg/dL, IgG: 934 (870–1700) mg/dL (9.34 g/L), IgA: 148 (110–410) mg/dL (1480 mg/L), and IgM: 70 (35–220) mg/dL (700 mg/L). His serum IgG4 level was elevated to 377 (8–105) mg/dL.In January 2011, a renal biopsy was performed. Light microscopy showed normocellular glomeruli with mild thickening of the glomerular capillary walls and normal mesangial matrix  per well as an antigen. The patient’s plasma diluted 1:1000 was incubated with the antigen. After washout, horseradish peroxidase-conjugated anti-human IgG1–G4 diluted 1:1500 was added . Anti-PLA2R antibodies were not detected in the patient’s serum. Absorbance at 450 nm was 0.093 for IgG4 . Absorbance at 450 nm for IgG1–3 was not significantly elevated compared with the negative control.The patient was treated with prednisolone  and cyclosporine. There was improvement in the urine protein to creatinine ratio, but serum albumin remained low Fig. . Three mWe reported a case of MN associated with IgG4-RD. This case has several distinct features compared with previous reports: (1) the severity of proteinuria was associated with the serum IgG4 level; (2) anti-PLA2R antibody was negative; (3) immunofluorescence staining showed IgG4-dominant deposition among IgG subclasses in the glomerular capillary wall; (4) no associated TIN was observed.A literature review revealed 10 cases of biopsy-proven MN associated with IgG4-RD –24. A clA pathological feature of renal involvement of IgG4-RD is usually TIN, and MN was observed in only 8 % of cases . As far MN could be idiopathic or secondary to various diseases. The diagnosis of IMN is usually by the exclusion of secondary causes, but several features could help the differentiation of idiopathic from secondary MN. Anti-PLA2R antibody was positive in 68.5–81.7 % of IMN, whereas it was positive in only 0–12.2 % of secondary MN –28. HoweIn summary, we reported a case of MN without TIN associated with IgG4-RD. The association of serum IgG4 and proteinuria and negative anti-PLA2R antibodies suggested that MN was secondary to IgG4-RD. There was dominant IgG4 deposit in the glomeruli. Our case suggests the possible role of IgG4 in the pathogenesis of MN more clearly than previous cases. The antigen against IgG4 in our case needs to be identified in a future study.


Experimental investigation of GFRP (glass fiber reinforced polymer) tubes that were filled with magnetically driven concrete was carried out to study the flexural behavior of specimens under bending. Specimens having different cross section and lengths were tested. The test specimens were fabricated by filling magnetically driven concrete into the GFRP tubes and the concrete was vibrated using magnetic force. Specimens vibrated using vibrating tube were also tested for comparison. In addition, specimens having steel reinforcing bars and GFRP bars were both tested to study the effect of reinforcing bars on the magnetically driven concrete. The load-displacement curves, load-strain curves, failure mode, and ultimate strengths of test specimens were obtained. Design methods for the flexural stiffness of test specimens were also discussed in this study. Since the application of concrete from the nineteenth century, it is always vibrated by mechanical force. Recently, the magnetically driven concrete was proposed by Chen et al. . In the Due to its excellent performance for high strength, light weight, and corrosion resistance, fiber reinforced polymer (FRP) has promising application in structural engineering. There are many researches towards the GFRP tubes filled with normal concrete under bending. Mirmiran and Shahawy  investigIn this study, concrete-filled GFRP tubes filled with magnetically driven concrete were fabricated. The filled concrete was vibrated by a magnetic vibration device developed by authors. Bending test was conducted to investigate the flexural behavior of the test specimens. In addition, the effect of reinforcing bars was also investigated.In the magnetically driven concrete, the coarse aggregates were totally replaced by steel slag (in weight). The mix proportions of the concrete are presented in Three series of GFRP tubes, namely series A, B, and C were used in this study. Series A has the nominal diameter of 300 mm and nominal thickness of 12 mm. Series B has the nominal diameter of 300 mm and nominal thickness of 8 mm. Series C has the nominal diameter of 180 mm and nominal thickness of 8 mm. GFRP tubes of Series A and B were fabricated by winding method with the direction of 45°, while the Series C was fabricated by extrusion and winding method with the directions of 45° and 0°.The reinforcing bars used in this study are Grade HRB335 with the nominal diameter of 6 mm. The nominal diameter of GFRP bars is also 6 mm. The stirrups used are Grade HPB300 with the nominal diameter of 6 mm. The layout of the bars and stirrups is shown in A magnetic vibration device was developed for concrete vibration, as shown in The four solenoid coils could move along the length of specimen in order to vibrate the concrete of whole specimen, as shown In order to vibrate the concrete from 360 degree, the base of the device is able to rotate during the vibrating process with the speed of 30 r/min. In this case, the magnetically driven concrete filled inside the GFRP tube could be vibrated evenly and the coarse aggregates are well-distributed. Each time about 200 mm height concrete were filled and vibrated for about 1.5 min. The magnetic field was controlled reversed at the interval of 0.25 s.In total, 13 test specimens were fabricated and specimen lengths are 1500 mm and 2000 mm, respectively. Size and number of the specimens are given in The first letters indicate that the type of the bars, where the prefix letter “G” refers to GFRP bars while the letter “R” refers to steel reinforcing bars.The following four digits “2000” indicate the length of the specimen in mm.The following three digits “300” indicate the outer diameter of the specimen in mm.The following two digit “12” is the nominal thickness of the GFRP tube in mm.The last character “A” refers to the specimen was vibrated using vibrating tube while the character “B” refers to the specimen was vibrated using magnetic method.For example, the label “G2000-300-12A” defines the specimen as follow:Four points bending test was conducted and the test specimens were loaded by means of a spreader. Each specimen was simply supported with a 100 mm extent portion overhang at each edge support. Some unloading–reloading cycles with small amplitude of load was initially applied in the elastic domain. Then, the load was slowly applied at the constant speed of 1.0 mm/min. A 10,000 kN YAW-10000F hydraulic testing machine  was used to apply the load with displacement control, which allowed for the tests to be continued to the post-ultimate stage. The test setup is shown in cuf) obtained on 150 mm cubic specimens at 28 days for concrete vibrated by magnetic method and vibrating tube were 44 MP and 46 MPa, respectively. Concrete cubic specimens used the same materials as the beam specimens. The compressive test of concrete cubic followed the Chinese Standard [Tensile coupon tests were performed on the same batch of materials to obtain the properties of GFRP used in the test specimens. The measured material properties of GFRP tubes are shown in The load-displacement curves at the mid-span of test specimens were presented in Generally, it is shown that the mid-span deflections of specimens vibrated using magnetic method are slightly larger than those specimens vibrated using vibrating tubes. This may mean that the elastic stiffness of concrete vibrated using magnetic method is slightly small than concrete vibrated using vibrating tubes. However, the difference is very small and the effect of magnetic vibration method is acceptable.There is an obvious slip between the GFRP tubes and concrete for specimen under bending, as shown in Typical load-strain curves of test specimen series G2000-300-8 and R2000-300-12 are shown in uM = 0.6 uN/2 and uM = 0.5 uN/2 for 2000 mm and 1500 mm specimens, respectively. Specimen G2000-300-12A has the maximum ultimate strength of moment (uM), while specimen G2000-180-8 has the minimum maximum ultimate strength of moment (uM). It is shown the 1500 mm specimens have relative large uN value when compared with 2000 mm specimens having the same cross section, which may due to the failure mode. The comparison between specimen series A and B indicates that the vibration method has a negligible effect on the ultimate strength of test specimens.All of the test specimens failed in the crack of outer GFRP tubes and typical failure modes are shown in Two methods were used to calculate the flexural stiffness in this study. The first method is specified in GB 50936  for concscmB of concrete-filled steel tubes specified in GB 50936 [The flexural stiffness GB 50936  could bescI could be calculated as Equations (4) and (5)sA is the cross section area of GFRP tube (mm2); cA is the cross section area of concrete (mm2).When there is tensile stress, EI)eff of concrete-filled steel tubes specified in Eurocode [The effective flexural stiffness (Eurocode  could beEI)eff are smaller than the flexural stiffness scmB, since the confined effect on concrete is not considered in Eurocode [The calculated results using Equations (1)–(7) are presented in Eurocode . The loaEurocode  agree wiBending test on GFRP tubes filled with magnetically driven concrete was conducted in this study. Test results of load-displacement curves, load-strain curves, failure mode, and ultimate strengths were obtained. It is shown that the flexural behavior of specimens vibrated using magnetic method and specimens vibrated using vibration tube is similar. The steel reinforcing bars have almost no effect on the magnetic vibration. There is obvious slip between the GFRP tube and concrete at the end of test specimens. Design methods that are specified in current Eurocode and GB for concrete-filled steel tubes were used to calculated the flexural stiffness of test specimens. It is shown that the predictions from GB agree with test results well for specimen series 2000-180-8 and specimens having length of 1500 mm. For other test specimens, the predictions from Eurocode agree with the test results well.


Biotechnol. Prog., 33:854–866, 2017This paper presents a systems approach to evaluating the potential of integrated continuous bioprocessing for monoclonal antibody (mAb) manufacture across a product's lifecycle from preclinical to commercial manufacture. The economic, operational, and environmental feasibility of alternative continuous manufacturing strategies were evaluated holistically using a prototype UCL decisional tool that integrated process economics, discrete‐event simulation, environmental impact analysis, operational risk analysis, and multiattribute decision‐making. The case study focused on comparing whole bioprocesses that used either batch, continuous or a hybrid combination of batch and continuous technologies for cell culture, capture chromatography, and polishing chromatography steps. The cost of goods per gram (COG/g), E‐factor, and operational risk scores of each strategy were established across a matrix of scenarios with differing combinations of clinical development phase and company portfolio size. The tool outputs predict that the optimal strategy for early phase production and small/medium‐sized companies is the integrated continuous strategy  perfusion, continuous capture, continuous polishing). However, the top ranking strategy changes for commercial production and companies with large portfolios to the hybrid strategy with fed‐batch culture, continuous capture and batch polishing from a COG/g perspective. The multiattribute decision‐making analysis highlighted that if the operational feasibility was considered more important than the economic benefits, the hybrid strategy would be preferred for all company scales. Further considerations outside the scope of this work include the process development costs required to adopt continuous processing. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers  A major challenge facing the biopharmaceutical industry centers on how best to improve research and development (R&D) productivity while reducing R&D and manufacturing costs.In recent years, continuous bioprocessing has seen a resurgence of interest related to its potential to challenge the established position of the batch platform for the production of biopharmaceuticals such as monoclonal antibodies (mAbs). As a result, several companies have been evaluating continuous bioprocessing technologiesA holistic assessment of continuous bioprocessing requires evaluation of the potential of integrated continuous bioprocesses across multiple products moving through the development pathway. This paper aims to achieve this by developing and applying a prototype decisional tool to address the following topical questions. Is there a business case for continuous bioprocessing for early phase manufacture? How does the business case change for commercial multiproduct manufacture? Will tomorrow's process be a hybrid of batch and continuous technologies? This study builds upon previous studies at UCLFed‐batch cell culture has been established as the platform choice for most mAbs in recent years given the increase in fed‐batch titres combined with their ease of operation.® and Stelara®, Janssen Biotech).Pollock et al.On the process economics front, there are few contributions that examine the economic feasibility of filtration‐based perfusion culture systems. Lim et al.The current and future mAb purification platforms rely on a series of orthogonal chromatography steps with Protein A as the preferred primary capture step.Semicontinuous chromatography has been shown to be an effective way to increase resin utilization and hence reduce resin volumes required by 40–50%, step buffer consumption by 40–50% and overall process buffer consumption by 12–15%.Financial implications of adopting continuous chromatography have been presented by Pollock et al.Recent papers have detailed successful implementation of integrated, closed, and continuous bioprocesses linking an alternating tangential flow perfusion process to two semicontinuous chromatography steps for capture and polishing.This paper scopes out a vision for a number of integrated continuous manufacturing processes and utilizes a decisional tool to provide novel insights on the suitability of these future manufacturing strategies across a matrix of industry scenarios. The paper presents a framework for evaluating integrated continuous processes rather than stepwise comparisons carried out in previous work. The analysis is extended to derive and incorporate operational risk scores considering the adoption of continuous technologies. These scores are combined with the calculated economic and environmental performance metrics so as to give an overall manufacturing strategy ranking. The impact of both development phase and company size is explored on the optimal manufacturing strategy in terms of batch and continuous unit operations. In all scenarios, the models spanned the whole bioprocess from cell culture through to polishing so as to capture the full impact of any upstream or downstream choice on the overall process economics and the impact of multiproduct facilities explored at each phase of development.A key concept of an integrated continuous process is that continuous, steady‐state processing extends from the bioreactor to the final purification operation. However, this concept is currently not possible in biopharmaceutical manufacture due to the lack of suitable technology and strict regulatory requirements. Continuous perfusion bioreactors for example do generate a continuous stream of harvested cell culture fluid (HCCF), but they can only achieve this in a batch operation. First, the cell culture has to reach the desired steady‐state cell density to achieve a constant concentration of HCCF and then can only produce a continuous stream of HCCF for a defined period, before a new cell culture batch is required. This semicontinuous mode of operation is also found in the search for continuous downstream processing operations where chromatography systems that are described as continuous are capable of continual loading but only generate discrete elution pools of product. The challenge is further complicated by the stringent quality and regulatory requirements that dominate biopharmaceutical manufacture. The ability of the manufacturing process to demonstrate viral clearance is critical, with a mandatory inclusion of two dedicated viral clearance operations . Both of these operations are currently achieved in a batch operation; for example, in a viral inactivation step the product stream is held at a low pH for a defined period of time, before processing continues. A further regulatory complication is batch traceability, a key area of debate surrounding continuous processing, with the principal concern being “how do you define a batch?.”These factors highlight how continuous processing is not currently possible in biopharmaceutical manufacture. However, the use of semicontinuous unit operations can lead to a semicontinuous manufacturing process, potentially capturing some of the economic advantages seen in continuous processing. The upstream can be operated in a semicontinuous manner by using perfusion culture, which is fed and bled at a constant rate to generate a constant stream of HCCF when steady‐state cell density is achieved. The downstream is more complicated due to the number of orthogonal purification operations. The initial capture of the product from HCCF can be achieved in a continuous manner, using semicontinuous chromatography. The resulting process stream is now being created in discrete elution volumes, which can either be pooled into larger volumes or processed individually before moving to the subsequent purification steps. These subbatches can be processed in the conventional batch manner for the remaining purification steps or can be processed in a continuous manner.−2 h−1)) seen in the batch processes A precalculation and optimization module was created to run prior to the simulation of a particular scenario so as to assess all the possible equipment sizing strategies before selecting the optimum process configuration  for the given demand in terms of both production time and cost. Alongside equipment scaling the precalculation module also evaluated whether single‐use technologies could be implemented in the form of single‐use bioreactors and product holding bags, based on the process volumes generated and utilized by each unit operation.The key economic metrics calculated by the tool were the capital investment and the cost of goods per gram (COG/g) for the complete manufacturing process (fermentation to bulk drug substance), as well as the manufacturing cost per launch . The capital investment was determined using the Lang factor method as a function of the total equipment purchase cost. The COG/g comprised the annual direct  and indirect (depreciation and facility‐dependent overheads) operating costs divided by the annual product output as detailed in Farid et al.The weighted sum method provides a systematic mechanism for explicitly capturing qualitative and nonfinancial performance indicators in the evaluation. Its use has been illustrated in several fields including bioprocessingSj is generated by the weighted sum method, using the following equation:ijecononmicr, ijenvironmentalr, ijoperationarl represent the weighted scores for the economic, environmental and operational groups respectively (prior to combination) and R1, R2, and R3 represent the economic, environmental, and operational combination ratios, respectively.All attribute values were standardizedThe decision‐support framework was used to compare the cost‐effectiveness of the five alternative manufacturing strategies throughout the development pipeline for a range of company sizes, exploring the trade‐offs between reduced equipment scales versus increased manufacturing risk. Table The base case manufacturing strategy used in the case study was based on a generic two‐column mAb process.Table The decision‐support framework was used to assess the cost‐effectiveness of five manufacturing strategies with different combinations of batch and continuous operations for cell culture, capture, and polishing steps throughout the drug development pipeline. This was initially carried out by determining the direct  and indirect (depreciation and facility‐dependent overheads) costs. These were used to establish the cost of goods per gram across combinations of different development phases  and company sizes . Each development phase required different manufacturing scales, batch numbers and material reuse strategies, and each company size resulted in different numbers of drug candidates at each development phase. The economic outputs were then considered alongside operational and environmental metrics using a multiattribute decision‐making technique for all the company sizes investigated.Figure The labor and QCQA costs are scale‐independent and rise in proportion to the increase in number of batches required per product across the development phases rather than the kg output Figure a. As a rTable Figure The base case is still dominated by indirect costs in the later manufacturing scales in the development pipeline . For example, the Phase III COG/g is dominated (70% of COG/g) by the batch suite cost (indirect costs), due to the costs only being spread over four batches in a commercial GMP facility. The continuous strategy sees a shift from the direct to indirect costs dominating COG/g for the later manufacturing scales. However, the labor costs still account for a third of the COG/g. Traditionally the direct costs are expected to dominate with an increase in kg output, however, due to the relatively low kg output in this scenario (200 kg) the indirect costs continue to dominate COG/g. The indirect COG/g does decrease in significance with increasing company size with direct costs starting to dominate as kg output increases (large company—2 × 200 kg products).The smaller batch sizes seen earlier in the development pipeline have higher direct manufacturing costs per gram because some of the costs are scale independent. For example the QCQA batch release costs  are constant between a Pre‐Clinical and Commercial batch, but due to the difference in kg output (0.5–200 kg) the batch strategies Pre‐Clinical QCQA cost per gram is $73/g compared to $3.4/g for the Commercial manufacturing scale. The same trend is seen in the batch strategy`s labour costs which accounts for nearly half the Pre‐Clinical direct manufacturing costs at $128/g compared to the Commercial manufacturing scale with labour costs of $6/g. The overall percentage of direct costs increases for the Commercial manufacturing of the batch strategy, even with the real decrease in direct costs per gram. This is due to the significant drop seen in batch suite costs, from the Phase III ($600K per batch) to Commercial ($240K per batch) manufacturing scale.For the medium‐sized company, the tool outputs predict that the integrated continuous ATF‐CC strategy offers cost savings for Pre‐Clinical and Clinical production, but becomes less economically attractive at the Commercial scale. This is due to the requirement for a second manufacturing production line given the kg output per batch being matched to the Phase III requirement 10 kg) and maximum number of batches per year, kg and mThe impact of both manufacturing scale  and company size  on the competiveness of the five alternative manufacturing strategies was investigated. The contour plots in Figures Figure Figure The manufacturing cost per launch of a successful drug candidate (DC) was a key economic metric used to compare the alternative manufacturing strategies encompassing the total risk‐adjusted clinical development manufacturing cost. The manufacturing cost per launch captures the costs of all the unsuccessful DCs incurred alongside the development of a successfully commercialized DC . Figure Figure This section extends the analysis beyond economic metrics to include the environmental and operational benefits of each strategy.The tool was also used to capture the water and consumable usage of the alternative manufacturing strategies to assess the environmental impact of the strategies across a range of manufacturing and company scales. E‐factor values were derived for the usage of water  and consumables  within the manufacturing process. Typical mAb manufacturing strategies (base case) consume water from 3,000 to over 7,000 kg water per kilogram product. The cell culture steps consume between 20% and 25% of the total, with the chromatographic operations often surpassing 50% of the total.As expected, all the alternative manufacturing strategies have a lower water E‐Factor value in comparison to the base case strategy, where the difference in water usage can be directly related to the use of the continuous capture step and the resulting lower buffer requirement. Table The consumable E‐Factor values are highly dependent on the amount of single‐use technologies and resin volume employed by the strategies. Table A risk score was assigned to each manufacturing strategy to assess the operational feasibility with respect to strategy robustness (likelihood of batch failure). The risk score is a ranking value used to compare the alternate strategies and does not capture a true value for batch failure risk. Table R1 = 0.6, R3 = 0.3) the ATF‐CB and FB‐CB strategies are equally attractive. The ability of the ATF‐CB strategy to offer a superior ranking as the importance of the economic benefits increases is due to superior savings offered in the cost per launch and capital expenditure at this company scale. Figure This section discusses the results from deriving a single multiattribute score so as to reconcile any conflicting outputs. The impact of the relative importance of the economic, environmental, and operational scores on the ranking of the manufacturing strategies was explored by determining the overall aggregate strategy score for a range of combination ratios. Figure This paper has presented a feasibility evaluation of continuous biopharmaceutical manufacturing strategies utilising perfusion cell culture and semicontinuous chromatography throughout the product life cycle from Pre‐Clinical to Commercial manufacture. The decision‐support framework was configured to cope not only with individual continuous unit operations but also integrated continuous processes. The framework enabled a rigorous analysis of the feasibility of mAb manufacturing facilities based on the standard batch platform compared to alternative integrated and hybrid continuous manufacturing strategies across a range of manufacturing and company scales so as to represent scenarios of relevance to industry. The analysis determined the key cost contributors in each strategy, as well as the robustness via an operational risk score and environmental indices that act as useful E‐factor benchmarks for continuous processes. This enabled the economic, environmental and operational outputs to be assessed simultaneously. The tool predicts that the complete continuous strategy (ATF‐CC) may find it hard to compete on economic, environmental and robustness fronts for Commercial manufacture especially when multiple trains are required, but offers savings for material supply for product development stages  due to the higher productivities and smaller footprint facilities. In contrast, the hybrid batch and continuous strategies (FB‐CB and ATF‐CB) outperform the continuous strategy for all manufacturing and company scales when weighing up multiple economic, operational and environmental perspectives. The FB‐CB strategy was shown to be the most consistent strategy offering savings at all manufacturing and company scales and is always the preferred manufacturing strategy for the large‐sized company. The ATF‐CB strategy is predicted to offer superior economic benefits for material supply during product development that outweigh its lower robustness and increased Commercial manufacturing costs for the medium and small sized companies as demonstrated in the multi‐attribute analysis. However, the analysis highlighted that if the operational feasibility is considered more important than the economic benefits the hybrid FB‐CB strategy is found to be the preferred strategy for all company scales. This paper demonstrates how the simulation framework acts as a valuable test bed for assessing the potential of novel continuous strategies to cope with different scales of operation and decisional drivers.The analysis demonstrates scenarios where continuous processing can offer COG and operational robustness benefits. Industrial uptake of continuous technologies will also be affected by further considerations that include the the development effort required, technology readiness for large‐scale manufacture , protocols and training to deal with the extra operational complexity, and regulatory concerns.